2,3-Diaminopyridine as a platform for designing structurally unique nonpeptide bradykinin B1 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.02.077

A novel class of 2,3-diaminopyridine bradykinin B₁ receptor antagonists is disclosed. Structure-activity relationship studies (SARs) that led to compounds with significantly improved potency and pharmacokinetic properties relative to the lead compound are described.

Full text of DOI:10.1016/j.bmcl.2005.02.077

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2385–2388
2,3-Diaminopyridine as a platform for designing structurally
unique nonpeptide bradykinin B1 receptor antagonists
Dong-Mei Feng,a,* Jenny M. Wai,a Scott D. Kuduk,a Christina Ng,a Kathy L. Murphy,b
Richard W. Ransom,b Duane Reiss,b Raymond S. L. Chang,b Charles M. Harrell,b
Tanya MacNeil,c Cuyue Tang,d Thomayant Prueksaritanont,d Roger M. Freidinger,a
Douglas J. Pettiboneb and Mark G. Bocka
aDepartments of Medicinal Chemistry, Merck Research Laboratories, West Point, PA 19486, USA
bNeuroscience Drug Discovery, Merck Research Laboratories, West Point, PA 19486, USA
cDepartment of Metabolic Disorders, Merck Research Laboratories, Rahway, NJ 07065, USA
dDrug Metabolism, Merck Research Laboratories, West Point, PA 19486, USA
Received 14 January 2005; accepted 25 February 2005
Available online 28 March 2005
Abstract—A novel class of 2,3-diaminopyridine bradykinin B1 receptor antagonists is disclosed. Structure–activity relationship stud-
ies (SARs) that led to compounds with significantly improved potency and pharmacokinetic properties relative to the lead com-
pound are described.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ists have therapeutic potential in treating inflammatory
pain such as osteoarthritis, as well as in ameliorating
neuropathic pain conditions.2,7,8 The identification of
potent and selective, small molecule BK B1 receptor
antagonists is currently an area of intense research. In
this context, we have previously disclosed the discovery
of nonpeptide BK B1 receptor antagonists that avidly
bind the human B1 receptor and exhibit in vivo efficacy
in animal models of pain.5e,f In our continuing search
for diverse chemical structures that exhibit affinity for
the BK B1 receptor and which also have the potential
for improved pharmacokinetic properties, we uncovered
a novel 2,3-diaminopyridine, compound 1 (Fig. 1), by
means of a receptor binding screen. It is the chemical
elaboration of this lead compound 1, which afforded
Kinins are a group of peptides that include the non-
apeptide, bradykinin (BK) (Arg1-Pro2-Pro3-Gly4-Phe5-
Ser6-Pro7-Phe8-Arg9) and the decapeptide, kallidin
(Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9-Arg10).
The kinins are formed in plasma and various tissues in
response to inflammatory insults, infection, or tissue
trauma.1 Once released, kinins exert most of their bio-
logical effects by activating at least two subtypes of spe-
cific G-protein coupled cell surface receptors, designated
as B1 and B2.2 The B2 receptors appear to be constitu-
tively expressed in most peripheral and central tissues
under normal physiological conditions.3 On the other
hand, the B1 receptors are typically expressed only at
low levels, but can be functionally upregulated in the
periphery and CNS by pro-inflammatory and noxious
stimuli.4 The role of BK B1 receptors to mediate re-
sponses to pain in animals has been established using
selective B1 receptor antagonists and B1 knockout
mice.5,6 These findings imply that B1 receptor antagon-
CH3
H
N
O
N
NH
O
OCH3
Keywords: Bradykinin B1; Receptor; Antagonists; 2,3-Diamino-
pyridine.
*
Corresponding author. Tel.: +1 2156527838; fax: +1 2156523971;
e-mail: dongmei_feng@merck.com
Figure 1. B1 receptor binding screening lead, 1.
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.077

2386
D.-M. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2385–2388
analogs with improved human BK B1 receptor binding
potencies and pharmacokinetic properties in rodents,
that forms the basis of this communication.
Accordingly, the commercially available 40-methyl-
biphenyl-2-carboxylic acid methyl ester A was trans-
formed via the four-step sequence of bromination,
2-amino-3-nitropyridine alkylation, reduction, and acyla-
tion to afford analogs 2–9, 11, and 13. Alternatively,
intermediate C could be diverted to give the methyl
amides 10 and 15 following standard procedures. The
compounds listed in Table 2 were derived according to
the synthetic routes outlined in Scheme 2. In this way,
2-chloro-3-nitro-4-methylpyridine was reacted with
(R)-4-bromo-a-methylbenzylamine to give the 2-amino-
pyridine adduct, which yielded intermediate E after
reduction with stannous chloride. Subsequent boronate
ester formation of E and Suzuki-coupling with methyl
2-bromobenzoate yielded intermediate F. The latter
was acylated in the standard fashion to give 12 and 14
or saponified and converted to the methyl amide 16 in
the usual manner.
