Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. Part 1: Synthesis and biological evaluation of dihydropyridin-2-imines

Article abstract of DOI:10.1016/S0960-894X(02)00455-9

Dihydropyridin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide inhibition assay. Compounds 1, 4, 5 and 7-11 exhibited potent activity in the inducible nitric oxide (iNOS) inhibition assay. Of these 5, 6,

Full text of DOI:10.1016/S0960-894X(02)00455-9

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2291–2294
Design and Synthesis of Orally Bioavailable Inhibitors of
Inducible Nitric Oxide Synthase. Part 1: Synthesis and
Biological Evaluation of Dihydropyridin-2-imines
a,
b
b
b
Yasufumi Kawanaka, * Kaoru Kobayashi, Shinya Kusuda, Tadashi Tatsumi,
b
b
b
b
Masanori Murota, Toshihiko Nishiyama, Katsuya Hisaichi, Atsuko Fujii,
b
b
b
b
b
Keisuke Hirai, Masao Naka, Masaharu Komeno, Hisao Nakai and Masaaki Toda
aFukui Research Institute, Ono Pharmaceutical Co., Ltd., Technoport, Yamagishi, Mikuni, Sakai, Fukui 913-8538, Japan
bMinase Research Institute, Ono Pharmaceutical Co., Ltd., Shimamoto, Mishima, Osaka 618-8585, Japan
Received 19 April 2002; accepted 7 June 2002
Abstract—Dihydropyridin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide inhi-
bition assay. Compounds 1, 4, 5 and 7–11 exhibited potent activity in the inducible nitric oxide (iNOS) inhibition assay. Of these 5,
6, 9 and 10 showed 5- to 11-fold increases in isoform selectivity. Compounds 1, 5, 9 and 10 showed potent inhibitory activity in the
NOx accumulation assay in mice. Compounds 1 and 5 also showed good bioavailability (BA) when given orally. # 2002 Elsevier
Science Ltd. All rights reserved.
Introduction
the overexpression of NO.4,5 The natural substrate for
NOS, arginine, has been the obvious basis for the
molecular design of NOS inhibitors.
Nitric oxide (NO) is a mediator of normal and patho-
physiological processes.
1ꢀ3
NO is synthesized by two
major classes of nitric oxide synthase (NOS): con-
stitutive and inducible. The constitutive enzymes require
G
Structural analogues of l-arginine such as N -methyl-
6
,7
G
8
l-arginine (l-NMA), N -nitro-l-arginine, N-imino-
2
+
9
10
Ca /calmodulin for their activity and are further divi-
ded into the neuronal and endothelial isoforms. The
endothelial isoform (eNOS) is found predominantly in
the vascular endothelium and it produces low con-
centrations of NO, which reduce the blood pressure and
inhibit platelet aggregation. NO produced by the neu-
ronal enzyme (nNOS) appears to function as a neuro-
transmitter which regulates neuronal transmission. The
inducible isoform is found in activated macrophages as
well as many other cells and it produces NO, which
plays a role in the host defense system. The over-
expression of NO which destroys functional tissue dur-
ing acute and chronic inflammation is caused by this
inducible NOS (iNOS). Identification of potent and
selective inhibitors for the above-mentioned iNOS has
been the subject of intense interest because of their thera-
peutic potential in the treatment of diseases mediated by
ethyl-l-ornithine (l-NIO), and l-thiocitrulline have
been shown to inhibit the various forms of NOS. Much
pharmacology and biology data have been determined
utilizing some of the early relatively nonspecific inhibi-
tors of arginine analogues as described above. A num-
ber of non-amino acid inhibitors of iNOS such as
1
1
12
aminoguanidine, isothioureas, 2-amino-5,6-dihydro-
1
3
14
6-methyl-4H-1,3-thiazine,
2-iminopiperidine
and
1
5
2-aminopyridine have also been described. As illu-
strated in Chart 1, our molecular design started with a
structural hybridization of 2-amino-4-methylpyridine
1
5
14
13
and (4R)-4-methylpiperidin-2-imine 14.
