Discovery of a series of aminopiperidines as novel iNOS inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.10.073

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS (eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor, with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.

Full text of DOI:10.1016/j.bmcl.2007.10.073

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 336–343
Discovery of a series of aminopiperidines as novel iNOS inhibitors
Bertrand Le Bourdonnec,a,* Lara K. Leister,a Christopher A. Ajello,a Joel A. Cassel,b
Pamela R. Seida,a Heather O’Hare,a Minghua Gu,a Guo-Hua Chu,a Paul A. Tuthill,a
Robert N. DeHavenb and Roland E. Dollea
aDepartment of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
bDepartment of Pharmacology, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
Received 31 August 2007; revised 19 October 2007; accepted 20 October 2007
Available online 25 October 2007
Abstract—Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes
of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS
(eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as
potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor,
with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.
Published by Elsevier Ltd.
Nitric oxide (NO) is a small reactive molecule with an
important role in various physiological processes,
including modulation of inflammatory responses and
regulation of vessel tone.1 Nitric oxide synthase (NOS)
catalyzes the formation of NO and L-citrulline from L-
arginine (Arg) and oxygen. The NOS family consists
of three known mammalian isoforms. The neuronal
NOS (nNOS) and endothelial NOS (eNOS) are constitu-
tively expressed under non-inflammatory conditions,
and their activity is tightly regulated by Ca2+-dependent
calmodulin. The third, inducible isoform (iNOS) is a key
mediator of inflammation and host defense systems. NO
generated by eNOS has been shown to be critical for
angiogenesis and for maintaining proper vascular tone.
Although there may be pathologies associated with
overactivity of eNOS, blood pressure homeostasis is so
critical that therapeutically useful NOS inhibitors must
not inhibit eNOS. Expression of iNOS is induced at a
transcriptional level by inflammatory stimuli, including
interferon (IFN), interleukin (IL)-1, tumor necrosis fac-
tor (TNF), and bacterial lipopolysaccharide (LPS). Pro-
duction of excess NO due to the prolonged induction of
iNOS has been observed in various inflammatory and
autoimmune diseases. Because of the dual nature of
NO, the development of selective inhibitors for the
inducible NOS isoform is a highly desirable goal. Ani-
mal studies suggest that inhibitors of iNOS are useful
in treating septic shock, stroke, and many inflammatory
diseases including rheumatoid arthritis, osteoarthritis,
inflammatory bowel disease and multiple sclerosis.1–7
Some of the earliest inhibitors of nitric oxide synthase
to be reported were direct analogs of the natural sub-
strate L-arginine such as L-NIL (1).8,9 A prodrug of L-
NIL, that is, L-N6-(1-iminoethyl)lysine 5-tetrazole
amide (SC-51), was evaluated in humans.10 This clinical
study demonstrated that SC-51 produces marked inhibi-
tion of exhaled breath NO in normal and asthmatic sub-
jects without producing the side effects observed
following the systemic administration of non-selective
NOS inhibitors. GW274150 (2), a close analog of L-
NIL, has also been reported to potently and selectively
inhibit iNOS and is currently undergoing Phase II clin-
ical evaluation for the potential treatment of rheumatoid
arthritis and migraine.11,12 Non-amino acid-based inhib-
itors of iNOS have also been described. In particular,
AR-C102222 (3), representative of the 1,2-dihydro-4-
quinazolinamine series of iNOS inhibitors, demon-
strated efficacy in the rat adjuvant-induced arthritis
model.13
In our search for a novel class of iNOS inhibitors using
high throughput screening, we found that the N-(5-
chloro-2-nitrobenzyl)-N,1-dimethylpiperidin-4-amine 4
strongly inhibits human iNOS with an IC50 value of
87 nM. Further modification of this lead resulted in
Keywords: iNOS; Inhibitors; Aminopiperidines.
