Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents against LNCaP, PC-3 and DU-145 cells (Dedicated to Prof. Mushtaq A. Qureishi for his pioneering contribution to Natural Product Research at University of Kashmir)

Article abstract of DOI:10.1016/j.steroids.2014.05.009

Two series of novel steroidal isoxazolines and oxazolines were synthesized through different routes from dehydroepiandrosterone acetate and pregnenolone acetate, respectively. The synthesis of the analogs of both series is multistep and proceeds in good overall yields. While the key step in the synthesis of former is the cycloaddition of aromatic nitrile oxides across α,β-unsaturated olefins, it is the condensation of α,β-azidoalcohols with aromatic aldehydes in the later. Compounds of both the series were tested for their cytotoxic activities against LNCaP, PC-3 and DU-145 prostate cancer cell lines. Amongst all the compounds of both the series screened for their prostate cancer activity, compound 6a, 6e and 12a are the most active especially against LNCaP and DU-145 cancer cell lines.2014 Elsevier Inc. All rights reserved.

Full text of DOI:10.1016/j.steroids.2014.05.009

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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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Design and synthesis of D-ring steroidal isoxazolines and oxazolines
as potential antiproliferative agents against LNCaP, PC-3 and DU-145
cells
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Abid H. Banday a,b, , S.M.M. Akram a,c, Rifat Parveen a, Nusrat Bashir a
⇑
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a Department of Chemistry, Islamia College of Science and Commerce, Srinagar 190009, India
b Department of Chemistry and Biochemistry, University of Arizona, Tucson 85721, USA
c Department of Chemistry, University of Kashmir, Srinagar 190002, India
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a r t i c l e i n f o
a b s t r a c t
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Article history:
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Two series of novel steroidal isoxazolines and oxazolines were synthesized through different routes from
dehydroepiandrosterone acetate and pregnenolone acetate, respectively. The synthesis of the analogs of
both series is multistep and proceeds in good overall yields. While the key step in the synthesis of former
Received 2 February 2014
Received in revised form 25 March 2014
Accepted 12 May 2014
Available online xxxx
is the cycloaddition of aromatic nitrile oxides across
a,b-unsaturated olefins, it is the condensation of
a,b-azidoalcohols with aromatic aldehydes in the later. Compounds of both the series were tested for
their cytotoxic activities against LNCaP, PC-3 and DU-145 prostate cancer cell lines. Amongst all the
compounds of both the series screened for their prostate cancer activity, compound 6a, 6e and 12a are
the most active especially against LNCaP and DU-145 cancer cell lines.
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Keywords:
Dehydroepiandrosterone
Pregnenolone
Isoxazolines
Oxazolines
Azidoalcohols
Cytotoxicity
Ó 2014 Elsevier Inc. All rights reserved.
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1. Introduction
six- membered 17b-exo-heterocycles (preferably nitrogen contain-
ing) have been found to cause the inhibition of 17a-hydroxylase/
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Steroids represent a pharmacologically active class of molecules
associated with variety of physiological functions. Steroids as well
as their derivatives have been found to have the potential to be
developed as drugs for the treatment of a large number of diseases
including cardiovascular [1], autoimmune diseases [2], brain
tumors, breast cancer, prostate cancer, osteoarthritis, etc. [3]. The
promise of using steroids for development of lead molecules lies
in their regulation of a variety of biological processes and being a
fundamental class of signaling molecules [4]. Though steroid and
steroid based molecules have been used as active pharmaceutical
agents against various diseases, there has recently been a surge in
the exploitation of these molecules against cancer. Despite the
recent advances in the early diagnosis, prevention and therapy, can-
cer still remains a challenge as it affects millions of people world
over and is one of the leading causes of death [5,6].
C17-20-lyase (P45017 ) which can block adrenal androgen synthesis
a
at an early stage and may therefore be useful in the treatment of
prostatic carcinoma [7]. The preliminary structure activity rela-
tionship (SAR) reveals that such activity is related to the presence
of nitrogen in the heterocyclic moiety on ring D, with the nitrogen
coordinating with the heme iron atom at the active site of the
enzyme [8]. Taking inputs from these literature precedents, we,
in continuation of our program toward the development of steroid
based lead molecules [9], designed synthesis of two series of novel
isoxazoline and oxazoline analogs from dehydroepiandrosterone
and pregnenolone respectively. The compounds of both the series
were evaluated for their antiproliferative activity toward PC-3,
DU-145 (androgen-independent) and LNCaP (androgen-depen-
dent) prostate cancer cell lines. It was observed that compounds
6a, 6e and 12a exhibit excellent cytotoxicity especially against
LNCaP and DU-145 cell lines.