This
modification led us to the discovery of 4-methyl-5,6-
dihydropyridin-2-imine 1, which is structurally new and
possesses no asymmetric center. A series of 5,6-dihydro-
pyridin-2-imines were synthesized and biologically eval-
uated with the expectation of identifying a new selective
inhibitor of human iNOS (hiNOS). We now report a ser-
ies of 5,6-dihydropyridin-2-imines which are structurally
new inhibitors of iNOS.
*Corresponding author. Tel.: +81-776-82-6161; fax: +81-776-82-8420;
e-mail: kawanaka@ono.co.jp
0
960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00455-9

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Y. Kawanaka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2291–2294
Chemistry
Synthesis of the compounds described in Table 1 is
outlined in Schemes 1–3. As described in Scheme 1, the
common intermediates, substituted 5,6-dihydropyridin-
2-ones 18a–i, were prepared by the conventional Beck-
mann rearrangement reaction of oximes prepared from
the corresponding cyclopentenones 15a–i via 16a–i and
17a–i (Method A: 22–48% yield) or directly from 15a–i
(Method B: 44–60% yield), respectively. The common
intermediates 18a–i were used to prepare 1 and 8–12 as
well as 4–5 and 7 according to Methods A and B,
respectively. As described in Scheme 2, another inter-
mediate 21 used to prepare 6 was obtained by the oxi-
dation of 20 with a 70% yield, which was prepared by
the Beckmann rearrangement of an oxime prepared
from the cyclopentanone 19 with a 52% yield (Method
C). As described in Scheme 3, 18a–i and 21 were con-
verted to the test compounds 1 and 4–12 (8–70% yield)
via their corresponding ethyl imidates 22a–i and 23,
respectively.
Chart 1. Molecular design of 5,6-dihydropyridin-2-imine.
Table 1. Inhibitory activities of 1–12 against hiNOS and heNOS and
e
their isoform selectivity
Structure
Compd
synthetic method)
hiNOS IC50 heNOS IC50
Selectivity
heNOS/hiNOS
a
a
b
(
(mM)
(mM)
1
2
R=Me (A)
R=Et
0.20
1.2
0.23
1.5
1.2
1.3
c
Results and Discussion
As described in Table 1, the compounds were evaluated
for their ability to inhibit the two isoforms of human
NOS. As expected, 4-methyl-5,6-dihydropyridin-2-imine
3c
0.70
0.31
0.44
0.32
0.6
1.0
1
demonstrated potent inhibitory activity against both
the human iNOS (hiNOS) and human eNOS (heNOS)
isoforms without any selectivity.
4
(B)
5
6
R=Me (B)
R=H (C)
0.42
2.8
2.1
31.0
5.0
11.1
7
(B)d
0.51
0.52
1.0
8
9
R=Me (A)d
R=n-Pr (A)
0.18
0.06
0.05
0.24
0.58
0.36
1.3
9.7
7.2
d
1
0 R=Allyl (A)d
1
1
1 n=1 (A)d
2 n=2 (A)d
0.17
1.4
0.51
7.4
3.0
5.3
Scheme 1. Synthesis of 5,6-dihydropyridin-2-ones. Method A: (a)
.
ꢁ
2
OH HCl, NaOAc, MeOH, reflux; (b) p-TsCl, pyridine, 0 C; (c)
NH
concd HCl, MeOH, refux (18a, 18e–i: 22–48% in three steps); Method
aPreparation of partially purified enzyme and determination of K
values.
i
3
B: (d) NaN , TFA, reflux (18b–d: 44–60%).
