5-(3-Cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-methyl-1H- pyrrole-2-carbonitrile: A novel, highly potent, selective, and orally active non-steroidal progesterone receptor agonist

Article abstract of DOI:10.1016/j.bmc.2007.07.011

We have recently discovered 5-(3-cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-methyl-1H- pyrrole-2-carbonitrile (14) as a potent, selective, and orally active non-steroidal progesterone receptor (PR) agonist. Compound 14 and its analog 13 poss

Full text of DOI:10.1016/j.bmc.2007.07.011

Bioorganic & Medicinal Chemistry 15 (2007) 6556–6564
5-(3-Cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-
methyl-1H-pyrrole-2-carbonitrile: A novel, highly potent, selective,
and orally active non-steroidal progesterone receptor agonist
Puwen Zhang,a,* Eugene Terefenko,a Jeffrey Kern,a Andrew Fensome,a Eugene Trybulski,a
Ray Unwalla,a Jay Wrobel,a Susan Lockhead,b Yuan Zhu,c Jeffrey Cohen,c
Margaret LaCava,c Richard C. Winnekerc and Zhiming Zhangc
aChemical and Screening Sciences, Wyeth Research, S-2250B, 500 Arcola Road, Collegeville, PA 19426, USA
bDrug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
cWomen’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
Received 15 May 2007; revised 6 July 2007; accepted 9 July 2007
Available online 26 July 2007
Abstract—We have recently discovered 5-(3-cyclopentyl-2-thioxo-2,3-dihydro-1H-benzimidazol-5-yl)-1-methyl-1H-pyrrole-2-carbo-
nitrile (14) as a potent, selective, and orally active non-steroidal progesterone receptor (PR) agonist. Compound 14 and its analog 13
possessed sub-nanomolar in vitro potency (EC50 0.1–0.5 nM) in the T47D alkaline phosphatase assay, similar to that of the steroidal
PR agonist medroxyprogesterone acetate (MPA). In contrast to MPA, 14 was highly selective (>500-fold) for the PR over both glu-
cocorticoid and androgen receptors. In the rat uterine decidualization and complement component C3 models, 14 had oral ED50
values of 0.02 and 0.003 mg/kg, respectively, and was from 6- to 20-fold more potent than MPA. In the monkey ovulation inhibition
model, compound 14 was also highly efficacious and potent with an oral ED100 of 0.03 mg/kg.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
cardiovascular complications. A tissue selective proges-
terone receptor (PR) agonist would potentially reduce
these side effects. In addition, many of the steroid-based
agonists have demonstrated undesirable activities at
other steroid receptors such as glucocorticoid receptor
(GR) and androgen receptor (AR). Novel non-steroidal
PR ligands may offer greater potential for tissue selectiv-
ity and receptor selectivity.
Progesterone (P4, 1) plays a critical role in the regula-
tion of female reproductive function. Synthetic proges-
tins are commonly used in oral contraceptives (OC)
and their contraceptive efficacy is achieved via inhibition
of ovulation and thickening of the cervical mucus.1
Progestins can also protect the endometrium from estro-
gen induced endometrial hyperplasia in women with an
intact uterus. This protective function has been used in
the estrogen-based hormone therapy in postmenopausal
women for the relief of vasomotor symptoms2,3 and pre-
vention of osteoporosis.4 Furthermore, progestins are
used for the treatment of reproductive disorders such
as dysmenorrhea and dysfunctional uterine bleeding.5
However, combination steroidal progestin and estrogen
therapy is often associated with side effects such as mas-
talgia, nausea, headaches, as well as some metabolic and
In our effort to search for potent and selective non-ste-
roidal PR agonists, we discovered a number of chemical
scaffolds,6–9 such as 6-aryl benzoxazin-2-ones and ben-
zoxazine-2-thiones, that afforded compounds with po-
tent PR agonist activity. Recently, we reported
another template, 6-aryl benzimidazolones such as 7,10
that demonstrated moderate to potent PR antagonist
activity. Unlike the 5-aryl oxindole and 6-aryl benzoxaz-
inone scaffolds,6 the modification of 6-aryl moiety for
the benzimidazolone template did not render PR ago-
nists. Only weak agonist activity for several potent benz-
imidazolone PR antagonists10 was observed at a high
concentration of 3 lM. After further SAR examination
of the benzimidazolone scaffold, gratifyingly, we found
Keywords: Progesterone receptor (PR); PR agonists; PR antagonists;
Non-steroidal.
*
Corresponding author. Tel.: +1 484 865 3856; fax: +1 484 865
9398; e-mail: Zhangp@wyeth.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.011

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
6557
that a number of 6-aryl benzimidazole-2-thiones were
potent PR agonists. The synthesis, in vitro SAR, and
in vivo activity of novel 6-aryl benzimidazoloe-2-thiones
(8–15) are the subject of this report.
recently reported several series of non-steroidal PR
modulators such as 5-aryl oxindoles and 6-aryl benzox-
azin-2-ones.15,16 Examination of the SAR results from
the oxindole and benzoxazinone scaffolds revealed
two PR agonist structural motifs, that is, a pendent
50-cyanopyrrol-2-yl group and a 2-thiocarbonyl moi-
ety.6,8,9,15,16 These structural motifs caused the func-
tional activity of 6-aryl benzoxazinones and 5-aryl
oxindoles to switch from PR antagonist to PR agonist
activity in the alkaline phosphatase assay using the hu-
man T47D breast carcinoma cell line.6,8,9 In addition,
incorporation of both structural motifs (50-cyanopyr-
rol-2-yl and thiocarbonyl) onto the benzoxazinone tem-
plate demonstrated synergistic effects on PR agonist
activity and led to the discovery of the potent PR
agonist, tanaproget.7,17 In contrast, when placing the
50-cyanopyrrol-2-yl group onto the benzimidazolone
scaffold, the corresponding 6-(50-cyanopyrrol-2-yl)-ben-
zimidazolones remained as PR antagonists (i.e., the
activity did not switch to PR agonism).10 Intrigued by
this finding, we decided to examine the impact of the
2-thiocarbonyl moiety on the SAR of the 6-aryl benzim-
idazolone template.
