Regio-and stereoselective synthesis of pregnane-fused isoxazolines by nitril-oxide/alkene 1,3-dipolar cycloaddition and an evaluation of their cell-growth inhibitory effect in vitro

Article abstract of DOI:10.1016/j.molstruc.2016.01.050

Efficient syntheses of some pregnane-fused isoxazolines from 16-dehydropregnenolone acetate with different arylnitrile oxides were carried out by 1,3-dipolar cycloadditions. The intermolecular ring-closures occurred in a highly regio- and stereoselective manner permitting the formation of a single 16α,17α-condensed diastereomer in which the O terminus of the nitrile oxide dipole is attached to C-17 of the sterane core. The conversions were found to be affected significantly by the electronic character of the substituents on the aromatic moiety of the 1,3-dipoles. Deacetylation of the primary products resulted in the corresponding 3β-OH analogs. All of the synthesized compounds were subjected to in vitro pharmacological studies for the determination of their antiproliferative effects on four breast cancer cell lines (MCF7, T47D, MDA-MB-231 and MDA-MB-361).

Full text of DOI:10.1016/j.molstruc.2016.01.050

Accepted Manuscript
Regio- and stereoselective synthesis of pregnane-fused isoxazolines by nitril-oxide/
alkene 1,3-dipolar cycloaddition and an evaluation of their cell-growth inhibitory effect
in vitro
Gergő Mótyán, Ádám Baji, István Zupkó, Éva Frank
PII:
S0022-2860(16)30050-3
10.1016/j.molstruc.2016.01.050
MOLSTR 22166
DOI:
Reference:
To appear in: Journal of Molecular Structure
Received Date: 2 November 2015
Revised Date: 19 January 2016
Accepted Date: 20 January 2016
Please cite this article as: G. Mótyán, Á. Baji, I. Zupkó, É. Frank, Regio- and stereoselective synthesis
of pregnane-fused isoxazolines by nitril-oxide/alkene 1,3-dipolar cycloaddition and an evaluation
of their cell-growth inhibitory effect in vitro, Journal of Molecular Structure (2016), doi: 10.1016/
j.molstruc.2016.01.050.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Regio- and stereoselective synthesis of pregnane-fused isoxazolines by nitril-oxide/alkene
1,3-dipolar cycloaddition and an evaluation of their cell-growth inhibitory effect in vitro
Gergő Mótyán, Ádám Baji, István Zupkó, Éva Frank*

ACCEPTED MANUSCRIPT
Regio- and stereoselective synthesis of pregnane-fused isoxazolines by nitril-oxide/alkene
1,3-dipolar cycloaddition and an evaluation of their cell-growth inhibitory effect in vitro
Gergő Mótyána, Ádám Bajia, István Zupkób, Éva Franka,*
aDepartment of Organic Chemistry, University of Szeged, H-6720 Szeged, Hungary;
*corresponding author: Fax: +36-62-544200; E-mail: frank@chem.u-szeged.hu
bDepartment of Pharmacodynamics and Biopharmacy, University of Szeged, H-6720 Szeged,
Hungary
Supporting Information is available in ………..
1

ACCEPTED MANUSCRIPT
Abstract:
Efficient syntheses of some pregnane-fused isoxazolines from 16-dehydropregnenolone
acetate with different arylnitrile oxides were carried out by 1,3-dipolar cycloadditions. The
intermolecular ring-closures occurred in a highly regio- and stereoselective manner permitting
the formation of a single 16α,17α-condensed diastereomer in which the O terminus of the
nitrile oxide dipole is attached to C-17 of the sterane core. The conversions were found to be
affected significantly by the electronic character of the substituents on the aromatic moiety of
the 1,3-dipoles. Deacetylation of the primary products resulted in the corresponding 3β-OH
analogs. All of the synthesized compounds were subjected to in vitro pharmacological studies
for the determination of their antiproliferative effects on four breast cancer cell lines (MCF7,
T47D, MDA-MB-231 and MDA-MB-361).
Keywords: steroids, 1,3-dipolar cycloaddition, nitrile oxides, isoxazoline, stereostructure,
antiproliferative effect
2

ACCEPTED MANUSCRIPT
1. Introduction
1,3-Dipolar cycloadditions of nitrile oxides with alkene or alkyne dipolarophiles leading to 2-
isoxazoline or isoxazole derivatives [1, 2] have been extensively studied and have found
general application in organic synthesis owing to the great importance of these heterocycles as
structural building blocks of several biologically active molecules [3, 4] and versatile
intermediates in the synthesis of numerous bifunctional compounds [5].
The propargyl/allenyl-type nitrile oxide 1,3-dipoles, containing a N atom in the center can
readily undergo cyclization with multiple-bond systems under thermal or catalytic conditions
[6]. The reactivity of the dipolarophile as well as the regio- and stereoselectivity of the
thermally-induced process depend on certain structural features. Thus, monosubstituted
alkenes are generally more reactive than disubstituted olefins or alkynes, and the reactions
with nitrile oxides occur with almost complete regiospecificity to furnish exclusively
5-substituted isoxazolines. However, mixtures of regioisomers are usually obtained with 1,2-
disubstituted dipolarophiles, although steric effects may have an influence on the
regioisomeric ratio, especially in case of polycyclic systems [1, 6]. π-Conjugation of the
double bond has a strong promoting effect on the reactivity of the alkene and site-selectivity
often predominates when different olefinic moieties compete for the dipole. Ring-strain also
destabilizes dipolarophiles and enhances their activity toward nitrile oxides. The
stereochemical outcome of the cycloadditions is affected by steric and geometric features of
the dipolarophile.
Attention to the incorporation of five-membered N,O-heterocyclic moiety into steroids
either connected to or condensed with ring A or D of the sterane core is caused by the fact that
some of these modified compounds have been reported to exhibit various biological activities,
including anabolic, antibacterial, antiproliferative, hypocholesteremic and anti-inflammatory
properties [5, 7−10]. Moreover, steroids are attractive chiral models with relatively rigid
3