Prostate cancer is the second most common cancer worldwide
and in the absence of any effective treatments available, this
disease remains to be a challenge for researchers across the globe.
Recently a large no of steroidal derivatives containing five- or
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2. Experimental
2.1. General methods
⇑
Corresponding author at: Department of Chemistry, Islamia College of Science
and Commerce, Srinagar 190009, India. Fax: +91 194 2429014.
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Melting points were recorded on Buchi Melting point apparatus
D-545; IR spectra (KBr discs) were recorded on Bruker Vector 22
Q2
E-mail address: abidrrl@gmail.com (A.H. Banday).
http://dx.doi.org/10.1016/j.steroids.2014.05.009
0039-128X/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Banday AH et al. Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.009

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instrument. NMR spectra were recorded on Bruker DPX200 instru-
ment in CDCl3 with TMS as internal standard for protons and sol-
vent signals as internal standard for carbon spectra. Chemical
shift values are mentioned in d (ppm) and coupling constants are
given in Hz. Mass spectra were recorded on EIMS (Shimadzu)
and ESI-esquire 3000 Bruker Daltonics instrument. The progress
of all reactions was monitored by TLC on 2 ꢀ 5 cm pre-coated silica
gel 60 F254 plates of thickness of 0.25 mm (Merck). The chromato-
grams were visualized under UV 254–366 nm and iodine.
J = 15.6), 5.33 (br, 1H), 7.50 (m, 4H), 7.85 (d, 2H, J = 8.9), 8.05 (d,
2H, J = 8.9), 8.54 (m,1H), 9.83 (s, 1H); 13C NMR (d, ppm, CDCl3):
13.5, 201.9, 22.3, 24.0, 28.9, 30.5, 38.7, 32.2, 36.2, 39.0, 41.3,
46.4, 54.2, 58.7, 71.7, 75.7, 76.2, 82.4, 109.9, 120.7, 122.32,
115.05, 126.5, 126.0, 128.4, 138.5, 152.6, 162.1, 199.9.; MS [ESI,
548 (M+ + H)], Anal. Calcd. for C37H41NO3, C, 81.14; H, 7.54; N,
2.56. Found C, 81.29; H, 7.67; N, 2.48.
153
154
155
156
157
158
159
160
161
162
163
2.2.1.4. 3b-Hydroxy-5-pregnene-17-(3-(p-fluoro-phenyl)-4’,5’-dihyd-
roisoxazole carboxaldehyde (6d). Yield: 77%, mp 186–188 °C;
2.2. Chemical synthesis
[a]
20 + 12.2 (c 0.20, CHCl3); IR (KBr): 3500, 2857, 2703,
D
94
1683 cmꢁ1 1H NMR (d, ppm, CDCl3): 0.78 and 1.05 (s, angular
,
95
96
2.2.1. General procedure for the synthesis of compounds 6a–f
The preparation of compound 5 is already reported in the liter-
ature [10]. To a solution of compound 5 (0.10 g, 0.30 mmol) in THF
was added p-methyl phenyl nitrile oxide (0.092 g, 0.60 mmol, gen-
erated in situ from the corresponding chlorooxime in presence of
Et3N). The mixture was stirred for 1hr at 0 °C and then at room
temperature for further 5 h. The reaction mixture was then filtered
and extracted with ethyl acetate (3 ꢀ 10 mL). The organic solvent
was dried over Na2SO4 and then evaporated in vacuo. Purification
was performed over silica gel (100–200 mesh; elution, hexane:
EtOAc) to yield the cycloaddition product 6a (0.114 g, 0.24 mmol,
83% yield). Though the cycloaddition could conceptually lead to
the formation of two regioisomers, we fortunately got the reported
regioisomer as the major product. The regiochemistry was
assigned on the basis of NMR chemical shift values of the methy-
lene protons in the isoxazoline ring which differ markedly for the
two regioisomers [16]. The stereochemistry at C-17 was assigned
on the basis of reported literature precedents [7]. The spectral
details of various such analogs (6a–f) are given as follows (most
of the peaks belonging to steroidal skeleton were merged and
could not be differentiated. Thus d values of only those peaks that
could easily be differentiated are reported):
CH3), 3.18 (d, 1H, J = 17.4), 3.42–4.32 (m, 1H, ACHOA), 3.68 (d,
1H, J = 17.4), 5.35 (br, 1H), 7.23 (d, 2H, J = 8.0), 7.54 (d, 2H,
J = 8.0), 9.67(s, 1H); 13C NMR (d, ppm, CDCl3): 15.8, 20.9, 22.3,
24.0, 28.9, 30.5, 30.7, 32.2, 36.2, 39.0, 41.3, 46.4, 54.2, 58.7, 71.7,
75.7, 76.2, 82.6, 109.6, 120.7, 126.5, 126.0, 129.4, 138.9, 151.6,
162.1, 200.4; MS [ESI, 488 (M+ + Na)], Anal. Calcd. for C29H36FNO3,
C, 74.81; H, 7.79; N, 3.01. Found C, 74.99; H, 7.62; N, 3.29.
