Multistep Synthesis and in Vitro Anticancer Evaluation of 2-Pyrazolyl-Estradiol Derivatives, Pyrazolocoumarin-Estradiol Hybrids and Analogous Compounds

Article abstract of DOI:10.3390/molecules25184039

Although the hormone independent cytotoxic activity of several estradiol derivatives endowed with a simple substituent at C-2 has been reported so far, 2-heterocyclic and 2,3-condensed analogs are less investigated from both synthetic and pharmacological points of view. Therefore, novel A-ring-connected 2-pyrazoles of estradiol and, for comparison, their structurally simplified non-steroidal pairs were synthesized from estradiol 3-methyl ether and 6-methoxy-1,2,3,4-tetrahydronaphthalene. Friedel-Crafts acetylation of the protected phenolic compounds and subsequent O-demethylation led to ortho-substituted derivatives regioselectively, which were converted to arylhydrazones with phenylhydrazine, 4-tolylhydrazine and 4-chloro-phenylhydrazine, respectively, under microwave conditions. The hydrazones were subjected to cyclization with the Vilsmeier-Haack reagent immediately after preparation and the ring closure/formylation sequence resulted in steroidal and non-steroidal 4⁰-formylpyrazoles in moderate to good yields. During reductive transformations, 4-hydroxymethyl-pyrazoles were obtained, while oxidative lactonization of the 4-formylpyrazole moiety with the phenolic OH in the presence of the Jones reagent afforded A-ring-integrated pyrazolocoumarin hybrids and related analogs. Steroidal pyrazoles, which were produced as C-17 acetates due to acetylation of C-17 OH during the primary Friedel-Crafts reaction, underwent deacetylation in alkaline methanol to furnish 2-heterocyclic estradiol derivatives. Pharmacological studies revealed the overall and cancer cell-specific cytotoxicity of the derivatives and the half maximal inhibitory concentrations were obtained for the most promising compounds.

Full text of DOI:10.3390/molecules25184039

molecules
Article
Multistep Synthesis and In Vitro Anticancer
Evaluation of 2-Pyrazolyl-Estradiol Derivatives,
Pyrazolocoumarin-Estradiol Hybrids and
Analogous Compounds
Barnabás Molnár 1, Mohana Krishna Gopisetty 2 , Dóra Izabella Adamecz 2, Mónika Kiricsi 2
and Éva Frank 1,
*
1
Department of Organic Chemistry, Doctoral School of Chemistry, University of Szeged, Dóm tér 8,
H-6720 Szeged, Hungary; barnabas.molnar@chem.u-szeged.hu
Department of Biochemistry and Molecular Biology, Doctoral School of Biology, University of Szeged,
Közép fasor 52, H-6726 Szeged, Hungary; gmohanakrishna@bio.u-szeged.hu (M.K.G.);
doraadamecz@gmail.com (D.I.A.); kiricsim@bio.u-szeged.hu (M.K.)
2
*
Correspondence: frank@chem.u-szeged.hu; Tel.: +36-62-544-275
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Jacques Lebreton
Received: 6 August 2020; Accepted: 3 September 2020; Published: 4 September 2020
Abstract: Although the hormone independent cytotoxic activity of several estradiol derivatives
endowed with a simple substituent at C-2 has been reported so far, 2-heterocyclic and
2,3-condensed analogs are less investigated from both synthetic and pharmacological points
of view. Therefore, novel A-ring-connected 2-pyrazoles of estradiol and, for comparison,
their structurally simplified non-steroidal pairs were synthesized from estradiol 3-methyl
ether and 6-methoxy-1,2,3,4-tetrahydronaphthalene. Friedel-Crafts acetylation of the protected
phenolic compounds and subsequent O-demethylation led to ortho-substituted derivatives
regioselectively, which were converted to arylhydrazones with phenylhydrazine, 4-tolylhydrazine
and 4-chloro-phenylhydrazine, respectively, under microwave conditions. The hydrazones were
subjected to cyclization with the Vilsmeier-Haack reagent immediately after preparation and the
ring closure/formylation sequence resulted in steroidal and non-steroidal 40-formylpyrazoles in
moderate to good yields. During reductive transformations, 4-hydroxymethyl-pyrazoles were
obtained, while oxidative lactonization of the 4-formylpyrazole moiety with the phenolic OH in
the presence of the Jones reagent afforded A-ring-integrated pyrazolocoumarin hybrids and related
analogs. Steroidal pyrazoles, which were produced as C-17 acetates due to acetylation of C-17
OH during the primary Friedel-Crafts reaction, underwent deacetylation in alkaline methanol to
furnish 2-heterocyclic estradiol derivatives. Pharmacological studies revealed the overall and cancer
cell-specific cytotoxicity of the derivatives and the half maximal inhibitory concentrations were
obtained for the most promising compounds.
Keywords: steroids; heterocyclic estradiol derivatives; Friedel-Crafts acetylation; arylhydrazones;
Vilsmeier-Haack reaction; 4-formyl-pyrazoles; pyrazolocoumarins; anticancer activity
1. Introduction
Natural steroids play an important role in several fields of medicine, including contraception,
treatment of inflammation, asthma, cardiovascular disease, osteoporosis, cancer, and other
disorders [
1,2]. During chemical modifications, steroids are often combined with other drugs or
relevant pharmacophores via covalent bonds either acting as an effective agent or inactive carrier,
or through domain integration of key structural units [
3,
4]. These multifunctional hybrid drugs or
Molecules 2020, 25, 4039; doi:10.3390/molecules25184039
www.mdpi.com/journal/molecules

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“chimeras”, which primarily serve to combat drug resistance, reduce unwanted side effects and enrich
the arsenal of existing medications, offer novel therapeutic options for many diseases, including cancer,
and are usually more active and specific than conventional treatments [5]. Nevertheless, steroids
are privileged structures for molecular hybridization approaches due to their optically pure chiral
character, conformational diversity with a varying possibility of functionalization, a hydrophobic
nature that facilitates cell membrane penetration, and a wide spectrum of bioactivity [6].
In addition to their normal physiological functions, endogenous sex hormones are also involved
in the development and progression of breast, ovarian, and prostate cancers [7]. The incorporation
or connection of different heterocyclic scaffolds to the relatively rigid sterane framework of
natural sex hormones can furnish a dramatic change in the biological properties of the parent
compound [7–9]. Modulation of the binding ability provided by the heteroring via hydrogen bonding
or coordinative interactions is considered a pivotal factor in the development of altered pharmacological
effects [10]. Furthermore, certain pharmacokinetic properties, like water solubility, intestinal absorption,
and stability against metabolic degradation can also be improved. Some heteroaromatic rings are
frequently used as bioisosteres of hydrolysis-sensitive functional groups due to structural mimicry.
In contrast to the effect of natural sex hormones on cell proliferation in hormone-dependent tumors,
the anticancer properties of several sterane-based molecular hybrids [11,12], most of which have been
obtained by modifying the D-ring of the steroid backbone with a heterocycle [7], have been previously
reported. Amongst them, the most well investigated heterocyclic steroids, suitable for the treatment
of advanced prostate cancer, are the androstane-based inhibitors of CYP17, a key regulatory enzyme
involved in the androgen biosynthesis [2,13]. Some D-ring-modified estrone derivatives were also
studied in hormone-dependent breast cancers as inhibitors of the aromatase enzyme responsible for
estrogen production [14]. Besides enzyme inhibitors, a number of androstane- and estrane-based
hybrids containing different heterocyclic motifs connected to, condensed with, or structurally integrated
in the D-ring of the sterane framework were reported to exert direct antiproliferative action in tumor
cells by affecting the apoptotic machinery [15–17]. Nevertheless, whether it is an enzyme inhibitor or a
direct cytotoxic agent, the primary hormonal effect of the parent compound is unfavorable and should
be reduced or eliminated.
Since the functional groups responsible for hormone receptor binding are at C-3 and C-17 in the
A- and D-rings of sex hormones, the chemical modifications made here or at nearby positions seem
reasonable to eliminate the undesirable side effects. Interestingly, far fewer derivatives have been
studied, mainly in the estrone series that contain a heterocyclic moiety attached to the A- instead of
the D-ring [18
including the best-known 2-methoxyestradiol, have been demonstrated to display cytotoxic activity
exempt from hormonal effects (Figure 1) [ 20 22]. The low hormone receptor-binding affinity of these
ligands can be attributed to steric and electronic factors, altered pKa value and disrupted intermolecular
H-bonding to the target protein of the phenolic A-ring [23 24]. The incorporation of a heterocycle
,19]. However, a number of estradiol derivatives having a simple substituent at C-2,
7,
–
,
condensed to the 2,3-position of the estrane skeleton, which permanently abolishes the acidic and
H-donor phenolic OH group, is further expected to result in compounds with diminished estrogenic
activity. Surprisingly, there are only a few literature examples of this type of compound (Figure 1) [19
]
in spite of the excellent reactivity of phenols, especially in aromatic electrophilic substitution reactions,
which may serve as a basis for heterocycle construction.

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Figure 1. A-ring modified derivatives of estradiol displaying anticancer activity and/or reduced
estrogenic action.
In recent years, considerable attention has been devoted to the synthesis of sex hormone-derived
pyrazoles, which have proved to be the most promising of all heterocyclic derivatives for inhibiting
one of the enzymes of steroidogenesis or influencing the cell cycle and inducing apoptosis in tumor
cells [16
open new perspectives in the research of anticancer agents, as the pyrazole ring is a very common
building block in non-steroidal anticancer compounds as well [31 32]. In addition, the incorporation of
,25–30]. The introduction of such a structural element into the 2-position of estradiol may
,
a formyl functionalized pyrazole moiety allows intramolecular cyclization with the phenolic 3-OH
group of the A-ring, leading to domain-integrated pyrazolocoumarin-estradiol hybrid compounds.
Both coumarins and pyrazolocoumarins are valuable heterocyclic units in a number of marketed drugs
and experimental agents with anticancer, antimicrobial, and anti-inflammatory activities [33–35].
Given the aforementioned literature background, the aim of the current study was to develop
an efficient synthetic route for the preparation of novel pyrazole-containing estradiol derivatives
by modifying the phenolic A-ring, and thus to extend the chemical space towards the synthesis
and investigation of a less studied family of compounds. Structurally similar molecules without
C- and D-rings of the sterane core starting from 5,6,7,8-tetrahydro-2-naphthol were also synthesized for
pharmacological comparison. To evaluate the anticancer efficiency of the new derivatives, their cytotoxic
activity was screened in vitro on two prostate (DU145, PC-3) and two gynecological (HeLa, MCF-7)
cell lines as well as on non-cancerous fibroblasts (MRC-5) using MTT assays [36]. Several compounds
showing cancer cell-specific cytotoxicity were identified and the half maximal inhibitory concentrations
for the most promising compounds were finally determined.