ACCEPTED MANUSCRIPT
framework for controlling the regio- and/or stereoselectivity of nitrile oxide/alkene
cycloadditions [11]. However, sterane-based dipolarophiles, containing an unactivated and/or
sterically less hindered double bond are usually not reactive enough necessitating elevated
temperature and/or prolonged reaction time for sufficient conversion and the reactions often
suffer from a lack of selectivity [5, 12, 13]. The introduction of an enone moiety into the
vicinity of sterically interfering groups has been found to have a strong driving effect on the
reactivity of such alkenes and also on the regio- and stereochemical outcome of the thermally-
induced ring-closures [1]. In this regard, we recently reported the regio- and stereoselective
synthesis of some novel 5α-androstanes containing an 2-isoxazoline moiety condensed to ring
A or D by intermolecular 1,3-dipolar cycloaddition of aryl nitrile oxides to steroidal α,β-
unsaturated ketones [14]. We have demonstrated that the cyclic enone moiety of the five-
membered ring D is more reactive than that of the six-membered ring A presumably due to
ring strain and conformation effects.
As a continuation of our research for the construction of steroidal ring-condensed
heterocycles [10, 14−18], our aim was to extend the ring closure reactions to the pregnane
skeleton to make available some additional ring D-fused isoxazolines. The present work
describes the transformations of 16-dehydropregnenolone acetate (16-DPA) containing a
reactive dipolarophilic enone moiety, with nitrile oxides via thermal 1,3-dipolar
cycloaddition. The regio- and stereoselectivity of the process and the influence of steric and
electronic factors on the ring-closure reactions were also investigated. Since several steroidal
isoxazoles and their saturated analogs have been reported to possess cell-growth-inhibitory
effect on malignant cell lines of diverse origins [7, 9−11], our further goal was to investigate
our compounds for such activities against four human adherent breast cancer cell lines
(MCF7, T47D, MDA-MB-231 and MDA-MB-361).
4

ACCEPTED MANUSCRIPT
2. Experimental
2.1. General
Melting points (mp-s) were determined on an SMS Optimelt digital apparatus. Elemental
analysis data were obtained with a Perkin Elmer CHN analyzer model 2400 and FT-IR
spectra were recorded on a FT/IR-4700 spectrometer (Jasco) using ATR. Infrared absorbance
1
is reported in reciprocal centimeters (cm-1). H NMR spectra were obtained at room
13
temperature in CDCl3 solution at 500 MHz (Brucker DRX 500) and the C NMR spectra at
125 MHz with the same instrument. Chemical shifts are reported in ppm (δ scale) relative to
1
TMS; coupling constants (J) are given in Hz. Multiplicity of the H resonance peaks are
1
indicated as singlet (s), doublet (d), triplet (t) and multiplet (m). 13C NMR spectra are H-
decoupled. 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 comprised an Agilent 1100 micro vacuum degasser, a quaternary pump, a micro-well
plate autoinjector and a 1946A MSD equipped with an electrospray ion (ESI) source operated
in positive ion mode. The ESI parameters: nebulizing gas N2, at 35 psi; drying gas N2, at
350 °C and 12 L min-1; capillary voltage 3000 V; and fragmentor voltage 70 V. The MSD was
operated in scan mode with the mass range m/z 60−620. Samples (0.2 ꢀL) were injected with
an automated needle wash directly into the solvent flow (0.3 mL min-1) of MeCN/H2O 70:30
(v/v) supplemented with 0.1% formic acid. The system was controlled by Agilent LC/MSD
Chemstation software. All solvents were distilled immediately prior to use. Reagents and
materials were obtained from reliable commercial suppliers and were used without
purification. The reactions were monitored by TLC on Kieselgel-G (Merck Si 254 F) layers
(0.25 mm thick). The spots were detected by spraying with 5% phosphomolybdic acid in 50%
aqueous phosphoric acid. The Rf values were determined for the spots observed by
5

ACCEPTED MANUSCRIPT
illumination at 254 and 365 nm. Solvent systems (ss): CH2Cl2 (A); EtOAc/CH2Cl2 (10/80,
v/v) (B). Flash chromatography: Merck silica gel 60, 40–63 ꢀm.
2.2. General procedure for the preparation of steroidal isoxazolines (6a−e)
16-DPA (1, 357 mg, 1.00 mmol) and the appropriate aromatic hydroximidoyl chloride (4a−e,
1.50 mmol) were dissolved in toluene (15 mL), and DIPEA (0.52 mL, 3.00 mmol) was added
dropwise to the reaction mixture at room temperature, with subsequent refluxing for 2 h. The
solvent was then evaporated off in vacuo and the resulting crude product was purified by
column chromatography with CH2Cl2.
2.2.1. 3β-Acetoxy-3′-phenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one (6a)
In accordance with the general procedure, N-hydroxybenzenecarboximidoyl chloride (4a, 233
mg) was used for the synthesis. The product 6a (452 mg) was obtained as a white precipitate.
Mp 201−204 °C (Mp 196−198 °C [22]); Rf = 0.25 (ss A); IR: 1730, 1708, 1593, 1442, 1193
1
cm-1, H NMR (CDCl3, 500 MHz):
δ 0.79 (s, 3H, 18-H3), 1.01 (s, 3H, 19-H3), 1.04 (m, 1H,
9α-H), 1.14 (m, 1H, 1α-H), 1.45-1.61 (overlapping m, 5H, 2α-H, 8β-H, 11β-H, 14α-H, 15α-
H ), 1.69-1.77 (overlapping m, 4H, 7β-H, 15β-H, 11α-H, 12β-H), 1.87 (m, 3H, 1β-H, 2β-H,
7α-H), 2.02 (s, 3H Ac-CH3), 2.08 (m, 1H, 12α-H), 2.28 (m, 2H, 4-H2), 2.31 (s, 3H, 21-H3),
4.42 (t-like m, 1H, 16-H), 4.58 (m, 1H, 3-H), 5.30 (m, 1H, 6-H), 7.39 (m, 3H, 3″-H, 4″-H and
13
5″-H), 7.66 (m, 2H, 2″-H and 6″-H); C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-
19), 20.5 (C-11), 21.4 (Ac-CH3), 27.4 (C-21), 27.6 (C-2), 31.2 (C-8), 31.6 (2C, C-7 and C-
12), 31.8 (C-15), 35.8 (C-10), 36.9 (C-1), 38.0 (C-4), 48.3 (C-13), 49.1 (C-9), 50.4 (C-14),
51.0 (C-16), 73.7 (C-3), 106.1 (C-17), 121.8 (C-6), 127.1 (2C, C-2″ and C-3″), 128.6 (C-1″),
128.7 (2C, C-3″ and C-5″), 130.1 (C-4″), 139.6 (C-5), 159.9 (C-3′), 170.4 (Ac-CO), 206.4 (C-
6