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
164
165
166
167
168
169
170
171
172
173
174
175
2.2.1.5. 3b-Hydroxy-5-pregnene-17-(3-(p-methoxy-phenyl)-4’,5’-dihy
droisoxazole carboxaldehyde (6e). Yield: 82%, mp 163–164 °C;
[a]
20 + 16.2 (c 0.20, CHCl3); IR (KBr): 3500, 2858, 2705,
D
1682 cmꢁ1 1H NMR (d, ppm, CDCl3): 0.77 and 1.08 (s, angular
,
CH3), 3.19 (d, 1H, J = 17.0), 3.49–3.56 (m, 1H, ACHOA), 3.69 (d,
1H, J = 17.0), 3.86 (s, 3H), 5.35 (br, 1H), 6.94 (d, 2H, J = 7.6), 7.72
(d, 2H, J = 7.6), 9.67 (s, 1H); 13C NMR (d, ppm, CDCl3): 14.4, 21.9,
22.3, 24.0, 29.9, 30.5, 30.7, 32.5, 36.4, 39.5, 41.4, 46.4, 54.2, 58.7,
71.8, 75.7, 76.2, 84.4, 109.9, 120.7, 125.5, 126.0, 128.4, 137.9,
152.6, 161.1, 200.6; MS [ESI, 500.1 (M+ + Na)], Anal. Calcd. for
C30H39NO4, C, 75.44; H, 8.23; N, 2.93. Found C, 75.63; H, 8.47; N,
2.61.
176
177
178
179
180
181
182
183
184
185
186
187
2.2.1.6. 3b-Hydroxy-5-pregnene-17-(3-(o-nitro-phenyl)-4’,5’-dihyd-
roisoxazole carboxaldehyde) (6f). Yield: 78%, mp 188–191 °C;
2.2.1.1. 3b-Hydroxy-5-pregnene-17-(3-(p-methyl-phenyl)-40,50-dihy
droisoxazole carboxaldehyde (6a). Yield: 83%, mp 170 °C;
[a]
20 + 6.4 (c 0.20, CHCl3); IR (KBr): 3500, 2856, 2700,
D
117
118
119
120
121
122
123
124
125
126
127
128
1675 cmꢁ1 1H NMR (d, ppm, CDCl3): 0.78 and 1.05 (s, angular
,
[a
]
D
20 + 6.5 (c 0.20, CHCl3); IR (KBr): 3500 (OH), 2700 (ACHO)
CH3), 3.28 (d, 1H, J = 17.1), 3.42–4.47 (m, 1H, ACHOA), 3.54 (d,
1H, J = 17.1), 5.35 (br, 1H), 7.46 (d, 1H, J = 6.89), 7.66 (m, 2H),
8.65 (d, 1H, J = 6.89), 9.66 (s, 1H); 13C NMR (d, ppm, CDCl3):
14.01, 19.38, 20.64, 21.63, 22.64, 23.88, 25.36, 29.31, 30.91,
31.50, 36.52, 37.22, 38.77, 39.22, 42.24, 49.94, 53.52, 56.07,
71.69, 121.30, 124.84, 130.80, 131.12, 133.52, 199.71; MS [ESI,
493 (M+ + H)], Anal. Calcd. for C29H36N2O5, C, 70.71; H, 7.37; N,
5.69. Found C, 70.94; H, 7.54; N, 5.92.
cmꢁ1 1H NMR (d, ppm, CDCl3): 0.78 and 1.05 (2s, angular CH3),
,
2.38–2.40 (s, 3H, CH3), 3.18 (d, 1H, J = 17.2), 3.42–4.32 (m, 1H,
ACHOA), 3.68 (d, 1H, J = 17.2), 5.36 (br, 1H), 7.21(d, 2H, J = 8.04),
7.56 (d, 2H, J = 8.04), 9.66 (s, 1H); 13C NMR (d, ppm, CDCl3): 15.5,
20.9, 22.3, 24.0, 28.9, 30.5, 30.7, 32.2, 36.2, 39.0, 41.3, 46.4, 54.2,
58.7, 71.7, 75.7, 76.2, 82.4, 109.9, 120,7, 125.5, 126.0, 128.4,
137.9, 152.6, 161.1, 200.3; MS [ESI, 484 (M+ + Na)], Anal. Calcd.
