Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

Article abstract of DOI:10.1016/S0960-894X(00)00389-9

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

Full text of DOI:10.1016/S0960-894X(00)00389-9

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1975±1978
Substituted 2-Aminopyridines as Inhibitors of Nitric
Oxide Synthases
William K. Hagmann,a,* Charles G. Caldwell,a Ping Chen,a Philippe L. Durette,a
Craig K. Esser,a Thomas J. Lanza,a Ihor E. Kopka,a Ravi Guthikonda,a
Shrenik K. Shah,a Malcolm MacCoss,a Renee M. Chabin,b Daniel Fletcher,d
Stephan K. Grant,b Barbara G. Green,b John L. Humes,c Theresa M. Kelly,b Sylvie Luell,
d Roger Meurer,d Vernon Moore,d Stephen G. Pacholok,c
Tony Pavia,b Hollis R. Williamsc and Kenny K. Wongb
aDepartment of Medicinal Chemistry, Merck Research Laboratories Rahway, NJ 07065, USA
bDepartment of Biochemistry, Merck Research Laboratories Rahway, NJ 07065, USA
cDepartment of In¯ammation Research, Merck Research Laboratories Rahway, NJ 07065, USA
dDepartment of Pharmacology Merck Research Laboratories Rahway, NJ 07065, USA
Received 22 May 2000; accepted 28 June 2000
AbstractÐA series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases
(NOS). 4,6-Disubstitution enhanced both potency and speci®city for the inducible NOS with the most potent compound having an
IC50 of 28 nM. # 2000 Elsevier Science Ltd. All rights reserved.
In recent years, nitric oxide (NO) has emerged as one of
the most interesting, and seemingly ubiquitous, mediators
of normal and pathophysiological processes.1 3 In
mammalian cells, NO is produced by the oxidation of
l-Arginine by nitric oxide synthase (NOS). There are
three isoforms of NOS: the constitutively expressed
neuronal NOS (n-NOS) and endothelial cell NOS
(e-NOS) and the inducible NOS (i-NOS). n-NOS is
believed to have a role in the production of NO as a
neurotransmitter. e-NOS is found primarily in vascular
endothelium where it regulates blood pressure and
vascular tone. i-NOS expression is induced in activated
macrophage and other cell types by numerous in¯am-
matory stimuli including endotoxin (LPS) and cytokines
(e.g. IL-1) and has a role in host defense and possibly
chronic in¯ammatory conditions. Transgenic mice that
have the NOS genes knocked-out con®rm many of the
biological roles of the respective NOS isoforms.4 8
associated with acute and chronic in¯ammation. As
such, i-NOS may have a role in a variety of diseases
including septic shock, arthritis, and in¯ammatory
bowel disease. The objective of our program was to
identify potent and selective inhibitors of i-NOS with
appropriate pharmacological properties. Several groups
have identi®ed 2-aminopyridines as NOS inhibitors. A
detailed report of the in vitro and in vivo properties of
2-amino-4-methylpyridine
structure±activity relationship for a variety of substi-
tuted 2-aminopyridines is described herein.
3
has appeared.9,10 The
Synthesis of 2-Aminopyridines
The synthesis of 2-aminopyridine derivatives has been
extensively reviewed.11 13 Several of the compounds in
Tables 1 and 2 are also commercially available. Among
the methods employed to prepare some of the com-
pounds described, the Chitchibabin reaction, Hofmann,
Curtius, Lossen and Neber-type rearrangements, and
halogen replacements were employed.14 The synthesis of
2-aminopyridine from pyridine-N-oxides has also been
described (Scheme 1).15 Pyridine-N-oxides were treated
with 4-chloro-2,2-dimethyl-1,3(2H)-benzoxazine to give
3-(2-pyridyl)-1,3-benzoxazinones. Subsequent treatment
with strong acid a€orded the 2-aminopyridine.
The induction of i-NOS by in¯ammatory stimuli and
the prolonged production of copious amounts of NO by
some activated in¯ammatory cells strongly suggest a role
for i-NOS in both host defense and tissue destruction
*Corresponding author. Tel.: +1-732-594-7249; fax: +1-732-594-
5966; e-mail: william_hagmann@merck.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00389-9

