Synthesis, characterization and biological evaluation of some novel 17-isoxazoles in the estrone series

Article abstract of DOI:10.1016/j.steroids.2012.05.003

Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to furnish novel steroidal 17α-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient catalyst, accelerating the intermolecula

Full text of DOI:10.1016/j.steroids.2012.05.003

Steroids 77 (2012) 1075–1085
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis, characterization and biological evaluation of some novel 17-isoxazoles
in the estrone series
a
a
a
a
a
b
}
Dóra Kovács , Zalán Kádár , Gergo Mótyán , Gyula Schneider , János Wölfling , István Zupkó ,
Éva Frank a,
⇑
a Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
b Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Regioselective 1,3-dipolar cycloadditions of different aryl nitrile oxides to mestranol were carried out to
furnish novel steroidal 17a-isoxazoles in good to excellent yields. Copper(I) was found to be an efficient
Received 20 March 2012
Received in revised form 2 May 2012
Accepted 3 May 2012
catalyst, accelerating the intermolecular ring-closures and leading exclusively to 3,5-disubstituted isox-
azoles. The yields of the cycloadducts, however, were influenced by the substituents on the aromatic moi-
ety of the 1,3-dipoles. Moreover, dehydration of the primary products resulted in the corresponding
Available online 18 May 2012
D
16,17 exo-heterocyclic derivatives. All the synthesized compounds were subjected to in vitro pharmaco-
Keywords:
Cycloaddition
Isoxazoles
Nitrile oxides
Cytotoxic activity
logical studies of their antiproliferative effects relative to three human malignant cell lines (HeLa, MCF7
and A2780).
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
Moreover, experimental results during the past few years have re-
vealed that some of these steroidal heterocycles play important
One of the major aims of steroid chemistry at present is the
development of novel derivatives containing a heterocyclic moiety,
as a part of, fused to or linked to the sterane framework, in order to
extend the already wide range of known biological activities of
these compounds. A number of semisynthetic molecules have been
described as potent inhibitors of specific enzymes involved in the
biosynthesis of endogenous hormones, allowing their potential
use in the medication of hormone-dependent diseases. Steroidal
exo-heterocycles, such as the therapeutically applied abiraterone,
roles in complex signal transduction mechanisms in a hormone
receptor-independent manner by the inhibition of angiogenesis,
tubulin polymerization, and the upregulation of apoptotic path-
ways [10,11]. Although the detailed mechanisms of action are
not very clear in these latter cases, the coordination of 17-hetero-
cycles of steroids to the target biomolecule and thus the better fit
of the ligand at the binding site of the receptor is thought to be an
important factor in the antiproliferative activity. The cell mem-
brane diffusion may be facilitated at the same time by the hydro-
phobic steroid unit.
can block androgen synthesis at an early stage by inhibiting 17a-
hydroxylase-C17,20-lyase (P45017 ) and can therefore be used in
Among the steroidal heterocycles, attention to derivatives of
isoxazole is caused by the fact that these compounds have been re-
ported to exhibit various biological activities, including anabolic,
myotrophic, hypochloesteremic and tumor-inhibiting properties
[12], and moreover they are convenient intermediates in the syn-
thesis of numerous polyfunctional compounds [13]. Danazol, prob-
ably the best-known member of this series, was approved as the
first drug for the specific treatment of endometriosis. Although
its clinical use is limited by its masculinizing side-effects, in vitro
bioassays have suggested that danazol also exerts a growth-inhib-
itory effect on human endometrial cancer cells [14].
One of the synthetic tools for the formation of five-membered
heterocyclic ring systems include 1,3-dipolar cycloaddition, which
has enjoyed a wide range of popularity for decades in view of the
large numbers of available dipoles and dipolarophiles [15–17].
In recent years, considerable attention has been focused on
a
the treatment of prostate cancer [1–4]. Some heterosteroids (e.g.
finasteride and dutasteride) are known to decrease the circulating
levels of dihydrotestosterone by exerting pronounced inhibitory
effects on 5a-reductase, and consequently to reduce the excessive
prostate growth in benign hyperplasia [5–7].
One of the most frequent modifications of the original steroid
molecule is performed at C-17, where the construction of a hete-
roring can be facilitated by the already existing functional groups.
Therefore, the introduction of different heterocyclic moieties (e.g.
imidazole, pyrazol, oxazole, thiazol or isoxazole) into this position
was found to be beneficial from a pharmacological aspect obtain-
ing several compounds as potent inhibitors of P45017 [1,8,9].
a
⇑
Corresponding author. Tel.: +36 62 544275; fax: +36 62 544200.
E-mail address: frank@chem.u-szeged.hu (É. Frank).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.05.003

1076
D. Kovács et al. / Steroids 77 (2012) 1075–1085
R2
Perkin Elmer CHN analyzer model 2400. NMR spectra were ob-
tained at room temperature with a Bruker DRX 500 instrument.
Chemical shifts are reported in ppm (d scale), and coupling con-
stants (J) in Hz. For the determination of multiplicities, the J-
MOD pulse sequence was used. Automated flow injection analyses
were performed by using an HPLC/MSD system. The system com-
prised an Agilent 1100 micro vacuum degasser, a quaternary
pump, a micro-well plate autoinjector and a 1946A MSD equipped
with an electrospray ion source (ESI) operated in positive ion
mode. The ESI parameters were: nebulizing gas N2, at 35 psi; dry-
ing gas N2, at 350 °C and 12 L/min; capillary voltage (VCap) 3000 V;
and fragmentor voltage 70 V. The MSD was operated in scan mode
N
N
R2
N
N
R2
N
N
N
5
N
1 N
1
R1
C
CH
+
+
4
"uncatalyzed"
R1
R1
Cu(I)
R1
C
C
Cu
O
"click"
"click"
R1
4
R1
Cu(I)
with the mass range m/z 60–620. Samples (0.2 lL) were injected
5
with automated needle wash directly into the solvent flow
(0.3 mL/min) of MeCN/H2O 70:30 (v/v) supplemented with 0.1%
formic acid. The system was controlled by Agilent LC/MSD Chem-
station software. All solvents were distilled immediately prior to
use. Reagents and materials were obtained from commercial sup-
pliers and were used without purification. The reactions were
monitored by TLC on Kieselgel-G (Merck Si 254 F) layers
(0.25 mm thick); solvent systems (ss): (A) CH2Cl2/EtOAc (98:2 v/
v), (B) CH2Cl2/EtOAc (95:5 v/v), (C) CH2Cl2/n-hexane (70:30 v/v),
(D) CH2Cl2/n-hexane (60:40 v/v); (E) CH2Cl2. The spots were de-
tected by spraying with 5% phosphomolybdic acid in 50% aqueous
phosphoric acid. The Rf values were determined for the spots ob-
served by illumination at 254 and 365 nm. Flash chromatography:
"uncatalyzed"
O
R1
C
CH
O
R3
R3
3
3
N
N
N
C
R3
Fig. 1. Synthesis of triazoles and isoxazoles via Huisgen versus catalytic 1,3-dipolar
cycloadditions.
Cu(I)-catalyzed azide–alkyne cycloaddition [18–20] as it meets all
of the criteria for 00click00 chemistry introduced by Sharpless [21] in
2001 (Fig. 1). As compared with the uncatalyzed (Huisgen) version,
the presence of the catalyst dramatically improves both the rate
and the regioselectivity of the process, leading exclusively to the
1,4-disubstituted 1,2,3-triazole, and eliminating the need for ele-
vated temperature and/or a prolonged reaction time. Although
the conventional cycloaddition occurs through a concerted mecha-
nism, the catalytic pathway follows a stepwise route with a copper
acetylide as the key intermediate [22,23].
Merck silica gel 60, 40–63 lm.
2.2. General procedure for the synthesis of 17b-hydroxy,17
a-isoxazolyl
derivatives (6a–k) in the estrone series
Cycloadditions between alkynes and nitrile oxides also provide
convenient possibilities for the construction of isoxazoles (Fig. 1).
Nitrile oxides are usually prepared from the corresponding aldox-
imes directly before application, by oxidative halogenation to imi-
doyl halides and subsequent base-induced dehydrohalogenation,
due to the instability of the precursor and the 1,3-dipole [24].
Although several syntheses of steroidal isoxazoles have been re-
ported, the two possible regioisomers are usually obtained during
different reaction times and at different temperatures. A further
disadvantage of the concerted reaction is the rapid formation of
furoxane-type by-products (especially for reactive nitrile oxide di-
poles), which can reduces the yields of the desired cycloadducts
[15]. The regiospecific stepwise sequence is not limited to azides,
however, but works with nitrile oxide dipoles as well. Thus, a
dramatic acceleration was observed experimentally when a Cu(I)
catalyst was applied, in good agreement with theoretical predic-
tions; more importantly, only the formation of the 3,5-disubsti-
tuted isoxazole occured [25].
As an extension of our program on the synthesis of steroidal
heterocycles [26–29] exerting antiproliferative activity, we set
out to develop an efficient route for the preparation of novel
17-exo-isoxazolyl derivatives from mestranol through use of the
‘‘click’’ chemistry approach. Since analogous compounds in the es-
trone series have been reported to have no estrogenic effect [12],
our aim was to investigate wheather the synthetized isoxazoles
display any effect on cell-growth. Therefore, all derivatives were
screened in vitro for their activities against a panel of three human
cancer cell lines (HeLa, MCF7 and A2780).