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2. Results and Discussion
2.1. Syntheses
For the synthesis of A-ring-modified pyrazole derivatives, 2-acetyl estradiol 17
β-acetate (4) was
first prepared as starting material. Friedel-Crafts regioselective ortho-acetylation of the aromatic
ring of estradiol 3-methyl ether ( ), obtained from its precursor by simple reduction, was carried
2
1
out under mild conditions using a one-pot procedure, further developing the available literature
method [37] (Scheme 1). Estradiol was previously reported to undergo diacetylation during treatment
with acetyl chloride (AcCl), and AlCl3 was found not to be a strong enough Lewis acid to initiate
a Fries rearrangement of the resulting diacetate to compound
moisture-sensitive ZrCl4 had to be used [38 39]. However, it was also demonstrated by Bubert et al.
that the acetylation could be performed in the presence of AlCl3 without difficulty when the phenolic
OH of estradiol was protected as a methyl ether ( ) [37]. Thanks to the strong ortho-directing effect
of the OMe group and because of the sterically less hindered character of C-2 over C-4, the aromatic
electrophilic substitution occurred regioselectively to furnish a 3-protected 2-acetyl derivative ( ) in
good yield. After purification, the aromatic methyl ether ( ) was cleaved in boiling dichloromethane
(DCM) using a chloroaluminate ionic liquid reagent [37]. According to our observation, however,
the acetylation/deprotection sequence can be carried out from with AlCl3 in a single step without
isolating the 2-acetyl-3-methyl ether intermediate ( ) (Scheme 1). Apart from the key role of low
4; thus, the more expensive and
,
2
3
3
2
3
reaction temperature during acetylation in order to access high regioselectivity, the amount of the
Lewis acid used was found to be of crucial importance; i.e., a twofold molar excess of AlCl3 compared
to the steroid favored the rapid formation and isolation of
for complete conversion of to within 3 h at 0
25 ◦C. In contrast to the two-step transformation,
which required two purification steps and an elevated temperature during deprotection, leading to the
desired product ( ) in a ca. 74% yield, our one-pot method proceeded under milder conditions in a
3, while a fourfold excess was needed
2
4
→
4
shorter time, resulting in 4 in 80% yield after recrystallization.
Scheme 1. Multistep synthesis of arylpyrazole derivatives and pyrazolocoumarin hybrids of estradiol.
Reagents and conditions: (i) NaBH4, EtOH, rt, 2 h; (ii) AcCl, AlCl3 (4 equiv.), DCM, N2 atm., 0
→
25 ◦C,
HCl, NaOAc,
4 h; (iii) KOH, MeOH, rt, 2 h (for
4
and 7a
–c
) or 40 ◦C, 3 h (for 10a
–
c); (iv) Ar-NHNH2
·
EtOH, MW, 100 ◦C, 30 min; (v) POCl3, DMF, 0 ◦C, 30 min, then 60 ◦C, 3 h (for 6a and 6b) or 4 h (for 6c);
(vi) Jones reagent, acetone, rt, 10 min.

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After optimization, the synthesis of the 2-acetylestradiol derivative (
4) was repeated on a larger
scale without significant changes to serve as starting material for heterocycle formation. Since the
hydrazones of methyl ketones are suitable precursors of pyrazole-4-carbaldehydes upon treatment
with the Vilsmeier-Haack reagent [26,40], compound 4 was next reacted with three arylhydrazines,
namely phenylhydrazine, 4-tolylhydrazine, and 4-chlorophenylhydrazine, respectively, differing in
the electron demand of their substituents on the aromatic ring (Scheme 1). The reagents were liberated
from their stable hydrochloride salts with NaOAc, and the ethanolic mixtures were irradiated by
microwave (MW) at 100 ◦C. Since the carbonyl-C of the acetophenone moiety has a diminished reactivity
toward nucleophilic attack due to steric and electronic reasons, hydrazone formation was found to
be very sluggish under conventional heating, and only moderate conversion was achieved during
5 h of reflux. However, the MW-assisted condensations took place within 30 min and the crystalline
hydrazones (6a–c) could be filtered off from the mixtures in yields around 75% (Table 1, entries
1–3); higher conversions could not be gained in these cases even by increasing the reaction time.
Although the nucleophilicity of the terminal nitrogen of the arylhydrazines is definitely affected by the
electron-donating CH3 group or electron-withdrawing Cl atom on the aromatic ring, a substituent
effect influencing the yield of the products could not be observed under MW conditions.
Table 1. Synthesis of arylhydrazones from steroidal and non-steroidal methyl ketones and their
conversion to 4-formylpyrazoles.
Starting
Material
Entry
R
Arylhydrazone Yield 1 (%)
4-Formylpyrazole
Yield 2 (%)
75 3
78 3
72 3
38 5
41 5
34 5
1
2
3
4
5
6
1
4
4
4
15
15
15
Ph
6a
6b
6c
16a
16b
16c
76
78
75
7a
7b
7c
17a
17b
17c
p-CH3-C6H4
p-Cl-C6H4
Ph
p-CH3-C6H4
p-Cl-C6H4
4
-
-
4
4
-
Crude isolated products without purification; 2 After chromatographic purification; 3 Yields refer to hydrazones
6a–c; 4 Could not be isolated; 5 Yields refer to 15.
The crude hydrazones were next treated with the Vilsmeier–Haack reagent generated in situ
from dimethylformamide (DMF) and POCl3 at 0 ◦C to afford 1-arylpyrazole-4-carbaldehydes (7a
–c)
in 72–78% yields via ring closure and simultaneous incorporation of two carbon atoms from t◦he
reagent [40] (Scheme 1 and Table 1, entries 1–3). The transformations were carried out at 60 C
for 3 h for 6a and 6b, while 4 h were needed for the conversion of 6c. Considering the proposed
mechanism of the Vilsmeier-Haack reaction of hydrazones [41], the lower reactivity of 6c may be
attributed to the electron-withdrawing effect of the Cl atom, which endows a molecule with reduced
nucleophilic character against electrophilic attack. Thereafter, oxidative lactonization of 7a
the Jones reagent in acetone occurred rapidly to give pyrazolocoumarin steroid hybrids 9a
reductive transformations led to 10a– in good yields. Moreover, intramolecular ether formation was
also tried for 10a under different conditions in order to obtain pyrazolopyran 12a; however, all synthetic
efforts failed. Contrarily, 17-OH analogs of 7a , and 10a were synthesized without difficulty in
–
c
with
–
c, while
c
4
,
–c
–c
alkaline methanol. Since similar deacetylation of lactone 9a did not result in a single product due to
partial ring-opening and parallel methyl ester formation, the idea of preparing 17-OH derivatives
was discarded.
To gain insights into the synthetic and pharmacological differences and to find structure–activity
relationships, thereactionsequencedescribedabovewasalsoperformedwith5,6,7,8-tetrahydro-2-naphthol
(
13), a simplified molecule that structurally mimics the A- and B-rings of the estrane backbone (Scheme 2).
Friedel-Crafts acetylation of the protected starting material (14) occurred regioselectively to furnish
15 42] in 69% yield after purification. In contrast to the similar reaction of the acetylated steroid
the subsequent condensation reactions with arylhydrazines did not result in isolable hydrazones, although
[
4,

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high conversions were observed by TLC monitoring; therefore, the intermediates (16a
–c) were subjected
to 4-formylpyrazole formation under Vilsmeier-Haack conditions. Consequently, the yield of products
17a could only be calculated from the starting material (15) for the two consecutive steps. Although
both transformations proceeded well, lower pyrazole yields (34–41%) were obtained compared to the
reactions on steroids (54–61% for ) due to the impossibility of removing excess reagents after the first step,
–c
4
the difficulty of purification, and the formation of byproducts due to the longer reaction times needed
for heterocyclizations (Table 1, entries 4–6). Reduction of the formyl group in the heteroaromatic ring
of 17a–c with NaBH4 in EtOH furnished hydroxymethyl-substituted derivatives 18a and 18b in good
yields (>80%), while the presence of the electron-withdrawing Cl atom in 17c allowed the formation of
18c only in a moderate yield (66%). A similar substituent effect, albeit to a lesser extent, was also observed
upon conversion of 7c to 10c. The behavior of 17a
from that of the analogous steroidal compounds (7a
–
c
under oxidative conditions was also found to differ
). The low solubility of 17a in acetone at room
–c
–c
temperature required heating of the mixtures, which led to the formation of unidentified byproducts,
with reduced yields of the desired lactones 19a–c.
Scheme 2. Multistep synthesis of non-steroidal arylpyrazole derivatives and pyrazolocoumarins.
Reagents and conditions: (i) Me2SO4, K2CO3, acetone, reflux, 3 h; (ii) AcCl, AlCl3 (4 equiv.), DCM, N2
atm., 0→
25 ◦C, 4 h; (iii) Ar-NHNH2 HCl, NaOAc, EtOH, MW, 100 ◦C, 20 min; (iv) POCl3, DMF, 0 ◦C,
·
30 min, then 60 ◦C, overnight; (v) NaBH4, EtOH, rt, 2 h; (vi) Jones reagent, acetone, reflux, 30 min
(for 17b and 17c).
Structural analyses of all synthesized compounds were carried out by NMR and ESI-MS
measurements. Hydrazones 6a
after formation due to their instability and/or difficulty of isolation. However, the signals in the
deshielded region (>6.8 ppm) of the 1H NMR spectra of 7a
and 17a confirmed the 2,3-disubstituted
–c and 16a–c were not characterized but were transformed immediately
–
c
–c
character of the condensed aromatic A-ring (two singlets for 1-H and 4-H and the appearance of the
phenolic OH), the heterocyclization (a singlet for 50-H at around 8.5 ppm), and the incorporation of a
formyl group into the pyrazole moiety (40-CHO peak at around 10.2 ppm) under the Vilsmeier-Haack
conditions (Table 2). The 13C NMR spectra and the determined molecular weights also provided
evidence for the chemical structures. Reduction of the formyl group by NaBH4 led to derivatives (10a
–c
and 18a ) containing a hydroxymethyl group instead of a formyl group on their heteroaromatic ring,
–
c
so instead of a signal of formyl proton, a singlet corresponding to two equivalent protons (CH2OH)
could be observed around 4.86 ppm in the 1H NMR spectra. Oxidative lactonization is associated
with the disappearance of both CHO and phenolic OH proton signals in 9a–c and 19a–c, while a
negative carbonyl signal around 158.5 ppm in the 13C NMR spectra (J-MOD) confirmed the cyclic
ester formation.

