Pyrazoles as non-classical bioisosteres in prolylcarboxypeptidase (PrCP) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.02.070

Bioisosteres are integral components of modern pharmaceutical research that allow structural optimization to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacodynamic, pharmacokinetic and physicochemical properties. A recent medicinal chemistry campaign focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investigation into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric replacement by incorporating subsequent structural modifications to maintain potency against the selected target. A heuristic model for an embedded pharmacophore is also described.

Full text of DOI:10.1016/j.bmcl.2014.02.070

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1657–1660
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrazoles as non-classical bioisosteres in prolylcarboxypeptidase
(PrCP) inhibitors
a
a
c
b
c
Thomas H. Graham a, , Min Shu , Andreas Verras , Qing Chen , Margarita Garcia-Calvo , Xiaohua Li ,
JeanMarie Lisnock d, Xinchun Tong c, Elaine C. Tung c, Judyann Wiltsie d, Jeffrey J. Hale a, Shirly Pinto b,
Dong-Ming Shen a
⇑
a Department of Medicinal Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Metabolic Disorders, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
c Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
d Department of In Vitro Sciences, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Bioisosteres are integral components of modern pharmaceutical research that allow structural optimiza-
tion to maximize in vivo efficacy and minimize adverse effects by selectively modifying pharmacody-
Received 12 January 2014
Revised 22 February 2014
Accepted 25 February 2014
Available online 6 March 2014
namic, pharmacokinetic and physicochemical properties.
A recent medicinal chemistry campaign
focused on identifying small molecule inhibitors of prolylcarboxypeptidase (PrCP) initiated an investiga-
tion into the use of pyrazoles as bioisosteres for amides. The results indicate that pyrazoles are suitable
bioisosteric replacements of amide functional groups. The study is an example of managing bioisosteric
replacement by incorporating subsequent structural modifications to maintain potency against the
selected target. A heuristic model for an embedded pharmacophore is also described.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Non-classical bioisostere
Pyrazole
Amide
Enzyme inhibitor
Prolylcarboxypeptidase (PrCP)
Angiotensinase C
Classical bioisosteres encompass the structural changes to a
molecule that include substituting atoms and the associated va-
lences or exchanging of ring equivalents. In contrast, non-classical
bioisosteres include the replacement of entire functional groups,
exchanging cyclic and non-cyclic forms and the inversion of func-
tional groups (retroisosteres).1 Modern pharmaceutical research
relies on bioisosteres to maximize in vivo efficacy and minimize
undesired effects by selectively modifying pharmacodynamic,
pharmacokinetic and physicochemical properties.2 A recent medic-
inal chemistry campaign that focused on discovering novel inhibi-
tors of prolylcarboxypeptidase (PrCP) investigated the use of a
pyrazole as a bioisostere for an amide. The study revealed that
the pyrazole initially attenuated the in vitro potency; however,
additional structural modifications restored in vitro potency.
Prolylcarboxypeptidase (PrCP) (angiotensinase C) is a single
chain serine peptidase that cleaves the amide bond between a
C-terminal amino acid and a proline residue (i.e. peptide-Pro-Xxx-
OH).3 Several hypotheses concerning the biological role of PrCP
are based on the ability of PrCP to degrade bioactive peptides4,5
Me
O
Me
N
O
N
N
Ph
Ph
Ph
Ph
N
H
O
1
2
Cl
PrCP IC50 (h,m): 3.5, 5.0 nM
PrCP IC50 (h,m): 2.5, 1.4 nM
Figure 1. Recently discovered structures of potent PrCP inhibitors.
as well as the extensive tissue distribution of PrCP.6,7 Studies have
suggested that PrCP may be involved in cardiovascular control,8
metabolism regulation,9,10 inflammation induction,11 angiogene-
sis12 and chemotherapy resistance.13 The possibility for numerous
therapeutic indications has resulted in the rapid development of
small molecule inhibitors of PrCP.14
A program initiated to identify small molecule inhibitors of
PrCP has produced several classes of potent inhibitors.15 The
aminocyclohexanes, represented by 1 and 2 (Fig. 1) were recently
discovered as potent inhibitors of PrCP.16,17 During lead-optimiza-
tion studies, a bioisosteric replacement of the amide functional
group was explored. Many heterocycles have been used as
⇑
Corresponding author. Tel.: +1 732 594 2918.