for C30H39NO3, C, 78.05; H, 8.52; N, 3.30. Found C, 78.02; H, 8.47;
N, 3.33,
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
2.2.2. General procedure for the synthesis of compounds 12a–g
Compound 11 [11] (2.4 g, 6 mmol) was dissolved in dry CH2Cl2
(50 ml) and treated with appropriate aromatic aldehydes (1.1
equivalent). The mixture was cooled to 0 °C followed by the drop-
wise addition of BF3ꢂOEt2 (50%) (12 mmol, 1.65 ml) which was
accompanied by evolution of gas. The reaction mixture was stirred
at room temperature for 6 h. After the disappearance of starting
material as monitored by TLC, saturated NaHCO3 solution was
added and the mixture was stirred until bubbling ceased. The
organic layer was washed with water, dried over anhydrous
Na2SO4 and concentrated in vacuo. The product was purified by
chromatography on silica gel with hexane/CH2Cl2 (30:70, v/v) to
give the 3b-acetylated oxazoline derivatives. Deacetylation was
performed using methanolic NaOCH3 to give the product 12a–g
(4–4.5 mmol, 67–75% yield) in the pure form after purification by
chromatography on silica gel using ethylacetate/CH2Cl2. The spec-
tral details of various such analogs (12a–g) are given as follows
(most of the peaks belonging to steroidal skeleton were merged
and could not be differentiated. Thus d values of only those peaks
that could easily be differentiated are reported):
129
130
131
132
133
134
135
136
137
138
139
140
2.2.1.2. 3b-Hydroxy-5-pregnene-17-(3-(p-chloro-phenyl)-4’,5’-dihyd-
roisoxazole carboxaldehyde (6b). Yield: 81%, mp 175–176 °C;
[a]
20 + 10.1 (c 0.20, CHCl3); IR (KBr): 3497, 2857, 2711, 1679.
D
cmꢁ1 1H NMR (d, ppm, CDCl3): 0.79 and 1.04 (s, angular CH3),
,
3.18 (d, 1H, J = 16.7), 3.50 (m, 1H, ACHOA), 3.67 (d, 1H, J = 16.7),
5.35 (br, 1H), 7.08 (d, 2H, J = 7.90), 7.55 (d, 2H, J = 7.90), 9.63 (s,
1H); 13C NMR (d, ppm, CDCl3): 14.13, 19.44, 20.69, 22.73, 23.88,
29.76, 31.56, 31.75, 36.56, 37.18, 37.26, 38.83, 42.27, 42.48,
49.98, 53.88, 56.10, 71.75, 94.74, 115.90, 116.07, 121.33, 125.37,
128.78, 140.90, 156.65 and 200.29; MS [ESI, 483 (M+ + H)], Anal.
Calcd. for C29H36ClNO3, C, 72.26; H, 7.53; N, 2.91. Found C, 72.41;
H, 7.32; N, 2.77.
141
142
143
144
145
2.2.1.3. 3b-Hydroxy-5-pregnene-17-(3-(anthracen-1-yl)-4’,5’-dihyd-
roisoxazole carboxaldehyde) (6c). Yield: 80%, mp 197–199 °C;
[a]
20 + 14.1 (c 0.20, CHCl3); IR (KBr): 3505, 2865, 2704,
D
,
1662 cmꢁ1 1H NMR (d, ppm, CDCl3): d 0.80 and 1.08 (s, angular
CH3), 3.39 (d, 1H, J = 15.6), 3.49 (m, 1H, ACHOA), 3.84 (d, 1H,
Please cite this article in press as: Banday AH et al. Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.009

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Table 1
IC50 values (
against human prostate cancer cell lines.
208
209
210
211
212
213
214
215
216
217
218
2.2.2.1. (5’R)-17b-[2-Phenyl)-4,5-dihydrooxazol-5-yl]androst-5-en-
20
lM) of steroidal isoxazolines (6a–f) and steroidal oxazolines (12a–g)
3b-ol (12a). Yield: 73%, mp 214–215 °C; [
a]
ꢁ 53 (c 1, CHCl3);
D
1H NMR (d, ppm, CDCl3): 0.87 (s, 3H), 1.04 (s, 3H), 3.51 (m, 1H),
3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz), 4.05 (dd, 1H, J = 14.5 Hz
and J = 9.5 Hz), 4.65 (m, 1H), 5.34 (d, 1H, J = 5.0 Hz), 7.39 (t, 2H,
J = 7.5 Hz), 7.46 (t, 1H, J = 7.5 Hz), 7.92 (d, 2H, J = 7.5 Hz). 13C
NMR (d, ppm, CDCl3): 12.7, 19.4, 20.9, 23.8, 24.7, 31.7, 31.7, 31.8,
31.9, 36.6, 37.3, 38.2, 39.0, 42.3, 42.7, 50.3, 55.0, 56.0, 59.7, 71.6,
81.7, 121.4, 128.1, 128.3, 131.1, 132.3, 140.9, 164.1; MS [ESI, 420
(M+ + H)], Anal. Calcd. for C28H37NO2, C, 80.15; H, 8.89; N, 3.34.