ACCEPTED MANUSCRIPT
20); ESI-MS: 476 [M+H]+; Anal. Calcd for C30H37NO4 C, 75.76; H, 7.84; Found: C, 75.92;
H, 7.69.
2.2.2. 3β-Acetoxy-3′-4″-tolyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one (6b)
In accordance with the general procedure, N-hydroxy-4-methylbenzenecarboximidoyl
chloride (4b, 255 mg) was used for the synthesis. The product 6b (480 mg) was obtained as a
white precipitate. Mp 164−166 °C (Mp 121−123 °C [22]); Rf = 0.26 (ss A); IR: 1727, 1712,
1593, 1456, 1197 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.78 (s, 3H, 18-H3), 1.02 (s, 3H, 19-
H3), 1.04 (m, 1H, 9α-H), 1.14 (m, 1H, 1α-H), 1.44-1.61 (overlapping m, 5H), 1.68-1.75
(overlapping m, 4H), 1.87 (m, 3H), 2.02 (s, 3H Ac-CH3), 2.07 (m, 1H), 2.28 (m, 2H, 4-H2),
2.30 (s, 3H, 21-H3), 2.36 (s, 3H, 4″-CH3), 4.40 (t-like m, 1H, 16-H), 4.58 (m, 1H, 3-H), 5.30
(m, 1H, 6-H), 7.19 (d, 2H, J = 7.8 Hz, 3″-H and 5″-H), 7.55 (d, 2H, J = 7.8 Hz, 2″-H and 6″-
H); 13C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-19), 20.5 (C-11), 21.4 (2C, Ac-CH3
and 4″-CH3), 27.4 (C-21), 27.6 (C-2), 31.2 (C-8), 31.6 (2C, C-7 and C-12), 31.8 (C-15), 36.5
(C-10), 36.9 (C-1), 38.0 (C-4), 48.3 (C-13), 49.1 (C-9), 50.4 (C-14), 51.1 (C-16), 73.7 (C-3),
105.9 (C-17), 121.9 (C-6), 125.7 (C-1″), 127.0 (2C, C-2″ and C-6″), 129.4 (2C, C-3″ and C-
5″), 139.6 (C-5), 140.4 (C-4″), 159.9 (C-3′), 170.4 (Ac-CO), 206.6 (C-20); ESI-MS: 490
[M+H]+; Anal. Calcd for C31H39NO4 C, 76.04; H, 8.03; Found: C, 76.16; H, 7.86.
2.2.3. 3β-Acetoxy-3′-4″-methoxyphenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one
(6c)
In accordance with the general procedure, N-hydroxy-4-methoxybenzenecarboximidoyl
chloride (4c, 279 mg) was used for the synthesis. The product 6c (490 mg) was obtained as a
white precipitate. Mp 187−189 °C (Mp 119−121 °C [22]); Rf = 0.20 (ss A); IR: 1722, 1712,
1609, 1465, 1190 cm-1; 1H NMR (CDCl3, 500 MHz):
δ
0.78 (s, 3H, 18-H3), 1.01 (s, 3H, 19-
7

ACCEPTED MANUSCRIPT
H3), 1.04 (m, 1H, 9α-H), 1.13 (m, 1H, 1α-H), 1.44-1.61 (overlapping m, 5H), 1.67-1.75
(overlapping m, 4H), 1.86 (m, 3H), 2.01 (s, 3H Ac-CH3), 2.07 (m, 1H), 2.28 (m, 2H, 4-H2),
2.30 (s, 3H, 21-H3), 3.82 (s, 3H, 4″-OMe), 4.39 (t-like m, 1H, 16-H), 4.57 (m, 1H, 3-H), 5.29
(m, 1H, 6-H), 6.90 (d, 2H, J = 7.2 Hz, 3″-H and 5″-H), 7.59 (d, 2H, J = 7.2 Hz, 2″-H and 6″-
13
H); C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-19), 20.5 (C-11), 21.4 (Ac-CH3),
27.4 (C-21), 27.6 (C-2), 31.2 (C-8), 31.6 (2C, C-7 and C-12), 31.8 (C-15), 36.5 (C-10), 36.9
(C-1), 38.0 (C-4), 48.2 (C-13), 49.1 (C-9), 50.4 (C-14), 51.2 (C-16), 55.3 (4″-OMe), 73.7 (C-
3), 105.7 (C-17), 114.2 (2C, C-3″ and C-5″), 121.0 (C-1″), 121.9 (C-6), 128.6 (2C, C-2″ and
C-6″), 139.6 (C-5), 159.9 (C-3′), 161.0 (C-4″), 170.4 (Ac-CO), 206.7 (C-20); ESI-MS: 506
[M+H]+; Anal. Calcd for C31H39NO5 C, 73.63; H, 7.77; Found: C, 73.45; H, 7.59.
2.2.4. 3β-Acetoxy-3′-4″-chlorophenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one
(6d)
In accordance with the general procedure, N-hydroxy-4-chlorobenzenecarboximidoyl chloride
(4d, 292 mg) was used for the synthesis. The product 6d (383 mg) was obtained as a white
precipitate. Mp 164−167 °C (Mp 114−116 °C [22]); Rf = 0.48 (ss A); IR: 1730, 1707, 1594,
1
1455, 1195 cm-1; H NMR (CDCl3, 500 MHz):
δ 0.78 (s, 3H, 18-H3), 1.01 (s, 3H, 19-H3),
1.04 (m, 1H, 9α-H), 1.14 (m, 1H, 1α-H), 1.45-1.61 (overlapping m, 5H), 1.68-1.76
(overlapping m, 4H), 1.86 (m, 3H), 2.02 (s, 3H Ac-CH3), 2.06 (m, 1H), 2.28 (m, 2H, 4-H2),
2.30 (s, 3H, 21-H3), 4.42 (dd, 1H, J = 8.5 Hz, J = 1.5 Hz, 16-H), 4.58 (m, 1H, 3-H), 5.29 (m,
1H, 6-H), 7.35 (d, 2H, J = 8.5 Hz, 3″-H and 5″-H), 7.59 (d, 2H, J = 8.5 Hz, 2″-H and 6″-H);
13C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-19), 20.5 (C-11), 21.4 (Ac-CH3), 27.3
(C-21), 27.6 (C-2), 31.2 (C-8), 31.5 (2C, C-7 and C-12), 31.8 (C-15), 36.5 (C-10), 36.9 (C-1),
37.9 (C-4), 48.3 (C-13), 49.1 (C-9), 50.5 (C-14), 50.8 (C-16), 73.6 (C-3), 106.4 (C-17), 121.8
(C-6), 127.1 (C-1″), 128.3 (2C, C-3″ and C-5″), 129.0 (2C, C-2″ and C-6″), 136.1 (C-4″),
8