Mestranol (1, 310 mg, 1.00 mmol) and the appropriate aro-
matic imidoyl chloride [25] (4a–k, 1.50 mmol) were dissolved in
toluene (15 mL), and Ph3P (52 mg, 0.20 mmol) and CuI (19 mg,
0.10 mmol) were then added to the solution. Finally, N,N-diido-
propyl ethylamine (DIPEA) (0.67 mL, 4.00 mmol) was added
dropwise to the gently heated reaction mixture, which was subse-
quently heated to reflux with stirring for 4 h. During the reaction,
the pale-yellow solution became dark-brown. After completion of
the reaction (TLC monitoring), the solvent was evaporated off in
vacuo. The resulting crude products were purified by flash
chromatography.
2.2.1. Synthesis of 3-methoxy-17a
-(30-phenylisoxazol-50-yl)estra-
1,3,5(10)-trien-17b-ol (6a)
According to Section 2.2, N-hydroxybenzenecarboximidoyl
chloride (4a, 233 mg) was used. After purification with CH2Cl2/
EtOAc (98:2) as eluent, 6a was obtained as
(399 mg, 93%). Mp 119–122 °C; Rf = 0.24 (ss A). Anal. Calcd for
28H31NO3: C, 78.29; H, 7.27. Found: C, 78.40; H, 7.43. 1H NMR
a white solid
C
(500 MHz, CDCl3): d = 0.93 (m, 1H), 1.06 (s, 3H, 18-H3), 1.36–1.64
(m, 4H), 1.72–1.79 (m, 2H), 1.91–2.01 (m, 2H), 2.05–2.15 (m,
2H), 2.22 (m, 1H), 2.50 (m, 1H), 2.86 (m, 2H, 6-H2), 3.77 (s, 3H,
3-OMe), 6.52 (s, 1H, 40-H), 6.62 (d, 1H, J = 2.2 Hz, 4-H), 6.68 (dd,
1H, J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.12 (d, 1H, J = 8.5 Hz, 1-H), 7.47
(overlapping m, 3H, 300-H, 400-H and 500-H), 7.84 (d, 2H, J = 8.2 Hz,
200-H and 600-H) ppm; 13C NMR (125 MHz, CDCl3): d = 14.0 (C-18),
23.6 (CH2), 26.2 (CH2), 27.4 (CH2), 29.7 (CH2), 33.1 (CH2), 37.3
(CH2), 39.4 (CH), 43.2 (CH), 48.3 (C-13), 48.8 (CH), 55.2 (3-OMe),
83.8 (C-17), 100.1 (C-40), 111.4 (C-2), 113.8 (C-4), 126.2 (C-1),
126.8 (2C, C-200 and C-600), 128.9 (2C, C-300 and C-500), 129.0 (C-100),
130.0 (C-400), 132.4 (C-10), 137.8 (C-5), 157.4 (C-3), 161.9 (C-30),
177.6 (C-50) ppm; ESI–MS: 430 (M+H)+.
2. Experimental
2.1. General
Melting points (mp) were determined on a Kofler block and are
uncorrected. Elementary analysis data were determined with a

D. Kovács et al. / Steroids 77 (2012) 1075–1085
1077
2.2.2. Synthesis of 3-methoxy-17
a
-[30-(200-methyl)phenylisoxazol-50-
2.2.5. Synthesis of 3-methoxy-17
a
-[30-(400-methoxy)phenylisoxazol-50-
yl]estra-1,3,5(10)-trien-17b-ol (6b)
yl]estra-1,3,5(10)-trien-17b-ol (6e)
According to Section 2.2, N-hydroxy-2-methylbenzenecarbox-
imidoyl chloride (4b, 254 mg) was added to the mixture. After
purification with CH2Cl2 as eluent, 6b was obtained as a white solid
(435 mg, 98%). Mp 141ꢀ143 °C; Rf = 0.40 (ss A). Anal. Calcd for
According to Section 2.2, N-hydroxy-4-methoxybenzenecarbox-
imidoyl chloride (4e, 279 mg) was added to the mixture. After
purification with CH2Cl2/EtOAc (98:2 v/v) as eluent, 6e was ob-
tained as a pale-yellow solid (445 mg, 97%). Mp 155–157 °C;
Rf = 0.28 (ss B). Anal. Calcd for C29H33NO4: C, 75.79; H, 7.24; Found:
C, 75.89; H, 7.05. 1H NMR (500 MHz, CDCl3): d = 0.93 (m, 1H), 1.06
(s, 3H, 18-H3), 1.35–1.66 (m, 4H), 1.71–1.79 (m, 2H), 1.90–1.99 (m,
2H), 2.04–2.15 (m, 2H), 2.22 (m, 1H), 2.52 (m, 1H), 2.87 (m, 2H, 6-
H2), 3.76 (s, 3H, 3-OMe), 3.86 (s, 3H, 400-OMe), 6.46 (s, 1H, 40-H),
6.62 (d, 1H, J = 2.3 Hz, 4-H), 6.68 (dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-
H), 6.98 (d, 2H, J = 8.4 Hz, 300-H and 500-H), 7.12 (d, 1H, J = 8.5 Hz,
1-H), 7.76 (d, 2H, J = 8.4 Hz, 200-H and 600-H) ppm; 13C NMR
(125 MHz, CDCl3): d = 14.0 (C-18), 23.6 (CH2), 26.2 (CH2), 27.3
(CH2), 29.8 (CH2), 33.1 (CH2), 37.2 (CH2), 39.4 (CH), 43.2 (CH),
48.3 (C-13), 48.8 (CH), 55.2 and 55.3: 3-OMe and 400-OMe, 83.7
(C-17), 100.0 (C-40), 111.4 (C-2), 113.8 (C-4), 114.3 (2C, C-300 and
C-500), 121.5 (C-100), 126.2 (C-1), 128.2 (2C, C-200 and C-600), 132.4
(C-10), 137.8 (C-5), 157.4 (C-3), 161.0 (C-400), 161.5 (C-30), 177.4
(C-50) ppm; ESI–MS: 460 (M+H)+.
C
29H33NO3: C, 78.52; H, 7.50; Found: C, 78.66; H, 7.37. 1H NMR
(500 MHz, CDCl3): d = 0.91 (m, 1H), 1.07 (s, 3H, 18-H3), 1.35–1.64
(m, 4H), 1.72–1.80 (m, 2H), 1.91–2.00 (m, 2H), 2.05–2.16 (m, 2H),
2.24 (m, 1H), 2.50 (m, 1H), 2.53 (s, 3H, 200-Me), 2.87 (m, 2H, 6-
H2), 3.77 (s, 3H, 3-OMe), 6.39 (s, 1H, 40-H), 6.63 (d, 1H, J = 2.2 Hz,
4-H), 6.69 (dd, 1H, J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.13 (d, 1H,
J = 8.5 Hz, 1-H), 7.27–7.36 (overlapping m, 3H, 300-H, 400-H and 500-
H), 7.54 (d, 1H, J = 7.9 Hz, 600-H) ppm; 13C NMR (125 MHz, CDCl3):
d = 14.1 (C-18), 21.2 (200-Me), 23.6 (CH2), 26.2 (CH2), 27.4 (CH2),
29.8 (CH2), 33.1 (CH2), 37.3 (CH2), 39.4 (CH), 43.2 (CH), 48.3
(C-13), 48.8 (CH), 55.2 (3-OMe), 83.8 (C-17), 102.7 (C-40), 111.4
(C-2), 113.8 (C-4), 125.9 (C-500), 126.2 (C-1), 128.7 (C-100), 129.4,
129.5, 131.1 (3C, C-300, C-400 and C-600), 132.3 (C-10), 136.9 (C-200),
137.8 (C-5), 157.4 (C-3), 162.5 (C-30), 176.6 (C-50) ppm; ESI–MS:
444 (M+H)+.
2.2.3. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6c)
a
-[30-(300-methyl)phenylisoxazol-50-
2.2.6. Synthesis of 3-methoxy-17a
-[30-(300,400-
dimethoxy)phenylisoxazol-50-yl]estra-1,3,5(10)-trien-17b-ol (6f)
According to Section 2.2, N-hydroxy-3,4-dimethoxybenzenecar-
boximidoyl chloride (4f, 323 mg) was used. After purification with
CH2Cl2/EtOAc (98:2 v/v) as eluent, 6f was obtained as a white solid
(465 mg, 95%). Mp 108–111 °C; Rf = 0.28 (ss B). Anal. Calcd for
According to Section 2.2, N-hydroxy-3-methylbenzenecarbox-
imidoyl chloride (4c, 254 mg) was used. After purification with
CH2Cl2 as eluent, 6c was obtained as a white solid (426 mg, 96%).
Mp 153–155 °C; Rf = 0.46 (ss A). Anal. Calcd for C29H33NO3: C,
78.52; H, 7.50; Found: C, 78.40; H, 7.68. 1H NMR (500 MHz, CDCl3):
d = 0.92 (m, 1H), 1.06 (s, 3H, 18-H3), 1.35–1.66 (m, 4H), 1.71–1.78
(m, 2H), 1.91–2.02 (m, 2H), 2.04–2.16 (m, 2H), 2.22 (m, 1H), 2.42 (s,
3H, 300-Me), 2.50 (m, 1H), 2.87 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OMe),
6.50 (s, 1H, 40-H), 6.62 (d, 1H, J = 2.4 Hz, 4-H), 6.68 (dd, 1H,
J = 8.5 Hz, J = 2.4 Hz, 2-H), 7.12 (d, 1H, J = 8.5 Hz, 1-H), 7.27 (d,
1H, J = 8.2 Hz, 400-H), 7.35 (t, 1H, J = 8.2 Hz, 500-H), 7.63 (d, 1H,
J = 8.2 Hz, 600-H), 7.67 (s, 1H, 200-H) ppm; 13C NMR (125 MHz,
CDCl3): d = 14.1 (C-18), 21.4 (300-Me), 23.6 (CH2), 26.2 (CH2), 27.3
(CH2), 29.8 (CH2), 33.1 (CH2), 37.3 (CH2), 39.4 (CH), 43.2 (CH),
48.3 (C-13), 48.8 (CH), 55.2 (3-OMe), 83.8 (C-17), 100.2 (C-40),
111.4 (C-2), 113.8 (C-4), 123.9 (C-600), 126.2 (C-1), 127.4 (C-200),
128.8 (CH), 128.9 (C-100), 130.8 (CH), 132.4 (C-10), 137.8 (C-5),
138.6 (C-300), 157.4 (C-10), 162.0 (C-30), 177.5 (C-50) ppm; ESI–
MS: 444 (M+H)+.