Found C, 80.87; H, 8.92; N, 3.33.
Entry
LNCaP
PC-3
DU-145
-Ar
6a
2.29 0.03 12.36
5.73 0.02
6b
8.21
11.51
4.63
Cl
6c
3.23 0.04 7.38
5.25 0.03 ND
14.84
6.84
219
220
221
222
223
224
225
226
227
228
229
2.2.2.2.
(5’R)-17b-[2-(4-Nitrophenyl)-4,5-dihydrooxazol-5-yl]and-
20
rost-5-en-3b-ol (12f). Yield: 70%, mp 237–239 °C; [
a]
ꢁ 46 (c
D
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.88 (s, 3H), 1.06 (s, 3H), 3.52
(m, 1H), 3.69 (dd, 1H, J = 14.6 Hz and J = 8.1 Hz), 4.11 (dd, 1H,
J = 14.1 Hz and J = 9.6 Hz), 4.73 (dd, 1H, J = 17.9, 8.9), 5.35 (d, 1H,
J = 5.0 Hz), 8.09 (d, 2H, J = 8.2 Hz), 8.26 (d, 2H, J = 8.2 Hz); 13C
NMR (d, ppm, CDCl3): 12.7, 19.4, 20.9, 23.7, 24.7, 31.6, 31.8, 31.7,
31.9, 36.6, 37.3, 39.1, 42.3, 42.7, 50.2, 55.0, 56.0, 59.9, 71.6, 82.5,
121.3, 123.5, 129.1, 132.2, 133.8, 140.9, 149.2, 162.3; MS [ESI,
487 (M+ + Na)], Anal. Calcd. for C28H36N2O4, Found C, 72.39; H,
7.81; N, 6.03, Found C, 72.37; H, 7.39; N, 6.06.
6d
F
6e
6f
2.32 0.04 5.69 0.02 5.11 0.03
OCH3
O2N
16.32
13.57
11.40
230
231
232
233
234
235
236
237
238
239
240
2.2.2.3. (5’R)-17b-[2-(4-Chlorophenyl)-4,5-dihydrooxazol-5-yl]and-
20
rost-5-en-3b-ol (12c). Yield: 65%, mp 215–217 °C; [
a
]
ꢁ 48 (c
D
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.88 (s, 3H), 1.05 (s, 3H), 3.50
(m, 1H), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz), 4.05 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz), 4.66 (m, 1H), 5.35 (d, 1H, J = 5.0 Hz),
7.38 (t, 2H, J = 8.5 Hz), 7.86 (dd, 2H, J = 8.5 Hz); 13C NMR (d, ppm,
CDCl3): 12.7, 19.4, 20.9, 23.8, 24.7, 31.7, 31.7, 31.9, 32.3, 36.6,
37.3, 39.3, 42.7, 42.8, 50.3, 55.0, 56.0, 59.7, 71.7, 81.1, 121.4,
126.7, 128.6, 129.4, 131.7, 137.3, 140.9, 163.3; MS [ESI, 454
(M+ + H)], Anal. Calcd. for C28H36ClNO2, C, 74.07; H, 7.99; N, 3.08.
Found C, 74.09; H, 8.01; N, 3.05.
12a
12b
12c
12d
3.24 0.02 10.11
3.10 0.03
21.91
26.38
13.46
24.28
33.75
NO2
Cl
32.90
241
242
243
244
245
246
247
248
249
250
251
2.2.2.4. (5’R)-17b-[2-(3-Chlorophenyl)-4,5-dihydrooxazol-5-yl]and-
20
rost-5-en-3b-ol (12d). Yield: 71%, mp 232–234 °C; [
a]
ꢁ 53 (c
4.32 0.03 7.62
11.34
D
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.86 (s, 3H), 1.04 (s, 3H), 3.51
(m, 1H), 3.63 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz), 4.05 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz), 4.66 (m, 1H), 5.35 (d, 1H, J = 5.0 Hz),
7.33 (t, 1H, J = 8.0 Hz), 7.43 (d, 1H, J = 8.0 Hz), 7.81 (d, 1H,
J = 8.0 Hz), 7.92 (s, 1H); 13C NMR (d, ppm, CDCl3): 12.7, 19.4, 20.9,
23.7, 24.7, 31.7, 31.7, 31.9, 36.6, 37.3, 39.0, 42.3, 42.7, 50.3, 55.0,
56.0, 59.7, 71.7, 82.1, 121.4, 126.7, 128.3, 129.5, 129.9, 134.3,
137.2, 140.9, 163.1; MS [ESI, 454 (M+ + H)], Anal. Calcd. for C28H36
ClNO2, C, 74.07; H, 7.99; N, 3.08. Found C, 74.06; H, 8.01; N, 3.06.