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Table 2. Selected 1H-NMR spectral parameters for steroidal (7a
(17a–c) in CDCl3 at 500 MHz.
–c) and non-steroidal 4-formylpyrazoles
δ (ppm)
Compound
Ar
4-H
(s) 1
1-H
(s) 1
50-H
3-OH
(bs) 2
40-CHO
(s) 1
(s) 1
7a
7b
7c
17a
17b
17c
Ph
6.83
6.82
6.83
6.83
6.82
6.82
7.88
7.88
7.84
7.53
7.55
7.51
8.56
8.51
8.53
8.56
8.50
8.52
9.84
9.89
9.65
9.68
9.69
9.45
10.19
10.17
10.18
10.20
10.19
10.18
p-CH3-C6H4
p-Cl-C6H4
Ph
p-CH3-C6H4
p-Cl-C6H4
1
Singlet; 2 Broad singlet.
2.2. Evaluation of In Vitro AnticancerActivity
Cytotoxicity screens were performed to assess the in vitro anticancer activity of the synthesized
A-ring-linked 2-pyrazoles of estradiol, the pyrazolocoumarin-estradiol hybrids, and their structurally
simplified non-steroidal analogs on various cancerous cell lines such as MCF-7, PC-3, DU145, and HeLa
and on non-cancerous MRC-5 fibroblasts. The 2-acetyl-phenol starting materials (
4, 5, and 15) were also
included for comparison. All compounds were applied in 2.5 M concentration for 72 h. The obtained
µ
cell viability data were used to construct a heat map (Figure 2) in order to select compounds that exert
cancer cell line-specific cytotoxicity. Overall, 10–15 potential molecules (steroids and non-steroids)
were found to be competent against cisplatin-resistant PC-3 and a similar number of compounds were
active on HeLa cells (Figure 2). Furthermore, some compounds discriminated only DU145 cells and
could eliminate these type of prostate cancer cells.
Based on the heat map, two steroidal 4-formylpyrazoles (7c and 8a), the reduced derivative of the
latter compound (11a), and some tetrahydronaphthol-derived molecules (15, 17a, 17c, 18b, and 19a)
were identified that exhibited a minimum of 40% cytotoxicity on at least one cancer cell line and had
no or very mild effect on non-cancerous fibroblasts. To understand structure-function relationships,
we also included the structural analogs of the selected compounds, namely 7a (C-17 acetate of 8a),
8c (C-17 OH counterpart of 7c), and 9a (steroidal pair of 19a) into subsequent analyses. All these
11 compounds (7a, 7c, 8a, 8c, 9a, 11a, 15, 17a, 17c, 18b, and 19a) were further examined to determine
their IC50 concentrations on all the cell lines previously mentioned and their efficacy was compared to
the reference drug cisplatin. For this, compounds were applied on MCF-7, PC-3, DU145, and HeLa
and on non-cancerous MRC-5 fibroblasts in various concentrations for 72 h or were treated with
cisplatin at different concentrations. On the viability data, dose–response curves were fitted (Figure S1)
and IC50 values were calculated accordingly (Table 3). In agreement with the primary cytotoxicity
screen (Table S1), the obtained IC50 concentrations clearly indicated which compound was selectively
effective on one or more cancer cell lines. Non-steroid small molecules 15 and 19a were very potent and
selective on HeLa or DU145 cells, respectively; however, the steroidal compounds 8a and 8c exhibited
a similar selective effect on PC-3 and on HeLa cells, respectively. On the other hand, we identified
compounds, like 7c, which were effective on three cancer cell lines, i.e., on PC-3, DU145, and MCF-7
cells, in significantly lower concentrations than cisplatin, albeit affecting non-cancerous fibroblasts as
well. When the activity of structurally related molecules was compared, we realized that often the
simplified small molecules themselves were able to induce significant toxicity (17a
, 17c); however,
when the same structural motif was incorporated into an estrane backbone, the resulting compounds

Molecules 2020, 25, 4039
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were either more effective on the cancer cells (8a, PC-3) or were able to target a different cell line
8c, HeLa). Typically, acetylated steroidal pyrazoles were less favorable than deacetylated counterparts
due to reduced cancer cell selectivity (7c 10a). As a whole, potent and tumor cell-selective compounds
(
,
were found on cervical and prostate cancer cell lines (9a—HeLa, 11a—PC-3, and 19a—DU145), each of
which is a phenyl-containing derivative on the pyrazole ring, i.e., the substitution of the aromatic ring
proved to be unfavorable in terms of biological effect or selectivity.
Figure 2. Heat map representation of the primary cytotoxic effects of novel A-ring-modified estradiol
derivatives and their structurally simplified non-steroidal pairs on different cell lines (compound
concentration = 2.5 µM; incubation time = 72 h). Control represents the viability of untreated cells.
Table 3. IC50 values (
on non-cancerous MRC-5 cells.
µ
M
±
SD) of the selected compounds determined on various cancer cell lines and
MRC-5
PC-3
DU145
MCF-7
HeLa
7a
7c
8a
8c
9a
5.90 ± 1.0
4.76 ± 1.1
5.12 ± 1.0
5.53 ± 1.0
6.41 ± 1.1
>7.00 ± 1.0
>7.00 ± 1.2
6.26 ± 1.1
5.72 ± 1.1
>7.00 ± 1.2
>7.00 ± 1.5
34.3 ± 1.02
3.63 ± 1.0
2.14 ± 1.1
3.05 ± 1.0
5.08 ± 1.0
4.22 ± 1.0
2.90 ± 1.3
>7.00 ± 1.4
4.30 ± 1.0
4.39 ± 1.0
>7.00 ± 1.5
4.70 ± 1.0
>330 ± 1.01
6.93 ± 1.0
3.37 ± 1.0
6.46 ± 1.0
4.82 ± 1.0
5.66 ± 1.0
>7.00 ± 1.0
>7.00 ± 1.2
5.62 ± 1.0
7.45 ± 1.0
2.70 ± 1.1
1.41 ± 1.1
116.1 ± 1.06
4.49 ± 1.0
1.99 ± 1.1
5.03 ± 1.0
5.34 ± 1.0
4.94 ± 1.0
6.80 ± 1.0
>7.00 ± 1.2
5.17 ± 1.0
4.12 ± 1.0
>7.00 ± 1.0
6.83 ± 1.1
>330 ± 1.03
4.18 ± 1.0
5.32 ± 1.1
4.09 ± 1.0
3.39 ± 1.0
1.55 ± 1.0
4.30 ± 1.0
3.60 ± 1.2
2.20 ± 1.1
6.23 ± 1.0
>7.00 ± 1.1
>7.00 ± 1.1
297.1 ± 1.02
11a
15
17a
17c
18b
19a
cisplatin

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3. Materials and Methods
3.1. Chemistry
3.1.1. General
Chemicals, reagents, and solvents were purchased from commercial suppliers (Sigma-Aldrich
Corporation, St. Louis, MO, USA; TCI, Tokyo, Japan and Alfa Aesar, Haverhill, MA, USA) and
used without further purification. Melting points (Mp) were determined on an SRS Optimelt digital
apparatus (Stanford Research Systems Inc., Sunnyvale, CA, USA) and are uncorrected. For MW-assisted
syntheses, a CEM Discover SP (CEM Corporation, Matthews, NC, USA) laboratory MW reactor was
used with a maximum power of 200 W (running a dynamic control program). Elementary analysis
data were obtained with a PerkinElmer CHN analyzer model 2400 (PerkinElmer Inc, Waltham, MA,
USA). The transformations were monitored by TLC using 0.25 mm thick Kieselgel-G plates (Si 254 F
,
Merck KGaA, Darmstadt, Germany). The compound spots were detected by spraying with 5%
phosphomolybdic acid in 50% aqueous phosphoric acid. Flash chromatographic purifications were
carried out on silica gel 60, 40–63 µm (Merck KGaA, Darmstadt, Germany). NMR spectra were recorded
with a Bruker DRX 500 (Bruker, Billerica, MA, USA) instrument at room temperature in CDCl3 or
DMSO-d6 using residual solvent signals as an internal reference. Chemical shifts are reported in ppm
(δ
scale), and coupling constants (J) are given in Hz. Multiplicities of the 1H signals are indicated as
a singlet (s), a broad singlet (bs), a doublet (d), a triplet (t), or a multiplet (m). 13C NMR spectra are
1H-decoupled and the J-MOD pulse sequence was used for multiplicity editing. In this spin-echo type
experiment, the signal intensity is modulated by the different coupling constants J of carbons depending
on the number of attached protons. Both protonated and unprotonated carbons can be detected
(CH3 and CH carbons appear as positive signals, while CH2 and C carbons as negative signals).
Automated flow injection analyses were performed with an HPLC/MSD system. System accessories:
a micro-well plate autoinjector, an Agilent 1100 micro vacuum degasser (Agilent Technologies, Santa Clara,
CA, USA), a quaternary pump, and a 1946A MSD equipped with an electrospray ion source (ESI) operated
in positive ion mode. ESI parameters were: nebulizing gas N2, at 35 psi; drying gas N2, at 350 ◦C and
12 L/min; capillary voltage 3000 V; fragmentor voltage 70 V. The MSD was operated with a mass range
of m/z 60 620 in scan mode. Samples (0.2 µL) were injected directly into the solvent flow (0.3 mL/min)
−
of acetonitrile/H2O = 70:30 (v/v) with the simultaneous addition of 0.1% formic acid with an automated
needle wash. Agilent LC/MSD Chemstation (C.01.08, Agilent Technologies Inc., Santa Clara, CA, USA)
was used as software to control the system.
3.1.2. General Procedure for the Friedel-Crafts Acetylation/Demethylation of Estradiol 3-Methyl Ether
(2) or 6-Methoxy-1,2,3,4-Tetrahydronaphthalene (14)
Anhydrous AlCl3 (10.5 g, 78.6 mmol) was suspended in dry CH2Cl2 (75 mL) under N2 atmosphere,
and the mixture was cooled to 0 ◦C. Acetyl chloride (3.1 mL, 43.6 mmol) was added slowly, then the
mixture was stirred for 15 min. A solution of
2 (5 g, 17.5 mmol) or 14 (2.6g, 17.6 mmol) in dry CH2Cl2
(25 mL) was added dropwise to the mixture over a period of 10 min, then stirred for 15 min at 0 ◦C,
after which it was allowed to warm to room temperature and stirred for another 3 h. The reaction
mixture was poured onto crushed ice and stirred vigorously for 10 min. The organic layer was
separated, and the remaining aqueous layer was extracted with CH2Cl2 (2
organic layers were washed with brine, dried with anhydrous Na2SO4, and concentrated in vacuo.
×
50 mL). The combined
2-Acetyl-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (4). The crude product was recrystallized from
MeOH. Yield: 4.95 g (80%, white solid); Mp 200–202 ◦C (198–200 ◦C [37]); Anal. Calcd. for C22H28O4
1
(356.46) C, 74.13; H, 7.92; O, 17.95. Found C 74.25; H 7.86. H NMR (CDCl3, 500 MHz):
δ 0.84 (s, 3H,
18-CH3), 1.27–1.58 (overlapping m, 7H), 1.76 (m, 1H), 1.87–1.92 (overlapping m, 2H), 2.07 (s, 3H,
AcO-CH3), 2.15–2.32 (overlapping m, 3H), 2.60 (s, 3H, 2-Ac-CH3), 2.87 (m, 2H, 6-H2), 4.70 (t, 1H,
J = 8.5 Hz, 17-H), 6.69 (s, 1H, 4-H), 7.59 (s, 1H, 1-H), 12.04 (s, 1H, OH); 13C NMR (CDCl3, 125 MHz):