C
30H35NO5: C, 73.59; H, 7.21; Found: C, 73.70; H, 7.34. 1H NMR
(500 MHz, CDCl3): d = 0.88 (m, 1H), 1.06 (s, 3H, 18-H3), 1.35–1.64
(m, 4H), 1.70–1.80 (m, 2H), 1.90–1.99 (m, 2H), 2.02–2.15 (m, 2H),
2.23 (m, 1H), 2.50 (m, 1H), 2.86 (m, 2H, 6-H2), 3.77 (s, 3H, 3-
OMe), 3.93 (s, 3H) and 3.96 (s, 3H, 300-OMe and 400-OMe), 6.47 (s,
1H, 40-H), 6.62 (d, 1H, J = 2.3 Hz, 4-H), 6.68 (dd, 1H, J = 8.5 Hz,
J = 2.3 Hz, 2-H), 6.93 (d, 1H, J = 8.4 Hz, 500-H), 7.12 (d, 1H,
J = 8.5 Hz, 1-H), 7.33 (dd, 1H, J = 8.4 Hz, J = 1.6 Hz, 600-H), 7.44 (d,
1H, J = 1.6 Hz, 200-H) ppm; 13C NMR (125 MHz, CDCl3): d = 14.1
(C-18), 23.6 (CH2), 26.2 (CH2), 27.4 (CH2), 29.7 (CH2), 33.1 (CH2),
37.3 (CH2), 39.4 (CH), 43.2 (CH), 48.3 (C-13), 48.8 (CH), 55.2 (3-
OMe), 55.9 and 56.0: 300-OMe and 400-OMe, 83.8 (C-17), 100.0 (C-
40), 109.3 and 111.1: C-200 and C-500, 111.4 (C-2), 113.8 (C-4),
119.9 (C-600), 121.7 (C-100), 126.2 (C-1), 132.3 (C-10), 137.8 (C-5),
149.3 and 150.6: C-300 and C-400, 157.4 (C-3), 161.6 (C-30), 177.5
(C-50) ppm; ESI–MS: 490 (M+H)+
.
2.2.4. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6d)
a
-[30-(400-methyl)phenylisoxazol-50-
2.2.7. Synthesis of 3-methoxy-17a
-[30-(300,500-dichloro-200,400,600-
According to Section 2.2, N-hydroxy-4-methylbenzenecarbox-
imidoyl chloride (4d, 254 mg) was added to the mixture. After
purification with CH2Cl2/EtOAc (97:3 v/v) as eluent, 6d was ob-
tained as a white solid (430 mg, 97%). Mp 139–142 °C; Rf = 0.25
(ss A). Anal. Calcd for C29H33NO3: C, 78.52; H, 7.50; Found: C,
78.71; H, 7.43. 1H NMR (500 MHz, CDCl3): d = 0.93 (m, 1H), 1.06
(s, 3H, 18-H3), 1.36–1.66 (m, 4H), 1.71–1.79 (m, 2H), 1.90–2.00
(m, 2H), 2.04–2.14 (m, 2H), 2.21 (m, 1H), 2.41 (s, 3H, 400-Me),
2.53 (m, 1H), 2.86 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OMe), 6.49 (s, 1H,
40-H), 6.62 (d, 1H, J = 2.0 Hz, 4-H), 6.68 (dd, 1H, J = 8.5 Hz,
J = 2.0 Hz, 2-H), 7.12 (d, 1H, J = 8.5 Hz, 1-H), 7.27 (d, 2H,
J = 8.1 Hz, 300-H and 500-H), 7.72 (d, 2H, J = 8.1 Hz, 200-H and 600-H)
trimethoxy)phenylisoxazol-50-yl]estra-1,3,5(10)-trien-17b-ol (6g)
According to Section 2.2, N-hydroxy-3,5-dichloro-2,4,6-tri-
methoxybenzenecarboximidoyl chloride (4g, 472 mg) was added
to the mixture. After purification with CH2Cl2 as eluent, 6g was ob-
tained as a white solid (577 mg, 98%). Mp 131–134 °C; Rf = 0.52 (ss
B). Anal. Calcd for C31H35Cl2NO6: C, 63.27; H, 5.99; Found: C, 63.15;
H, 6.08. 1H NMR (500 MHz, CDCl3): d = 0.84 (m, 1H), 1.07 (s, 3H, 18-
H3), 1.35–1.69 (m, 4H), 1.76–1.82 (m, 2H), 1.90–1.97 (m, 2H), 2.00–
2.06 (m, 1H), 2.11–2.17 (m, 1H), 2.23 (m, 1H), 2.49 (m, 1H), 2.86
(m, 2H, 6-H2), 3.74 (s, 6H, 2 ꢁ OMe), 3.77 (s, 3H, 3-OMe), 3.96 (s,
3H, OMe), 6.37 (s, 1H, 40-H), 6.63 (d, 1H, J = 2.6 Hz, 4-H), 6.69
(dd, 1H, J = 8.5 Hz, J = 2.6 Hz, 2-H), 7.14 (d, 1H, J = 8.5 Hz, 1-H)
ppm; 13C NMR (125 MHz, CDCl3): d = 14.0 (C-18), 23.5 (CH2), 26.2
(CH2), 27.4 (CH2), 29.7 (CH2), 33.2 (CH2), 37.3 (CH2), 39.4 (CH),
43.5 (CH), 48.4 (C-13), 48.9 (CH), 55.2 (3-OMe), 60.9 (OMe), 62.1
(2C, 2 ꢁ OMe), 83.7 (C-17), 104.2 (C-40), 111.5 (C-2), 113.8 (C-4),
116.9 (2C, C-300 and C-500), 120.2 (C-100), 126.2 (C-1), 132.1 (C-10),
137.8 (C-5), 154.3 (2C, C-200 and C-600), 154.9 (C-30), 155.6 (C-400),
157.5 (C-3), 176.5 (C-50) ppm; ESI–MS: 589 (M+H)+.
13
ppm; C NMR (125 MHz, CDCl3): d = 14.0 (C-18), 21.4 (400-Me),
23.6 (CH2), 26.2 (CH2), 27.3 (CH2), 29.8 (CH2), 33.1 (CH2), 37.2
(CH2), 39.4 (CH), 43.2 (CH), 48.3 (C-13), 48.8 (CH), 55.2 (3-OMe),
83.7 (C-17), 100.1 (C-40), 111.4 (C-2), 113.8 (C-4), 126.1 (C-100),
126.2 (C-1), 126.7 (2C, C-200 and C-600), 129.6 (2C, C-300 and C-500),
132.4 (C-10), 137.8 (C-5), 140.1 (C-400), 157.4 (C-3), 161.8 (C-30),
177.4 (C-50) ppm; ESI–MS: 444 (M+H)+.

1078
D. Kovács et al. / Steroids 77 (2012) 1075–1085
2.2.8. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6h)
a
-[30-(400-fluoro)phenylisoxazol-50-
cation with CH2Cl2 as eluent, 6k was obtained as a yellow solid
(299 mg, 63%). Mp 92–95 °C; Rf = 0.38 (ss A). Anal. Calcd for
28H30N2O5: C, 70.87; H, 6.37; Found: C, 71.06; H, 6.20. 1H NMR
According to Section 2.2, N-hydroxy-4-fluorobenzenecarboxim-
idoyl chloride (4h, 260 mg) was used. After purification with
CH2Cl2 as eluent, 6h was obtained as a white solid (363 mg,
81%). Mp 140–142 °C; Rf = 0.49 (ss A). Anal. Calcd for C28H30FNO3:
C, 75.14; H, 6.76; Found: C, 75.29; H, 6.94. 1H NMR (500 MHz,
CDCl3): d = 0.91 (m, 1H), 1.06 (s, 3H, 18-H3), 1.36–1.64 (m, 4H),
1.71–1.78 (m, 2H), 1.90–2.00 (m, 2H), 2.05–2.14 (m, 2H), 2.22
(m, 1H), 2.52 (m, 1H), 2.86 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OMe),
6.48 (s, 1H, 40-H), 6.62 (d, 1H, J = 2.3 Hz, 4-H), 6.68 (dd, 1H,
J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.11–7.18 (overlapping m, 3H, 1-H, 300-
H and 500-H), 7.82 (dd, 2H, J = 8.2 Hz, J = 4.9 Hz, 200-H and 600-H)
ppm; 13C NMR (125 MHz, CDCl3): d = 14.1 (C-18), 23.6 (CH2), 26.2
(CH2), 27.3 (CH2), 29.8 (CH2), 33.1 (CH2), 37.3 (CH2), 39.4 (CH),
43.2 (CH), 48.3 (C-13), 48.8 (CH), 55.2 (3-OMe), 83.8 (C-17),
100.0 (C-40), 111.4 (C-2), 113.8 (C-4), 116.0 (2C, J = 21.8 Hz, C-300
and C-500), 125.2 (C-100), 126.2 (C-1), 128.7 (2C, J = 8.4 Hz, C-200
and C-600), 132.3 (C-10), 137.8 (C-5), 157.4 (C-3), 161.0 (C-30),
163.8 (C-400, J = 248.1 Hz), 177.8 (C-50) ppm; ESI–MS: 448 (M+H)+.