O
Me
Me
O
O
O
Me
Me
Me
Me
Me
O
O
Me
Medroxyprogesterone
acetate (MPA, 2)
Progesterone (P4, 1)
R1
N
R1
N
NC
Ar
N
O
S
N
H
N
H
7f, R1 = c-C4H7
7g, R1 = c-C5H9
8-15
7h, R1 = 3-C5H11
We have previously demonstrated that cyclic butyl, pen-
tyl, and 3-pentyl were among the best substituents at the
1-position for the 6-aryl benzimidazolones.10 Thus, a
number of novel 6-aryl benzimidazole-2-thiones (8–15)
were prepared using these optimized substituents. These
compounds were evaluated for PR agonist and antago-
nist activities in the T47D cell alkaline phosphatase as-
say.18,19 The results are listed in Tables 1 and 2.
2. Synthetic chemistry
The preparation of the target compounds 8–15 as shown
in Scheme 1 was described previously.11,12 In brief,
alkylation of the BOC protected benzimidazolones 411
to yield 5 was readily achieved via a Mitsunobu proto-
col. Removal of protecting groups from benzimidazo-
lone 5 under acidic conditions afforded 6 which was
cross-coupled with an appropriate aryl boronic acid to
provide the 6-aryl benzimidazolones 7 in good yields.
Refluxing a mixture of Lawesson’s reagent and ben-
zimidazolones 7 in toluene provided the desired 6-aryl
benzimidazole-2-thiones 8–15.
Table 1 lists the alkaline phosphatase activity data of the
6-phenyl based-benzimidazole-2-thiones. As illustrated,
the 2-thiocarbonyl compounds 8–10 showed good PR
agonist potency compared to the PR antagonist activity
exhibited by their corresponding 2-carbonyl ben-
zimidazolones (IC50 3.3–134.7 nM).10 Interestingly, the
3-pentyl analog 11 exhibited PR antagonist activity with
moderate potency. Congener 12 was a potent antagonist
at a lower concentration while a weak agonist at a high-
er concentration. These results indicated that the PR
functional activity of benzimidazole-2-thiones was not
completely controlled by the 2-thiocarbonyl moiety
and dependent on the 1-substituent as well.
3. Results and discussion
There have been a number of non-steroidal PR modula-
tors disclosed over the last few years.13,14 We have
R1
N
R1
N
H
Br
Br
Br
N
a
b
O
O
O
N
N
H
6a-c
N
Boc
Boc
4
5
c
R1
N
R1
N
Ar
Ar
d
O
S
N
H
7a-h
N
H
8-15
Scheme 1. 6-Aryl benzimidazole-2-thiones. Reagents and conditions: (a) R1OH, DIAD, Ph3P, THF, rt, 30–80%; (b) CH2Cl2, TFA, rt, 80–95%; (c)
ArB(OH)2, Pd(Ph3P)4, K2CO3, toluene, EtOH/H2O, 90 ꢁC, 30–85%; (d) Lawesson’s reagent, toluene, reflux, 30–80%.

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P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
Table 1. PR agonist and antagonist activity in T47D cell alkaline
phosphatase assay
pyrrol-2-yl)-benzimidazole-2-thiones 13–15 (Table 2). In
the T47D alkaline phosphatase assay, compounds 13
and 14 had sub-nanomolar in vitro potency (EC50 0.1–
0.5 nM), similar to that of the steroidal PR agonist
medroxyprogesterone acetate (2) and more potent than
progesterone (1). In comparison with their correspond-
ing 2-carbonyl analogs (7f–h), compounds 13–15 not
only switched from PR antagonist to PR agonist they
were also more potent in the T47D alkaline phosphatase
assay. Consistent with the functional activity, com-
pounds 13–15 had higher binding affinity compared to
their 2-carbonyl analogs. For example, compound 14
had a binding IC50 of 0.4 nM while the IC50 of its 2-car-
bonyl analog 7g was only 26.2 nM when tested in a PR
competition binding assay using cytosol from the human
T47D breast carcinoma cell line.19
R1
N
R2
S
N
H
8-12
Compound
R1
R2
PR alk. phos. PR alk. phos.
a
EC50 (nM) IC50 (nM)
b
Progesterone (1)
MPA (2)
0.9
0.1
NAd
NA
8
c-C4H7 3-CN, 5-F
c-C5H9 3-Cl, 4-F
c-C5H9 3-F, 4-F
3-C5H11 3-Cl, 4-F
2.7 (60%)c NA
49.3 (55%) NA
62.5 (75%) NA
9
10
11
12
NA
33.7 (60%)
3-C5H11 3-CN, 5-F 269.8 (70%) 3.6 (50%)
a 50% effective concentration of tested compounds on alkaline phos-
phatase activity in the human T47D breast carcinoma cell line.
b 50% inhibitory concentration of tested compounds on 1 nM pro-
gesterone induced alkaline phosphatase activity in the human T47D
breast carcinoma cell line. Values represent the average of at least
duplicate determinations. The standard deviations for these assays
were typically 20% of the mean or less.
To understand why subtle structural changes (2-car-
bonyl in 7f–h vs 2-thiocarbonyl in 13–15) switched PR
functional activities, 7f and 13 were closely examined
for their interaction in the PR binding pocket using
docking studies.20 Recently, several co-crystal structures
of PR LBD/steroidal and non-steroidal PR agonists
have been reported.7,21,22 Using the PR LBD crystal
structure derived from PR LBD/tanaproget (a non-ste-
roidal PR agonist), PR agonist 13 was docked into the
PR LBD and was shown to demonstrate the same key
interactions within PR binding site as tanaproget
(Fig. 1). Similar to tanaproget,7 the nitrile group of 13
makes important hydrogen bond interactions to residues
Gln725 from helix-3 and Arg766 from helix-5, which are
held in position by a water molecule. The NH group
of the benzimidazole-2-thione moiety in 13 acts as a
hydrogen bond donor by forming an interaction with
the side chain oxygen of Asn719 within helix-3. Clearly,
compound 13 has the same favorable agonist mode
interactions with PR as tanaproget and as such it was
a potent PR agonist.
c Data in parentheses represent compounds’ efficacy compared to
progesterone (1) of 100%.
d NA, not active.