2. Chemistry
The compounds described in this study are tabulated in
Tables 1 and 2. The general preparative method used to
access the compounds in Table 1 is outlined in Scheme 1.
Table 1. Effects of 3-amidopyridine modifications on BK B1 receptor
binding affinities and PK properties
H
R2
N
O
N
NH
O
R4
3. Biological results and discussion
Compounda
R2
R4
hBK1
Ki (nM)b
Rat PKc
Ki values (nM) were determined radiometrically using
the appropriate radioligand and Chinese hamster ovary
(CHO) cells stably expressing the human BK B1 recep-
tor.9 The protocol for determining pharmacokinetic
properties using Sprague–Dawley rats (n = 3) wherein
the oral dose is 10 mg/kg and the IV dose is 2 mg/kg,
was identical to that previously described. Interanimal
variability was less than 20%.5e
F % t1/2 CL
1
2
n-C4H9
n-C3H7
i-C3H7
n-C2H5
OCH3
OCH3
OCH3
OCH3
200
88
2
0.2 30
15 0.4 23
ND
ND
3
230
119
1280
390
40
4
5
CH2N(CH3)2 OCH3
CH2OCH3 OCH3
CH2SO2CH3 OCH3
ND
ND
6
7
ND
0.2 35
8
CH2CF3
CH2CN
CH2CF3
OCH3
OCH3
NHCH3
12
11
9
The initial objective in optimizing the pharmacological
profile of the screening lead compound 1 was to increase
its human BK B1 receptor binding affinity. Therefore,
our attention was immediately drawn to the 3-amide
function following the observation that shortening the
chain length of the n-pentanoyl amide in 1, by one car-
bon atom, resulted in a 2-fold boost in potency (cf. 1
and 2). An extensive analoging effort ensued, which
9
10
28 0.8 18
95 0.3 15
33
a All compounds were >98% pure by HPLC and characterized by 1H
NMRand HRMS.
b Values represent the numerical average of at least two experiments.
Inter-assay variability was 20% for the binding assays.
c F % oral bioavailability, half-life is represented in hours, and CL in
mL/min/kg.
Table 2. 4-Methyl pyridine analogs
CH3
H
R2
N
O
N
NH
O
R4
R3
Compounda
R2
R3
R4
hBK1 Ki (nM)b
Rat PKc
F %
t1/2
CL
14
11
12
13
14
15
16
CH2CF3
CH2CF3
CH2CN
CH2CN
CH2CF3
CH2CF3
H
OCH3
OCH3
OCH3
OCH3
7.1
1.8
11.5
2.6
11
40
17
19
27
79
25
0.4
0.9
2.0
0.2
1.0
0.5
(R)CH3
H
8.0
4.3
18.5
30
(R)CH3
H
(R)CH3
NHCH3
NHCH3
6.0
31
a All compounds were >98% pure by HPLC and characterized by 1H NMRand HRMS.
b Values represent the numerical average of at least two experiments. Inter-assay variability was 20% for the binding assays.
c F % oral bioavailability, half-life is represented in hours, and CL in mL/min/kg.

D.-M. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2385–2388
2387
position in an attempt to mitigate this metabolic path-
way (Table 2). The latter modification yielded the in-
tended result. The 3,3,3-trifluoropropionamido methyl
ester 11 and cyanoacetamido methyl ester 13 were essen-
tially equipotent with their respective des-methyl ana-
logs, 8 and 9. Moreover, improvements in oral bio-
availability, half life, and clearance were also realized.
A similar trend was observed with the 3,3,3-trifluoro-
propionamido methyl amides 10 and 15, effectively mak-
ing the 2,3-diamino-4-methylpyridine ring the new lead
template.
