Identification and SAR of selective inducible nitric oxide synthase (iNOS) dimerization inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.08.112

We have identified and synthesized a series of imidazole containing dimerization inhibitors of inducible nitric oxide synthase (iNOS). The necessity of key imidazole and piperonyl functionality was demonstrated and SAR studies led to the identification of compound 35, which showed a dose dependant inhibition in multiple pain models, including tactile allodynia induced by spinal nerve ligation (Chung model).

Full text of DOI:10.1016/j.bmcl.2011.08.112

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6888–6894
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and SAR of selective inducible nitric oxide synthase (iNOS)
dimerization inhibitors
Timothy C. Gahman a, , Mark R. Herbert a, Henk Lang a, Angie Thayer a, Kent T. Symons b,
⇑
Phan Manh Nguyen b, Mark E. Massari b, Sara Dozier c, Yan Zhang c, Marciano Sablad c, Tadimeti S. Rao c,
Stewart A. Noble a, Andrew K. Shiau b, Christian A. Hassig b
a Department of Medicinal Chemistry, Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
b Department of Biology, Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
c Department of Pharmacology, Kalypsys Inc., 10420 Wateridge Circle, San Diego, CA 92121, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have identified and synthesized a series of imidazole containing dimerization inhibitors of inducible
nitric oxide synthase (iNOS). The necessity of key imidazole and piperonyl functionality was demon-
strated and SAR studies led to the identification of compound 35, which showed a dose dependant inhi-
bition in multiple pain models, including tactile allodynia induced by spinal nerve ligation (Chung
model).
Received 31 October 2010
Revised 20 August 2011
Accepted 25 August 2011
Available online 8 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
iNOS
Dimerization inhibitor
Tactile allodynia
Nitric oxide (NO) is involved in the regulation of many physio-
chemical processes as well as the pathophysiology of a broad range
of human disease indications including inflammation, pain, asthma,
periodentitis and neurodegenerative disorders.1,2 It is synthesized
enzymatically from L-arginine and molecular oxygen via a five elec-
tron transfer process by three distinct isoforms of the heme-contain-
ing enzyme nitric oxide synthase (NOS).3 The three isoforms of NOS,
inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) differ in
CO2H
CO2H
NH2
NH2
S
H2N
H
N
NH
NH
NH
NH
NH
NH
O2N
CO2H
1 - L-NNA
2 - L-NIL
3 - GW271450
O
O
N
N
N
O
F
H
N
H
N
N
O
O
N
O
N
N
F
NH2
N
N
N
4 - AR-C102222
5 - BBS-2
Figure 1. Representative iNOS inhibitors.
⇑
Corresponding author. Tel.: +1 760 815 9120; fax: +1 858 534 7750.
E-mail address: tgahman@gmail.com (T.C. Gahman).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.112

T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
6889
R3
N
their tissue distribution and biological role but are similar in that
they are only active in a homo-dimeric form.4 Two of these isoforms,
eNOS and nNOS are expressed in a constitutive manner and are cal-
cium/calmodulin dependent.4 Under normal physiological condi-
tions, these constitutive isoforms generate low, transient levels of
NO in response to increases in intracellular Ca2+ concentrations.
These low levels of NO are essential in aiding the regulation of blood
pressure, gastrointestinal motility, platelet adhesion and neuromo-
tor transmission.1 The third isoform, iNOS, is expressed and induced
at a transcriptional level by inflammatory stimuli, including inter-
R1
S
R2
O
O
R1
N
O
O
R2
N
N
N
N
N
N
N
N
Pyrimidine Series
1,2,4-Thiadiazole Series
Figure 2. Imidazole based iNOS dimerization inhibitor scaffolds.
feron (IFN-c), interleukin (IL)-1b, tumor necrosis factor (TNF-a)
and bacterial lipopolysaccharide (LPS).5 iNOS is calcium indepen-
dent and generates high, sustained levels of NO. These elevated lev-
els of NO and resulting NO metabolites cause cellular cytotoxicity
and tissue damage and are thought to contribute to the pathophys-
iology of a number of diseases.1 This suggests that selective inhibi-
tion of iNOS may have therapeutic potential for the treatment of
diseases mediated by the overproduction of NO.1 Therefore, given
the double-edged nature of NO in both basic physiological functions
and various pathological conditions, our focus was to develop a
selective iNOS inhibitor.