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δ
12.2 (C-18), 21.4 (AcO-CH3), 23.4 (CH2), 26.3 (CH2), 26.7 (2-Ac-CH3), 26.9 (CH2), 27.7 (CH2), 30.0
(CH2), 36.8 (CH2), 38.4 (CH), 43.0 (C-13), 43.5 (CH), 49.9 (CH), 82.7 (C-17), 117.7 (C-4), 118.0 (C-2), 127.4
(C-1), 131.5 (C-10), 147.3 (C-5), 160.2 (C-3), 171.4 (AcO-CO), 204.2 (2-Ac-CO); ESI-MS 357 [M + H]+.
3-Acetyl-5,6,7,8-tetrahydro-2-naphthol (15).
chromatography with hexane/CH2Cl2 = 1:1. Yield: 2.29 g (69%, white solid); Mp 70
The crude product was purified by column
−
72 ◦C
◦
(71
−
72 C [42]); Anal. Calcd. for C12H14O2 (190.24) C 75.76; H 7.42. Found C 75.62; H 7.35.
1H NMR (CDCl3, 500 MHz):
δ
1.78 (t-like m, 4H, 6-H2 and 7-H2), 2.59 (s, 3H, Ac-CH3), 2.71 (m, 2H) and
2.76 (m, 2H): 5-H2 and 8-H2, 6.67 (s, 1H, 1-H), 7.40 (s, 1H, 4-H), 11.97 (s, 1H, OH); 13C NMR (CDCl3,
125 MHz): 22.8 and 23.3: C-6 and C-7, 26.7 (Ac-CH3), 28.7 and 30.1: C-5 and C-8, 117.7 (C-1), 118.1
(C-3), 128.0 (C-4a), 131.0 (C-4), 147.6 (C-8a), 160.0 (C-2), 204.1 (Ac-CO); ESI-MS 191 [M + H]+.
δ
3.1.3. General Procedure for the MW-Assisted Synthesis of Arylhydrazones (6a
–c) and Their Conversion
to Steroidal 4-Formylpyrazoles (7a–c) under Vilsmeier–Haack Conditions
To a solution of
(246 mg, 3.0 mmol) and (p-substituted) phenylhydrazine hydrochloride (3.0 mmol) were added, and the
mixture was irradiated at 100 ◦C for 30 min in a closed tube. Hydrazones 6a
can be obtained by
chilling the reaction vessel, filtering off the yellow precipitate and washing it with ice-cold methanol.
16a could not be isolated; thus, the reaction mixture was evaporated to dryness and was used as-is in
the next step. POCl3 (10.73 mmol, 1.0 mL) was added to DMF (10 mL) cooled to 0 ◦C. After stirring for
15 min, a solution of 6a or 16a (obtained in the previous step) in DMF (5 mL) was added dropwise
to the reaction mixture and was then heated to 60 ◦C and stirred for another 3 h (7a
) or 16 h (17a ).
The mixture was then poured onto crushed ice, extracted with EtOAc (3 25 mL), and the combined
organic phases were washed with brine, dried with anhydrous Na2SO4, and concentrated in vacuo.
4 (713 mg, 2.0 mmol) or 15 (380 mg, 2.0 mmol) in EtOH (5 mL), anhydrous NaOAc
–
c
–
c
–
c
–c
–c
–c
×
The crude products were purified by column chromatography with EtOAc/CH2Cl2 = 1:99 (7a
hexane/CH2Cl2 = 2:8 (17a–c).
–c) or
2-(40-Formyl-10-phenyl-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (7a)
According to the general procedure, phenylhydrazine hydrochloride (435 mg) was used. Yield
of the phenylhydrazone (6a, yellow solid): 678 mg (76%). Yield of 7a (yellow solid): 550 mg (75%).
1
Mp 230
(CDCl3, 500 MHz):
−
232 ◦C; Anal. Calcd. for C30H32N2O4 (484.60) C 74.36; H 6.66. Found C 74.48; H 6.52. H NMR
δ
0.85 (s, 3H, 18-CH3), 1.26–1.61 (overlapping m, 7H), 1.76 (m, 1H), 1.92 (m, 2H),
2.07 (s, 3H, Ac-CH3), 2.20–2.30 (overlapping m, 2H), 2.39 (m, 1H), 2.90 (m, 2H, 6-H2), 4.69 (t, 1H,
J = 8.4 Hz, 17-H), 6.83 (s, 1H, 4-H), 7.43 (t-like m, 1H, 400-H), 7.54 (t-like m, 2H, 300-H and 500-H), 7.88
(d, 2H, J = 7.9 Hz, 200-H and 600-H), 7.88 (s, 1H, 1-H), 8.56 (s, 1H, 50-H), 9.84 (bs, 1H, 3-OH), 10.19 (s, 1H,
CHO); 13C NMR (CDCl3, 125 MHz):
δ 12.2 (C-18), 21.4 (Ac-CH3), 23.4 (CH2), 26.5 (CH2), 27.3 (CH2),
27.8 (CH2), 29.7 (CH2), 36.9 (CH2), 38.6 (CH), 43.0 (C-13), 43.9 (CH), 49.9 (CH), 82.9 (C-17), 113.2 (C-40),
117.1 (C-4), 119.7 (2C, C-200 and C-600), 123.1 (C-2), 126.9 (C-1), 128.5 (C-400), 130.0 (2C, C-300 and C-500),
132.2 (C-10), 132.7 (C-50), 138.4 (C-100), 140.7 (C-5), 153.2 (C-30), 153.8 (C-3), 171.4 (Ac-CO), 184.5 (CHO);
ESI-MS 485 [M + H]+.
2-(40-Formyl-10-(400-tolyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (7b)
According to the general procedure, 4-tolylhydrazine hydrochloride (475 mg) was used. Yield
of the 4-tolylhydrazone (6b, yellow solid): 717 mg (78%). Yield of 7b (white solid): 607 mg (78%).
1
Mp 229
(CDCl3, 500 MHz):
−
231 ◦C; Anal. Calcd. for C31H34N2O4 (498.62) C 74.67; H 6.87. Found C 74.53; H 6.99. H NMR
δ
0.85 (s, 3H, 18-CH3), 1.27–1.59 (overlapping m, 7H), 1.76 (m, 1H), 1.91 (m, 2H),
2.07 (s, 3H, Ac-CH3), 2.20–2.29 (overlapping m, 2H), 2.39 (m, 1H), 2.43 (s, 3H, 400-CH3), 2.90 (m, 2H,
6-H2), 4.69 (t, 1H, J = 8.4 Hz, 17-H), 6.82 (s, 1H, 4-H), 7.32 (d, 2H, J = 8.4 Hz, 300-H and 500-H), 7.59
(d, 2H, J = 8.8 Hz, 200-H and 600-H), 7.88 (s, 1H, 1-H), 8.51 (s, 1H, 50-H), 9.89 (bs, 1H, 3-OH), 10.17 (s,
1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ
12.2 (C-18), 21.2 (400-CH3), 21.4 (Ac-CH3), 23.4 (CH2), 26.4
(CH2), 27.3 (CH2), 27.8 (CH2), 29.7 (CH2), 36.9 (CH2), 38.6 (CH), 43.0 (C-13), 43.9 (CH), 49.9 (CH), 82.9

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(C-17), 113.2 (C-40), 117.1 (C-4), 119.6 (2C, C-200 and C-600), 122.9 (C-2), 126.9 (C-1), 130.5 (2C, C-300 and
C-500), 132.1 (C-10), 132.6 (C-50), 136.1 (C-400), 138.6 (C-100), 140.6 (C-5), 153.0 (C-30), 153.8 (C-3), 171.4
(Ac-CO), 184.5 (CHO); ESI-MS 499 [M + H]+.
2-(10-(400-Chlorophenyl)-40-formyl-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (7c)
According to the general procedure, 4-chlorophenylhydrazine hydrochloride (539 mg) was used.
Yield of the 4-chlorophenylhydrazone (6c, white solid): 722 mg (75%). Yield of 7c (white solid): 560 mg
(72%). Mp 268–270 ◦C; Anal. Calcd. for C30H31ClN2O4 (519.04) C 69.42; H 6.02. Found C 69.29; H
1
6.11. H NMR (CDCl3, 500 MHz):
δ 0.85 (s, 3H, 18-CH3), 1.26–1.59 (overlapping m, 7H), 1.76 (m, 1H),
1.92 (m, 2H), 2.07 (s, 3H, Ac-CH3), 2.20–2.29 (overlapping m, 2H), 2.37 (m, 1H), 2.90 (m, 2H, 6-H2),
4.69 (t, 1H, J = 8.4 Hz, 17-H), 6.83 (s, 1H, 4-H), 7.51 (d, 2H, J = 8.8 Hz, 300-H and 500-H), 7.67 (d, 2H,
J = 8.8 Hz, 200-H and 600-H), 7.84 (s, 1H, 1-H), 8.53 (s, 1H, 50-H), 9.65 (bs, 1H, 3-OH), 10.18 (s, 1H, CHO);
13C NMR (CDCl3, 125 MHz):
δ 12.2 (C-18), 21.4 (Ac-CH3), 23.4 (CH2), 26.5 (CH2), 27.2 (CH2), 27.8
(CH2), 29.7 (CH2), 36.9 (CH2), 38.6 (CH), 43.0 (C-13), 43.8 (CH), 49.9 (CH), 82.9 (C-17), 113.0 (C-40),
117.2 (C-4), 120.9 (2C, C-200 and C-600), 123.3 (C-2), 126.9 (C-1), 130.2 (2C, C-300 and C-500), 132.3 (C-10),
132.6 (C-50), 134.3 (C-400), 136.9 (C-100), 140.9 (C-5), 153.4 (C-30), 153.7 (C-3), 171.4 (Ac-CO), 184.5 (CHO);
ESI-MS 519 [M + H]+.
30-(3-Hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-10-phenyl-10H-pyrazole-40-carbaldehyde (17a)
According to the general procedure, phenylhydrazine hydrochloride (435 mg) was used.
Phenylhydrazone (16a) could not be isolated. Yield of 17a (white solid): 242 mg (38%). Mp 137–138
1
◦C; Anal. Calcd. for C20H18N2O2 (318.38) C 75.45; H 5.70. Found C 75.32; H 5.55. H NMR (CDCl3,
500 MHz):
δ 1.82 (t-like m, 4H, 6-H2 and 7-H2), 2.79 (d-like m, 5-H2 and 8-H2), 6.83 (s, 1H, 4-H),
7.42 (t-like m, 1H, 400-H), 7.53 (m, 3H, 1-H, 300-H and 500-H), 7.72 (d, 2H, J = 7.7 Hz, 200-H and 600-H),
8.56 (s, 1H, 50-H), 9.68 (bs, 1H, 3-OH), 10.20 (s, 1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ 23.1 and 23.5:
C-6 and C-7, 28.8 and 29.6: C-5 and C-8, 113.3 (C-40), 117.2 (C-4), 119.7 (2C, C-200 and C-600), 123.0 (C-2),
128.5 (C-1), 128.8 (C-8a), 130.0 (2C, C-300 and C-500), 130.1 (C-400), 132.0 (C-50), 138.4 (C-100), 140.7 (C-4a),
153.1 (C-30), 153.6 (C-3), 184.9 (CHO); ESI-MS 319 [M + H]+.
30-(3-Hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-10-(400-tolyl)-10H-pyrazole-40-carbaldehyde (17b)
According to the general procedure, 4-tolylhydrazine hydrochloride (475 mg) was used.
4-tolylhydrazone (16b) could not be isolated. Yield of 17b (white solid): 273 mg (41%). Mp 151–153 ◦C;
Anal. Calcd. for C21H20N2O2 (332.40) C 75.88; H 6.06. Found C 76.02; H 5.95. 1H NMR (CDCl3,
500 MHz):
δ
1.82 (t-like m, 4H, 6-H2 and 7-H2), 2.42 (s, 3H, 400-CH3), 2.79 (d-like m, 5-H2 and 8-H2),
6.82 (s, 1H, 4-H), 7.32 (d, 2H, J = 8.5 Hz, 300-H and 500-H), 7.55 (s, 1H, 1-H), 7.59 (d, 2H, J = 8.9 Hz,
200-H and 600-H), 8.50 (s, 1H, 50-H), 9.69 (bs, 1H, 3-OH), 10.19 (s, 1H, CHO); 13C NMR (CDCl3, 125
MHz):
δ
(400-CH3), 23.2 and 23.6: C-6 and C-7, 28.8 and 29.7: C-5 and C-8, 113.5 (C-40), 117.2 (C-4),
119.7 (2C, C-200 and C-600), 122.9 (C-2), 128.8 (C-8a), 130.1 (C-1), 130.5 (2C, C-300 and C-500), 131.9 (C-50),
136.2 (C-400), 138.6 (C-100), 140.9 (C-4a), 153.0 (C-30), 153.7 (C-3), 184.8 (CHO); ESI-MS 333 [M + H]+.
10-(400-Chlorophenyl)-30-(3-hydroxy-5,6,7,8-tetrahydronaphthalen-2-yl)-10H-pyrazole-400-carbaldehyde (17c
)
According to the general procedure, 4-chlorophenylhydrazine hydrochloride (540 mg) was used.
4-chlorophenylhydrazone (16c) could not be isolated. Yield of 17c (white solid): 240 mg (34%).
◦
Mp 182–184 C; Anal. Calcd. for C20H17ClN2O2 (352.82) C 68.09; H 4.86. Found C 67.95; H 4.95.
1H NMR (CDCl3, 500 MHz):
δ
1.82 (t-like m, 4H, 6-H2 and 7-H2), 2.79 (d-like m, 5-H2 and 8-H2), 6.82
(s, 1H, 4-H), 7.49 (d, 2H, J = 8.9 Hz, 300-H and 500-H), 7.51 (s, 1H, 1-H), 7.67 (d, 2H, J = 8.9 Hz, 200-H and
600-H), 8.52 (s, 1H, 50-H), 9.45 (bs, 1H, 3-OH), 10.18 (s, 1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ 23.1
and 23.5: C-6 and C-7, 28.8 and 29.7: C-5 and C-8, 113.3 (C-40), 117.3 (C-4), 120.9 (2C, C-200 and C-600),
123.4 (C-2), 129.0 (C-8a), 130.1 (C-1), 130.2 (2C, C-300 and C-500), 132.0 (C-50), 134.3 (C-400), 137.0 (C-100),
141.3 (C-4a), 153.4 (C-30), 153.6 (C-3), 184.8 (CHO); ESI-MS 353 [M + H]+.

