Development of an in vivo active, dual EP1 and EP3 selective antagonist based on a novel acyl sulfonamide bioisostere

Article abstract of DOI:10.1016/j.bmcl.2012.11.046

Recent preclinical studies demonstrate a role for the prostaglandin E ₂ (PGE₂) subtype 1 (EP1) receptor in mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide and N-acylsulfonamid

Full text of DOI:10.1016/j.bmcl.2012.11.046

Bioorganic & Medicinal Chemistry Letters 23 (2013) 37–41
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Development of an in vivo active, dual EP1 and EP3 selective antagonist
based on a novel acyl sulfonamide bioisostere
Jason D. Downey a, Sam A. Saleh b, Thomas M. Bridges a,c,d, Ryan D. Morrison a,c,d, J. Scott Daniels a,c,d
,
Craig W. Lindsley a,c,d,e, Richard M. Breyer a,f,
⇑
a Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
b Vanderbilt University Institute of Imaging Science, Vanderbilt University Medical Center, Nashville, TN 37232, USA
c Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
d Vanderbilt Specialized Chemistry Center for Probe Development (MLPCN), Nashville, TN 37232, USA
e Department of Chemistry, Vanderbilt University, Nashville, TN 37232, USA
f Division of Nephrology and Hypertension, Department of Medicine, Vanderbilt University Medical Center, Nashville, TN 37232, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Recent preclinical studies demonstrate a role for the prostaglandin E2 (PGE2) subtype 1 (EP1) receptor in
mediating, at least in part, the pathophysiology of hypertension and diabetes mellitus. A series of amide
and N-acylsulfonamide analogs of a previously described picolinic acid-based human EP1 receptor antag-
onist (7) were prepared. Each analog had improved selectivity at the mouse EP1 receptor over the mouse
thromboxane receptor (TP). A subset of analogs gained affinity for the mouse PGE2 subtype 3 (EP3) recep-
tor, another potential therapeutic target. One analog (17) possessed equal selectivity for EP1 and EP3, dis-
played a sufficient in vivo residence time in mice, and lacked the potential for acyl glucuronide formation
common to compound 7. Treatment of mice with 17 significantly attenuated the vasopressor activity
resulting from an acute infusion of EP1 and EP3 receptor agonists. Compound 17 represents a potentially
novel therapeutic in the treatment of hypertension and diabetes mellitus.
Received 20 September 2012
Revised 9 November 2012
Accepted 14 November 2012
Available online 24 November 2012
Keywords:
Prostaglandin E2
EP1
EP3
Antagonist
Ó 2012 Elsevier Ltd. All rights reserved.
Prostanoids are a family of oxidative metabolites of arachidonic
acid that act in a autocrine and paracrine fashion. The cyclooxygen-
ase activity of COX-1 and COX-2 converts arachidonic acid to pros-
taglandin G2 (PGG2) and the peroxidase activity of the same
enzymes reduces PGG2 to prostaglandin H2 (PGH2). PGH2 is then
isomerized to the five principal prostanoids by their respective
synthases. Prostanoids bind to and activate a family of cell surface
G-protein coupled receptors.1 Prostaglandin E2 (PGE2) is formed
kidney transplantation for survival. Elimination of PGE2 production
with COX inhibitors,7,8 like NSAIDs,9 is not a viable option as high-
lighted in a number of clinical trials. Recent studies in rodents and
humans have suggested a role for the EP1 receptor in mediating at
least part of the pathophysiology of diabetes mellitus10–12 and
hypertension.13–16 EP1 has been prosecuted as a potential thera-
peutic target for chronic pain.17–21 As such, small molecule, drug-
like antagonists of EP1 have been developed. Human prostanoid
receptor-targeting molecules are often nonselective,22 owing to
the evolution of the EP family of GPCRs to recognize the same
endogenous ligand, PGE2. The molecular pharmacology of these
compounds at mouse prostanoid receptors is less well known, of-
ten poorly selective, and not always comparable to human phar-
macology.23 In order to study these molecular targets more
precisely, we developed EP1 antagonists selective for the mouse
receptor to use in mouse models of hypertension and diabetes
mellitus.
To develop antagonists selective for the mouse EP1 receptor, we
started with compound 7 (Figure 1), synthesized as previously
described (Scheme 1).24 Diethyl dipicolinic acid (1) was reduced
with NaBH4 to 2. Parikh–Doering oxidation of 2 with sulfur triox-
ide–pyridine complex and DMSO produced the unstable aldehyde
3. 4-Chlorophenoxide was then reacted with 3, followed by neu-
tralization with HCl to form 4. Reduction of the secondary alcohol
from PGH2 by prostaglandin
E synthases (cPGES, mPGES-1,
mPGES-2) and is a major prostanoid produced by the kidney and
the vasculature.2 The bioactivity of PGE2 is mediated through four
subtypes of E-Prostanoid (EP) receptors, designated EP1-EP4.3 EP2
and EP4 couple to stimulatory G-proteins, which increase intracel-
lular cAMP when activated. EP3 canonically couples to inhibitory
G-proteins, suppressing cAMP accumulation. Both EP1 and EP3
are known to induce calcium flux into the cell.4,5 The tissue local-
ization of each of these EP receptors produces diverse and some-
times opposing biological activities of PGE2 in vivo.3
Hypertension and diabetes are the primary causes of 62% of
patients with end-stage renal disease (ESRD) and 72% of patients
that develop ESRD each year6, which requires life-long dialysis or
⇑
Corresponding author. Tel.: +1 615 343 0257; fax: +1 615 343 4704.
E-mail address: richard.m.breyer@vanderbilt.edu (R.M. Breyer).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.046