Cl
12e
12f
12g
3.15 0.02 ND
14.84
16.99
12.60
13.53
Br
10.34
11.23
12.69
ND
F
252
253
254
255
256
257
258
259
260
261
262
2.2.2.5. (5’R)-17b-[2-(4-Bromophenyl)-4,5-dihydrooxazol-5-yl]and-
20
rost-5-en-3b-ol (12e). Yield: 67%, mp 202–203 °C; [
a
]
D
ꢁ 37 (c
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.87 (s, 3H), 1.04 (s, 3H), 3.50
(m, 1H), 3.62 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz), 4.04 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz), 4.65 (m, 1H), 5.35 (d, 1H, J = 5.0 Hz),
7.54 (d, 2H, J = 7.5 Hz), 7.78 (d, 2H, J = 7.5 Hz); 13C NMR (d, ppm,
CDCl3): 12.7, 19.4, 20.9, 23.8, 24.7, 31.7, 31.7, 31.8, 31.9, 36.6,
37.3, 39.3, 42.3, 42.7, 50.3, 55.0, 56.0, 59.8, 71.7, 82.1, 121.4,
125.7, 127.1, 129.4, 131.6, 132.3, 140.9, 164.2; MS [ESI, 499
(M+ + H)], Anal. Calcd. for C28H36BrNO2, C, 67.46; H, 7.28; N, 2.81.
Found C, 67.45; H, 7.26; N, 2.82.
Cl
Finasteride 14.53
17.83
—
ND = not determined.
Prostate cancer cell lines: LNCaP (androgen dependant), PC-3 and DU-145 (andro-
gen independent).
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268
269
270
J = 14.5 Hz and J = 9.5 Hz), 4.67 (m, 1H), 5.34 (d, 1H, J = 5.0 Hz),
7.07 (t, 2H, J = 8.5 Hz), 7.92 (dd, 2H, J = 8.0 Hz); 13C NMR (d, ppm,
CDCl3): 12.7, 19.4, 20.9, 23.8, 24.7, 31.7, 31.7, 31.9, 36.6, 37.3,
39.2, 42.3, 42.7, 50.3, 55.0, 56.0, 59.6, 71.7, 81.6, 115.3, 121.4,
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264
265
266
2.2.2.6. (5’R)-17b-[2-(4-Fluorophenyl)-4,5-dihydrooxazol-5-yl]and-
20
rost-5-en-3b-ol (12f). Yield: 73%, mp 233–235 °C; [
a]
ꢁ 62 (c
D
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.86 (s, 3H), 1.04 (s, 3H), 3.51
(m, 1H), 3.62 (dd, 1H, J = 14.5 Hz and J = 8.0 Hz), 4.03 (dd, 1H,
Please cite this article in press as: Banday AH et al. Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.009

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313
314
315
124.4, 130.1, 128.3, 131.1, 132.3, 140.9, 165.6; MS [ESI, 460
(M+ + Na)], Anal. Calcd. for C28H36FNO2, C, 76.85; H, 8.29; N, 3.20.
Found C, 76.86; H, 8.27; N, 3.23.
steroidal derivatives (test material) were dissolved in a mixture
of DMSO:Water (1:1) and then introduced into the medium con-
taining the cancer cell lines.
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275
276
277
278
279
280
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282
283
284
285
316
2.2.2.7. (5’R)-17b-[2-(2-Chlorophenyl)-4,5-dihydrooxazol-5-yl]and-
2.4. Results and discussion
20
rost-5-en-3b-ol (12g). Yield: 69%, mp 164–167 °C; [
a
]
ꢁ 56 (c
D
1, CHCl3); 1H NMR (d, ppm, CDCl3): 0.82 (s, 3H), 1.01 (s, 3H), 3.50
(m, 1H), 3.68 (dd, 1H, J = 14.5 Hz and J = 8.5 Hz), 4.13 (dd, 1H,
J = 14.5 Hz and J = 9.5 Hz), 4.64 (m, 1H), 5.34 (d, 1H, J = 5.0 Hz),
7.28 (t, 1H, J = 8.0 Hz), 7.34 (d, 1H, J = 7.5 Hz), 7.43 (d, 1H,
J = 7.5 Hz), 7.75 (d, 1H, J = 7.5); 13C NMR (d, ppm, CDCl3): 12.6,
19.4, 20.9, 23.7, 24.7, 31.7, 31.7, 31.9, 36.6, 37.3, 39.0, 42.3, 42.6,
50.3, 55.0, 56.0, 60.3, 71.7, 81.6, 121.4, 126.7, 127.7, 130.7, 131.2,
131.3, 132.4, 140.9, 162.7; MS [ESI, 477 (M+ + Na)], Anal. Calcd.
for C28H36ClNO2, C, 74.07; H, 7.99; N, 3.08. Found C, 74.08; H,
8.01; N, 3.05.