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3.1.4. General Procedure for the Reduction of Steroidal and Non-Steroidal 4-Formylpyrazoles
(7a–c and 17a–c)
4-formylpyrazole (7a–c, 0.30 mmol or 17a–c, 0.50 mmol) was suspended in EtOH (10 mL),
and NaBH4 (45 mg, 1.20 mmol) was added. The solution was stirred at room temperature for 2 h,
then poured into water and neutralized with diluted HCl. The resulting precipitate was filtered,
washed with water, dried, and purified by flash chromatography if necessary.
3-(40-(Hydroxymethyl)-10-phenyl-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17
β-diol-17-acetate (10a)
According to the general procedure, compound 7a (145 mg) was used. After purification with
EtOAc/CH2Cl2 = 5:95 as eluent, 10a was obtained as a white solid (127 mg, 87%). Mp 257–259 ◦C; Anal.
1
Calcd. for C30H34N2O4 (486.61) C 74.05; H 7.04. Found C 73.91; H 7.18. H NMR (CDCl3, 500 MHz):
δ
0.84 (s, 3H, 18-CH3), 1.28–1.57 (overlapping m, 7H), 1.76 (m, 1H), 1.90 (m, 2H), 2.07 (s, 3H, Ac-CH3),
2.20–2.27 (overlapping m, 2H), 2.37 (m, 1H), 2.87 (m, 2H, 6-H2), 4.69 (t, 1H, J = 8.4 Hz, 17-H), 4.87 (s, 2H,
CH2-OH), 6.80 (s, 1H, 4-H), 7.31 (t-like m, 1H, 400-H), 7.47 (t-like m, 2H, 300-H and 500-H), 7.65 (d,
2H, J = 8.0 Hz, 200-H and 600-H), 7.68 (s, 1H, 1-H), 8.02 (s, 1H, 50-H), 10.56 (3-OH); 13C NMR (CDCl3,
125 MHz):
δ 12.2 (C-18), 21.4 (Ac-CH3), 23.4 (CH2), 26.4 (CH2), 27.4 (CH2), 27.7 (CH2), 29.6 (CH2), 37.1
(CH2), 38.7 (CH), 43.0 (C-13), 44.0 (CH), 49.9 (CH), 56.7 (CH2-OH), 82.9 (C-17), 114.4 (C-2), 116.8 (C-4),
118.7 (2C, C-200 and C-600), 120.9 (C-40), 125.0 (C-400), 126.9 (C-1), 127.9 (C-50), 129.8 (2C, C-300 and C-500),
131.6 (C-10), 138.8 (C-5), 139.1 (C-100), 150.6 (C-30), 153.9 (C-3), 171.4 (Ac-CO); ESI-MS 487 [M + H]+.
3-(40-(Hydroxymethyl)-10-(400-tolyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17β-diol-17-acetate (10b)
According to the general procedure, compound 7b (150 mg) was used. After purification with
EtOAc/CH2Cl2 = 5:95 as eluent, 10b was obtained as a white solid (128 mg, 85%). Mp 254–256 ◦C; Anal.
1
Calcd. for C31H36N2O4 (500.64) C 74.37; H 7.25. Found C 74.50; H 7.32. H NMR (CDCl3, 500 MHz):
δ
0.84 (s, 3H, 18-CH3), 1.26–1.57 (overlapping m, 7H), 1.74 (m, 1H), 1.89 (m, 2H), 2.06 (s, 3H, Ac-CH3),
2.16–2.25 (m, 2H), 2.36 (m, 1H), 2.39 (s, 3H, 400-CH3), 2.87 (m, 2H, 6-H2), 4.68 (t, 1H, J = 8.5 Hz, 17-H),
4.84 (d-like m, 2H, CH2OH), 6.79 (s, 1H, 4-H), 7.26 (d, 2H, J = 8.4 Hz, 300-H and 500-H), 7.52 (d, 2H,
J = 8.4 Hz, 200-H and 600-H), 7.67 (s, 1H, 1-H), 7.96 (s, 1H, 50-H); 13C NMR (CDCl3, 125 MHz):
δ 12.2
(C-18), 21.1 (400-CH3), 21.4 (Ac-CH3), 23.4 (CH2), 26.3 (CH2), 27.3 (CH2), 27.7 (CH2), 29.6 (CH2), 37.0
(CH2), 38.7 (CH), 43.0 (C-13), 44.0 (CH), 49.9 (CH), 56.7 (CH2-OH), 82.9 (C-17), 114.5 (C-2), 116.8 (C-4),
118.7 (2C, C-200 and C-600), 120.6 (C-40), 125.0 (C-1), 127.8 (C-50), 130.2 (2C, C-300 and C-500), 131.6 (C-400),
136.8 (C-10), 136.9 (C-100), 138.7 (C-5), 150.2 (C-30), 153.8 (C-3), 171.4 (Ac-CO); ESI-MS 501 [M + H]+.
3-(10-(400-chlorophenyl)-40-(hydroxymethyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-triene-3,17
β-diol-17-acetate (10c)
According to the general procedure, compound 7c (156 mg) was used. After purification with
EtOAc/CH2Cl2 = 5:95 as eluent, 10c was obtained as a white solid (120 mg, 77%). Mp 288–290 ◦C; Anal.
1
Calcd. for C30H33ClN2O4 C 69.15; H 6.38. Found C 69.03; H 6.27. H NMR (DMSO-d6, 500 MHz):
δ 0.79
(s, 3H, 18-CH3), 1.28–1.43 (overlapping m, 6H), 1.49 (m, 1H), 1.69 (m, 1H), 1.77 (m, 1H), 1.82 (m, 1H),
2.01 (s, 3H, Ac-CH3), 2.11 (m, 1H), 2.19 (m, 1H), 2.30 (m, 1H), 2.79 (m, 2H, 6-H2), 4.51 (d-like m, 2H,
CH2OH), 4.61 (t, 1H, J = 8.4 Hz, 17-H), 5.15 (t-like m, 1H, CH2OH), 6.65 (s, 1H, 4-H), 7.56 (s, 1H, 1-H),
7.57 (d, 2H, J = 8.9 Hz, 300-H and 500-H), 7.87 (d, 2H, J = 8.9 Hz, 200-H and 600-H), 8.54 (s, 1H, 50-H),
9.93 (3-OH); 13C NMR (DMSO-d6, 125 MHz):
δ 12.0 (C-18), 20.9 (Ac-CH3), 22.8 (CH2), 25.8 (CH2), 26.8
(CH2), 27.2 (CH2), 28.9 (CH2), 36.5 (CH2), 38.2 (CH), 42.5 (C-13), 43.3 (CH), 49.1 (CH), 54.7 (CH2-OH),
81.9 (C-17), 115.5 (C-2), 115.8 (C-4), 119.6 (2C, C-200 and C-600), 123.2 (C-40), 126.5 (C-1), 128.3 (C-50),
129.6 (2C, C-300 and C-500), 130.2 and 130.8: C-400 and C-10, 137.9 (2C): C-5 and C-100, 150.0 (C-30), 152.9
(C-3), 171.4 (Ac-CO); ESI-MS 521 [M + H]+.

