Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities

Article abstract of DOI:10.1016/j.bmcl.2011.07.103

The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.

Full text of DOI:10.1016/j.bmcl.2011.07.103

Bioorganic & Medicinal Chemistry Letters 21 (2011) 5849–5853
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of potent and novel S-nitrosoglutathione reductase inhibitors
devoid of cytochrome P450 activities
Xicheng Sun a, , Jian Qiu a, Sarah A. Strong a, Louis S. Green a, Jan W.F. Wasley b, Joan P. Blonder a,
⇑
Dorothy B. Colagiovanni a, Sarah C. Mutka a, Adam M. Stout a, Jane P. Richards a, Gary J. Rosenthal a
a N30 Pharmaceuticals LLC, 3122 Sterling Circle, Suite 200, Boulder, CO 80301, USA
b Simpharma LLC, 1 Stone Fence Lane, Guilford, CT 06437, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitro-
soglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the
treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates
the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of
GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases
including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endoge-
nous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We
describe here the synthesis and structure–activity relationships (SAR) of novel pyrrole based analogues of
N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel
GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbu-
min (OVA) model of asthma.
Received 8 July 2011
Revised 26 July 2011
Accepted 26 July 2011
Available online 3 August 2011
Keywords:
GSNO
GSNOR
GSNOR inhibitor
Nitric oxide
Cytochrome P450
N6022
Ó 2011 Elsevier Ltd. All rights reserved.
Endogenous S-nitrosothiols (SNOs) are important conductors of
the biological influence of nitric oxide, from bronchodilation and
vasodilation to controlling inflammation.1–3 S-Nitrosoglutathione
reductase (GSNOR), also known as the human ADH class III enzyme
and formaldehyde dehydrogenase,4,5 catalyzes the metabolism of
the most abundant low molecular weight SNO, S-nitrosoglutathione
(GSNO),6,7 and has been identified as a potential drug target for the
treatment of a broad range of diseases.8–13 The therapeutic potential
of GSNOR inhibitors has been demonstrated in animal models of
asthma,14–16 chronic obstructive pulmonary disease (COPD),17
inflammatory bowel disease (IBD)18 and high salt induced
hypertension.19
We have recently reported the identification of N6022 as a po-
tent S-nitrosoglutathione reductase inhibitor in clinical develop-
ment20,21 for the treatment of acute asthma, and extended these
observations with a report on structure–activity relationship of
GSNOR inhibitors based on a pyrrole scaffold.22 As a continuation
of the series communication of the pyrrole based GSNOR inhibi-
tors, we report here the studies to further improve the overall
pharmacological properties of GSNOR inhibitors, focusing on the
substitution of the imidazole moiety and replacement of this moi-
ety with a variety of heterocycles in an attempt to eliminate the
cytochrome P450 inhibitory activities previously reported GSNOR
inhibitors.20
Most of the GSNOR inhibitors presented here were synthesized
according to Scheme 1. The synthesis started from either commer-
cially available ketones or the ketones prepared as described in the
Supplementary data. In Scheme 1, condensation of ketone 1 and 2-
furanaldehyde provided intermediate 2 in good yield.23 Furan ring
opening of intermediate 2 by HBr in ethanol under reflux condi-
tions provided diketone 3.24 Pyrrole formation was achieved by
condensation of the diketone 3 and 4-amino-3-methylbenzamide
under acidic conditions to afford intermediate 4.25 For the synthe-
sis of compounds 6a–6s, where the Ar is phenyl, compounds 5a–5s
were synthesized either using the Suzuki coupling conditions for
the C–C connected aromatic heterocyclic analogs, or coupling of
heterocycles such as morpholine (6o) with intermediate 4 in the
presence of copper iodide (I) and potassium carbonate in DMSO
to form N–C connected compounds. The N–C connected analogs
can also be synthesized using similar conditions, but using amides
instead. Compounds 8a–8i were prepared using substituted imida-
zoles as starting materials to couple with intermediate 4 either
Abbreviations:
GSNOR,
S-nitrosoglutathione reductase;
GSNO,
using L
-proline26 or N,N-dimethyl-cyclohexane-1,2-diamine27 as a
catalyst in the presence of copper iodide(I) and potassium carbon-
ate in DMSO followed by hydrolysis of ester in aqueous lithium
S-nitrosoglutathione; NO, nitric oxide; SNOs, nitrosothiols.
⇑
Corresponding author. Tel.: +1 720 945 7706; fax: +1 303 440 8339.
E-mail address: xicheng.sun@n30pharma.com (X. Sun).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.07.103