ACCEPTED MANUSCRIPT
139.6 (C-5), 159.1 (C-3′), 170.4 (Ac-CO), 206.1 (C-20); ESI-MS: 510 [M+H]+; Anal. Calcd
for C30H36ClNO4 C, 70.64; H, 7.11; Found: C, 70.81; H, 6.98.
2.2.5. 3β-Acetoxy-3′-4″-nitrophenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one (6e)
In accordance with the general procedure, N-hydroxy-4-nitrobenzenecarboximidoyl chloride
(4e, 300 mg) was used for the synthesis. The product 6e (312 mg) was obtained as a white
precipitate. Mp 226−228 °C (Mp 109−111 °C [22]); Rf = 0.42 (ss A); IR: 1730, 1709, 1596,
1
1515, 1455, 1351, 1235, 852 cm-1; H NMR (CDCl3, 500 MHz):
δ 0.79 (s, 3H, 18-H3), 1.02
(s, 3H, 19-H3), 1.04 (m, 1H, 9α-H), 1.13 (m, 1H, 1α-H), 1.46-1.63 (overlapping m, 5H), 1.69-
1.81 (overlapping m, 4H), 1.86 (m, 3H), 2.01 (s, 3H Ac-CH3), 2.06 (m, 1H), 2.28 (m, 2H, 4-
H2), 2.32 (s, 3H, 21-H3), 4.43 (d, 1H, J = 9.2 Hz, 16-H), 4.57 (m, 1H, 3-H), 5.29 (m, 1H, 6-
13
H), 7.83 (d, 2H, J = 8.6 Hz, 2″-H and 6″-H), 8.24 (d, 2H, J = 8.6 Hz, 3″-H and 5″-H); C
NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-19), 20.4 (C-11), 21.4 (Ac-CH3), 27.3 (C-
21), 27.6 (C-2), 31.2 (C-8), 31.5 (2C, C-7 and C-12), 31.8 (C-15), 36.5 (C-10), 36.9 (C-1),
37.9 (C-4), 48.3 (C-13), 49.1 (C-9), 50.3 (C-14), 50.6 (C-16), 73.6 (C-3), 107.4 (C-17), 121.6
(C-6), 124.0 (2C, C-3″ and C-5″), 127.8 (2C, C-2″ and C-6″), 134.7 (C-1″), 139.7 (C-5),
148.4 (C-4″), 158.5 (C-3′), 170.4 (Ac-CO), 205.4 (C-20); ESI-MS: 521 [M+H]+; Anal. Calcd
for C30H36N2O6 C, 69.21; H, 6.97; Found: C, 69.07; H, 7.08.
2.3. General procedure for the preparation of 3-deacetylated steroidal isoxazolines
(10a−e)
The 3β-acetoxy derivative (6a−e) (0.50 mmol) was dissolved in MeOH (10 mL) and KOH
(56 mg, 1.00 mmol) was added. The mixture was stirred for 2 h at room temperature, and then
diluted with water. The resulting precipitate was filtered off, washed with water and dried.
9

ACCEPTED MANUSCRIPT
2.3.1. 3β-Hydroxy-3′-phenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one (10a)
Compound 6a (238 mg) was used for the synthesis as described in the General procedure.
Yield (10a): 199 mg (92%, white solid); mp 189−192 °C; Rf = 0.38 (ss B); IR: 3322, 1709,
1593, 1456, 1356, 1194 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.79 (s, 3H, 18-H3), 1.00 (m, 1H
and s, 3H, 19-H3), 1.08 (m, 1H), 1.46-1.60 (overlapping m, 5H), 1.66-1.76 (overlapping m,
5H), 1.86 (m, 3H), 2.07 (m, 1H), 2.18-2.28 (overlapping m, 2H), 2.31 (s, 3H, 21-H3), 3.51 (m,
1H, 3-H), 4.42 (t-like m, 1H, 16-H), 5.27 (m, 1H, 6-H), 7.39 (m, 3H, 3″-H, 4″-H and 5″-H),
7.67 (m, 2H, 2″-H and 6″-H); 13C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18), 19.2 (C-19), 20.5
(C-11), 27.4 (C-21), 31.2 (C-8), 31.5 (CH2), 31.6 (2C, 2 × CH2), 31.9 (C-15), 36.4 (C-10),
37.2 (C-1), 42.1 (C-4), 48.3 (C-13), 49.2 (C-9), 50.5 (C-14), 51.0 (C-16), 71.5 (C-3), 106.1
(C-17), 120.9 (C-6), 127.1 (2C, C-3″ and C-5″), 128.6 (C-1″), 128.7 (2C, C-2″ and C-6″),
130.1 (C-4″), 140.7 (C-5), 160.0 (C-3′), 206.4 (C-20); ESI-MS: 434 [M+H]+; Anal. Calcd for
C28H35NO3 C, 77.56; H, 8.14; Found: C, 77.72; H, 8.22.
2.3.2. 3β-Hydroxy-3′-4″-tolyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one (10b)
Compound 6b (245 mg) was used for the synthesis as described in the General procedure.
Yield (10b): 199 mg (89%, white solid); mp 217−220 °C; Rf = 0.37 (ss B); IR: 3325, 1708,
1592, 1456, 1192 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.78 (s, 3H, 18-H3), 1.00 (s, 3H, 19-
H3), 1.03 (m, 1H), 1.08 (m, 1H), 1.45-1.59 (overlapping m, 5H), 1.66-1.75 (overlapping m,
4H), 1.87 (m, 3H), 2.06 (m, 1H), 2.25 (m, 2H), 2.30 (s, 3H, 21-H3), 2.36 (s, 3H, 4″-CH3), 3.51
(m, 1H, 3-H), 4.40 (t-like m, 1H, 16-H), 5.26 (m, 1H, 6-H), 7.19 (d, 2H, J = 7.9 Hz, 3″-H and
5″-H), 7.55 (d, 2H, J = 7.9 Hz, 2″-H and 6″-H); 13C NMR (CDCl3, 125 MHz):
δ 15.2 (C-18),
19.3 (C-19), 20.5 (C-11), 21.4 (4″-CH3), 27.4 (C-21), 31.2 (C-8), 31.5 (CH2), 31.6 (CH2, 2 ×
CH2), 31.8 (C-15), 36.4 (C-10), 37.2 (C-1), 42.1 (C-4), 48.3 (C-13), 49.2 (C-9), 50.4 (C-14),
51.1 (C-16), 71.5 (C-3), 105.9 (C-17), 120.9 (C-6), 125.7 (C-1″), 127.0 (2C, C-2″ and C-6″),
10