*
Corresponding author. Tel.: +1 484 595 1061; fax: +1 484 595
1551; e-mail: blebourdonnec@adolor.com
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2007.10.073

B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
337
the discovery of aminopiperidine derivatives as novel
iNOS inhibitors. Here, we report the synthesis of these
new compounds (Formula A, Fig. 1) and their struc-
ture–activity relationships (SARs).
which is a toxicological concern because of the possible
mutagenic and carcinogenic potential of the aromatic ni-
tro group. Many nitroaromatic compounds have been
shown to bind covalently to DNA. The reactive forms
are metabolically generated through nitro reduction
and, in many cases, through oxidative pathways.16,17
Therefore, one of the objectives of the SAR campaign
was to identify a surrogate for the nitro functionality
of 4. Replacement of the 5-chloro-2-nitrobenzyl moiety
of 4 with a benzyl (compound 5), a 2-nitrobenzyl (com-
pound 6) or a 2-chloro-5-nitrobenzyl (compound 7)
group resulted in a complete loss of the binding toward
the iNOS enzyme (Table 1). This demonstrated that the
substitution pattern at the benzylic moiety of 4 is of cru-
cial importance for iNOS inhibitory activity. Isosteric
The target compounds 4–36, prepared according to
Schemes 1–3, were evaluated for their abilities to inhibit
human iNOS, eNOS, and nNOS mediated conversion of
[3H]arginine to [3H]citrulline.14 The potency of these
compounds in intact cells was determined by measuring
inhibition of cytokine mediated induction of iNOS
activity in DLD-1 cells.15 The screening lead 4, despite
its potent iNOS inhibitory activity, demonstrated no
selectivity over the other NOS isoforms (Table 1). In
addition, this compound is a nitroaromatic derivative,
O
F
O
H
N
N
O
H
N
H
N
HO
S
N
CN
HO
N
NH2
NH
NH2
NH
F
NH2
AR-C102222 (3)
GW274150 (2)
L-NIL (1)
R1
NO2
R4
N
N
() n
R2
Cl
N
R3
N
Formula A
4
Figure 1. iNOS inhibitors (1–3), structure of HTS lead (4) and general formula of the new compounds prepared (Formula A).
Cl
NO2
Br
NH
N
N
R2
R2
a
b
R1
R1
N
37
37a-h
:
4
R1 = CN, CF3, Br, Cl
R2 = Cl, Br, OCH3, F, CF3
R2
R3
R2
O
N
R1
R1
f
c-e
R3
N
N
O
O
N
R4
N
H
8-12,14-20
13, 21-28
Scheme 1. Reagents and conditions: (a) 5-chloro-2-nitrobenzaldehyde, borane–pyridine complex, EtOH, 25 °C, 32%; (b) NBS, benzoyl peroxide or
AIBN, CCl4, reflux, 36–55%; (c) CH3COOH, NaBH(OAc)3, R3NH2, ClCH2CH2Cl, 25 °C, 45–100%; (d) 37a–h, K2CO3, NaI, DMF or K2CO3,
acetone, 25 °C, 26–77%; (e) anhyd HCl/Et2O, MeOH/CH2Cl2, 25 °C, or anhyd HCl/dioxane, 25 °C, or CF3COOH/CH2Cl2, 25 °C, or MeOH/H2O/
microwave, 175 °C, 28–100%; (f) Et3N, CH3COOH, NaBH(OAc)3, aldehyde, ClCH2CH2Cl, 25 °C, 32–89%.

338
B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
Cl
(IC50 = 270 nM) and displayed a better selectivity profile
Cl
over eNOS (16-fold) and nNOS (7-fold) when compared
to its N-methyl analog 8 (3-fold selective over eNOS;
2-fold selective over nNOS). In contrast, compound
20, the N-phenethyl analog of 19, did not show any
selectivity over the other iNOS isoforms.
O
CN
CN
a-d
a, c-d
N
32
N
N
O
N
O
NH
NH
Further, modification of compound 13 was explored to
increase the potency and selectivity for iNOS (Table 3).