1976
W. K. Hagmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1975±1978
Table 1. Inhibition of nitric oxide synthases by substituted 2-aminopyridinesa
Compound
R
i-NOS
IC50 (mM)
e-NOS
IC50 (mM)
Selectivity
e-NOS/i-NOS
n-NOS
IC50 (mM)
Selectivity
n-NOS/i-NOS
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
H
3-CH3
4-CH3
5-CH3
6-CH3
3,4-(CH3)2
3,5-(CH3)2
4,5-(CH3)2
4,6-(CH3)2
5,6-(CH3)2
4-C2H5
4-n-C3H7
4-Cl
4-CF3
1.9
0.94
0.17
0.6
2.8
1.20
0.072
3.1
0.82
0.15
3.6
0.6
0.045
2.8
0.23
ND
ND
72.4
3.3
1.5
1.3
0.4
5.2
0.4
2.0
0.2
0.7
0.4
0.4
1.0
ND
ND
5.5
1.2
ND
1.4
2.6
0.15
9.1
1.8
>2.7
5.4
25
4.8
ND
0.075
ND
ND
ND
2.0
0.34
ND
2.2
ND
ND
ND
ND
ND
ND
ND
2.5
ND
0.4
ND
ND
ND
0.1
0.4
ND
0.3
ND
ND
ND
ND
ND
ND
ND
0.5
0.3
0.8
10.9
<2.2
3.6
2.0
0.076
14.6
0.81
0.11
7.2
0.23
>50
>50
13.2
2.7
3-C2H5, 4-CH3
3-n-C3H7, 4-CH3
3-NH2, 4-CH3
5-C2H5, 4-CH3,
4-CH3, 6-C2H5
4-CH3, 6-n-C3H7
4-CH3, 6-i-C3H7
4-CH3, 6-n-C4H9
4-CH3, 6-i-C4H9
4-CH3, 6-i-C5H11
4-CH3, 6-(CH2)3Ph
34.5
0.059
1.3
ND
0.081
3.4
0.049
1.0
0.2
0.61
42% @ 0.1
0.09
1.2
59% @ 0.1
0.10
0.51
3.3
0.33
0.11
0.11
0.046
0.028
0.076
12.7
10
40% @ 0.1
0.15
1.9
6.7
0.3
0.3
13.1
1.1
25
17.2
8.7
7.9
1.4
0.9
5.6
5.0
Ng-Methyl-l-arginine
2.7
18.4
118
N-Iminoethyl-l-lysine
Aminoguanidine
1.4
101
500
aND=not determined. NOS activity was determined by comparing conversion of 3H-(l)-arginine to 3H-(ll)-citrulline in the presence of compound
with control. The assay mixture (pH 7.5) containing 1 mM 3H-(l)-arginine (2 mCi), cofactors and inhibitor or aq DMSO (control) was incubated for
30 min at room temperature. The reaction was quenched by adding a slurry of Dowex 50W-X8 resin which removed unreacted substrate. The con-
centration of 3H-(l)-citrulline in the supernatant was determined on a scintillation counter. For each inhibitor, the percent inhibition was determined
(2Â) at 10 di€erent concentrations and an IC50 calculated using SIGMAPLOT.
Scheme 1.
Scheme 2.
A procedure for the selective alkylation of 2-amino-4,6-
dimethylpyridine is outlined in Scheme 2.16 18 2-Amino-
4,6-dimethylpyridine A was treated with acetonylacetone
with the removal of water to form pyridylpyrrole B.
Reaction of B with one equiv of n-butyllithium in
diethylether formed the anion on the 6-methyl group
which was subsequently allowed to react with an
electrophile R-X to form C. However, if LDA is used as
the base in THF solvent, alkylation occurs primarily
on the 4-methyl group. The pyrrole protecting group
was removed by reaction with hydroxylamine hydro-
chloride in re¯uxing aqueous ethanol to form the
6-substituted methyl product D. This method allowed
the preparation of 19±25. Compounds were evaluated as
inhibitors of the three human NOS isoforms (Tables 1
and 2).