ACCEPTED MANUSCRIPT
129.4 (2C, C-3″ and C-5″), 140.4 (C-4″), 140.7 (C-5), 160.0 (C-3′), 206.6 (C-20); ESI-MS:
448 [M+H]+; Anal. Calcd for C29H37NO3 C, 77.82; H, 8.33; Found: C, 77.65; H, 8.19.
2.3.3. 3β-Hydroxy-3′-4″-methoxyphenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one
(10c)
Compound 6c (253 mg) was used for the synthesis as described in the General procedure.
Yield (10c): 202 mg (87%, white solid); mp 191−192 °C; Rf = 0.33 (ss B); IR: 3333, 1709,
1609, 1594, 1455, 1354, 1193 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.78 (s, 3H, 18-H3), 1.00
(s, 3H, 19-H3), 1.04 (m, 1H), 1.08 (m, 1H), 1.44-1.60 (overlapping m, 5H), 1.68-1.75
(overlapping m, 4H), 1.85 (m, 3H), 2.06 (m, 1H), 2.28 (m, 2H), 2.30 (s, 3H, 21-H3), 3.50 (m,
1H, 3-H), 3.82 (s, 3H, 4″-OMe), 4.39 (t-like m, 1H, 16-H), 5.27 (m, 1H, 6-H), 6.90 (d, 2H, J
13
= 8.6 Hz, 3″-H and 5″-H), 7.59 (d, 2H, J = 8.6 Hz, 2″-H and 6″-H); C NMR (CDCl3, 125
MHz):
δ 15.2 (C-18), 19.3 (C-19), 20.5 (C-11), 27.3 (C-21), 31.0 (C-8), 31.4 (CH2), 31.5 (2C,
2×CH2), 31.8 (C-15), 36.4 (C-10), 37.2 (C-1), 42.1 (C-4), 48.2 (C-13), 49.2 (C-9), 50.4 (C-
14), 51.2 (C-16), 55.3 (4″-OMe), 71.5 (C-3), 105.8 (C-17), 114.2 (2C, C-3″ and C-5″), 120.9
(C-6), 121.0 (C-1″), 128.6 (2C, C-2″ and C-6″), 140.7 (C-5), 159.6 (C-3′), 161.0 (C-4″), 206.7
(C-20); ESI-MS: 464 [M+H]+; Anal. Calcd for C29H37NO4 C, 75.13; H, 8.04; Found: C,
75.20; H, 7.92.
2.3.4. 3β-Hydroxy-3′-4″-chlorophenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one
(10d)
Compound 6d (255 mg) was used for the synthesis as described in the General procedure.
Yield (10d): 199 mg (85%, white solid); mp 205−207 °C; Rf = 0.40 (ss B); IR: 3331, 1709,
1593, 1455, 1193 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.78 (s, 3H, 18-H3), 1.00 (m, 1H és s,
3H, 19-H3), 1.08 (m, 1H), 1.45-1.61 (overlapping m, 5H), 1.66-1.77 (overlapping m, 5H),
11

ACCEPTED MANUSCRIPT
1.86 (m, 3H), 2.06 (m, 1H), 2.18-2.28 (overlapping m, 2H), 2.30 (s, 3H, 21-H3), 3.51 (m, 1H,
3-H), 4.38 (dd, 1H, J = 8.7 Hz, J = 1.4 Hz, 16-H), 5.27 (m, 1H, 6-H), 7.36 (d, 2H, J = 8.6 Hz,
13
3″-H and 5″-H), 7.59 (d, 2H, J = 8.6 Hz, 2″-H and 6″-H); C NMR (CDCl3, 125 MHz):
δ
15.2 (C-18), 19.3 (C-19), 20.5 (C-11), 27.3 (C-21), 31.2 (C-8), 31.5 (CH2), 31.6 (CH2), 31.8
(CH2), 36.4 (C-10), 37.2 (C-1), 42.1 (C-4), 48.3 (C-13), 49.2 (C-9), 50.5 (C-14), 50.8 (C-16),
71.5 (C-3), 106.4 (C-17), 120.8 (C-6), 127.1 (C-1″), 128.3 (2C, C-3″ and C-5″), 129.0 (2C, C-
2″ and C-6″), 136.1 (C-4″), 140.7 (C-5), 159.1 (C-3′), 206.2 (C-20); ESI-MS: 468 [M+H]+;
Anal. Calcd for C28H34ClNO3 C, 71.85; H, 7.32; Found: C, 72.02; H, 7.23.
2.3.5. 3β-Hydroxy-3′-4″-nitrophenyl-2′-isoxazolino[4′,5′-d:16α,17α]-pregn-5-en-20-one
(10e)
Compound 6e (260 mg) was used for the synthesis as described in the General procedure.
Yield (10e): 215 mg (90%, white solid); mp 250−253 °C; Rf = 0.38 (ss B); IR: 3341, 1711,
1607, 1568, 1455, 1349, 1198, 851 cm-1; 1H NMR (CDCl3, 500 MHz):
δ 0.79 (s, 3H, 18-H3),
1.00 (s, 3H, 19-H3), 1.04 (m, 1H), 1.08 (m, 1H), 1.45-1.63 (overlapping m, 5H), 1.70-1.81
(overlapping m, 4H), 1.86 (m, 3H), 2.05 (m, 1H), 2.26 (m, 2H), 2.32 (s, 3H, 21-H3), 3.48 (m,
1H, 3-H), 4.44 (d, 1H, J = 9.1 Hz, 16-H), 5.26 (m, 1H, 6-H), 7.84 (d, 2H, J = 8.4 Hz, 2″-H
and
6″-H),
8.24
(d,
2H,
J
=
8.4
Hz,
3″-H
and
5″-H);
13C NMR (CDCl3, 125 MHz):
δ
15.2 (C-18), 19.3 (C-19), 20.5 (C-11), 27.3 (C-21), 31.2 (C-
8), 31.5 (2C, 2×CH2), 31.8 (CH2), 36.4 (C-10), 37.1 (C-1), 42.0 (C-4), 48.3 (C-13), 49.1 (C-
9), 50.3 (C-14), 50.7 (C-16), 71.5 (C-3), 107.4 (C-17), 120.7 (C-6), 124.0 (2C, C-3″ and C-
5″), 127.7 (2C, C-2″ and C-6″), 134.7 (C-1″), 140.8 (C-5), 148.4 (C-4″), 158.5 (C-3′), 205.5
(C-20); ESI-MS: 479 [M+H]+; Anal. Calcd for C28H34N2O5 C, 70.27; H, 7.16; Found: C,
70.42; H, 7.02.
2.4. Determination of antiproliferative activities
12

