Bicyclic amidine inhibitors of nitric oxide synthase: Discovery of perhydro-iminopyrindine and perhydro-iminoquinoline as potent, orally active inhibitors of inducible nitric oxide synthase

Article abstract of DOI:10.1016/j.bmcl.2005.02.067

Syntheses and nitric oxide synthase inhibitory activity of cyclic amidines containing 5,6- 6,6- and 7,6-fused systems are described. X-ray structure determination facilitated the assignment of the stereochemistry of the most active compounds perhydro-2-im

Full text of DOI:10.1016/j.bmcl.2005.02.067

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1997–2001
Bicyclic amidine inhibitors of nitric oxide synthase: discovery of
perhydro-iminopyrindine and perhydro-iminoquinoline as
potent, orally active inhibitors of inducible nitric oxide synthase
Ravindra N. Guthikonda,a Shrenik K. Shah,a,* Stephen G. Pacholok,b
John L. Humes,b Richard A. Mumford,b Stephan K. Grant,c Renee M. Chabin,c
Barbara G. Green,c Nancy Tsou,a Richard Ball,a Daniel S. Fletcher,d Silvi Luell,d
D. Euan MacIntyred and Malcolm MacCossa
aDepartment of Medicinal Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Immunology and Rheumatology, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Biochemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
dDepartment of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 20 January 2005; revised 18 February 2005; accepted 22 February 2005
Abstract—Syntheses and nitric oxide synthase inhibitory activity of cyclic amidines containing 5,6- 6,6- and 7,6-fused systems are
described. X-ray structure determination facilitated the assignment of the stereochemistry of the most active compounds perhydro-
2-iminoisoquinoline (8a) and perhydro-2-iminopyrindine (10a). Both 8a and 10a are very potent inhibitors of iNOS, with excellent
selectivity over eNOS and they are orally active in rats with long duration suitable for once or twice a day dosing.
Ó 2005 Elsevier Ltd. All rights reserved.
The physiological roles of nitric oxide (NO), a reactive,
free radical gas, have been extensively studied in many
laboratories in recent years.1 In mammalian systems
NO is produced by a two-step oxidation of the terminal
guanidine group of L-arginine by nitric oxide synthase
(NOS).2 Three distinct NOS isoforms have been identi-
fied in humans. The activities of the two constitutive
enzymes, neuronal NOS (nNOS, or NOS-1) and endo-
thelial NOS (eNOS or NOS-3) are regulated by Ca2+
calmodulin complex and they play important roles in
neurotransmission and blood pressure regulation,
respectively. The third isoform, inducible NOS (iNOS
or NOS-2) is expressed in many cell types after stimula-
tion by numerous inflammatory mediators such as cyto-
kines and endotoxin. The NO produced by iNOS is
thought to be critical in host defense mechanisms such
as killing of intracellular pathogens.3 The biological
roles for individual NOS enzymes have been verified
by producing transgenic mice lacking the gene for the
respective NOS isoforms.
NO may also be involved in certain pathophysiological
conditions. Since iNOS is induced by inflammatory
stimuli which results in sustained production of NO by
inflammatory cells, it has been suggested that iNOS
could be responsible for the tissue damage observed in
chronic inflammation.4 NO derived from iNOS may
also be involved in the organ damage and rapid drop
of blood pressure seen in septic shock.5 It is hoped that
a selective iNOS inhibitor may be able to stop or reduce
the NO mediated damage in such conditions and may be
clinically useful in the treatment of septic shock and
inflammatory diseases.
N
H
NH
Keywords: Nitric oxide synthase; Amidine; iNOS.
*
N
H
NH
Corresponding author. Tel.: +1 732 594 7988; fax: +1 732 594
5790; e-mail: shrenik_shah@merck.com
1
2
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.067