The compounds incorporating two PR agonist struc-
tural motifs were potent agonists irrespective of the
substituent at the 1-position as depicted by 6-(50-cyano-
Table 2. PR agonist and antagonist activity in T47D cell alkaline
phosphatase assay
R
N
NC
N
X
N
H
In contrast to PR agonists, the co-crystal structure of
PR LBD/PR antagonists (in particular non-steroidal
13-15
Compound
X
R
PR alk. phos. PR alk. phos.
a
EC50 (nM)
b
IC50 (nM)
Progesterone (1)
MPA (2)
0.9
0.1
NAd
NA
10.9
5.6
7f
O
O
O
S
c-C4H7
c-C5H9
>3000c
>3000c
7g
7h
13
14
15
3-C5H11 >3000c
11.7
NA
NA
NA
c-C4H7
c-C5H9
3-C5H11 1.0 (70%)
0.5 (70%)e
0.1 (75%)
S
S
a 50% effective concentration of tested compounds on alkaline phos-
phatase activity in the human T47D breast carcinoma cell line.
b 50% inhibitory concentration of tested compounds on 1 nM pro-
gesterone induced alkaline phosphatase activity in the human T47D
breast carcinoma cell line. Values represent the average of at least
duplicate determinations. The standard deviations for these assays
were typically 20% of mean or less.
c Compounds showed weak agonist activity in the absence of 1 nM
progesterone (agonist mode) at 3000 nM.
Figure 1. Docked poses of PR antagonist 7f (magenta) overlaid with
agonist 13 (cyan) in PR LBD binding site. (Only key residues and a
Connolly surface of the binding site are shown for simplicity.
Hydrogen bonds are shown as yellow dotted lines.)
d NA, not active.
e Data in parentheses represent compounds’ efficacy compared to
progesterone (1) of 100%.

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
6559
Table 3. Relative potencies of P4 (1), MPA (2), and 14 at AR and GR
PR antagonists) has not been reported to date. More re-
cently, a co-crystal structure of PR LBD/asoprisnil, a
steroidal selective PR modulator (SPRM), in the
presence of co-repressor was disclosed.23 Asoprisnil pos-
sesses a large 11-b pendent aryl group (likewise, RU-486
also has a large 11-b pendent aryl moiety) and induced a
conformational shift in the helix-12 region and favor-
ably permits co-repressor binding.23 However, smaller
antagonists 7f–h lack the structural moiety similar to
that of 11-b pendent aryl group from asoprisinil and
RU-486 and are unlikely to induce helix-12 conforma-
tional shift that leads to PR antagonist activity. Consid-
ering the close structure similarity between agonists 13–
15 versus antagonists 7f–h, we decided to choose 7f, the
counterpart of agonist 13, and docked it into the PR
LBD binding pocket from PR LBD/tanaproget
(Fig. 1). As illustrated in Figure 1, 7f and 13 overlaid
well in the binding pocket and have the same key inter-
actions with PR although 13 has slightly favorable dock-
ing score (ꢀ0.7 kcal/mol). While we are unable to
explain the functional activity switch for 7f and 13 from
docking study with PR LBD, it is possible that non-ste-
roidal PR antagonists such as 7f have a unique mecha-
nism that induces PR antagonism. Compared to other
steroidal receptors such as estrogen receptor (ER), PR
has much smaller and less stable dimmer interface22 that
presumably could influence PR functional activity in dif-
ferent ways. Therefore, the PR conformational shifts
that may result in the functional activity switch, due to
the nature of PR dimmer interface, may be more suscep-
tible to a subtle structural change induced by PR ligands
such as non-steroidal PR antagonists 7f–h versus ago-
nists 13–15. Further study is needed to shed light on
why PR functional activities were switched when there
is only a small structural change in these non-steroidal
ligands.
Compound PR EC50 AR EC50 AR IC50 GR EC50 GR IC50
(nM)
(nM)
(nM)
37 (46%) ND
ND
(nM)
(nM)
1a
2a
0.9
0.1
NDc
6.1
>1000
10 (157%) ND
(159%)
>10,000 ꢀ1000
14b
0.1
>10,000
99
a Data from Ref. 6.
b Experimental values represent the average of at least duplicate
determinations. The standard deviation for these assays was typically
15% of mean or less.
c ND, not determined.
Table 4. Oral activities of P4 (1), MPA (2), and 14 in rat decidual-
ization and complement component C3 model
Compound
1
2
14
5.62b
NDc
0.40b
0.03b
0.02
0.003
a
Decidualization ED50 (mg/kg)
a
Component C3 ED50 (mg/kg)
a Experimental values represent the average of at least duplicate
determinations. The standard deviation for the decidualization and
C3 assays was typically 15% of mean or less.
b Dosed via subcutaneous injection.
c ND, not determined.
its pharmacokinetics profile. In a separate PK study
using female rats, compound 14 had moderate clearance
(25 mL/min/kg), high volume of distribution (7.6 L/kg),
a reasonable terminal half-life (4.4 h), and AUC0–1
(0.68 lg h/mL) following a single IV bolus injection of
1 mg/kg dose. When administered orally at the dose of
1 mg/kg, its exposure (AUC0–1) was 0.48 lg h/mL
which resulted in an excellent bioavailability of 71%.
Compound 14 was further evaluated for ovulation inhi-
bition by treating cynomolgus monkeys once daily
throughout a normal menstrual cycle. In this model,
14 inhibited ovulation-induced progesterone levels with
an ED100 of 0.03 mg/kg when dosed orally, which was
similar to that of tanaproget (ED100 0.01 mg/kg, po).
In comparison, single subcutaneous injection of MPA
at the dose of 15 mg/kg26 also completely blocked pro-
gesterone level in cynomolgus monkey for a prolonged
time. Levonorgestrel, another steroidal PR ligand evalu-
ated in this model, achieved complete blockage of pro-
gesterone level at 0.9 lg/kg via intramuscular
injection.27 Limited data have been reported in this
model for non-steroidal PR ligands. However, a tetra-
hydropyridazine27 was described to completely block
progesterone level at 0.9 mg/kg when dosed intramuscu-
larly. While comparison of these PR ligands is difficult
because of different routes of administration, 14 was
clearly a potent and orally active non-steroidal PR ago-
nist in the cynomolgus monkeys.