ACCEPTED MANUSCRIPT
Human breast cancer cell lines (MCF7, T47D, MDA-MB-231 and MDA-MB-361) were
maintained in minimal essential medium supplemented with 10% fetal bovine serum, 1% non-
essential aminoacids and an antibiotic-antimycotic mixture. All cell lines were purchased
from the European Collection of Cell Cultures (Salisbury, UK) and were grown in a
humidified atmosphere of 5% CO2 at 37 °C. For pharmacological investigations, 10 mM stock
solutions of the tested compounds were prepared with DMSO. The highest applied DMSO
concentration of the medium (0.3%) did not have a substantial effect on the proliferation of
the cells. All the chemicals, if otherwise not specified, were purchased from Sigma–Aldrich
Ltd. (Budapest, Hungary). Cells were seeded onto 96-well plates at a density of 5,000/well,
except for MDA-MB-361 cells which was seeded 10000/well and allowed to stand overnight,
after which the medium containing the tested compound was added. After a 72-h incubation,
viability was determined by the addition of 20 µL of MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide]) solution (5 mg/mL). The precipitated formazan crystals were
solubilized in DMSO and the absorbance was determined at 545 nm with an ELISA reader
[19]. Two independent experiments were performed with 5 parallel wells; cisplatin, an agent
administered clinically in the treatment of certain gynecological malignancies, was used as a
positive control. All tested agents were screened at 10 and 30 µM.
3. Results and discussion
3.1. Synthesis
For the transformations, 16-DPA (1) was used as steroidal dipolarophile (Scheme 1). The
relatively stable aromatic hydroximidoyl chloride precursors (4a−e) of nitrile oxide 1,3-
dipoles (5a−e) [20] were prepared in two steps by the condensation of benzaldehyde (2a) or
13

ACCEPTED MANUSCRIPT
its p-substituted derivatives (2b−e) with hydroxylamine hydrochloride in alkaline medium
and subsequent chlorination of the aldoximes 3a−e with N-chlorosuccinimide (NCS) [21].
The 1,3-dipolar cycloaddition of the steroidal enone 1 was first carried out in toluene
using an excess of N-hydroxybenzenecarboximidoyl chloride (4a), which underwent almost
immediate dehydrohalogenation by the slow addition of N,N-diisopropylethylamine (DIPEA),
leading to the in situ formation of benzonitrile oxide 1,3-dipole (5a). After stirring of the
mixture for 5 min at room temperature, the solution was refluxed to achieve complete
conversion for compound 1 within 2 h to afford a single product 6a in a yield of 95% after
chromatographic purification (Table 1, entry 1). Similar intermolecular ring closures of 1 with
different benzonitrile oxides (5b−e), obtained from the corresponding aryl aldehydes (2b−e)
by the general protocol mentioned above, were then performed under the same conditions to
furnish ring-condensed isoxazolines (6b−e) in moderate to excellent yields (Table 1, entries 2-
5). The yields of the cycloadducts 6b−e were found to depend on the electronic character of
the substituents on the aromatic moiety of the dipoles 5b−e. The electron-donating CH3 and
OMe groups in 5b and 5c favored cycloaddition to 1, in consequence of the higher resistance
of these dipoles to undergo dimerization to furoxanes [1, 2], while the yields of the desired
products 6d and 6e were decreased in case of electron-withdrawing Cl and NO2 substituents
on the aromatic ring in 5d and 5e. Although the synthesis of 6a−e was reported earlier in
moderate-yields by a single step procedure from 1 with aldoximes 3a−e in the presence of
chloramine-T in refluxing ethanol [22], the exact stereostructure of the products was not
supported unambiguously. Moreover, neither the spectral data nor the melting points
determined by the authors for the ring D-fused isoxazolines 6a−e are in agreement with those
obtained by us.
14

ACCEPTED MANUSCRIPT
Scheme 1. Regio- and stereoselective formation of ring D-fused 2-isoxazolines from 16-DPA
(1) and their deacetylations
Table 1. Cycloaddition products of 1 with different aromatic nitrile oxides
Entry
Hydroximidoyl chloride/Nitrile oxide
Ar
Ph
Product
6a
Yielda (%)
1
2
3
4
5
4a/5a
4b/5b
4c/5c
4d/5d
4e/5e
95
98
97
75
60
p-CH3-C6H4
p-MeO-C6H4
p-Cl-C6H4
p-NO2-C6H4
6b
6c
6d
6e
aDetermined after purification by column chromatography
In principle, the formation of four isoxazolines (6−9) can be conceived as possible
products in the cycloadditions of 1 with aromatic nitrile oxides (5a−e), as depicted in Scheme
1. The orientation of the 1,3-dipole relative to the double bond of the enone moiety can be of
two kinds; the negatively charged O terminus may interact with either the α or the β carbon of
15