38
J. D. Downey et al. / Bioorg. Med. Chem. Lett. 23 (2013) 37–41
O
O
EP1 receptor by Schild Analysis (Figure 2). 7 had no detectable
O2
S
Cl
N
Cl
N
affinity for mouse EP3 or EP4 receptors by radioligand binding as-
says. 7 had poor, but detectable affinity at mouse EP2, and sup-
pressed signaling through mouse TP receptor at concentrations
100-fold higher than at the human receptor (Table 1), confirming
the off-target activity of 7 at mouse TP.
N
H
OH
O
F
O
F
Cl
Cl
Cl
Results from in vivo pharmacokinetics experiments (Table 2)
revealed compound 7 to possess a moderate systemic plasma
clearance (CLp) and volume of distribution predicted at steady-
state (Vss), subsequently displaying a short half-life (t1/2, >60 min)
in mice receiving a parenteral administration of the EP1 receptor
antagonist. We observed a bioavailability (%F) of approximately
14% following the oral administration (10 mg/kg) of 7 to mice.
Recently, Ostenfeld et al. have shown that in rats 7 is cleared
primarily by glucuronidation and sequestration into the bile.25
With the goal of inhibiting glucuronidation while improving
molecular pharmacology of 7, a series of carboxylic acid bioisoster-
es of 7 were pursued. N-acylsulfonamides are common carboxylic
acid bioisosteres that have been successfully implemented in
antagonists of angiotensin II AT1 receptors26 as well as EP3 recep-
tors.27 A series of analogs (8–21) resulting from the amidation of 7
was prepared (Table 3). Tertiary amide analogues of 7 (9 and 14)
were included to evaluate a structure–activity role of an acidic
proton in ligand-receptor interactions. Each was synthesized by
17
7
Figure 1. Lead picolinic acid-based human EP1 antagonist 7, and 4-chloro-N-
acylsulfonamide analog 17.
of 4 under H2 and Pd/C with the addition of H2SO4 and ZnBr2 gave
5. Alkylation of 5 with 2-fluoro-4-chlorobenzyl bromide and cleav-
age of the ester by refluxing with NaOH produced the sodium salt
(6) of the lead (7) which was formed by protonation of 6. The lead
compound was shown to have good affinity for the human EP1
receptor and was stable in microsomes and S9 fractions of several
species. However, 7 was previously reported to have a high-affinity
interaction with the human thromboxane (TP) receptor.24 We eval-
uated the molecular pharmacology of 7 at the mouse EP receptors
as well as the mouse TP receptor.
Compound 7 was confirmed to be a functional antagonist of
mEP1 in vitro and to have submicromolar affinity for the mouse
O
O
OH
O
O
O
(a)
(b)
(c)
N
N
N
O
O
O
O
1
2
3
OH
O
O
(d)
(e), (f)
Cl
N
Cl
N
O
O
OH
OH
O
4
5
O
O
Cl
N
Cl
N
Cl
N
ONa
OH
R
(h)
(g)
O
O
O
F
F
Cl
F
Cl
Cl
6
7
8 - 21
Scheme 1. Reagents: (a) NaBH4, EtOH (47%); (b) PyrÁSO3, DMSO, DCM, (c) 4-chlorophenol, EtMgBr, DCM (54%); (d) H2, Pd/C, H2SO4, ZnBr2, EtOAc (69%); (e) 2-fluoro-4-
chlorobenzyl bromide, K2CO3, EtOH; (f) NaOH, reflux; (g) HOAc, PhMe (68%); (h) RNH2, R2NH, or RSO2NH2, EDCÁHCl, HOBt, DIPEA, DMF (10%–20%)
Figure 2. Concentration response curves (A) and transformed Schild regression; (B) for mEP1-expressing CHOk1 cells treated with six concentrations of 7 before being
challenged with a range of concentrations of 17-phenyl-x
-trinor PGE2 (17PTPGE2), an EP1 agonist (m = 0.953 0.085, pKD = 7.283, r2 = 0.9689).