Since compound 14 was the most potent analog in the
T47D alkaline phosphatase assay it was evaluated for
its selectivity over other steroidal receptors and in sev-
eral animal models. Many steroids such as MPA were
known to cross-react with GR and AR.24 The selectivity
of 14 with GR and AR was examined using an HRE-tk-
luciferase assay in the human lung carcinoma cell line
A549 for GR and in mouse fibroblast cell line L929
for AR.19 In contrast to MPA, 14 was over 500-fold
selective for the PR over both GR and AR (Table 3)
suggestive of less potential AR or GR-related side
effects.
Excellent oral potency was observed for 14 in the ovari-
ectomized female rat decidualization model (Table 4).24
In this model it was 20-fold more potent than MPA and
over 200-fold than progesterone with both steroids
administered by subcutaneous injection. Compound 14
was also tested in an additional in vivo PR agonist mod-
el, the adult ovariectomized rat uterine component C3
assay.25 PR agonists are known to down-regulate the
estrogen induced synthesis of complement component
C3 in the epithelial cells of the rat uterus. As shown in
Table 4, 14 had oral potency of 0.003 mg/kg and was
six times more potent than MPA (2). The oral activities
demonstrated in rat models for 14 were substantiated by
In conclusion, the SAR examination of 6-aryl benzimid-
azole-2-thiones has led to discovery of novel potent PR
agonists. Among the benzimidazole-2-thiones prepared,
compound 14 was the most potent analog and, in con-
trast to MPA, highly selective (>500-fold) for the PR
over both glucocorticoid and androgen receptors. In
the rat uterine decidualization and complement compo-
nent C3 models, 14 was from 6- to 20-fold more potent
than MPA. In the monkey ovulation inhibition model,

6560
P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
compound 14 was also highly efficacious and potent. Ta-
ken together, compound 14 is potentially useful as a no-
vel contraceptive and for the treatment of reproductive
disorders.
4.2. 3-(3-Cyclobutyl-2-oxo-2,3-dihydro-1H-benzimidazol-
5-yl)-5-fluorobenzonitrile (7a)
A mixture of 6-bromo-1-cyclobutyl-1,3-dihydro-2H-
benzimidazol-2-one (6a) (1.0 g, 3.7 mmol), 3-cyano-5-
fluorophenyl boronic acid (0.92 g, 5.6 mmol), and so-
dium carbonate (0.78 g, 7.4 mmol) in DME (60 mL)
and water (30 mL) was subject to a blanket of nitro-
gen for 15 min at 50 ꢁC. Tetrakis(triphenylphos-
phine)-palladium (0) (0.43 g, 0.37 mmol) was added
and the reaction mixture was heated to 85 ꢁC for
1 h. The reaction mixture was then cooled to room
temperature and ethyl acetate (100 mL) added. The
organic layer was separated and aqueous layer ex-
tracted with ethyl acetate (2· 50 mL). The combined
organic layers were washed with a saturated aqueous
ammonium chloride solution (2· 100 mL), dried over
magnesium sulfate, and concentrated. The residue
was purified via a silica gel column (40% ethyl ace-
tate in hexane) to give 7a (0.34 g, 31%) as a white
solid. 1H NMR (DMSO-d6) d 11.01 (s, 1H), 8.10
(m, 1H), 7.97 (m, 1H), 7.77 (m, 1H), 7.68 (d, 1H,
J = 8.0, 1.6 Hz), 7.43 (dd, 1H, J = 8.0, 1.6 Hz), 7.06
(d, 1H, J = 8.0 Hz), 4.89 (m, 1H), 2.94 (m, 2H),
2.26 (m, 2H), 1.87 (m, 1H), 1.78 (m, 1H). MS
(ESI) m/z 308 ([M+H]+). HRMS: Calcd for
C18H14FN3O+H+, 308.11937. Found (ESI [M+H]+):
308.12050.
4. Experimental
1H NMR spectra were recorded on a Bruker DPX300,
Varian INOVA 400, or Varian INOVA 500 instrument.
Chemical shifts are reported in d values (parts per mil-
lion, ppm) relative to an internal standard of tetrameth-
ylsilane in CDCl3 or DMSO-d6. Electrospray (ESI) mass
spectra were recorded using a Hewlett-Packard 5989B
MS engine or Waters Alliance-ZMD mass spectrometer.
Electron Impact ionization (EI, EE = 70 eV) mass spec-
tra were recorded on a Finnigan Trace mass spectrome-
ter. Elemental analyses were carried out on a modified
PerkinElmer model 2400 series II CHN analyzer or sent
to Robertson Microlit. Analytical thin-layer chromatog-
raphy (TLC) was carried out on pre-coated plates (silica
gel, 60 F-254), and spots were visualized with UV light
and stained in iodine. Preparative HPLC purifications
were performed on a preparative Gilson HPLC system
using a CombiPrep Pro C18 column with acetonitrile
(0.1% TFA) and water (0.1% TFA) as solvents at a flow
rate of 20 mL/min. Solvents were purchased as
anhydrous grade and were used without further
purification.
4.3. 3-(3-Cyclobutyl-2-thioxo-2,3-dihydro-1H-benzimida-
zol-5-yl)-5-fluorobenzonitrile (8)
4.1. 6-Bromo-1-cyclobutyl-1,3-dihydro-2H-benzimidazol-
2-one (6a)
A mixture of 3-(3-cyclobutyl-2-oxo-2,3-dihydro-1H-
benzimidazol-5-yl)-5-fluorobenzonitrile (7a) (80 mg,
0.26 mmol) and Lawesson’s reagent (110 mg,
0.27 mmol) in anhydrous toluene was heated at reflux
under nitrogen for 18 h. The solvent was removed in
vacuo and residue was purified on a silica gel column
(30% ethyl acetate in hexane) to afford 8 as an off-
white solid (28 mg, 33%). 1H NMR (DMSO-d6) d
12.94 (s, 1H, D2O exchangeable), 8.19 (s, 1H), 8.04–
8.16 (m, 1H), 7.97 (s, 1H), 7.82–7.86 (m, 1H), 7.61
(dd, 1H, J = 8.27, 1.55 Hz), 7.28 (d, 1H, J = 8.28 Hz),
5.62–5.69 (m, 1H), 3.09–3.19 (m, 2H), 2.28–2.35 (m,
2H), 2.01–2.11 (m, 1H), 1.82–1.92 (m, 1H). MS (ESI)
m/z 324 ([M+H]+). Anal. Calcd for C18H14FN3SÆ0.1-
H2O: C, 66.48; H, 4.4; N, 12.92. Found: C, 66.81; H,
4.33; N, 12.57.