O
N
N
H
N
N
H
O
S
N
N
S
N
N
N
N
N
N
6
7
hiNOS IC50 = 0.007 µM
hiNOSIC 50 > 100 µM
Initial work by other groups concentrated on direct enzyme inhi-
bition, specifically compounds that mimic arginine, the substrate for
all NOS isoforms, or molecules that directly interact with the heme
(Fig. 1). Compounds such as L-NNA (1) and L-NIL (2), inhibited the
enzyme, but were poorly selective for iNOS over the other isoforms.6
The structurally related iNOS inhibitor, GW271450 (3) with im-
proved selectivity for iNOS and demonstrated efficacy in preclinical
models of pain,7 is currently in clinical development.8 Other small
molecule guanidine mimetics include AR-C102222 (4), a selective
iNOS inhibitor that is efficacious in a rat adjuvant-induced model
of arthritis.9 More recently, inhibitors of iNOS dimerization were re-
ported by Berlex (5) with marked selectivity and efficacy in rodent
models of neuropathic pain.10
O
O
O
O
N
N
N
H
N
N
H
N
N
N
N
N
N
8
9
hiNOS IC50 = 0.010 µM
hiNOS IC50 > 100 µM
Figure 3. Deletion of piperonyl methylenedioxy or key imidazole nitrogen abolish
inhibitory activity against hiNOS.
We sought to expand on the existing knowledge of these iNOS
dimerization inhibitors and capitalize on the inherent selectivities
and non-arginine character of these small molecules.
Two series of molecules were identified as iNOS inhibitors
( Fig. 2).11 Analogous to the molecules from Berlex, these com-
pounds contained both an imidazole and a piperonyl sub-unit that
were each necessary for activity.
A deletion strategy was first employed to understand the key
functionality required for maintenance of iNOS inhibitory activity.
As shown in Figure 3, removal of the methylenedioxy portion of
the piperonyl in 6 to afford the parent phenyl compound 7, com-
pletely eliminated iNOS inhibitory activity. Similarly, removal of
Figure 4. Western blot of iNOS dimerization.
O
R1
(a)
R1
R1
(b)
R3 NH2
13
R3
H
n
Br
R2
N
N
H
R2
N
n
n
10
Boc
Boc
+
14
12
Br
R2 NH2
11
R1
R1
R3
S
R3
S
N
R2
N
N
R2
N
n
n
(c)
(d)
NNa
Boc
H
S
N
N
N
Cl
N
N
N
N
16
15
Cl
N
Cl
N
Scheme 1. Reagents and conditions: (a) i. IPA, AcOH, NaHB(OAc)3, rt, 3 h, 41–53%, ii. Boc2O, Et3N, MeOH, rt, 1 h, 90–97%. (b) EtOH, rt, 14 h, 99% (c) Et3N, CH2Cl2, 0–20 °C,
90–95%. (d) i. DMSO, 80 °C, 6 h, 99%, ii. HCl, EtOH, 70 °C, 1 h, 75%.

6890
T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
R5
R5
R5
O
O
O
Bz
Bz
Bz
N
O
N
O
N
N
(a)
(b)
O
O
O
O
O
Cl
OtBu
O
N
n
NH2
19
17
18
R5
R5
R1
H
O
N
N
R2
N
Bz
(c)
(d)
12
O
N
N
N
N
N
N
N
N
20
21
Scheme 2. Reagents and conditions: (a) i. CH3MgBr, THF, 4 °C, 15 h, 99%. ii. cat. pTSA, toluene, 80 °C, 4 h, 95%. (b) urea, Na, EtOH, 80 °C, 12 h, 40–52%. (c) gloxal, (CHO)n, NH4Cl,
80–100 °C, 62–77%. (d) 10% Pd/C, H2 (1 atm), EtOH, 13 h, 88–95%, ii. 12, KI, DMF, DIEA, 80 °C, 3 h, 70%, ii. HCl, EtOH, 70 °C, 1 h, 75%.