In spite of the tremendous pharmacological potential of the het-
erocyclic analogs of D-ring modified steroids [12], there are limited
literature precedents available for their efficient synthesis and
evaluation as potential pharmacological agents. This is especially
true of the five membered heterocyclic oxazoline and isoxazoline
analogs which are pharmacologically very interesting species
[13]. Though there are reports available about the synthesis of
isoxazole and oxazole analogs condensed with the steroid skeleton
[14], same is not true for the synthesis of such analogs in the side
chain at ring D. Thus we, in continuation of our interest in develop-
ing pharmacologically active steroidal D-ring heterocyclic analogs,
herein report the efficient synthesis of novel side chain D-ring isox-
azoline and oxazoline derivatives of 17-androstanes and pregnen-
olone respectively. The synthetic strategies are discussed below.
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330
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2.3. Cell culture and bio-assays
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301
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304
305
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312
The human prostate cancer cell lines used for the test were
LNCaP, PC-3 and DU-145. All these cancer cell lines were obtained
from National cancer institute (NCI), biological testing branch,
Federick Research and Development centre, USA. Cellular viability
in the presence and absence of experimental agents was deter-
mined using the standard Sulforhodamine B assay. Briefly, cells
in their log phase of growth were harvested, counted and seeded
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353
354
2.4.1. Synthesis of isoxazoline derivatives 6 (a–f)
The starting ketone 1 upon condensation with ethyl cyanoace-
tate in boiling toluene, in the presence of ammonium acetate
was transformed to Knoevenagel adduct 2 presumably as a
mixture of E and Z isomers (89% yield) after purification using
silica-gel column chromatography. The condensation product
was reduced with an excess of sodium borohydride in methanol
to the saturated alcohol 3 (97% yield). Hydroxy group in compound
3 was then protected as its tetrahydropyranyl (THP) ether and the
derivative 4 was subjected to reduction with an excess of neat di-
isobutylaluminum hydride (DIBAL) for prolonged period in toluene
at ꢁ78 °C resulting in the formation of two products as indicated
by the TLC. After purification and spectral analysis of the products,
it was seen that the major product was the required one i.e. 5
formed in almost 50% yield. The intermediate 5 served as an
activated olefin having a great potential for the construction of
large number of carbocyclic and heterocyclic analogs across ring
D preferably through dipolar cycloadditions. The same was done
for the preparation of isoxazoline derivatives 6 (a–f) by employing
the cycloaddition of aromatic nitrile oxides across the olefin 5.
Though the cycloaddition could conceptually lead to the formation
of two regioisomers, we fortunately obtained only one regioisomer
as the sole isolable product. The regiochemistry was assigned on
the basis of NMR chemical shift values of the methylene protons
(104 cells/well in 100
lL medium) in 96-well microtitre plates.
After 24 h of incubation at 37 °C and 5% CO2 to allow cell
attachment, cultures were treated with varying concentrations
(10ꢁ9–10ꢁ4 M) of experimental agents i.e., the steroidal analogs
kept in six series of tubes. Four replicate wells were set up for each
experimental condition. Test samples were left in contact with the
cells for 48 h under same conditions. Thereafter, cells were fixed
with 50% chilled trichloroacetic acid (TCA) and kept at 4 °C for
1 h, washed and air dried. Cells were stained with Sulforhodamine
B dye. The adsorbed dye was dissolved in Tris-Buffer and plates
were gently shaken for 10 min on a mechanical shaker. The optical
density (OD) was recorded on ELISA reader at 540 nm. The cell
growth was calculated by subtracting mean OD value of respective
blank from the mean OD value of experimental set. Percent growth
in presence of test material was calculated considering the
growth in absence of any test material as 100% and in turn percent
growth inhibition in presence of test material was calculated.