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3-(40-(Hydroxymethyl)-10-phenyl-10H-pyrazol-30-yl)-5,6,7,8-tetrahydro-2-naphthol (18a)
According to the general procedure, compound 17a (159 mg) was used. After purification with
EtOAc/CH2Cl2 = 2:98 as eluent, 18a was obtained as a white solid (133 mg, 83%). Mp 135–137 ◦C;
Anal. Calcd. for C20H20N2O2 (320.39) C 74.98; H 6.29. Found C 75.12; H 6.16. 1H NMR (CDCl3,
500 MHz):
δ 1.80 (m, 4H, 6-H2 and 7-H2), 2.76 (d-like m, 4H, 5-H2 and 8-H2), 4.87 (s, 2H, CH2-OH),
6.79 (s, 1H, 1-H), 7.31 (t-like m, 1H, 400-H), 7.37 (s, 1H, 4-H), 7.46 (t-like m, 2H, 300-H and 500-H), 7.64
(d, 2H, J = 7.9 Hz, 200-H and 600-H), 8.00 (s, 1H, 50-H); 13C NMR (CDCl3, 125 MHz):
δ 23.3 and 23.6: C-6
and C-7, 29.0 and 29.5: C-5 and C-8, 56.8 (CH2-OH), 114.6 (C-3), 116.9 (C-1), 118.7 (2C, C-200 and C-600),
121.1 (C-40), 126.9 (C-4), 127.8 (C-50), 128.1 (C-400), 128.2 (C-4a), 129.7 (2C, C-300 and C-500), 139.1 (C-100),
139.2 (C-8a), 150.1 (C-30), 153.7 (C-2); ESI-MS 321 [M + H]+.
3-(40-(Hydroxymethyl)-10-(400-tolyl)-10H-pyrazol-3-yl)-5,6,7,8-tetrahydro-2-naphthol (18b)
According to the general procedure, compound 17b (166 mg) was used. After purification with
EtOAc/CH2Cl2 = 2:98 as eluent, 18b was obtained as a white solid (139 mg, 83%). Mp 163–165 ◦C; Anal.
1
Calcd. for C21H22N2O2 (334.42) C 75.42; H 6.63. Found C 75.55; H 6.51. H NMR (CDCl3, 500 MHz):
δ
1.80 (m, 4H, 6-H2 and 7-H2), 2.39 (s, 3H, 400-CH3), 2.76 (d-like m, 4H, 5-H2 and 8-H2), 4.86 (s, 2H,
CH2-OH), 6.79 (s, 1H, 1-H), 7.26 (d, 2H, J = 8.5 Hz, 300-H and 500-H), 7.37 (s, 1H, 4-H), 7.52 (d, 2H,
J = 8.5 Hz, 200-H and 600-H), 7.96 (s, 1H, 50-H); 13C NMR (CDCl3, 125 MHz):
δ
21.1 (400-CH3), 23.3 and
23.7: C-6 and C-7, 29.0 and 29.6: C-5 and C-8, 56.8 (CH2-OH), 114.8 (C-3), 116.9 (C-1), 118.8 (2C, C-200
and C-600), 120.8 (C-40), 127.8 (C-4), 128.1 (C-50), 128.2 (C-4a), 130.2 (2C, C-300 and C-500), 136.8 (C-400),
137.0 (C-100), 139.1 (C-8a), 149.9 (C-30), 153.8 (C-2); ESI-MS 335 [M + H]+.
3-(10-(400-Chlorophenyl)-40-(hydroxymethyl)-10H-pyrazol-30-yl)-5,6,7,8-tetrahydro-2-naphthol (18c)
According to the general procedure, compound 17c (176 mg) was used. After purification with
EtOAc/CH2Cl2 = 2:98 as eluent, 18c was obtained as a white solid (117 mg, 66%). Mp 154–156 ◦C; Anal.
1
Calcd. for C20H19ClN2O2 (354.83) C 67.70; H 5.40. Found C 67.82; H 5.27. H NMR (CDCl3, 500 MHz):
δ
1.80 (m, 4H, 6-H2 and 7-H2), 2.76 (d-like m, 4H, 5-H2 and 8-H2), 4.86 (s, 2H, CH2-OH), 6.79 (s, 1H,
1-H), 7.34 (s, 1H, 4-H), 7.43 (d, 2H, J = 9.0 Hz, 300-H and 500-H), 7.57 (d, 2H, J = 9.0 Hz, 200-H and 600-H),
7.97 (s, 1H, 50-H); 13C NMR (CDCl3, 125 MHz):
δ 23.3 and 23.7: C-6 and C-7, 29.0 and 29.6: C-5 and C-8,
56.8 (CH2-OH), 114.5 (C-3), 117.0 (C-1), 119.9 (2C, C-200 and C-600), 121.6 (C-40), 127.7 (C-4), 128.2 (C-50),
128.4 (C-4a), 129.9 (2C, C-300 and C-500), 132.4 (C-400), 137.8 (C-100), 139.5 (C-8a), 150.4 (C-30), 153.8 (C-2);
ESI-MS 355 [M + H]+.
3.1.5. General Procedure for the Oxidative Lactonization of Steroidal and Non-steroidal 4-Formylpyrazoles
(7a–c and 17a–c)
4-formylpyrazole (7a
Jones reagent was added dropwise into the solution until its color remained. The mixture was then
stirred at room temperature for 10 min (7a ) or kept at reflux temperature for 30 min (17a ). After
the given reaction time, the solution was diluted with water (15 mL). The precipitate that formed was
extracted with EtOAc (3 10 mL), and the combined organic phases were washed with brine (10 mL),
–c, 0.30 mmol or 17a–c, 0.50 mmol) was suspended in acetone (10 mL) and
–c
–c
×
then dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product (9a
–c or 19a–c) was
purified by column chromatography with EtOAc/hexane = 20:80.
11β-Acetoxy-2-phenylpyrazolo [3,4:4,5]pyrano [2,3:3,2]estra-1,3,5(10)-triene-4(2H)-one (9a)
According to the general procedure, compound 7a (145 mg) was used. Compound 9a was obtained
as a white solid (106 mg, 73%). Mp 198–200 ◦C; Anal. Calcd. for C30H30N2O4 (482.58) C 74.67; H 6.27.
1
Found C 74.80; H 6.18. H NMR (CDCl3, 500 MHz):
δ 0.86 (s, 3H, 11a-CH3), 1.28–1.64 (overlapping m,
7H), 1.78 (m, 1H), 1.95 (m, 2H), 2.07 (s, 3H, 11-OAc-CH3), 2.19–2.35 (overlapping m, 2H), 2.51 (m, 1H),
2.97 (m, 2H, 7-H2), 4.71 (t, 1H, J = 8.5 Hz, 11-H), 7.10 (s, 1H, 6-H), 7.44 (t-like m, 1H, 40-H), 7.55 (t-like

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m, 2H, 30-H and 50-H), 7.84 (d, 2H, J = 8.0 Hz, 20-H and 60-H), 8.05 (s, 1H, 14-H), 8.65 (s, 1H, 3-H);
13C NMR (CDCl3, 125 MHz):
12.2 (11a-CH3), 21.4 (11-OAc-CH3), 23.4 (CH2), 26.4 (CH2), 27.1 (CH2),
δ
27.7 (CH2), 29.9 (CH2), 36.9 (CH2), 38.3 (CH), 43.0 (C-11a), 44.1 (CH), 50.0 (CH), 82.8 (C-11), 109.4
(C-3a), 112.1 (C-14a), 117.3 (C-6), 119.6 (C-40), 120.6 (2C, C-20 and C-60), 128.6 (C-14), 129.6 (C-3), 130.0
(2C, C-30 and C-50), 137.5 (C-13b), 139.4 (C-10), 140.7 (C-6a), 150.4 and 151.3 (C-5a, C-14b), 158.5 (C-4),
171.4 (11a-OAc-CO); ESI-MS 483 [M + H]+.
11β-Acetoxy-2-(40-tolyl)-pyrazolo[3,4:4,5]pyrano[2,3:3,2]estra-1,3,5(10)-triene-4(2H)-one (9b)
According to the general procedure, compound 7b (150 mg) was used. Compound 9b was
obtained as a white solid (103 mg, 69%). Mp 264–266 ◦C; Anal. Calcd. for C31H32N2O4 (496.61) C
1
74.98; H 6.50. Found C 75.12; H 6.38. H NMR (CDCl3, 500 MHz):
δ 0.86 (s, 3H, 11a-CH3), 1.28–1.65
(overlapping m, 7H), 1.77 (m, 1H), 1.95 (m, 2H), 2.07 (s, 3H, 11-OAc-CH3), 2.20–2.33 (overlapping m,
2H), 2.43 (s, 3H, 40-CH3), 2.50 (m, 1H), 2.97 (m, 2H, 7-H2), 4.71 (t, 1H, J = 8.5 Hz, 11-H), 7.09 (s, 1H,
6-H), 7.34 (d, 2H, J = 8.4 Hz, 30-H and 50-H), 7.71 (d, 2H, J = 8.4 Hz, 20-H and 60-H), 8.04 (s, 1H, 14-H),
8.60 (s, 1H, 3-H); 13C NMR (CDCl3, 125 MHz): 12.2 (11a-CH3), 21.2 (40-CH3), 21.4 (11-OAc-CH3), 23.4
(CH2), 26.4 (CH2), 27.1 (CH2), 27.7 (CH2), 29.9 (CH2), 36.9 (CH2), 38.3 (CH), 43.0 (C-11a), 44.1 (CH),
50.0 (CH), 82.8 (C-11), 109.2 (C-3a), 112.1 (C-14a), 117.2 (C-6), 119.6 (C-14), 120.4 (2C, C-20 and C-60),
129.4 (C-3), 130.4 (2C, C-30 and C-50), 137.1 (C-40), 137.4 (C-13b), 138.8 (C-10), 140.5 (C-6a), 150.3 and
151.3 (C-5a, C-14b), 158.5 (C-4), 171.4 (11a-OAc-CO); ESI-MS 497 [M + H]+.
11β )
-Acetoxy-2-(40-chlorophenyl)-pyrazolo[3,4:4,5]pyrano[2,3-3,2]estra-1,3,5(10)-triene-4(2H)-one (9c
According to the general procedure, compound 7c (156 mg) was used. Compound 9c was obtained
as a white solid (112 mg, 72%). Mp 283–285 ◦C; Anal. Calcd. for C30H29ClN2O4 (517.02) C 69.69; H 5.65.
1
Found C 69.81; H 5.52. H NMR (CDCl3, 500 MHz):
δ 0.86 (s, 3H, 11a-CH3), 1.27–1.65 (overlapping m,
7H), 1.78 (m, 1H), 1.95 (m, 2H), 2.06 (s, 3H, 11-OAc-CH3), 2.21–2.34 (overlapping m, 2H), 2.49 (m, 1H),
2.97 (m, 2H, 7-H2), 4.71 (t, 1H, J = 8.5 Hz, 11-H), 7.09 (s, 1H, 6-H), 7.52 (d, 2H, J = 8.8 Hz, 30-H and
50-H), 7.79 (d, 2H, J = 8.8 Hz, 20-H and 60-H), 8.03 (s, 1H, 14-H), 8.62 (s, 1H, 3-H); 13C NMR (CDCl3,
125 MHz):
δ 12.2 (11a-CH3), 21.4 (11-OAc-CH3), 23.4 (CH2), 26.4 (CH2), 27.0 (CH2), 27.7 (CH2), 29.9
(CH2), 36.9 (CH2), 38.3 (CH), 43.0 (C-11a), 44.1 (CH), 50.0 (CH), 82.7 (C-11), 109.4 (C-3a), 111.8 (C-14a),
117.3 (C-6), 119.6 (C-14), 121.6 (2C, C-20 and C-60), 129.5 (C-3), 130.1 (2C, C-30 and C-50), 134.3 (C-40),
137.6 (C-13b), 137.9 (C-10), 140.9 (C-6a), 150.6 and 151.3 (C-5a, C-14b), 158.2 (C-4), 171.4 (11a-OAc-CO);
ESI-MS 518 [M + H]+.
2-Phenyl-7,8,9,10-tetrahydrobenzo[6,7]chromeno[4,3-c]pyrazol-4(2H)-one (19a)
According to the general procedure, compound 17a (159 mg) was used. Compound 19a was
obtained as a white solid (101 mg, 64%). Mp 221–223 ◦C; Anal. Calcd. for C20H16N2O2 (316.36) C
1
75.93; H 5.10. Found C 76.09; H 5.19. H NMR (CDCl3, 500 MHz):
δ 1.84 (m, 4H, 8-H2 and 9-H2), 2.87
(d-like m, 4H, 7-H2 and 10-H2), 7.09 (s, 1H, 6-H), 7.44 (t-like m, 1H, 40-H), 7.55 (t-like m, 2H, 30-H and
50-H), 7.83 (d, 2H, J = 7.9 Hz, 20-H and 60-H), 7.85 (s, 1H, 11-H), 8.64 (s, 1H, 3-H); 13C NMR (CDCl3,
125 MHz):
δ 22.9 and 23.2: C-8 and C-9, 29.0 and 29.9: C-7 and C-10, 109.5 (C-3a), 112.0 (C-11a), 117.4
(C-6), 117.4 (2C, C-20 and C-60), 122.8 (C-40), 128.6 (C-11), 129.6 (C-3), 129.9 (2C, C-30 and C-50), 134.1
(C-10a), 139.4 (C-10), 140.9 (C-6a), 150.3 (C-5a), 151.2 (C-11b), 158.5 (C-4); ESI-MS 317 [M + H]+.
2-(40-Tolyl)-7,8,9,10-tetrahydrobenzo[6,7]chromeno[4,3-c]pyrazol-4(2H)-one (19b)
According to the general procedure, compound 17b (166 mg) was used. Compound 19b was
obtained as a white solid (89 mg, 54%). Mp 224–226 ◦C; Anal. Calcd. for C21H18N2O2 (330.39) C 76.34;
1
H 5.49. Found C 76.48; H 5.36. H NMR (CDCl3, 500 MHz):
δ
1.84 (m, 4H, 8-H2 and 9-H2), 2.43 (s, 3H,
40-CH3), 2.86 (d-like m, 4H, 7-H2 and 10-H2), 7.08 (s, 1H, 6-H), 7.33 (d, 2H, J = 8.5 Hz, 30-H and 50-H),
7.69 (d, 2H, J = 8.5 Hz, 20-H and 60-H), 7.84 (s, 1H, 11-H), 8.59 (s, 1H, 3-H); 13C NMR (CDCl3, 125 MHz):
δ
21.2 (40-CH3), 23.0 and 23.2: C-8 and C-9, 29.0 and 29.9: C-7 and C-10, 109.3 (C-3a), 112.2 (C-11a),