J. D. Downey et al. / Bioorg. Med. Chem. Lett. 23 (2013) 37–41
39
Table 1
Molecular pharmacology of 7 at mouse EP and TP receptors
a
b
mEP1 pKD
mEP1 KD (nM)
mEP2 pKIb
mEP3 pKIb
mEP4 pKIb
mTP pIC50
6.04 0.18
7.32 0.08
47.9
5.76 0.21
<6
<6
a
Value represents mean SEM of at least two independent experiments measured in duplicate.
Values represent mean SEM of at least three independent experiments measured in triplicate.
b
Table 2
coupling 7 to a series of primary and secondary amines (8–16) and
sulfonamides (17–21) employing common activators such as
EDCÁHCl, HOBt, and DIPEA in DMF (Scheme 1).
Pharmacokinetic parameters for compound 7 following IV (1 mg/kg) or PO (10 mg/kg)
dosing
t1/2 (min)
CLp (mL/min/kg)
59.2 6.4
Vss (L/kg)
F (%)
The molecular pharmacology observed for 8–21 was deter-
mined at mEP1–mEP4 and mTP (Table 3). Generally, N-acylsulf-
65 8.2
0.90 0.1
13.8 2.1
Table 3
Molecular pharmacology of amide and acylsulfonamide analogues of 7 at mouse EP and TP receptors
O
Cl
N
R
O
F
Cl
8 - 21
a
b
Compd.
R
mEP1 pKD
mEP1 KD (nM)
mEP2 pKIb
mEP3 pKIb
mEP4 pKIb
mTP pIC50
7
OH
7.32 0.08
6.26 0.02
47.9
549
5.76 0.21
<6
<6
6.04 0.18
<6
NH
8
<6
<6
<6
N
9
4.80 0.05
6.04 0.06
6.52 0.36
15800
912
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
10
11
NH
NH
<6
302
6.67 0.03
12
13
14
5.86 0.01
5.36 0.12
6.35 0.34
1380
4360
447
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
NH
NH
N
15
16
17
5.66 0.13
5.58 0.30
7.39 0.39
2190
2630
40.7
<6
<6
<6
<6
<6
<6
<6
<6
<6
<6
N
H
NH2
<6
O2
S
N
H
6.97 0.22
Cl
Cl
O2
S
N
H
18
7.25 0.32
53.7
<6
6.69 0.13
<6
<6
Cl
O2
S
19
20
6.67 0.14
N.D.c
214
<6
<6
<6
<6
<6
<6
<6
<6
N
H
O2
N.D.c
S
HN
O2
S
Cl
Cl
N
H
21
6.67 0.16
214
<6
7.18 0.05
<6
<6
a
b
c
Values represent mean SEM of at least two independent experiments measured in duplicate.
Values represent mean SEM of at least three independent experiments measured in triplicate.
No functional antagonism was evident at concentrations exceeding 100 lM.

