Investigation of piperazine benzamides as human β3 adrenergic receptor agonists for the treatment of overactive bladder

Article abstract of DOI:10.1016/j.bmcl.2016.12.033

The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human β₃-adrenergic receptor for the treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established β₃ potency and selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.

Full text of DOI:10.1016/j.bmcl.2016.12.033

Accepted Manuscript
Investigation of Piperazine Benzamides as Human β3 Adrenergic Receptor Ag-
onists for the Treatment of Overactive Bladder
Bart H. Harper, Liping Wang, Cheng Zhu, Nam F. Kar, Bing Li, Christopher R.
Moyes, Stephen D. Goble, Melissa Costa, Karen Dingley, Jerry Di Salvo,
Sookhee N. Ha, Amanda Hurley, Xiaofang Li, Randy R. Miller, Hiroshi
Nagabukuro, Gino M. Salituro, Sean Smith, Mary Struthers, Jeffrey J. Hale,
Scott D. Edmondson, Richard Berger
PII:
S0960-894X(16)31300-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.033
BMCL 24522
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 November 2016
7 December 2016
9 December 2016
Please cite this article as: Harper, B.H., Wang, L., Zhu, C., Kar, N.F., Li, B., Moyes, C.R., Goble, S.D., Costa, M.,
Dingley, K., Di Salvo, J., Ha, S.N., Hurley, A., Li, X., Miller, R.R., Nagabukuro, H., Salituro, G.M., Smith, S.,
Struthers, M., Hale, J.J., Edmondson, S.D., Berger, R., Investigation of Piperazine Benzamides as Human β3
Adrenergic Receptor Agonists for the Treatment of Overactive Bladder, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.033
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Investigation of Piperazine Benzamides as Human 3 Adrenergic
Receptor Agonists for the Treatment of Overactive Bladder
Bart H. Harper*, Liping Wang, Cheng Zhu, Nam F. Kar, Bing Li, Christopher R. Moyes,
Stephen D. Goble, Melissa Costa, Karen Dingley, Jerry Di Salvo, Sookhee N. Ha,
Amanda Hurley, Xiaofang Li, Randy R. Miller, Hiroshi Nagabukuro, Gino M. Salituro,
Sean Smith, Mary Struthers, Jeffrey J. Hale, Scott D. Edmondson, and Richard Berger
Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065, United States
Abstract— The synthesis of a novel class of piperazine benzamide (reverse amides) targeting the human 3-adrenergic receptor for the
treatment of overactive bladder (OAB) is described. The SAR studies directed towards maintaining well established 3 potency and
selectivities while improving the overall pharmacokinetic profile in the reverse amide class will be evaluated. The results and
consequences associated with functional activity at the norepinephrine transporter (NET) will also be discussed.
Keywords: 3-adrenergic receptor agonist; reverse amide;
pyrrolidine scaffold; overactive bladder; norepinephrine
transporter
and nocturia. It is an underdiagnosed condition that
negatively affects the quality of life of millions of
people.
Pharmacological agents for treating OAB have
evolved over the past two decades resulting in clinically
approved drugs such as mirabegron (YM-178, 1, Fig.
1).11 Our group’s research efforts over the past several
years has primarily focused on constraining the linear
ethanolamine moiety present in many of the first
generation 3-AR agonists such as 1, resulting in a
novel pyrrolidine scaffold.12a Extensive SAR (structure-
activity relationship) evaluations in this series led to the
discovery of many potent and selective human 3-AR
agonists with improved metabolic stabilities.12b Our
key discovery, vibegron (2, Fig. 1), is emblematic of
this research and has recently progressed into Phase 3
clinical trials.13 Compounds 1 and 2 as well as other
lead compounds in that series are all aniline-derived
amides.
In search for continued structural diversity, our
group focused on a series of RHS (right-hand side)
benzamide (reverse amides) while keeping the
pyrrolidine core intact.14 Initial efforts leveraging
parallel synthesis resulted in promising leads in potent
and selective analogs such as piperidine ethyl ester 3
(Fig. 2) with modest potency (human 3-AR EC50 = 63
nM). Subsequent optimization further improved the
overall profile of these compounds to afford the highly
The human gene which encodes the 3-adrenergic
receptor (3-AR) was discovered in the 1980s and
opened a field of exploration around its function in
metabolic processes.1 Highly expressed in adipose
tissue, it is involved in regulating lipolysis and
thermogenesis.2,3 Due to its role in these processes, 3-
adrenergic receptor agonists were investigated as a
potential treatments for obesity and diabetes.4 As a
result of this research, several compounds entered
clinical trials but failed to demonstrate the sustained
weight loss efficacy exhibited in rodents which resulted
in diminished interest in the pursuit of these
pharmacological indications.5
Subsequent research exposed evidence of 3-AR
expression in the bladder detrusor muscle in multiple
species including humans.6 Furthermore, 3-AR
agonists were demonstrated to relax bladder detrusor
muscle strips in vitro as well as increase bladder
capacity in rodent cystometry studies.7-9 In light of
these discoveries, 3-AR agonists became desirable
targets throughout the pharmaceutical industry for the
treatment of overactive bladder (OAB).6, 10 OAB is a
highly prevalent disorder, particularly among the elderly
population, with symptoms of urinary urgency, with or
without urgency incontinence, and often with frequency

