Aromatic β-amino-ketone derivatives as novel selective non-steroidal progesterone receptor antagonists

Article abstract of DOI:10.1016/j.bmc.2010.04.092

A novel class of non-steroidal progesterone receptor antagonists with aromatic β-amino-ketone scaffold have been synthesized and characterized with high binding affinity and great selectivity for the cognate receptors. Among them, compound 22 was shown to be the most potent progesterone receptor antagonist in cotransfection assay and a murine model of ligand-induced decidualization.

Full text of DOI:10.1016/j.bmc.2010.04.092

Bioorganic & Medicinal Chemistry 18 (2010) 4255–4268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Aromatic b-amino-ketone derivatives as novel selective non-steroidal
progesterone receptor antagonists
Yongli Du a, , Qunyi Li b, , Bing Xiong a, Xin Hui b, Xin Wang a, Yang Feng b, Tao Meng a, Dingyu Hu a,
a,
Datong Zhang c, Mingwei Wang a,b, , Jingkang Shen
*
*
a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, PR China
b The National Center for Drug Screening, 189 Guo Shou Jing Road, Shanghai 201203, PR China
c School of Chemical Engineering, Shandong Institute of Light Industry, Jinan, Shandong 250353, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of non-steroidal progesterone receptor antagonists with aromatic b-amino-ketone scaffold
have been synthesized and characterized with high binding affinity and great selectivity for the cognate
receptors. Among them, compound 22 was shown to be the most potent progesterone receptor antago-
nist in cotransfection assay and a murine model of ligand-induced decidualization.
Ó 2010 Published by Elsevier Ltd.
Received 23 March 2010
Revised 28 April 2010
Accepted 28 April 2010
Available online 24 May 2010
Keywords:
Progesterone receptor (PR)
Progesterone receptor antagonist
Non-steroidal progesterone receptor
antagonist
Aromatic b-amino-ketone
Mannich reaction
1. Introduction
ones,12 benzoxazin-2-ones,12 3-aryl-1,2-diazepines,13 5-aryl inden-
1-ols14, and 5-aryl indanones,14 as exemplified by compounds 1–7
Progesterone receptor (PR) is a member of the nuclear hormone
receptor super family which is a group of ligand-dependent
transcription factors.1 Small molecule antagonists to PR have impor-
tant roles in healthcare, including the potential use as therapeutic
agents for treatment of leiomyomas,2 endometriosis,3 breast can-
cer,4 and meningiomas,5 as well as application in fertility control.6
However, the most well-known PR antagonist, namely, mifepristone
(RU486), also possesses potent activity at other steroid receptors
such as glucocorticoid receptor (GR), which limits its broad clinical
utility, especially for chronic administration. Non-steroidal PR
antagonists with distinct structural features may therefore circum-
vent the liabilities manifested by their steroidal counterparts.
During the past decade, several classes of non-steroidal PR antag-
onists have been described in the literature, and their pharmacolog-
ical properties characterized.7,8 Some of the most frequently
reported structural features of non-steroidal PR antagonists
included 6-aryl-1,3-dihydrobenzoimidazol-2-ones,9,10 5-Aryl-1,3-
dihydro-indol-2-ones,11 6-aryl-1,4-dihydrobenzo[d][1,3]oxazin-2-
shown in Figure 1.
In our pursuit to discover non-steroidal modulators to nuclear
hormone receptors, a series of b-amino-ketone analogues were
identified as androgen receptor (AR) modulators through a high-
throughput screening campaign described in a recent report.15
During the process of modifying the b-amino-ketone derivatives
(8, Fig. 2) against AR, compound 9 (Fig. 2) was shown to possess
moderate PR (IC50 = 180 nM) and high potent AR binding affinities
(IC50 = 2.9 nM).15 Although it is well documented that the steroidal
ligands of nuclear hormone receptors have a common fused four-
ring system, little is known relative to shared non-steroidal scaf-
folds for different nuclear hormone receptors. Thus, the finding
that b-amino-ketone derivatives affect both AR and PR activities
instigated us to further study this novel non-steroidal template
that led to the discovery of a series of PR antagonists with high po-
tency and selectivity. In this report we describe in detail the syn-
theses and structure–activity relationship (SAR) analyses of these
novel non-steroidal PR antagonists.
* Corresponding authors. Tel.: +86 21 50806600 5407; fax: +86 21 50807088
(J.S.), tel.: +86 21 50800598; fax: +86 21 50800721 (M.W.).
E-mail addresses: wangmw@mail.shcnc.ac.cn (M. Wang), jkshen@mail.shcn-
c.ac.cn (J. Shen).
2. Chemistry
The syntheses of aromatic b-amino-ketone analogues 13–55
were accomplished through Mannich reactions, and the mixture
These two authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.04.092

4256
Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
N
N
O
NO2
OH
N
F
O
O
N
H
N
H
Mifepristone
2
1
N
N
F
F
F
N
N
S
O
F
F
O
O
F
N
O
N
H
O
H
Cl
5
3
4
N
N
Cl
HO
O
7
6
Figure 1. The PR antagonist Mifepristone and representative examples of non-steroidal PR antagonists (1–7) previously reported.
O
O
R2
O
N
N
NO2
H
R1
H
R3
Cl
8
9
Figure 2. General structure of aromatic b-amino-ketone 8 and selective AR modulator 9.
of aromatic ketones 10, aldehydes 11 and aromatic amines 12 in an
ethanolic hydrogen chloride solution (Scheme 1)16 were conducted
over periods of 12–24 h at room temperature. The crude products
13–55 collected by filtration and washed with absolute EtOH and
10% NaHCO3 were purified by recrystallization from absolute EtOH
(Table 1).
Compounds 13–55 were obtained as racemic products which
were employed to detect biological activities unless further resolu-
tion was performed. To investigate chiral isomer-induced effect on
biological activities HPLC separation of the enantiomers were ex-
plored for racemates 22 (having best PR antagonist activity
in vivo) and 32 (for determining the absolute configuration of
enantiomers of 22). After a survey of a number of chiral stationary
phases, it was found that the ‘Chiralpak IA’ column cleanly sepa-
rated the enantiomers of 22 and 32. Using a 20 mm id ꢀ 250 mm
length ‘Chiralpak IA’ column, optimization of the mobile phase
afforded conditions (hexane/THF 70/30 at a flow rate of 10 ml/
min) suitable for separation of up to 10 mg racemate per 20-min
injection, and each enantiomer could be separated to a purity of
greater than 98% ee.
As shown in Figure 3 the absolute configuration of (+)-32 was
determined to be (+)-(S)-32 isomer by X-ray crystallography. Com-
paring circular dichroism curves of the enantiomers of compound
22 with that of (+)-(S)-32, we found that isomer (+)-22 was similar
to (+)-32 in CD curves and the absolute configuration of compound
(+)-22 was determined to be S. Isomer (ꢁ)-22 that has the opposite
CD curves with compound (+)-(S)-32 was determined to be R
(shown in Fig. 4).
3. Results and discussion
To investigate the effects of different b-aromatic groups (R2) on
the binding affinities to AR and PR, compounds 13–22 (Table 2)
were designed and prepared, respectively. Compounds 13 and 14
with a 3-thiophenyl group were found to be more potent for AR,
exhibiting a 10- to 33-fold more selectivity over PR. While the
O
R2
O
O
R3
N
a, b
R1
H
R1
NH2
R3
R2
11
13-55
12
10
Scheme 1. Reagents and conditions: (a) concd HCl/EtOH, rt, 12–24 h; (b) 10% NaHCO3.

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4257
Table 1
oxyl group. Similarly, b-3-fluorophenyl substituted analogues 20–
22 produced a more potent PR binding profile over that of AR.
These results suggesting that steric variance of group R2 can mod-
ulate the selectivity for PR and AR.
Preparation of aromatic b-amino-ketone analogues
No.
R1
R2
R3
Yield (%)
13
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
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
4-Me
4-CN
4-Me
4-Me
4-Me
3-Me
3-F
Thiophen-3-yl
Thiophen-3-yl
Ph
4-NO2
4-NO2
4-NO2
4-MeO
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
3-NO2
4-CF3
83
53
61
62
70
67
72
96
89
80
51
83
59
48
86
83
67
51
90
93
68
82
89
59
81
48
74
76
80
59
89
49
62
48
28
36
43
40
48
58
49
36
51
Next, 3-(phenylamino)-3-(3-fluorophenyl)-1-p-tolylpropan-1-
one was chosen as general structure and a number of substituted
aniline analogues (e.g., 23–26, Table 3) were synthesized to study
the structure–activity relationships. Both 4-nitroaniline and 3-
nitroaniline derivatives (22 and 23) displayed higher PR binding
affinity than AR, but replacement of the 4-nitro group (22) with a
4-trifluoromethyl group in 24 resulted in a dramatic decrease in
PR binding (Ki >10,000 nM), substitution by a 4-fluoro atom in 25
produced a similar outcome (Ki = 444 nM), and the introduction
of an additional fluoro atom in compound 26 did not alter the sit-
uation (Ki = 574 nM). So the preliminary optimization experiments
(24, 25, 26) above suggest that nitro aniline analogues (e.g., 22, 23)
are better for PR binding, indicating the importance of this struc-
tural moiety.
To further improve the binding affinity and selectivity for PR
versus AR, several substituted groups were introduced into the ring
of aromatic ketone to produce analogues 27–55 (Table 4), and the
structure–activity relationships (SAR) of b-(p-nitrophenylamino)-
ketones were studied. All the compounds (27–55) showed high
PR binding affinity (Ki <100 nM). The trend observed for the
b-(3-fluorophenyl) analogues (21, 22, 27–35) was that the substi-
tuted group (R1) at the 30-position was preferred to 40-position
(e.g., 21 vs 30, 27 vs 31) for PR binding potency and selectivity
against AR. The data of b-(3-fluorophenyl) analogues 31–33 clearly
demonstrated that a large steric group (R1 = Ph, Cy, morpholino) at
the 40-position was tolerated for PR binding activity and good for
PR selectivity. 3-chlorine analogue 27 (Ki = 23 nM) was the most
potent compound in the b-(3-fluorophenyl) analogues in PR bind-
ing assay. Removing the fluoro atom in b-phenyl group (R2) from 30
(27) to 40-position (36) resulted in threefold increase in the selec-
tivity for PR against AR. The replacement of 4-fluoro atom (36)
with 4-trifluoromethyl moiety (37) in b-phenyl group (R2) further
improved the PR binding selectivity (hAR/hPR >100-fold). The
excellent selectivity could be accounted for the increase of steric
hindrance from 4-trifluoromethyl moiety in 37 compared to 4-flu-
oro group in 36. Most of the b-p-trifluoromethylphenyl analogues
(e.g., 37, 39, 41 and 44) exhibited significant selectivity for PR
when optimizing the R1 group. b-Cyclohexyl (R2) analogues
45–53 displayed high PR binding affinity ranging from 6.7 to
Ph
3,4,5-(MeO)3Ph
3,4,5-(MeO)3Ph
3,4,5-(MeO)3Ph
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-Me
3-F
4-Me
4-Me
4-Me
4-Me
4-Me
3-Cl
3-Br
H
4-F
4-Cl
4-F
3,4-di-F
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-Br
4-Ph
4-Cy
4-Morpholino
3-Cl
3-Cl
3-F
3-Me
3-CN
3-MeO
4-MeO
4-Cl
3-FPh
4-FPh
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
4-Br
4-Me
4-MeO
4-Br
4-Cl
4-F
3-F
3-MeO
3,4-di-Cl
H
4-Ph
4-Cy
two b-phenyl substituted products, compounds 15 and 16, were
less selective, compounds 17, 18 and 19 displayed a inverse trend
when the b-phenyl ring was substituted by a bulky 3,4,5-trimeth-
Table 2
Effects of b-aromatic substituted groups on hPR-B and hARa binding characteristics
O
R2
N
H
R1
R3
Compound
R1
R2
R3
hPR-B Binding
hAR Binding
Selectivity hAR/hPR
Ki (nM) mean SEM
Ki (nM) mean SEM
Progesterone
5.1 0.6
35
7
DHT
13
14
15
16
17
18
19
20
21
22
4.7 0.6
10.4 1.3
2.3 0.2
4-Me
4-CN
4-Me
4-Me
4-Me
3-Me
3-F
3-Me
3-F
4-Me
Thiophen-3-yl
Thiophen-3-yl
Ph
4-NO2
4-NO2
4-NO2
4-MeO
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
103
75
8
8
0.1
0.03
0.3
4
2.1
8.8
26.1
1.8
1.1
1.8
66 12
18
3
Ph
1119b
4523 1021
490 79
1531 312
3178 881
3,4,5-(MeO)3Ph
3,4,5-(MeO)3Ph
3,4,5-(MeO)3Ph
3-FPh
3-FPh
3-FPh
236 23
173 21
121 12
35
46
54
2
4
6
65
51 13
96
1
9
a
Values represent triplicate determinations.
Assayed once.
b