40
J. D. Downey et al. / Bioorg. Med. Chem. Lett. 23 (2013) 37–41
Figure 3. Concentration response curves (A) and transformed Schild regression; (B) for mEP1-expressing CHOk1 cells treated with six concentrations of 17 before being
challenged with a range of concentrations of 17PTPGE2 (m = 1.20 0.12, pKD = 7.063, r2 = 0.9487).
that approached the hepatic blood flow in mice (QH, 90 mL/min/
kg).
Table 4
Intrinsic clearance of amide and N-acylsulfonamide analogs of 7 by mouse liver
microsomes
Results from metabolite identification studies in hepatic subcel-
lular fractions indicated extensive biotransformation of the amide
11 and the N-acylsulfonamide 17, including NADPH-independent
hydrolysis (i.e., esterases) and NADPH-dependent oxidation (i.e.,
P450) of these analogs. Figure 4 depicts the metabolism of 17,
including the hydrolysis of the sulfonamide (M1), and P450-medi-
ated oxidation of the methylene linker (M2) and benzylic oxidation
(M3). The extent of plasma protein binding (fraction unbound, Fu)
in mouse was determined to be extensive for three compounds as-
sessed (Fu: 7 = 0.005, 11 = 0.010, 17 = 0.004).
Compd.
Clint (mL/min/kg)
CLHEP (mL/min/kg)
7
8
84.7
43.6
89.3
89.2
88.9
86.6
88.4
87.4
11382
9806
7157
2260
5039
2994
11
14
17
19
21
Given the molecular pharmacology and in vitro metabolism
data, we proceeded to evaluate the in vivo pharmacokinetics of
17. Mice (n = 3) were subsequently administered a subcutaneous
dose (5 mg/kg) with intermittent plasma collections to measure
systemic levels of 17 (Figure 5). Compound 17 achieved a maxi-
mum plasma concentration (Cmax) of 504 nM ( 167) 2 h (tmax) fol-
lowing subcutaneous administration and displayed an area-under-
the-curve (AUC) of 7508 nM⁄h.
To evaluate 17 as an antagonist of EP1 and EP3 in vivo, we mea-
sured blockade of mEP1 and mEP3 acute vasopressor activity in
mice. Left common carotid arteries and right jugular veins of
anesthetized mice were cannulated. Direct arterial pressure was
measured via carotid catheter. Vasoactive substances were admin-
istered via jugular catheter. 17PTPGE2 was used to acutely raise
mean arterial pressure (MAP) via mEP1 and sulprostone was used
for mEP3 (Figure 6). Agonists were administered IV through the
onamides retained mEP1 affinity similar to 7 (representative data
for 17, Figure 3) while the amide series had reduced affinity for
mEP1. Each analog displayed reduced affinity for mEP2 and mTP.
Interestingly, four analogs (11, 17, 18, and 21) displayed enhanced
affinity for mEP3, a potential therapeutic target for hypertension-
and diabetes mellitus-related ESRD. EP3 is of particular interest
as it shares signaling pathways and endogenous ligands with EP1
and may represent a compensatory signaling pathway in the event
of EP1 blockade.3,5,16,28,29 These dual-selectivity compounds were
confirmed to be functional antagonists of mEP3 by Schild analysis
(data not shown).
We subsequently determined the intrinsic clearance (Clint) of
several potent amide and N-acylsulfonamide analogs (Table 4). Re-
sults indicated an exceptional instability to metabolism in vitro,
displaying estimated predicted hepatic clearance (CLHEP) values
Figure 4. Metabolism of 17 in hepatic subcellular fractions.

J. D. Downey et al. / Bioorg. Med. Chem. Lett. 23 (2013) 37–41
41
for their technical expertise. This work was supported by National
Institutes of Health grants R01 DK46205 (R.M.B.), R01 DK37097
(R.M.B.), P50 GM015431 (R.M.B.) and the Integrative Training in
Therapeutic Discovery program T90 DA022873 (J.D.D.). R.M.B. has
a Merit Award from the Department of Veterans Affairs.
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jugular catheter 2 h after subcutaneous administration of 17. Pre-
treatment of mice with 5 mg/kg 17 administered subcutaneously
significantly attenuated the pressor activity of an IV bolus of
20
Pretreatment with 17 also significantly suppressed pressor activity
of an IV bolus of 10 g/kg sulprostone ( MAP 53.3 2.3 mmHg vs
32.0 3.5 mmHg). To ensure the observed effect was selective for
EP-mediated vasoconstriction, phenylephrine (10 g/kg) was
lg/kg 17PTPGE2 (DMAP 50.3 5.5 mmHg vs 27.0 3.6 mmHg).
l
D
l
shown to be unaffected by pretreatment with 17 (data not shown).
In conclusion, we have identified a novel, dual-selectivity antag-
onist (17) of the mouse EP1 and mouse EP3 receptors possessing an
acylsulfonamide bioisostere for the prototypical carboxylic acid
moiety of EP ligands. 17 was found to have indistinguishable affin-
ity for mEP1 as for mEP3 (mEP1 pKD vs mEP3 pKI, P = 0.40, Stu-
dent’s two-tailed t test). 17 had improved selectively over mEP2
and mTP. 17 was less stable in mouse hepatic microsomes than
7, due in part to hydrolysis of 17 to 7, a problem effectively circum-
vented by subcutaneous administration of 17. Finally, we con-
firmed 17 is a functional antagonist of mEP1 and mEP3 in vivo
by blocking mEP1/mEP3-mediated acute vasopressor activity in
anesthetized mice. While the attenuation of pressor activity ap-
pears to be incomplete, these results recapitulate experiments per-
formed in mice with genetic disruptions of EP1.13 Dual specificity
EP1/EP3 antagonists represent a novel class of potential ESRD ther-
apeutics we hypothesize will be more beneficial than blocking
either receptor alone.
Acknowledgements
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The authors acknowledge Kwangho Kim of the Vanderbilt
Institute of Chemical Biology Synthesis Core and Christina Bartlett