Replacement of the N-methyl group of 13 with an N-
ethyl functionality (compound 21) resulted in a 3-fold
increase in potency for iNOS. Furthermore, the selec-
tivity profile of 21 was improved when compared to
the selectivity profile of 13. In addition, compound 21
was found to be 3-fold more potent than its N-methyl
analog in the whole cell assay. This finding suggested
that introduction of an appropriately sized hydropho-
bic alkyl group at the R4-position of compounds of
Formula A (Fig. 1) would enhance the potency for
iNOS, eNOS, and nNOS selectivity, and potency in
the iNOS cellular assay. Substitution of the N-ethyl
group of 21 with an N-propyl moiety (compound 22)
resulted in an increase in potency in the cellular assay.
This might be attributed to a concurrent increase in the
overall lipophilicity of the molecule resulting in im-
proved cell membrane permeability. Further extension
of this key alkyl chain to a butyl moiety (compound
23) led to a significant decrease in the potency for iNOS
as well as potency in the cellular assay. The N-isopropyl
analog 24 showed strong inhibitory activity against
iNOS. This derivative, which was the most active com-
pound in this study, also displayed moderate selectivity
over the eNOS (18-fold) and nNOS (6-fold) isoforms.
In order to explore the size of the lipophilic pocket in
which the isopropyl of 24 is thought to interact (see
molecular modeling section), we prepared the N-isobu-
tyl (compound 25), N-cyclopropyl methyl (compound
26), N-cyclobutyl (compound 27), and N-phenyl (com-
pound 28) analogs of 24. These structural modifications
led to a decrease in the potency for the iNOS enzyme,
indicating that the hydrophobic cavity in which the iso-
propyl group of 24 interacts is relatively small. The N-
isobutyl derivative 25, which retained good potency for
29
O
Cl
( )n
CN
a-d
N
O
O
( )n
NH
n = 1, 3
30 (n = 1)
31
(n = 3)
Scheme 2. Reagents and conditions: (a) CH3COOH, NaBH(OAc)3,
C6H5CH2NHCH3 or (CH3)2CHCH2NH2, CH2Cl2, 25 °C, 68–86%; (b)
H2, Pd/C, EtOH, 25 °C, 78–94%; (c) 2-bromomethyl-4-chloro-benzo-
nitrile, K2CO3, NaI, DMF, 25 °C, 64–91%; (d) anhyd HCl/dioxane,
25 °C, 90–97%.
Cl
Cl
CN
CN
NH2
*
N
*
N
f
a-e
*
N
O
NH
N
O
33*
35*
,
36*,
,
S
R
S
R
34*,
Scheme 3. Reagents and conditions: (a) 2,4-dinitrobenzenesulfonyl
chloride, 2,6-lutidine, CH2Cl2, THF, 25 °C, 60–81%; (b)
(CH3)2CHCH2OH, PPh3, DEAD, THF, 25 °C, 80–90%; (c)
CH3(CH2)3NH2, CH2Cl2, 25 °C, 90%; (d) 2-bromomethyl-4-chloro-
benzonitrile, K2CO3, NaI, DMF, 25 °C, 50–69%; (e) anhyd HCl/
dioxane, 25 °C, 92–99%; (f) formaldehyde, CH3COOH, NaBH(OAc)3,
CH2Cl2, 25 °C, 82–87%.