Human eNOS was overexpressed in Sf-21 cells, by infecting the cells with bacu-
lovirus carrying heNOS cDNA. The hiNOS was overexpressed in A549 by sti-
mulation with LPS (10 mg/mL) plus cytokines (10 ng/mL TNF-a, 5 ng/mL IL-1b
and 100 ng/mL interferon-g). Human eNOS and iNOS were partially purified by
Compd
R
1
R
2
R
3
R
4
R
5
0
0
chromatography on 2 , 5 -ADP-Sepharose gels. NOS activity was determined by
the method for the conversion of [14C]-l-arginine to l-citrulline with a minor
modification. The conversion rates for various concentrations of the test com-
pounds and l-arginine were measured. Dixon and Lineweaver–Bulk plots were
a
b
c
d
e
f
g
h
i
H
Me
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
H
H
Me
H
H
H
H
Me
n-Pr
Allyl
–
–
(H,Me)
constructed to determine the K
i
values and the mode of inhibition.
H
H
H
H
H
H
H
H
b
Selectivity was evaluated as the rate of the IC50 values for heNOS and hiNOS.
Compounds 2 and 3 were prepared according to a modified Birch reduction of
c
16
2
-amino-4-methylpyridine and 2-amino-4-ethylpyridine, respectively.
Synthesized as a mixture of racemates.
–(CH
–(CH
2
)
)
3
d
2
4
e
All compounds were prepared as their hydrochlorides.

Y. Kawanaka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2291–2294
2293
2 3 2
Scheme 2. Synthesis of 5,5-dimethyl-5,6-dihydropyridin-2-one (21). Method C: (a) NH OSO H, HCO H, reflux (52%); (b) DDQ, BSTFA, dioxane,
reflux (70%).
O+BF ,CH
ꢀ
4
3 2 2 3
Scheme 3. Synthesis of 5,6-dihydropyridine-2-imines (1, 4–12) Reagents: (a) Et Cl ; (b) NH , EtOH; (c) 4 M HCl dioxane (8–70% in
three steps)
Compd
R
1
R
2
R
3
R
4
R
5
a
b
c
d
e
f
g
h
i
H
Me
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
H
H
Me
H
H
Me
H
H
H
H
Me
n-Pr
Allyl
(H,Me)
H
H
H
H
H
H
H
H
–(CH
–(CH
2
)
)
3
–
–
2
4
Replacement of the 4-methyl group with a 4-ethyl group
NOS. The cis-fused 5,6-bicyclic analogue 11 demon-
strated nearly the same potency of hiNOS inhibition as
compared with 1 but with a 2.2-fold decrease in the
potency of heNOS inhibition, while the isoform selec-
tivity of 11 was higher than that of 1. This was also true
for the cis-fused 6,6-bicyclic analogue 12, although its
IC50 values for the isoforms were much less potent than
those of 11.
1
6
afforded 2 with a nearly 6-fold reduction in the inhi-
bitory activity and still no isoform selectivity. Shifting
the double bond of 1 from position 3,4- to 4,5- provided
1
6
3
with reduced inhibitory activity against both of the
isoforms as well as a reduced selectivity. Based on this
information, further chemical modification was carried
out by fixing the double bond in position 3,4- as illu-
strated in compounds 4–12. Introduction of another
methyl group into position 3 of 1 afforded 4 with little
change in the inhibitory activity or isoform selectivity.
