Discovery of a new class of potent prolylcarboxypeptidase inhibitors derived from alanine

Article abstract of DOI:10.1016/j.bmcl.2011.12.064

Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarboxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.

Full text of DOI:10.1016/j.bmcl.2011.12.064

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1774–1778
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of a new class of potent prolylcarboxypeptidase inhibitors
derived from alanine
Zhicai Wu a, , Cangming Yang a, Yusheng Xiong a, Zhe Feng a, Matthew Lombardo a, Andreas Verras a,
⇑
Renee M. Chabin b, Suoyu Xu c, Xinchun Tong c, Dan Xie d, Mike E. Lassman d, Urmi R. Bhatt d,
Margarita M. Garcia-Calvo d, Wayne Geissler d, Zhu Shen d, Qing Chen d, Ranabir Sinharoy d,
Jeffrey J. Hale a, James R. Tata a, Shirly Pinto d, Dong-Ming Shen a, Steven L. Colletti a
a Department of Discovery Chemistry, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065-0900, USA
b Department of Research Operations, 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 Diabetes, 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:
Efforts to modify the central proline portion of lead compound 4 lead to the discovery of novel prolylcarb-
oxypeptidase (PrCP) inhibitors. Especially, replacement with alanine afforded compound 19 displaying
more potent human and mouse PrCP inhibitory activity than 4 and an overall comparable profile.
Ó 2011 Published by Elsevier Ltd.
Received 26 October 2011
Revised 9 December 2011
Accepted 12 December 2011
Available online 24 December 2011
Keywords:
Prolylcarboxypeptidase (PrCP)
Obesity
Inhibitor
Prolylcarboxypeptidase (PrCP) is a member of serine proteases
that specifically cleaves C-terminal amino acid residue connected
to proline in peptides such as angiotensin II (AngII), angiotensin
III (AngIII) and des-Arg9-bradykinin.1,2 More recently PrCP was
As part of an effort to develop potent PrCP inhibitors with good
physical properties and pharmacokinetic profiles, we focused on
modifying the central proline portion which occupies the S1 pock-
et. In this Letter, we report the replacement of proline with other
amino acids affording new classes of PrCP inhibitors.
First, a group of amino acid derivatives from our sample collec-
tion was tested for their PrCP inhibitory activity9 at two concentra-
tions and the results for selected compounds 5, 6, and 7 were
shown in Chart 2. The alanine derived compound 5 gave a promis-
found to metabolize
a
-melanocyte-stimulating hormone
(a-
MSH).3 Via the regulation of active
a
-MSH, PrCP might be an
important component of melanocortin signaling and weight main-
tenance.3–6 Indeed it is reported that PrCPÀ/À mice demonstrated
reduced body weight and food intake, as well as fat mass reduction
versus the control WT mice.3 Our in-house effort7 in phenotyping
PrCPÀ/À mice confirmed these findings. Thus, effort was initiated
to validate PrCP as a potential target for obesity treatment using
small molecule PrCP inhibitors.8 A series of noncovalent PrCP
inhibitors such as 3 (Chart 1) was identified from presumably
covalent binders 1 and 2 based on rational design by our co-work-
ers.8a Optimization of left hand, the purported P2 and P3 binding
pockets (S2 and S3 sites, respectively) and right hand imidazole
(assumed to interact with catalytic triad) provided reversible,
potent and selective PrCP inhibitors represented by compound 4,
which led to a moderate body weight loss and reduced food intake
in WT eDIO mice with subchronic dosing at 100 mpk.8a
ing activity of 65% inhibition at 0.5 lM. The analogous phenylala-
nine or cyclohexylglycine derivatives 6 and 7 showed very little
PrCP inhibitory activity. We also tried to modify the pyrrolidine
ring of the central proline portion. The piperidine carboxylic acid
derived analog 8 and pyridylcarboxlic acid derivatives 9 and 10
were prepared. Unfortunately, these six-membered ring analogs
were not active against PrCP.
Encouraged by the PrCP inhibitory activity of compound 5, we
further investigated the alanine derivatives and its close analogs.
Shown in Chart 3 are those compounds with the optimized dichlo-
robenzimidazole on the right side and a typical biphenylpropionic
amide5a on the left side for the convenience of comparison. Clearly,
alanine derived compound 12 display the most potent activity
against PrCP. The glycine derivative 11 was not active and methyl-
alanine derived compound 13 lost about six fold in activity. It was
⇑
Corresponding author.
E-mail address: zhicai_wu@merck.com (Z. Wu).
0960-894X/$ - see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.12.064

Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1774–1778
1775
O
O
O
N
O
N
S
N
O
N
O
N
NH
N
NH
Boc
NH
NH
Fmoc
O
1
2
3
h-PrCP IC50 5 µM
h-PrCP IC50 1.2 µM
h-PrCP IC50 0.3 µM
Cl
Cl
O
N
N
NH
4 h-PrCP IC501 nM
m-PrCP IC50 2 nM
m-AngIII IC50 26 nM
HN
O
NH2
Chart 1.
OMe
OMe
N
N
H
H
N
N
N
O
N
H
O
H
O
Me
O
6
5
h-PrCP:
h-PrCP:
31% inh. @ 5 uM
7% inh. @ 0.5 uM
90% inh. @ 5 uM
65% inh. @ 0.5 uM
Cl
Cl
N
H
N
NH
O
N
N
F
O
H
O
N
7
NH
h-PrCP:
Boc
13% inh. @ 5 uM
3% inh. @ 0.5 uM
8
h-PrCP IC50 > 10 uM
Cl
Cl
Cl
Cl
N
N
O
N
NH
NH
NH
9
10
h-PrCP IC50 > 10 uM
N
h-PrCP IC50 > 10 uM
Chart 2.
mouse whole plasma AngIII assay.9 In the presence of 100% mouse
serum, mouse AngIII assay assesses serum shift of the compounds.
Overall, the alanine series displayed SAR similar to proline analogs.
With regard to the biphenylpropyl group at the P2 position, meta-
biphenyl (16) lowered the activity against PrCP 15-fold compared
to para-biphenyl (12). As discovered in proline series,8a incorpora-
tion of an alpha amino isobutyrate (AIB) and a beta methyl group
led to compound 19, which is much more potent against h- and
m-PrCP than the corresponding compound 4 of proline series.
These two compounds have comparable activity in mouse AngIII
assay. In addition, the terminal phenyl group can be removed
and replaced with a simple acyclic substituent such as cyano (21
and 22) and trifluoroethoxy groups (23 and 24). In fact, compounds
23 and 24 are comparable to 19 and 4 based on their h-PrCP activ-
ity and serum shift. Moreover, polar groups are tolerated at the
biphenyl position. The end phenyl group can also be replaced with
aromatic heterocycles (15, 25) or substituted with polar groups. As
for compound 25, even though its h-PrCP activity reduced about
10-fold, its activity in mouse AngIII assay is comparable to com-
pound 19. Most likely the added polarity by the end polar group
further reduced its serum shift.
Cl
Cl
N
H
N
N
2
1
H
R
R
O
11, R1 = H, R2 = H, h-PrCP IC50 > 10 µM
12, R1 = H, R2 = Me, h-PrCP IC50 = 60 nM
13, R1 = Me, R2 = Me, h-PrCP IC50 = 374 nM
14, R1 = H, R2 = CH2OH, h-PrCP IC50 = 225 nM
Chart 3.
worth noting that the serine derivatives 14 have a reasonable
activity with an IC50 of 225 nM.
Efforts were then focused on the investigation of alanine deriv-
atives. We fixed the 5,6-dichlorobenzimidazole on the right hand
side to take advantage of its improvements in PrCP potency and
pharmacokinetic profile. Optimization was carried out at the pur-
ported P2 and P3 positions. Showed in Table 1 are selected com-
pounds with their activities on h- and m-PrCP inhibition8a and

1776
Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1774–1778
Table 1
In vitro SAR of alanine derivatives
Cl
Cl
N
H
N
R
N
H
O
Me
Compound
R
h-PrCP IC50 (nM)
m-PrCP IC50 (nM)
Mouse AngIII IC50 (nM)
N
15
48
—
—
16
17
998
160
—
—
—
—
NH2
NH2
18
19
2.6
2.6
638
Me
NH2
O
HN
0.37
0.039
22
Me
NH2
20
21
1
—
—
585
HN
NH2
102
5000
NC
NH2
22
23
6.6
128
1600
O
HN
NC
NH2
O
F3C
O
0.17
—
92
HN
Me
OH
F3C
O
O
24
25
HN
0.74
—
—
45
78
Me
OH
HN
N
O
HN
ME
5.6
It has been reported that the biphenyl propionic amide could be
replaced with phenylpiperidyl propionic amide in the proline ser-
ies.8b The possibility of incorporating phenylpiperidyl onto the
newly discovered acyclic series was examined. Selected com-
pounds are shown in Table 2. Again, the alanine derivative 28
was more potent against PrCP than glycine derivative 26 and

Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1774–1778
1777
Table 2
Cl
SAR of phenylpiperidinyl or phenylpiperazinyl amides
O
H2N
Cl
Cl
H
N
a
b
Cl
N
Cl
+
Boc
OH
H
N
H2N
Me
N
H
Boc
Cl
N
33
H
N
1R
34
35
Me
d
R
N
H
Cl
R2
O
Cl
N
c
Compound
R
R1 R2 h-PrCP
IC50 (nM) IC50 (nM)
Mouse AngIII
H2N
N
H
NHBoc
OH
Me
36
37
Me
O
26
H
H
427
—
Cl
N
Boc
Cl
HN
Me
N
e
f
H
N
27
28
29
30
Me Me 181
—
N
H
N
N
N
O
Me
38
O
Cl
BocHN
OH
H
H
H
Me
Me
Me
14
38
5
—
40
Me Me
g
Cl
NH2
N
H
N
N
H
N
Me
O
93
Me
—
N
F
NH2
Me
Cl
Me
5000
F
F
N
N
N
O
Cl
HN
H
N
N
H
31
32
H
H
Me
Me
16
10
20,000
Me
O
19
Me
N
N
Scheme 1. Reagents and conditions: (a) EDC, HOBt, Et3N, DMF, rt, 16 h; (b) acetic
acid, 60 °C, 16 h; 70% for (a) and (b); (c) trifluoroacetic acid, CH2Cl2, rt, 1 h, 100%; (d)
37, EDC, HOBt, Et3N, DMF, rt, 16 h, 43%; (e) trifluoroacetic acid, CH2Cl2, rt, 1 h, 100%;
(f) 40, EDC, HOBt, Et3N, DMF, rt, 16 h, 48%; (g) trifluoroacetic acid, CH2Cl2, rt, 1 h,
22%.
F
methylalanine derivative 27. The piperazine analogs of alanine ser-
ies (29, 30 and 31) were also potent. 3-Fluorophenylpiperazine on
the right hand side gave a compound with h-PrCP IC50 5 nM. Even
though its activity in mouse AngIII assay shifted 1000-fold, it was
believed that replacement of the end phenyl with polar group
might decrease the serum shift significantly as discovered in the
proline series.
Initial pharmacokinetic studies were done in mice. Shown in Ta-
ble 3 were the results for the most promising alanine derivative 19.
Compared to proline compound 4, the closely related alanine ana-
log 19 has a slightly improved overall pharmacokinetic profile with
regard to half life and oral exposure.
moval of Boc group in 38 gave amine 39. Compound 39 coupled
with N-Boc protected AIB (40) followed by deprotection of the N-
Boc group generated the desired compound 19.
In conclusion, a class of highly potent PrCP inhibitors derived
from alanine has been discovered. A representative compound 19
displayed excellent human, mouse PrCP and mouse whole serum
PrCP inhibitory activities (hIC50 = 0.37 nM, mIC50 = 0.039 nM,
mAngIII IC50 = 22 nM), and reasonable pharmacokinetic properties.
Its overall profile is comparable to the benchmark prolinyl com-
pound 4. Further improvement of physical properties and pharma-
cokinetic profile will be reported in due course.
The synthesis of the representative compound 19 is illustrated
in Scheme 1. Benzimidazole 35 was made from N-Boc protected
alanine 33 with 4,5-dichlorobenzene-1,2-diamine 34 in a two-step
reaction. Deprotection of the amine 35 gave intermediate 36,
which reacted with biphenylaminoacid 37 to afford amide 38. Re-
References and notes
1. (a) Yang, H. Y. T.; Erdos, E. G.; Chiang, T. S. Nature 1968, 218, 1224; (b) Yang, H. Y.
T.; Erdos, E. G.; Chiang, T. S.; Jenssen, T. A.; Rodgers, J. G. Biochem. Pharmcol.
1970, 19, 1201; (c) Odya, C. E.; Marinkovic, D. V.; Hammon, K. J.; Stewart, T. A.;
Erdos, E. G. J. Biol. Chem. 1978, 253, 5927; (d) Skidgel, R. A.; Erdos, E. G. Immun.
Rev. 1998, 161, 129; (e) Kumamoto, K.; Stewart, T. A.; Johnson, A. R.; Erdos, E. G.
J. Clin. Invest. 1981, 67, 210.
2. (a) Shariat-Madar, Z.; Mhdi, F.; Schmaier, A. H. J. Biol. Chem. 2002, 277, 17962;
(b) Moreira, C. R.; Schmaier, A. H.; Mahdi, F.; da Motta, G.; Nader, H. B.; Shariat-
Madar, Z. FEBS Lett. 2002, 523, 167; (c) Shariat-Madar, Z.; Mhdi, F.; Schmaier, A.
H. Blood 2004, 103, 4554.
3. Wallingford, N.; Perround, B.; Gao, Q.; Coppola, A.; Gyengesi, E.; Liu, Z. W.; Gao,
X. B.; Diament, A.; Haus, K. A.; Shariat-Madar, Z.; Hahdi, F.; Wardlaw, S. L.;
Schmaier, A. H.; Warden, C. H.; Diano, S. J. Clin. Invest. 2009, 119, 2291.
4. (a) Mallela, J.; Yang, J.; Shariat-Madar, Z. Int. J. Biochem. Cell Biol. 2009, 41, 477;
(b) Wang, L.; Feny, Y.; Zhang, Y.; Zhou, H.; Jiang, S.; Niu, T.; Wei, J. L.; Xu, X.; Xu,
X. P.; Wang, X. Am. J. Obster. Gynecol. 2006, 195, 162.
Table 3
Mouse PK of selected PrCP inhibitorsa
Compds
F%
Cl (mL/min/kg)
Vdss (L/kg)
T1/2(h)
AUCNpo
M h kg/mg)
(l
4
19
13
17
23
58
4.4
14
2.3
4
0.18
0.52
a
Formulations: 1 mg/mL ethanol/PEG/water (20:40:40). IV dose: 1 mg/kg (n = 2).
PO dose: 2 mg/kg (n = 3). Blood concentration was determined by LC/MS/MS fol-
lowing protein precipitation with acetonitrile.
5. (a) Shariat-Madar, B.; Kolte, D.; Verlangiert, A.; Shariat-Madar, Z. Diab. Metab.
Syndr. Obes.: Targets Ther. 2010, 3, 67.