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Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
Figure 3. Single crystal X-ray structures of compound (+)-(S)-32.
15
Table 3
hPR-B and hARa binding characteristics of non-steroidal compounds 22–26 and SAR of
substituted anilines
F
10
O
N
H
R1
R3
R3
0
Compound
R1
hPR-B Binding hAR Binding Selectivity
Ki (nM)
Ki (nM)
hAR/hPR
mean SEM
mean SEM
Progesterone
DHT
22
23
24
5.1 0.6
35
4.7 0.6
96
7
4-Me 4-NO2
4-Me 3-NO2
4-Me 4 -CF3
4-Me 4-F
54
99
6
4
9
1.8
2.0
-10
-13
203 46
NAb
NAb
25
26
444 191
4-Me 3,4-di-F 574 109
322 48
1214 397
0.7
2.1
200
250
300
350
400
Wavelength [nm]
a
Values represent triplicate determinations.
Not active, defined as Ki >10,000 nM.
Figure 4. CD curves of compounds (ꢁ)-22 (green), (+)-22 (purple) and (+)-(S)-32
b
(blue).
0.1 mg/day intraluminally, resulting in net increases of only 3.1%
of the control value. Compound 22 inhibited deciduomata forma-
tion by 83% at a dose of 5.0 mg/day, 65% at a dose of 1.0 mg/day,
45% at a dose of 0.5 mg/day, thereby establishing a dose-dependent
effect that nearly reached the maximum at 5.0 mg/day. Compound
26 is structurally similar molecule but is less efficacious/potent
than compound 22 in cotransfection/binding assay. It blocked
decidualization by 52% at a dose of 5.0 mg/day as well, though it
was ineffective at 1.0 mg/day (data not shown), which indicate
the effectiveness of this type of scaffold.
13 nM. The significant improvement in PR binding affinity of
b-cyclohexyl analogue 45, compared with phenyl analogue 15,
may be explained by the steric effect caused by the change from
b-phenyl group to b-cyclohexyl moiety. The b-cyclohexyl ana-
logues with a large group (R1 = Ph, Cy) substituted at the 40-posi-
tion, such as 54 and 55, also displayed excellent PR selectivity.
The SAR from compounds 27–55 above suggests that steric effect
of group R1 coordinating with group R2 can also modulate the
potency and selectivity for PR and AR.
According to the binding activity results, compounds 22, 26, 27,
30, 41, 42, 44, 46, 47, 48 and 50 were chosen for functional charac-
terization (Table 5). In the hPR-B cotransfection assay with CV-1
cells, none of these compounds showed any PR agonist activity.
Compound 22 appears to be the most efficacious PR antagonist
in the assayed compound with an IC50 of 79 nM. The cross-reactiv-
ity profile of compound 22 was studied with a panel of steroid
receptors. Apart from some moderate AR binding or activation
properties, it did not react with hER, hGR and hMR (data shown
in Table 6).
An in vivo experiment was conducted to verify the PR antago-
nist activity of compound 22 and 26 using a murine uterine decid-
ulization assay.17,18 As shown in Figure 5, control mice showed a
dramatic increase in net uterine wet weight gain in response to
the decidual stimulus. RU486 blocked this response at a dose of
There was no significant difference between the R and corre-
sponding
S enantiomers for PR binding affinities (Table 7).
Compound (ꢁ)-(R)-22 was slightly more active than (+)-(S)-22
enantiomer for binding to both PR and AR.
The docking simulation on PR/mifepristone, PR/(ꢁ)-(R)-22 bind-
ing was carried out using human PR crystal structure (PDB entry
2OVH) as the model. According the docking prediction (Fig. 6A),
the docking model of mifepristone strongly resembled the binding
model of Asoprisnil on the PR ligand-binding domain of 2OVH. The
key features of the binding mode of mifepristone are summarized
as follows: first, hydrogen bonds existed between the A-ring car-
bonyl group of mifepristone and the side-chains of Gln725 and
ARG766 of the receptor. Second, N,N-dimethyl-aminophenyl group
attached to C12 in mifepristone would probably force a displace-
ment of helix 12 and the C-terminal extension, accounting for

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4259
Table 4
hPR-B and hARa binding characteristics of non-steroidal compounds 27–55
O
R2
N
NO2
H
R1
Compound
R1
R2
hPR-B Binding
hAR Binding
Selectivity hAR/hPR
Ki (nM) mean SEM
Ki (nM) mean SEM
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
3-Cl
3-Br
H
4-F
4-Cl
4-Br
4-Ph
4-Cy
4-Morpholino
3-Cl
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
3-FPh
4-FPh
4-CF3Ph
4-CF3Ph
4-CF3Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
4-CF3 Ph
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
Cy
23
36
49
4
1
7
110
124
63 21
30
18
39
417 60
1010 260
283 23
493 66
>10,000
1314 224
>10,000
883 259
>10,000
3
6
4.8
3.4
1.3
0.4
0.4
1.0
8.3
13.3
3.6
68 14
9
1
1
43
39
50
76 23
78 14
58 16
87 15
47 11
85 13
56
31 10
18
80 19
66
13
10.8 1.4
10.2 0.6
6.7 0.8
8.5 0.3
8.5 0.9
11.2 0.3
9.1 1.5
13.0 0.9
5
5
4
8.5
3-Cl
3-F
>100
28.0
>100
15.8
>300
17.8
7.9
>150
8.8
3.4
3.4
6.7
7.5
7.5
36.2
15.3
5.7
>350
>196
3-Me
3-CN
3-MeO
4-MeO
4-Cl
8
4
320 43
636 142
>10,000
4-Br
7
1
4-Me
4-MeO
4-Br
4-Cl
4-F
115 15
37
35 10
45
64 10
64
406 78
140 34
74 10
1
1
3-F
6
3-MeO
3,4-di-Cl
H
4-Ph
4-Cy
Cy
Cy
28
51
2
1
>10,000
>10,000
a
Values represent triplicate determinations.
the protease sensitivity19 and increased accessibility of the PR to a
presumed repressor.20–22 The docking of (ꢁ)-(R)-22 into the
ligand-binding domain of PR together with mifepristone is shown
in Figure 6B. It is interesting to note that the p-nitro group of
(ꢁ)-(R)-22 is networked to Gln725 and Arg766, and the m-fluoro-
phenyl group and the p-methylphenyl group in (ꢁ)-(R)-22 are
superimposed with the D ring and N,N-dimethyl-aminophenyl
group of mifepristone, respectively. All these factors demonstrate
that the isomer (ꢁ)-(R)-22 can mimic the interaction mode of mife-
pristone in the PR ligand-binding pocket.
4. Conclusions
A novel structural class of non-steroidal PR antagonists, aro-
matic b-amino-ketone analogues, were discovered using PR bind-
ing and cotransfection assays. After optimizing the substitution
groups at aromatic ketone and b-aromatic ring the resulted aro-
matic b-amino-ketone template significantly improved selectivity
Table 6
Binding affinities, agonist and antagonist cross-reactivities on hAR, hER, hGR, and
hMRa
Table 5
Compound
Activities of non-steroidal compounds on hPR-B in CV-1 cell cotransfection assaya,d
RU486
22
Compound
Agonist
EC50 (nM) mean SEM
Antagonist
IC50 (nM) mean SEM
Binding affinities Ki (nM) mean SEM
Agonist EC50 (nM) mean SEM
hAR
8.4 1.4
NAb
96
9
hERa
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
NAb
Progesterone
RU486
22
26
27
30
41
42
44
46
47
48
50
15.2 4.8
NAb
NAb
0.6 0.1
79 58
hERb
hGR
hMR
hAR
2876c
0.84 0.11
NAb
NAb
NAb
6064 2368
3538 1048
805c
1831 811
2696 873
1213 688
139 13
1555 197
216 85
193 43
10
2
NAb
hERa
NAb
NAb
hERb
hGR
hMR
hAR
hERa
hERb
NAb
NAb
872 106
NAb
NAb
NAb
Antagonist IC50 (nM) mean SEM
1.0 0.2
>1000
140 19
NAb
NAb
NAb
812b
NAb
NAb
hGR
hMR
0.95 0.26
>1000
NAb
NAb
NAb
a
b
c
a
b
c
Values represent triplicate determinations.
Not active; defined as efficacy < 20%, potency >10,000 nM.
Assayed once.
Values represent triplicate determinations.
Not active; defined as efficacy <20%, potency >10,000 nM.
Assayed once.