E-mail address: thomas.graham@merck.com (T.H. Graham).
http://dx.doi.org/10.1016/j.bmcl.2014.02.070
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

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T. H. Graham et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1657–1660
Table 1
Table 3
Initial attempts to incorporate a pyrazole bioisostere into the lead molecules
Optimization of the lead with the pyrazole bioisostere
PrCP IC50 h,ma (nM)
F
Compd
N
HN
N
N
Me
N
HN
HN
Cl
3b
860,—
N
R
PrCP IC50 h,ma (nM)
74, 20
O
O
Compd
R=
8b
Me
N
Cl
4b
940,—
Cl
Cl
Cl
9
3.5, 2.2
92,—
114,—
40, 24
4-epi-9
ent-9
4-epi-(ent-9)
Br
OH
10
11
13, 5.9
69, 30
N
HN
5
270, 220
O
N
O
O
Cl
Br
12b
13b
14b
15b
150,—
F
F
N
HN
40, 56
1500,—
66, 180
epi-5
580
F
F
F
F
N
Me
Me
Me
Me
a
Compounds were tested as the trifluoroacetate salt; values are based on one or
two experiments, each in triplicate; h = human, m = mouse.
Me
b
Compound was tested as the racemate.
16b
17b
150,—
1000,—
H
bioisosteres of amides including oxazoles, thiazoles, triazoles, tet-
razoles and oxadiazoles; however, the use of pyrazoles as amide
bioisosteres, is less common.18 The present study was designed
a
Compounds were tested as the trifluoroacetate salt; values are based on one or
two experiments, each in triplicate; h = human, m = mouse.
b
Compound was tested as the racemate.
Table 2
Data supporting the heuristic model of a linear pharmacophore
to explore the possibility that a pyrazole can replace an amide
functional group in a series of PrCP inhibitors.19
Compd
PrCP IC50
h,ma (nM)
Replacement of the amide in 1 with a pyrazole afforded 3; how-
ever, the potency was significantly attenuated by the structural
modification.20 Attempts to regain the potency with additional
structural modifications afforded compounds 4 and 5 that also
proved to have attenuated inhibitory activity. In addition, com-
pounds 5 and epi-5 indicated that the relative stereochemistry of
the morpholine substituent has minimal effect on the in vitro inhi-
bition of PrCP.21
The data in Table 1 indicated that the incorporation of the pyr-
azole as an amide bioisostere resulted in a substantial loss in po-
tency. An additional set of compounds suggested a possible
reason for the decrease in activity (Table 2). Compound 6, the fluo-
rophenyl analog of 2, displayed similar potency to 2; however, 7
and ent-7, as structural isomers of 6 with the nitrogen shifted by
one position, demonstrated a substantial loss of potency despite
the retained amide functional group. The results in Table 2 sug-
gested that the distance between the basic nitrogen and the amide
hydrogen bond acceptor (HBA) may be a crucial parameter for the
inhibition of the PrCP enzyme.
F
O
O
O
6
10.4, 4.7
N
Ph
Ph
N
H
Cl
Cl
Cl
F
7
990,—
N
Ph
Ph
N
F
ent-7
850,—
The hypothesis of a linear pharmacophore embedded within 1
and 2 suggested that the compounds in Table 1 lost potency be-
cause of an expansion of the distance between the basic nitrogen
and the HBA (e.g. 1 vs 4), while the compounds in Table 2 lost po-
tency because of a contraction of the basic nitrogen-HBA distance
(e.g. 6 vs 7). Combining the pyrazole bioisostere shown in Table 1
N
Ph
Ph
N
a
Compounds were tested as the trifluoroacetate salt; values are based on one or
two experiments, each in triplicate; h = human, m = mouse.