1998
R. N. Guthikonda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1997–2001
Many different types of NOS inhibitors have been de-
scribed in the past few years. Some of them are analogs
of the substrate L-arginine.6 Potent non-amino acid
inhibitors from isothiourea and amidine classes have
also been reported.7 We have been interested in potent
and selective iNOS inhibitors with good pharmacoki-
netic properties that will allow their evaluation in animal
models of inflammation.8 Previously 2-aminopyridines
as NOS inhibitors have been described from these labo-
ratories wherein methyl substitution at C-4 and C-6 of
the pyridine ring improved activity.9 Selective nNOS
inhibitors containing this scaffold were recently
reported.10
exploited. The synthesis and activity of such fused 2-
iminopiperidines is reported herein.
The desired bicyclic amidines were prepared from the
corresponding lactams via their imino ethers (Scheme
1). Thus, commercially available 3,4-dihydroquinolone
was converted to its methyl imino ether by reaction with
trimethyloxonium fluoroborate and the imino ether was
subsequently treated with NH4Cl in refluxing ethanol to
afford the hydrochloride of amidine 3 as a crystalline so-
lid. Similarly 4-methyl-3,4-dihydroquinolone was con-
verted to 4.
In order to prepare the saturated analogs 5 and 6, hexa-
hydro-2(1H)-quinolone (7) was first reduced by reaction
with sodium formate in refluxing formic acid to afford a
9:1 mixture of trans and cis lactams, which were sepa-
rated by chromatography.12 The trans lactam was con-
verted to amidine 5 via its imino ether. The cis lactam
was more conveniently obtained by catalytic hydrogena-
tion of 7 using 10% Pd/C13 which was then carried
through the two steps to give 6.
The 2-aminopyridine leads were reduced to their satu-
rated analogs 2-iminopiperidine (1) and 4-methyl-2-
iminopiperidine (2) which are more active inhibitors.11
Concurrently compounds 1 and 2 were also indepen-
dently discovered as orally active NOS inhibitors.7d,e
Bicyclic amidines were targeted to improve on the selec-
tivity of 2. It was hoped that by presenting the amidine
pharmacophore in a more rigid framework subtle differ-
ences in the active sites of the three enzymes might be
The methyl substituted analog 8 was synthesized by
hydrogenation of 2-hydroxy-4-methylquinoline (Scheme
2). In this case more forcing conditions (PtO2 in acetic
acid) were required to saturate the phenyl ring. The
reduction was highly stereoselective and only the cis,syn
isomer, 9, was isolated. Reaction of 9 with MeerweinÕs
reagent and amidine formation with NH4Cl yielded 8.
Since 8 exhibited good activity and selectivity (see
below) the individual enantiomers 8a and b were
prepared. It was found that the racemic lactam 9 could
be separated into the two enantiomers (slower, 9a
and faster, 9b) on a Chiralcel OD column using 10%
isopropanol/hexane as eluent. Each isomer was then
converted to the amidine (8a14a and 8b) as described.
The stereochemistry of 8a (aD À12.5 (c 0.2, EtOH))
derived from the slower lactam isomer (9a) was determined
to be 4(R),4a(R),8a(R) by X-ray crystallography.15
R
R
a,b
N
H
O
N
H
NH
3 R = H
4 R = Me
H
H
H
a,b
c
N
H
NH
N
H
O
N
H
O
O
H
5
7
H
H
H
a,b
d
N
O
N
H
NH
N
H
H
H
The 5,6 fused analog 10, and 7,6 fused analog 11, were
synthesized from cyclopentanone and cycloheptanone,
respectively, as shown in Scheme 3. Condensation of
cyclopentanone with ethyl acetoacetate and NH4OAc
by the method of Sakurai furnished 12,16 which was
6
7
Scheme 1. Reagents and conditions: (a) Me3OBF4, CH2Cl2, rt; (b)
NH4Cl, EtOH, 80 °C; (c) HCOONa, HCOOH, 100 °C; (d) H2, Pd/C,
dioxane, ethanol.
Me
Me
H
H
H
a
b,c
N
OH
N
H
O
N
H
NH
H
8 (racemic)
9 (racemic)
d
b,c
b.c
Me
Me
H
H
H
H
N
H
NH
N
H
NH
8b (S,S,S)
8a (R,R,R)
Scheme 2. Reagents and conditions: (a) H2, PtO2, HOAc; (b) Me3OBF4, CH2Cl2, rt; (c) NH4Cl, EtOH, 80 °C; (d) resolution on Chiralcel OD
column, 10% isopropanol/hexane.

