New biaryl-chalcone derivatives of pregnenolone via Suzuki-Miyaura cross-coupling reaction. Synthesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study

Article abstract of DOI:10.1016/j.steroids.2015.05.011

A new class of steroids is being synthesized for its ability to prevent intratumoral androgen production by inhibiting the activity of CYP17 hydroxylase enzyme. The scheme involved the synthesis of chalcone derivative of pregnenolone 5 which was further m

Full text of DOI:10.1016/j.steroids.2015.05.011

STE 7790
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Steroids xxx (2015) xxx–xxx
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Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
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New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura
cross-coupling reaction. Synthesis, CYP17 hydroxylase inhibition
activity, QSAR, and molecular docking study
Najim A. Al-Masoudi a, , Rawaa A. Kadhim , Nabeel A. Abdul-Rida , Bahjat A. Saeed , Mathias Engel
⇑
b
c
d
e
8
a
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0
1
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3
Department of Chemistry, College of Science, University of Basrah, Basrah, Iraq
Department of Chemistry, College of Education, University of Qadisiya, Qadisiya, Iraq
Department of Chemistry, College of Science, University of Qadisiya, Qadisiya, Iraq
Department of Chemistry, College of Education, University of Basrah, Basrah, Iraq
b
1
1
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1
c
d
e
Institut für Pharmazeutische und Medizinische Chemie, Universität des Saarlandes, Saarbrücken, Germany
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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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A new class of steroids is being synthesized for its ability to prevent intratumoral androgen production by
inhibiting the activity of CYP17 hydroxylase enzyme. The scheme involved the synthesis of chalcone
derivative of pregnenolone 5 which was further modified to the corresponding biaryl-chalcone pregne-
nolone analogs 16–25 using Suzuki–Miyaura cross-coupling reaction. The synthesized compounds were
2
2
2
2
Received 20 February 2015
Received in revised form 14 May 2015
Accepted 19 May 2015
Available online xxxx
tested for activity using human CYP17
most active inhibitor in this series, with IC50 values of 0.61
a
hydroxylase expressed in Escherichia coli. Compounds 21 was the
M and selectivity profile of 88.7% inhibition
l
2
2
2
2
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5
6
7
8
Keywords:
of hydroxylase enzyme. Molecular docking study of 21 was performed and showed the hydrogen bonds
and hydrophobic interaction with the amino acid residues of the active site of CYP17.
Ó 2015 Published by Elsevier Inc.
CYP17 hydroxylase enzyme
Molecular docking study
Pregnenolone
QSAR
29
30
Suzuki–Miyaura cross-coupling reaction
4
2
4
3
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4
1. Introduction
(CRPC) can clearly benefit from the newly approved drug abi-
raterone acetate (Zytiga) 3 [20,21]. Hartmann and co-workers
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59
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A number of steroids and their derivatives possess divers phar-
macological activities 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].
Recently, a large number of steroidal derivatives containing five- or
six-membered 17b-exo-heterocycles (preferably nitrogen contain-
ing), such as steroidal azoles [4,5] have been found to cause the
[22–24] have reported the synthesis of several CYP17 inhibitors
as a new strategy for the treatment of prostate carcinoma. In
2014, we have synthesized novel 17-pregnenolone-imine deriva-
tives as well as the 3-O-sulfonate and ester analogs at C-3,
designed as new CYP17A1 inhibitors [25]. Banday et al. have
reported recently some D-ring substituted steroidal chalcone [26]
and isoxazolines and oxazolines [27] derivatives with remarkable
activity against breast cancer and potential antiproliferative agents
against LNCaP, PC-3 and DU-145 cells, respectively.
CYP17 catalyzes two reactions, the 17R-hydroxylation of preg-
nenolone and progesterone to the corresponding 17R alcohols
and the subsequent 17,20-lyase reaction cleaving the C17AC20
bond. This yields the 17-keto androgens androstenedione and
dehydroepiandrosterone, precursors of all other androgens, includ-
ing testosterone.
inhibition of 17a-hydroxylase/C17-20-lyase (P45017a) which can
block adrenal androgen synthesis at an early stage and may there-
fore be useful in the treatment of prostatic carcinoma [6–11]. In
1996, Njar et al. [12] reported the first steroidal inhibitors of
CYP17 bearing a heterocyclic moiety bound to C17 by a nitrogen
atom, among which the imidazolyl derivative 1 was found to be
the most promising [12–15]. Later, in 2005, the same group
reported the synthesis of galeterone 2 and its
[15–17], where 2 is currently undergoing phase I/II clinical trials
for the treatment of chemotherapy naive CRPC [18,19]. However,
patients suffering from castration-resistant prostate cancer
4
D
-3-keto derivative
In continuation of our program on the synthesis of D-ring of
steroidal inhibitors analog, we investigated the synthesis of
biaryl-chalcone pregnenolone derivatives via Suzuki cross-cou-
pling reaction together with the CYP17 hydroxylase enzyme inhi-
bition activity, QSAR and the molecular modeling study.
⇑
Corresponding author at: Am Tannenhof 8, 78464 Konstanz, Germany.
E-mail address: najim.al-masoudi@gmx.de (N.A. Al-Masoudi).
http://dx.doi.org/10.1016/j.steroids.2015.05.011
039-128X/Ó 2015 Published by Elsevier Inc.
0
Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
thesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.05.011

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N
N
N
N
N
HO
. 17-(1H-Imidazol-1-yl)-
HO
AcO
1
2. Galeterone TOK-001
3. Abiraterone acetate
pregnen-16-ene-3-ol (VN/85-1)
8
8
5
6
2. Experimental
stirred for 15 min at ambient temperature followed by addition
of Pd(0)(PPh (22 mg, 5% mmol) and aq. solution of 2 M Na CO
(5 ml). The reaction mixture was heated under reflux for 12–
4 h. After cooling, water (5 ml) was added and the mixture was
partitioned with EtOAc (3 ꢀ 10 ml). The combined organic extracts
were washed with aq. solution of 5% Na CO
(3 ꢀ 10 ml), dried
(Na SO ) and evaporated in vacuo. The residue was purified on a
short SiO column using hexane: EtOAc (3:2) as eluent to give
the desired product.
134
135
136
137
138
139
140
141
142
3
)
4
2
3
2.1. General methods
1
8
8
8
9
9
9
9
9
9
9
9
9
9
7
8
9
0
1
2
3
4
5
6
7
8
9
Melting points are uncorrected and were measured on a Büchi
melting point apparatus B-545 (Büchi Labortechnik AG,
Switzerland). Microanalytical data were obtained with a Vario
Elemental Analyzer (Shimadzu, Japan). NMR spectra were recorded
on 400 and 600 MHz ( H) and on 150:91 MHz ( C) spectrometers
(Bruker, Germany) with TMS as internal standard and on the d
scale in ppm. Signal assignments for protons were performed by
selective proton decoupling or by COSY spectra. Heteronuclear
assignments were verified by HSQC, HMBC and DFQ-COSY experi-
ments. Mass spectra (EI, 70 eV, and FAB) were recorded on MAT
8200 spectrometers (Finnegan MAT, USA). TLC plates 60 F254 were
purchased from Merck. The chromatograms were visualized under
UV 254–366 nm and iodine.