ACCEPTED MANUSCRIPT
the dipolarophile 1 leading to two regioisomeric pairs, each involving two diastereomers (6
and 7 or 8 and 9). The regiochemical mode of addition could easily be determined on the
13
basis of the C NMR spectra of 6a−e indicating the presence of the C-17 signal at around
106 ppm, which is consistent with its linkage to an oxygen. Similar regioselectivity was
found to predominate during the thermal cycloaddition of α-aryl-N-phenylnitrones to the
C16-C17 π-bond of 1 [23]. The attack of the nitrile oxide dipole 5 from above the general
plane of the sterane framework (the β side) is unfavorable in 1 in consequence of the same
spatial orientation of the 18-CH3. Therefore, the most facilitated isomer is undoubtedly 6, in
which the isoxazoline ring is located in the 16α,17α position opposite to the angular methyl
group on C-13. In the 1H NMR spectra of the products, the coupling pattern of 16-H (
δ
4.41
ppm) is informative in respect of the connection mode of the heteroring. The triplet-like signal
(3J 5.2 Hz) for 6a−c and 10a−c, the double doublet (3J 8.6 Hz, 1.5 Hz) for 6d and 10d or
the doublet (3J 9.2 Hz) for 6e and 10e all indicate dihedral angles of about 40° and 110°
between 16-H and the 15-protons, respectively. This is in good agreement with the torsion
angles calculated for the 16α,17α-isoxazolines. In the case of the 16β,17β-fused heteroring
(7), the θ(H16,C16,C15,H15α) = 150° and θ(H16,C16,C15,H15β) = 20° would manifest in
two large ( 10 Hz) coupling constants. The stereochemistry was supported with the aid of
homonuclear 2D NMR (COSY and NOESY) and heteronuclear 2D NMR (HSQC and
HMBC) measurements. The NOESY spectrum of 6a showed cross-peaks between the C-18
methyl protons and 16β-H and 15β-H, respectively, while the spatial vicinity of 15β-H and
16β-H was also supported (Scheme 2).
16

ACCEPTED MANUSCRIPT
18
19
13
11
12
17
16
1
10
2
9
15
8
14
3′
6″
5
4
7
6
3
5″
1″
4″
2″
3″
Scheme 2. Partial NOESY spectra and 3D representation of 6a
For the enlargement of the compound library suitable for pharmacological studies and in
the hope of finding structure-activity relationships, further derivatives of 6a−e were
synthesized by simple deacetylations to furnish the corresponding 3β-OH analogs (10a−e)
(Scheme 1).
3.2. Pharmacological studies
Since a number of compounds containing isoxazoline moiety have been reported to exert
noteworthy antiproliferative activities [24, 25] and some sterane-based N,O-heterocycles have
also been demonstrated to inhibit cell proliferation [11, 26], the synthesized isoxazolines
(6a−e and 10a−e) were subjected to in vitro pharmacological studies in order to characterize
17

ACCEPTED MANUSCRIPT
their cell-growth inhibitory effects on four malignant human breast cell lines, MCF7, T47D,
MDA-MB-231 and MDA-MB-361 (Table 2). Their antiproliferative activities were
determined by a microplate-based MTT colorimetric assay [19], in comparison with cisplatin
as reference agent. The cell-proliferation inhibitory potencies, expressed as growth inhibition,
revealed that some of the 3β-OH analogs exhibited marked effects on cell proliferation,
especially at 30 µM (Table 2).
The cell growth-inhibitory potencies of the 3-acetate analogs (6a−e) were generally found
to be lower than those of their 3β-hydroxy counterparts (10a−e). Hence, the esters (6a−e) may
be considered to be practically ineffective. Among the hydroxylated compounds, p-
substitution of the aromatic ring with a CH3 (10b), Cl (10d) or NO2 group (10e) caused
relevant changes in the biological effects as compared with the unsubstituted derivative 10a.
The introduction of OMe substituent in 10c was, however, less favorable. The p-nitro-
substituted derivative (10e) proved to be the most promising compound in the currently
presented set.
18

ACCEPTED MANUSCRIPT
Table 2 Antiproliferative effects of the synthesized compounds
Growth inhibition % ± (SEM)
Entry
Product
MCF7
T47D
MDA-MB-231
MDA-MB-361
10 µM
<25a
30 µM
10 µM
30 µM
10 µM
30 µM
10 µM
30 µM
1
2
<25
<25
<25
<25
<25
<25
<25
6a
6b
28.7
(1.1)
58.9
(1.7)
28.3
(2.3)
<25
<25
<25
<25
<25
3
4
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
6c
6d
75.4
(1.4)
29.0
(1.5)
45.4
(2.2)
32.3
(0.8)
5
6
7
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
6e
40.6
(1.0)
45.2
(0.8)
70.5
(2.4)
49.0
(2.2)
10a
10b
71.3
(0.6)
70.6
(1.4)
41.8
(0.4)
84.1
(1.0)
67.5
(0.8)
36.1
(2.4)
8
9
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
<25
10c
10d
10e
72.8
(0.6)
62.7
(1.3)
85.8
(1.2)
76.7
(1.4)
59.8
(2.1)
82.5
(1.4)
72.6
(2.15)
93.3
(0.6)
60.3
(0.9)
90.7
(2.0)
70.5
(0.7)
85.4
(0.9)
10
66.9
(1.8)
96.8
(0.4)
51.0
(2.0)
57.9
(1.4)
71.7
(1.2)
67.5
(1.0)
87.7
(1.1)
cisplatin
<25
aCompounds eliciting less than 25% inhibition of proliferation were considered ineffective and the exact results
are not given, for simplicity.
4. Conclusions
In summary, an improved method for the efficient synthesis of steroidal ring D-fused
2-isoxazolines in the pregnane series by thermally-induced 1,3-dipolar cycloaddition of a
steroidal enone with nitrile oxide 1,3-dipoles was developed. The intermolecular ring-closures
afforded the heterocyclic products regio- and stereoselectively in good to excellent yields.
The conversions were observed to increase by the presence of electron-donating groups on the
aromatic ring of the 1,3-dipole in consequence of the lower tendency of these species to
undergo dimerization. The stereostructure of the synthesized compounds was established by
NMR measurements. The library of compounds was expanded by further deacetylations to
furnish the 3-OH analogs of the primarily formed cycloadducts. The antiproliferative
activities of the synthesized derivatives on breast cancer cell lines depended mainly on both
19