potent and selective 3.1.0 azabicyclic ethyl oxadiazole 4
(Fig. 2) (3 EC50 = 3.1 nM,1 and 2 IC50 > 20 M).
Compound 4 was thoroughly profiled and exhibited
acetylene intermediate 10 as presented in Scheme 1.
The first step in the synthesis involved installation of
chiral auxiliary, (S)-4-phenyl-2-oxazolidinone, via acid
chloride coupling with hexynoic acid 5 resulting in the
N-acyloxazolidinone 6. A subsequent magnesium
chloride catalyzed anti-Aldol reaction15 with
benzaldehyde converted 6 into intermediate 7 with
excellent diastereoselectivity. The chiral oxazolidinone
auxiliary was then removed via hydrolysis in aqueous
LiOH/H2O2 to furnish acid intermediate 8. The alcohol
of 8 was then protected as a TBS ether in the presence
of DBU and a subsequent Curtius rearrangement with 4-
methoxybenzyl alcohol yielded the key 4-
methoxybenzyloxycarbonyl (Moz) protected amino
acetylene intermediate 10. Coupling of 4-
iodomethylbenzoate with the terminal alkyne under
Sonogashira conditions afforded the methyl ester
acetylene 11. Deprotection of the Moz group with TFA
set up amine 12 for a palladium catalyzed cyclization to
a 2,3-dihydropyrrole intermediate which was
Figure 1. Example of 3-AR agonists
subsequently reduced via platinum catalyzed
hydrogenation resulting in the highly diastereoselective
formation of cis pyrollidine isomer 13. The pyrrolidine
was then sequentially Boc protected (14) and then TBS
deprotected with TBAF to furnish intermediate 15.
Finally, the desired benzoic acid handle was formed
upon hydrolysis of the methyl ester under standard
aqueous lithium hydroxide conditions to afford the
desired coupling key intermediate 16.
This chemistry paved way for multi-gram synthesis
of 16 which was utilized for extensive piperazine
benzamide SAR exploration. Depicted in Scheme 2, the
two-step operation towards final targets involved
standard amide coupling conditions with EDC or HATU
followed by Boc deprotection under acidic conditions.
Some of the SAR in the piperazine series was
Figure 2. Example of 3-AR agonists reverse amide benzamides.
developed in conjunction with non-piperazine
benzamides that were being investigated at the time. As
previously mentioned, one of our initial objectives was
to improve PK while maintaining potency and
improvements within the series in PK across species
(Table 2). Despite these advances, however, the overall
profile exhibited a low probability of achieving our
target of QD dosing in humans. In our search for more
optimal PK profiles in the benzamide series, a unique
class of piperazine derivatives was discovered and will
be the focus of this report.
Our group previously published synthetic approaches
towards the pyrrolidine aniline core which allowed
efficient parallel synthesis of compounds leading up to,
and including, vibegron.12, 13 This work provided the
foundation for modifications resulting in the synthesis
of the benzoic acid core which in turn was utilized to
evaluate the SAR of benzamides such as 3 and 4.14 In
an effort to further streamline the synthetic sequence,
the group developed a more convergent, high yielding
and diastereoselective route whereby either the aniline
or benzoic acid could be installed from a common
selectivity. Earlier disclosures from our laboratories
describe the importance of reducing risk for inducing
phospholipidosis (PLDS) and poor selectivities over the
serotonin transporter (SERT).12b, 13 In order to identify
any potential cardiac liabilities the analogs were also
screened against the human Ether-a-go-go Related Gene
(hERG) potassium channel and the human cardiac
sodium channel (hNav1.5).