2
3
2
4
2
1
13
0
0
0
2
.2.3. (E)-3-(2 ,3 -difluoro[1,1 -biphenyl]4-yl)-1-(3b-hydroxy-pregnen
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
-
17-yl)prop-2-en-1-one (16)
From 2,3-difluorophenylboronic acid 6 (63 mg). Yield: 81 mg
(
cm
7
39%) as a colorless powder. M.p.: 170–172 °C; R
f
= 0.50; IR (KBr)
ꢁ1
1
;
H NMR (DMSO-d
6
) d 7.89 (d, 1H, J21,22 = 16.1 Hz, H-22),
0
0
.75 (d, 2H,
J
2
0
,3
0
= 8.3 Hz,
Harom.-2 + Harom.-6 ), 7.61 (d, 1H,
0
J21,22 = 16.1 Hz, H-21), 7.49 (d, 2H, J
2
0
,3
0
= 8.3 Hz, Harom.-3 + Harom.
-
0
5
), 5.27 (t, 1H, J6,7 = 2.3 Hz, H-6), 4.61 (br s., 1H, OH), 3.26 (m,
1
00
2.2. Chemical synthesis
1H, H-3), 2.56 (m, 1H, H-17), 2.16 (m, 1H, H-16a), 2.11 (m, 2H,
CH -4), 1.94 (m, 1H, H-7a), 1.91 (m, 1H, H-12a), 1.78 (m, 1H, H-
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2.2.1. 17-((1-(4-Chlorophenyl)prop-1-en-2-yl)-5-pregnen-3b-ol (5)
To a stirred solution of pregnenolone 3 (100 mg, 0.32 mmol) in
EtOH (10 ml) were added 4-chlorobenzaldehyde
0.32 mmol) and aq. solution of 2 M NaOH (5 ml). After stirring at
ambient temperature for 24 h, the mixture was neutralized with
1 M HCl and partitioned with EtOAc (3 ꢀ 15 ml). The combined
organic extracts were washed with brine, dried over Na SO and
evaporated in vacuo. The residue was purified on a short SiO col-
umn using the eluent hexane: EtOAc (3:2) as eluent to give 5
(82 mg, 58%) as a yellow powder. M.p.: 98–92 °C; IR (KBr) cm
3675 (OH), 2927 (CH ), 1698, 1508 (C@C), 1049 (CAO); H NMR
1a), 1.68 (m, 1H, H-2a), 1.60 (m, 1H, H-15a), 1.55 (m, 1H, H-16b),
1.53 (m, 1H, H-7b), 1.51 (m, 3H, H-11a + H-12b), 1.43–1.35 (m,
2H, H-2b + H-8 + H-11b), 1.14 (m, 2H, H-14 + H-15b), 1.02 (m,
1H, H-1b), 0.98 (m, 1H, H-9), 0.94 (s, 3H, Me-19), 0.53 (s, 3H,
4 (50 mg,
1
3
Me-18); C NMR (DMSO-d
6
,): d 208.9 (C-20), 141.8 (C-5), 140.0
0
0
0
0
(C-22), 131.6 (Carom.-1 + Carom.-4 ), 130.6 (Carom.-2 + Carom.-6 ),
0
0
129.5 (Carom.-3 + Carom.-5 ), 129.2 (C-21), 120.7 (C-6), 70.5 (C-3),
56.6 (C-14 + C-17), 50.0 (C-9), 43.8 (C-13), 42.7 (C-4), 38.5 (C-12),
37.4 (C-1), 36.6 (C-10), 31.9 (Me-21), 31.8, 31.7 (C-2 + C-7 + C-8),
24.5 (C-15), 22.7 (C-16), 21.1 (C-11), 19.6 (Me-19), 13.4 (Me-18);
2
4
2
ꢁ1
1
Anal. calc. for C34
78.83; H, 7.30.
28 2 2
H F O (516.67): C, 79.04; H, 7.41. Found: C,
2
(DMSO-d ) d 7.89 (d, 1H, H, J
6
21,22
0
= 16.1 Hz, H-22), 7.75 (d, 2H,
-6 ), 7.61 (d, 1H, J
0
J2
0
,3
H-21), 7.49 (d, 2H, J2
0
= 8.3 Hz, Harom.-2 + H
arom.
21,22
= 16.1 Hz,
0
0
0
0
= 8.3 Hz, Harom.-3 + H
-5 ), 5.27 (t, 1H,
0
,3
arom.
2.2.4. 4 -(E)-3-hydroxy-pregenen-17-yl)-3-oxyprop-1-en-1-yl)-5-nit-
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
J6,7 = 2.3 Hz, H-6), 4.61 (br s., 1H, OH), 3.26 (m, 1H, H-3), 2.56 (m,
1H, H-17), 2.16 (m, 1H, H-16a), 2.11 (m, 2H, CH -4), 1.94 (m, 1H,
H-7a), 1.91 (m, 1H, H-12a), 1.78 (m, 1H, H-1a), 1.68 (m, 1H, H-
2a), 1.60 (m, 1H, H-15a), 1.55 (m, 1H, H-16b), 1.53 (m, 1H, H-7b),
1.51 (m, 3H, H-11a + H-12b), 1.43–1.35 (m, 2H, H-2b + H-8 + H-
11b), 1.14 (m, 2H, H-14 + H-15b), 1.02 (m, 1H, H-1b), 0.98 (m,
0
ro-[1,1 -biphenyl]-2-carboxylic acid (17)
2
From 3-carboxy-5-nitrophenylboronic acid 7 (84 mg). Yield:
1
02 mg (45%) as a yellow powder. M.p.: 139–141 °C; R
f
= 0.57; IR
);
H), 8.68 (d, 2H, J = 3.1 Hz,
ꢁ1
(
KBr) cm 3402 (OH), 2939 (CH
H NMR (DMSO-d
2
), 1651(C@C), 1412 (CANO
2
1
6
) d 11.89 (s, 1H, CO
2
00
00
00
H-6 ), 8.59 (d, 1H, J = 3.1 Hz, H-2 ), 8.51 (d, 1H, J = 3.1 Hz, H-4 ),
7
J21,22 = 16.1 Hz, H-22), 7.49 (d, 2H,
Harom.-6 ), 6.93 (d, 1H, J21,22 = 16.1 Hz, H-21), 5.29 (t, 1H,
J6,7 = 2.5 Hz, H-6), 4.68 (br s, 1H, OH), 3.26 (m, 1H, H-3), 2.95 (m,
13
1H, H-9), 0.94 (s, 3H, Me-19), 0.53 (s, 3H, Me-18); C NMR
0
0
2 ,3
.75 (d, 2H, J = 8.5 Hz, Harom.-3 + Harom.-5 ), 7.63 (d, 1H,
0 0
(DMSO-d ,): d 208.9 (C-20), 141.8 (C-5), 140.0 (C-22), 131.6
6
0
J
5 ,6
= 8.5 Hz, Harom.-2 +
0
0
0
0
0
0
(Carom.-1 + C
(Carom.-3 + C
arom.
-4 ),
130.6
(Carom.-2 + C
arom.
-6 ),
129.5
0
0
0
arom.
-5 ), 129.2 (C-21), 120.7 (C-6), 70.5 (C-3), 56.6
(C-14 + C-17), 50.0 (C-9), 43.8 (C-13), 42.7 (C-4), 38.5 (C-12), 37.4
(C-1), 36.6 (C-10), 31.9 (Me-21), 31.8, 31.7 (C-2 + C-7 + C-8), 24.5
(C-15), 22.7 (C-16), 21.1 (C-11), 19.6 (Me-19), 13.4 (Me-18);
Anal. calc. for C28H35ClO (439.04): C, 76.60; H, 8.04. Found: C,
76.48; H, 7.95.