R. N. Guthikonda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1997–2001
1999
Me
line as previously described.17 The observed NOS IC50
are listed in Table 1 and from these values selectivity
for eNOS and nNOS defined as IC50(eNOS)/IC50(iNOS)
and IC50(nNOS)/IC50(iNOS), respectively, were calcu-
lated and are shown in Table 1. Since the constitutive
enzymes seems to be involved in important physiological
processes our aim was to find a potent and selective
iNOS inhibitor.
O
a
CO2Et
+
O
( )n
( )n
N
H
O
12 n = 1
13 n = 3
b
Me
Me
H
H
H
Although the benzo fused analogs 3 and 4 lost activity
against all three enzymes; they had better eNOS selec-
tivity than 1 and 2. The selectivity was even better for
the saturated cyclohexane fused amidines 5 and 6.
The cis isomer 6 (iNOS IC50 = 186 nM) was as good
iNOS inhibitor as 1 but was 20-fold less active against
eNOS. As we had observed for 1 and 2, the activity
of 6 was further improved by introduction of C-4
methyl (8, iNOS IC50 = 38 nM) and the selectivity was
retained. Since 8 was a mixture of two enantiomers, it
was resolved and the R,R,R isomer 8a (iNOS
IC50 = 22 nM,) was responsible for most of the inhibi-
tory activity. Interestingly, the enantiomer 8b was a
selective nNOS inhibitor but it is not very potent. The
5,6-fused system, 10, was somewhat more active than
8 but 11 with a fused cycloheptane ring lost activity
against all three enzymes indicating a sterically re-
stricted binding site. Separation of the enantiomers of
10 again provided the potent R,R,R isomer 10a with
iNOS IC50 = 9 nM. However, in this case the enantio-
mer, 10b (iNOS IC50 = 133 nM) still had significant
activity possibly due to its smaller size. Both 8a and
10a were 35-40x selective for iNOS over eNOS, indicat-
ing that the initial hypothesis about using a rigid bicy-
clic framework to discriminate between the active sites
is probably valid in this case. The binding region for
nNOS seems to be very similar to iNOS and it was very
difficult to achieve selectivity in this series. This was
also true for the monocyclic amidines. Nevertheless,
the best compounds 8a and 10a, were 2-fold selective,
while the leads 1 and 2 were more powerful inhibitors
of nNOS than iNOS.
c,d
Me
( )n
( )n
N
H
O
N
H
NH
H
14 n = 1
15 n = 3
10 n = 1
11 n = 3
Me
H
H
H
N
H
NH
N
H
NH
H
10b (S,S,S)
10a (R,R,R)
Scheme 3. (a) NH4OAc, EtOH, 80 °C; (b) H2, PtO2, HOAc;
(c) Me3OBF4, CH2Cl2, rt; (d) NH4Cl, EtOH, 80 °C.
hydrogenated using PtO2 in acetic acid. The reduction in
this case was also highly stereoselective and only 14 (cis,
syn) was isolated. This lactam was converted to the race-
mic amidine 10 via its imino ether. The individual
enantiomers 10a and b were obtained after resolution
of 14 on Chiralcel OD column and the slower lactam,
14a, gave 10a14b (aD À54.5 (c 0.2, EtOH)). The stereo-
chemistry of 10a as 4(R),4a(R),7a(R), was also
confirmed by X-ray crystallography.15 Analogous con-
densation of cycloheptanone with ethyl acetoacetate fol-
lowed by reduction gave 15, which was converted to
racemic amidine 11 in two steps.
The inhibitory activities of these amidines against the
three recombinant human NOS isoforms were measured
by following the conversion of 3H-arginine to 3H-citrul-
Table 1. NOS inhibitory activity and selectivity of bicyclic amidines
Compound
iNOS IC50 (nM)a
eNOS IC50 (nM)a
nNOS IC50 (nM)a
Selectivityb eNOS
Selectivityc nNOS
1
300
37
630
146
240
16
2.1
3.9
6.1
15.7
29.6
67
0.8
0.43
0.35
0.25
1
2
3
14,000
974
528
186
38
85,000
15,300
5500
12,500
2000
830
4900
240
530
246
190
53
4
5
6
1.3
5
8
52
37
NDd
8a
8b
10
10a
10b
11
22
>5000
15
2.4
NDd
2.1
2.3
0.27
1.5
>5000
470
360
610
31
31
40
9
21
36
133
860
870
4500
6.5
5.2
1300
a Inhibitory activity for human iNOS, eNOS, and nNOS was measured as described in Ref. 17 with a final L-arginine concentration of 1 lM. For
each compound, the percent inhibition was determined in duplicate at 10 different concentrations and IC50 was calculated using SIGMAPLOT.
b Selectivity against eNOS is the ratio of eNOS IC50/iNOS IC50
.
c Selectivity against nNOS is the ratio of nNOS IC50/iNOS IC50
d ND = not determined.
.