Scheme 1. Synthesis of the pyrrolidine benzoic acid core. Reagents and conditions: (a) Trimethylacetyl chloride, TEA, THF ; (b) (S)-4-phenyl-2-
oxazolidinone, LiCl, THF; (c) PhCHO, MgCl2, TMS-Cl, TEA, EtOAc; (d) LiOH, H2O2, THF, H2O; (e) TBS-Cl, DBU, CH3CN; (f) K2CO3, MeOH, H2O;
o
(g) DPPA, TEA, 4-Methoxybenzyl alcohol, toluene, 110 C; (h) Pd(OAc)2, (t-Butyl)3P-tetrafluoroborate salt, Cs2CO3, Methyl 4-iodobenzoate, CH3CN, 50
oC; (i) TFA, DCM (j) Pd(OAc)2, toluene, 80 oC; (k) PtCl2, Pd/C, DCM/MeOH, H2 50 psi; (l) Boc2O, TEA, DCM (m) TBAF, THF (n) NaOH,
THF/MeOH/H2O.
The group reasoned that with the shorter C-N bond
length associated with piperazine in comparison to
piperidine it might be necessary to homologate
The rest of the compounds highlighted in this report
center around ester bioisosteres which also proved
beneficial in the non-piperazine series with enhanced
3-AR potency and improvements in PK. In general,
the relevant off-target activities continued to be well
maintained throughout the series (Table 1). Utilizing a
similar oxadiazole substituent as 4, a significant
improvement in 3-AR potency was observed with 19b
(EC50 = 2.5 nM). Compound 19c, the 1, 2, 4-oxadiazole
regioisomer of 19b, further improved 3-AR potency
into the sub-nanomolar range (EC50 = 0.32 nM).
Unfortunately, 19c suffered from suboptimal rat PK
(Table 2). Difluoro (19d) and trifluoro (19e) ethyl
substituted triazoles were also well tolerated with 3-AR
EC50s of 2.9 nM and 1.6 nM respectively. Despite
having longer half-lives in rat, these triazoles exhibited
high clearance (> 105 mL/min/kg) and low oral
substituents by one carbon in order to emulate 3-AR
activity observed in the piperidine class. Initiated with
these objectives in mind, one of the first compounds
synthesized was a relatively simple homologated 2-
pyridyl piperazine 19a. This compound proved to be an
excellent starting point in the series. Although its 3-
AR potency (EC50 = 20 nM) was about 7 times less
potent than 4, the compound exhibited excellent
selectivities over binding to 1/2-ARs, cardiac ion
channels, SERT and also showed minimal risk for
phospholipidosis (A50 = 54 M).16 The preclinical
pharmacokinetic results for 19a are shown in Table 2
and illustrate high unbound plasma clearance (148
mL/min/kg) and a moderate half-life (t1/2 = 2.6 h) in rat.
In addition, its profile was improved in dog and rhesus
exhibiting much lower unbound clearances and
increased half-lives in dog t1/2 (12h) and rhesus (5.8h).
bioavailabilities (< 1%) and were not further developed.