1
(
1
H, H-17), 2.18 (m, 1H, H-16a), 2.13 (m, 2H, CH
m, 1H, H-7a), 1.84 (m, 1H, H-12a), 1.76 (m, 1H, H-1a), 1.68 (m,
H, H-2a), 1.60 (m, 1H, H-15a), 1.57 (m, 1H, H-16b), 1.55 (m, 1H,
H-7b), 1.54 (m, 1H, H-11a + H-12b), 1.43 (m, 1H, H-8), 1.41
m, 1H, H-11b), 1.36 (m, 1H, H-2b), 1.24 (m, 2H, H-14 + H-15b),
2
-4), 1.96
2
(
1
.01 (m, 1H, H-1b), 0.97 (m, 1H, H-9), 0.93 (s, 3H, Me-19), 0.53
1
3
1
1
1
1
30
31
32
33
2.2.2. General procedure for the synthesis of diaryl derivatives of
chalconyl pregnenolone via Suzuki cross-coupling reaction (16–25)
To a solution of 5 (200 mg, 0.40 mmol) in 1-propanol (15 ml)
was added arylboronic acid (0.40 mmol) and the mixture was
(s, 3H, Me-18);
(CO H), 147.8 (C
135.3 (Carom.-1 ), 134.0 (C
C NMR (DMSO-d ,): d 200.2 (C-20), 173.9
6
0
2
arom.
ANO ), 141.8 (C-5), 140.0 (C-22 + C
2
arom.
-4 ),
0
0
0
arom.
-1 ), 133.6, 132.5, 132.0, 130.6,
129.5 (Carom.), 129.2 (C-21), 120.7 (C-6), 70.5 (C-3), 60.9 (C-17),
Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
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1
1
1
1
86
87
88
89
56.8 (C-14), 50.1 (C-9), 44.8 (C-13), 42.7 (C-4), 38.6 (C-12), 37.4 (C-
1), 36.6 (C-10), 32.1, 31.9, 31.8 (C-2 + C-7 + C-8), 24.7 C-15), 22.8
(C-16), 21.1 (C-11), 19.6 (Me-19), 13.8 (Me-18); Anal. calc. for
21), 5.28 (t, 1H, J6,7 = 2.6 Hz, H-6), 3.26 (m, 1H, H-3), 2.99 (m, 1H,
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
H-17), 2.16 (m, 1H, H-16a), 2.12 (m, 2H, CH -4), 1.95 (m, 1H, H-
2
7a), 1.82 (m, 1H, H-12a), 1.75 (m, 1H, H-1a), 1.69 (m, 1H, H-2a),
1.66 (m, 1H, H-15a), 1.63 (m, 1H, H-16b), 1.57 (m, 1H, H-7b),
C35H39NO (569.70): C, 73.79; H, 6.90. Found: C, 78.83; H, 7.30.
6
1
(
1
.55 (m, 1H, H-11a), 1.53 (m, 1H, H-12b), 1.43 (m, 1H, H-8), 1.39
m, 1H, H-11b), 1.36 (m, 1H, H-2b), 1.23 (m, 2H, H-14 + H-15b),
.02 (m, 1H, H-1b), 0.99 (m, 1H, H-9), 0.97 (s, 3H, Me-19), 0.53
0
0
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
90
91
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
2.2.5. (E)-3-(4 -fluoro-[1,1 -biphenyl]-4-yl)-1-(3-hydroxy-prenen-17-
yl)prop-2-en-1-one) (18)
13
(
(
(
s, 3H, Me-18);
C
C
C NMR (DMSO-d
arom.-OH), 141.8 (C-5), 140.0 (C-22), 138.4 (Carom.-4 ), 135.3
6
,): d 200.0 (C-20), 155.2
From 4-fluorophenylboronic acid 8 (56 mg). Yield: 92 mg (46%)
0
ꢁ
1
as a semi-solid; R = 0.40; IR (KBr) cm
f
3402 (OH), 2939 (CH2),
0
arom.-1 ), 133.6, 132.9, 132.5, 131.9, 129.4 (Carom.), 129.2 (C-21),
1
1651 (C@C); H NMR (DMSO-d ) d 7.69 (d, 2H, J
6
3 ,4
0
0
= 7.8 Hz,
1
20.6 (C-6), 70.5 (C-3), 60.9 (C-17), 56.8 (C-14), 50.0 (C-9), 44.9
0
0
00
00
Harom.-3 + H
arom.
-5 ), 7.65–7.61 (m, 2H, H
arom.
-2 + H
arom.
-6 ), 7.54
(
3
(
C-13), 42.7 (C-4), 38.6 (C-12), 37.4 (C-1), 36.6 (C-10), 32.1, 31.9,
1.7 (C-2 + C-7 + C-8), 25.5 (C-15), 22.6 (C-16), 21.1 (C-11), 19.6
Me-19), 13.8 (Me-18); Anal. calc. for C34 (496.69): C,
00
00
(m, 2H, Harom.-3 + H
arom.
-5 ), 7.48 (d, 1H, J
= 7.8 Hz, Harom.-2 + H
21,22
= 16.0 Hz, H-22),
0
0
7.45 (d, 2H, J2
0
,3
0
arom.
-6 ), 6.87 (d, 1H,
40 3
H O
J2
1,22
= 16.0 Hz, H-21), 5.28 (br s, 1H, H-6), 4.29 (br s, 1H, OH),
8
2.22; H, 8.12. Found: C, 81.97; H, 7.90.
3.29 (m, 1H, H-3), 2.95 (m, 1H, H-17), 2.18 (m, 1H, H-16a), 2.11
(m, 2H, CH -4), 1.96 (m, 1H, H-7a), 1.87 (m, 1H, H-12a), 1.77 (m,
2
1H, H-1a), 1.70 (m, 1H, H-2a), 1.58 (m, 1H, H-15a), 1.57 (m, 2H,
0
0
0
2
.2.8. (E)-3-(2 ,4 -dimethoxy-[1,1 -biphenyl]1–4-y)-1-(3-hydroxy-
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
H-7b + H-16b), 1.56 (m, 1H, H-11a), 1.53 (m, 1H, H-12b), 1.45
(m, 1H, H-8), 1.40 (m 1H, H-11b), 1.38 (m, 1H, H-2b), 1.25 (m,
2H, H-14 + H-15b), 1.00 (m, 1H, H-1b), 0.99 (m, 1H, H-9), 0.95 (s,
pregenen-17-yl)prop-2-en-1-one (21)
From 3,4-dimethoxyphenylboronic acid 11 (73 mg). Yield:
1
21 mg (56%) as a dark brown semi-solid; R
f
= 0.52; IR (KBr)
3H, Me-19), 0.58 (s, 3H, Me-18); 13C NMR (DMSO-d ,): d 200.2
ꢁ1
1
6
cm 3402 (OH), 2932 (CH
2
), 1620 (C@C), 1026 (CAO); H NMR
(C-20), 159.1 (d, J = 249 Hz, C400-F), 141.8 (C-5), 140.0 (C-22),
(
H
H
DMSO-d
6
)
d
7.63 (1H,
J
21,22 = 15.6 Hz, H-22), 7.61 (m, 3H,
0
135.3 (Carom.-1 ), 134.1, 133.5, 132.5, 131.0, 129.3 (Carom.), 129.2
0
0
00
arom.-3 + Harom.-5 + Harom.-6 ), 7.55 (d, 2H,
J2 ,3 = 8.5 Hz,
0
0
00
00
(C-21), 120.7 (C-6), 110.8 (Carom.-3 + C
arom.
