Discovery of orally active, pyrrolidinone-based progesterone receptor partial agonists

Article abstract of DOI:10.1016/j.bmcl.2009.06.081

We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial agonists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and in vivo potency compared to the lead compound. In addi

Full text of DOI:10.1016/j.bmcl.2009.06.081

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4664–4668
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of orally active, pyrrolidinone-based progesterone receptor
partial agonists
a
a
a
a
David G. Washburn a, , Tram H. Hoang , James S. Frazee , Latisha Johnson , Marlys Hammond ,
Sharada Manns a, Kevin P. Madauss d, Shawn P. Williams d, Chaya Duraiswami e, Thuy B. Tran d,
Eugene L. Stewart d, Eugene T. Grygielko b, Lindsay E. Glace b, Walter Trizna b, Rakesh Nagilla c,
Jeffrey D. Bray b, Scott K. Thompson a
*
a Department of Chemistry, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
b Department of Biology, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406, USA
c Department of Drug Metabolism and Pharmacokinetics, Metabolic Pathways Centre for Excellence in Drug Discovery, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road,
King of Prussia, PA 19406, USA
d Department of Computational and Structural Chemistry, Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals, PO Box 13398, Research Triangle Park, NC 27709, USA
e Department of Computational and Structural Chemistry, Molecular Discovery Research, GlaxoSmithKline Pharmaceuticals, 1250 South Collegeville Road, PO Box 5089, Collegeville,
PA 19426, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and synthesized a novel series of pyrrolidinones as progesterone receptor partial ago-
nists. Compounds from this series had improved AR selectivity, rat pharmacokinetic properties, and
in vivo potency compared to the lead compound. In addition, these compounds had improved selectivity
against hERG channel inhibition.
Received 17 February 2009
Revised 12 June 2009
Accepted 22 June 2009
Available online 25 June 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Progesterone receptor
Agonist
Partial agonist
Endometriosis
Nuclear receptors
Hormone receptors
hERG binding
Androgen receptor
OVX rat model
Progestin
The progesterone receptor (PR) is a member of the nuclear
receptor superfamily of ligand-activated transcription factors.1 In
addition to its numerous other physiological actions, progesterone
(P4, 1)-bound PR attenuates estrogen (E2)-stimulated proliferation
of endometrial tissue (Chart 1).2 Progesterone and other progestins
such as medroxyprogesterone acetate (MPA, 2) are commonly used
in hormone replacement therapy, secondary amenorrhea, and oral
contraception; moreover, because of their ability to block the pro-
liferation of endometrial tissue, they are also effective treatments
for endometriosis.3–5 Unfortunately, these therapies have multiple
side effects associated with their full agonist activity and/or poor
nuclear hormone receptor (NHR) selectivity such as weight gain,
breakthrough bleeding, and mood disturbances.3–5
Recently, a number of selective non steroidal PR agonists de-
signed to treat endometriosis have been reported.6,7 While these li-
gands will likely reduce selectivity-related side effects, those
associated with full agonism will persist. In an effort to diminish
PR-related side effects, we initiated a chemistry effort to develop
a selective PR partial agonist. It is hypothesized that a partial
O
Me
O
Me
OAc
Me
Me
H
H
H
H
H
H
O
O
Me
2
1
* Corresponding author. Tel.: +1 610 270 4941.
E-mail address: dave.g.washburn@gsk.com (D.G. Washburn).
Chart 1. Steroidal PR ligands.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.06.081