Scheme 2. Synthesis of piperazine benzamides. Reagents and conditions: (a) amine, HATU or EDC, HOAt or HOBt, DIEA, DMF ; (b) TFA, DCM or
HCl, dioxane/DCM.
Table 1
SAR of piperazine benzamides synthesized via Scheme 2.
Compd
Human EC50 (nM)a (%Act)b
Human IC50 (nM)c
1 2
hERG IC50 (nM)d
hNav1.5 IC50 (nM)e
SERT IC50 (nM)f
PLDS A50 (M)g
3
19a
19b
19c
19d
20 (87)
> 20,000 > 20,000
> 20,000 > 20,000
> 20,000 > 20,000
> 20,000 > 20,000
> 60,000
> 60,000
> 60,000
34,010
> 30,000
> 30,000
> 30,000
> 30,000
> 10,000
> 10,000
8794
54
68
2.5 (109)
0.32 (99)
2.9 (108)
> 10,000
19e
19f
19g
19h
19i
1.6 (113)
5.1 (91)
5.2 (99)
1.5 (101)
1.9 (98)
> 20,000 > 20,000
> 20,000 > 20,000
> 20,000 > 20,000
> 20,000 > 20,000
> 20,000 > 20,000
> 60,000
> 60,000
39,380
> 30,000
> 30,000
> 30,000
> 30,000
> 30,000
> 10,000
> 10,000
> 10,000
> 10,000
> 10,000
70
> 100
72
> 60,000
29,890
a3 values reported as the mean from a minimum of twoexperiments. bPercent activation defined as the fitted maximal value of the test compound
concentration response expressed as a percent of the maximal response to R-(–)-isoproterenol. cn=1. dIKr binding assay, ref 17a eReference 17b.
fReference 18. gReference 16.
A group of substituted tetrazoles was also
19a, compounds 19h and 19i showed PK improvements
in dog relative to rat, highlighted by lower unbound
clearance and longer half-lives,. The clearance of 19h
in dog was low (22 mL/min/kg) with a long half-life
(t1/2 = 8.5 h). Dog unbound exposure and bioavailability
investigated resulting in a slight loss in 3-AR activity
observed with aliphatic substituents, 19f (EC50 = 5.1
nM) and 19g (EC50 = 5.2) compared to their difluoro-
and trifluoroethyl counterparts, 19h (EC50 = 1.5 nM)
and 19i (EC50 = 1.9 nM) respectively. As observed with
of 19h were modest (PO AUCnorm, u
=

Table 2
Pharmacokinetic profiles of 3 adrenergic agonists in non-clinical speciesa,, b
Compd
Species
Clp (mL/min/kg)
Clu (mL/min/kg)
Vd (L/kg)
t1/2 (h)
PO AUCnorm, total
(M•h/mpk)
0.21
PO AUCnorm, u
(M•h/mpk)
F (%)
% unbound
4
Rata
Doga
Rhesusa
Rata
56
36
9.3
9.2
5.2
19
6.4
11
19
57
38
21
13
17
11
5.8
3.0
3.7
2.4
2.6
12
33
44
5
0.46
0.06
0.35
1.3
30
19a
71
148
17
0.17
0.62
0.14
71
27
16
48
48
42
Doga
Rhesusa
Ratb
7.9
18
43
5.8
1.8
7.3
5.4
2.6
8.5
2.8
6.9
6.5
0.34
19c
19d
19e
19h
181
111
105
102
22
Rata
0.29
0.3
< 1
< 1
16
Rata
Rata
182
34
0.05
0.29
0.14
1.9
0.03
0.19
0.09
1.2
56
65
65
63
44
Doga
Rata
Doga
Rhesusa
20
19i
98
151
35
46
22
100
5.4
19
43
0.09
0.04
aRat iv: 1 mpk, po: 2 mpk; Dog iv: 0.5 mpk, po: 2 mpk; Rhesus: 0.5 mpk, po: 2 mpk. bRat iv (cassette): 0.5 mpk.
0.19 M•h/mpk and F = 20%). Trifluoroethyl tetrazole
19i showed further improvement in dog PK with higher
unbound exposure (PO AUCnorm, u = 1.2 M•h/mpk) and
bioavailability (F = 100%) in conjunction with a
Table 3
Norepinephrine transportor (NET) binding acitvities of selected 3
relatively long half-life (t1/2 = 6.9 h). Unfortunately,
however, 19i also exhibited poor oral exposure and
bioavailability in rhesus. Evaluation of 19i in cross-
species liver microsome incubations at least partially
explains this trend, including the sub-optimal rhesus PK
observed, with the order of stability being dog > rat >
human > rhesus (% parent @ 45 min. = 85% dog, 71%
rat, 42% human, 19% rhesus). In addition, compound
19i proved stable up to 60 minute hepatocyte
incubations in human (92% parent remaining) and dog
(100%) with moderate stabilities in rat (73%) and
rhesus (50%). As a result, 19i was deemed a lead
compound in the series with an overall preclinical
pharmacokinetic profile projected to be suitable for QD
dosing in humans.
The excellent in vitro potencies, selectivities and
improved PK associated with the piperazine class
triggered more extensive off-target screenings of the
highlighted compounds. A screen of more than 100
enzymes, receptors, and ion channels resulted in activity
at the norepinephrine transporter (NET). This modest
NET binding activity justified a follow-up to better
understand its impact on the series. Therefore, a
functional in vitro NET reuptake inhibition screening
assay was performed.19
adrenergic agonists
Compd
Human EC50 (nM)a (%Act)b
NET EC50 (nM)c
3
20 (88)
19a
19b
19c
19d
339
254
251
26
2.5 (109)
0.32 (99)
2.9 (108)
19e
19f
19g
19h
19i
1.6 (113)
5.1 (91)
5.2 (99)
1.5 (101)
1.9 (98)
65
1,520
1,390
827
627
a3 values reported as the mean from a minimum of two experiments.
bPercent activation defined as the fitted maximal value of the test
compound concentration response expressed as a percent of the maximal
response to R-(–)-isoproterenol. cReference 19.
The results of this assay, documented in Table 3,
indicate functional activity among the lead compounds