Table 1
Piperonyl amine optimization
R2
N
H
N
S
N
N
N
N
Compound
R2
hiNOS
EC50
hnNOS
EC50
heNOS
EC50 (l
M)a
(
l
M)a
(
l
M)a
O
O
6
0.008(0.001)
17.3(2.1)
>100b
>100b
>100b
b
17
>
100
O
O
22
23
0.048 (0.010)
0.36 (0.058)
24 (12)
>100b
>100b
>100b
OCH3
OCH3
24
0.76 (0.21)
>100b
>100b
OCH3
F
25
26
>100b
>100b
>100b
>100b
>100b
>100b
F
N
27
28
>100b
>100b
>100b
>100b
>100b
N
H
O
O
O
6.3 (4.1)
O
F
29
30
8.9 (4.5)
>100b
>100b
>100b
F
O
O
2
0.23 (0.060)
47 (42)
O
a
Value represents mean of three experiments with standard deviations shown in parentheses.
<50% inhibition at 100 lM.
b

T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
6891
Table 2
Backbone linker optimization
O
O
R4
N
N
N
N
Compound
R4
hiNOS
EC50
hiNOS
EC50
heNOS
EC50 (l
M)a
(
l
M)a
(
l
M)a
H
N
31
32
0.029 (0.007)
0.020 (0.007)
6.0 (4.3)
>100b
>100b
>100b
N
N
H
H
H
N
N
33
0.014 (0.002)
>100b
>100b
O
O
H
N
34
35
36
N
H
0.293 (0.08)
0.004 (0.001)
0.030 (0.008)
>100b
>100b
>100b
>100b
N
3.9 (3.3)
6.6 (2.1)
N
N
N
N
H
O
H
N
37
38
0.009 (0.003)
0.036 (0.010)
2.7 (0.36)
9.5 (2.6)
>100b
O
O
b
N
>
100
N
H
H
N
39
40
41
2.3 (1.4)
>100b
>100b
>100b
>100b
N
H
N
1.8 (0.87)
N
H
N
>100b
>100b
>100b
>100b
>100b
O
N
H
N
42
0.29 (0.095)
O
a
Value represents mean of three experiments with standard deviations shown in parentheses.
<50% inhibition at 100 lM.
b
the key nitrogen in imidazole 8 to provide pyrrole analog 9, also
completely abrogated the inhibition of iNOS.
deprotection provided the disubstituted-1,2,4-thiadiazole (16) in
excellent overall yields. Alternatively, the pyrimidine scaffold
was obtained by condensation of the appropriate amino acid chlo-
ride (17) with the magnesium anion of tert-butyl acetoacetate, fol-
lowed by decarboxylation to afford diketone (18). This diketone
was condensed with urea to give the aminopyrimidine (19), which
was subjected to cyclodehydrative conditions using glyoxal, form-
aldehyde and ammonium chloride to provide the imidazole (20).
Deprotection and sidechain coupling with alkylbromide (12) affor-
ded the appropriately substituted pyrimidine (21) in moderate to
excellent overall yields.