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Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
for PR. Compound 22 manifests itself as a potent PR antagonist
both in vitro and in vivo, without observable cross-reactivities with
ER, GR and MR. Preliminary structure–activity relationship analysis
suggests that this scaffold of aromatic b-amino-ketone may serve
as a starting point for development of novel PR antagonists.
2H); 13C NMR (CDCl3): d 197.4, 152.2, 144.8, 142.3, 138.3, 133.9,
129.5 (2C), 128.2 (2C), 127.0, 126.2 (2C), 125.7, 121.4, 111.9 (2C),
50.2, 44.2, 21.7; Anal. (C20H18N2O3S) C, H, N.
5.1.2. 3-(4-Nitrophenylamino)-1-(4-cyanophenyl)-3-(thiophen-
3-yl)propan-1-one (14)
4-Cyanoacetophenone (145 mg, 1 mmol), thiophen-3-carboxal-
5. Experimental section
5.1. Chemistry
dehyde (88
l
L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
L) were subjected to conditions similar to
(3 mL), EtOH/HCl (30
l
those employed in the procedure for 13 to give 14 (199 mg, 53%).
1H NMR (DMSO-d6, 300 MHz): d 8.14 (d, 2H, J = 8.2 Hz), 7.94–
8.03 (m, 4H), 7.45–7.52 (m, 2H), 7.19 (m, 1H), 6.64–6.70 (m, 2H),
5.30 (dd, 1H, J = 8.9, 4.1 Hz), 3.80 (dd, 1H, J = 17.9, 9.1 Hz), 3.56
(dd, 1H, J = 17.9, 4.1 Hz); 13C NMR (CD3COCD3): d 196.5, 153.9,
143.9, 140.4, 137.9, 133.1 (2C), 129.2 (2C), 126.9, 126.8, 126.3
(2C), 122.1, 118.3, 116.6, 112.2 (2C), 49.5, 45.8; HRMS (M+, EI)
calcd for C20H15N3O3S: 377.0834; found, 377.0817. HPLC pur-
ity = 98.4% (system A), 97.3% (system B).
Unless otherwise noted, all materials were obtained from com-
mercial suppliers and used without further purification. All final
compounds were purified to >95% purity, as determined by high-
performance liquid chromatography (HPLC: A, YMC ODS column,
4.6 ꢀ 50 mm, 8 min; flow rate = 2.0 mL/min; gradient = 10–95%
0.1% TFA in CH3CN/90–5% 0.1% TFA in water; B, YMC ODS column,
4.6 ꢀ 50 mm, 8 min; flow rate = 2.0 mL/min; gradient = 10–90%
0.1% TFA in CH3OH/90–10% 0.1% TFA in water). Chiralpak IA,
AD-H columns were produced by Daicel Chemical Industries,
LTD. Column chromatography was carried out on silica gel (200–
300 mesh). All reactions were monitored using thin-layer chroma-
tography (TLC) on silica gel plates. NMR spectra were determined
using a Varian 300 or 400 MHz spectrometer. High-resolution mass
spectra (HRMS) were recorded at an ionizing voltage of 70 eV on a
Finnigan/MAT95 spectrometer. Elemental analyses were per-
formed on a CE 1106 elemental analyzer. Optical rotations were
determined on a Perkin–Elmer 341 polarimeter. Yields were of
purified compounds and were not optimized.
5.1.3. 3-(4-Nitrophenylamino)-3-phenyl-1-p-tolylpropan-1-one
(15)
4-Methylacetophenone (133
1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH (3 mL), EtOH/HCl
(30 L) were subjected to conditions similar to those employed in
lL, 1 mmol), benzaldehyde (101 lL,
l
the procedure for 13 to give 15 (219 mg, 61%). 1H NMR (DMSO-d6,
300 MHz): d 7.93 (d, 2H, J = 8.7 Hz), 7.88 (d, 2H, J = 8.2 Hz), 7.45 (d,
2H, J = 7.3 Hz), 7.31–7.36 (m, 4H), 7.24 (t, 1H, J = 7.3 Hz), 6.61 (d,
2H, J = 9.1 Hz), 5.16 (dd, 1H, J = 9.0, 4.0 Hz), 3.71 (dd, 1H, J = 16.6,
9.1 Hz), 3.37 (dd, 1H, J = 17.6, 4.2 Hz), 2.37 (s, 3H); 13C NMR
(CDCl3): d 197.5, 152.2, 144.8, 141.2, 138.3, 133.8, 129.5 (2C),
129.1 (2C), 128.3 (2C), 127.8, 126.1 (2C), 126.1 (2C), 112.1 (2C),
54.3, 45.4, 21.7; Anal. (C22H20N2O3) C, H, N.
5.1.1. 3-(4-Nitrophenylamino)-3-(thiophen-3-yl)-1-p-tolylpropan-
1-one (13)
4-Nitroaniline (138 mg, 1 mmol) was dissolved or suspended in
a absolute EtOH (3 mL), then 4-methylacetophenone (133
1 mmol) and thiophen-3-carboxaldehyde (88 L, 1 mmol) was
added, followed by saturation ethanol solution of HCl (30 L) un-
der stirring at room temperature for about 8–24 h. The product
was collected by filtration and washed with absolute EtOH and
10% NaHCO3, respectively. The pure product compound 13
(303 mg, 83%) was obtained by recrystallization from absolute
EtOH. 1H NMR (DMSO-d6, 300 MHz): d 7.99–8.04 (m, 2H), 7.80
(d, 2H, J = 8.5 Hz), 7.29 (m, 1H), 7.23 (s, 1H), 7.18 (m, 1H), 7.06
(m, 1H), 6.53–6.60 (m, 2H), 5.25 (t, 1H, J = 5.8 Hz), 3.45–3.61 (m,
lL,
5.1.4. 3-(4-Methoxyphenylamino)-3-phenyl-1-p-tolylpropan-1-
one (16)
l
l
p-Anisidine (3 mmol, 369 mg), 4-methylacetophenone (3 mmol,
399
(60
l
L), benzaldehyde (3 mmol, 310
lL) EtOH (8 mL), EtOH/HCl
l
L) were subjected to conditions similar to those employed
in the procedure for 13 to give 16 (641 mg, 62%). 1H NMR (CDCl3,
400 MHz): d 7.70–7.72 (m, 2H), 7.43–7.46 (m, 2H), 7.31–7.37 (m,
4H), 7.23 (m, 1H), 6.66–6.69 (m, 2H), 6.51–6.54 (m, 2H), 4.92
(dd, 1H, J = 7.9, 5.2 Hz), 3.68 (s, 3H), 3.47 (dd, 1H, J = 16.2,
5.0 Hz), 3.38 (dd, 1H, J = 16.2, 7.8 Hz), 2.39 (s, 3H); 13C NMR (CDCl3,
100 MHz): d 198.6, 152.2, 143.3, 141.2, 138.5, 136.7, 134.1, 128.8
(2C), 128.7, 128.5, 127.2, 126.4 (2C), 125.4, 115.3 (2C), 114.6
(2C), 55.6 (2C), 46.5, 21.3; HRMS (M+, EI) calcd for C23H23NO2:
345.1729; found: 345.1729; HPLC purity = 99.2% (system A),
99.1% (system B).
120
90
5.1.5. 3-(4-Nitrophenylamino)-3-(3,4,5-trimethoxyphenyl)-1-p-
tolylpropan-1-one (17)
**
60
4-Methylacetophenone (133
benzaldehyde (196 mg, 1 mmol), 4-nitro-benzenamine (138 mg,
1 mmol), EtOH (2 mL), EtOH/HCl (30 L) were subjected to condi-
lL, 1 mmol), 3,4,5-trimethoxy-
***
***
l
tions similar to those employed in the procedure for 13 to give
17 (315 mg, 70%). 1H NMR (DMSO-d6, 300 MHz): 7.95–7.98 (d,
2H, J = 9.1 Hz), 7.88–7.91 (d, 2H, J = 7.8 Hz), 7.33–7.36 (d, 2H,
J = 8.2 Hz), 6.80 (s, 2H), 6.65–6.68 (d, 2H, J = 9.2 Hz), 5.10 (dd, 1H,
J = 9.0, 3.3 Hz), 3.76 (s, 6H), 3.70 (dd, 1H, J = 17.7, 9.4 Hz), 3.62 (s,
3H), 3.35 (dd, 1H, J = 17.5, 3.8 Hz), 2.38 (s, 3H); Anal.
(C25H26N2O6) C, H, N.
30
0
++
***
***
Control
22
5 mg
22
1 mg 0.5 mg
(6)
(6)
22
22
0.1 mg
26
5 mg
RU486
0.1 mg
Sham
(8)
(6)
(6)
(6)
(6)
(6)
Figure 5. Effect of RU486 (0.1 mg, intrauterine), 22 (0.1 mg, 0.5 mg, 1 mg and 5 mg
intrauterine), and 26 (5 mg, intrauterine) on sesame oil-induced uterine decidual-
5.1.6. 3-(4-Nitrophenylamino)-3-(3,4,5-trimethoxyphenyl)-1-m-
tolylpropan-1-one (18)
**
***
P
ization (single dose, day 4 of pseudopregnancy) in BALB/c mice; P <0.01;
<0.001 versus control, ++P <0.01 versus 26 (5 mg, intrauterine). Numbers of mice in
3-Methylacetophenone (133
lL, 1 mmol), 3,4,5-trimethoxy-
each group are given in parentheses.
benzaldehyde (196 mg, 1 mmol), 4-nitro-benzenamine (138 mg,

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4261
Table 7
113.6 (d, Jc–f = 21.8 Hz), 111.8 (2C), 52.0, 45.8; Anal. (C21H16F2N2O3)
C, H, N.
Binding affinities of R and S enantiomers for hPR-B and hARa
O
R2
5.1.10. 3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-p-tolylpro-
pan-1-one (22)
N
NO2
H
R1
4-Methylacetophenone (2.67 mL, 20 mmol), 3-fluorobenzalde-
hyde (2.12 mL, 20 mmol), 4-nitroaniline (2.76 g, 20 mmol), EtOH
(60 mL), EtOH/HCl (300 lL) were subjected to conditions similar
Compound
R1
R2
hPR-B Binding
Ki (nM)
mean SEM
hAR Binding
Ki (nM)
mean SEM
Selectivity
hAR/hPR
to those employed in the procedure for 13 to give 22 (6.08 g,
80%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.96 (d,
2H, J = 9.1 Hz), 7.89 (d, 2H, J = 8.2 Hz), 7.29–7.43 (m, 5H), 7.04–
7.11 (m, 1H), 6.63–6.68 (m, 2H), 5.21 (dd, 1H, J = 9.1, 4.2 Hz),
3.73 (dd, 1H, J = 17.5, 9.1 Hz), 3.41 (dd, 1H, J = 17.8,4.4 Hz), 2.38
(ꢁ)-(R)-22
(+)-(S)-22
4-Me
4-Me
3-FPh
3-FPh
35
4
24.2 0.8
342 36
0.69
4.1
84 15
a
Values represent triplicate determinations.
(s, 3H); 13C NMR (DMSO-d6, 100 MHz): d 196.0, 162.4 (d, Jc–f
=
1 mmol), EtOH (2 mL), EtOH/HCl (30
l
L) were subjected to condi-
242.7 Hz), 153.5, 145.8 (d, Jc–f = 6.9 Hz), 143.9, 136.3, 134.1, 130.7
(d, Jc–f = 7.8 Hz), 129.4 (2C), 128.3 (2C), 126.2 (2C), 122.8, 114.1
(d, Jc–f = 21 Hz), 113.5 (d, Jc–f = 21.9 Hz), 111.7 (2C), 51.9, 45.5,
21.3; Anal. (C22H19FN2O3) C, H, N.
tions similar to those employed in the procedure for 13 to give
18 (301 mg, 67%). 1H NMR (DMSO-d6, 300 MHz): 7.96–7.99 (d,
2H, J = 9.3 Hz), 7.78–7.80 (d, 2H, J = 7.1 Hz), 7.39–7.49 (m, 2H),
6.79 (s, 2H), 6.65–6.68 (d, 2H, J = 9.5 Hz), 5.10 (dd, 1H, J = 9.3,
3.8 Hz), 3.80 (s, 6H), 3.79 (s, 3H), 3.69 (d, 1H, J = 9.4 Hz), 3.38 (dd,
1H, J = 17.6, 3.8 Hz), 2.38 (s, 3H); Anal. (C25H26N2O6) C, H, N.
5.1.11. 3-(3-Nitrophenylamino)-3-(3-fluorophenyl)-1-p-tolylpro-
pan-1-one (23)
4-Methylacetophenone (133
hyde (106 L, 1 mmol), 3-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), 3-fluorobenzalde-
5.1.7. 3-(4-Nitrophenylamino)-1-(3-fluorophenyl)-3-(3,4,5-trime-
thoxyphenyl)propan-1-one (19)
l
l
3-Fluoroacetophenone (123
benzaldehyde (196 mg, 1 mmol), 4-nitro-benzenamine (138 mg,
1 mmol), EtOH (2 mL), EtOH/HCl (30 L) were subjected to condi-
tions similar to those employed in the procedure for 13 to give
19 (327 mg, 72%). 1H NMR (DMSO-d6, 300 MHz): 7.96–7.99 (m,
2H), 7.86 (d, 1H, J = 6.9 Hz), 7.77 (m, 1H), 7.49–7.64 (m, 2H), 6.81
(s, 2H), 6.66–6.69 (d, 2H, J = 9.1 Hz), 5.11 (dd, 1H, J = 9.2, 3.6 Hz),
3.77 (s, 6H), 3.75 (dd, 1H, J = 17.7, 9.6 Hz), 3.63 (s, 3H), 3.45 (d,
1H, J = 3.5 Hz); Anal. (C24H23FN2O6) C, H, N.
l
L, 1 mmol), 3,4,5-trimethoxy-
those employed in the procedure for 13 to give 23 (195 mg, 51%)
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 8.27 (d, 1H,
J = 7.7 Hz), 8.21 (s, 1H), 8.02 (d, 1H, J = 8.1 Hz), 7.92–7.96 (m 2H),
7.79 (t, 1H, J = 8.0 Hz), 7.35 (d, 2H, J = 7.5 Hz), 7.15 (d, 2H,
J = 7.5 Hz), 6.59–6.62 (t, 2H, J = 8.4 Hz), 5.13 (dd, 1H, J = 9.0,
4.0 Hz), 3.80 (dd, 1H, J = 18.0, 9.2 Hz), 3.51 (d, 1H, J = 3.6 Hz), 2.26
l
(s, 3H); 13C NMR (CDCl3, 100 MHz): d 197.2, 163.2 (d, Jc–f
=
245.4 Hz), 149.1, 147.6, 144.9, 144.6 (d, Jc–f = 6.3 Hz), 133.8, 130.6
(d, Jc–f = 8.2 Hz), 129.7, 129.5 (2C), 128.3 (2C), 121.9, 119.5, 114.6
(d, Jc–f = 21.4 Hz), 113.1 (d, Jc–f = 21.9 Hz), 112.6, 107.8, 54.3, 45.7,
21.7; Anal. (C22H19FN2O3) C, H, N.
5.1.8. 3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-m-tolylpro-
pan-1-one (20)
3-Methylacetophenone (340
hyde (265 L, 2.5 mmol), 4-nitroaniline (345 mg, 2.5 mmol), EtOH
(8 mL), EtOH/HCl (60 L) were subjected to conditions similar to
those employed in the procedure for 13 to give 20 (907 mg, 96%).
1H NMR (DMSO-d6, 300 MHz): d 7.93–7.97 (d, 2H, J = 9.2 Hz),
7.74–7.80 (m, 2H), 7.36–7.48 (m, 3H), 7.29–7.33 (t, 2H,
J = 9.3 Hz), 7.05–7.11 (q, 1H, J = 7.8, 1.9 Hz), 6.62–6.65 (d, 2H,
J = 9.4 Hz), 5.20 (dd, 1H, J = 8.9, 3.9 Hz), 3.75 (dd, 1H, J = 18.0,
9.1 Hz), 3.42 (dd, 1H, J = 17.6, 4.0 Hz), 2.37 (s, 3H); 13C NMR
(DMSO-d6, 100 MHz): d 196.6, 162.4 (d, Jc–f = 241.8 Hz), 153.5,
l
L, 2.5 mmol), 3-fluorobenzalde-
5.1.12. 3-(4-(Trifluoromethyl)phenylamino)-3-(3-fluorophenyl)-
1-p-tolylpropan-1-one (24)
l
l
4-Methylacetophenone (333
hyde (265 L, 2.5 mmol), 4-(trifluoro-methyl)-benzenamine (308
L, 2.5 mmol), EtOH (6 mL), EtOH/HCl (60 L) were subjected to
lL, 2.5 mmol), 3-fluorobenzalde-
l
l
l
conditions similar to those employed in the procedure for 13 to
give 24 (831 mg, 83%) as a white solid. 1H NMR (DMSO-d6,
300 MHz): d 7.88–7.91 (d, 2H, J = 8.1 Hz), 7.29–7.41 (m, 7H), 7.05
(m, 1H), 6.63–6.66 (d, 2H, J = 8.6 Hz), 5.10 (dd, 1H, J = 8.9, 4.1 Hz),
3.67 (dd, 1H, J = 17.4, 8.9 Hz), 3.34 (dd, 1H, J = 17.1, 4.3 Hz), 2.39
145.8 (d, Jc–f = 6.4 Hz), 138.3, 136.6, 136.4, 134.1, 130.7 (d, Jc–f
8.1 Hz), 128.7, 128.6, 126.1 (2C), 125.3, 122.8, 114.1 (d, Jc–f
=
=
(3H, s); 13C NMR (CDCl3, 100 MHz): d 196.9, 162.8 (d, Jc–f
245 Hz), 148.9, 144.7 (d, Jc–f = 6.4 Hz), 144.5, 133.5, 130.2 (d, Jc–f
=
=
20.9 Hz), 113.5 (d, Jc–f = 21.8 Hz), 111.7 (2C), 51.9, 45.7, 20.9; Anal.
(C22H19FN2O3) C, H, N.
8.2 Hz), 129.2 (2C), 127.9 (2C), 126.1 (2C), 125.8, 121.5, 119.1 (q,
Jc–f = 32.4 Hz), 114.2 (d, Jc–f = 21.4 Hz), 112.8 (d, Jc–f = 21.9 Hz),
112.6 (2C), 53.7, 45.3, 21.4; HRMS (M+, EI) calcd for C23H19F4NO:
401.1403; found: 401.1405; HPLC purity = 100% (system A),
98.4% (system B).
5.1.9. 3-(4-Nitrophenylamino)-1,3-bis(3-fluorophenyl)propan-
1-one (21)
3-Fluoroacetophenone (308
hyde (266 L, 2.5 mmol), 4-nitroaniline (345 mg, 2.5 mmol), EtOH
(8 mL), EtOH/HCl (60 L) were subjected to conditions similar to
lL, 2.5 mmol), 3-fluorobenzalde-
l
5.1.13. 3-(4-Fluorophenylamino)-3-(3-fluorophenyl)-1-p-tolyl-
propan-1-one (25)
l
those employed in the procedure for 13 to give 21 (845 mg, 89%)
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.95 (d, 2H,
J = 9.0 Hz), 7.84 (d, 1H, J = 7.6 Hz), 7.76 (d, 1H, J = 9.2 Hz), 7.48–
7.63 (m, 2H), 7.29–7.44 (m, 3H), 7.05–7.11 (m, 1H), 6.64 (d, 2H,
J = 8.9 Hz), 5.20 (dd, 1H, J = 9.6, 4.9 Hz), 3.77 (dd, 1H, J = 18.4,
9.4 Hz), 3.42 (m, 1H); 13C NMR (DMSO-d6, 100 MHz): d 195.7,
162.5 (d, Jc–f = 242.3 Hz), 162.3 (d, Jc–f = 244.1 Hz), 153.6, 145.6
4-Methylacetophenone (133
hyde (106 L, 1 mmol), 4-fluoroaniline (96
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
l
L, 1 mmol), 3-fluorobenzalde-
l
l
L, 1 mmol), EtOH
l
those employed in the procedure for 13 to give 25 (173 mg, 59%)
as a white solid. 1H NMR (DMSO-d6, 300 MHz): d 7.86–7.88 (d,
2H, J = 8.1 Hz), 7.26–7.38 (m, 5H), 7.00 (m, 1H), 6.84 (t, 2H,
J = 8.7 Hz), 6.48–6.52 (m, 2H), 4.97 (dd, 1H, J = 8.6, 4.7 Hz), 3.58
(dd, 1H, J = 17.0, 8.8 Hz), 3.26 (dd, 1H, J = 17.2, 4.8 Hz), 2.38 (s,
(d, Jc–f = 5.9 Hz), 138.8 (d, Jc–f = 6.1 Hz), 136.5, 131.1 (d, Jc–f
=
7.3 Hz), 130.8 (d, Jc–f = 7.7 Hz), 126.2 (2C), 124.5, 122.9, 120.4 (d,
Jc–f = 21.4 Hz), 114.7 (d, Jc–f = 24.1 Hz), 114.2, (d, Jc–f = 20.5 Hz),
3H); 13C NMR (DMSO-d6, 100 MHz): d 196.8, 162.4 (d, Jc–f
=
241.8 Hz), 154.5 (d, Jc–f = 230 Hz), 147.2 (d, Jc–f = 6.4 Hz), 144.3,