C
(500 MHz, CDCl3): d = 0.89 (m, 1H), 1.07 (s, 3H, 18-H3), 1.36–1.67
(m, 4H), 1.72–1.79 (m, 2H), 1.92–2.02 (m, 2H), 2.04–2.16 (m,
2H), 2.22 (m, 1H), 2.51 (m, 1H), 2.86 (m, 2H, 6-H2), 3.76 (s, 3H,
3-OMe), 6.60 (s, 1H, 40-H), 6.62 (d, 1H, J = 2.1 Hz, 4-H), 6.68 (dd,
1H, J = 8.5 Hz, J = 2.1 Hz, 2-H), 7.11 (d, 1H, J = 8.5 Hz, 1-H), 8.02
(d, 2H, J = 8.4 Hz, 200-H and 600-H), 8.33 (d, 2H, J = 8.4 Hz, 300-H and
500-H) ppm; 13C NMR (125 MHz, CDCl3): d = 14.1 (C-18), 23.6
(CH2), 26.2 (CH2), 27.4 (CH2), 29.7 (CH2), 33.2 (CH2), 37.4 (CH2),
39.4 (CH), 43.2 (CH), 48.4 (C-13), 48.9 (CH), 55.2 (3-OMe), 83.9
(C-17), 100.2 (C-40), 111.5 (C-2), 113.8 (C-4), 124.2 (2C, C-300 and
C-500), 126.2 (C-1), 127.6 (2C, C-200 and C-600), 132.2 (C-10), 135.2
(C-100), 137.8 (C-5), 148.7 (C-400), 157.5 (C-3), 160.1 (C-30), 178.9
(C-50) ppm; ESI–MS: 475 (M+H)+.
2.3. Synthesis of 3-methoxy-17a
-(30-phenylisoxazol-50-yl)estra-
1,3,5(10)-trien-17b-ol (6a)
Mestranol (1, 310 mg, 1.00 mmol) was dissolved in CH2Cl2
(10 mL), and a solution of CuSO4ꢂ5H2O (12.5 mg, 0.05 mmol) and
sodium ascorbate (30 mg, 0.15 mmol) in water (10 mL) was poured
into the organic phase, and 4a (233 mg, 1.5 mmol) was then added
to the mixture under vigorous stirring. Finally, DIPEA (0.67 mL,
4.00 mmol) was added dropwise to the two-phase system, and
the reaction mixture was stirred for 4 h at ambient temperature.
After completion of the reaction (TLC monitoring), the mixture
was extracted with CH2Cl2 (2 ꢁ 10 mL). The combined organic
phase was washed with water (10 mL), dried over Na2SO4, and
evaporated in vacuo. The crude product was purified by flash chro-
matography with CH2Cl2/EtOAc (98:2) to give 6a as a white solid
(395 mg, 92%).
2.2.9. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6i)
a
-[30-(400-chloro)phenylisoxazol-50-
According to Section 2.2, N-hydroxy-4-chlorobenzenecarboxim-
idoyl chloride (4i, 285 mg) was used. After purification with CH2Cl2
as eluent, 6i was obtained as a white solid (376 mg, 81%). Mp 144–
147 °C; Rf = 0.43 (ss A). Anal. Calcd for C28H30ClNO3: C, 72.48; H,
6.52; Found: C, 72.60; H, 6.67. 1H NMR (500 MHz, CDCl3): d = 0.91
(m, 1H), 1.06 (s, 3H, 18-H3), 1.36–1.65 (m, 4H), 1.71–1.79 (m, 2H),
1.91–2.00 (m, 2H), 2.05–2.15 (m, 2H), 2.22 (m, 1H), 2.52 (m, 1H),
2.86 (m, 2H, 6-H2), 3.77 (s, 3H, 3-OMe), 6.49 (s, 1H, 40-H), 6.62 (d,
1H, J = 2.5 Hz, 4-H), 6.68 (dd, 1H, J = 8.5 Hz, J = 2.5 Hz, 2-H), 7.12
(d, 1H, J = 8.5 Hz, 1-H), 7.44 (d, 2H, J = 8.4 Hz, 300-H and 500-H), 7.77
(d, 2H, J = 8.4 Hz, 200-H and 600-H) ppm; 13C NMR (125 MHz, CDCl3):
d = 14.1 (C-18), 23.6 (CH2), 26.2 (CH2), 27.3 (CH2), 29.7 (CH2), 33.1
(CH2), 37.3 (CH2), 39.4 (CH), 43.2 (CH), 48.3 (C-13), 48.8 (CH),
55.2 (3-OMe), 83.8 (C-17), 100.0 (C-40), 111.4 (C-2), 113.8 (C-4),
126.2 (C-1), 127.5 (C-100), 128.1 (2C, C-200 and C-600), 129.2 (2C, C-
300 and C-500), 132.3 (C-10), 136.0 (C-400), 137.8 (C-5), 157.4 (C-3),
160.9 (C-30), 178.0 (C-50) ppm; ESI–MS: 465 (M+H)+.
2.4. General procedure for the synthesis of
D
16,17-17-isoxazolyl
derivatives (7a–k) in the estrone series
The appropriate 17b-hydroxy-17a-isozaxole derivative (6a–k,
0.50 mmol) was dissolved in pyridine (10 mL), and POCl3 (0.8 mL,
12 mmol) was added dropwise at 0 °C under vigorous stirring.
The reaction mixture was allowed to warm to room temperature
and after 24 h it was poured into a mixture of ice and concentrated
HCl (20 mL), and extracted with EtOAc (2 ꢁ 10 mL). The combined
organic phases were washed with water (10 mL), followed by sat-
urated NaHCO3 solution (10 mL), then dried over Na2SO4 and evap-
orated in vacuo. The resulting crude product was purified by flash
chromatography.
2.2.10. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6j)
a
-[30-(400-bromo)phenylisoxazol-50-
According to Section 2.2, N-hydroxy-4-bromobenzenecarbox-
imidoyl chloride (4j, 352 mg) was added to the mixture. After puri-
fication with CH2Cl2 as eluent, 6j was obtained as a white solid
(397 mg, 78%). Mp 98–100 °C; Rf = 0.53 (ss A). Anal. Calcd for
C
28H30BrNO3: C, 66.14; H, 5.95; Found: C, 66.01; H, 5.77. 1H NMR
(500 MHz, CDCl3): d = 0.90 (m, 1H), 1.06 (s, 3H, 18-H3), 1.35–1.65
(m, 4H), 1.71–1.78 (m, 2H), 1.91–2.00 (m, 2H), 2.04–2.15 (m,
2H), 2.22 (m, 1H), 2.48 (m, 1H), 2.86 (m, 2H, 6-H2), 3.77 (s, 3H,
3-OMe), 6.49 (s, 1H, 40-H), 6.62 (d, 1H, J = 2.2 Hz, 4-H), 6.68 (dd,
1H, J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.12 (d, 1H, J = 8.5 Hz, 1-H), 7.59
(d, 2H, J = 8.3 Hz, 200-H and 600-H), 7.70 (d, 2H, J = 8.3 Hz, 300-H and
500-H) ppm; 13C NMR (125 MHz, CDCl3): d = 14.1 (C-18), 23.6
(CH2), 26.2 (CH2), 27.4 (CH2), 29.7 (CH2), 33.1 (CH2), 37.4 (CH2),
39.4 (CH), 43.2 (CH), 48.3 (C-13), 48.8 (CH), 55.2 (3-OMe), 83.8
(C-17), 100.0 (C-40), 111.4 (C-2), 113.8 (C-4), 124.3 (C-400), 126.2
(C-1), 127.9 (C-100), 128.3 (2C, C-200 and C-600), 132.1 (2C, C-300 and
C-500), 132.3 (C-10), 137.8 (C-5), 157.5 (C-3), 161.0 (C-30), 178.1
(C-50) ppm; ESI–MS: 509 (M+H)+.
2.4.1. Synthesis of 3-methoxy-17a
-(30-phenylisoxazol-50-yl)estra-
1,3,5(10),16-tetraene (7a)
Compound 6a (215 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (25:75 v/v) as eluent, 7a was obtained as a yellow
solid (183 mg, 89%). Mp 167ꢀ170 °C; Rf = 0.52 (ss C). Anal. Calcd
for C28H29NO2: C, 81.72; H, 7.10; Found: C, 81.93; H, 6.96. 1H
NMR (500 MHz, CDCl3): d = 1.05 (s, 3H, 18-H3), 1.50 (m, 1H),
1.65–1.85 (m, 4H), 1.96 (m, 1H), 2.21 (m, 1H), 2.32–2.40 (m, 2H),
2.42–2.49 (m, 2H), 2.92 (m, 2H, 6-H2), 3.80 (s, 3H, 3-OMe), 6.52
(overlapping m, 2H, 40-H and 16-H), 6.67 (d, 1H, J = 2.0 Hz, 4-H),
6.74 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz, 2-H), 7.23 (d, 1H, J = 8.5 Hz, 1-
H), 7.47 (overlapping m, 3H, 300-H, 400-H and 500-H), 7.84 (d, 2H,
J = 7.2 Hz, 200-H and 600-H) ppm; 13C NMR (125 MHz, CDCl3):
d = 16.5 (C-18), 26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.7 (CH2),
35.2 (CH2), 37.1 (CH), 44.1 (CH), 47.1 (C-13), 55.2 (3-OMe), 55.9
(CH), 97.4 (C-40), 111.4 (C-2), 113.8 (C-4), 126.0 (C-1), 126.7 (2C,
C-200 and C-600), 128.8 (2C, C-300 and C-500), 129.3 (C-100), 129.8
2.2.11. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10)-trien-17b-ol (6k)
a
-[30-(400-nitro)phenylisoxazol-50-
According to Section 2.2, N-hydroxy-4-nitrobenzenecarboximi-
doyl chloride (4k, 300 mg) was added to the mixture. After purifi-

D. Kovács et al. / Steroids 77 (2012) 1075–1085
1079
(C-400) 132.5 (C-10), 133.0 (C-16), 137.8 (C-5), 141.9 (C-17), 157.5
(C-3), 162.4 (C-30), 167.4 (C-50) ppm; ESI–MS: 412 (M+H)+.