To investigate the SAR, three main areas were explored: (1)
replacement of the piperonyl sidechain connected to the pendant
backbone amine, (2) modification of the linker between the thia-
zole or pyrimidine core and the piperonyl sidechain, and (3) intro-
duction of novel alkyl and heteroaryl sidechains off of the
The role of compounds 6 and 8 as iNOS dimerization inhibitors
is illustrated in the Western blot shown in Figure 4. LT–SDS–PAGE
of extracts prepared from IFN-c
/LPS stimulated RAW cells12 dem-
onstrates the ability of compounds 6 and 8 to block iNOS dimer
formation, while Compounds 7 and 9 with altered piperonyl or
imidazole sub-units have no effect on iNOS dimerization.17
The compounds described herein were synthesized as outlined
in Schemes 1 and 2.13 Reductive amination of the appropriate alde-
hyde (10) and aminoalkylbromide (11), followed by Boc protection
afforded the substituted alkylbromide (12). Displacement of this
bromide with amine (13) provided secondary amine (14) which
was added to 3,5-dichloro-1,2,4-thiadiazole to afford the regiospe-
cifc 3-chlorothiadiazole (15). Finally, nucleophilic aromatic substi-
tution of the 3-chlorothiadiazole with sodium imidazole and

6892
T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
Table 3
Table 4
Alkyl sidechain optimization
Aryl and heteroaryl sidechain optimization
R1
R1
O
O
O
O
N
N
H
N
N
H
S
N
S
N
N
N
N
N
N
N
Compound R1
hiNOS
hiNOS
heNOS
EC50 (l
M)a
Compound
R1
hiNOS
EC50
hiNOS
heNOS
EC50 (l
M)a
M)a
EC50
(l
M)a
(
l
M)a
EC50
(
l
M)a
EC50
(l
>100b
>100b
CH3
6
0.008 (0.001)
0.009 (0.002)
3.9 (3.3)
N
52
0.005 (0.002) 0.48 (0.060) >100b
0.006 (0.002) 0.31 (0.053) >100b
43
2.3 (0.60)
CN
44
45
0.012 (0.007)
0.056 (0.007)
7.2 (1.3)
36 (15)
>100b
>100b
N
53
54
55
56
S
S
O
O
O
O
O
>100b
>100b
>100b
>100b
NH2
46
47
48
49
0.081 (0.007)
0.034 (0.009)
0.059 (0.007)
0.080 (0.009)
11 (2.6)
7.6 (1.4)
7.2 (5.1)
>100b
0.009 (0.001) 0.69 (0.16)
0.014 (0.007) 2.1 (0.20)
0.071 (0.020) 18 (1.5)
>100b
>100b
>100b
OEt
NH
N
N
N
OH
O
N
>100b
>100b
>100b
N
NH
N
O
57
58
59
0.042 (0.011) 11 (1.0)
0.090 (0.026) 7.9 (0.95)
0.163 (0.040) 6.8 (1.3)
50
51
4.0 (1.7)
>100b
>100b
>100b
>100b
>100b
OH
a
Value represents mean of three experiments with standard deviations shown in
parentheses.
N(CH3)2
b
<50% inhibition at 100 lM.
backbone linker. The SAR was driven by three in vitro assays that
measured inducible NOS (iNOS), endothelial NOS (eNOS) and neu-
ronal NOS (nNOS) inhibition.14
a
Value represents mean of three experiments with standard deviations shown in
parentheses.
b
<50% inhibition at 100 lM.
While the importance of the piperonyl group was demonstrated
by removing the methylenedioxy portion of the sidechain (6 and
7), we were interested in further exploring the effects of substitu-
tion on the benzylamine (Table 1). As shown, replacement of the
methylenedioxy portion of the piperonyl group of 6 with an ethyl-
enedioxy substituent (22) resulted in a modest (six-fold) reduction
of iNOS activity. Substitution with mono- or di-methoxy (23 and
24) had a more dramatic 45-and 95-fold reduction of iNOS activity,
respectively, and additional substitution or heterocycle replace-
ment (Compounds 25–27) completely eliminated iNOS inhibitory
activity. Oxidation of the benzylic amine (28) or introduction of
two fluorines to the piperonyl (29) resulted in a 1000-fold loss of
iNOS inhibitory potency. Additionally, one carbon homologation
of the benzylamine to the phenethylamine (30), also significantly
reduced inhibitory activity. This is presumably due to the improper
positioning of the essential piperonyl subunit within the enzyme.
From our studies a more suitable piperonyl replacement was not
identified.
the sidechain to four carbons (32) resulted in maintenance of iNOS
potency and selectivity. Oxidation could be introduced in the form
of urea (33) or amide (34) offering equivalent or a modest 10-fold
loss in potency, respectively. Rigidification of the sidechain by intro-
duction of a heteroalkyl ring (35–38) afforded significant improve-
ments in iNOS inhibitory potency with minor effects on selectivity.