1778
Z. Wu et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1774–1778
binding. Therefore LC/MS was used to monitor the cleavage of exogenous
6. Diament, A. L.; Warden, C. H. Int. J. Obes. 2004, 28, 199.
7. Pinto, S. et al. manuscript in preparation.
angiotensin III (AngIII, RVYIHP;F) by endogenous plasma PrCP. Since peptide
substrates are susceptible to cleavage by multiple proteases in plasma,
protease inhibitors cocktail was added and the pH of the assay medium was
optimized to pH 5.5 to minimize background proteases activity so that the rate
of AngIII cleavage is proportional to PrCP activity. In detail, Experiments were
8. (a) Zhou, C.; Garcia-Calvo, M.; Pinto, S.; Lombardo, M.; Feng, Z.; Bender, K.; Pryor,
K. D.; Bhatt, U. R.; Chabin, R. M.; Geissler, W. M.; Shen, Z.; Tong, X.; Zhang, Z.;
Wong, K. K.; Roy, R. S.; Chapman, K. T.; Yang, L.; Xiong, Y. J. Med. Chem. 2010, 53,
7251; (b) Shen, H. C.; Ding, F.-X.; Zhou, C.; Xiong, Y.; Verras, A.; Chabin, R. M.;
Xu, S.; Tong, X.; Xie, D.; Lassman, M. E.; Bhatt, U. R.; Garcia-Calvo, M.; Geissler,
W. M.; Shen, Z.; Chen, D.; Sinha, R. R.; Hale, J. J.; Tata, J. R.; Pinto, S.; Shen, D.;
Colletti, S. L. Bioorg. Med. Chem. Lett. 2011, 21, 1299.
carried out at 37 °C under standard reaction conditions defined as 48
mouse plasma, of 5 M ammonium acetate, of protease inhibitor
cocktail and 4 L of 50 M AngIII for 8 min. Reaction was stopped with 100 lL
lL of DIO
3
l
L
3 lL
l
l
9. Mouse whole plasma AngIII assay measures PrCP activity/inhibition in mouse
of 8 M urea. Peptides were extracted and then analyzed with LC/MS. IC50
values are reported as single determinations or as the average of at least two
determinations.
plasma. The enzyme assay described in Ref. 8a utilizes
a FRET substrate
(Mca-Ala-Lys-Dnp) that is incompatible with plasma due to high protein