T. H. Graham et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1657–1660
1659
Table 4
simplicity of the heuristic model guided the rapid design of new
molecular entities during the lead optimization process.
The results of shifting the aryl substituent to remove the stereogenic centers
Having restored the potency with the pyrazole bioisostere in
place, a brief SAR survey of the nitrogen substituent was con-
ducted. Substitution on the b-carbon with a hydroxyl (10) lost 2-
to 4-fold potency relative to 9 and substitution with a ketone
(11) resulted in a more substantial decrease in inhibitory activity.
Removal of a methylene from 8 afforded analog 12 with a 2-fold
loss in potency. The trifluoropropyl analog 13 lost 11- and 25-fold
relative to 9. Trifluoroethyl analog 14 was substantially less potent.
The progressive loss in potency for 13 and 14 is likely attributable
to inductive effects reducing the basicity of the nitrogen rather
than steric effects because the tert-butyl (15) and isopropyl (16)
analogs were more potent.24 The results for compounds 13–16
gave additional credence to the importance of a basic nitrogen in
the pharmacophore. Finally, the secondary amine 17 had an IC50
Compd
PrCP IC50
h,ma (nM)
X
F
18 (X = Cl)
19 (X = F)
105,—
30, 23
N
N
N
N
N
N
N
N
HN
20
21
22
7.0, 3.6
300,—
N
Me
F
F
value of 1 lM.
As mentioned earlier, the position of the p-fluorophenyl aro-
matic of 9 relative to the pharmacophore appears to be less re-
stricted to variations. To test this hypothesis, a series of analogs,
with the aromatic shifted by one carbon atom, was prepared
(Table 4). Compound 18 lost potency relative to the closest analog
9; however, substitution of the chloro- with a fluoro-group affor-
ded a modest improvement with compound 19. Methylation of
the pyrazole afforded 20 and 21, which demonstrated a greater
than 40-fold difference in potency against human PrCP and may
indicate the preferred pyrazole tautomer of 19. Finally, replace-
ment of the phenyl in 20 with a pyridyl afforded 22, which is the
most potent compound in the series.
N
Me
N
3.3, 0.7
N
Me
a
Compounds were tested as the trifluoroacetate salt; values are based on one or
two experiments, each in triplicate; h = human, m = mouse.
and the piperidine core shown in Table 2 would balance the linear
distance between the basic nitrogen and the HBA. The changes
would appear as a one bond frameshift of the original pharmaco-
phore that is in 1 and 2. Importantly, envisioning this model re-
quires selecting the proper tautomeric form of the pyrazole. To
test the heuristic model, the racemic pyrazole hybrid 8 was pre-
pared and demonstrated a greater than 10-fold improvement in
potency (Table 3). Replacing the p-chlorophenyl with a phenyl re-
stored the potency to the level exhibited by the initial leads 1 and
2. Thus, moving the nitrogen to an endocyclic position and
exchanging the chlorine atom for a hydrogen atom afforded potent
inhibitors of PrCP that incorporated a pyrazole as a replacement of
the amide in the lead structures. In addition, the results for the
stereoisomers of 9 suggested that the exact positioning of the
p-fluorophenyl aromatic relative to the core structure is less influ-
ential on the PrCP inhibition than the stereogenicity at the pyrazole
center.22,23 In general, additional geometric or electronic perturba-
tions could also explain differences in binding affinities, but the
The effect of the bioisosteric replacement on the mouse phar-
macokinetics was also evaluated (Table 5). Both amides 1 and 2
demonstrated high plasma protein binding (PPB) with the morpho-
line in 1 imparting a measurable free-fraction. The pharmacokinet-
ics of 1 and 2 reveal the differences in PPB because 1 has higher
total clearance and a consequently shorter half-life compared to
2. Compounds 9 and 12, like compound 2, were highly plasma pro-
tein bound and the pharmacokinetics parameters are generally
comparable to 2, with long half-life and improved oral bioavailabil-
ity. Similarly, compounds 15 and 16 also had improved oral bioav-
ailabilities relative to 1 and 2. Finally, compounds 18–22, with the
aromatic group shifted by one carbon atom thus removing the ste-
reogenic carbons, demonstrated high total clearances and modest
oral bioavailabilities in the mouse. The results in Table 5 demon-
strated that the pyrazole bioisostere can have mouse pharmacoki-
netic parameters that are comparable to those of the original
amides 1 and 2.