-5 ), 70.5 (C-3), 60.9
0
0
arom.-2 + Harom.-6 ), 7.17 (d, 1H, J21,22 = 15.6 Hz, H-21), 7.14 (s,
(C-17), 56.8 (C-14), 50.1 (C-9), 44.9 (C-13), 42.7 (C-4), 38.6 (C-
12), 37.4 (C-1), 36.6 (C-10), 32.1, 31.9, 31.8 (C-2 + C-7 + C-8), 24.7
00
00
1
H, Harom.-3 ), 6.99 (d, 1H, J
5
00,600 = 8.3 Hz, Harom.-5 ), 5.26 (t, 1H,
J
6,7 = 2.6 Hz, H-6), 4.62 (br s., 1H, OH), 3.84, 3.77 (2 ꢀ s, 6H,
1
9
(C-15), 22.8 (C-16), 21.1 (C-11), 19.6 (Me-19), 13.8 (Me-18);
F
2
1
1
1
1
ꢀ OMe), 3.26 (m, 1H, H-3), 2.99 (m, 1H, H-17), 2.15 (m, 1H, H-
NMR (DMSO-d ,): d ꢁ115.6; Anal. calc. for C34H39FO2 (498.68): C,
6
6a), 2.10 (m, 2H, CH -4), 1.94 (m, 1H, H-7a), 1.81 (m, 1H, H-
2
81.89; H, 7.88. Found: C, 81.77; H, 7.71.
2a), 1.76 (m, 1H, H-1a), 1.67 (m, 1H, H-2a), 1.65 (m, 1H, H-
5a + H-16b), 1.57 (m, 1H, H-7b), 1.56 (m, 1H, H-11a), 1.54 (m,
H, H-12b), 1.43 (m, 2H + H-2b + H-8), 1.39 (m, 1H, H-11b), 1.15
0
0
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
2.2.6. (E)-1-(3-hydroxy-pregenen-17-yl)-3-(4 -methylthio)-[1,1 -biph
enyl]-4-yl)prop-2-en-1-one (19)
(
0
m, 2H, H-14 + H-15b), 1.08 (m, 1H, H-1b), 0.98 (m, 1H, H-9),
.92 (s, 3H, Me-19), 0.51 (s, 3H, Me-18); C NMR (DMSO-d ,): d
6
1
3
From 4-methylthiophenylboronic acid 9 (67 mg). Yield: 110 mg
(52%) as a dark brown solid. M.p.: 84–87 °C; R = 0.50; IR (KBr)
f
00
00
208.9 (C-20), 162.3 (C -OMe), 160.2 (C -OMe), 141.7 (C-5),
2
4
ꢁ
1
1
cm 3395 (OH), 2932 (CH ), 1605 (C@C); H NMR (DMSO-d ) d
2
6
0
0
140.0 (C-22), 133.6 (Carom.-4 ), 132.5 (C
129.3 (Carom.), 129.2 (C-21), 119.0 (C-6), 112.7 (Carom.-5 ), 110.9
(Carom.-3 ), 70.5 (C-3), 63.1 (C-17), 56.6 (C-14), 56.1, (2xOMe),
arom.
-1 ), 132.0, 131.9,
00
00
7.75 (d, 2H, J200 00 = 8.5 Hz, Harom.-2 + H
-6 ), 7.62 (d, 1H,
00
,6
arom.
0
J2
1,22
= 16.0 Hz, H-22), 7.60 (d, 2H,
J
2 ,3
0
0
= 8.5 Hz,
H
arom.
-3 +
00
0
0
0
Harom.-5 ), 7.49 (d, 2H, J
2H,
2 ,3
= 8.7 Hz, H
Harom.-3 + H
0
0
arom.
-2 + H
arom.
-6 ), 7.34 (d,
50.0 (C-9), 43.7 (C-13), 42.7 (C-4), 38.4 (C-12), 37.4 (C-1), 36.6
(C-10), 31.9, 31.7 (C-2 + C-7 + C-8), 24.5 (C-15), 22.7 (C-16), 21.1
(C-11), 19.6 (Me-19), 13.4 (Me-18); Anal. calc. for C36H44O4
00
00
J2
0
,3
0
= 8.5 Hz,
arom.
-5 ),
6.92
(d,
1H,
J21,22 = 16.0 Hz, H-21), 5.27 (t, 1H, J6,7 = 2.5 Hz, H-6), 3.29 (m, 1H,
H-3), 2.98 (m, 1H, H-17), 2.38 (s, 3H, SMe), 2.20 (m, 1H, H-16a),
(
540.32): C, 79.96; H, 8.20. Found: C, 79.69; H, 8.02.
2.14 (m, 2H, CH -4), 1.95 (m, 1H, H-7a), 1.82 (m, 1H, H-12a),
2
1.75 (m, 1H, H-1a), 1.73 (m, 1H, H-2a), 1.63 (m, 1H, H-15a), 1.57
(m 1H, H-16b), 1.56 (m, 1H, H-7b + H-11a), 1.53 (m, 1H, H-12b),
1.42 (m, 1H, H-8), 1.40 (m, 1H, H-11b), 1.36 (m, 1H, H-2b), 1.23
(m, 2H, H-14 + H-15b), 1.01 (m, 1H, H-1b), 0.98 (m, 1H, H-9),
0
0
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
2
.2.9. (E)-1-(3-hydroxy-pregenen-17-yl)-3-(4 -trimethylsilyl)-[1,1 -bi
phenyl]-4-yl)prop-2-en-1-one (22)
From 4-trimethylsilylphenylboronic acid 12 (78 mg). Yield:
0.96 (s, 3H, Me-19), 0.53 (s, 3H, Me-18); 13C NMR (DMSO-d ,): d
6
67 mg (45%) as a gray powder; M.p.: 92–95 °C; R = 0.40; IR (KBr)
