Discovery of non-steroidal mifepristone mimetics: Pyrazoline-based PR antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.05.001

Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor (PR

Full text of DOI:10.1016/j.bmcl.2005.05.001

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3203–3206
Discovery of non-steroidal mifepristone mimetics:
Pyrazoline-based PR antagonists
David G. Jones, Xi Liang, Eugene L. Stewart, Robert A. Noe, Lara S. Kallander,
Kevin P. Madauss, Shawn P. Williams, Scott K. Thompson,
David W. Gray and William J. Hoekstra*
GlaxoSmithKline, Research Triangle Park, NC 27709-3398, USA
Received 28 March 2005; revised 28 April 2005; accepted 3 May 2005
Available online 31 May 2005
Abstract—Mifepristone is a non-selective antagonist of 3-oxosteroid receptors with both abortifacient and anti-endometriotic activ-
ities. Non-steroidal mimetics of mifepristone and progesterone are important templates for modulation of the progesterone receptor
(PR). For our PR program, we sought an unexplored, synthetically accessible non-steroidal mimetic of mifepristone, suitable for
parallel synthesis of analogues. Docking of compounds into a PR homology model identified 4-substituted pyrazolines, which, when
synthesized and tested, exhibited functional antagonism of PR.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Progesterone receptor (PR) antagonists have been tar-
gets of medicinal chemistry programs for decades.1
Mifepristone (RU-486), a clinically approved PR antag-
onist, effectively terminates pregnancy and offers thera-
peutic promise for endometriosis, uterine fibroids, and
breast cancer.2 PR, a member of the nuclear receptor
superfamily of ligand-dependent transcription factors,
undergoes a conformational change upon ligand binding
to initiate a cascade of regulatory events with its target
genes.3 Steroidal PR agonists, in combination with
estrogens, have been widely used in oral contraception
and hormone replacement therapy to oppose estrogen-
mediated endometrial cancer risk, yet present potential
breast cancer risk with long-term dosing.4 Thus, there
is a need for selective PR modulators to oppose uterine
proliferative events while improving safety indices. Non-
steroidal PR antagonists/modulators have been identi-
fied with desirable reproductive tissue safety profiles in
animal models.5,6
Mifepristone was docked into a PR antagonist homol-
ogy model generated from the crystal structure of PR
complexed to the endogenous agonist progesterone7
and ERa complexed to the antagonist tamoxifen8 using
the program MVP.9 The results suggested that the
N,N-dimethylaniline moiety of this compound was
responsible for driving the receptor into an antagonist
conformation through displacement of the AF2 helix.
*
Corresponding author. Tel.: +1 919 483 8212; fax: +1 919 315 0430; e-mail: william.j.hoekstra@gsk.com
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.05.001

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D. G. Jones et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3203–3206
In our medicinal chemistry program, we aimed to intro-
duce a variety of substituents to a suitable core, using
solution-phase parallel synthesis techniques, to mimic
the conformational changes induced by mifepristone.
The approach resulted in the design of diarylpyrazo-
lines, a previously unexplored PR chemotype. Related
pyridazine-based PR agonists have been disclosed with-
out description of their possible binding mode.10,11 Syn-
thetic, array-based approaches to the diarylpyrazolines
were developed to provide rapid access to both 4- and
5-substituted targets that probe the PR antagonist pock-
et (e.g., 1).
Docking of these pyrazoline target molecules into PR
co-crystal structures (with agonists progesterone,6 nor-
ethindrone,12 and mometasone furoate12) and the mife-
pristone homology model suggested that substituted
pyrazolines might yield antagonist activity through dis-
placement of the AF2 helix. 4- and 5-Aryl-pyrazolines
(R2 and R3 of 1, respectively) were initially proposed.
Based on our in silico results, the 3-aryl ring of the
pyrazoline overlays suitably with the A-ring of mifepri-
stone, the pyrazoline ring acts as a C-ring moiety, and
Figure 1. Progesterone receptor ligand binding domain overlay of
mifepristone (magenta) with pyrazoline 7b (cyan) docked into the PR
antagonist homology model.
geted pyrazolines required an a-substituted-a,b-unsatu-
rated aryl ketone (e.g., 5) to furnish variably
substituted tricyclic intermediate 6 (Eq. 1). Synthetic
details are presented below.
the benzenesulfonamide portion extends into
a
17a-pocket previously observed in the crystal structure
of PR bound with mometasone furoate12 (Fig. 1). The
R-configured R2 group occupies a position in the recep-
tor binding pocket very similar to that observed with the
N,N-dimethylaniline group of mifepristone (Fig. 1).