D. G. Washburn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4664–4668
4665
agonist could reduce the level of PR-regulated gene products to
such a level to oppose E2 but not to induce side effects associated
with full agonism. Consequently, these ligands would fully sup-
press the action of E2 on endometrial tissue but minimize the side
effects associated with both poor NHR selectivity and full PR
agonism.
The pyrrolidine 3, identified from a structure-based design
strategy, was the starting point for our chemistry effort (Chart
2).7 Compound 3 had excellent PR binding potency and partial ago-
nist activity as measured by a T47D-based alkaline phosphatase PR
agonist assay and >100-fold selectivity over several nuclear recep-
tors, including, estrogen (ER), mineralocorticoid (MR), and gluco-
corticoid (GR) receptors.8 Unfortunately, 3 and its N-alkyl analogs
all suffered from potent androgen receptor (AR) binding affinity,
potent hERG channel blockade, and poor rat and monkey PK
parameters including high clearance, large volumes of distribu-
tions and low oral exposure. The latter two (hERG and PK), we
attributed to the inherent lipophilic, basic nature of the pyrrolidine
ring.9
Figure 1. Binding pocket surface of compound 3. Compound 3 shown in purple. Key
Examination of the binding pocket surface of an X-ray crystal
structure of 3 bound to the PR ligand binding domain (LBD)
showed an unoccupied region of the protein corresponding to the
carbon 5 of the pyrrolidine ring (Fig. 1).7 It was believed that incor-
poration of a carbonyl group at this position would adequately fill
this region of the LBD and possibly interact with Cys891. Further-
more, a carbonyl group at this position would have an additional
benefit of reducing the basicity of the ring nitrogen. Toward this
end, we investigated this novel pyrrolidinone core template.
The pyrrolidinone derivatives 10–22 were prepared according
to Scheme 1. Aryl fluoride displacement of 4 with (2S)-2-amino-
4-[(1,1-dimethylethyl)oxy]-4-oxobutanoic acid 5 followed by a
reduction of the resultant acid afforded the primary alcohol 6.
Tosylation of 6 followed by nucleophilic displacement with sodium
azide and then hydrogenation of the resultant azide provided the
desired amine 7. Incorporation of an alkyl group onto the amine
7 was accomplished via a 3-step process consisting of nosylation
of the amine, alkylation, and then removal of the nosyl group with
thiophenol. Alternatively, incorporation of more sterically hin-
dered alkyl groups was accomplished either through reaction with
a cyanohydrin and NaBH4 or reductive amination to afford 8.
Transesterification of 8 to its methyl ester followed by cyclization
under basic conditions yielded pyrrolidinone 9 which was then
benzylated to provide the target analogs 10–23.
backbone residues are highlighted as ball and stick.
O
H
H
H2N
+
F
NC
Cl
Cl
N
Cl
N
OH
O
a,b
c-e
OH
O
NH2
O
NC
NC
Ot-Bu
Ot-Bu
Ot-Bu
6
4
7
5
f-h or i
R2
NHR1
H
N
H
N
Cl
NC
Cl
j,k
l
R1
N
Cl
N
R1
N
O
NC
O
Ot-Bu
NC
O
10-22
9
8
Scheme 1. Reaction conditions: (a) NaHCO3, DMSO, H2O,
D; (b) CDI, NaBH4, THF;
(c) TsCl, pyr; (d) NaN3, DMF; (e) H2, PtO2, EtOH; (f) o-NO2C6H4SO2Cl, Et3N, CH2Cl2;
(g) R1X, K2CO3, DMF; (h) PhSH, K2CO3, DMF; (i) acetone cyanohydrin, 4 Å molecular
sieves, NaBH4; or NaB(OAc)3H, DCE, 2-butanone or 3-pentanone; (j) 4 N HCl/
dioxane, MeOH; (k) K2CO3, CH3CN/CH3OH,
D
or HMDS, CH3CN; (l) NaH, DMF,
R2BnX.
Table 1
Selected in vitro data for compounds 10-15a
The N-methyl pyrrolidinones synthesized were tested for PR
and AR binding (AR selectivity is shown as a ratio of AR to PR bind-
ing) and for functional activity in the human T47D cell alkaline
phosphatase assay (Table 1).8 The unsubstituted benzyl analog
10 showed poor PR binding, AR selectivity, and weak activity in
the T47D cellular assay. Incorporation of a halogen at the 2-posi-
tion of the benzyl substituent produced partial agonists with im-
proved PR binding affinity, AR selectivity, and cellular potency
(11 and 12). Interestingly, the 2,3 di-fluoro substituted benzyl ana-
log 14 demonstrated increased cellular potency relative to the 2,4
di-fluoro substituted benzyl analog 13.
R2
Cl
N
CH3
N
NC
O
Compd
R2
PR binding IC50, nM
AR/PR
PR T47D EC50, nM (% P4)
10
11
12
13
14
15
—
2-Cl
2-F
2-F,4-F
2-F,3-F
2-CF3
200
25
20
40
25
25
50
250
330
205
100
80
1265 (39)
0.3 (62)
135 (50)
625 (46)
35 (53)
We next examined the effect of varying not only the benzyl
group substitution but the nature of the alkyl group on the pyrro-
70 (65)
a
Values are the mean of P2 determinations.
F3C
PR binding IC50= 15 nM
PR T47D EC50 (%P4) = 30 nM (61%
AR binding IC50 = 250 nM
hERG IC50 = 0.78 µM
lidinone nitrogen (Table 2). Incorporation of either an ethyl or iso-
propyl group onto the pyrrolidinone nitrogen produced a number
of partial agonists with excellent PR binding affinity (with several
analogs reaching the tight binding limit of the assay), AR selectiv-
ity, and cellular potency. Compounds with larger alkyl groups
(than ethyl or isopropyl) at this position demonstrated reduced
N
N
Me
NC
Cl
3
Chart 2. Profile of lead alkylpyrrolidine 3.