A
mixture of 5-bromo-2-oxo-2,3-dihydro-benzimid-
azole-1-carboxylic acid tert-butyl ester11 (10 g,
31.9 mmol), cyclobutanol (4.9 mL, 48.4 mmol), and tri-
phenylphosphine (16.7 g, 48.4 mmol) in anhydrous
THF (320 mL) was treated with a solution of diethyla-
zodicarboxylate (10 mL, 48.4 mmol) in THF (60 mL)
in a dropwise manner at room temperature under nitro-
gen. After stirred for 30 min, to the reaction mixture was
added ethyl acetate (100 mL). The organic layer was
separated and aqueous layer was extracted with ethyl
acetate (2· 50 mL). The combined organic layers were
washed with a saturated aqueous sodium bicarbonate
solution (100 mL), dried with anhydrous sodium sulfate,
concentrated, and purified via a silica gel column (5%
ethyl acetate in hexane) to give tert-butyl 5-bromo-3-
cyclobutyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-1-
carboxylate (2.29 g, 20%) as a clear oil. The oil was ta-
ken up in dichloromethane (40 mL) and treated with tri-
fluoroacetic acid (2.4 mL, 31 mmol). After stirred for
30 min, the solvent was removed under a reduced pres-
sure and the residue was taken up in ethyl acetate
(50 mL) and washed with saturated aqueous sodium
bicarbonate solution (50 mL). The organic layer was
separated, dried over anhydrous sodium sulfate, and
concentrated to give 6a (1.5 g, 88%) as a white solid.
1H NMR (DMSO-d6) d 11.02 (s, 1H, D2O exchange-
able), 7.48 (d, 1H, J = 1.77 Hz), 7.14 (dd, 1H, J = 8.25,
1.80 Hz), 6.91 (d, 1H, J = 8.24 Hz), 4.77 (m, 1H), 2.8
(m, 2 H), 2.23 (m, 2H), 1.79 (m, 2H). MS (ESI) m/z
267 ([M+H]+).
The following compounds (9–12) were prepared by fol-
lowing the procedures of compound 8.
4.4. 6-Bromo-1-cyclopentyl-1,3-dihydro-2H-benzimidazol-
2-one (6b)
The title compound was prepared from 5-bromo-2-
oxo-2,3-dihydro-benzimidazole-1-carboxylic acid tert-
butyl ester and cyclopentanol as an off-white solid
(2.1 g, 85%). 1H NMR (DMSO-d6) d 11.0 (s, 1H),
7.32 (d, 1H, J = 1.79 Hz), 7.13 (dd, 1 H, J = 8.22,
1.87 Hz), 6.92 (d, 1H, J = 8.25 Hz), 4.68 (m, 1H), 2.0
(m, 2H), 1.88 (m, 4H), 1.64 (m, 2H). MS (ESI) m/z
281 ([M+H]+).

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
6561
4.5. 6-(3-Chloro-4-fluorophenyl)-1-cyclopentyl-1,3-dihy-
dro-2H-benzimidazol-2-one (7b)
64%). 1H NMR (DMSO-d6) d 10.92 (s, 1H, D2O
exchangeable), 7.88 (dd, 1H, J = 7.15, 2.34 Hz), 7.67
(m, 1H), 7.48 (m, 2H), 7.28 (dd, 1H, J = 8.19,
1.69 Hz), 7.04 (d, 1H, J = 8.06 Hz), 4.25 (m, 1H),
2.08–2.18 (m, 2H), 1.73–1.82 (m, 2H), 0.76 (t, 6H,
J = 7.40 Hz). MS (ESI) m/z 333 ([M+H]+).
The title compound was prepared from 6b and 3-chloro-4-
fluorophenyl boronic acid as a white solid (0.67 g, 44%).
1H NMR (DMSO-d6) d 10.93 (s, 1H, D2O exchangeable),
7.87 (dd, 1H, J = 7.15, 2.34 Hz), 7.64–7.68 (m, 1H), 7.47
(t, 1H, J = 9.10 Hz), 7.41 (d, 1H, J = 1.55 Hz), 7.28 (dd,
1H, J = 8.06, 1.69 Hz), 7.04 (d, 1H, J = 8.06 Hz), 4.72–
4.82 (m, 1H), 2.09–2.18 (m, 2H), 1.83–1.92 (m, 4H),
1.60–1.73 (m, 2H). MS (ESI) m/z 331 ([M+H]+).
4.11. 6-(3-Chloro-4-fluorophenyl)-1-(1-ethylpropyl)-1,3-
dihydro-2H-benzimidazole-2-thione (11)
The title compound was prepared from 7d and Lawes-
son’s reagent as an off-white solid (0.11 g, 55%). 1H
NMR (DMSO-d6) d 12.86 (s, 1H, D2O exchangeable),
7.93 (d, 1H, J = 5.20 Hz), 7.72 (m, 2H), 7.48 (m, 2H),
8.18 (d, 1H, J = 8.18 Hz), 5.17 (m, 1H), 2.12–2.21 (m,
2H), 1.90–1.99 (m, 2H), 0.74 (t, 6H, J = 7.27 Hz). MS
(ESI) m/z 349 ([M+H]+). Anal. Calcd for
C18H18ClFN2S: C, 61.97; H, 5.20; N, 8.03. Found: C,
61.76; H, 5.09; N, 7.84.
4.6. 6-(3-Chloro-4-fluorophenyl)-1-cyclopentyl-1,3-dihy-
dro-2H-benzimidazole-2-thione (9)
The title compound was prepared from 7b and Lawes-
son’s reagent as an off-white solid (89 mg, 46%). 1H
NMR (DMSO-d6) d 12.88 (s, 1H, D2O exchangeable),
7.91 (dd, 1H, J = 7.02, 2.34 Hz), 7.68–7.72 (m, 1H),
7.45–7.56 (m, 3H), 7.27 (d, 1H, J = 8.31 Hz), 5.48–5.56
(m, 1H), 2.22–2.33 (m, 2H), 1.92–2.06 (m, 4H), 1.67–
1.78 (m, 2H). MS (ESI) m/z 347 ([M+H]+).