Introduction of a gem-dimethyl group into position 5 of
Among the tested compounds described in Table 1, the
compound 1, which is a structurally representative
compound of this series with potent iNOS inhibitory
activity was selected together with 5, 9 and 10 for fur-
ther biological and pharmacodynamic evaluations in
animal models because of their in vitro potency and
isoform selectivity, as well as their mouse acute toxicity
(MTD: maximum tolerated dose) and structural fea-
tures. Compounds 9 and 10 exhibited greater potency
and more isoform selectivity than the above-mentioned
compounds 1 and 5. As shown in Table 2, compounds 9
and 10 demonstrated higher acute toxicity than 1 and 5,
as evaluated by the ratio between their maximum toler-
1
provided 5 with a 2-fold reduction in the inhibitory
activity against hiNOS and 10-fold reduction against
heNOS. As a result, the isoform selectivity was 5 times
more than that of 1. Removal of the 4-methyl group
from 5 afforded 6 with a 14-fold reduction in the
potency of the hiNOS inhibition and a 135-fold reduc-
tion in the heNOS inhibition. As a result, the isoform
selectivity (heNOS/hiNOS) of 6 increased to nearly 10
times greater than that of 1. Introduction of another
methyl group into position 5 of 1 afforded 7 with a
nearly 2-fold reduction in potency against both the
hiNOS and heNOS isoforms but with the isoform
selectivity retained. Introduction of another methyl
group into position 6 of 1 afforded 8 which retained the
inhibitory activity for both isoforms as well as the
selectivity. Replacement of the 6-methyl group of 8 with
a 6-propyl group afforded 9 with a 3-fold increase in
potent inhibitory activity against hiNOS and a 2.4-fold
reduction against heNOS resulting in an isoform selec-
tivity which was 7.5-fold higher than 8. A similar result
was obtained in the corresponding allyl derivative 10.
1
7
ated doses (MTDs) and the ID50 values of NOx accu-
mulation (MTD/NOx).
In order to assess compounds 1, 5, 9 and 10 for their
ability to inhibit iNOS in vivo, mice were given a single
compound subcutaneously (sc) 3 h after lipopoly-
saccharide (LPS) injection. The plasma NOx accumula-
tion from 3 to 6 h after the LPS injection was then
determined. As described in Table 2, these test com-
pounds 1, 5, 9 and 10 inhibited NOx accumulation in
plasma and their ID50 values were 0.16, 0.28, 0.36 and
0.16 mg/kg, sc, respectively. To assess the acute toxicity,
the MTDs were determined. As shown in Table 2, the
MTDs of 1 and 5, when a single intravenous (iv) dose
Finally, two of the cis-fused bicyclic analogues were
prepared and biologically evaluated as inhibitors of

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Y. Kawanaka et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2291–2294
Table 2. Pharmacological evaluation of 1, 5, 9 and 10
and 10 exhibited greater potent inhibitory activity and
isoform selectivity than those of 1 and 5 but their acute
toxicity in mice was also higher. Full details will soon be
reported in Bioorganic Medicinal Chemistry.
Compd
Mouse iNOS
NOx
MTD
MTD/NOx
IC50
a
ID50
(mg/kg, sc)
mg/kg, ivc
b
(
mM)
1
5
9
0.022
0.10
0.10
0.025
3.5
0.16
0.28
0.36
0.16
26
20
20
5
5
3000
130
71
14
31
References and Notes
1
0
1. Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Pharmacol.
Rev. 1991, 43, 109.
l-NMMA
120
2
3
. Nathan, C.; Xie, Q.-W. Cell 1994, 78, 915.
. Kerwin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L.
aEnzyme assay with recombinant mouse iNOS. Recombinant mouse
iNOS was purchased from Cayman Chemical (Cat. No. 60862) and
the inhibitory activities of the test compounds were evaluated by
J. Med. Chem. 1995, 38, 4343.
4. Kerwin, J. F., Jr.; Heller, M. Med. Res. Rev. 1994, 14, 23.
measuring the conversion rate from [14C]-l-arginine to [14C]-l–citrul-
5
. Marletta, M. A. J. Med. Chem. 1994, 37, 1899.
line, and then the IC50 values were determined.
b
6. Olken, N. M.; Marletta, M. A. Biochemistry 1993, 32, 9677.
7. Feldman, P. L.; Griffith, O. W.; Hong, H.; Stuehr, D. J.
J. Med. Chem. 1993, 36, 491.
8. Furfine, E. S.; Harmon, M. F.; Paith, J. E.; Garvey, E. P.
Biochemistry 1993, 32, 8512.