4666
D. G. Washburn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4664–4668
Table 2
Selected in vitro data for compounds 15–22a
R2
Cl
N
R1
N
NC
O
Compd
R2
R1
PR binding IC50 (nM)
AR/PR
PR T47D EC50 (nM) (% P4)
16
17
18
19
20
21
22
2-CH3
2-CH3
2-CH3
2-CH3
2-Cl
CH2CH3
CH(CH3)2
Propyl
CH(CH3)CH2CH3
CH(CH3)2
CH2CH3
15b
20
80
125
10b
10b
10b
170
265
80
0.8 (57)
2 (71)
1415 (49)
750 (18)
0.3 (63)
0.8 (52)
0.3 (53)
80
830
210
525
2-Cl,5-Cl
2-Cl,5-F
CH2CH3
a
Values are the mean of P2 determinations.
Tight-binding limit of the assay. ND = no data.
b
of 12 alters the orientation of Cys891 compared to its position
when compounds 1 and 3 (structure not shown) are bound to
the PR LBD.7 The movement of Met909 could be significant since
this residue is part of AF2 region of the c-terminal helix 12 which
has been associated with ligand functionality.10 In addition, 2-F
benzyl group of 12 extends towards the 17-a pocket which is
unoccupied by 1 and subsequently displaces Tyr890 and Leu797
relative to their position when 1 is bound. Since the corresponding
17-a pocket of AR is smaller and less flexible, this interaction is a
possible explanation for the improved AR selectivity of 12 over 1.11
The pharmacokinetic properties (rat and monkey) of selected
analogs are shown in Tables 3 and 4, respectively. In the rat, ana-
logs 14, 20, and 22 demonstrated lower oral plasma clearances, re-
duced volumes of distributions, and improved oral bioavailability
with respect to pyrrolidine 3. In addition, the N-methyl and N-ethyl
analogs 14 and 22 exhibited increased oral exposures, while the N-
isopropyl analog 20 showed no significant improvement with re-
spect to 3. In the monkey, pyrrolidinone analogs 14 and 21 dis-
played reduced volumes of distribution and lower oral plasma
clearances compared to 3; however, only the N-methyl analog 14
showed significantly improved oral exposure.
To further profile these partial agonists, hERG channel blocking
data via whole-cell patch clamp assay format were collected for se-
lected analogs (Table 5).12 Both of these compounds were mark-
edly less potent than the lead 3, with compound 12 having the
most significant improvement.
With a set of PR ligands having both potent partial agonism
and attractive pharmacokinetic properties in hand, we next mea-
sured their E2 opposition effect in an ovariectomized rat utero-
trophic model.11,13 It has been previously reported that in this
model progestins inhibit the E2-induced increase of uterine
wet weight. Along with the wet weight measurement, gene
expression of complement C3 (an E2-regulated gene) was also
Figure 2. Superposition of 12 (cyan)
backbone and key backbone residues are shown in the color of the ligand to which
they correspond.
& 1 (green) crystal structures. Enzyme
PR binding affinity, AR selectivity, and cellular potency (e.g., 18–
19).
Figure 2 shows overlay of the crystal structures of 1 and 12
bound to PR-LBD. These ligands share similar protein/ligand con-
tacts, most notably the benzonitrile group of 12 interacting with
the same residues (Gln725 and Arg766) as the A-ring carbonyl oxy-
gen of 1.7,10,11 However, compound 12 differs from 1 such that the
pyrrolidinone ring resides essentially perpendicular to the back-
bone of 1 which appears to shift the positioning of the binding
pocket residues Met909 and Trp755. Likewise, the carbonyl group
Table 3
Rat pharmacokinetic properties for 3, 14, 20, 21 and 22a
Parameter
Compound
3
14
20
21
22
Dose (iv; po; mg/kg)
Oral CLp (mL/min/kg)
Vdss (L/kg)
1.1; 1.9
510 (210)
55.9 (22)
13 (6)
0.05 (0.02)
5.8 (3.3)
ꢀ29
1.0; 2.2
16 (2)
1.1; 0.12
40 (14)
42 (10)
10 (4)
0.03 (0.01)
5.8 (2.3)
ꢀ100
1.1;2.0
230 (27)
35 (30
84 (33)
0.15 (0.02)
1.8 (0.2)
ꢀ100
1.0; 1.3
75 (14)
15 (3)
97 (20)
0.29 (0.06)
2.0 (0.1)
ꢀ100
3.4 (0.6)
660 (160)
2.3 (0.2)
6.2 (0.6)
ꢀ 100
Oral Cmax (ng/mL)
Oral AUC0-t, (
l
g h/mL)
t
, po (h)
½
Oral%F
a
Values are means of three experiments, standard deviation is given in parentheses.