Modification of the carbon directly attached to the core significantly
affected iNOS potency. The quaternization by introduction of a gem
dimethyl group (39) or sp2 hybridization (40 and 41) of this carbon
greatly reduced or eliminated iNOS inhibition. Finally, replacement
of the piperonyl benzyl amine with oxygen (42) reduced the potency
by 10-fold. So, while exceptions were identified, as long as the linker
length was consistent between the core and piperonyl sidechain,
significant modification was tolerated.
An additional area of allowed substitution was identified off of
the linker and is illustrated by the thiadiazole analogs in Table 3.
The methyl group of compound 6 could be extended (43 and 44)
with little to no loss in iNOS potency and more polar groups could
be introduced (46–49) with only modest losses in iNOS potency
One area where significant modification was tolerated was in
the backbone linker (Table 2). One of the early leads (31) had a four
atom spacer between the core and the pendant benzyl amine.
Keeping the length of the sidechain constant, simplification of

T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
6893
Figure 7. Profile of compound 35 in Brennan model of post-surgical pain.
Figure 5. Compound 35 attenuated changes in plasma nitrates in the rat endotox-
emia model.
and little effect on selectivity. Only when the polarity was in-
creased to the tetrazole (50) or carboxylic acid (51), was a signifi-
cant loss of inhibitory activity observed.
Finally, introduction of novel aryl and heteroaryl sidechains off
of the backbone linker provided very active iNOS inhibitors, but the
selectivity of many of these analogs versus nNOS was diminished,
as illustrated in Table 4. Thus, while addition of the 3-pyridyl (52),
2-thiazole, (53), 2-thiophene (54), 2-furan (55) and N-methyl-
imidazole (56) to the linker provided compounds with potencies
equal or even better than the parent compound 6, this effect was
often concomitant with a loss in selectivity over nNOS. Three
substituted phenyl analogs (57–59) showed the toleration of addi-
tional polar groups in this region, illustrating the potential for opti-
mization of both potency and physicochemical properties through
analogs at this site.
Compound 35 was selected for evaluation of its in vivo efficacy
due to its significant iNOS inhibitory activity and selectivity over
both hnNOS and heNOS. Injection of bacterial lipopolysaccharide
(LPS) in rodents induces systemic inflammation (endotoxemia)
Figure 8. Profile of compound 35 in the Chung neuropathic pain model.
Figure 6. Profile of compound 35 in carrageenin model.

6894
T. C. Gahman et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6888–6894
Parkinson, J.; Polokoff, M.; Saionz, K.; Santos, C.; Subramanyam, B.; Vergona, R.;
Wei, R. G.; Whitlow, M.; Ye, B.; Zhao, Z. S.; Devlin, J. J.; Phillips, G. J. Med. Chem.
2007, 50, 1146; (b) Blasko, E.; Glaser, C. B.; Devlin, J. J.; Xia, W.; Feldman, R. I.;
Polokoff, M. A.; Phillips, G. B.; Whitlow, M.; Auld, D. S.; McMillan, K.; Ghosh, S.;
Stuehr, D. J.; Parkinson, J. F. J. Biol. Chem. 2002, 277, 295.
accompanied by increases in cytokines and induction of iNOS lead-
ing to accumulation of nitrates in circulation.11 The endotoxemia-
associated plasma nitrate accumulation is completely absent in
mice with genetic deletion of iNOS (data not shown) directly pro-
viding evidence for the utility of LPS induced plasma nitrates as a
direct readout of iNOS activity. Compound 35, upon oral adminis-
tration to female Lewis rats (140–160 g), inhibited plasma nitrates
with an ED50 of 9.3 mg/kg (Fig. 5)11 thus demonstrating in vivo tar-
get engagement. Compound 35 was further evaluated in acute
inflammation/hyperalgesia (Fig. 6), a post-operative pain model15
(Fig. 7) and in a model of chronic neuropathic pain, that is, spinal
nerve ligation-induced tactile allodynia (Chung) model16 (Fig. 8).
Oral or parenteral administration of Compound 35 demonstrated
attenuated inflammation, inflammatory hyperalgesia, incision-in-
duced paw weight bearing response and chronic nerve-ligation in-
duced tactile allodynia.