2 2 0
2 0 0
1 8 0
1 6 0
1 4 0
1 2 0
1 0 0
N orepinephrine
C m p d 19i (100m p k P O )
C m p d 19i (10m p k P O )
V e h icle (3% aq . H C l)
* *
1 6 0 0 0
1 2 0 0 0
8 0 0 0
4 0 0 0
0
**
* *
* *
8 0
0
-6 0
-3 0
0
3 0
6 0
9 0
1 2 0 1 5 0 1 8 0
V e h ic le
1 0 m p k
1 0 0 m p k
T im e (m in )
C m p d 19i
Figure 3. Effects of orally dosed compound 19i on norepinephrine in the rat medial prefrontal cortex.
in the series. Overall, a notable improvement in NET
selectivity was observed with the piperazine tetrazoles
relative to other compounds in the series. To better
define the relevance of this activity, 19i was evaluated
in an in vivo rat microdialysis study, depicted in Figure
3, in which norepinephrine (NE) levels in the brain were
monitored after oral dosing at 10 and 100 mpk.
Compound 19i exhibited weak activity of 3-AR at 100
mpk in rat (EC50 = 4500 nM) indicating that any
observed increase in NE is not 3-AR mediated. The
results in Figure 3 illustrate a dose dependent trend with
no significant effect at 10 mpk and a sustained, ~2-fold
increased efflux of norepinephrine in the medial
prefrontal cortex after 1 hour post-dose at 100 mpk.
Norepinephrine reuptake inhibitors designed for the
treatment of CNS indications such as depression and
anxiety generally show a 3-5-fold increase in this
model.20 Unfortunately, the liabilities associated with
regard to NE activity in this piperazine class therefore
warranted further investigation to identify a new series
that is more selective over the NET.
In summary, the piperazine class of benzamides
afforded a promising new lead class for further
opimization for the treatment of OAB by providing
compounds with excellent 3-AR potencies, selectivities
and PK profiles suitable for QD dosing in humans.
However, due to the risk of CNS-mediated effects
associated with NET reuptake inhibition, further
progression of 19i as well as other compounds in this
series was halted. Compound designs leading to
enhanced NET selectivity will be described in due
course.
Acknowledgements
The authors thank Drs. Christopher Sinz and Dong-
Ming Shen for their helpful comments in the
preparation of this manuscript. In addition, we’d like to
acknowledge Judy Morris, Regina Black, and Lisa Frey
for purification assistance and Alka Bansal, Donna
Hreniuk, and Scott Feigner for in vitro assay support.
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