W. K. Hagmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1975±1978
Table 2. Inhibition of nitric oxide synthases by fused-ring aminopyridinesa
1977
Compound
Structure
i-NOS
IC50 (mM)
e-NOS
IC50 (mM)
Selectivity
e-NOS/i-NOS
n-NOS
IC50 (mM)
Selectivity
n-NOS/i-NOS
32
33
4.6
1.4
0.3
6.6
1.4
>5 0
N D
N D
N D
N D
34
35
1.7
29
ND
24
ND
0.8
ND
11
ND
0.4
36
4.1
16.6
4.0
10.6
2.6
37
38
>50
4.0
>50
19.8
ND
5.0
>50
2.4
ND
0.6
aND=not determined. See footnote to Table 1 for assay conditions.
Discussion
Finally, a series of fused bicyclic analogues were
prepared and evaluated as inhibitors of NOS (Table 2).
3,4-Dimethyl substitution (6) was much more potency
enhancing than were its fused unsaturated (32) and
saturated (35) analogues. This was also true for 4,5-
disubstitution (8 versus 33 and 36). However, 2-amino-
quinoline (34) was more potent than the 5,6-dimethyl-
pyridyl (10) analogue as well as the saturated derivatives
(37 and 38).
The introduction of a single methyl group to the
4-position of 2-aminopyridine, as in 3, signi®cantly
enhanced the inhibition of all three NOS isoforms, in
particular the constitutive enzymes (Table 1). The other
single positional isomers were less impressive. Interest-
ingly, 2-amino-4-methylpyridine (3) is available by pre-
scription in some parts of the world as an analgesic and
for the treatment of hypotension; possibly associated
with its inhibition of the constitutive NOS's. Intro-
duction of a second methyl group suggested that the
6-position may enhance selectivity and/or potency with
respect to 3. Substitution at the 5-position looked to be
deleterious in this series. As seen with 3 versus 11±14,
at least for i-NOS, the 4-methyl group was optimal.
The 4-Cl (13) and 4-CF3 (14) groups, which would be
expected to be nearly isosteric with methyl, lost nearly
all the activity and may suggest that the pKa of the
aminopyridine system plays a role in inhibition.
Similar patterns of potency enhancement for the
saturated 2-iminopiperidine analogues have been
reported.19 4-Methyl and 4,6-dimethyl substitution
enhanced potency for all three isoforms of NOS. An
in vivo comparison of aminopyridine 3 and imino-
piperidine 39 revealed certain shortcomings of the
2-aminopyridine series (Table 3). Pyridine 3 was
more potent in raising mean arterial pressure (MAP)
in rats, a result of its greater potency versus e-NOS.
This hypertensive e€ect is consistent with the clinical
use of 3 to treat hypotension.20 On the other hand,
3 was less potent than 39 in a model of LPS-induced
nitrate production in mice which re¯ects either its
lesser potency versus i-NOS or poorer oral bioavail-
ability.
Despite the initial results obtained with a 4,6-dimethyl
substitution (9), larger alkyl groups at the 6-position
along with the 4-CH3 group o€ered the most potent and
selective compounds in this series (19±24). As the size of
the alkyl group increased from methyl to isopentyl,
potency for e-NOS decreased while i-NOS potency
remained relatively constant, thus increasing i-NOS
selectivity. The 6-iso-butyl (23) and 6-iso-pentyl (24)
analogues were the most potent and selective inhibitors
of i-NOS that were obtained in this series. The larger
phenylpropyl (25) was much less potent.
In summary, we have explored the SAR of a series
of substituted 2-aminopyridines and obtained sig-
ni®cant increases in selectivity for inhibition of i-NOS.
4-Alkyl and 4,6-dialkyl substituents provided the
most potent and selective inhibitors of the inducible
NOS isoform. However, the 2-aminopyridine analogues

1978
W. K. Hagmann et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1975±1978
Table 3. In vivo comparison of aminopyridine 3 and iminopiperidine
39
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0.17
0.072
25
0.11
0.016
0.22
0.15
1.8
e-NOS: IC50 (mM)
LPSa: ED50 (mg/kg po)
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respect to control).
bBP=Blood pressure elevation in anesthetized rats: ec-NOS mediated
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their saturated counterparts, the 2-iminopiperidines.
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