4262
Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
Figure 6. A The docking model of mifepristone (cyan) on the progesterone receptor ligand-binding domain of 2OVH bound with asoprisnil (yellow). Hydrogen bonding
interactions are shown with dotted red lines. B Superimposition of the docking models of mifepristone (cyan) and (ꢁ)-(R)-22 (pink) on the progesterone receptor ligand-
binding domain of 2OVH. Hydrogen bonding interactions are shown with dotted yellow lines. These images were generated using the PyMol program (http://
www.pymol.org/).
143.8, 134.3, 130.3 (d, Jc–f = 8.2 Hz), 129.4 (2C), 128.3 (2C), 123.0,
115.3 (2C, d, Jc–f = 21.9 Hz), 113.8 (2C, d, Jc–f = 6.9 Hz), 113.6,
113.5, 53.1, 46.1, 21.2; Anal. (C22H19F2NO) C, H, N.
245.4 Hz), 150.6 (dd, Jc–f = 230 Hz), 145.3 (d, Jc–f = 6.4 Hz), 144.7,
143.9 (d, J = Hz), 143.3 (dd, Jc–f = 223.1 Hz), 133.9, 130.5 (d, Jc–f
7.8 Hz), 129.5 (2C), 128.3 (2C), 121.9, 117.3 (d, Jc–f = 16.9 Hz), 114.5
(d, Jc–f = 20.9 Hz), 113.2 (d, Jc–f = 21.9 Hz), 109.0, 102.6 (d, Jc–f
=
=
5.1.14. 3-(3,4-Difluorophenylamino)-3-(3-fluorophenyl)-1-p-
tolylpropan-1-one (26)
20.9 Hz), 54.9, 45.8, 21.7; HRMS (M+, EI) calcd for C22H18F3NO:
369.1341; found: 369.1345; HPLC purity = 100% (system A), 98.9%
(system B).
4-Methylacetophenone (133
hyde (106 L, 1 mmol), 3,4-difluoroaniline (99
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), 3-fluorobenzalde-
l
lL, 1 mmol), EtOH
l
5.1.15. 3-(4-Nitrophenylamino)-1-(3-chlorophenyl)-3-(3-fluoro-
phenyl)propan-1-one (27)
those employed in the procedure for 13 to give 26 (177.6 mg,
48%) as a pale white solid. 1H NMR (DMSO-d6, 300 MHz): d 7.85–
7.88 (d, 2H, J = 8.2 Hz), 7.27–7.39 (m, 5H), 6.99–7.08 (m, 2H),
6.46 (m, 1H), 6.30 (t, 1H, J = 5.3 Hz), 4.97 (dd, 1H, J = 8.8, 4.3 Hz),
3.59 (dd, 1H, J = 16.9, 8.7 Hz), 3.28 (dd, 1H, J = 16.9, 4.4 Hz), 2.37
3-Chloroacetophenone (325
hyde (266 L, 2.5 mmol), 4-nitroaniline (345 mg, 2.5 mmol), EtOH
(8 mL), EtOH/HCl (60 L) were subjected to conditions similar to
lL, 2.5 mmol), 3-fluorobenzalde-
l
l
those employed in the procedure for 13 to give 27 (859 mg, 86%)
(s, 3H); 13C NMR (CDCl3, 100 MHz): d 197.3, 162.2 (d, Jc–f
=
as a yellow solid. 1H NMR (CD3COCD3, 400 MHz): d 7.97–8.01 (m,