ACCEPTED MANUSCRIPT
the substitution pattern of the incorporated heteroring and the nature of substituents on C-3 of
the sterane core. Although the effects of the tested compounds on cell division are moderate,
the results suggest that steroidal isoxazolines can be promising candidates motivating the
search for further derivatives with better activities.
Acknowledgements
The financial support by the Hungarian Scientific Research Fund (OTKA K-109107 and
K-109293) is gratefully acknowledged. The research by G. Mótyán was supported by the
European Union and the State of Hungary, co-financed by the European Social Fund in
the framework of TÁMOP 4.2.4. A/2-11/1-2012-0001 ″National Excellence Program″.
The work of Á. Baji was supported by a PhD Fellowship of the Talentum Fund of Richter
Gedeon Plc.
References
[1] P. Caramella, P. Grünanger, In 1,3-Dipolar Cycloaddition Chemistry, A. Padwa, Ed.;
John Wiley & Sons, New York, 1984, Vol. 1. Chapter 3.
[2] L. I. Belen’kii, N. D. Zeninksy, In Nitrile oxides, Nitrones, and Nitronates in Organic
Synthesis: Novel Strategies in Synthesis, H. Feuer, Ed.; John Wiley & Sons, Hoboken,
New Jersey 2008, 1.
[3] S. Cicchi, F. M. Cordero, D. Giomi, Prog. Heterocycl. Chem. 24 (2012) 317.
[4] L. Rahbœk, C. Christophersen, Alkaloids Chem. Biol. 57 (2001) 185.
[5] S. V. Drach, R. P. Litvinovskaya, V. A. Khripach, Chem. Heterocycl. Compds. 36
(2000) 233.
20

ACCEPTED MANUSCRIPT
[6] V. Jäger, P. Colinas, A. In Synthetic Applications of 1,3-Dipolar Cycloaddition
Chemistry Toward Heterocycles and Natural Products, A. Padwa, W. H. Pearson, Eds.;
John Wiley & Sons, Hoboken, New Jersey, 2003, Chapter 6.
[7] Ch. Camoutsis, S. Nikolaropoulos, J. Heterocycl. Chem. 35 (1998) 731.
[8] A. H. Banday, S. Singh, M. S. Alam, D. M. Reddy, B. D. Gupta, H. M. S. Kumar,
Steroids 73 (2008) 370.
[9] D. Kovács, Z. Kádár, G. Mótyán, Gy. Schneider, J. Wölfling, I. Zupkó, É. Frank,
Steroids 77 (2012) 1075.
[10] É. Frank, Z. Mucsi, M. Szécsi, I. Zupkó, J. Wölfling, Gy. Schneider, New J. Chem. 34
(2010) 2671.
[11] É. Frank, D. Kovács, Gy. Schneider, J. Wölfling, T. Bartók, I. Zupkó, Mol. Divers. 18
(2014) 521.
[12] J. Kalvoda, H. Kaufmann, J. Chem. Soc., Chem. Commun. (1976) 209.
[13] R. P. Litvinovskaya, S. V. Drach, Yu. I. Lapchinskaya, V. A. Khripach, Russ. J. Org.
Chem. (2001) 46.
[14] G. Mótyán, Z. Kádár, D. Kovács, J. Wölfling, É. Frank, Steroids 87 (2014) 76.
[15] É. Frank, J. Wölfling, B. Aukszi, V. König, T. R. Schneider, Gy. Schneider,
Tetrahedron 58 (2002) 6843.
[16] É. Frank, Gy. Schneider, J. Steroid Biochem. Mol. Biol. 137 (2013) 301.
[17] É. Frank, Z. Mucsi, I. Zupkó, B. Réthy, G. Falkay, Gy. Schneider, J. Wölfling, J. Am.
Chem. Soc. 131 (2009) 3894.
[18] G. Mótyán, I. Zupkó, R. Minorics, Gy. Schneider, J. Wölfling, É. Frank, Mol. Divers.
19 (2015) 511.
[19] T. Mosmann, J. Immunol. Methods 65 (1983) 55.
21

ACCEPTED MANUSCRIPT
[20] F. Himo, T. Lovell, R. Hilgraf, V. V. Rostovtsev, L. Noodleman, K. B. Sharpless, V.
V. Fokin, J. Am. Chem. Soc. 127 (2005) 210.
[21] K. C. Liu, B. R. Shelton, R.K. Howe, J. Org. Chem. 45 (1980) 3916.
[22] P. Chowdhury, A. M. Das, P. Goswami, Steroids 70 (2005) 494.
[23] N. K. Girdhar, M. P. S. Ishar, Tetrahedron Lett. 41 (2000) 7551.
[24] S. Castellano, D. Kuck, M. Viviano, J. Yoo, F. López-Vallejo, P. Conti, L. Tamborini,
A. Pinto, J. L. Medina-Franco, G. Sbardella, J. Med. Chem. 54 (2011) 7663.
[25] J. Khazir, P. P. Singh, D. M. Reddy, I. Hyder, S. Shafi, S. D Sawant, G. Chashoo, A.
Mahajan, M. S. Alam, A. K. Saxena, S. Arvinda, B. D. Gupta, H. M. Kumar, Eur. J.
Med. Chem. 63 (2013) 279.
[26] A. H. Banday, A. K. Giri, R. Parveen, N. Bashir, Steroids 87 (2014) 93.
22

ACCEPTED MANUSCRIPT
Highlights
> Isoxazoline hetero rings were introduced into ring D of the sterane core.
> Intermolecular alkene−nitrile oxide cycloadditions were carried out.
> The stereo- and regioselectivity of the ring-closures were investigated.
> The structures of all novel compounds were confirmed by NMR measurements.
> Antiproliferative effects were determined in vitro on 4 malignant human cell lines.