Based on SAR reported for the related pyridazine
Racemic pyrazoline target 7a was prepared as follows
(Eq. 1).14 Acid 2 was converted to its corresponding acid
chloride with oxalyl chloride followed by Weinreb
amide preparation to furnish 3. The amide 3 was
converted to the benzyl ketone 4 by treatment with
3,4-dichlorophenylmagnesium bromide in THF. Con-
densation of 4 with aqueous formaldehyde in methanol
afforded the a,b-unsaturated cyclization substrate 5. The
desired pyrazoline 7a was then obtained by cyclo-
condensation of 5 with aqueous hydrazine, followed
by in situ sulfonylation of the crude pyrazoline product
series,10,11
prototype
4-chlorophenylsulfonamide
pyrazoline analogues were synthesized bearing the
3,4-dichlorophenyl substituent to mimic the A-ring of
progesterone.
The cyclocondensation of a,b-unsaturated aryl ketones
with hydrazine to afford pyrazolines is well known.13
The key cyclization step in the synthesis of our PR-tar-
ð1Þ

D. G. Jones et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3203–3206
Table 1. PR binding and functional profile of pyrazolines
3205
those of the steroidal PR antagonist mifepristone. The
parallel synthesis of substituted pyrazolines represents
a general methodology for the discovery of PR antago-
nist analogues. 4-Aryl-pyrazolines were proposed to mi-
mic the antagonistic interaction of mifepristoneÕs N,N-
dimethylaniline in the PR ligand binding pocket. This
hypothesis was later confirmed through solution of a
PR co-crystal structure bound with a representative pyr-
azoline (data not shown).19 X-ray crystallographic anal-
ysis and homology modeling of these new agents
indicate that interaction of the AF2 helix with R-config-
ured appendages at four positions of the pyrazoline core
can modulate functional PR antagonist efficacy.
Compound R2
R3
PR
bindinga
pKi
CV-1 cellb
pIC50
7a
7b
(R/S)-4-Me2NPh
H
H
H
7.4
7.0
6.1 (95%)
5.8 (96%)
<5.5
R-4-Me2NPh
S-4-Me2NPh
H
7c
<5.0
8
9a
(R/S)-Me 7.4
6.7 (72%)
NT
(R/S)-Me
R-Me
S-Me
H
H
H
7.4
7.7
5.7
Acknowledgments
9b
9c
7.6 (51%)
5.3 (48%)
6.4 (75%)
NT
10
11
H
(R/S)-Ph
(R/S)-Ph 5.1
We thank Karl Erhard for assistance in chromato-
graphic separation of enantiomers and Douglas Minick
for determinations of absolute configuration.
H
7.0
8.0
RU-486
9.6 (100%)
a Assay measures compound interaction with the ligand binding
domain of PR by displacement of a proprietary fluorescent ligand
(n = 2, SD = 0.25).17
b Assay measures inhibition of progesterone-stimulated (4 nM) trans-
activation of BacMam expressed human PR-B in CV-1 cells using an
MMTV-Luc reporter (n = 2, SD = 0.20). Mifepristone antagonist
efficacy = 100%.18
References and notes
1. Ashok, P. W.; Wagaarachchi, P. T.; Templeton, A. Curr.
Med. Chem. Immun. Endocr. Metab. Agents 2002, 2, 71.
2. Hess-Stumpp, H.; Hoffmann, J.; Fuhrmann, U. Drugs
Future 2002, 27, 1113.
3. Spitz, I. M.; Chwalisz, K. Steroids 2000, 65, 807.
4. Pike, M. C.; Ross, R. K. Steroids 2000, 65, 659.
5. Zhi, L.; Tegley, C. M.; Pio, B.; Edwards, J. P.; Motamedi,
M.; Jones, T. K.; Marschke, K. B.; Mais, D. E.; Risek, B.;
Schrader, W. T. J. Med. Chem. 2003, 46, 4104.