4.12. 3-[3-(1-Ethylpropyl)-2-oxo-2,3-dihydro-1H-benzim-
idazol-5-yl]-5-fluorobenzonitrile (7e)
4.7. 1-Cyclopentyl-6-(3,4-difluorophenyl)-1,3-dihydro-2H-
benzimidazol-2-one (7c)
The title compound was prepared from 6c and 3-cyano-
5-fluorophenyl boronic acid as a white solid (0.55 g,
44%). 1H NMR (DMSO-d6) d 11.04 (s, 1H, D2O
exchangeable), 8.10 (s, 1H), 7.95 (m, 1H), 7.77 (m,
1H), 7.62 (s, 1H), 7.42 (dd, 1H, J = 8.13, 1.51 Hz),
7.07 (d, 1H, J = 8.15 Hz), 4.16 (m, 1H), 2.05–2.15 (m,
2 H), 1.72–1.81 (m, 2H), 0.76 (t, 6H, J = 7.32 Hz). MS
(ESI) m/z 324 ([M+H]+). Anal. Calcd for
C19H18FN3OÆ0.2EtOH: C, 70.1; H, 5.76; N, 12.64.
Found: C, 70.28; H, 5.73; N, 12.39.
The title compound was prepared from 6b and 3,4-diflu-
orophenyl boronic acid as a white solid (0.87 g, 54%).
1H NMR (DMSO-d6) d 10.93 (s, 1H, D2O exchange-
able), 7.75 (m, 1H), 7.5 (m, 2H), 7.40 (d, 1H, J = 1.56
Hz), 7.29 (dd, 1H, J = 8.06, 1.69 Hz), 7.04 (d, 1H,
J = 8.06 Hz), 4.78 (m, 1H), 2.13 (m, 2H), 1.91 (m, 4H),
1.65 (m, 2H). MS (ESI) m/z 315 ([M+H]+).
4.8. 1-Cyclopentyl-6-(3,4-difluorophenyl)-1,3-dihydro-
2H-benzimidazole-2-thione (10)
4.13. 3-[3-(1-Ethylpropyl)-2-thioxo-2,3-dihydro-1H-ben-
zimidazol-5-yl]-5-fluorobenzonitrile (12)
The title compound was prepared from 7c and Lawes-
son’s reagent as an off-white solid (0.12 g, 67%). 1H
NMR (DMSO-d6) d 12.88 (s, 1H, D2O exchangeable),
7.81 (m, 1H), 7.47–7.56 (m, 4H), 7.27 (d, 1H,
J = 8.18 Hz), 5.53 (m, 1H), 2.23 (m, 2H), 1.97 (m, 4H),
1.73 (m, 2H). MS (ESI) m/z 331 ([M+H]+). Anal. Calcd
for C18H16F2N2S: C, 65.44; H, 4.88; N, 8.48. Found: C,
65.07; H, 4.98; N, 8.30.
The title compound was prepared from 7e and Lawes-
son’s reagent as an off-white solid (0.13 g, 46%). 1H
NMR (DMSO-d6) d 12.91 (s, 1H, D2O exchangeable),
8.14 (s, 1H), 8.00 (d, 1H, J = 10.50 Hz), 7.85 (m, 2H),
7.61 (dd, 1H, J = 8.32, 1.15 Hz), 7.29 (d, 1H,
J = 8.20 Hz), 5.17 (m, 1H), 2.12–2.21 (m, 2H), 1.91–
2.03 (m, 2H), 0.72 (t, 6H, J = 7.31 Hz). MS (ESI) m/z
340 ([M+H]+). Anal. Calcd for C19H18FN3SÆ0.5H2O:
C, 65.49; H, 5.5; N, 12.06. Found: C, 65.17; H, 5.2; N,
11.76.
4.9. 6-Bromo-1-(1-ethylpropyl)-1,3-dihydro-2H-benz-
imidazol-2-one (6c)
4.14. 5-(3-Cyclopentyl-2-oxo-2,3-dihydro-1H-benzim-
idazol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (7g)
The title compound was prepared from 5-bromo-2-oxo-
2,3-dihydro-benzimidazole-1-carboxylic acid tert-butyl
1
ester and 3-pentanol as a white solid (1.9 g, 73%). H
To a stirred solution of N-methyl-2-cyanopyrrole
(0.25 g, 2.32 mmol) and triisopropylborate (0.55 mL,
2.38 mmol) in THF (5 mL) at 0 ꢁC under nitrogen was
added lithium diisopropyl amide (2.0 M in THF,
1.5 mL, 3 mmol) in a dropwise manner. After stirred
for 1 h, the solution was treated with 6-bromo-1-cyclo-
penyl-1,3-dihydro-2H-benzimidazol-2-one (6b, 0.33 g,
1.16 mmol), glyme (5 mL), sodium carbonate (0.37 g,
3.48 mmol) dissolved in water (2 mL), and tetrakis(tri-
phenylphosphine) palladium (0) (0.07 g, 0.06 mmol).
The resulting mixture was heated at 70 ꢁC for 3 h and
cooled to ambient temperature. A saturated ammonium
NMR (DMSO-d6) d 11.03 (s, 1H, D2O exchangeable),
7.39 (d, 1H, J = 1.80 Hz), 7.12 (dd, 1H, J = 8.24,
1.81 Hz), 6.93 (d, 1H, J = 8.24 Hz), 4.05 (m, 1H),
1.90–2.02 (m, 2 H), 1.72–1.83 (m, 2H), 0.72 (t, 6H,
J = 7.36 Hz). MS (ESI) m/z 283 ([M+H]+).
4.10. 6-(3-Chloro-4-fluorophenyl)-1-(1-ethylpropyl)-1,3-dihy-
dro-2H-benzimidazol-2-one (7d)
The title compound was prepared from 6c and 3-chloro-
4-fluorophenyl boronic acid as a white solid (0.77 g,

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P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
chloride aqueous solution (50 mL) and ethyl acetate
(100 mL) was added. The organic layer was separated,
dried over magnesium sulfate, and concentrated. The
residue was purified via a silica gel column (50% ethyl
acetate in hexane) to afford 7g as an off-white solid
2H), 1.98–2.07 (m, 1H), 1.82–1.91 (m, 1H). MS (ESI)
m/z 309 ([M+H]+).