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117.3 (C-6), 120.4 (2C, C-20 and C-60), 122.8 (C-11), 129.4 (C-3), 130.4 (2C, C-30 and C-50), 134.0 (C-10a),
137.2 (C-40), 138.7 (C-10), 140.8 (C-6a), 150.2 (C-5a), 151.2 (C-11b), 158.5 (C-4); ESI-MS 331 [M + H]+.
2-(40-Chlorophenyl)-7,8,9,10-tetrahydrobenzo[6,7]chromeno[4,3-c]pyrazol-4(2H)-one (19c)
According to the general procedure, compound 17c (176 mg) was used. Compound 19c was
obtained as a white solid (89 mg, 51%). Mp 291–293 ◦C; Anal. Calcd. for C20H15ClN2O2 (350.80) C
1
68.48; H 4.31. Found C 68.60; H 4.22. H NMR (CDCl3, 500 MHz): δ1.85 (m, 4H, 8-H2 and 9-H2), 2.87
(d-like m, 4H, 7-H2 and 10-H2), 7.09 (s, 1H, 6-H), 7.52 (d, 2H, J = 9.0 Hz, 30-H and 50-H), 7.79 (d, 2H,
J = 9.0 Hz, 20-H and 60-H), 7.83 (s, 1H, 11-H), 8.61 (s, 1H, 3-H); 13C NMR (CDCl3, 125 MHz):
δ 22.9 and
23.2: C-8 and C-9, 29.0 and 30.0: C-7 and C-10, 109.9 (C-3a), 111.9 (C-11a), 117.4 (C-6), 121.6 (2C, C-20
and C-60), 122.9 (C-11), 129.5 (C-3), 130.1 (2C, C-30 and C-50), 134.2 (C-10a), 134.4 (C-40), 138.0 (C-10),
141.2 (C-6a), 150.5 (C-5a), 151.3 (C-11b), 158.3 (C-4); ESI-MS 351 [M + H]+.
3.1.6. General Procedure for the Deacetylation of Compounds 4, 7a–c and 10a–c
Compound
(50 mg, 0.89 mmol) was added. The solution was stirred at room temperature for 2 h (for
or 40 ◦C for 3 h (for 10a
), then poured into water and neutralized with diluted HCl. The resulting
4
(0.50 mmol), 7a
–c
or 10a
–c
(0.30 mmol) was dissolved in MeOH (10 mL), and KOH
4
and 7a–c)
–c
precipitate was filtered off, washed with water, and dried.
2-Acetyl-estra-1,3,5(10)-triene-3,17β-diol (5)
According to the general procedure, compound 4 (178 mg) was used. Yield (5): 141 mg (90%, white
solid); Mp 189–191 ◦C (192–195 ◦C [38]); Anal. Calcd. for C20H26O3 (314.43) C 76.40; H 8.34. Found C
1
76.56; H 8.25. H NMR (CDCl3, 500 MHz):
δ 0.79 (s, 3H, 18-CH3), 1.17–1.59 (overlapping m, 7H), 1.71
(m, 1H), 1.89 (m, 1H), 1.99 (m, 1H), 2.10–2.19 (overlapping m, 2H), 2.34 (m, 1H), 2.60 (s, 3H, Ac-CH3),
2.85 (m, 2H, 6-H2), 3.74 (t, 1H, J = 8.6 Hz, 17-H), 6.69 (s, 1H, 4-H), 7.61 (s, 1H, 1-H), 12.04 (s, 1H, OH);
13C NMR (CDCl3, 125 MHz):
δ 11.2 (C-18), 23.2 (CH2), 26.4 (CH2), 26.6 (Ac-CH3), 26.9 (CH2), 30.1
(CH2), 30.7 (CH2), 36.7 (CH2), 38.7 (CH), 43.3 (C-13), 43.6 (CH), 50.2 (CH), 81.9 (C-17), 117.7 (C-4), 118.0
(C-2), 127.4 (C-1), 131.7 (C-10), 147.4 (C-5), 160.1 (C-3), 204.2 (Ac-CO); ESI-MS 315 [M + H]+.
2-(40-Formyl-10-phenyl-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (8a)
According to the general procedure, compound 7a (145 mg) was used. Yield (8a): 118 mg
(89%, white solid); Mp 172–174 ◦C; Anal. Calcd. for C28H30N2O3 (442.56) C 75.99; H 6.83. Found
1
C 76.08; H 6.74. H NMR (CDCl3, 500 MHz):
δ 0.81 (s, 3H, 18-CH3), 1.30–1.43 (overlapping m, 4H),
1.45–1.63 (overlapping m, 3H), 1.72 (m, 1H), 1.92 (m, 1H), 1.99 (m, 1H), 2.13 (m, 1H), 2.25 (m, 1H), 2.43
(m, 1H), 2.90 (m, 2H, 6-H2), 3.76 (t, 1H, J = 8.5 Hz, 17-H), 6.83 (s, 1H, 4-H), 7.43 (t-like m, 1H, 400-H), 7.55
(t-like m, 2H, 300-H and 500-H), 7.73 (d, 2H, J = 7.9 Hz, 200-H and 600-H), 7.90 (s, 1H, 1-H), 8.57 (s, 1H,
50-H), 10.20 (s, 1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ 11.1 (C-18), 23.2 (CH2), 26.5 (CH2), 27.2
(CH2), 29.6 (CH2), 30.6 (CH2), 36.7 (CH2), 38.8 (CH), 43.3 (C-13), 43.9 (CH), 50.1 (CH), 81.9 (C-17), 113.0
(C-40), 117.0 (C-4), 119.6 (2C, C-200 and C-600), 123.0 (C-2), 126.8 (C-1), 128.4 (C-400), 129.9 (2C, C-300 and
C-500), 132.2 (C-10), 132.7 (C-50), 138.2 (C-100), 140.7 (C-5), 153.1 (C-30), 153.7 (C-3), 184.4 (CHO); ESI-MS
443 [M + H]+.
2-(40-Formyl-10-(400-tolyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (8b)
According to the general procedure, compound 7b (150 mg) was used. Yield (8b): 123 mg (91%,
white solid); Mp > 190 ◦C (decomp.); Anal. Calcd. for C29H32N2O3 (456.59) C 76.29; H 7.06. Found
1
C 76.43; H 6.95. H NMR (CDCl3, 500 MHz):
δ 0.80 (s, 3H, 18-CH3), 1.30–1.43 (overlapping m, 4H),
1.44–1.62 (overlapping m, 3H), 1.71 (m, 1H), 1.91 (m, 1H), 1.99 (m, 1H), 2.13 (m, 1H), 2.25 (m, 1H), 2.42
(m, 1H), 2.43 (s, 3H, 400-CH3), 2.90 (m, 2H, 6-H2), 3.75 (t, 1H, J = 8.5 Hz, 17-H), 6.83 (s, 1H, 4-H), 7.32
(d, 2H, J = 8.5 Hz, 300-H and 500-H), 7.59 (d, 2H, J = 8.5 Hz, 200-H and 600-H), 7.90 (s, 1H, 1-H), 8.52