Table 5
Mouse pharmacokinetic parameters for selected compounds
PPBb
Mouse pharmacokinetics (C57Bl/6)c
Compda
h
m
Cl (mL/min/kg)
Vd (L/kg)
t
(h)
AUCNpo
(l
Mhkg/mg)
%F
½
1d
2
2
<1
3
<1
—
—
3
7
<1
1
4
72.2 5.5
31.8 8.0
28.9 7.5
15.4 0.7
44.7 4.0
50.1 3.0
59.2 13.1
77.1 6.6
51.9 4.3
74.4 16.3
10.8 1.5
12.1 3.5
13.3 3.3
9.4 0.7
18.1 0.5
18.3 4.3
11.4 3.3
17.2 4.2
19.9 2.8
14.1 4.0
1.7 0.2
4.4 2.0
4.8 0.4
7.7 0.8
5.4 0.5
4.6 1.3
1.8 0.7
2.7 0.7
4.7 0.5
2.0 0.3
0.037 0.011
0.276 0.131
0.413 0.088
0.877 0.229
0.313 0.100
0.453 0.043
0.057 0.014
0.043 0.013
0.163 0.017
0.076 0.016
7.8
28
33
39
36
54
8.7
12
22
14
<1
<1
<1
—
9e
12
15
16
18
19
20
22
—
<1
<1
<1
4
a
b
c
Compounds were tested as the trifluoroacetate salt.
Plasma protein binding data are reported as % unbound in 100% plasma; h = human, m = mouse.
Unless otherwise noted, mixture PK dosing (4 compounds + 1 standard, n = 3): iv 0.5 mg/kg (0.1 mg/mL in EtOH/PEG/water for each compound); po 1 mg/kg, (0.1 mg/mL
in EtOH/PEG/water for each compound).
d
Single compound PK dosing (n = 3): iv 2 mg/kg (1 mg/mL in EtOH/PEG/water); po 10 mg/kg, (1 mg/mL in EtOH/PEG/water).
Single compound PK dosing (n = 3): iv 1 mg/kg (0.2 mg/mL in EtOH/PEG/water); po 2 mg/kg, (0.2 mg/mL in EtOH/PEG/water).
e

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E.; Lisnock, J.; Li, X.; Shen, Z.; Tong, X.; Tung, E. C.; Wiltsie, J.; Xiao, J.; Xie, D.;
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isostere afforded a new series of PrCP inhibitors. Initially, the incor-
poration of the pyrazole decreased the in vitro potency; however,
the SAR revealed that exchanging an aminocyclohexane with a
piperidine restored the in vitro activity. Simplified analogs, with
the stereogenic centers removed by shifting the aromatic group,
were also potent in vitro inhibitors of PrCP. The pharmacokinetics
of selected compounds was comparable to the original amides.
While other heterocycles may also be suitable bioisosteres, the
present study demonstrates that pyrazoles are satisfactory bioisos-
teric replacements of amide functional groups. In addition, the
present study is an example of managing bioisosteric replacement
by incorporating subsequent structural modifications to maintain
potency against the selected target. The heuristic model for an
embedded pharmacophore is also described and may prove to be
useful for future inhibitor design. Additional studies on structurally
related inhibitors of PrCP will be reported in subsequent
communications.