6. Zhi, L.; Ringgenberg, J. D.; Edwards, J. P.; Tegley, C. M.;
West, S. J.; Pio, B.; Motamedi, M.; Jones, T. K.;
Marschke, K. B.; Mais, D. E.; Schrader, W. T. Bioorg.
Med. Chem. Lett. 2003, 13, 2075.
7. Williams, S. P.; Sigler, P. B. Nature 1998, 393, 392.
8. Shiau, A. K.; Barstad, D.; Loria, P. M.; Cheng, L.; Kushner,
P. J.; Agard, D. A.; Greene, G. L. Cell 1998, 95, 927.
9. Lambert, M. H. Practical Application of Computer-Aided
Drug Design; Marcel-Dekker: New York, 1997; pp. 243–
303.
10. Palmer, S.; Campen, C. A.; Allan, G. F.; Rybczynski, P.;
Johnson, D. H.; Hutchins, A.; Kraft, P.; Kiddoe, M.; Lai,
M.-T.; Lombardi, E.; Pedersen, P.; Hodgen, G.; Combs,
D. W. J. Steroid Biochem. Mol. Biol. 2001, 75, 33.
11. Combs, D. W.; Reese, K.; Cornelius, L. A. M.; Gunnett, J.
W.; Cryan, E. V.; Granger, K. S.; Jordan, J. J.; Demarest,
K. T. J. Med. Chem. 1995, 38, 4880.
12. Protein Data Bank number 1sr7. For a reference, see:
Madauss, K. P.; Deng, S.-J.; Austin, R. J. H.; Lambert,
M. H.; McLay, I.; Pritchard, J.; Short, S.; Stewart, E. L.;
Uings, I.; Williams, S. P. J. Med. Chem. 2004, 47, 3381.
13. Levai, A. J. Heterocyclic Chem. 2002, 39, 1, and references
cited therein.
14. Compound 7a was synthesized from 2 as follows: 4-N,N-
dimethylaminophenylacetic acid (2, 3.0 g, 16.8 mmol) was
dissolved in DCM (100 ml). Oxalyl chloride (3.2 g,
25.1 mmol) and two drops of DMF were added and the
reaction was stirred for 1 h at rt. The solvent was
evaporated and the residue was re-dissolved in DCM
(20 ml) and evaporated. N,O-dimethylhydroxylamine
hydrochloride (1.79 g, 18.4 mmol) was suspended in
DCM (50 ml) with addition of pyridine (7 ml). The acid
chloride was dissolved in DCM (50 ml) and added
dropwise to the amine solution at 0 ꢁC. The resulting
6 with 4-chlorophenylsulfonyl chloride. Select racemic
products were purified on a chiral column to isolate
the respective enantiomers (Table 1).15 The absolute
configurations of enantiomers 9b and 9c were deter-
mined by vibrational circular dichroism.16
Pyrazoline sulfonamides 7a–11 are representatives of ar-
rays of 4- and 5-substituted targets that were synthesized
and tested for receptor binding as well as functional
activity in CV-1 cells (Table 1). Racemic 4-substituted
analogues 7a, 9a, and 11 (R2 = 4-Me2NPh, Me, and
Ph) exhibited strong affinity for PR (pKi = 7.0–7.4). Fur-
thermore, enantiomerically enriched, R-R2 configured
products bound PR with high affinity in agreement with
the proposed overlay with the N,N-dimethylaniline of
the steroid RU-486; S-R2-substituted products (7c, 9c)
showed low PR binding affinity. Since PR affinity of
racemate 9a resides predominantly in the assigned R-
enantiomer 9b, by analogy, we proposed that the enan-
tiomer 7b also bears the N,N-dimethylaniline group in
the R-configuration (Table 1). Also as predicted by
homology modeling, these compounds profiled as antag-
onists, inhibiting progesterone-stimulated PR activity in
cells consistent with their binding affinities (pIC50 = 5.8–
7.6). 5-substituted analogues 8 and 10 (R3 = Me, Ph)
exhibited partial antagonism of PR reporter activity
(72–75% efficacy of RU-486). High affinity pyrazolines
7b and 9b exhibited >10-fold steroid receptor selectivity
over the androgen and glucocorticoid receptors (data
not shown).