4.18. 5-[3-(1-Ethylpropyl)-2-oxo-2,3-dihydro-1H-benzim-
idazol-5-yl]-1-methyl-1H-pyrrole-2-carbonitrile (7h)
1
(0.12 g, 34%). H NMR (DMSO-d6) d 11.02 (s, 1H),
7.23 (d, 1H, J = 1.17 Hz), 7.06–7.12 (m, 2H), 7.03 (d,
1H, J = 4.03 Hz), 6.31 (d, 1H, J = 4.03 Hz), 4.72 (q,
1H, J = 8.6 Hz), 3.72 (s, 3H), 2.05–2.10 (m, 2H), 1.85–
1.93 (m, 4H), 1.62–1.66 (m, 2H). MS (ESI) m/z 307
([M+H]+). HRMS: Calcd for C18H18N4O + H+,
307.15534. Found (ESI_FT [M+H]+): 307.15508.
The title compound was prepared from 6c and N-
methyl-2-cyanopyrrole as an off-white solid (0.36 g,
48%). H NMR (DMSO-d6) d 11.00 (s, 1H), 7.30 (s,
1H), 7.06–7.11 (m, 2H), 7.01 (d, 1H, J = 4.03 Hz), 6.30
(d, 1H, J = 4.03 Hz), 4.02–4.13 (m, 1H), 3.71 (s, 3H),
1.97–2.05 (m, 2H), 1.74–1.81 (m, 2H), 0.73 (t, 6H,
J = 7.41 Hz). MS (ESI) m/z 309 ([M+H]+). HRMS:
Calcd for C18H20N4O + H+, 309.17099. Found (ESI_FT
[M+H]+): 309.17076.
1
4.15. 5-(3-Cyclopentyl-2-thioxo-2,3-dihydro-1H-benzim-
idazol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (14)
To a stirred solution of 5-(3-cyclopentyl-2-oxo-2,3-
dihydro-1H-benzimidazol-5-yl)-1-methyl-1H-pyrrole-2-
carbonitrile ( 0.10 g, 0.33 mmol) in toluene (7 mL) was
added Lawesson’s reagent (0.19 g, 0.47 mmol) and the
resulting solution was heated to 100 ꢁC overnight.
The solution was allowed to cool, partitioned between
saturated ammonium chloride solution (50 mL) and
ethyl acetate (80 mL). The organic layer was separated,
dried over magnesium sulfate, and concentrated. The
residue was purified via a silica gel column using a
10–20% ethyl acetate in hexane gradient to afford 14
4.19. 5-[3-(1-Ethylpropyl)-2-thioxo-2,3-dihydro-1H-ben-
zimidazol-5-yl]-1-methyl-1H-pyrrole-2-carbonitrile (15)
The title compound was prepared from 7h and Lawes-
son’s reagent a yellowish solid (0.088g, 63%). 1H
NMR (DMSO-d6) d 12.92 (s, 1H), 7.60 (s, 1H), 7.29
(s, 2H), 7.05 (d, 1H, J = 4.03 Hz), 6.36 (d, 1H, J = 4.03
Hz), 5.10–5.20 (m, 1H), 3.72 (s, 3H), 2.02–2.10 (m,
2H), 1.89–1.95 (m, 2H), 0.71 (t, 6H, J = 7.41 Hz). MS
(ESI) m/z 325 ([M+H]+). HRMS: Calcd for
C18H20N4S + H+,
325.14814.
Found
(ESI_FT
as
a
yellowish solid (0.035 g, 33%). 1H NMR
[M+H]+): 325.14805.
(DMSO-d6) d 12.95 (s, 1H), 7.44 (s, 1H), 7.28–7.32
(m, 2H), 7.05 (d, 1H, J = 4.03 Hz), 6.39 (d, 1H,
J = 4.03 Hz), 5.54 (q, 1H, J = 9.0 Hz), 3.73 (s, 3H),
2.10–2.19 (m, 2H), 1.95–1.99 (m, 4H), 1.70–1.73 (m,
2H). MS (ESI) m/z 323 ([M+H]+). HRMS: Calcd for
4.20. Rat decidualization assay
4.20.1. Reagents. Test compounds were dissolved in
100% ethanol and mixed with corn oil (vehicle). Stock
solutions of the test compounds in oil (MazolaTM) were
then prepared by heating (ꢀ80 ꢁC) the mixture to evap-
orate ethanol. Test compounds were subsequently di-
luted with 100% corn oil or 10% ethanol in corn oil
prior to the treatment of animals. No difference in decid-
ual response was found when these two vehicles were
compared.
C18H18N4S + H+,
323.13249.
Found
(ESI_FT
[M+H]+): 323.13252. Anal. Calcd for C18H18N4S: C,
67.05; H, 5.63; N, 17.38. Found: C, 66.71; H, 5.43;
N, 17.29.
The following compounds (13 and 15) were prepared by
following the procedures of compound 14.
4.16. 5-(3-Cyclobutyl-2-oxo-2,3-dihydro-1H-benzimida-
zol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (7f)
4.20.2. Animals. Ovariectomized mature female Spra-
gue–Dawley rats (ꢀ60-day-old and 230 g) were obtained
from Taconic (Taconic Farms, NY) following surgery.
Ovariectomy was performed at least 10 days prior to
treatment to reduce circulating sex steroids. Animals
were housed under 12 h light/dark cycle and given stan-
dard rat chow and water ad libitum.
The title compound was prepared from 6a and N-
methyl-2-cyanopyrrole as an off-white solid (0.23g,
44%). 1H NMR (DMSO-d6) d 11.02 (s, 1H, D2O
exchangeable), 7.40 (s, 1H), 7.02–7.12 (m, 3H), 6.32
(dd, 1H, J = 4.03 Hz), 4.82–4.88 (m, 1H), 3.73 (s, 3H),
2.81–2.91 (m, 2H), 2.40–2.49 (m, 2H), 1.82–1.92 (m,
1H), 1.72–1.81 (m, 1H). MS (ESI) m/z 293 ([M+H]+).
Anal. Calcd for C17H16N4OÆ0.5H2O: C, 67.76; H, 5.69;
N, 18.59. Found: C, 67.82; H, 5.42; N, 18.21.