iNOS
(IC50 = 390 nM)
and
cellular
activity
replacement of the nitro functionality of 4 with a cyano
group (compound 8), which retained electron-withdraw-
ing and hydrogen-bond acceptor properties, resulted in
a 3-fold decrease in the potency for the iNOS enzyme
and a 4-fold decrease in the cellular iNOS inhibitory
activity. Additional benzylic substitutions were explored
(i.e., 9–12) but these modifications did not result in any
improvement in potency for iNOS. SAR at the piperi-
dine nitrogen of 8 was then investigated (Table 2). The
unsubstituted derivative 13inhibited iNOS with a po-
tency comparable (IC50 = 330 nM) to its N-methyl ana-
log (compound 8; IC50 = 290 nM). Replacement of the
N-methyl group of 12 with ethyl, propyl, butyl, isobutyl
or cyclopropylmethyl substituents (compounds 14–18,
respectively) was found to be well tolerated. However,
these modifications did not result in a significant
improvement in the potency for iNOS, selectivity or cel-
lular iNOS inhibitory activity. The N-benzyl derivative
19, however, retained good iNOS potency
(IC50 = 570 nM), was nonetheless of special interest
for further optimization, since this compound interacts
weakly with the eNOS isoform even at high concentra-
tions (20% inhibition at 10 lM). As indicated in Table
4, the 3-substituted piperidine analog 29 also displayed
good potency for iNOS (IC50 = 180 nM). In contrast,
substitution of the piperidine template of 13 with a pyr-
rolidine (compound 30) or azepine (compound 31) scaf-
fold resulted in a significant decrease in iNOS potency.
Based on these results, we prepared the 3-substituted
piperidine analog of 25, that is, compound 32, and
found that this compound retained good potency for
iNOS and displayed high selectivity against eNOS
(15% inhibition at 10 lM). Since 32 was racemic, the
synthesis of enantiomers 33 and 34 was undertaken to
see if additional potency/selectivity were to be gained.
As indicated in Table 4, the S-isomer 33 was superior
to the R-isomer 34, in terms of iNOS cellular activity
and selectivity over eNOS and nNOS. Compound 35,

B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
Table 1. NOS inhibitory activity and selectivity of compounds 1–12
339
R1
N
N
R2
R1
R2
Selectivityb
Selectivity
iNOS cellular assay
a
IC50 (nM)
c
Compound
d
iNOS
eNOS
nNOS
eNOS/iNOS
nNOS/iNOS
IC50 (nM) or % inhibition at 10 lM
4
NO2
H
Cl
H
90
nsf
180
nde
nde
nde
770
810
nde
1400
820
2500
nsf
80
2
1
1100
nsf
5
nde
nde
nde
540
530
nde
780
590
6000
8400
840
nde
nde
nde
3
nde
nde
nde
2
6
NO2
Cl
H
nsf
nsf
7
NO2
Cl
nsf
nsf
8
CN
CN
290
210
5100
700
420
280
220
170
3700
3700
nsf
9
Br
4
3
10
11
12
CN
CF3
OCH3
Cl
Cl
nde
2
nde
1
48%
51%
31,000
26%
210
Br
1 (L-NIL)
2
1
9
21
38
5
2 (GW274150)
3 (AR-C102222)
>45
>70
nsf
a Inhibition of human iNOS, eNOS, and nNOS mediated conversion of [3H]arginine to [3H]citrulline by tested compounds. IC50 values were
determined by testing each compound at eight concentrations. IC50 values are geometric means computed from at least three separate
determinations.
b Selectivity was defined as the ratio of IC50 value of eNOS to that of iNOS.
c Selectivity was defined as the ratio of IC50 value of nNOS to that of iNOS.
d Inhibition of cytokine mediated induction of iNOS activity in DLD-1 cells by tested compounds. IC50 values were determined by testing each
compound at eight concentrations. IC50 values are geometric means computed from at least three separate determinations.
e nd, not determined.
f ns, no significant effect (<15% inhibition at 10 lM).