2000
R. N. Guthikonda et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1997–2001
Table 2. In vivo activity of NOS inhibitors in LPS treated rats
References and notes
Compound
Plasma NOx inhibition
ED50, mg/kg iva
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0.19
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a The ability of test compounds to inhibit elevation of plasma nitrate/
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78
57
69
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95
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52
39
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The ability of 8a and 10a to inhibit iNOS activity in vivo
was measured in a rat endotoxin assay. It has been
shown that the increase in plasma nitrate/nitrite (NOx)
levels following LPS administration to animals is medi-
ated by iNOS and this model has been used to evaluate
other iNOS inhibitors.18 The results from an iv study are
listed in Table 2. Both 8a and 10a are 2-fold more active
than 2, when they were dosed 2 h after LPS challenge.
To assess the oral bioavailability and pharmacokinetics
of these agents a time course of activity was performed.
Thus 10 mg/kg po dose of the drug was administered to
rats at various times before LPS challenge and the inhi-
bition in plasma NOx levels measured 5 h after LPS
challenge is shown in Table 3. Both 8a and 10a were or-
ally efficacious and at early times (0 and 1 h before LPS)
almost complete inhibition was achieved with 10a. The
activity was sustained and when dosed 16 h before
LPS, both 8a and 10a reduced plasma nitrite by 40%
with a 10 mg/kg po dose. This result clearly demon-
strated that high levels of 8a and 10a can be maintained
in circulation for 16–20 h and therefore they are suitable
for once or twice a day dosing for chronic studies in
rats.
In summary, modification of the 2-iminopiperidine has
lead to the identification of perhydro-iminoquinoline,
8a, and perhydro-iminopyrindine, 10a, as very potent
iNOS inhibitors. The stereochemistry of the active enan-
tiomer in each case was confirmed by X-ray crystallo-
graphy. Both 8a and 10a are much poorer inhibitors
of eNOS and are orally bioavailable in rats. Their
potency against iNOS, selectivity, and pharmacokinetic
properties make them useful tools to evaluate the role of
iNOS in models of chronic inflammation.
11. Guthikonda, R. N. Abstract #90, National Medicinal
Chemistry Symposium, Ann Arbor, MI, 1996.

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2001
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tallographic Data Centre as supplementary publication
numbers CCDC 260319 for 8a and CCDC 260320 for 10a.
Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK [fax:+44 (0)1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
13. Murahashi, S.-I.; Sasao, S.; Saito, E.; Naota, T. J. Org.
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1
14. (a) Compound 8a: H NMR (CD3OD): d (ppm) 1.05 (d,
J = 7 Hz, 3H), 1.1–2.2 (m, 10H), 2.30 (dd, J = 19 and
12 Hz, 1H), 2.63 (dd, J = 19 and 6 Hz, 1H), 3.72 (d,
J = 3 Hz, 1H); 13C NMR (CD3OD): d (ppm) 16.93, 18.55,
19.96, 24.58, 29.34, 29.59, 29.98, 37.71, 53.5, 167.63. (b)
Compound 10a: 1H NMR (CD3OD): d (ppm) 1.07 (d,
J = 7 Hz, 3H), 1.4–2.4 (m, 9H), 2.50 (dd, J = 18 and 5 Hz,
1H), 3.95 (t, J = 6 Hz, 1H); 13C NMR (CD3OD): d (ppm)
18.05, 22.21, 22.49, 26.15, 28.17, 33.40, 41.38, 57.73,
166.71.
16. Sakurai, A.; Midorikawa, H. Bull. Chem. Soc. Jpn. 1968,
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M . G.Eur. J. Pharmacol. 1995, 273, 15; (b) Tracey, W. R.;
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15. Crystallographic data (excluding structure factors) for 8a
and 10a have been deposited with the Cambridge Crys-