f
0
ꢁ1
1
200.1 (C-20), 141.8 (C-5), 140.0 (C-22), 137.7 (Carom.-4 +
cm 3402 (OH), 2932 (CAH), 1622 (C@C), 1026 (CAO); H NMR
(DMSO-d6) 7.63–7.45 (m, 9H, Harom. + H-22), 6.92 (d, 1H,
J21,22 = 16.0 Hz, H-21), 5.26 (t, 1H, J6,7 = 2.5 Hz, H-6), 4.62 (br s,
00
0
Carom.-SMe), 134.0 (Carom.-1 ), 132.0 (Carom.-1 ), 129.3 (C-21),
128.8, 128.5, 127.2, 126.9 (Carom.), 120.7 (C-6), 70.5 (C-3), 60.9
(C-17), 56.8 (C-14), 50.1 (C-9), 44.8 (C-13), 42.7 (C-4), 38.6
(C-12), 37.4 (C-1), 36.6 (C-10), 32.1, 31.9, 31.8 (C-2 + C-7 + C-8),
24.7 (C-15), 22.8 (C-16), 21.1 (C-11), 19.6 (Me-19), 15.2 (SMe),
d
1
1
1
H, OH), 3.27 (m, 1H, H-3), 2.97 (m, 1H, H-17), 2.17 (m, 1H, H-
6a), 2.11 (m, 2H, CH -4), 1.93 (m,1H, H-7a), 1.82 (m, 1H, H-
2a), 1.74 (m, 1H, H-1a), 1.69 (m, 1H, H-2a), 1.67 (m, 1H, H-
2
13.8 (Me-18); Anal. calc. for C35H42O S (526.29): C, 79.80; H,
2
15a + H-16b), 1.58 (m, 1H, H-7b + H-11a), 1.56 (m, 1H, H-12b),
.41 (m, 1H, H-8), 1.39 (m, 1H, H-11b), 1.37 (m, 1H, H-2b), 1.24
m, 2H, H-14 + H-15b), 1.06 (m, 1H, H-1b), 1.00 (m, 1H, H-9),
0.98 (s, 3H, Me-19), 0.52 (s, 3H, Me-18), 0.26 (s, 9H, SiMe ); 0.52
8.04. Found: C, 79.39; H, 7.88.
1
(
0
0
2
2
2
2
2
2
2
2
2
39
40
41
42
43
44
45
46
47
2.2.7. (E)-1-(3-hydroxy-pregenen-17-yl)-3-(4 -hydroxy)-[1,1 -biphen-
yl]-4-yl)prop-2-en-1-one (20)
3
1
3
(s, 3H, Me-18), 0.26 (s, 9H, SiMe ); C NMR (DMSO-d ,): d 199.5
3
6
0
0
From 4-hydroxyphenylboronic acid 10 (55 mg). Yield: 115 mg
(C-20), 141.2 (C-5), 140.6 (C-22), 139.5 (C
arom.
-1 ), 138.8 (Carom.-
0
00
(58%) as a colorless powder. M.p.: 150–152 °C; R = 0.57; IR (KBr)
f
4 ), 134.5 (C-4 ), 133.7, 133.0, 131.5, 128.7 (Carom.), 128.6 (C-21),
120.2 (C-6), 70.0 (C-3), 62.7 (C-17), 56.3 (C-14), 49.6 (C-9), 44.3
(C-13), 42.2 (C-4), 37.9 (C-12), 36.9 (C-1), 36.1 (C-10), 31.4, 31.1
(C-2 + C-7 + C-8), 25.5 (C-15), 24.0 (C-16), 21.2 (C-11), 19.1 (Me-
19), 13.2 (Me-18), ꢁ1.2 (SiMe ); Anal. calc. for C37H48O Si
ꢁ
1
1
cm 3433 (OH), 2932 (CH ), 1605 (C@C); H NMR (DMSO-d ) d
2
6
0
0
7.55 (d, 2H, J2
0
,6
0
= 8.5 Hz, Harom.-2 + H
= 16.1 Hz, H-22), 7.49 (d, 2H, J
arom.
-6 ), 7.52 (d, 1H,
= 8.5 Hz, H
= 8.5 Hz, Harom.-2 + H
0
J2
1,22
2 ,3
0
0
arom.
-3 + Harom.-
-6 ), 7.35
= 16.1 Hz, H-
0
00
00
5 ), 7.43 (d, 2H, J2
0
,3
0
arom.
3
2
3
00
00
J2
0
,3
0
= 8.5 Hz, Harom.-3 + H
arom.
-5 ), 6.92 (d, 1H, J
21,22
(552.34): C, 80.38; H, 8.75. Found: C, 80.11; H, 8.66.
Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
thesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.05.011

STE 7790
June 2015
No. of Pages 8, Model 5G
5
4
N.A. Al-Masoudi et al. / Steroids xxx (2015) xxx–xxx
0
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
2.2.10. (E)-1-(3-hydroxy-pregenen-17-yl)-3-(4 -(trifluoromethyl)-
[1,1 -biphenyl]-4-yl)prop-2-en-1-one (23)
4), 1.96 (m, 1H, H-7a), 1.84 (m, 1H, H-12a), 1.75 (m, 1H, H-1a),
1.68 (m, 1H, H-2a), 1.65 (m, 1H, H-16b), 1.60 (m, 1H, H-15a),
1.56 (m, 1H, H-7b + H-11a), 1.54 (m, 1H, H-12b), 1.42 (m, 1H, H-
8), 1.34 (m, 1H, H-11b), 1.32 (t, 3H, OCH CH ), 1.30 (m, 1H, H-
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
0
From 2-trifluoromethylphenylboronic acid 13 (76 mg). Yield:
90 mg (41%) as a brown semi-solid; R = 0.43; IR (KBr) cm 3395
(OH), 2932 (CAH), 1682 (C@Carom.); 1026 (CAO);
(DMSO-d ) d 7.74 (d, H, J 00 00 = 8.0 Hz, Harom.-6 ), 7.63 (d, 1H,
ꢁ
1
f
2
3
1
H
NMR
2b), 1.25 (m, 2H, H-14 + H-15b), 1.00 (m, 1H, H-1b), 0.97 (m, 1H,
00
13
6
5 ,6
H-9), 0.96 (s, 3H, Me-19), 0.56 (s, 3H, Me-18); C NMR (DMSO-
0
0
0
J21,22 = 16.1 Hz, H-22), 7.61 (m, 4H, Harom.-3 + Harom.-5
d6,): d 200.0 (C-20), 158.2 (C
5), 140.0 (C-22), 135.3 (Carom.-1 ), 132.0 (C
arom.
-OEt), 141.6 (C
arom.
00
-4 ), 141.5 (C-
0
0
00
0
+ H
2 + H
arom.
-3 + H
arom.
-5 ), 7.55 (d, 2H,
J
2 ,3
0
0
= 8.0 Hz, Harom.
-
arom.
-1 ), 129.1 (C-21),
129.1, 127.7, 126.0 (Carom.), 120.5 (C-6), 115.6 (Carom.-3 + Carom.-
5 ), 70.8 (C-3), 60.9 (C-17), 64.6 (OCH CH ), 61.3 (C-17), 56.1 (C-
2 3
0
0
00
00
-6 ), 7.47 (d, 1H, J 00 00 = 8.0 Hz, Harom.-4 ), 6.92 (d, 1H,
arom.
4 ,5
0
0
J21,22 = 16.1 Hz, H-21), 5.26 (t, 1H, J6,7 = 2.6 Hz, H-6), 3.27 (m, 1H,
H-3), 2.99 (m, 1H, H-17), 2.16 (m, 1H, H-16a), 2.10 (m, 2H, CH2-
4), 1.94 (m, 1H, H-7a), 1.82 (m, 1H, H-12a), 1.77 (m, 1H, H-1a),
1.74 (m, 1H, H-2a), 1.66 (m, 2H, H-15a + H-16b), 1.57 (m, 1H, H-
7b), 1.55 (m, 1H, H-11a), 1.53 (m, 1H, H-12b), 1.40 (m, 1H, H-8),
1.35 (m 1H, H-11b), 1.33 (m, 1H, H-2b), 1.21 (m, 2H, H-14 + H-
15b), 1.03 (m, 1H, H-1b), 0.98 (m, 1H, H-9), 0.96 (s, 3H, Me-19),
14), 50.2 (C-9), 44.2 (C-13), 42.4 (C-4), 38.6 (C-12), 37.3 (C-1),
36.2 (C-10), 32.0, 31.7, 31.4 (C-2 + C-7 + C-8), 25.7 (C-15), 24.8
(C-16), 22.2 (C-11), 19.2 (Me-19), 14.5 (OCH CH ), 13.2 (Me-18);
2
3
Anal. calc. for C36H44O3 (524.33): C, 82.40; H, 8.45. Found: C,
82.21; H, 8.36.