Table 2. NOS inhibitory activity and selectivity of compounds 13–20
CN
N
Cl
N
R
a
IC50 (nM)
Compound
R
Selectivityb
eNOS/iNOS
3
Selectivityc
nNOS/iNOS
1
iNOS cellular assay
d
IC50 (nM)
iNOS
330
eNOS
nNOS
350
13
14
15
16
1100
5900
4500
4400
3800
H
370
670
340
450
1100
680
370
810
420
1
2
2
1
1
1
17
18
400
250
540
860
600
530
1
3
2
2
3200
3500
19
20
270
300
4300
220
1800
150
16
1
7
3500
3300
0.5
a Inhibition of human iNOS, eNOS, and nNOS mediated conversion of [3H]arginine to [3H]citrulline by tested compounds. IC50 values were
determined by testing each compound at eight concentrations. IC50 values are geometric means computed from at least three separate
determinations.
b Selectivity was defined as the ratio of IC50 value of eNOS to that of iNOS.
c Selectivity was defined as the ratio of IC50 value of nNOS to that of iNOS.
d Inhibition of cytokine mediated induction of iNOS activity in DLD-1 cells by tested compounds. IC50 values were determined by testing each
compound at eight concentrations. IC50 values are geometric means computed from at least three separate determinations.

340
B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
Table 3. NOS inhibitory activity and selectivity of compounds 21–28
CN
Cl
R
N
N
H
a
IC50 (nM) or % inhibition at
Compound
R
Selectivityb
Selectivityc
iNOS cellular assay
10 lM
d
IC50 (nM)
iNOS
eNOS
1000
nNOS
eNOS/iNOS
10
nNOS/iNOS
4
21
22
100
360
580
130
1700
nde
140
13
1
280
23
24
570
32
nde
190
nde
18
nde
6
5000
380
570
25
26
27
28
390
160
1500
660
20%
2100
1%
1900
748
>25
13
5
570
5
1400
6700
8000
24%
33%
>7
>6
>15
0%
>15
a Inhibition of human iNOS, eNOS, and nNOS mediated conversion of [3H]arginine to [3H]citrulline by tested compounds. IC50 values were
determined by testing each compound at eight concentrations. IC50 values are geometric means computed from at least three separate
determinations.
b Selectivity was defined as the ratio of IC50 value of eNOS to that of iNOS.
c Selectivity was defined as the ratio of IC50 value of nNOS to that of iNOS.
d Inhibition of cytokine mediated induction of iNOS activity in DLD-1 cells by tested compounds. IC50 values were determined by testing each
compound at eight concentrations. IC50 values are geometric means computed from at least three separate determinations.
e nd, not determined.
the N-methylated analog of 33, resulted in an improve-
ment in cellular potency, while the potency and selectiv-
ity for nNOS was reduced when compared to 33.
tate interaction, is stabilized in the iNOS active site by
p-stacking with the heme group.20 The induced-fit
docking protocol of the Schro¨dinger suite24 was used
to dock 13, a presumably p-stacking ligand, into the
iNOS active site. As shown in Figure 2a and b, there
are several key interactions between the inhibitor and
the enzyme: (1) the cyano group of 13 forms a hydro-
gen-bond with the amide hydrogen of Met374; (2) the
basic nitrogen of the piperidine ring, protonated at
physiological pH, forms two hydrogen bonds with
the iNOS active site, one with Asp382 and another
with one of the propionate tails of the heme. These
interactions are consistent with those seen in crystal
structures of other p-stacking iNOS ligands; (3) the
methyl group of 13 interacts with a small hydrophobic
pocket of the active site; and (4) the cyano phenyl
moiety of 13 interacts by p stacking with the heme.
Several crystal structures of various ligands co-crystal-
lized with the mouse or human iNOS dimer have been
published.18–24 The active form of the enzyme is a
homodimer with two independent active sites, each
containing a heme group and a tetrahydrobiopterin
cofactor. Each L-arginine binding site is located at
the interface between the two monomers and is com-
prised of residues from both monomers. An extensive
network of hydrogen bonds between L-arginine, the
carboxylate sidechain of Glu377 (human iNOS num-
bering), the backbone amide of Met374, the backbone
carbonyl of Trp372, the propionate tails of the heme,
and the pterin co-factor stabilizes the active site struc-
ture when L-arginine is bound.20 Examination of the
crystal structures of various ligands which bind com-
petitively with L-arginine reveals two distinct classes.