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(s, 1H, 50-H), 9.90 (bs, 1H, 3-OH), 10.20 (s, 1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ
11.2 (C-18), 21.2
(400-CH3), 23.3 (CH2), 26.6 (CH2), 27.3 (CH2), 29.7 (CH2), 30.7 (CH2), 36.8 (CH2), 38.9 (CH), 43.4 (C-13),
44.0 (CH), 50.2 (CH), 82.0 (C-17), 113.2 (C-40), 117.1 (C-4), 119.6 (2C, C-200 and C-600), 122.9 (C-2), 126.9
(C-1), 130.5 (2C, C-300 and C-500), 132.2 (C-10), 132.6 (C-50), 136.1 (C-400), 138.7 (C-100), 140.7 (C-5), 153.1
(C-30), 153.8 (C-3), 184.6 (CHO); ESI-MS 457 [M + H]+.
2-(10-(400-Chlorophenyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (8c)
According to the general procedure, compound 7c (156 mg) was used. Yield (8c): 124 mg (87%,
white solid); Mp > 190 ◦C (decomp.); Anal. Calcd. for C28H29ClN2O3 (477.00) C 70.50; H 6.13. Found
1
C 70.62; H 6.04. H NMR (CDCl3, 500 MHz):
δ 0.80 (s, 3H, 18-CH3), 1.30–1.43 (overlapping m, 4H),
1.44–1.62 (overlapping m, 3H), 1.72 (m, 1H), 1.91 (m, 1H), 1.99 (m, 1H), 2.13 (m, 1H), 2.24 (m, 1H),
2.41 (m, 1H), 2.90 (m, 2H, 6-H2), 3.75 (t, 1H, J = 8.5 Hz, 17-H), 6.83 (s, 1H, 4-H), 7.50 (d, 2H, J = 8.9
Hz, 300-H and 500-H), 7.66 (d, 2H, J = 8.9 Hz, 200-H and 600-H), 7.85 (s, 1H, 1-H), 8.53 (s, 1H, 50-H), 9.66
(bs, 1H, 3-OH), 10.18 (s, 1H, CHO); 13C NMR (CDCl3, 125 MHz):
δ 11.2 (C-18), 23.3 (CH2), 26.6 (CH2),
27.3 (CH2), 29.7 (CH2), 30.7 (CH2), 36.8 (CH2), 38.9 (CH), 43.4 (C-13), 44.0 (CH), 50.2 (CH), 82.0 (C-17),
113.0 (C-40), 117.2 (C-4), 120.8 (2C, C-200 and C-600), 123.3 (C-2), 126.9 (C-1), 130.2 (2C, C-300 and C-500),
132.4 (C-10), 132.7 (C-50), 134.2 (C-400), 136.9 (C-100), 141.0 (C-5), 153.5 (C-30), 153.7 (C-3), 184.5 (CHO);
ESI-MS 478 [M + H]+.
3-(40-(Hydroxymethyl)-10-phenyl-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (11a)
According to the general procedure, compound 10a (146 mg) was used. Yield (11a): 121 mg (91%,
white solid); Mp 141–143 ◦C; Anal. Calcd. for C28H32N2O3 (444.58) C 75.65; H 7.26. Found C 75.74;
1
H 7.13. H NMR (DMSO-d6, 500 MHz):
δ 0.68 (s, 3H, 18-CH3), 1.10–1.44 (overlapping m, 7H), 1.59
(m, 1H), 1.80–1.90 (overlapping m, 3H), 2.14 (m, 1H), 2.33 (m, 1H), 2.77 (m, 2H, 6-H2), 3.54 (m, 1H,
17-H), 4.51 (d, 1H, J = 4.8 Hz, 17-OH), 4.56 (d, 2H, J = 4.9 Hz, CH2-OH), 5.15 (t, 1H, J = 4.9 Hz, CH2-OH),
6.65 (s, 1H, 4-H), 7.31 (t-like m, 1H, 400-H), 7.53 (t-like m, 2H, 300-H and 500-H), 7.65 (s, 1H, 1-H), 7.83
(d, 2H, J = 8.0 Hz, 200-H and 600-H), 8.60 (s, 1H, 50-H), 10.14 (3-OH); 13C NMR (DMSO-d6, 125 MHz):
δ
11.3 (C-18), 22.8 (CH2), 26.0 (CH2), 26.9 (CH2), 28.9 (CH2), 29.9 (CH2), 36.6 (CH2), 38.6 (CH), 42.8
(C-13), 43.6 (CH), 49.6 (CH), 54.7 (CH2-OH), 80.1 (C-17), 115.2 (C-2), 115.8 (C-4), 118.0 (2C, C-200 and
C-600), 122.6 (C-40), 126.2 (2C, C-400 and C-1), 128.4 (C-50), 129.7 (2C, C-300 and C-500), 131.1 (C-10), 137.9
(C-5), 139.0 (C-100), 149.8 (C-30), 152.9 (C-3); ESI-MS 445 [M + H]+.
3-(40-(Hydroxymethyl)-10-(400-tolyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (11b)
According to the general procedure, compound 10b (150 mg) was used. Yield (11b): 122 mg (89%,
white solid); Mp 201–203 ◦C; Anal. Calcd. for C29H34N2O3 (458.60) C 75.95; H 7.47. Found C 76.07;
1
H 7.39. H NMR (DMSO-d6, 500 MHz):
δ 0.69 (s, 3H, 18-CH3), 1.11–1.43 (overlapping m, 7H), 1.60
(m, 1H), 1.81–1.94 (overlapping m, 3H), 2.16 (m, 1H), 2.34 (m, 1H), 2.36 (s, 3H, 400-CH3), 2.78 (m, 2H,
6-H2), 3.55 (m, 1H, 17-H), 4.52 (d, 1H, J = 4.9 Hz, 17-OH), 4.56 (d, 2H, J = 4.9 Hz, CH2OH), 5.18 (t, 1H,
J = 4.9 Hz, CH2OH), 6.65 (s, 1H, 4-H), 7.34 (d, 2H, J = 8.5 Hz, 300-H and 500-H), 7.67 (s, 1H, 1-H), 7.72
(d, 2H, J = 8.5 Hz, 200-H and 600-H), 7.96 (s, 1H, 50-H), 9.95 (3-OH); 13C NMR (DMSO-d6, 125 MHz):
δ
11.3 (C-18), 20.4 (400-CH3), 22.8 (CH2), 26.0 (CH2), 26.9 (CH2), 28.9 (CH2), 29.9 (CH2), 36.6 (CH2),
38.6 (CH), 42.8 (C-13), 43.6 (CH), 49.6 (CH), 54.7 (CH2-OH), 80.1 (C-17), 115.2 (C-2), 115.8 (C-4), 117.9
(2C, C-200 and C-600), 122.2 (C-40), 126.1 (C-1), 128.3 (C-50), 130.1 (2C, C-300 and C-500), 131.0 (C-400), 135.6
(C-10), 136.8 (C-100), 137.8 (C-5), 149.5 (C-30), 152.9 (C-3); ESI-MS 459 [M + H]+.
3-(10-(400-Chlorophenyl)-40-(hydroxymethyl)-10H-pyrazol-30-yl)-estra-1,3,5(10)-trien-3,17β-diol (11c)
According to the general procedure, compound 10c (156 mg) was used. Yield (11c): 126 mg
(88%, white solid); Mp 240–242 ◦C; Anal. Calcd. for C28H31ClN2O3 (479.02) C 70.21; H 6.52. Found C
1
70.09; H 6.43. H NMR (DMSO-d6, 500 MHz):
δ 0.68 (s, 3H, 18-CH3), 1.10–1.44 (overlapping m, 7H),
1.59 (m, 1H), 1.79–1.93 (overlapping m, 3H), 2.14 (m, 1H), 2.32 (m, 1H), 2.77 (m, 2H, 6-H2), 3.54 (m, 1H,

Molecules 2020, 25, 4039
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17-H), 4.50 (d, 1H, J = 4.9 Hz, 17-OH), 4.53 (d, 2H, J = 4.9 Hz, CH2OH), 5.18 (t, 1H, J = 4.9 Hz, CH2OH),
6.64 (s, 1H, 4-H), 7.57 (d, 2H, J = 9.0 Hz, 300-H and 500-H), 7.59 (s, 1H, 1-H), 7.87 (d, 2H, J = 8.9 Hz, 200-H
and 600-H), 8.55 (s, 1H, 50-H), 9.95 (3-OH); 13C NMR (DMSO-d6, 125 MHz):
δ 11.3 (C-18), 22.8 (CH2),
26.0 (CH2), 26.9 (CH2), 28.9 (CH2), 29.9 (CH2), 36.6 (CH2), 38.6 (CH), 42.8 (C-13), 43.6 (CH), 49.6 (CH),
54.7 (CH2-OH), 80.1 (C-17), 115.3 (C-2), 115.8 (C-4), 119.6 (2C, C-200 and C-600), 123.1 (C-40), 126.4 (C-1),
128.4 (C-50), 129.6 (2C, C-300 and C-500), 130.2 and 131.1: C-400 and C-10, 137.9 (C) and 138.0 (C): C-5 and
C-100, 150.0 (C-30), 152.9 (C-3); ESI-MS 479 [M + H]+.
3.2. Pharmacology
To evaluate the in vitro pharmacological effects of the synthesized molecules, each compound
was first dissolved in cell culture grade DMSO (Sigma) to a final concentration of 10 mM. Using MTT
colorimetric assay [36], the cytotoxicity of these compounds was evaluated on cancerous (MCF-7, HeLa,
DU145, PC-3) and non-cancerous (MRC-5) cell lines. Briefly, 5000 cells/well of each cell line were
seeded in 96-well plates and were left to grow overnight in a 5% CO2 atmosphere at 37 ◦C. On the next
day, cells were treated with each derivative separately, in case of the primary screen, or only with the
selected compounds upon IC50 determination. Cells were incubated with the test compounds for 72 h
in a 5% CO2 atmosphere at 37 ◦C. End point was measured by incubating cells for 1 h with 0.5 mg/mL
of MTT reagent (Serva) dissolved in serum free culture medium. The formazan crystals formed by
metabolically active cells were later dissolved in DMSO. Optical density of the obtained solutions
was measured at 570 nm using a Synergy HTX plate reader. For the overall screen, all compounds
were used at 2.5 µM concentration; for the determination of IC50 values, the selected compounds were
applied in 1, 2, 3, 4, 5, 6, and 7
24 h in 20, 40, 80, 160, and 330
µ
M concentrations, and the positive control cisplatin was applied for
µM concentrations. The analysis and conclusions were made based on
three independent experiments. Normalized data were analyzed and the heat map was constructed
using GraphPad Prism 7 software.
4. Conclusions
In summary, an AlCl3-induced one-pot process has been developed for the regioselective
ortho-acetylation/demethylation of O-protected tetracyclic steroidal and bicyclic non-steroidal phenols
under mild conditions. The acetylated phenols were then successfully subjected to MW-assisted
arylhydrazone formation with three arylhydrazines having different p-substituted groups, which were
then treated with the Vilsmeier-Haack reagent to give pyrazolyl estradiol and tetrahydronaphthol
derivatives via cyclization and subsequent formylation. The resulting 4-formylpyrazoles were further
converted by reduction to give 4-hydroxymethylpyrazoles, while pyrazolocoumarins were obtained
by lactonization under oxidative conditions. Significant differences were observed during the similar
reactions of the steroid and the smaller analogue modelling the A- and B-rings of estradiol, and a
substituent effect mainly due to the presence of an electron-withdrawing Cl atom was also noticed in
certain reaction steps. Among the synthesized molecules (steroid and non-steroid), we found 10–15
potent cancer cell-selective molecules, many of them proved to be competent against HeLa, DU145,
or cisplatin-resistant PC-3 cells. Structure-activity considerations suggested that several simplified
small molecules were able to induce significant toxicity; however, when the same structural motif was
incorporated into an estrane framework, the resulting compounds were either more effective on the
cancer cells or were able to target a different cell line. Moreover, acetylated steroidal pyrazoles proved
to be less advantageous than their deacetylated counterparts due to reduced cancer cell selectivity.
The unsubstituted phenyl ring on the pyrazole moiety proved to be the most favorable for selective
antiproliferative effect.
Supplementary Materials: The following are available online, 1H NMR and 13C NMR spectra of the synthesized
compounds, Table S1: Mean
used to construct the heat map, Figure S1: Dose–response curves used to evaluate IC50 concentrations of
selected compounds.
±
SD values of primary growth inhibitory screen (given as cell viability)

Molecules 2020, 25, 4039
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.F. and
Author Contributions: Conceptualization, resources, supervision, and writing—review and editing,
É
M.K.; chemical synthesis and methodology, B.M.; pharmacological studies and methodology, M.K.G. and D.I.A.;
writing—original draft preparation, B.M. and M.K.G. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Acknowledgments: Financial support by National Research, Development and Innovation Office—NKFIH
through projects GINOP-2.3.2-15-2016-00038 is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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α1
Sample Availability: Samples of the compounds are not available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).