139.9 (C-400), 141.9 (C-17), 157.5 (C-3), 162.3 (C-30), 167.2 (C-50)
ppm; ESI–MS: 426 (M+H)+.
2.4.2. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7b)
a
-[30-(200-methyl)phenylisoxazol-50-
2.4.5. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7e)
a
-[30-(400-methoxy)phenylisoxazol-50-
Compound 6b (222 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7b was obtained as a white
solid (196 mg, 92%). Mp 145–147 °C; Rf = 0.51 (ss C). Anal. Calcd for
Compound 6e (230 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (20:80 v/v) as eluent, 7e was obtained as a yellow
solid (194 mg, 88%). Mp 171–174 °C; Rf = 0.26 (ss C). Anal. Calcd for
29H31NO2: C, 81.85; H, 7.34; Found: C, 81.98; H, 7.51. 1H NMR
C
29H31NO3: C, 78.88; H, 7.08; Found: C, 79.02; H, 7.29. 1H NMR
C
(500 MHz, CDCl3): d = 1.06 (s, 3H, 18-H3), 1.52 (m, 1H), 1.64–1.86
(m, 4H), 1.97 (m, 1H), 2.22 (m, 1H), 2.32–2.38 (m, 2H), 2.41–2.48
(m, 2H), 2.52 (s, 3H, 200-Me), 2.92 (m, 2H, 6-H2), 3.80 (s, 3H, 3-
OMe), 6.40 and 6.53 (bs, 2H, 40-H and 16-H), 6.67 (d, 1H,
J = 2.1 Hz, 4-H), 6.74 (dd, 1H, J = 8.5 Hz, J = 2.1 Hz, 2-H), 7.23 (d,
1H, J = 8.5 Hz, 1-H), 7.27–7.36 (overlapping m, 3H, 300-H, 400-H and
500-H), 7.54 (d, 1H, J = 7.2 Hz, 600-H) ppm; 13C NMR (125 MHz,
CDCl3): d = 16.5 (C-18), 21.1 (200-Me), 26.5 (CH2), 27.7 (CH2), 29.6
(CH2), 31.8 (CH2), 35.2 (CH2), 37.1 (CH), 44.1 (CH), 47.1 (C-13),
55.2 (3-OMe), 56.0 (CH), 100.2 (C-40), 111.4 (C-2), 113.8 (C-4),
125.9 (C-500), 126.0 (C-1), 129.0 (C-100), 129.3, 129.4, 131.0 (3C, C-
300, C-400 and C-600), 132.5 (C-10), 132.9 (C-16), 136.8 (C-200), 137.8
(C-5), 141.9 (C-17), 157.5 (C-3), 163.0 (C-30), 166.5 (C-50) ppm;
ESI–MS: 426 (M+H)+.
(500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.50 (m, 1H), 1.63–1.84
(m, 4H), 1.96 (m, 1H), 2.20 (m, 1H), 2.31–2.39 (m, 2H), 2.40–2.48
(m, 2H), 2.91 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OMe), 3.86 (s, 3H, 400-
OMe), 6.46–6.50 (overlapping m, 2H, 40-H and 16-H), 6.67 (d, 1H,
J = 2.1 Hz, 4-H), 6.73 (dd, 1H, J = 8.5 Hz, J = 2.1 Hz, 2-H), 6.99 (d,
2H, J = 8.2 Hz, 300-H and 500-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H), 7.78
(d, 2H, J = 8.2 Hz, 200-H and 600-H) ppm; 13C NMR (125 MHz, CDCl3):
d = 16.5 (C-18), 26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.7 (CH2), 35.2
(CH2), 37.1 (CH), 44.1 (CH), 47.1 (C-13), 55.2 and 55.3: 3-OMe and
400-OMe, 55.9 (CH), 97.2 (C-40), 111.4 (C-2), 113.8 (C-4), 114.2 (2C,
C-300 and C-500), 121.8 (C-100), 126.0 (C-1), 128.1 (2C, C-200 and C-600),
132.5 (C-10), 132.8 (C-16), 137.8 (C-5), 142.0 (C-17), 157.5 (C-3),
160.8 (C-400), 162.0 (C-30), 167.2 (C-50) ppm; ESI–MS: 442 (M+H)+
.
2.4.6. Synthesis of 3-methoxy-17a
-[30-(300,400-
2.4.3. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7c)
a
-[30-(300-methyl)phenylisoxazol-50-
dimethoxy)phenylisoxazol-50-yl]estra-1,3,5(10),16-tetraene (7f)
Compound 6f (245 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
CH2Cl2/EtOAc (98:2 v/v) as eluent, 7f was obtained as a white solid
(207 mg, 87%). Mp 188–191 °C; Rf = 0.31 (ss C). Anal. Calcd for
Compound 6c (222 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7c was obtained as a white
solid (192 mg, 90%). Mp 165–167 °C; Rf = 0.33 (ss D). Anal. Calcd for
30H33NO4: C, 76.41; H, 7.05; Found: C, 76.27; H, 6.86. 1H NMR
C
29H31NO2: C, 81.85; H, 7.34; Found: C, 81.70; H, 7.45. 1H NMR
C
(500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.50 (m, 1H), 1.65–1.84
(m, 4H), 1.97 (m, 1H), 2.22 (m, 1H), 2.31–2.39 (m, 2H), 2.41–2.48
(m, 2H), 2.92 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OMe), 3.94 (s, 3H) and
3.97 (s, 3H): 300-OMe and 400-OMe, 6.47 and 6.51 (bs, 2H, 40-H and
16-H), 6.66 (d, 1H, J = 2.3 Hz, 4-H), 6.74 (dd, 1H, J = 8.5 Hz,
J = 2.3 Hz, 2-H), 6.94 (d, 1H, J = 8.4 Hz, 500-H), 7.22 (d, 1H,
J = 8.5 Hz, 1-H), 7.33 (dd, 1H, J = 8.4 Hz, J = 1.6 Hz, 600-H), 7.45 (d,
1H, J = 1.6 Hz, 200-H) ppm; 13C NMR (125 MHz, CDCl3): d = 16.5
(C-18), 26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2),
37.1 (CH), 44.1 (CH), 47.1 (C-13), 55.2 (3-OMe), 55.8 and 55.9:
2 ꢁ OMe, 56.0 (CH), 97.3 (C-40), 109.3 and 111.0 (2C, C-200 and C-
500), 111.5 (C-2), 113.8 (C-4), 119.8 (C-600), 122.0 (C-100), 126.0 (C-
1), 132.4 (C-10), 132.9 (C-16), 137.8 (C-5), 141.9 (C-17), 149.3
and 150.5 (2C, C-300 and C-400), 157.5 (C-3), 162.1 (C-30), 167.3 (C-
(500 MHz, CDCl3): d = 1.05 (s, 3H, 18-H3), 1.51 (m, 1H), 1.65–1.85
(m, 4H), 1.97 (m, 1H), 2.21 (m, 1H), 2.32–2.39 (m, 2H), 2.42 (s,
3H, 300-Me), 2.43–2.50 (m, 2H), 2.92 (m, 2H, 6-H2), 3.80 (s, 3H, 3-
OMe), 6.51 (overlapping m, 2H, 40-H and 16-H), 6.67 (d, 1H,
J = 2.4 Hz, 4-H), 6.74 (dd, 1H, J = 8.5 Hz, J = 2.4 Hz, 2-H), 7.23 (d,
1H, J = 8.5 Hz, 1-H), 7.27 (d, 1H, J = 7.8 Hz, 400-H), 7.36 (t, 1H,
J = 7.8 Hz, 500-H), 7.63 (d, 1H, J = 7.8 Hz, 600-H), 7.67 (s, 1H, 200-H)
13
ppm; C NMR (125 MHz, CDCl3): d = 16.5 (C-18), 21.4 (300-Me),
26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2), 37.1
(CH), 44.1 (CH), 47.1 (C-13), 55.2 (3-OMe), 55.9 (CH), 97.5 (C-40),
111.4 (C-2), 113.8 (C-4), 123.9 (C-600), 126.0 (C-1), 127.4 (C-200),
128.7 (CH), 129.1 (C-100), 130.6 (CH), 132.5 (C-10), 132.9 (C-16),
137.8 (C-5), 138.6 (C-300), 141.9 (C-17), 157.5 (C-3), 162.5 (C-30),
167.3 (C-50) ppm; ESI–MS: 426 (M+H)+.
50) ppm; ESI–MS: 472 (M+H)+
.