1
3
2.3. Biological evaluations
389
0.52 (s, 3H, Me-18); C NMR (DMSO-d ,): d 199.6 (C-20), 141.3
6
0
0
(C-5), 139.5 (C-22), 139.4 (C
arom.
-4 ), 136.7 (C
arom.
-1 ), 135.3
00
2.3.1. CYP17 enzyme preparation
390
391
392
393
394
395
(Carom.-5 ), 133.0, 132.5, 132.3, 132.0, 131.5, 0, 131.0 (Carom.),
128.7 (C-21), 125.4 (d, JC,F = 249 Hz, CF ), 120.2 (C-6), 70.0 (C-3),
62.6 (C-17), 56.3 (C-14), 50.0 (C-9), 44.3 (C-13), 42.2 (C-4), 38.1
(C-12), 36.9 (C-1), 36.1 (C-10), 31.5, 31.4, 31.2 (C-2 + C-7 + C-8),
25.5 (C-15), 24.1 (C-16), 22.2 (C-11), 19.1 (Me-19), 13.3 (Me-18);
Anal. calc. for C35H39F O (548.69): C, 76.62; H, 7.16. Found: C,
The coexpression of human CYP17 and rat NADPH-P450-reduc-
tase in Escherichia coli and the isolation of the membrane fractions
were performed as described by Hartmann and co-workers [28].
Membrane fractions were diluted to a concentration that gives a
3
1
5–25% conversion in the controls for the different assays.
3
2
76.42; H, 7.02.
2
.3.2. 17
Determination of the hydroxylase activity of CYP17 was per-
formed by measurement of the conversion of P5 to 17OHP . An
a
-Hydroxylase enzyme assay
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
0
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
2.2.11. 4 -((E)-3-(3-hydroxy-pregenen-17-yl)-3-oxoprop-1-en-1-yl)-
0
[1,1 -biphenyl]3-carbonitrile (24)
5
assay mixture consisting of 140
7.4, 1 M MgCl , 0.1 M EDTA, and 0.1
NADPH generating system (in phosphate buffer with 50
l
l phosphate buffer (0.05 M, pH
M dithiothreitol), 50
M glu-
M NADP , and 27.5 U/ml 5 glucose-6-
phosphate dehydrogenase) and 5 l substrate solution (25
[3H]-P ) was preincubated at 37 °C for 5 min. The reaction was
From 3-cyanophenylboronic acid 14 (59 mg). Yield: 59 mg
(39%) as a brown solid; M.p.: 88–91 °C; R = 0.45; IR (KBr) cm
3402 (OH); 2932 (CAH), 1358 (CAN), 1682 (C@Carom.); H NMR
-2 ), 8.12 (d, 1H, J 00 00 = 7.9 Hz,
Harom.-6 ), 7.89 (d, 1H, J 00 00 = 7.9 Hz, Harom.-4 ), 7.70 (t, 1H,
ꢁ
1
l
l
l
ll
2
f
1
l
+
00
cose-6-phosphate, 5.75 l
(DMSO-d ) d 8.29 (s, 1H, H
6
arom.
5 ,6
0
0
00
l
lM
5 ,6
0
0
J400 00 = 7.9 Hz, Harom.-5 ), 7.62 (d, 1H, J
= 16.1 Hz, H-22), 7.55
5
,5
21,22
0
0
started by addition of 50 ll enzyme preparation. Compared to
(d, 2H, J5
0
,6
0
= 8.4 Hz, Harom.-3 + H
arom.
-5 ), 7.49 (d, 2H,
= 16.1 Hz,
0
0
the lyase assay, however, enzyme concentration had to be reduced
to keep control conversion in the favorable range of 15–25% and to
prevent DHEA formation. After a 30 min incubation at 37 °C, the
J2
0
,3
0
= 8.4 Hz, Harom.-2 + H
arom.
-6 ), 6.91 (d, 1H, J
21,22
H-21), 5.27 (t, 1H, J6,7 = 2.5 Hz, H-6), 3.27 (m, 1H, H-3), 4.21 (d,
1H, J = 6.2 Hz, OH), 3.25 (m, 1H, H-3), 2.97 (m, 1H, H-17), 2.13
(m, 1H, H-16a), 2.10 (m, 2H, CH -4), 1.93 (m, 1H, H-7a), 1.81 (m,
enzyme reaction was stopped by addition of 50
Extraction of the steroids was performed by addition of 1000
ethyl acetate and vigorous shaking for 10 min. After a centrifuga-
tion step (5 min, 15,000g), 900 l of the organic phase were
removed and transferred into a fresh tube containing 250 l phos-
phate buffer and 50 l 1 N HCl. Shaking and centrifugation was
repeated as described above. 800 l ethyl acetate was evaporated
ll 1N HCl.
2
ll
1H, H-12a), 1.77 (m, 1H, H-1a), 1.70 (m, 1H, H-2a), 1.65 (m, 2H,
H-15a + H-16b), 1.56 (m, 1H, 1H-7b), 1.54 (m, 1H, H-11a), 1.52
(m, 1H, H-12b), 1.40 (m, 1H, H-8), 1.34 (m 1H, H-11b), 1.32
(m, 1H, H-2b), 1.23 (m, 2H, H-14 + H-15b), 1.10 (m, 1H, H-1b),
l
l
1
3
l
0.98 (m, 1H, H-9), 0.92 (s, 3H, Me-19), 0.54 (s, 3H, Me-18);
C
00
l
6
arom.
NMR (DMSO-d ,): d 200.2 (C-20), 142.3 (C -1 ), 141.8 (C-5),
0
0
to dryness in a fresh tube and re-dissolved in 40
ll acetoni-
140.0 (C-22), 139.6 (Carom.-4 ), 135.3 (Carom.-1 ), 132.5
0
0
00
00
00
trile/water (1:1) for HPLC analysis.
(Carom.-4 + C
arom.-
6 ), 132.0 (C
arom.
0
-2 ), 130.8 (C
arom.
-5 ), 129.3 (C-
0
0
0
21), 128.3 (Carom.-3 + C
arom.
-5 ), 127.4 (C
arom.
-2 + C
arom.
-6 ), 120.7
2.3.3. HPLC methods
418
419
420
421
422
423
424
425
(C-6), 119.1 (CN), 112.8 (C300-CN), 70.5 (C-3), 63.1 (C-17), 56.8 (C-
14), 50.1 (C-9), 44.9 (C-13), 42.7 (C-4), 38.4 (C-12), 37.4 (C-1),
36.6 (C-10), 31.9, 31.7 (C-2 + C-7 + C-8), 25.6 (C-15), 24.3 (C-16),
22.7 (C-11), 19.6 (Me-19), 13.8 (Me-18); Anal. calc. for
C35H39NO2 (505.70): C, 83.13; H, 7.77; N, 2.77. Found: C, 82.93;
H, 7.65; N, 2.56.