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4263
4H), 7.68 (m, 1H), 7.57 (m, 1H), 7.33–7.43 (m, 3H), 7.04 (m, 1H),
6.72–6.74 (m, 2H), 5.36 (dd, 1H, J = 8.7, 4.2 Hz), 3.88 (dd, 1H,
J = 18.0, 9.0 Hz), 3.59 (dd, 1H, J = 17.9, 4.2 Hz); 13C NMR
(CD3COCD3, 100 MHz): d 195.6, 163.5 (d, Jc–f = 243.2 Hz), 153.8,
146.2 (d, Jc–f = 6.4 Hz), 139.1, 138.0, 134.8, 133.5, 131.0 (3C),
4H), 7.76 (d, NH), 7.59–7.62 (t, 2H, J = 8.5 Hz), 7.40 (m, 1H), 7.29–
7.33 (m, 2H), 7.05–7.11 (m, 1H), 6.62–6.65 (d, 2H, J = 9.1 Hz),
5.20 (dd, 1H, J = 8.7, 4.0 Hz), 3.75 (dd, 1H, J = 17.8, 9.0 Hz), 3.45
(dd, 1H, J = 17.8, 3.9 Hz); 13C NMR (DMSO-d6, 100 MHz): d 195.6,
162.5 (d, Jc–f = 242.3 Hz), 153.5 (d, Jc–f = 9.1 Hz), 145.7 (d, Jc–f
=
128.3 (d, Jc–f = 13.6 Hz), 127.1, 126.3, 123.2, 114.5 (d, Jc–f
=
6.4 Hz), 138.5, 136.3, 135.2, 130.7 (d, Jc–f = 8.2 Hz), 130.2 (2C),
20.9 Hz), 114.0 (d, Jc–f = 21.8 Hz), 112.3 (2C), 53.0, 46.4; Anal.
128.9 (2C), 126.2, 122.8, 114.2 (d, Jc–f = 20.5 Hz), 113.5 (d, Jc–f =
(C21H16ClFN2O3) C, H, N.
21.9 Hz), 111.6 (2C), 51.9, 45.7; Anal. (C21H16ClFN2O3) C, H, N.
5.1.16. 3-(4-Nitrophenylamino)-1-(3-bromophenyl)-3-(3-fluoro-
phenyl)propan-1-one (28)
5.1.20. 3-(4-Nitrophenylamino)-1-(4-bromophenyl)-3-(3-fluoro-
phenyl)propan-1-one (32)
3-Methylacetophenone (134
hyde (106 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
l
L, 1 mmol), 3-fluorobenzalde-
3-bromoacetophenone (498 mg, 2.5 mmol), 3-fluorobenzalde-
l
hyde (266
lL, 2.5 mmol), 4-nitroaniline (345 mg, 2.5 mmol), EtOH
l
(8 mL), EtOH/HCl (60
lL) were subjected to conditions similar to
those employed in the procedure for 13 to give 28 (363 mg, 83%)
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 8.13 (s, 1H),
7.95–8.00 (m, 3H), 7.86 (m, 1H), 7.51 (t, 1H, J = 8.0 Hz), 7.39 (q,
1H, J = 8.1, 6.1 Hz), 7.30–7.33 (d, 2H, J = 7.9 Hz), 7.08 (m, 1H),
6.62–6.65 (d, 2H, J = 9.3 Hz), 5.19 (dd, 1H, J = 9.4, 4.0 Hz), 3.78
(dd, 1H, J = 18.3, 9.8 Hz), 3.46 (dd, 1H, J = 18.0, 3.9 Hz); 13C NMR
(DMSO-d6, 100 MHz) d 195.5, 162.4 (d, Jc–f = 242.3 Hz), 153.4,
145.6 (d, Jc–f = 6.3 Hz), 138.5, 136.3, 136.0, 131.0 (2C), 130.6 (d,
those employed in the procedure for 13 to give 32 (1.03 g, 93%)
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.92–7.98 (t,
4H, J = 9.1 Hz), 7.74–7.77 (d, 2H, J = 8.5 Hz), 7.39 (m, 1H), 7.29–
7.33 (t, 2H, J = 9.4 Hz), 7.09 (t, 1H, J = 8.6 Hz), 6.61–6.66 (d, 2H,
J = 9.2 Hz), 5.19 (dd, 1H, J = 9.1, 4.4 Hz), 3.75 (dd, 1H, J = 18.1,
9.3 Hz), 3.43 (dd, 1H, J = 17.2, 3.6 Hz); 13C NMR (CD3COCD3,
100 MHz): d 195.9, 163.5 (d, Jc–f = 242.7 Hz), 153.8, 146.2 (d, Jc–f
=
6.3 Hz), 137.9, 136.2, 132.3 (2C), 131.1131.0, 130.5, 128.2126.3,
123.1 (2C), 114.5 (d, Jc–f = 21.4 Hz), 114.0 (d, Jc–f = 22.3 Hz), 112.3
(2C), 53.0, 46.3; Anal. (C21H16BrFN2O3) C, H, N.
Jc–f = 8.6 Hz), 127.1, 126.1 (2C), 122.7, 122.2, 114.1 (d, Jc–f
=
21.4 Hz), 113.5 (d, Jc–f = 21.4 Hz), 111.6 (2C), 51.7, 45.7; Anal.
(C21H16BrFN2O3) C, H, N.
5.1.21. 3-(4-Nitrophenylamino)-1-(4-phenyl-phenyl)-3-(3-fluoro-
phenyl)propan-1-one (33)
5.1.17. 3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-phenylpro-
pan-1-one (29)
4-acetylbiphenyl (134
(106 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH (3 mL),
EtOH/HCl (30 L) were subjected to conditions similar to those
employed in the procedure for 13 to give 33 (298 mg, 68%) as a yel-
low soild. 1H NMR (DMSO-d6, 300 MHz):
8.08 (d, 2H,
lL, 1 mmol), 3-fluorobenzaldehyde
Acetophenone (117
1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH (3 mL), EtOH/HCl
(30 L) were subjected to conditions similar to those employed in
lL, 1 mmol), 3-fluorobenzaldehyde (106
lL,
l
l
l
the procedure for 13 to give 29 (245 mg, 67%) as a yellow soild. 1H
NMR (DMSO-d6, 300 MHz): d 7.95–8.01 (q, 4H, J = 9.3, 6.9 Hz), 7.67
(m, 1H), 7.53–7.57 (t, 2H, J = 8.0 Hz), 7.31–7.44 (m, 3H), 7.09 (m,
1H), 6.64–6.67 (d, 2H, J = 9.5 Hz), 5.23 (dd, 1H, J = 9.4, 4.1 Hz),
3.77 (dd, 1H, J = 18.0, 9.5 Hz), 3.48 (d, 1H, J = 4.1 Hz); 13C NMR
(DMSO-d6, 100 MHz) d 196.6, 162.4 (d, Jc–f = 242.3 Hz)), 153.5,
145.8 (d, Jc–f = 6.3 Hz), 136.5, 136.4, 133.5, 130.6 (d, Jc–f = 7.8 Hz),
128.8 (2C), 128.2 (2C), 126.2 (2C), 122.8, 114.1 (d, Jc–f = 20.9 Hz),
113.5 (d, Jc–f = 21.4 Hz), 111.7 (2C), 51.9, 45.6; HRMS (M+, EI) calcd
for C21H17N2FO3: 364.1223 (M+); found: 364.1208; HPLC purity =
100% (system A), 95.2% (system B).
d
J = 8.4 Hz),7.97 (d, 2H, J = 9.2 Hz),7.85(d, 2H, J = 8.4 Hz), 7.76 (d,
2H, J = 7.7 Hz), 7.31–7.54 (m, 6H), 7.08 (t, 1H, J = 9.0 Hz), 6.66 (d,
2H, J = 9.0 Hz), 5.25 (dd, 1H, J = 9.0, 3.9 Hz), 3.80 (dd, 1H, J = 17.5,
9.0 Hz), 3.50 (d, 1H, J = 3.9 Hz); 13C NMR (CDCl3, 100 MHz): d
197.0, 163.1 (d, Jc–f = 245.9 Hz), 151.9, 146.5, 144.0 (d, Jc–f
=
6.4 Hz), 139.4, 138.5, 134.8, 130.7 (d, Jc–f = 7.8 Hz), 129.0
(2C),128.8 (2C), 128.5, 127.4 (2C), 127.3 (2C), 126.2 (2C), 121.8,
114.8 (d, Jc–f = 21 Hz), 113.0 (d, Jc–f = 21.9 Hz), 112.2 (2C), 53.8,
45.3; Anal. (C27H21FN2O3) C, H, N.
5.1.22. 3-(4-Nitrophenylamino)-1-(4-cyclohexylphenyl)-3-(3-
fluorophenyl)propan-1-one (34)
5.1.18. 3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-(4-fluoro-
phenyl)propan-1-one (30)
4-Cyclohexylacetophenone (202 mg, 1 mmol), 3-fluorobenzal-
4-Flluoroacetophenone (63
hyde (55 L, 0.5 mmol), 4-nitroaniline (69 mg, 0.5 mmol), EtOH
(1.5 mL), EtOH/HCl (20 L) were subjected to conditions similar
to those employed in the procedure for 13 to give 30 (97 mg,
51%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 8.05–8.10
(m, 2H), 7.96 (d, 2H, J = 9.5 Hz), 7.28–7.41 (m, 5H), 7.04–7.11 (m,
1H), 6.64 (d, 2H, J = 9.1 Hz), 5.20 (dd, 1H, J = 9.0, 3.9 Hz), 3.75 (dd,
1H, J = 17.8, 9.3 Hz), 3.41 (d, 1H, J = 4.1 Hz); 13C NMR (CDCl3,
l
L, 0.5 mmol), 3-fluorobenzalde-
dehyde (106
(3 mL), EtOH/HCl (30
l
L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
l
lL) were subjected to conditions similar to
l
those employed in the procedure for 13 to give 34 (367 mg, 82%)
as a yellow soild. 1H NMR (DMSO-d6, 300 MHz): d 7.94–7.97 (d,
2H, J = 9.3 Hz), 7.90–7.93 (d, 2H, J = 8.2 Hz), 7.29–7.41 (m, 5H),
7.07 (m, 1H), 6.62–6.65 (d, 2H, J = 9.6 Hz), 5.21 (dd, 1H, J = 8.8,
3.8 Hz), 3.73 (dd, 1H, J = 17.8, 9.2 Hz), 3.37 (d, 1H, J = 4.3 Hz),
1.69–1.80 (m, 5H), 1.25–1.44 (m, 6H); 13C NMR (CDCl3, 100 MHz)
100 MHz):
245.6 Hz), 151.8, 143.7 (d, Jc–f = 6.4 Hz), 138.6, 132.6, 130.9,
130.8, 130.7, 126.2 (2C), 121.8, 116.1, 115.9, 114.9 (d, Jc–f
20.9 Hz), 113.1 (d, Jc–f = 21.9 Hz), 112.1 (2C), 53.7, 45.1; Anal.
(C21H16F2N2O3) C, H, N.
d
195.8, 166.1 (d, Jc–f = 255.1 Hz), 163.2 (d, Jc–f
=
d 197.1, 163.1 (d, Jc–f = 245.4 Hz), 154.8, 151.9, 144.0 (d,
Jc–f = 6.4 Hz), 138.5, 134.0, 130.7 (d, Jc–f = 8.2 Hz), 128.4 (2C),
127.3 (2C), 126.1 (2C), 121.8, 114.8 (d, Jc–f = 20.9 Hz), 113.1
(d, Jc–f = 21.8 Hz), 112.2 (2C), 53.9, 45.1, 44.7, 33.9 (2C), 26.6 (2C),
25.9; Anal. (C27H27FN2O3) C, H, N.
=
5.1.19. 3-(4-Nitrophenylamino)-1-(4-chlorophenyl)-3-(3-fluoro-
phenyl)propan-1-one (31)
5.1.23. 3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-(4-morpho-
linophenyl)propan-1-one (35)
4-Chloroacetophenone (325
hyde (266 L, 2.5 mmol), 4-nitroaniline (345 mg, 2.5 mmol), EtOH
(8 mL), EtOH/HCl (60 L) were subjected to conditions similar to
l
L, 2.5 mmol), 3-fluorobenzalde-
4-morpholinoacetophenone (205 mg, 1 mmol), 3-fluorobenzal-
l
dehyde (106
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
l
(3 mL), EtOH/HCl (30
lL) were subjected to conditions similar to
those employed in the procedure for 13 to give 31 (891 mg, 90%)
those employed in the procedure for 13 to give 35 (400 mg, 89%)
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.94–8.02 (m,
as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.94–7.97 (d,