2.4.4. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7d)
a
-[30-(400-methyl)phenylisoxazol-50-
2.4.7. Synthesis of 3-methoxy-17a
-[30-(300,500-dichloro-200,400,600-
Compound 6d (222 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7d was obtained as a yellow
solid (187 mg, 88%). Mp 197–199 °C; Rf = 0.30 (ss D). Anal. Calcd for
trimethoxy)phenylisoxazol-50-yl]estra-1,3,5(10),16-tetraene (7g)
Compound 6g (294 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7g was obtained as a white
solid (260 mg, 91%). Mp 65–68 °C; Rf = 0.33 (ss C). Anal. Calcd for
C
29H31NO2: C, 81.85; H, 7.34; Found: C, 82.01; H, 7.17. 1H NMR
(500 MHz, CDCl3): d = 1.03 (s, 3H, 18-H3), 1.49 (m, 1H), 1.62–1.84
(m, 4H), 1.95 (m, 1H), 2.18 (m, 1H), 2.31–2.38 (m, 2H), 2.39 (s,
3H, 400-Me), 2.40–2.48 (m, 2H), 2.90–2.91 (m, 2H, 6-H2), 3.78 (s,
3H, 3-OMe), 6.46–6.51 (overlapping m, 2H, 40-H and 16-H), 6.66
(d, 1H, J = 2.0 Hz, 4-H), 6.73 (dd, 1H, J = 8.5 Hz, J = 2.0 Hz, 2-H),
7.21 (d, 1H, J = 8.5 Hz, 1-H), 7.26 (d, 2H, J = 8.0 Hz, 300-H and 500-
H), 7.72 (d, 2H, J = 8.0 Hz, 200-H and 600-H) ppm; 13C NMR
(125 MHz, CDCl3): d = 16.5 (C-18), 21.4 (400-Me), 26.5 (CH2), 27.7
(CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2), 37.1 (CH), 44.1 (CH),
47.1 (C-13), 55.2 (3-OMe), 55.9 (CH), 97.4 (C-40), 111.5 (C-2),
113.8 (C-4), 126.0 (C-1), 126.4 (C-100), 126.6 (2C, C-200 and C-600),
129.5 (2C, C-300 and C-500), 132.5 (C-10), 132.8 (C-16), 137.8 (C-5),
C
31H33Cl2NO5: C, 65.26; H, 5.83; Found: C, 65.38; H, 5.95. 1H
NMR (500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.51 (m, 1H),
1.64–1.86 (m, 4H), 1.97 (m, 1H), 2.21 (m, 1H), 2.28–2.37 (m, 2H),
2.40–2.50 (m, 2H), 2.92 (m, 2H, 6-H2), 3.76 (s, 6H, 2 ꢁ OMe), 3.79
(s, 3H, OMe), 3.97 (s, 3H, OMe), 6.42 and 6.54 (bs, 2H, 40-H and
16-H), 6.66 (d, 1H, J = 2.6 Hz, 4-H), 6.73 (dd, 1H, J = 8.5 Hz,
J = 2.6 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H) ppm; 13C NMR
(125 MHz, CDCl3): d = 16.5 (C-18), 26.5 (CH2), 27.7 (CH2), 29.6
(CH2), 31.8 (CH2), 35.1 (CH2), 37.1 (CH), 44.1 (CH), 47.1 (C-13),
55.2 (3-OMe), 56.0 (CH), 60.9 (OMe), 62.1 (2 ꢁ OMe), 101.5
(C-40), 111.5 (C-2), 113.9 (C-4), 117.2 (2C, C-300 and C-500), 120.2
(C-100), 126.0 (C-1), 132.5 (C-10), 133.0 (C-16) 137.8 (C-5), 141.7

1080
D. Kovács et al. / Steroids 77 (2012) 1075–1085
(C-17), 154.3 (2C, C-200 and C-600), 154.8 (C-30), 156.1 (C-400), 157.5
(C-3), 166.5 (C-50) ppm; ESI–MS: 571 (M+H)+.
2.4.11. Synthesis of 3-methoxy-17
yl]-estra-1,3,5(10),16-tetraene (7k)
a
-[30-(400-nitro)phenylisoxazol-50-
Compound 6k (237 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
CH2Cl2 as eluent, 7j was obtained as a pale-yellow solid (199 mg,
87%). Mp 196ꢀ198 °C; Rf = 0.52 (ss C). Anal. Calcd for
2.4.8. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7h)
a
-[30-(400-fluoro)phenylisoxazol-50-
Compound 6h (224 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7h was obtained as a white
solid (191 mg, 89%). Mp 157ꢀ160 °C; Rf = 0.34 (ss D). Anal. Calcd
for C28H28FNO2: C, 78.30; H, 6.57; Found: C, 78.17; H, 6.73. 1H
NMR (500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.51 (m, 1H),
1.65–1.85 (m, 4H), 1.97 (m, 1H), 2.21 (m, 1H), 2.32–2.40 (m, 2H),
2.42–2.49 (m, 2H), 2.92 (m, 2H, 6-H2), 3.80 (s, 3H, 3-OMe), 6.48
and 6.53 (bs, 2H, 40-H and 16-H), 6.67 (d, 1H, J = 1.8 Hz, 4-H),
6.74 (dd, 1H, J = 8.5 Hz, J = 1.8 Hz, 2-H), 7.16 (t, 2H, J = 8.1 Hz, 300-
H and 500-H) ppm, 7.23 (d, 1H, J = 8.5 Hz, 1-H), 7.83 (dd, 2H,
J = 8.1 Hz, J = 5.5 Hz, 200-H and 600-H) ppm; 13C NMR (125 MHz,
CDCl3): d = 16.5 (C-18), 26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.8
(CH2), 35.2 (CH2), 37.1 (CH), 44.1 (CH), 47.1 (C-13), 55.2 (3-OMe),
55.9 (CH), 97.3 (C-40), 111.4 (C-2), 113.8 (C-4), 116.0 (2C,
J = 21.8 Hz, C-300 and C-500), 125.5 (C-100, J = 3.1 Hz,), 126.1 (C-1),
128.7 (2C, J = 8.3 Hz, C-200 and C-600), 132.4 (C-10), 133.2 (C-16),
137.8 (C-5), 141.8 (C-17), 157.5 (C-3), 161.5 (C-30), 163.7 (C-400,
J = 248.1 Hz), 167.6 (C-50) ppm; ESI–MS: 430 (M+H)+.
C
28H28N2O4: C, 73.66; H, 6.18; Found: C, 73.82; H, 5.98. 1H NMR
(500 MHz, CDCl3): d = 1.05 (s, 3H, 18-H3), 1.50 (m, 1H), 1.66–1.85
(m, 4H), 1.97 (m, 1H), 2.22 (m, 1H), 2.32–2.40 (m, 2H), 2.42–2.50
(m, 2H), 2.92 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OMe), 6.58 (overlapping
m, 2H, 40-H and 16-H), 6.66 (d, 1H, J = 2.6 Hz, 4-H), 6.74 (dd, 1H,
J = 8.5 Hz, J = 2.6 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H), 8.02 (d,
2H, J = 8.7 Hz, 200-H and 600-H), 8.33 (d, 2H, J = 8.7 Hz, 300-H and 500-
H) ppm; 13C NMR (125 MHz, CDCl3): d = 16.5 (C-18), 26.5 (CH2),
27.7 (CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2), 37.1 (CH), 44.1
(CH), 47.1 (C-13), 55.2 (3-OMe), 56.0 (CH), 97.4 (C-40), 111.5 (C-
2), 113.8 (C-4), 124.1 (2C, C-300 and C-500), 126.0 (C-1), 127.6 (2C,
C-200 and C-600), 132.3 (C-10), 134.1 (C-16), 135.4 (C-100), 137.8 (C-
5), 141.5 (C-17), 148.6 (C-400), 157.5 (C-3), 160.6 (C-30), 168.4 (C-
50) ppm; ESI–MS: 457 (M+H)+.
2.5. Synthesis of 3-methoxy-17-ethynylestra-1,3,5(10),16-tetraene (8)
Compound 1 (310 mg, 1.00 mmol) was dissolved in pyridine
(20 mL), and POCl3 (1.6 mL, 24 mmol) was added dropwise at
0 °C under vigorous stirring. The reaction mixture was allowed to
warm to room temperature and after 24 h it was poured into a
mixture of ice and HCl, and extracted with EtOAc (2 ꢁ 15 mL).
The combined organic phases were washed with water (15 mL),
followed by saturated NaHCO3 solution (15 mL), then dried over
Na2SO4 and evaporated in vacuo. The resulting crude product was
purified by flash chromatography with CH2Cl2 as eluent to give 8
as a pale-yellow solid (257 mg, 88%).
2.4.9. Synthesis of 3-methoxy-17
yl]estra-1,3,5(10),16-tetraene (7i)
a
-[30-(400-chloro)phenylisoxazol-50-
Compound 6i (232 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (50:50 v/v) as eluent, 7i was obtained as a white
solid (190 mg, 85%). Mp 179–182 °C; Rf = 0.37 (ss D Anal. Calcd for
C
28H28ClNO2: C, 75.41; H, 6.33; Found: C, 75.64; H, 6.24. 1H NMR
(500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.50 (m, 1H), 1.65ꢀ1.85
(m, 4H), 1.97 (m, 1H), 2.21 (m, 1H), 2.31ꢀ2.38 (m, 2H), 2.40–2.48
(m, 2H), 2.92 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OMe), 6.48–6.53 (over-
lapping m, 2H, 40-H and 16-H), 6.67 (d, 1H, J = 2.3 Hz, 4-H), 6.74
(dd, 1H, J = 8.5 Hz, J = 2.3 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H),
7.44 (d, 2H, J = 8.4 Hz, 300-H and 500-H), 7.78 (d, 2H, J = 8.4 Hz, 200-
H and 600-H) ppm; 13C NMR (125 MHz, CDCl3): d = 16.5 (C-18),
26.5 (CH2), 27.7 (CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2), 37.1
(CH), 44.1 (CH), 47.1 (C-13), 55.2 (3-OMe), 55.9 (CH), 97.3 (C-40),
111.5 (C-2), 113.8 (C-4), 126.0 (C-1), 127.8 (C-100), 128.0 (2C, C-200
and C-600), 129.1 (2C, C-300 and C-500), 132.4 (C-10), 133.3 (C-16),
135.8 (C-400), 137.8 (C-5), 141.8 (C-17), 157.5 (C-3), 161.4 (C-30),
167.7 (C-50) ppm; ESI–MS: 447 (M+H)+.