HPLC separation of the steroids was performed using an Agilent
100 HPLC system with PDA detector (Böblingen, Germany), a CC
25/3 Nucleodur 100–3 C-18 ec column (Macherey–Nagel,
1
1
Düren, Germany) and a Berthold radioflow detector LB509 with
scintillator pump (Bad Wildbad, Germany). Quickszint Flow 302
LSC Cocktail (Zinsser Analytic, Frankfurt/Main, Germany) was used
as scintillator fluid. More details are reported in [29].
0
0
363
364
365
366
367
368
369
370
371
372
373
2.2.12. (E)-3-(4 -ethoxy-[1,1 -biphenyl]-4-yl)-1-(3-hydroxypregenen-
17-yl)prop-2-en-1-one (25)
From 4-ethoxyphenylboronic acid 15 (67 mg). Yield: 88 mg
3. Results and discussion
426
427
ꢁ1
(42%) as a brown semi-solid; R = 0.40; IR (KBr) cm 3339 (OH),
f
1
2932 (CAH), 1736 (C@O), 1574 (C@Carom.); H NMR (DMSO-d ) d
6
3.1. Chemistry
0
0
7.63–7.60 (m, 3H, H-22 + H
arom.
0
-3 + H
arom.
-5 ), 7.54 (d, 2H,
-6 ), 7.48 (d, 2H,
200,300 = 8.6 Hz, Harom.-3
+ Harom.-5 ), 6.85 (d, 1H, J21,22 = 16.0 Hz, H-21), 5.25 (t, 1H,
J6,7 = 2.5 Hz, H-6), 4.05 (q, 2H, J = 6.9 Hz, OCH CH ), 3.28 (m, 1H,
0
J200 00 = 8.6 Hz,
H
-2 + H
-6 ), 6.95 (d, 2H,
J
00 00 = 8.6 Hz,
Treatment of pregnenolone 3 with p-chlorobenzaldehyde (4)
gave the chalcone derivative 5 (58%). The Suzuki–Miyaura cross-
coupling reaction [30] has been employed in the preparation of
new pregnenolone analogs. Thus, treatment of 5 with the appropri-
ate arylboronic acids (e.g.: 2-fluorophenyl-, 3-fluorophenyl-, 4-fluor
428
429
430
431
432
,6
arom.
00
arom.
5 ,6
0
0
00
Harom.-2 + H
arom.
J
00
2
3
H-3), 3.02 (m, 1H, H-17), 2.18 (m, 1H, H-16a), 2.07 (m, 2H, CH2-
Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
thesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.05.011

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5
Me
O
5
O
Cl
CHO
i
+
HO
Cl
HO
3
4
O
B(OH)2
R
ii
R
6-15
HO
16-25
Entry
16 2,3-F 2
17 3-NO ,5-CO H
R
R
Entry
11, 21
6
7,
,
2,4-OMe2
4-SiMe3 12, 22
2
2
8
,
9,
18 4-F
19 4-SMe
0, 20 4-OH
4
3
4
-CF3
-CN
-OEt
13, 23
14, 24
15, 25
1
3 4 2 3
Scheme 1. Reagents and conditions: (i) EtOH, 2 M aq. NaOH, 24 h, r.t.; (ii) Pd(Ph P) , K CO ,1-PrOH, reflux, 12–14 h.
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
ophenyl-, 3,4-difluorophenyl-, 3,4-dimethoxy phenyl-, 2-methylth-
iophenyl-, 4-nitrophenyl-, 4-ethoxycarbonylphenyl-, 4-hydrox-
yphenyl-, 3-cyanophenyl-, and 4-triflouromethylphenyl boronic
acids 6–15, using palladium(0) tetrakis-triphenylphosphine
2''
2
2
SMe
H
1'
4
''
4
'
6''
2
1 H
2
0
6
(Pd(0)Ph P) ) and sodium bicarbonate as catalysts in 1-propanol
3
4
O
Me
afforded 16–25 in 72–78% yield (Scheme 1).
18
1
9
The structures of 5 and 17–28 were assigned on the basis of
H
Me
1
13
H
their NMR ( H, C and 2D), which showed rather similar patterns
1
1
of the proton and carbon atoms of pregnen scaffold. The H NMR
spectrum of 5 showed beside the expected signals for preg-
nenolone moiety two doublets at d 7.61 and 7.89 ppm with a large
J coupling (ꢂ16.0 Hz) were assigned for the olefin protons H-21
HO
Fig. 1. JC,H correlations in the HMBC (double head arrow), and NOESY (single head
arrow) correlations of (19).
1
and H-22 of chalcone moiety. The H NMR spectra of 17–28 were
characterized by the presence of additional aromatic protons and
carbon atoms, indicative for arylation of the chalconyl preg-
nenolone analogs. The aromatic protons protons appeared as dou-
blets or multiplets at the regions d 8.59–6.95 ppm (J = 8.5–7.9 or
3.1 Hz). The triplets at d 5.29–5.25 ppm (J ꢂ 2.6 Hz) were assigned
between H-21 at d
tion for the coupling between H-22 at d
.20 ppm. These data confirmed that both H-21 and H-22 are in
a trans-configuration (Fig. 1). All the compounds have been identi-
H
6.92 ppm and Me-18 at d
H
0.53 ppm in addi-
475
476
477
478
479
480
481
482
483
484
485
486
487
H
7.62 ppm H-16a at d
H
2
to H-6, while H-3 and CH -4 appeared as multiplets at the regions d
2
1
13
fied by their H, C HSQC NMR spectra [33].
2.16–2.07 and 1.58–1.53 ppm, respectively. The other aliphatic
Further, the configuration of the olefinic protons (C21@C22) of
6–25 has been calculated by the DFT method (GGA) at the level
PBE/DNP [34]. The results in Table 1 demonstrated that the trans
configuration of these analogs is the more stable. Fig. 2 represents
the orbitals of 16, which revealed that the HOMO in both trans and
cis isomers is located at the olefinic bond (C @C ) of the pregnen
5 6
part, while the LUMO is separated on the carbon atoms of the ole-
finic linkage (C21@C22) and the oxygen atom of the carbonyl group.
protons were fully analyzed (c.f. Experimental section). H-21 and
H-22 were resonated as two doublets at the regions d 7.17–6.85
and 7.63–7.50 ppm, respectively with a large J ꢂ 16.0 Hz, indica-
tive for the trans-configuration of the chalcone protons (H-21
1
1
3
and H-22). In the C NMR spectra of 5 and 17–28, the resonances
at d 208.9–199.5 ppm were assigned for C-20, whereas the olefinic
carbon atoms (C-21, C-22) were appeared at the regions d 129.3–
128.6 and 140.0.139.5 ppm, respectively. The aromatic, other preg-
nen aliphatic carbon atoms and the substituents have been fully
identified (c.f. Experimental section). Compound 19 was selected
for further NMR studies, since its gradient HMBC spectrum [31]
3.2. In vitro CYP17 hydroxylase enzyme activity
488
2
allowed the identification of H-21 at dH 6.92 ppm from the JC,H
The hydroxylase substrate, 17-hydroxypregnenolone is formed
489
490
491
492
493
494
495
496
497
498
499
couplings to the chalconyl atom (C-22) at dC 135.2 ppm as well
in situ by the cytochrome P450 17
a 10 min preincubation. At this time point, almost all pregnenolone
substrate has been transformed into 17 -hydroxypregnenolone
a-hydroxylase enzyme during
2
as the carbonyl carbon atom (C-20) at dC 200.1 ppm. A JC,H cou-
pling between H-22 at dH 7.62 ppm and the aromatic carbon atom
a
0
(C-1 ) at d 132.0 ppm was observed. Furthermore, SMe protons at
C
(>95%). The incubation of an aliquot is stopped using HCl. At this
time point, the hydroxylase assay is initiated by addition of our test
compounds 5 and 16–25 in different concentrations dissolved in
3
dH 2.38 ppm showed a J coupling to the aromatic carbon atom
C,H
0
0
3
(C-4 ) at d 137.7 ppm. Additionally, JC,H couplings between C-20
and H-16a and H-16b at dH 2.20 and 1.57 ppm, respectively, were
distinguished (Fig. 1). The configuration of the chalcone protons
(H-21 and H-22) was further confirmed by the 1H, H NOESY
NMR spectrum [32]. Thus, the spectrum of 19 revealed coupling
DMSO (final concentration usually between 0 and 10 lM). The
results are presented in Table 2. The steroidal CYP17A1 inhibitor
abiraterone acetate (3) was used as a reference compound.