The ID50 value was determined from a log–logit transformation of
the dose–response curves (1, 5, 9 and 10; 0.1, 0.3, 1 mg/kg, sc l-
NMMA; 10, 30 and 100 mg/kg, sc). The ID50 value was defined as the
dose of test compound that produced a 50% inhibition in the NOx
accumulation induced by LPS treatment alone. The MTD was defined
as the maximum dose at which no death was observed within 24 h
after an intravenous injection administration. The doses used were 5,
9
. Rees, D. D.; Palmer, R. M. J.; Schulz, R.; Hodson, H. F.;
Moncada, S. Br. J. Pharmacol. 1990, 101, 746.
0. Narayanan, K.; Griffith, O. W. J. Med. Chem. 1994, 37,
885.
10, 20, 30, 40, and 50 mg/kg for 1, 5, 9 and 10 and 1000, 2000, 3000,
1
4
000, and 5000 mg/kg for l-NMMA.
c
Inhibition of NOx accumulation and the maximum tolerated dose
MTD) in mice. The test compounds or saline were administered sub-
1
1. Misko, T. P.; Moore, W. M.; Kasten, T. P.; Nickols,
(
cutaneously 3 h after the LPS (10 mg/kg, iv) injection into 7 week old
Balb/c mice (Charles River Japan, Inc.). Blood was collected by veni-
puncture from the abdominal aorta under light anesthesia at 6 h after
LPS treatment. Plasma was obtained by centrifugation and the con-
centration of accumulated NOx over 3 h was determined by the
method described below. To evaluate the acute toxicity, the MTD (iv
maximum dose where no death was observed within 24 h after the
administration) of the test compound was determined.
G. A.; Corbett, J. A.; Tilton, R. G.; McDaniel, M. L.; Wil-
liamson, J. R.; Currie, M. G. Eur. J. Pharmacol. 1993, 233,
119.
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1
3. Nakane, M.; Klinghofer, V.; Kuk, J. E.; Donnelly, J. L.;
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Pharmacol. 1995, 47, 831.
1
4. Webber, R. K.; Metz, S.; Moore, W. M.; Connor, J. R.;
was given to normal mice, were 20 and 20 mg/kg,
respectively. The MTD values of all four compounds
were lower than that of l-NMMA (3000 mg/kg),
although the ratio of MTD/ID for NOx accumulation
Currie, M. G.; Fok, K. F.; Hagen, T. J.; Hansen, D. W., Jr.;
Jerome, G. M.; Manning, P. T.; Pitzele, B. S.; Toth, M. V.;
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41, 96.
5
0
in mice was 130 for 1, 71 for 5, 14 for 9 and 31 for 10,
respectively. The oral bioavailability of 1 and 5 in rats
was excellent (1: 88% and 5: 84%).
15. Hagmann, W. K.; Galdwell, C. G.; Chen, P.; Durette,
P. L.; Esser, C. K.; Lanza, T. J.; Kopka, I. E.; Guthikonda,
R.; Shah, S. K.; MacCoss, M.; Chabin, R. M.; Fletcher, D.;
Grant, S. K.; Green, B. G.; Humes, J. L.; Kelly, T. M.; Luell,
S.; Meurer, R.; Moore, V.; Pacholok, S. G.; Pavia, T.; Wil-
liams, H. R.; Wong, K. K. Bioorg. Med. Chem. Lett. 2000, 10,
In summary, we have explored the structure–activity rela-
tionship (SAR) for a series of substituted 5,6-dihydro-
pyridin-2-imines and obtained significant increases in
the isoform selectivity and potency of iNOS inhibition.
1
1
975.
6. Kawanaka, Y.; Kobayashi, K.; Kusuda, S.; Tatsumi, T.;
Murota, M.; Nishiyama, T.; Hisaichi, K.; Fujii, A.; Hirai, K.;
Naka, M.; Komeno, M.; Nakai, H.; Toda, M. Synlettt. In
press.
4
-Methyl and 4-methyl-5,5-dimethyl substituents pro-
vided compounds 1 and 5 which were potent and/or
selective inhibitors of the iNOS isoform. Compounds 9
17. Mixture of nitric oxides.