In summary, we have described the discovery of novel
pyrazolines as progesterone receptor ligands. Mifepri-
stone mimetic 7b exhibits high affinity for the progester-
one receptor and a functional profile comparable to

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D. G. Jones et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3203–3206
mixture was warmed to rt and stirred for 2 h. The solvent
solution was washed with 1 N HCl (aq), satd NaHCO3
(aq), and brine, dried over MgSO4, and concentrated in
vacuo. The residue was purified by chromatography on
silica gel eluted with EtOAc/hexane (15/85) to give
pyrazoline 7a.
was then evaporated and the residue was re-dissolved in
EtOAc. The organic solution was washed with 1 N HCl
(aq), satd NaHCO3 (aq), and brine, dried over MgSO4,
and concentrated in vacuo. The residue was purified by
chromatography on silica gel eluted with EtOAc/hexane
(40/60) to give 3. To a solution of 3 (1.6 g, 7.2 mmol) in
THF (50 ml) was added 3,4-dichlorophenylmagnesium
bromide (0.5 M in THF, 17.3 ml, 8.6 mmol) dropwise over
30 min at 0 ꢁC. The resulting mixture was stirred for 1 h,
poured into 1 N HCl (aq), and extracted with three
portions of EtOAc. The combined organic layers were
washed with satd NaHCO3 (aq) and brine, dried over
MgSO4, and concentrated in vacuo. The residue was
purified by chromatography on silica gel eluted with
EtOAc/hexane (7/93) to give 4. To a solution of 4 (0.30 g,
4.5 mmol) in MeOH (15 ml) were added formaldehyde
(37% aq. solution, 4 ml) and piperidine (1 ml). The
solution was heated at reflux for 1 h. The solvent was
evaporated and the residue was re-dissolved in a small
amount of EtOAc and purified by chromatography on
silica gel eluted with EtOAc/hexane (5/95) to give 5. To a
solution of 5 (180 mg, 0.56 mmol) in EtOH (25 ml) was
added hydrazine hydrate (0.113 g, 2.25 mmol). The reac-
tion was heated at reflux for 3 h. After cooling to rt, the
solvent was evaporated and the crude product 6 was re-
dissolved in DCM (25 ml). Pyridine (0.09 g, 1.12 mmol),
catalytic DMAP, and 4-chlorobenzenesulfonyl chloride
(0.24 g, 1.12 mmol) were added, and the resulting solution
was stirred at rt for 2 h. The solvent was then evaporated
and the residue was dissolved in EtOAc. The organic
15. Enantiomerically enriched products were isolated from
racemates using a 10-lm Chiralpak AD stationary phase
(21 · 250 mm) and 10% MeOH/water mobile phase at a
flow rate of 50 ml/min with detection at 254 nm.
16. The absolute configurations of 9b (assigned as R-enantio-
mer) and 9c (assigned as S-enantiomer) were determined
on a Bomem Chiral-IR Vibrational Circular Dichroism
spectrometer operated at 4 cmꢀ1 resolution in carbon
tetrachloride at a concentration of 20 mg/ml. For a
reference, see: Devlin, F. J.; Stephens, P. J.; Osterle, C.;
Wiberg, K. B.; Cheeseman, J. R.; Frisch, M. J. J. Org.
Chem. 2002, 67, 8090, and references cited therein.
17. The PR fluorescence polarization binding assay measures
compound interaction with the ligand binding domain of
PR (590 nm) by displacement of a fluorescent steroidal
progesterone mimetic (Panvera catalog number P2964;
Kd = 10 nM). The response is expressed as a pKi.
18. The functional assay measures compound-mediated inter-
action of the PR-B isoform with the MMTV luciferase
reporter to calculate compound potency and efficacy in
BacMam transduced, progesterone-stimulated (4 nM)
CV-1 cells. The antagonist response is expressed as a
pIC50 (RU-486 pIC50 = 9.8, 100% efficacy). Comparable
potencies and efficacies were observed for a limited set of
compounds in a T47D cell alkaline phosphatase assay.
19. Details will be reported in a forthcoming full paper.