Br
H3C
O
OCH3
O
OCH3
a
d
A
B
b, c
R1
N
H
N
R2
R1
N
NH2
NH
O
NH
O
OCH3
O
OCH3
During the course of optimizing the newly identified
lead compounds, 11 and 13, additional points of metabo-
lism were identified. Not surprisingly, among these, the
benzylic position of the central aromatic ring appeared
especially vulnerable. In response, a methyl group was
installed to yield 12 and 14, respectively. Both com-
pounds display improved human B1 receptor binding
affinity relative to their progenitors and in the case of
the 3,3,3-trifluoromethylpropionamide 12, a modest
improvement in clearance and half life was observed
compared to 11. The (S)-configured enantiomers of clo-
sely related analogs were also examined but in each in-
stance, showed substantially reduced human B1
C
R1 = H; 2-9, 11, 13
e, .f, g
R1
N
R2
H
N
O
NH
O
NHCH3
R1 = CH3; 10, 15
CH3
Scheme 1. Reagents and conditions: (a) NBS, AIBN, CCl4, 85%; (b) 2-
amino-3-nitropyridine, NaH, 0 °C, DMF; (c) SnCl2, MeOH, 70 °C,
72% (for b, c); (d) carboxylic acid, EDCI, HOBt, 95%; (e) 4 N NaOH,
MeOH, H2O, 50 °C; (f) carboxylic acid, EDCI, HOBt; (g) NH2CH3,
EDCI, HOBt, 77% (for e, f, g).
CH3
NH2
NH
a, b
NO2
Cl
N
N
H3C
Br
D
E
was designed to flesh out the SARrelated specifically to
the 3-amide function. As the BK B1 receptor binding
data for selected compounds in Table 1 indicate, binding
affinity is clearly influenced by steric and electronic fac-
tors. Thus, increased steric bulk (1, 3), as well as the
incorporation of electron donating groups (5, 6) at the
3-amide group have a detrimental effect on potency.
Conversely, the introduction of electron withdrawing
groups on the 3-amide side chain resulted in improved
binding affinities. Among the functional groups, which
were surveyed, the 3,3,3-trifluoropropionamide (8) and
cyanoacetamide (9) were optimum. While these minor
structural changes to 1 had a significant effect on BK
B1 receptor binding affinity, these modifications alone
had no marked influence on pharmacokinetic (PK)
properties. We surmised that the methyl ester on the dis-
tal aromatic ring of compounds 1–9 was, in part,
responsible for their poor PK profile. The latter assump-
tion was born out by the improved bioavailability and
reduced clearance rate of the corresponding N-methyl
amide 10.
c, d
CH
N
CH3
R2
3H
N
NH2
NH
e
O
NH
N
O
OCH3
O
OCH3
H3C
H3C
F
12, 14
f, g, h
CH
3H
R2
N
O
N
NH
O
NHCH3
H3C
16
To gain further insight toward improving the PK prop-
erties of lead compounds exemplified by structures 8–10,
we examined their in vitro stabilities in rat liver micro-
somal preparations (data not shown). Metabolite profil-
ing indicated that the 4-position of the pyridine ring was
oxidized in all compounds, which were examined. Con-
sequently, we installed a methyl group at the 4-pyridine
Scheme 2. Reagents and conditions: (a) (R)-4-bromo-a-methylbenzyl-
amine, TEA, n-BuOH, 110 °C; (b) SnCl2, MeOH, 70 °C, 75% (for a,
b); (c) Pd(dppf)Cl2, KOAc, pinacoldiboron ester, DMSO, 80 °C; (d)
methyl 2-bromobenzoate, Pd(dppf)Cl2, K2CO3, DMSO, 80 °C, 75%
(for c, d); (e) carboxylic acid, EDCI, HOBt, 95%; (f) 4 N NaOH,
MeOH, H2O, 50 °C; (g) carboxylic acid, EDCI, HOBt; (h) NH2Me,
EDCI, HOBt, 77% (for f, g, h).

2388
D.-M. Feng et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2385–2388
3. Bock, M. G.; Hess, J. F.; Pettibone, D. J. In Annual
Reports in Medicinal Chemistry; Doherty, A. M., Ed.;
Elsevier: New York, 2003; Vol. 38, pp 111–120.
4. (a) Calixto, J. B.; Cabrini, D. A.; Ferreira, J.; Campos, M.
M. Pain 2000, 87, 1–5; (b) Calixto, J. B.; Cabrini, D. A.;
Ferreira, J.; Campos, M. M. Curr. Opin. Anaesthesiol.
2001, 14, 519–526.
receptor binding affinity (data not shown). Similarly,
replacement of the (R)-benzylic methyl group in 14 with
ethyl or methoxymethyl groups resulted in a significant
diminution of human B1 receptor binding affinity. It is
apparent that the installation of a benzylic methyl group
benefits human B1 receptor binding potency in this series
of compounds but its effects on their PK properties is
not as definitive. This observation also obtains for the
methyl amides 15 and 16.