In conclusion, a series of substituted 1,2,4-thiadiazoles and 2,6-
pyrimidines containing both imidazole and piperonyl subunits
have been shown to interrupt protein–protein interactions, as evi-
denced by inhibition the dimerization of iNOS monomers. These
series have been optimized for their potency and selectivity against
other NOS isoforms. Compound 35 is a potent, selective molecule
that shows efficacy in both acute inflammation and chronic neuro-
pathic pain. These results are consistent with profile of inhibitors
of iNOS active site and those that target dimerization of iNOS7,9
in preclinical rodent or primate models of pain. Inhibition of iNOS
11. Zhang, Y.; Sablad, M.; Rozenkrants, N.; Gahman T. C.; Hassig, C.; Rao, T.
Inhibitors of Inducible Nitric Oxide Synthase Dimerization (iNOS-DI) are
Effective Anti-nociceptive Agents in Rodent Models. Keystone Symposium,
Steamboat, CO, June 12, 2006.
12. Gel-based dimer assay (Low Temperature SDS–PAGE) for iNOS: RAW 264.7
cells were seeded into 6-well dishes at a density of 1.8 Â 106 cells/well. Cells
were incubated for 5 h and 1 mL of media was removed from each well and
replaced with 1 mL of a 2X cocktail containing murine IFN
Roche Diagnostics), LPS (2 g/mL final; Sigma) and 10 M compound or vehicle
(0.1% DMSO final) diluted in DMEM with 10% serum. Cells were incubated
overnight. Cells were washed twice with ice cold PBS and then 200 L of ice-
c (100 U/mL final;
l
l
l
cold lysis buffer (250 mM sucrose, 10 mM Tris pH 7.5, 1 mM EDTA) containing
protease inhibitors was added to each well. Cells were scraped from the dish
and transferred to micro-centrifuge tubes on ice. Samples were sonicated for
6 s at setting 4 (Branson) and centrifuged at 16,000 g for 10 min at 4 °C. The
concentration of protein was normalized using a Bradford assay (BioRad). An
equal volume of ice-cold 2X loading buffer (63 mM Tris pH 6.8, 10% glycerol, 3%
SDS, 1% b-mercaptoethanol, 0.002% Bromophenyl Blue) was added to each
sample and loaded onto a 4–20% Tris–glycine polyacrylamide gel. The gel was
run in pre-chilled 1X SDS running buffer in a cold room for 2.5 h at 125 V.
Proteins were transferred to nitrocellulose for 2 h at 80 V in 1X transfer buffer
and were blocked overnight in Odyssey buffer (LI-COR Biosciences) at 4 °C. The
membranes were incubated in 1:2,500 of mouse anti-iNOS (BD Biosciences) in
Odyssey buffer with 0.15% Tween-20 for 1 h at room temperature followed by
4 Â 5 min washes in PBS + 0.1% Tween-20. The blot was then incubated in a
1:10,000 dilution of goat anti-mouse IRDye 800 (Rockland Immunochemicals)
secondary antibody in Odyssey buffer with 0.15% Tween-20 for 1 h at room
temperature. Following 4 Â 5 minute washes, the blot was dried in the dark
and the proteins were visualized on the Odyssey infrared imaging system (LI-
COR Biosciences).
may represent potentially
therapeutics.
a useful approach for novel pain
13. All final compounds displayed spectral data (NMR, MS) that was consistent
with the assigned structure.
14. Nitric Oxide Detection (DAN Assay): HEK 293 cells were plated into 15 cm
dishes and grown to 70% confluency in DMEM containing 10% FBS, 100 U/ml
Acknowledgement
penicillin, and 100
a CMV-driven plasmid expressing human iNOS, human nNOS, or human eNOS
by addition of 30 l Fugene and 10 g/dish of the appropriate NOS plasmid for
lg/ml streptomycin. Cells were transiently transfected with
We would like to acknowledge Christine Kao for her diligent
profiling assistance.
l
l
4 h. Transfected cells were plated in 384- or 1536-well plates at a density of
5e5 cells/ml and compounds were added immediately using a proprietary pin-
transfer technology. Cells for the iNOS assay were incubated for 18 h for nitrite
accumulation. NO production was determined indirectly by measuring nitrite
accumulation in cell culture supernatants using the 2,3-diaminonapthalene
References and notes
(DAN) assay. Supernatants were mixed with (DAN 10 lg/ml final; Invitrogen)
1. (a) Vallance, P.; Leiper, J. Nat. Rev. Drug Discov. 2002, 1, 939; (b) Alderton, W. K.;
Cooper, C. E.; Knowles, R. G. Biochem. J. 2001, 357, 593.