4264
Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
2H, J = 9.2 Hz), 7.85–7.88 (d, 2H, J = 8.9 Hz), 7.28–7.43 (m, 3H), 7.07
(m, 1H), 6.98–7.01 (d, 2H, J = 8.7 Hz), 6.63–6.66 (d, 2H, J = 9.0 Hz),
5.20 (dd, 1H, J = 8.5, 4.1 Hz), 3.72–3.75 (t, 4H, J = 4.4 Hz), 3.65
(dd, 1H, J = 17.2, 8.8 Hz), 3.34 (d, 1H, J = 4.4 Hz), 3.27–3.32 (t, 4H,
74%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.95 (d, 2H,
J = 9.3 Hz), 7.65–7.80 (m, 6H), 7.39–7.50 (m, 2H), 6.59–6.65 (m,
2H), 5.28 (dd, 1H, J = 9.1, 3.8 Hz), 3.78 (dd, 1H, J = 9.0, 17.9 Hz),
3.39–3.48 (dd, 1H, J = 17.9, 3.9 Hz), 2.32 (s, 3H); 13C NMR
(CD3COCD3, 100 MHz): d 196.6, 153.7, 148.1, 138.9, 138.0, 137.3,
134.5, 129.4 (q, Jc–f = 32 Hz), 129.1, 129.0, 128.1 (2C), 126.3 (2C),
126.1, 126.0, 125.8, 123.6, 112.3 (2C), 53.1, 46.3, 20.8; HRMS
(M+, EI) calcd for C23H19F3N2O3: 428.1348; found: 428.1345. HPLC
purity = 99.4% (system A), 99.5% (system B).
J = 4.8 Hz); 13C NMR (DMSO-d6, 100 MHz):
d 194.2, 162.4
(d, Jc–f = 242.3 Hz), 154.3, 153.5, 146.0 (d, Jc–f = 6.3 Hz), 136.3,
130.7 (d, Jc–f = 7.7 Hz), 130.1 (2C), 126.6, 126.2, 122.8, 114.1
(d, Jc–f = 21 Hz), 113.5 (d, Jc–f = 21.9 Hz), 113.1(2C), 111.7 (2C),
65.9 (2C), 52.2, 46.9 (2C), 44.9; Anal. (C25H24FN3O4) C, H, N.
5.1.24. 3-(4-Nitrophenylamino)-1-(3-chlorophenyl)-3-(4-fluoro-
phenyl)propan-1-one (36)
5.1.28. 3-(4-Nitrophenylamino)-1-(3-cyanophenyl)-3-(4-(trifluo-
romethyl)phenyl)propan-1-one (40)
3-chloroacetophenone (130
(108 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH (3 mL),
EtOH/HCl (30 L) were subjected to conditions similar to those
l
L, 1 mmol), 4-fluorobenzaldehyde
3-cyanoacetophenone (145 mg, 1 mmol), 4-trifluoromethyl-
l
benzaldehyde (137
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
l
EtOH (3 mL), EtOH/HCl (30
lL) were subjected to conditions simi-
employed in the procedure for 13 to give 36 (235 mg, 59%) as a yel-
low solid. 1H NMR (DMSO-d6, 400 MHz): d 8.02 (t, 1H, J = 2.0 Hz),
7.96–7.98(m, 3H), 7.80 (br, NH), 7.74 (m, 1H), 7.59 (t, 1H,
J = 7.9 Hz), 7.52–7.55 (m, 2H), 7.18–7.22 (m, 2H), 6.64 (d, 2H,
J = 9.3 Hz), 5.19 (dd, 1H, J = 9.1, 4.0 Hz), 3.79 (dd, 1H, J = 18.0,
9.3 Hz), 3.45 (dd, 1H, J = 17.9, 3.8 Hz); 13C NMR (DMSO-d6,
100 MHz): d 195.7, 161.4 (d, Jc–f = 241.8 Hz), 153.4, 138.5, 138.4,
136.2, 133.8, 133.1, 130.8, 128.6 (2C, d, Jc–f = 8.2 Hz), 127.9,
126.7, 126.1 (2C), 115.3 (2C, d, Jc–f = 21.4 Hz), 111.6 (2C), 51.6,
45.9; Anal. (C21H16ClFN2O3) C, H, N.
lar to those employed in the procedure for 13 to give 40 (331 mg,
76%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 8.49(s, 1H),
8.27(d, 1H, J = 8.0 Hz), 8.12 (1H, d, J = 8.0 Hz), 7.96(d, 2H,
J = 8.7 Hz), 7.69–7.78 (m, 5H), 6.63 (d, 2H, J = 9.3 Hz), 5.27 (dd,
1H, J = 9.5, 3.4 Hz), 3.85 (dd, 1H, J = 18.3, 9.6 Hz), 3.52 (dd, 1H,
J = 17.95, 3.4 Hz); 13C NMR (DMSO-d6, 100 MHz): d 195.2, 153.4,
147.3, 137.1, 136.6, 136.4, 132.4, 132.2, 130.2, 127.8 (q, Jc–f
=
30.9 Hz), 127.6 (2C), 126.1 (2C), 125.6 (2C), 122.9, 118.2, 112.1,
111.7 (2C), 51.8, 45.7; Anal. (C23H16F3N3O3) C, H, N.
5.1.29. 3-(4-Nitrophenylamino)-3-(4-(trifluoromethyl)phenyl)-
1-(3-methoxyphenyl)propan-1-one (41)
5.1.25. 3-(4-Nitrophenylamino)-1-(3-chlorophenyl)-3-(4-(trifluo-
romethyl)phenyl)propan-1-one (37)
3-Methoxyacetophenone (138
benzaldehyde (137 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30 L) were subjected to conditions simi-
lL, 1 mmol), 4-trifluoromethyl-
3-Choloroacetophenone (324
benzaldehyde (343 L, 2.5 mmol), 4-nitroaniline (345 mg, 2.5
mmol), EtOH (8 mL), EtOH/HCl (60 L) were subjected to condi-
tions similar to those employed in the procedure for 13 to give
37 (326 mg, 81%) as
yellow solid. 1H NMR (DMSO-d6,
lL, 2.5 mmol), 4-trifluoromethyl-
l
l
l
l
lar to those employed in the procedure for 13 to give 41 (356 mg,
80%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.95–7.98
(d, 2H, J = 9.3 Hz), 7.69–7.75 (m, 4H), 7.60 (d, 1H, J = 7.5 Hz),
7.43–7.49 (q, 2H, J = 7.9, 2.6 Hz), 7.23 (dd, 1H, J = 17.7, 2.7 Hz),
6.62–6.66 (d, 2H, J = 9.3 Hz), 5.29 (dd, 1H, J = 9.0, 3.6 Hz), 3.82 (s,
3H), 3.78 (dd, 1H, J = 18.1, 9.0 Hz), 3.49 (dd, 1H, J = 18.0, 4.0 Hz);
13C NMR (DMSO-d6, 100 MHz) d 196.2, 159.5, 153.4, 147.5, 137.9,
136.4, 130.0, 128.0 (q, Jc–f = 31.4 Hz), 127.6 (2C), 126.2 (2C), 125.6
(2C), 122.9, 120.7, 119.6, 112.5, 111.6 (2C), 55.5, 51.9, 45.7; Anal.
(C23H19F3N2O4) C, H, N.
a
300 MHz): d 7.94–8.01 (m, 4H), 7.69–7.75 (m, 5H), 7.58 (t, 1H,
J = 7.8 Hz), 6.62–6.65 (d, 2H, J = 9.2 Hz), 5.27 (dd, 1H, J = 9.2,
3.6 Hz), 3.81 (dd, 1H, J = 17.7, 9.0 Hz), 3.50 (dd, 1H, J = 18.0,
3.8 Hz); 13C NMR (CD3COCD3, 100 MHz): d 195.5, 153.6, 147.8,
139.0, 138.1, 134.8, 133.5, 131.0, 129.4 (q, Jc–f = 32 Hz), 128.3,
128.1 (2C), 127.0, 126.3 (2C), 126.1, 126.0, 123.6, 112.3 (2C),
53.1, 46.3; HRMS (M+, EI) calcd for C22H16ClF3N2O3: 448.0802;
found: 448.0799. HPLC purity = 98.1% (system A), 99.0% (system B).
5.1.30. 3-(4-Nitrophenylamino)-3-(4-(trifluoromethyl)phenyl)-
1-(4-methoxyphenyl)propan-1-one (42)
5.1.26. 3-(4-Nitrophenylamino)-3-(4-(trifluoromethyl)phenyl)-
1-(3-fluorophenyl)propan-1-one (38)
4-methoxyacetophenone (150 mg, 1 mmol), 4-trifluoromethyl-
3-Fluoroacetophenone (123
benzaldehyde (137 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30 L) were subjected to conditions simi-
l
L, 1 mmol), 4-trifluoromethyl-
benzaldehyde (137
EtOH (3 mL), EtOH/HCl (30
l
L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
l
lL) were subjected to conditions simi-
l
lar to those employed in the procedure for 13 to give 42 (262 mg,
59%). 1H NMR (DMSO-d6, 300 MHz): d 8.00–8.05 (m, 4H), 7.74–
7.80(dd, 4H, J = 11.0, 8.7 Hz), 7.15 (d, 2H, J = 9.3 Hz), 6.69 (d, 2H,
J = 9.1 Hz), 5.33 (dd, 1H, J = 8.3, 4.2 Hz), 3.90 (s, 3H), 3.79 (dd, 1H,
J = 17.7, 9.3 Hz), 3.45 (dd, 1H, J = 17.6, 4.8 Hz); 13C NMR (CDCl3,
100 MHz): d 195.7, 164.2, 151.9, 145.4, 138.6, 130.5 (2C), 130.1 (d,
Jc–f = 31.9 Hz), 129.1, 126.6 (2C), 126.2 (2C), 126.0 (2C), 125.2,
113.9 (2C), 112.2 (2C), 55.5, 54.0, 44.7; Anal. (C23H19F3N2O4) C, H, N.
lar to those employed in the procedure for 13 to give 38 (205 mg,
48%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 7.95–7.98
(d, 2H, J = 9.6 Hz), 7.86 (d, 1H, J = 7.6 Hz), 7.69–7.79 (m 5H),
7.49–7.64 (m, 2H), 6.62–6.65 (d, 2H, J = 9.3 Hz), 5.27 (t, 1H,
J = 8.7 Hz), 3.81 (dd, 1H, J = 17.8, 9.1 Hz), 3.51 (dd, 1H, J = 17.9,
3.5 Hz); 13C NMR (DMSO-d6, 100 MHz): d 195.5, 162.3 (d, Jc–f
=
243.7 Hz), 153.4, 147.4, 138.6 (d, Jc–f = 5.9 Hz), 136.4, 131.0 (d,
Jc–f = 8.2 Hz), 128.5(q, Jc–f = 31.5 Hz), 127.6 (2C), 126.2 (2C), 125.6
(2C), 124.4, 122.9, 120.5 (d, Jc–f = 21.4 Hz), 114.6 (d, Jc–f
=
5.1.31. 3-(4-Nitrophenylamino)-1-(4-chlorophenyl)-3-(4-(trifluo-
romethyl)phenyl)propan-1-one (43)
21.8 Hz), 111.6 (2C), 51.9, 45.7; HRMS (M+, EI) calcd for
C22H16N2F4O3: 432.1097 (M+); found: 432.1093; HPLC pur-
ity = 96.3% (system A), 97.8% (system B).
3-Chloroacetophenone (130
benzaldehyde (137 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30 L) were subjected to conditions simi-
lL, 1 mmol), 4-trifluoromethyl-
l
l
5.1.27. 3-(4-Nitrophenylamino)-3-(4-(trifluoromethyl)phenyl)-
1-m-tolylpropan-1-one (39)
lar to those employed in the procedure for 13 to give 43 (398 mg,
89%) as a yellow solid. 1H NMR (DMSO-d6, 300 MHz): d 8.38 (d, 2H,
J = 8.3 Hz), 8.25 (d, 2H, J = 8.7 Hz), 7.99 (d, 2H, J = 9.2 Hz), 7.72–7.78
(m, 4H), 6.65 (d, 2H, J = 9.3 Hz), 5.31 (dd, 1H, J = 8.9, 3.4 Hz), 3.88
(dd, 1H, J = 18.1, 9.1 Hz), 3.60 (1H, dd, J = 18.2, 3.7 Hz); 13C NMR
(CDCl3, 100 MHz) d 195.8, 152.0, 145.3, 140.4, 138.3, 134.3, 130.0
3-Methylacetophenone (136
benzaldehyde (137 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30 L) were subjected to conditions simi-
lar to those employed in the procedure for 13 to give 39 (315 mg,
lL, 1 mmol), 4-trifluoromethyl-
l
l