One class mimics a critical bidentate interaction be-
tween the guanidinium group of arginines and the car-
boxylate of Glu377 in the iNOS active site. A second
class of ligands, without the ability to form this biden-
Electron-withdrawing substituents on the benzylic ring
are essential for the inhibitor to form a strong p-stack-
ing interaction with the heme. Removal of either elec-
tron-withdrawing substituent from the ring disrupts
the p-stacking interaction by making the benzylic p-
system a poorer acceptor. The docking pose of 13 high-

B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
Table 4. NOS inhibitory activity and selectivity of compounds 29–36
341
CN
R1
N
R2
Cl
R1
R2
Selectivityb
Selectivity
iNOS cellular assay
a
IC50 (nM) or %
c
Compound
inhibition at 10 lM
d
iNOS
eNOS
nNOS
eNOS/iNOS
5
nNOS/iNOS
1
IC50 (nM) or % inhibition at 10 lM
NH
NH
29
30
31
180
983
190
4400
1200
660
4000
4400
1200
1200
3
7
1
2
33%
31%
NH
NH
NH
32
33
290
330
15%
0%
2500
5300
>15
>25
9
1200
570
16
NH
34
35
36
260
720
470
23%
0%
1200
6500
4900
>25
>50
>15
5
1500
320
N
N
9
0%
10
660
a Inhibition of human iNOS, eNOS, and nNOS mediated conversion of [3H]arginine to [3H]citrulline by tested compounds. IC50 values were
determined by testing each compound at eight concentrations. IC50 values are geometric means computed from at least three separate
determinations.
b Selectivity was defined as the ratio of IC50 value of eNOS to that of iNOS.
c Selectivity was defined as the ratio of IC50 value of nNOS to that of iNOS.
d Inhibition of cytokine mediated induction of iNOS activity in DLD-1 cells by tested compounds. IC50 values were determined by testing each
compound at eight concentrations. IC50 values are geometric means computed from at least three separate determinations.
lights the need for a hydrogen-bond acceptor at the cy-
ano-position on the benzylic ring (position R1 of For-
mula A). The cyano group is oriented toward Met374
and this hydrogen-bond interaction is important for
the stability of the ligand–enzyme interaction. Addi-
tionally, the N-methyl group at the R4-position pro-
jects into a small hydrophobic pocket of the enzyme
active site. Slightly larger R4 substituents (ethyl, pro-
pyl, isopropyl, cyclopropylmethyl) improve binding
by increasing favorable hydrophobic interactions with
the enzyme. Bulkier substituents, however, encounter
steric restrictions in this pocket which reduce the ligand
affinity for the enzyme.
ethylpiperidin-4-amine 4, displaying strong iNOS inhib-
itory activity but no selectivity over the other NOS
isoforms. Further modification of this lead resulted in
the discovery of aminopiperidines as a novel chemical
class of iNOS inhibitors. The substitution pattern in
the phenyl ring of these classes of compounds was found
to be of key importance for iNOS inhibitory activity. In
addition, introduction of an isopropyl group at the R4-
position was critical to disrupt the interaction of the
compounds with eNOS while maintaining potent iNOS
inhibitory activity in the enzyme assay as well as in the
cellular assay. This work ultimately led to the identifica-
tion of compound 33, which displayed better activity in
the iNOS whole cell assay and a much improved selec-
tivity profile over eNOS and nNOS when compared to
the original lead.