4.20.3. Treatment. Rats were weighed and randomly as-
signed to groups of 4 or 5 before treatment. Test com-
pounds in 0.2 mL vehicle were administered by
subcutaneous injection in the nape of the neck or by ga-
vage using 0.5 mL. The animals were treated once daily
for seven days. For testing antiprogestins, animals were
given the test compounds and an EC50 dose of proges-
terone (5.6 mg/kg) during the first 3 days of treatment.
Following decidual stimulation, animals continued to
receive progesterone until necropsy 4 days later.
4.17. 5-(3-Cyclobutyl-2-thioxo-2,3-dihydro-1H-benzimi-
dazol-5-yl)-1-methyl-1H-pyrrole-2-carbonitrile (13)
The title compound was prepared from 7f and Lawes-
son’s reagent a yellowish solid (0.11 g, 67%). H NMR
1
(DMSO-d6) d 12.95 (s, 1H, D2O exchangeable), 7.79
(d, 1H, J = 0.91 Hz), 7.27–7.34 (m, 2H), 7.06 (d, 1H,
J = 4.03 Hz), 6.41 (d, 1H, J = 4.03 Hz), 5.62–5.71 (m,
1H), 3.75 (s, 3H), 2.91–3.03 (m, 2H), 2.38–2.49 (m,
4.20.4. Dosing. Doses were prepared based upon mg/kg
mean group body weight. In all studies, a control group
receiving vehicle was included. Determination of dose–

P. Zhang et al. / Bioorg. Med. Chem. 15 (2007) 6556–6564
6563
response curves was carried out using doses with half-
log increases (e.g., 0.1, 0.3, 1.0, 3.0 mg/kg. . .).
4.21.4. Assays uterine complement component C3. Fol-
lowing euthanasia, the uteri are removed from the ani-
mals, stripped of remaining fat and mesentary, and
weighed. The uteri are snap-frozen on dry ice in groups
of two. Total RNA is extracted using TRIzol reagent
(Gibco-BRL). RNA samples are run on a 1% agarose/
formaldehyde gel. The nucleic acids are then transferred
to a nylon membrane overnight by capillary action. The
nucleic acids are crosslinked to the membrane using UV
light, the 28S rRNA is quantitated using the IP Lab Gel
software (Signal Analytics Corp.), and then the blot is
hybridized with a cDNA probe for complement compo-
nent C3. Following hybridization, the blot is exposed to
a phosphorscreen. Quantitation of C3 message is per-
formed using a phosphorimager (Molecular Dynamics).
4.20.5. Decidual induction. Approximately 24 h after the
third injection, decidualization was induced in one of the
uterine horns by scratching the antimesometrial luminal
epithelium with a blunt 21 G needle. The contralateral
horn was not scratched and served as an unstimulated
control. Approximately 24 h following the final treat-
ment, rats were sacrificed by CO2 asphyxiation and
body weight measured. Uteri were removed and
trimmed of fat. Decidualized (D-horn) and control (C-
horn) uterine horns were weighed separately.
4.20.6. Analysis of results. The increase in weight of the
decidualized uterine horn was calculated by D-horn/C-
horn and logarithmic transformation was used to maxi-
mize normality and homogeneity of variance. The Huber
M-estimator was used to down weight the outlying
transformed observations for both dose–response curve
fitting and one-way analysis of variance. JMP software
(SAS Institute, Inc.) was used for both one-way ANO-
VA and non-linear dose–response analyses.
4.21.5. Analysis of results. Results are reported as the
ratio C3/28S. These ratios are transformed by loga-
rithms to normalize the data. The Huber M-estimator
is used to down weight the outlying transformed obser-
vations. The JMP software (SAS Institute, Inc.) is used
to analyze the transformed and weighted data for both
the one-way ANOVA and the non-linear dose–re-
sponse curves. The IC50 values with 95% confidence
intervals are calculated using a four parameter logistic
model that calculates minimum, maximum, slope, and
IC50.
4.21. Rat uterine C3 model
4.21.1. Reagents. Stock solutions of the test compounds
are prepared in 100% ethanol or 100% DMSO if they are
not soluble in ethanol. The compounds are prepared for
dosing in 10% ethanol in corn oil (MazolaTM) vehicle.
4.22. Primate ovulation inhibition assay
The ovulation inhibition assay in cynomolgus monkeys
was carried out as described27 with modifications.
Briefly, animals were treated with vehicle (2% Tween
80/0.5% methylcellulose) or test compounds orally from
day 2 to 28 of the menstrual cycle. Blood samples were
taken every Monday, Wednesday, and Friday until next
menses for progesterone measurement. The occurrence
of ovulation was inferred from the pattern of progester-
one during the menstrual cycles.
4.21.2. Animals. Ovariectomized-female, 60-day-old
Sprague–Dawley rats are obtained following surgery
(Harlan). The surgeries are to be done a minimum of
8 days prior to the first treatment. The animals are
housed under 12 h light/dark cycle. In some of the assay
validation studies, the animals were fed standard rat
chow and water ad libitum, while in others the animals
were fed the casein-based Laboratory Rodent Diet 5K96
(Purina) and water ad libitum. All future studies will be
run using the 5K96 diet.
Acknowledgments
4.21.3. Treatment. Upon arrival the rats are random-
ized, placed in groups of six, and given a minimum
of 72 h to acclimate to the surroundings. They are then
treated once a day for two days with the compound(s)
of interest or vehicle (vehicle control group). Adminis-
tration of the compound is either by subcutaneous
injection of 0.2 mL in the nape of the neck or orally
by gavage of 0.5 mL. On the second day, the animals
are co-treated with 17a-ethinyl estradiol (EE) or vehicle
(vehicle control group), orally by gavage, 0.5 mL per
dose dosing: two control groups are included in all
analyses: a vehicle group and an EE group. Doses
are prepared based on mg/kg mean group body weight.
Initial screening of test compounds is done at three
doses (e.g., 0.03, 0.3, 3 mg/kg body weight). Dose–re-
sponse curves may be run on active compounds using
doses with half-log increments over a dose range deter-
mined from the initial screening data. Approximately
24 h following the final treatment the animals are killed
by CO2 asphyxiation and the body weight and uterine
wet weight determined.
We thank Drs. Ron Magolda and Magid Abou-Gharbia
for their support, Department of Analytical Chemistry
for analytical data, Mei Wu and Wyeth Pearl River pri-
mate center for their technical assistance.
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