2. Levy, D.; Zochodne, D. W. Pain Practice 2004, 4, 11.
3. Li, H.; Poulos, T. L. J. Inorg. Biochem. 2005, 99, 293.
4. Wang, Y.; Marsden, P. A. Adv. Pharmacol. 1995, 34, 71.
diluted in DMEM supplemented with hydrochloric acid (0.1 N final) and
incubated for 20 min at room temperature. Fluorescence (Excitation 360 nm/
Emission 425 nm) was measured after increasing pH with sodium hydroxide
(0.12 N final) on an Acquest plate reader. Cells for eNOS and nNOS were
incubated 24 h with compound and then activated by ionophore (0.8
lM final
5. Zamora, R.; Vodovotz, Y.; Billiar, T. R. Mol. Med. 2000, 6, 347.
6. Paige, J. S.; Jaffrey, S. R. Curr. Top. Med. Chem. 2007, 7, 97.
A23187 for eNOS or 30 M final ionomycin for nNOS) and incubated an
l
additional 18 h for nitrite accumulation followed by the DAN assay.
15. Brennan, T. J. Pain 2011, 152, S33.
7. (a) Alderton, W. K.; Angell, A. D. R.; Craig, C.; Dawson, J.; Garvey, E.; Moncada,
S.; Monkhouse, J.; Rees, D.; Russell, L. J.; Russell, R. J.; Schwartz, S.; Waslidge, N.;
Knowles, R. G. Br. J. Pharm. 2005, 145, 301; (b) Alba, D. A.; Clayton, N. M.;
Collins, S. D.; Colthup, P.; Chessell, I.; Knowles, R. G. Pain 2006, 120, 170.
8. Edwards, R. M.; Stack, E. J.; Trizna, W. J. Pharmacol. Exp. Ther. 1998, 285, 1019.
9. (a) LaBuda, C. J.; Koblish, M.; Tuthill, P.; Dolle, R. E.; Little, P. J. Eur. J. Pain 2006,
10, 505; (b) Tinker, A. C.; Beaton, H. G.; Boughton-Smith, N.; Cook, T. R.; Cooper,
S. L.; Fraser-Rae, L.; Hallam, K.; Hamley, P.; McInally, T.; Nicholls, D. J.; Pimm, A.
D.; Wallace, A. V. J. Med. Chem. 2003, 46, 913.
16. (a) Chaplan, S. R.; Bach, F. W.; Pogrel, J. W.; Chung, J. M.; Yaksh, T. L. J. Neurosci.
Methods 1994, 53, 55; (b) Kim, S. H.; Chung, J. M. Pain 1992, 50, 355.
17. The requirement of the imidazole nitrogen was anticipated based on the
reported role of this atom in direct coordination of the heme iron. McMillan, K.;
Adler, M.; Auld, D. S.; Baldwin, J. S.; Blasko, E.; Browne, L. J.; Chelsky, D.; Davey,
D.; Dolle, R. E.; Eagen, K. A.; Erickson, S.; Feldman, R. I.; Glaser, C. B.; Mallari, C.;
Morrissey, M. M.; Ohlmeyer, M. H. J.; Gonghua, P.; Parkinson, J. F.; Phillips, G.
B.; Polokoff, M. A.; Sigal, N. H.; Vergona, R.; Whitlow, M.; Young, T. A. PNAS
2000, 97, 1506.
10. (a) Davey, D. D.; Adler, M.; Arnaiz, D.; Eagen, K.; Erickson, S.; Guilford, W.;
Kenrick, M.; Morrissey, M. M.; Ohlmeyer, M.; Pan, G.; Paradkar, V. M.;