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4265
(q, Jc–f = 32.3 Hz), 129.4 (2C),129.1 (2C), 126.6 (2C), 126.2 (2C),
126.1 (2C), 123.9 (d, Jc–f = 270.5 Hz), 112.2 (2C), 53.4, 45.2; Anal.
(C22H16ClF3N2O3) C, H, N.
6.63 (d, 2H, J = 9.4 Hz), 4.03 (dd, 1H, J = 11.6, 5.9 Hz), 3.27 (d, 2H,
J = 6.2 Hz), 1.57–1.79 (m, 6H), 1.04–1.79 (m, 5H); 13C NMR (CDCl3,
100 MHz): d 197.3, 153.0, 140.0, 137.6, 134.9, 129.4 (2C), 129.1
(2C), 126.5 (2C),111.2 (2C), 54.3, 42.1, 40.1, 29.9, 29.5, 26.1, 26.0
(2C); Anal. (C21H23ClN2O3) C, H, N.
5.1.32. 3-(4-Nitrophenylamino)-1-(4-bromophenyl)-3-(4-(trifluo-
romethyl)phenyl)propan-1-one (44)
4-bromoacetophenone (199 mg, 1 mmol), 4-trifluoromethyl-
5.1.37. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-(4-fluorophenyl)-
propan-1-one (49)
benzaldehyde (137
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30
l
L) were subjected to conditions simi-
4-Fluoroacetophenone (122
dehyde (122 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), cyclohexanecarboxal-
lar to those employed in the procedure for 13 to give 44 (240 mg,
49%). 1H NMR (DMSO-d6, 300 MHz): d 7.92–7.98 (m, 4H), 7.68–
7.77 (m, 6H), 6.63 (d, 2H, J = 9.2 Hz), 5.26 (dd, 1H, J = 9.1, 3.9 Hz),
3.77(dd, 1H, J = 17.6, 9.3 Hz), 3.48 (dd, 1H, J = 17.5, 4.3 Hz); 13C
NMR (CD3COCD3, 100 MHz): d 195.7, 153.7, 147.9, 138.1, 136.2,
132.4 (2C), 130.5 (2C), 129.4 (q, Jc–f = 32 Hz), 128.2, 128.1 (2C),
126.3 (2C), 126.1, 126.0123.6, 111.7 (2C), 51.8, 45.6; HRMS (M+,
EI) calcd for C22H16BrN2O3F3: 492.0296; found: 492.0281. HPLC
purity = 99.4% (system A), 98.2% (system B).
l
l
those employed in the procedure for 13 to give 49 (160 mg, 43%).
1H NMR (DMSO-d6, 300 MHz): d 8.03–8.08 (m, 2H), 7.95 (d, 2H,
J = 9.0 Hz), 7.35 (t, 2H, J = 8.9 Hz), 6.63 (d, 2H, J = 9.1 Hz), 4.04
(dd, 1H, J = 11.5, 6.0 Hz), 3.27 (d, 2H, J = 6.2 Hz), 1.57–1.80 (m,
6H), 1.05–1.21 (m, 5H); 13C NMR (CDCl3, 100 MHz): d 196.9,
165.9 (d, Jc–f = 254 Hz), 153.0, 137.6, 133.0, 130.6 (d, Jc–f = 9.1 Hz),
126.6 (2C), 115.89 (d, Jc–f = 21.9 Hz), 111.1 (2C), 54.3, 42.1, 39.9,
29.9, 29.5, 26.1, 26.0, 25.9; HRMS (M+, EI) calcd for C21H23FN2O3:
370.1693; found: 370.1681. HPLC purity = 100% (system A), 98.7%
(system B).
5.1.33. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-p-tolylpropan-
1-one (45)
4-Methylacetophenone (133
aldehyde (122 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), cyclohexanecarbox-
l
5.1.38. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-(3-fluorophenyl)-
propan-1-one (50)
l
those employed in the procedure for 13 to give 45 (226 mg, 62%).
1H NMR (CDCl3, 400 MHz): d8.02–8.06 (m, 2H), 7.82 (d, 2H,
J = 8.3 Hz), 7.25 (d, 2H, J = 7.9 Hz), 6.51–6.55 (m, 2H), 4.87(d, 1H,
J = 9.8 Hz), 3.96 (m, 1H), 3.15–3.26 (m, 2H), 2.42 (s, 3H), 1.62–
1.93 (m, 6H), 0.97–1.28 (m, 5H); 13C NMR (CDCl3, 100 MHz): d
198.2, 153.1, 144.5, 137.5, 134.1, 129.4 (2C), 128.1 (2C), 126.5
(2C), 111.1 (2C), 54.5, 42.1, 39.8, 29.9, 29.6, 26.2, 26.0 (2C), 21.7;
Anal. (C22H26N2O3) C, H, N.
3-Fluoroacetophenone (108
dehyde (122 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), cyclohexanecarboxal-
l
l
those employed in the procedure for 13 to give 50 (149 mg, 40%).
1H NMR (DMSO-d6, 300 MHz): d 7.95 (d, 2H, J = 8.4 Hz), 7.83 (d,
1H, J = 7.3 Hz), 7.74 (d, 1H, J = 9.2 Hz), 7.59 (dd, 1H, J = 13.9,
7.9 Hz), 7.50 (t, 1H, J = 8.6 Hz), 6.65 (d, 2H, J = 8.6 Hz), 4.05
(m, 1H), 3.30 (d, 2H, J = 5.9 Hz), 1.60–1.81 (m, 6H), 1.06–1.21
(m, 5H); 13C NMR (CDCl3, 100 MHz):
d
197.3, 162.8
5.1.34. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-(4-methoxyphe-
nyl)propan-1-one (46)
(d, Jc–f = 247.3 Hz), 153.0, 138.6 (d, Jc–f = 5.9 Hz), 137.6, 130.4
(d, Jc–f = 7.8 Hz), 126.5 (2C), 123.7, 120.5 (d, Jc–f = 21.4 Hz), 114.7
(d, Jc–f = 21.8 Hz), 111.2 (2C), 54.3, 42.1, 40.3, 29.9, 29.5, 26.1,
26.0, 25.9; HRMS (M+, EI) calcd for C21H23FN2O3: 370.1693; found:
370.1703. HPLC purity = 98.8% (system A), 97.3% (system B).
4-Methoxyacetophenone (150 mg, 1 mmol), cyclohexanecarb-
oxaldehyde (122
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30
l
L) were subjected to conditions simi-
lar to those employed in the procedure for 13 to give 46 (182 mg,
48%). 1H NMR (DMSO-d6, 300 MHz): d 7.93–7.96 (m, 4H), 7.04 (d,
2H, J = 8.8 Hz), 6.63 (d, 2H, J = 9.4 Hz), 4.04 (m, 1H), 3.84 (s, 3H),
3.20 (d, 2H, J = 6.2 Hz), 1.51–1.80 (m, 6H), 1.00–1.21 (m, 5H); 13C
NMR (CDCl3, 100 MHz): d 197.1, 163.8, 153.2, 137.4, 130.3 (2C),
129.7, 126.5 (2C), 113.9 (2C), 111.1 (2C), 55.5, 54.6, 42.1, 39.5,
29.9, 29.6, 26.2, 26.0 (2C); Anal. (C22H26N2O4) C, H, N.
5.1.39. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-(3-methoxy-
phenyl)propan-1-one (51)
4-Methoxyacetophenone (150 mg, 1 mmol), cyclohexanecarb-
oxaldehyde (122
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30
lL) were subjected to conditions simi-
lar to those employed in the procedure for 13 to give 51 (182 mg,
48%). 1H NMR (DMSO-d6, 300 MHz): d 7.92 (d, 2H, J = 9.1 Hz), 7.57
(d, 1H, J = 7.7 Hz), 7.41–6.46 (m, 2H), 7.20 (m, 1H), 6.63 (d, 2H,
J = 9.6 Hz), 4.04 (dd, 1H, J = 11.7, 5.8 Hz), 3.80 (s, 3H), 3.26 (d, 2H,
J = 6.2 Hz), 1.55–1.79 (m, 6H), 0.98–1.14 (m, 5H); 13C NMR (CDCl3,
100 MHz): d 198.4, 159.9, 153.1, 137.9, 137.6, 129.7 (2C), 126.5
(2C), 120.5, 119.9, 112.3, 111.2, 55.4, 54.4, 42.1, 40.2, 29.9, 29.5,
26.2, 26.1 (2C); Anal. (C22H26N2O4) C, H, N.
5.1.35. 3-(4-Nitrophenylamino)-1-(4-bromophenyl)-3-cyclohexyl-
propan-1-one (47)
4-Bromoacetophenone (398 mg, 2 mmol), cyclohexanecarbox-
aldehyde (242
(7 mL), EtOH/HCl (50
l
L, 2 mmol), 4-nitroaniline (276 mg, 2 mmol), EtOH
L) were subjected to conditions similar to
l
those employed in the procedure for 13 to give 47 (245 mg, 28%).
1H NMR (DMSO-d6, 300 MHz): d 7.89–7.96 (q, 4H, J = 8.7, 8.7 Hz),
7.73 (d, 2H, J = 8.3 Hz,), 6.64 (d, 2H, J = 9.1 Hz), 4.03 (dd, 1H,
J = 11.7, 5.8 Hz), 3.26 (d, 2H, J = 6.3 Hz), 1.56–1.79 (m, 6H), 1.03–
1.21 (m, 5H); 13C NMR (CDCl3, 100 MHz): d 197.5, 153.0, 137.6,
135.3, 132.0 (2C), 129.5 (2C), 128.8, 126.5 (2C), 111.2 (2C), 54.3,
42.1, 40.0, 29.9, 29.5, 26.1, 26.0 (2C); Anal. (C21H23BrN2O3) C, H, N.
5.1.40. 3-(4-Nitrophenylamino)-1-(3,4-dichlorophenyl)-3-cyclo-
hexylpropan-1-one (52)
3,4-Dichloroacetophenone (189 mg, 1 mmol), cyclohexanecarb-
oxaldehyde (122
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
EtOH (3 mL), EtOH/HCl (30
lL) were subjected to conditions simi-
lar to those employed in the procedure for 13 to give 52 (243 mg,
58%). 1H NMR (DMSO-d6, 300 MHz): d 8.16 (d, 1H, J = 2.2 Hz), 7.88–
7.95 (m, 3H), 7.79 (d, 1H, J = 8.6 Hz), 6.62 (d, 2H, J = 9.1 Hz), 4.01
(dd, 1H, J = 11.7, 5.6 Hz), 3.28 (d, 2H, J = 6.1 Hz), 1.52–1.79 (m,
6H), 1.00–1.17 (m, 5H); 13C NMR (CDCl3, 100 MHz): d 196.2,
152.9, 138.2, 137.8, 136.1, 133.5, 130.9, 130.0, 126.9, 126.6 (2C),
111.2 (2C), 54.2, 42.1, 40.2, 29.9, 29.5, 26.1, 26.0, 25.9; Anal.
(C21H22Cl2N2O3) C, H, N.
5.1.36. 3-(4-Nitrophenylamino)-1-(4-chlorophenyl)-3-cyclohexyl-
propan-1-one (48)
4-Chloroacetophenone (130
dehyde (122 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30 L) were subjected to conditions similar to
lL, 1 mmol), cyclohexanecarboxal-
l
l
those employed in the procedure for 13 to give 48 (137 mg, 36%).
1H NMR (DMSO-d6, 300 MHz): d 7.93–7.99 (m, 4H), 7.59 (m, 2H),