D. G. Washburn et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4664–4668
4667
Table 4
125
Monkey pharmacokinetic properties for 3, 14, and 21a
14
22
100
75
50
25
0
Parameter
Compound
3
14
21
Dose (iv; po; mg/kg)
Oral CLp (mL/min/kg)
Vdss (L/kg)
Oral Cmax (ng/mL)
Oral AUC0-t, (
2.1; 4.0
1.0; 1.6
110 (38)
1.7 (0.2)
92 (20)
0.27 (0.08)
1.4 (0.4)
15%
1.0; 1.3
480 (110)
2.8 (0.5)
15 (1)
0.032 (0.002)
1.5 (0.5)
4.9%
1351 (100)
9.3 (1.7)
12 (2)
0.039 (0.006)
1.9 (0.3)
2.8%
Maximum Repression
l
g h/mL)
t
, po (h)
½
Oral%F
a
Values are means of three experiments, standard deviation is given in
0.0001 0.001
0.01
0.1
1
10
100
parentheses.
(mg/kg)
Figure 5. Full dose response study in ovariectomized rat uterotrophic model.
Table 5
hERG IC50 data for 3, 12, 22
Compound
hERG, IC50 (lM)
3
12
22
0.78
6.5
3.1
reduction in complement C3 gene expression (Figs. 3 and 4). Un-
der the same dosing conditions, the N-ethyl and N-isopropyl
analogs 22 and 20 also demonstrated a decrease in uterine
wet weight and reduction of complement C3 gene expression
comparable to the full agonist 1.
To obtain a better measure of the in vivo efficacy of this series of
compounds, a full dose response study was conducted on 14 and
22 (Fig. 5). Both compounds showed a robust and dose-dependent
decrease in uterine wet weight; however, the N-ethyl analog 22
(ED50 = 0.007 mg/kg) proved to be approximately 350-fold more
potent than 14 (ED50 = 2.5 mg/kg).
In summary, we have described a series of selective and potent
PR partial agonists. In general this series has improved AR selectiv-
ity, reduced hERG channel blocking, and improved PK parameters
in the rat and monkey relative to lead pyrrolidine 3. Optimization
of several of these analogs demonstrated attractive rat pharmaco-
kinetic properties and in vivo efficacy in an estrogen-induced
uterotrophic model.
110
100
90
80
70
60
50
40
30
20
10
***
0
None
14
22
20
1
Compound (10 mg/kg)
Acknowledgments
Figure 3. Estrogen-induced wet weight model. ***P <0.001 by One-way ANOVA with
The authors would like to thank Melanie Nord and Harvey
(Rusty) Fries for conducting PK studies. We would also like to
acknowledge our colleagues in the Screening and Compound Pro-
filing group for performing the PR and AR binding assays.
Tukey’s post-test. Compounds were orally dosed at 10 mg/kg.
**
110
100
90
Supplementary data
80
70
60
50
40
30
20
10
0
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.06.081.
*
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None
14
22
20
1
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