5. (a) Rupniak, N. M. J.; Longmore, J.; Hill, R. G. In
Molecular Basis of Pain Induction; Wood, J. J., Ed.; Wiley:
New York, 2000; pp 149–174; (b) Stewart, J. M.; Gera, L.;
Chan, D. C.; Whalley, E. T.; Hanson, W. L.; Zuzack, J. S.
Can. J. Physiol. Pharmacol. 1997, 75, 719–724; (c) Ferre-
ira, J.; Campos, M. M.; Arau´jo, R.; Bader, M.; Pesquero,
J. B.; Calixto, J. B. Neuropharmacology 2002, 43, 1188–
1197; (d) Mason, G. S.; Cumberbatch, M. J.; Hill, R. G.;
Rupniak, N. M. J. Can. J. Physiol. Pharmacol. 2002, 80,
264–268; (e) Wood, M. R.; Kim, J. J.; Han, W.; Dorsey, B.
D.; Homnick, C. F.; DiPardo, R. M.; Kuduk, S. D.;
MacNeil, T.; Murphy, K. L.; Lis, E. V.; Ransom, R. W.;
Stump, G. L.; Lynch, J. J.; OÕMalley, S. S.; Miller, P. J.;
Chen, T.-B.; Harrell, C. M.; Chang, R. S. L.; Sandhu, P.;
Ellis, J. D.; Bondiskey, P. J.; Pettibone, D. J.; Freidinger,
R. M.; Bock, M. B. J. Med. Chem. 2003, 46, 103–1806; (f)
Su, D.-S.; Markowitz, M. K.; DiPardo, R. M.; Murphy,
K. L.; Harrell, C. M.; OÕMalley, S. S.; Ransom, R. W.;
Chang, R. S. L.; Ha, S.; Hess, J. F.; Pettibone, D. J.;
Mason, G. S.; Boyce, S.; Freidinger, R. M.; Bock, M. G.
J. Am. Chem. Soc. 2003, 125, 7516–7517.
In summary, we have disclosed the novel 2,3-diamino-
pyridine screening lead 1, which displays modest recep-
tor binding affinity for the human bradykinin B1
receptor and described its structural elaboration to give
compounds with up to 100-fold enhanced binding po-
tency and significantly improved PK properties. These
SARstudies, guided by in vitro metabolism experiments
using rat liver microsomes, demonstrate that the 2,3-di-
amino-4-methylpyridine ring system is a viable platform
for the design of a new generation of low molecular
weight, selective BK B1 receptor antagonists with desir-
able PK properties.10 These studies are continuing and
their outcomes will be reported in due course.
Acknowledgements
6. Pesquero, J. B.; Arau´jo, R.; Heppenstall, P. A.; Stuck, C.
L.; Silva, J. A.; Walther, T.; Oliveira, S. M.; Pesquero, J.
L.; Paiva, A. C.; Calixto, J. B.; Lewin, G. R.; Bader, M.
Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 8140–8145.
7. Bock, M. G.; Longmore, J. Curr. Opin. Chem. Biol. 2000,
4, 401–406.
We would like to acknowledge the assistance of Dr. P.
Sandhu, Ms. Y. M. Leonard, B.-L. Wan, J. Murphy,
and Mr. C. F. Homnick. We are grateful to Drs. D.-S.
Su and M. R. Wood for useful discussions.
8. Marceau, F.; Regoli, D. Nat. Rev. Drug Disc. 2004, 3,
845–852.
9. All test compounds in this report were selective for the BK
References and notes
B1 receptor (BK B2 IC50 > 10 lM).
1. Couture, R.; Harrisson, M.; Vianna, R. M.; Cloutier, F.
Eur. J. Pharmacol. 2001, 429, 161–176.
2. Calixto, J. B.; Medeiros, R.; Fernandes, E. S.; Ferreira, J.;
Carini, D. A.; Campos, M. M. Br. J. Pharmacol. 2004,
143, 803–818.
10. Kuduk, S. D.; Ng, C.; Feng, D.-M.; Wai, J. M.-C.; Chang,
R. S. L.; Harrell, C. M.; Murphy, K. L.; Ransom, R. W.;
Reiss, D.; Ivarsson, M.; Mason, G.; Boyce, S.; Tang, C.;
Prueksaritanont, T.; Freidinger, R. M.; Pettibone, D. J.;
Bock, M. G. J. Med. Chem. 2004, 47, 6439–6442.