4266
Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
5.1.41. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-phenylpropan-
1-one (53)
1H), 7.24 (d, 2H, J = 8.1 Hz), 7.18 (d, J = 7.9 Hz), 7.10 (m, 1H), 6.96
(m, 1H), 6.50 (m, 2H), 5.65 (br, NH), 5.08 (dd, 1H, J = 12.1,
6.0 Hz), 3.51 (dd, J = 17.4, 4.4 Hz), 3.46 (dd, J = 16.6, 5.1 Hz),
Acetophenone (117
(122 L, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH (3 mL),
EtOH/HCl (30 L) were subjected to conditions similar to those
lL, 1 mmol), cyclohexanecarboxaldehyde
l
2.40 (s, 3H); 13C NMR (CDCl3, 100 MHz):
d 197.1, 163.0
l
(d, Jc–f = 245.9 Hz), 151.9, 145.0, 144.0 (d, Jc–f = 6.4 Hz), 138.5,
133.7, 130.7 (d, Jc–f = 8.2 Hz), 129.5 (2C), 128.3 (2C), 126.1 (2C),
121.8, 114.8 (d, Jc–f = 21.4 Hz), 113.1 (d, Jc–f = 22.4 Hz), 112.2 (2C),
53.9, 45.1, 21.7; Anal. (C22H19FN2O3) C, H, N.
employed in the procedure for 13 to give 53 (173 mg, 49%). 1H
NMR (DMSO-d6, 300 MHz): d 7.92–7.97 (t, 4H, J = 8.3 Hz), 7.65
(m, 1H), 7.50–7.55 (m, 2H), 6.63 (d, 2H, J = 8.8 Hz), 4.05 (dd, 1H,
J = 12.0, 5.7 Hz), 3.28 (d, J = 6.3 Hz), 1.55–1.80 (m, 6H), 1.05–1.22
(m, 5H); 13C NMR (CDCl3, 100 MHz): d 198.6, 153.1, 137.5, 136.6,
133.6, 128.8 (2C), 128.0 (2C), 126.5 (2C), 111.2 (2C), 54.4, 42.1,
40.0, 29.9, 29.5, 26.2, 26.1 (2C); Anal. (C21H24N2O3) C, H, N.
5.1.45. (+)-(S)-3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-p-
tolylpropan-1-one ((+)-22)
Compound (+)-22 was prepared and analyzed in a manner iden-
tical to that described above for (ꢁ)-22, tR = 68.1 min, 99.7% ee;
5.1.42. 3-(4-Nitrophenylamino)-1-(4-phenyl-phenyl)-3-cyclohexyl-
propan-1-one (54)
½
a 2D0 = +96.0° (c 0.57 g/100 mL, CH3CN). 1H NMR (CDCl3,
ꢂ
400 MHz): d 7.97–8.01 (m, 2H), 7.78 (m, 2H), 7.31 (m, 1H), 7.24
(d, 2H, J = 7.9 Hz), 7.18 (d, 1H, J = 7.9 Hz), 7.10 (m, 1H), 6.95 (m,
1H), 6.49 (m, 2H), 5.64 (br, NH), 5.07 (dd, 1H, J = 11.9, 5.8 Hz),
3.50 (dd, 1H, J = 16.4, 6.8 Hz), 3.45 (dd, 1H, J = 16.4, 5.3 Hz),
4-acetylbiphenyl (196 mg, 1 mmol), cyclohexanecarboxalde-
hyde (122
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol), EtOH
(3 mL), EtOH/HCl (30
lL) were subjected to conditions similar to
those employed in the procedure for 13 to give 54 (154 mg, 36%).
1H NMR (DMSO-d6, 300 MHz): d 8.05 (d, 2H, J = 8.4 Hz), 7.95 (d,
2H, J = 9.0 Hz), 7.83 (d, 2H, J = 8.4 Hz), 7.75 (m, 2H), 7.41–7.55
(m, 3H), 6.65 (d, 2H, J = 9.6 Hz), 4.07 (dd, 1H, J = 12.0, 6.2 Hz),
3.30 (d, 2H, J = 6.1 Hz), 1.54–1.82 (m, 6H), 1.06–1.25 (m, 5H); 13C
NMR (CDCl3, 100 MHz): d 198.1, 153.1, 146.2, 139.6, 137.6, 135.3,
128.9 (2C), 128.6 (2C), 128.4, 127.4 (2C), 127.2 (2C), 126.6 (2C),
111.2 (2C), 54.5, 42.1, 40.0, 29.9, 29.6, 26.2, 26.1 (2C); Anal.
(C27H28N2O3) C, H, N.
2.38 (s, 3H); 13C NMR (CDCl3, 100 MHz):
d 197.1, 163.1
(d, Jc–f = 245.5 Hz), 151.9, 145.0, 144.0 (d, Jc–f = 6 Hz), 138.5,
133.7, 130.7 (d, Jc–f = 8.2 Hz), 129.5 (2C), 128.3 (2C), 126.1 (2C),
121.8, 114.8 (d, Jc–f = 20.9 Hz), 113.1 (d, Jc–f = 21.9 Hz), 122.2 (2C),
53.9, 45.1, 21.7; Anal. (C22H19FN2O3) C, H, N.
5.1.46. (+)-(S)-3-(4-Nitrophenylamino)-1-(4-bromophenyl)-3-
(3-fluorophenyl)propan-1-one((+)-32)
Compound (+)-32 was prepared on an ‘Chiralpak IA’ preparative
column (20 mm id ꢀ 250 mm length) in a manner identical to that
described above for (ꢁ)-22 and analyzed on an ‘Chiralpak AD-H’
column (4.6 mm id ꢀ 150 mm length), 1.0 ml/min, (hexane/2-pro-
5.1.43. 3-(4-Nitrophenylamino)-3-cyclohexyl-1-(4-cyclohexyl-
phenyl)propan-1-one (55)
4-Cyclohexylacetophenone (202 mg, 1 mmol), cyclohexanecarb-
panol = 82/18), tR = 13.9 min, 98.1% ee; ½a D20
ꢂ
= +107.5 (c 0.48 g/100
oxaldehyde (122
lL, 1 mmol), 4-nitroaniline (138 mg, 1 mmol),
mL, CH3CN). 1H NMR (CD3COCD3, 400 MHz): 7.92–7.98 (m, 4H),
7.68–7.72 (m, 2H), 7.38–7.40 (m, 2H), 7.32 (m, 1H), 7.02 (m, 1H),
6.68–6.72 (m, 2H), 5.32 (dd, 1H, J = 8.8, 4.3 Hz), 3.82 (dd, 1H,
J = 17.7, 8.7 Hz), 3.54 (dd, 1H, J = 17.4, 4.5 Hz). Anal. (C21H16Br-
FN2O3) C, H, N.
EtOH (3 mL), EtOH/HCl (30
l
L) were subjected to conditions simi-
lar to those employed in the procedure for 13 to give 55 (221 mg,
51%). 1H NMR (DMSO-d6, 300 MHz): d 7.85–7.95 (q, 4H, J = 9.1,
8.2 Hz), 7.35 (d, 2H, J = 8.2 Hz), 6.62 (d, 2H, J = 8.9 Hz), 4.04 (dd,
1H, J = 11.6, 6.4 Hz, CH), 3.23 (d, 2H, J = 5.6 Hz), 1.03–1.79 (m,
22H); 13C NMR (CDCl3, 100 MHz): d 198.2, 154.3, 153.1, 137.5,
134.4, 128.2 (2C), 127.2 (2C), 126.5 (2C), 111.1 (2C), 54.4, 44.6,
42.0, 39.8, 34.0 (2C), 29.9, 29.5, 26.6 (2C), 26.2, 26.1 (2C), 25.9;
Anal. (C27H34N2O3) C, H, N.
5.2. Receptor binding assay
Steroid receptor binding assays were performed as previously
described.23 An appropriate amount of baculovirus derived nuclear
HPLC separation of enantiomers of 22 and 32: An ‘Chiralpak IA’
preparative column (20 mm id ꢀ 250 mm length) on a YMC K-Prep
was equilibrated with an eluent of hexane/THF (80:20) at a flow
rate of 10 ml/min. A 1 mg/ml solution of 22 in hexane/THF
(70:30) was prepared, and 4.0 mL of this solution was injected
per run. Eluent was monitored by absorbance detection at
280 nm. The retention time of the first peak was approximately
15 min. The first eluting enantiomer (levorotatory) was collected
until the absorbance began to decline at which point the mixed
fractions were collected separately. Once the absorbance had re-
turned to a fixed height, the second (dextrorotatory) enantiomer
was collected until all of the second enantiomer had been col-
lected, then another injection was made. The mixed fractions were
combined and resubjected to the HPLC conditions. The enantio-
mers of 32 were resolved in an identical manner to that described
above for 22.
receptor protein extract (hPR-B, hAR, hERa, hERb, hGR and hMR)
was loaded into each well of Isoplate containing the assay buffer
followed by addition of 5 nM [3H] progesterone, 5 nM [3H]DHT,
5 nM [3H]E2, 5 nM [3H]E2, 5 nM [3H]dexamethasone, and 5 nM
[3H] aldosterone, respectively. Increasing concentrations of test
compounds (1 pM to 10
a final volume of 100
L wellꢁ1. The plates were sealed and incu-
bated for 16 h at 4 °C. Hydroxyapatite [HA 25% (v/v), 25 L] was
lM) were added thereafter (2.5 lL) to give
l
l
added to each well the next day and the plates were gently agi-
tated twice for 5 min each. Following centrifugation at 2500 rpm
for 3 min at 4 °C, the supernatant was decanted and 100
cold assay buffer added to each well. This washing procedure
was repeated twice before adding 150 L scintillation liquid,
lL ice-
l
gently agitating the plates to resuspend HA and counting bound
radioactivity with a MicroBeta counter. Non-linear regression anal-
yses were performed to generate dose–response curves. Ki values
were calculated from IC50 using the equation of Cheng and Prusoff
[Ki = IC50/(1 + [radioligand]/Kd)], where Kd is the dissociation con-
stant of the radioligand.24
5.1.44. (ꢁ)-(R)-3-(4-Nitrophenylamino)-3-(3-fluorophenyl)-1-p-
tolylpropan-1-one ((ꢁ)-22)
Compound (ꢁ)-22 was prepared on an ‘Chiralpak IA’ prepara-
tive column (20 mm id ꢀ 250 mm length) with 98% recovery and
analyzed on an ‘Chiralpak AD-H’ column (4.6 mm id ꢀ 250 mm
length), 0.6 ml/min, (hexane/ethanol, 90:10), tR = 60.9 min, 99.9%
5.3. Cotransfection assay
Transient cotransfection assay was performed in CV-1 cells
according to the method described previously.23 CV-1 cells (African
green monkey kidney fibroblasts) were cultured in the presence of
ee; ½a 2D0
ꢂ
= ꢁ95.5° (c 0.53 g/100 mL, CH3CN). 1H NMR (CDCl3,
400 MHz): d 7.97–8.01 (m, 2H), 7.78 (d, 2H, J = 8.3 Hz), 7.31 (m,

Y. Du et al. / Bioorg. Med. Chem. 18 (2010) 4255–4268
4267
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
10% CDT-FBS and seeded 24 h before transfection in 6 cm dish
(6 ꢀ 105 cells per dish). Two micrograms of the reporter plasmid
the receptor suitable for docking studies, the PDB structure 2OVH
was processed in graphic software AutoDockTools. According the
default setting in AutodockTools, the hydrogens were added and
the atoms were typing as the rules in AutoDock. The Gasteiger
charges were calculated and assigned for the atoms of the struc-
ture. The coordinates along with the charge, salvation information
of the receptor were saved in pdbqt format for later docking stud-
ies. The initial structures of mifepristerone, distomers of (ꢁ)-(R)-22
was optimized using the Cerius2 software with OPEN force field.
The conjugated gradient method was used for energy minimization
with an energy convergence gradient value of 0.001 kcal/(mol Å).
The advance docking program AUTODOCK 4.0 was used to dock li-
gands to the progesterone receptor ligand-binding domain. The
Lamarckian genetic algorithm25 was applied to analyze protein–li-
gand interactions. A Solis and Wets local search25 was performed
for energy minimization on a user-specified proportion of the pop-
ulation. The docked structures of the ligands were generated after a
reasonable number of evaluations. The whole docking operation
could be stated as follows:
(MMTV-Luc) and 0.4 lg of pSG5-hPR-B were introduced simulta-
neously into cells with a ratio of 5 to 1. Cells were transfected for
8 h, and harvested with 0.05% trypsin and 0.02% EDTA prior to
reseeding onto a 96-well microtiter plate (8000 cells wellꢁ1). They
were incubated for 24 h with or without various concentrations of
control or test compounds. For antagonist assay, test samples were
added 30 min ahead of progesterone at its EC50 concentration. Cell
extracts were prepared and the expressed luciferase activity was
determined in a Wallac 1420 multi label counter (VICTOR2, Perkin-
Elmer) using a Steady-Glo luciferase kit from Promega. To detect
potential cytotoxicity of compounds, treated cells were reacted
with AlamarBlue™ (United States Biological) for 4 h and the fluo-
rescence is monitored at 540 nm excitation wavelength and
590 nm emission wavelength on a FlexStation 384II (Molecular De-
vices, Sunnyvale, CA, USA) prior to luciferase activity measure-
ment. The relative luciferase activity was normalized against cell
viability (% growth) assessed with AlamarBlue in the same well.
To determine cross-reactivity of test compounds, cotransfection
assays with hAR, hMR, and hGR with the MMTV-Luc reporter and
1. Each ligand molecule was checked for polar hydrogens and
assigned atom types, the partial atomic charges with Gasteiger
method and the atomic solvation parameters.
hERa, hERb with the ERE-MMTV-Luc reporter were performed.
2. The three-dimensional grid with 60 ꢀ 60 ꢀ 60 points and a
spacing of 0.375 Å was created by the AutoGrid4 algorithm to
evaluate the binding energies between the ligands and the pro-
teins. In this stage, the protein was embedded in the three-
dimensional grid, and a probe atom was placed at each grid
point. The affinity and electrostatic potential grid were calcu-
lated for each type of atom in the ligands. The energetics of a
particular ligand configuration was found by trilinear interpola-
tion of affinity values and electrostatic interaction of the eight
grid points surrounding each of the atoms in a ligand.
3. A series of the docking parameters were set on. Not only the
atom types but also the generations and the number of runs
for the Lamarckian genetic algorithm were edited and properly
assigned according to the requirement of the Amber force field.
The number of generations, energy evaluations, and docking
runs were set to 27 ꢀ 103, 2.5 ꢀ 106, and 10, respectively.
5.4. Murine uterine decidulization assay
Murine uterine decidulization assay was performed as de-
scribed in the literature.17,18 BALB/c mice were used in the exper-
iment. The animals were housed at 22 0.8 °C in
a 12 h
light:dark cycle and kept on a standard laboratory diet and drink-
ing water ad libitum. 7-week old male BALB/c mice were vasecto-
mized 3 weeks before decidulization assay. Mature female virgin
BALB/c (21 g mean weight) were caged together with vasecto-
mized male BALB/c mice with a ratio of 2 to 1 between 17.00
and 10.00 h. It was regarded as pseudopregnant when a vaginal
plug was detected the next morning (day 1). Mating was presumed
to have taken place at 02.00 (time 0). Pseudopregnant mice were
treated intraluminally with either RU486 or test compounds (22,
26). RU486 and test compounds were first dissolved in 100% etha-
nol and then diluted with sesame oil. The sesame oil mixtures were
blown with nitrogen to make sure that no residual ethanol was left.
Acknowledgment
On day 4 (16.00 h), 10 lL of sesame oil containing RU486 or test
compounds was injected intraluminally into the right uterine horn
(stimulated) and the left horn served as an internal control (non-
stimulated). Control animals were injected 10 lL of sesame oil into
This work was supported in part by a grant from the Ministry of
Science and Technology of China (2009ZX09302-001).
the right uterine horns only. Seventy-two h after decidual stimula-
tion, the mice were killed and the uterine horns were removed and
weighed. For each mouse, uterine wet weight gain was calculated
by subtracting the weight of the non-stimulated horn from that of
the stimulated horn. Percent inhibition was calculated using the
formula: % inhibition = (1 ꢁ weight gain in treated group/weight
gain in control group) ꢀ 100.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.092.
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