Finally the IC50 values (Table 1) were calculated using Sigma Plot
software. Finasteride was used as a positive control. The different
H
NC
NC
COOEt
ii
O
OH
iii
H
i
AcO
AcO
AcO
3
1
2
N
O
H
OHC
Ar
NC
CHO
OTHP
H
H
H
iv
Ar-CNO
THF
6 (a-f)
4
5
HO
AcO
HO
Scheme 1. Synthesis of the D-ring Dehydroepiandrosterone isoxazolines. Reagents and conditions: (i) NCCH2COOEt, AcOH, AcNH4/toluene (90%); (ii) NaBH4/MeOH (98%);
(iii) DHP/p-TSA/DCM (95%); (iv) DIBAL neat/toluene/ꢁ78 °C (50%).
Please cite this article in press as: Banday AH et al. Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.009

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OAc
OH
OH
H3C
H2C
O
H2C
O
H
H
H
i
ii, iii
7
9
8
AcO
AcO
AcO
Ar
N
N3
OH
Cl
H2C
11
H2C
OH
O
H
H
H
H
vi, vii
v
iv
10
12 (a-g)
AcO
HO
AcO
Scheme 2. Synthesis of D-ring substituted pregnenolone oxazolines. Reagents and conditions: (i) Pb (OAc)4, BF3.OEt2, MeOH; (ii) KBH4, MeOH, rt; (iii) Al2O3, MW; (iv) P(Ph)3,
CCl4, reflux; (v) NaN3, DMF, 90 °C; (vi) aromatic aldehydes, CH2Cl2, BF3ꢂOEt2, rt, (vii) NaOH, MeOH, rt;
355
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359
396
397
398
399
400
401
402
403
404
405
in the isoxazoline ring which differ markedly for the two
regioisomers [16]. The stereochemistry at C-17 was assigned on
the basis of reported literature precedents [7]. The stereochemistry
of the hydroxyl at C-3 was not changed during any stage of the
synthesis (Scheme 1).
It is clear from the IC50 values, that the compounds 6a, 6e and
12a showed significant cytotoxic activity especially against LNCaP
and DU-145 prostate cancer cell lines. It is evident from the data
that the nature of substituent on the aromatic ring influences the
relative cytotoxicity which can be attributed to their differences
in either the bioavailability or the protein binding properties. As
already discussed it can be assumed that the electron donating
groups such as methyl- and methoxy- increase the in vitro cytotox-
icity. Overall the cytotoxicity of these compounds is cancer cell
specific as evidenced by the IC50 values.
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361
362
363
364
365
366
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368
369
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373
374
375
376
377
378
379
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381
382
383
384
385
386
387
388
389
2.4.2. Synthesis of the oxazoline derivatives 12 (a–g)
For the synthesis of oxazolines, we followed an efficient strat-
egy earlier described by Wolfing et al. [11]. This strategy involves
a one pot conversion of aldehydes to oxazolines upon reaction with
a,b-azidoalcohols. For this we chose 3b-acetoxypregn-5-en-20-one
(7) as the starting material. Oxidation with Pb(OAc)4 in the pres-
ence of BF3.OEt2 furnished 3b,21-diacetoxypregn-5-en-20-one (8).
Reduction with KBH4 gave two compounds, (20R)-3b,21-
diacetoxypregn-5-en-20-ol and its 20S epimer, latter in a very
small quantity. The required pure epimer was obtained by flash
chromatography. Selective deacetylation on alkaline alumina was
carried out by an earlier developed method to obtain the dihydroxy
derivative 9 [11]. Chlorination of 9 in the Appel reaction [15]
produced the (20R)-3b-acetoxy-21-chloropregn-5-en-20-ol (10).
Nucleophilic exchange with NaN3 in dimethylformamide led to
the required (20R)-3b-acetoxy-21-azidopregn-5-en-20-ol (11).
406
3. Conclusions
407
408
409
410
411
412
413
A series of novel D-ring substituted isoxazoline and oxazoline
derivatives of dehydroepiandrosterone and pregnenolone respec-
tively were synthesized and screened for anticancer activity against
a panel of human prostate cancer cell lines. From the data it was
found that all the compounds are having promising anticancer activ-
ity especially against LNCaP and DU-145 cell lines and the compound
6e was found to be the most active in this study.
414
References
Reaction of the a,b-azidoalcohol 11 with appropriately substituted
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proceeded cleanly to give the corresponding acetylated product
which upon deacetylation in presence of methanolic NaOCH3
yielded the deacetylated D-ring steroidal oxazolines 12 (a–g)
(Scheme 2).
The in vitro cytotoxicity studies of various steroidal isoxazoline
and oxazoline derivatives revealed that these derivatives are cell
specific as these were found to be active mostly against the LNCaP
(androgen dependant) compared to PC-3 and DU-145 (androgen
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Please cite this article in press as: Banday AH et al. Design and synthesis of D-ring steroidal isoxazolines and oxazolines as potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells. Steroids (2014), http://dx.doi.org/10.1016/j.steroids.2014.05.009