Aiming at the development of new drugs for the treatment of
1
Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
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N.A. Al-Masoudi et al. / Steroids xxx (2015) xxx–xxx
Table 1
Energies of the analogs 16–25 calculated at the theory level PBE/DN.
Compd.
cis-isomer
trans-isomer
Compd.
cis-isomer
trans-isomer
16
17
18
19
20
ꢁ1667.103153
ꢁ1667.103153
ꢁ1567.8899860
ꢁ1906.0519105
ꢁ1543.8767651
ꢁ1667.109300
ꢁ1861.5775018
ꢁ1567.8972197
ꢁ1906.0661973
ꢁ1543.8839474
21
22
23
24
25
ꢁ1697.5689582
ꢁ1877.1550518
ꢁ1560.8606047
ꢁ1805.5785310
ꢁ1622.3943784
ꢁ1697.5768487
ꢁ1877.1669258
ꢁ1560.8679660
ꢁ1805.5854794
ꢁ1622.4105373
Fig. 2. Represents the energies of compound 16: (A) trans HOMO = ꢁ0.106368 hartree; (B) trans LUMO = ꢁ0.106368 hartree, (C) cis HOMO = ꢁ0.186773 hartree; (D) cis
LUMO = ꢁ0.104875 hartree.
Table 2
hydrophobicity interactions of both the phenyl and C21@C22
Inhibition of CYP17 hydroxylase enzyme by pregnenolone analogs.
519
520
521
through
p–p interactions. Such argument is in agreement with
Compd. Inhibition %a IC50 M)b Compd. Inhibition %a IC50 M)b
(l (l
the results reported previously [35].
5
1
1
1
1
78.2
78.7
76.2
83.8
70.1
92.93
3.10 ± 0.8
1.53 ± 0.2
2.80 ± 0.5
1.10 ± 0.1
2.85 ± 0.4
0.072
20
21
22
23
24
25
68.4
88.7
60.0
75.6
80.1
58.9
nd
0.61 ± 0.0
nd
1.25 ± 0.6
1.91 ± 0.3
2.97 ± 0.7
6
7
8
9
3
.3. Molecular docking study
522
c
The structural common features of compounds 5 and 16–25 to
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
546
547
548
ABT
abiraterone acetate (3) (pregnenolone scaffold) with some inhibi-
tory activity against CYP17, prompted us to study their interactions
with the active sites of the enzyme via the molecular computa-
tional studies. In the docking study, the X-ray crystal structure of
human microsomal cytochrome protein P450 (CYP17; PDBID:
a
Data shown were obtained by performing the tests in duplicate. IC50: the
concentration of the inhibitor that is required for 50% inhibition in vitro.
b
Hydroxylase enzyme inhibition measured at 10
ABT: abiraterone acetate; nd: not determined.
lM.
c
3
ruk) was obtained from Protein Data Bank (http://www.rcsb.
org) [36].
AutoDock4.2 software [37] and AutoDock Tools were used for
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
prostate cancer, the effects of steroidal compounds on androgen
biosynthesis were evaluated in vitro.
Derivatization of the pregnenolone scaffold by a p-chlorophe-
nyl-chalcone group is readily tolerated in 17-position (compound
5) followed by introduction of substituted aryl groups via Suzuki
cross-coupling reaction as accomplished in compounds 16–25.
The inhibitory profile (Table 2) showed that 5, 16–19, and 23–25
exhibited inhibition of CYP17 with IC50 values of 0.10–3.10
Interestingly, compound 21 having 2,4-dimethoxyphenyl sub-
stituents, pronounced higher inhibitory activity with
IC50 = 0.61 M and inhibition of 88.7% in comparison for those car-
rying other substituents. Introduction of nitro and carboxy resi-
dues in meta positions, 2,3-difluoro, 4-fluoro 4-trifluomethyl or
3-cyano groups as accomplished in compounds 16–18, 23 and 24
decreased the inhibitory potency, which might be due to its elec-
docking experiments following Lamarckian Genetic algorithm
(hybrid of genetic and local search algorithm) [38]. Best docked
conformation of each ligand based on docking parameters and
binding site residues interactions and selected among top ten
docked conformations generated during docking study. The results
were visualized with AutoDock Tools software, whereas the auto-
mated docking experiments have been reported previously in
details [25].
Compound 21 has been selected for the docking modeling
study, since it is the most active candidate of the series, meanwhile
its binding energy score ꢁ13.02 indicating a selectivity and
potency profile of dimethoxy-biphenylchalcone-pregnenolone to
bind the active site of HIV-RT pocket (Fig. 3). Detailed analysis of
the binding mode showed an interesting complexation of heme
lM.
a
l
2
tron withdrawing effects reducing the electron density of the sp
hybrid of the C21@C22 and phenyl
the hydrophobicity interactions with the CYP17 amino residues.
However, the two methoxy group would enhance such the
+
2
p
bonds, which in turn decrease
iron (Fe ) with the lone pair of oxygen atom OH group at C-3.
Beside the complexation, the phenyl ring B point toward the aro-
matic ring of Phe300 residue apparently developing
p–p stacking
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7
+
2
Fig. 3. Computer model of human CYP17 (pdbid 3ruk) with 21. Complexation of heme-Fe (brown) with lone pair of oxygen atom of OH group at C-3. Three hydrogen bonds
00
00
are shown: Ile371 with OH at C-3; Gly297 and Leu242 with 2 and 4 -OMe groups of phenyl moiety (B). In addition, a hydrophobic interaction between phenyl group B and
Phe300 of the CYP17 enzyme amino acid residues is observed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of
this article.)
5
5
5
5
5
5
5
5
5
5
5
5
5
49
50
51
52
53
54
55
56
57
58
59
60
61
interactions with the phenyl ring B. In addition, the chalcone–preg-
nenolone backbone locate in the middle of the binding pocket,
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thesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.05.011

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Please cite this article in press as: Al-Masoudi NA et al. New biaryl-chalcone derivatives of pregnenolone via Suzuki–Miyaura cross-coupling reaction. Syn-
thesis, CYP17 hydroxylase inhibition activity, QSAR, and molecular docking study. Steroids (2015), http://dx.doi.org/10.1016/j.steroids.2015.05.011