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21. Human and mouse PrCP enzyme inhibition was determined using a standard
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22. The preparation of compound 9 began with the known piperidine that was
isolated as the earlier eluting isomer from chiral SFC separation using an AD-H
column:
10. (a) Chissoe, S. U.S. Pat. Appl. US 2008/0108080 A1, 8 May 2008; (b) Horvath, T.;
F
Diano, S.; Gao, Q.; Warden, C. H. Int. Pat. Appl. WO 2005/115446 A2,
8
December 2005.
11. (a) Chajkowski, S. M.; Mallela, J.; Watson, D. E.; Wang, J.; McCurdy, C. R.;
Rimoldi, J. M.; Shariat-Madar, Z. Biochem. Biophys. Res. Commun. 2011, 405, 338;
(b) Ngo, M.-L.; Mahdi, F.; Kolte, D.; Shariat-Madar, Z. J. Inflamm. 2009, 6, 3.
12. Adams, G. N.; Stavrou, E. X.; Fang, C.; Merkulova, A.; Alaiti, M. A.; Nakajima, K.;
Morooka, T.; Merkulov, S.; LaRusch, G. A.; Simon, D. I.; Jain, M. K.; Schmaier, A.
H. Blood 2013, 122, 1522.
13. Duan, L.; Motchoulski, N.; Danzer, B.; Davidovich, I.; Shariat-Madar, Z.;
Levenson, V. V. J. Biol. Chem. 2011, 286, 2864.
14. For recent reviews of PrCP inhibitors, refer to: (a) Scarry, S. C.; Rimoldi, J. M.
Annu. Rep. Med. Chem. 2013, 48, 91; (b) Jeong, J. K.; Diano, S. Trends Endocrinol.
Metab. 2013, 24, 61.
15. (a) Graham, T. H.; Liu, W.; Verras, A.; Reibarkh, M.; Bleasby, K.; Bhatt, U. R.;
Chen, Q.; Garcia-Calvo, M.; Geissler, W. M.; Gorski, J. N.; He, H.; Lassman, M. E.;
Lisnock, J.; Li, X.; Shen, Z.; Tong, X.; Tung, E. C.; Wiltsie, J.; Xie, D.; Xu, S.; Xiao, J.;
Hale, J. J.; Pinto, S.; Shen, D.-M. Bioorg. Med. Chem. Lett. 2012, 22, 2818; (b)
Graham, T. H.; Liu, W.; Verras, A.; Sebhat, I. K.; Xiong, Y.; Bleasby, K.; Bhatt, U.
R.; Chen, Q.; Garcia-Calvo, M.; Geissler, W. M.; Gorski, J. N.; He, H.; Lassman, M.
O
a, b, c, d
9 and 4-epi-9
OH
N
BOC
(a) CDI, THF, rt, 1 h then 4-chloroacetophenone, NaH, THF, 60 °C, 3 h; (b)
hydrazine, ethanol, reflux, 12 h; (c) TFA, CH2Cl2, rt, 12 h; (d) phenyl acetone,
NaBH(OAc)3, 4 Å mol sieves, 1,2-dichloroethane, rt, 12 h; separation of
diastereomers by HPLC (C-18, MeCN, water, TFA).
23. The basic conditions during the pyrazole synthesis caused
a fortuitous
epimerization of the stereogenic center at the 4-position of the piperidine
ring; thus compounds 4-epi-9 and 4-epi-(ent-9) are enantiomers
24. Morgenthaler, M.; Schweizer, E.; Hoffmann-Röder, A.; Benini, F.; Martin, R. E.;
Jaeschke, G.; Wagner, B.; Fischer, H.; Bendels, S.; Zimmerli, D.; Schneider, J.;
Diederich, F.; Kansy, M.; Müller, K. ChemMedChem 2007, 2, 1100.