2.6. Synthesis of 3-methoxy-17a
-[30-(200-methyl)phenylisoxazol-50-
yl]estra-1,3,5(10),16-tetraene (7b) from 3-methoxy-17-ethynylestra-
1,3,5(10),16-tetraene (8)
3-Methoxy-17-ethynylestra-1,3,5(10),16-tetraene (8, 250 mg,
0.85 mmol) and N-hydroxy-2-methylbenzenecarboximidoyl chlo-
ride (4b, 288 mg, 1.70 mmol) were dissolved in toluene (10 mL),
and Ph3P (44 mg, 0.17 mmol) and CuI (17 mg, 0.09 mmol) were
then added to the solution. DIPEA (0.57 ml, 3.40 mmol) was added
dropwise to the gently heated reaction mixture, which was subse-
quently heated to reflux under stirring for 4 h. After the disappear-
ance of the starting material (TLC monitoring), the solvent was
evaporated off in vacuo. The resulting crude product was purified
by flash chromatography with n-hexane/CH2Cl2 (50:50 v/v) as elu-
ent to give 7b as a white solid (137 mg, 38%).
2.4.10. Synthesis of 3-methoxy-17
yl]-estra-1,3,5(10),16-tetraene (7j)
a
-[30-(400-bromo)phenylisoxazol-50-
Compound 6j (254 mg) was used for the synthesis as described
in Section 2.4. After purification by column chromatography with
n-hexane/CH2Cl2 (20:80 v/v) as eluent, 7j was obtained as a white
solid (221 mg, 90%). Mp 187–190 °C; Rf = 0.56 (ss C). Anal. Calcd for
2.7. Determination of antiproliferative activities
C
28H28BrNO2: C, 68.57; H, 5.75; Found: C, 68.75; H, 5.93. 1H NMR
Antiproliferative effects were measured in vitro on three human
cancer cell lines (ECACC; Salisbury, UK): HeLa (cervix adenocarci-
noma), MCF7 (breast adenocarcinoma) and A2780 (ovarian carci-
noma). The cells were cultivated in minimal essential medium
(Sigma–Aldrich, Budapest, Hungary) supplemented with 10% fetal
bovine serum, 1% non-essential amino acids and an antibiotic-anti-
mycotic mixture. Near-confluent cells were seeded into a 96-well
plate (5000 cells/well) and, after overnight standing, the medium
(200 lL) containing the tested compound (at 10 or 30 lM) was
added. Following a 72-h incubation in a humidified atmosphere
of 5% CO2 at 37 °C, the living cells were assayed by the addition
of 20 lL of 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
(500 MHz, CDCl3): d = 1.04 (s, 3H, 18-H3), 1.50 (m, 1H), 1.64–1.84
(m, 4H), 1.96 (m, 1H), 2.21 (m, 1H), 2.31–2.38 (m, 2H), 2.41–2.48
(m, 2H), 2.92 (m, 2H, 6-H2), 3.79 (s, 3H, 3-OMe), 6.48 and 6.53
(bs, 2H, 40-H and 16-H), 6.66 (d, 1H, J = 2.2 Hz, 4-H), 6.74 (dd, 1H,
J = 8.5 Hz, J = 2.2 Hz, 2-H), 7.22 (d, 1H, J = 8.5 Hz, 1-H), 7.60 (d,
2H, J = 8.3 Hz, 200-H and 600-H), 7.71 (d, 2H, J = 8.3 Hz, 300-H and 500-
H) ppm; 13C NMR (125 MHz, CDCl3): d = 16.5 (C-18), 26.5 (CH2),
27.7 (CH2), 29.6 (CH2), 31.8 (CH2), 35.2 (CH2), 37.1 (CH), 44.1
(CH), 47.1 (C-13), 55.2 (3-OMe), 56.0 (CH), 97.3 (C-40), 111.5 (C-
2), 113.8 (C-4), 124.1 (C-400), 126.0 (C-1), 128.3 (2C, C-200 and C-
600), 132.1 (2C, C-300 and C-500), 132.5 (C-10), 133.4 (C-16), 137.8
(C-5), 141.8 (C-17), 157.5 (C-3), 161.5 (C-30), 167.7 (C-50) ppm;
ESI–MS: 491 (M+H)+.
nyltetrazolium bromide] solution [30]. During a 4-h contact period,
the MTT was converted by intact mitochondrial reductase and

D. Kovács et al. / Steroids 77 (2012) 1075–1085
1081
Table 1
Synthesis of steroidal isoxazoles by Cu(I)-catalyzed cycloaddition.
O
C
NH2OH.HCl
NCS
Ar
C
H
3a−k
N
OH
Ar
C
N
OH
Ar
H
OH−
Cl
2a−k
Ar
4a−k
N
O
CH
OH
Ar
C
N
O
OH
17
5a−k
H
H
CuI (0.1 equiv.)
PPh3 (0.2 equiv.)
DIPEA (4 equiv.)
toluene, 111 oC, 3 h
H
H
H
H
MeO
MeO
1
6a−k
Entry
1
Imidoyl chloride/nitrile oxide
Ar
Product
Yielda (%)
4a/5a
6a
93
2
3
4b/5b
4c/5c
6b
6c
98
96
4
5
6
4d/5d
4e/5e
4f/5f
6d
6e
6f
97
97
95
MeO
MeO
MeO
Cl
7
4gb/5g
6g
98
OMe
MeO
Cl
OMe
8
4h/5h
5i/5i
6h
6i
81
81
78
63
F
9
Cl
10
11
5j/5j
6j
Br
5k/5k
6k
O2N
a
After purification by column chromatography.
During treatment of 2,4,6-trimethoxybenzaldehyde oxime (3g) with NCS, bis-chlorination of the aromatic ring also occurred.
b
precipitated as blue crystals. The medium was then removed, the
precipitated formazan crystals were solubilized in DMSO
3. Results and discussion
(100 lL) during a 60-min period of shaking at 25 °C, and the absor-
For the preparation of novel steroidal isoxazoles, mestranol 1,
containing an -ethynyl group on C-17 of the sterane skeleton,
bance was read at 545 nm with a microplate reader. Wells with
untreated cells were utilized as controls. All in vitro experiments
were carried out on two microplates with at least five parallel
wells. Stock solutions of the tested substances (10 mM) were pre-
pared with DMSO. The DMSO concentration (0.3%) of the medium
did not have any significant effect on cell proliferation. Cisplatin
was used as reference compound.
a
was used as starting material. Preliminary Cu(I)-catalyzed ring-clo-
sure experiments on 1 with benzonitrile oxide 5a were first carried
out in order to optimize the reaction conditions (Table 1, entry 1).
The stable precursor 4a of the nitrile oxide dipole (5a) was synthe-
tized in a two-step pathway by condensation of benzaldehyde 2a
with hydroxylamine hydrochloride in alkaline medium and

1082
D. Kovács et al. / Steroids 77 (2012) 1075–1085
Table 2
Synthesis of D16,17 17-exo-isoxazolyl derivatives.
Ar
N
Ar
N
O
O
17
OH
17
POCl3
H
H
16
pyridine
rt, 24 h
H
H
H
H
MeO
MeO
6a−k
7a−k
Entry
1
Substrate
Ar
Product
Yielda (%)
89
6a
7a
2
3
6b
6c
7b
7c
92
90
4
5
6
6d
6e
6f
7d
7e
7f
88
88
87
MeO
MeO
MeO
7
6g
7g
91
Cl
Cl
OMe
OMe
MeO
8
6h
6i
7h
7i
89
85
90
87
F
9
Cl
10
11
6j
7j
Br
O2N
6k
7k
a
After purification by column chromatography.
subsequent chlorination of the aldoxime 3a with N-chlorosuccini-
mide (NCS) [31]. Benzonitrile oxide 5a can be generated in situ
from 4a by dehydrochlorination with a base in the presence of
the dipolarophile 1. A similar intermolecular ring-closure reaction
During optimization of the Cu(I)-catalyzed cycloaddition of 1
with benzonitrile oxide (5a), the best conversion was found to
occur on the use of a catalytic amount of CuI as Cu(I) source,
PPh3 as stabilizing ligand, and excess DIPEA as base in refluxing
toluene. The presence of Ph3P is important to stabilize the catalyt-
ically active Cu(I) by complexation so as to prevent its oxidation to
Cu(II) [32,33]. Furthermore, the solubility of Cu(I) in the organic
medium is also improved. The dual function of DIPEA is to facilitate
the formation of Cu(I)-acetylide in sufficient amount and the in situ
generation of the 1,3-dipole (5a) from its relatively stable precur-
sor (4a). Since dimerization of unhindered nitrile oxides to furox-
anes can reduce their proportion available for cycloaddition [34],
the amine base was added to the reaction mixture terminally in
order to ensure a low stationary concentration of the dipole and
to minimize the undesired side-reaction. Under the applied
of 17a-ethynylestradiol (the 3-demethylated derivative of mestra-
nol) with 4-methoxybenzonitrile oxide 5e in a 1:1 (v/v) mixture of
H2O/tBuOH, using CuSO4ꢂ5H2O/Na-ascorbate as Cu(I) source, was
reported previously by Sharpless et al. [25]. A single isoxazole reg-
ioisomer was obtained in 98% yield after 1 h at ambient tempera-
ture on the application of KHCO3 as base to release the 1,3-
dipole (5e) from its precursor (4e). However, mestranol 1 contains
a methoxy instead of a hydroxy group at C-3 and is therefore less
polar and cannot be dissolved in the above solvent system. Accord-
ingly, the reaction conditions needed modification in order to
avoid the solubility problems.