Screening of the Adolor compound collection provided
a
novel lead, N-(5-chloro-2-nitrobenzyl)-N,1-dim-

342
B. Le Bourdonnec et al. / Bioorg. Med. Chem. Lett. 18 (2008) 336–343
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14. In vitro method: human iNOS, nNOS, and eNOS med-
iated conversion of [3H]arginine to [3H]citrulline. Com-
pounds were evaluated for their abilities to inhibit human
iNOS (human iNOS endogenously expressed in colon
adenocarcinoma cells (DLD-1 cells)), eNOS (human
recombinant eNOS expressed in SF9 cells), and nNOS
(human recombinant eNOS expressed in SF9 cells) med-
iated conversion of [3H]arginine to [3H]citrulline. Series of
concentrations of compounds were incubated with 10 nM
[3H]arginine, [14C]citrulline (80–120 nM) as an internal
standard, cofactors (1.4 mM b-NADP+, 3.0 mM glucose-
6-phosphate (G6P), 3.4 mM MgCl2, 0.4 U/mL G6P dehy-
drogenase, 5.0 lM FAD, 5.0 lM FMN, 5.0 lM BH4), and
enzyme in 100 lL of 50 mM Hepes buffer for 1 h at 37 °C.
For eNOS and nNOS assays, additional cofactors 25 nM
calmodulin, 0.5 mM CaCl2, and 3.5 mM glutathione were
included in the reactions. The reactions were stopped by
the addition of 25 lL of 1 M MES and filtered through
AG-50W-X8 (200–400 mesh, Na form) cation exchange
resin that had been loaded onto 96-well filter plates using a
100 lL column loader (Millipore). The filters were then
washed serially with 75 lL and 25 lL of ddH2O, which
was collected in the collection plate, and the radioactivity
in the filtrate counted. Background activity was deter-
mined in the presence of 10 lM AMT. Data were fit to the
one-site competition model in GraphPad Prism (version
4.02 for Windows) and are from a single experiment that
was repeated at least four times with similar results.
15. In vitro method: cytokine mediated induction of iNOS in
DLD-1 cells. Human colon adenocarcinoma (DLD-1)
cells, obtained from the American Type Culture Collec-
tion (ATCC), were plated into 96-well plates at 40,000
cells per well 3 days before the experiment. On the day of
the experiment, the medium was removed and replaced
with 90 lL of fresh complete growth medium containing
20 ng/mL interferon c (INFc), 4 ng/mL tumor necrosis
factor a (TNFa), 2 ng/mL interleukin 1b (IL1b), and
1 mM L-arginine. Background iNOS activity was deter-
mined using complete growth medium in the absence of
cytokines and L-arginine. Titrations of test compounds
(10 lL) in 50 mM Hepes, pH 7.4, were then added to the
appropriate wells and the assay incubated for 24 h at
37 °C with 5% CO2. After incubation, an aliquot from
each well (75 lL) was transferred to a clear, untreated 96-
well plate containing an equal volume of modified Griess
reagent (Sigma). The absorbance at 540 nm was measured
for each well using the Fusion (Perkin-Elmer Life
Sciences). IC50 values were determined from non-linear
regression fits of the data in GraphPad Prism (version 4.02
for Windows).
Met
A374
Gln
Tyr
A263
A373
Pro
A350
Trp
HEM
N
A372
B510
H
+
+
N
NH
H
FE2
509
Asp
Cl
A382
Glu
Gly
A371
A377
Val
A352
Phe
A369
Figure 2. (a) Compound 13 (white) docked into the iNOS active site.
The aromatic ring exhibits p-stacking with the heme (orange). On the
surface, green represents hydrophobic regions, blue represents mildly
polar regions of the active site, and pink shows hydrogen-bonding
areas. The large green area surrounding the central amino group
underlies the enzyme’s preference for lipophilic substituents on the
amino nitrogen. Pink regions near the hydrogens on the piperidine
nitrogen highlight H-bond interactions of the ligand with one of the
heme propionate tails and Asp382. The pink region near the cyano
nitrogen indicates the presence of an H-bond interaction with the
amide nitrogen of Met374. (b) H-bond interactions with Asp382,
Met374, and the heme propionate tail are shown in the ligand
interaction diagram. Blue regions around ligand atoms indicate the
degree of solvent exposure.
References and notes
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