D. Kovács et al. / Steroids 77 (2012) 1075–1085
1083
N
O
CH
4b
17
POCl3
[5b]
1
H
16
pyridine
CuI (0.1 equiv.)
rt, 24 h
H
H
PPh3 (0.2 equiv)
DIPEA (4 equiv.)
toluene, 111 oC, 3 h
H
MeO
7b
8
+
possible by-products:
O
N
N
O
9
10
Scheme 1. Dehydration of mestranol (1) and the following Cu(I)-catalyzed cycloaddition with 2-methylbenzonitrile oxide (5b). The possible by-products (9 and 10) were not
isolated.
to furnish novel 17a-isoxazoles (6b–k) in good to excellent yields
(Table 1, entries 2–11). During the reactions of oximes (3b–k) with
NCS, bis-chlorination of the aromatic ring was observed for the
2,4,6-trimethoxybenzaldehyde oxime 3g, which is not surprising
in view of the additive ortho-directing effects of the electron-
donating methoxy-substituents. Although NMR characterizations
of the imidoyl chlorides (4a–k) were not carried out, and their for-
mation was monitored only by TLC, the 30,50-dichloro substitution
of product 6g was confirmed by NMR and MS methods. Otherwise,
both the oximes (3a–k) and their halogenated derivatives (4a–k)
appeared as double spots on the TLC plates, which indicated their
formation as mixtures of E and Z isomers. However, the imidoyl
chlorides (4a–k) were used directly after preparation without fur-
ther purification. The yields of the cycloadducts 6b–k were found
to depend on the electronic and steric features of the substituents
on the aromatic ring of the dipole 5b–k. The electron-donating
substituents in 5b–g (Table 1, entries 2–7) favored cycloaddition
to 1, in consequence of the lower tendency of these dipoles to un-
dergo dimerization to furoxanes, while the yields of the desired
products (6h–k) were decreased in the case of the electron-with-
drawing groups on the aromatic moiety in 5h–k (entries 8–11).
The lowest conversion was achieved for the reaction of 1 with
p-nitrobenzonitrile oxide 5k, owing to its shortest lifetime as a
monomer.
Table 3
Antiproliferative effects of the synthetized compounds.
Isoxazole
Growth inhibition % (SEM)
HeLa MCF7
10 10
A2780
10
lM
30
lM
lM
30
l
M
lM
30 lM
6a
6b
6c
6d
6e
6f
6g
6h
6i
<25a
<25
<25
<25
<25
<25
<25
<25
<25
<25
98 (0.1)
96 (0.7)
90 (0.5)
91 (0.3)
92 (0.2)
37 (2.4)
70 (1.8)
96 (0.4)
98 (0.1)
94 (0.5)
95 (0.4)
59 (2.0)
35 (2.4)
59 (0.7)
59 (0.6)
58 (0.9)
43 (2.4)
58 (0.9)
57 (1.2)
65 (1.3)
78 (0.3)
28 (1.7)
<25
<25
95 (0.2)
92 (1.2)
94 (0.4)
91 (0.4)
91 (0.5)
92 (0.3)
72 (1.0)
96 (0.8)
95 (0.5)
92 (0.2)
91 (0.6)
48 (1.8)
<25
26 (1.2)
50 (0.3)
57 (2.9)
<25
30 (2.9)
34 (1.7)
41 (2.7)
68 (1.1)
29 (2.1)
<25
98 (0.2)
94 (0.3)
91 (0.4)
81 (2.5)
78 (0.3)
71 (0.4)
78 (0.7)
98 (0.4)
97 (0.2)
84 (0.3)
96 (0.2)
45 (2.3)
38 (1.8)
38 (1.3)
45 (1.5)
65 (1.2)
39 (1.4)
33 (2.5)
40 (2.0)
43 (1.7)
79 (0.9)
29 (2.0)
28 (1.9)
32 (1.9)
43 (2.7)
49 (1.4)
40 (2.3)
29 (1.5)
28 (2.7)
41 (1.5)
29 (1.7)
<25
<25
<25
43 (2.9)
36 (2.5)
<25
33 (2.4)
54 (1.1)
35 (2.6)
<25
48 (1.3)
35 (1.9)
25 (1.9)
43 (1.0)
<25
6j
6k
7a
7b
7c
7d
7f
7g
7h
7i
28 (1.1)
32 (2.1)
<25
<25
<25
<25
35 (0.6)
58 (0.8)
31 (1.8)
25 (0.8)
32 (1.2)
37 (1.4)
68 (0.7)
27 (1.3)
36 (2.2)
<25
<25
<25
<25
<25
46 (2.5)
7j
7k
<25
63 (1.8)
Although novel isoxazoles (6a–k) were prepared in high yields
in the presence of CuI as catalyst under the optimized conditions
above, the reactions can also be performed at room temperature
by using CuSO4ꢂ5H2O/Na-ascorbate as Cu(I) source. The reaction
of 1 with 5a in a mixture of CH2Cl2 as solvent and water as co-sol-
vent resulted in the corresponding product 6a in 92% yield after
purification. Consequently, the two kinds of methods were found
to work similarly.
E2-type dehydration of 6a–k was carried out with POCl3 in pyr-
idine, to furnish D16,17 17-isoxazolyl derivatives 7a–k (Table 2).
The transformations were not accompanied by side-reactions
(e.g. chlorination or Wagner–Meerwein rearrangements [35])
under the applied conditions, and after separation by column chro-
matography the corresponding products (7a–k) were obtained in
yields of 85–92%.
Cisplatin
43 (2.3)
100 (0.3)
53 (2.3)
87 (1.2)
84 (1.2)
95 (0.3)
a
Compounds eliciting less than 25% inhibition of proliferation were considered
ineffective and the exact results are not given, for simplicity.
conditions, the reaction was complete within 3 h and the corre-
sponding isoxazole 6a was obtained in a yield of 93% after chro-
matographic purification. The conversion, however, was observed
to be significantly lower (by ca. 20%) when DIPEA was added to
the reaction mixture in an early stage, due to the greater extent
of dipole dimerization.
After determination of the optimal reaction parameters, similar
cycloadditions of 1 with different substituted benzonitrile oxides
(5b–k), synthetized from the corresponding aryl aldehydes (2b–
k) by the well-known sequence mentioned above, were performed

1084
D. Kovács et al. / Steroids 77 (2012) 1075–1085
The preparation of 17-isoxazole 7b was also investigated in a
series were prepared efficiently via Cu(I)-catalyzed regioselective
cycloadditions of different aryl nitrile oxides to mestranol. The
transformations were carried out under two kinds of conditions,
using different additives to furnish the desired products with sim-
ilar high yields. The ring closures were affected by the substitution
pattern of the nitrile oxide, the highest conversions being achieved
with sterically hindered dipoles containing electron-donating sub-
stituents. Efficient dehydrations of the 17b-hydroxy derivatives led
reverse synthetic sequence. Mestranol 1 was first dehydrated to
3-methoxy-17-ethynylestra-1,3,5(10),16-tetraene 8, and this was
followed by Cu(I)-catalyzed cycloaddition to 2-methylbenzonitrile
oxide 5b, generated in situ from 4b by using DIPEA (Scheme 1). The
effectiveness of the initial elimination reaction was found to be
quite similar to those of the preparations of 6a–k, and 8 was
obtained in 88% yield. However, the step of cycloaddition of 8 with
5b appeared to be less chemoselective since the formation of by-
products accompanying 7b was detected by TLC, this eventually
decreasing the isolated yield of 7b from 98% to 38%. The observed
side-reactions can be explained by the structural character of 8,
which contains both C@C and C„C bonds, which display similar
reactivities toward cycloaddition with nitrile oxides. The 1,3-cyclo-
addition of the dipole 5b to the olephinic moiety, affording isoxaz-
oline regioisomers (9 and 10), can therefore compete with the
alkyne–dipole reaction and reduce the yield of the desired product
7b. Accordingly, the 1,3-dipolar cycloaddition of mestranol 1 with
nitrile oxides followed by dehydration was demonstrated to be a
more efficient pathway for the preparation of 7a–k than the latter
method, with its low selectivity.
The structures of the synthetized compounds (6a–k and 7a–k)
were determined by NMR and MS measurements. In the 1H NMR
spectra of 6a–k, the signals of the protons on the aromatic moiety
introduced by the nitrile oxides (5a–k) appeared at 7.1–8.3 ppm,
with appropriate multiplicities; an exception was 6g, which con-
tains a fully-substituted ring. The 1H and 13C NMR spectra indi-
cated the 40-H singlet of the newly formed hetero ring at
6.5 ppm, while the signal of the quaternary C-50 was identified at
around 178 ppm. The 1H NMR spectra of the unsaturated deriva-
tives 7a–k confirmed the presence of the double bond between
C-16 and C-17 since the multiplets of 16-H2 had disappeared as
compared with 6a–k, and the signal of 16-H was at 6.5 ppm, to-
gether with the peak of 40-H. The elimination of water and hence
the development of conjugation with the isoxazole ring were also
proved by 13C NMR; the signals of C-16 and C-17 appeared at
around 133 and 142 ppm, respectively.
to
D
16,17 steroidal isoxazoles. All compounds were tested in vitro as
concerns their antiproliferative activities on three malignant cell
lines, and some hydroxylated analogs proved to exert promising
cell growth-inhibitory effects. Although the antiproliferative activ-
ities of the tested compounds are moderate, the results suggest
that steroidal isoxazoles may induce a disturbance in the cell divi-
sion by a mode other than hormone receptor-based action, moti-
vating the design of further anticancer lead candidates and
optimization for better activities.
Acknowledgments
This work was supported financially by the New Hungary
Development Plan (TÁMOP 4.2.1/B-09/1/KONV-2010-0005) and
the Hungarian Scientific Research Fund (K 101659). The project
00TÁMOP 4.2.1/B-09/1/KONV-2010-0005 – Creating the Center of
Excellence at the University of Szeged00 is supported by the Euro-
pean Union and co-financed by the European Regional Fund.
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