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X. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5849–5853
O
O
O
O
X
X
a
Ar
b
c
Ar
X
Ar
EtO2C
O
3
2
1
X = Cl or Br
X
CO2Et
N
CO2H
N
CO2Et
Ar
N
R1
R1
d or e or f
g
CONH2
CONH2
CONH2
5a-5s
6a-6s
4
h or i
N
N
N
N
Ar
CO2H
CO2Et
Ar
N
N
R2
R2
g
CONH2
CONH2
7a-8i
8a-8i
Scheme 1. Synthetic route of GSNOR inhibitors. Reagents and conditions: (a) furan-2-carbaldehyde/NaOMe/MeOH, room temperature, overnight; (b) HBr/EtOH, reflux, 8 h;
(c) aniline/pTsOH/EtOH, reflux, overnight; (d) boronic acid or ester, Suzuki coupling conditions; (e) heterocycles/CuI/K2CO3/DMSO; (f) amide/CuI/K3PO4/DMEDA/dioxane; (g)
LiOH; (h) imidazole/L-proline/CuI/K2CO3/DMSO; (i) imidazole/N,N-dimethyl-cyclohexane-1,2-diamine/CuI/K2CO3/DMSO, microwave.
hydroxide. The synthesis of compounds 9–24 are described in de-
tail in the Supplementary data.
Table 1
Imidazole replacement with five-membered rings
One issue we encountered during the lead optimization of
GSNOR inhibitors was cytochrome P450 enzyme inhibition with
compounds, such as N6022, containing a nitrogen linked imidazole
moiety.20 CYP inhibitions may be a concern in the drug develop-
ment due to potential drug–drug interaction. Our initial approach
was to replace the imidazole ring with other heterocycles in an at-
tempt to avoid the CYP inhibition. A large set of five-membered
heterocyclic analogs was synthesized and the GSNOR inhibitory
activity was evaluated (Table 1). Overall, none of the new analogs
exhibited better GSNOR inhibition than N6022 although a number
of compounds (6a, 6j, and 10) achieved sub-micromolar activity.
To our surprise, the nitrogen containing analogs 12–16 exhibited
substantially decreased GSNOR inhibitory activity. The thiadiazole
analog 9 was completely devoid of enzyme inhibition activity
while the thiazole comparator 10 achieved sub-micromolar po-
tency. Substitution of imidazole at 5-position (6k) resulted in
greater than a 2-log of loss in GSNOR inhibition activity. The best
compound in the series was compound 6a having GSNOR
Compd
6a
R1
GSNOR IC50
0.51
(lM)
O
S
6b
29.32
O
6c
6.52
6d
11.81
N
S
9
>100
0.76
N
N
S
10
N
O
O
N
11
6e
53.49
50.64
O
IC50 = 0.51 lM.
HN
Next, six-membered ring heterocycles (6l–6s) were also used to
replace imidazole. However, all of these compounds exhibited
markedly decreased GSNOR inhibitory activity as shown in Table 2.
As a second approach to circumvent the CYP inhibition, we eval-
uated the crystal structure of the antifungal agent, ketoconazole,
bound to CYP 3A428 in comparison with the crystal structure of
N6022 bound to the GSNOR enzyme.20 We hypothesized that sub-
stitution at the 2-position of imidazole may hinder the binding of
imidazole to CYP 3A4 enzyme, with the assumption that the imid-
azole moiety of N6022 binds similarly to CYP 3A4. In addition,
there is space in the N6022-GSNOR crystal structure to fit small
groups. We started the SAR exploration with substitution of the
imidazole ring at 2-position by a series of lower alkyl groups (Table
3). The overall CYP activity, including activity for CYP 3A4 and
2C19, was significantly reduced with 2-methyl (8a) and 2-ethyl
(8b) substituted analogs as compared to non-substituted N6022.
N
6f
N
N
10.15
4.74
2.66
N
6g
6h
HN
N
N
N
6i
5.14
0.68
6.25
N
6j
N
6k
N
N

X. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5849–5853
5851
binding in the presence of 10
l
M of test compound. Typical bind-
Table 1 (continued)
ing assays were performed with a minimum of 6-control wells
with/without vehicle for soluble compounds. Inhibition of 50% or
greater was considered a positive response. Limited off-target
activity was observed towards the d2 opiate receptor for com-
pound 8d, 8f, and 8h similar to N6022 as reported earlier.20 Com-
pound 8h demonstrated potent GSNOR inhibition and a clean
profile toward CYP inhibition, but exhibited 69% inhibition against
the opioid peptide receptor (Mu). Compounds 8d, 8f, and 8h were
also screened for cytotoxicity towards the A549 epithelial lung cell
Compd
R1
GSNOR IC50
4.16
(lM)
N
N
12
N
N
N
13
14
13.27
1.48
N
N
N
N
N
N
N
line. The IC50 values for 8f and 8h were >250
lM, and minimal
15
16
1.75
5.19
N
H
N
cytotoxicity for 8d (IC50 = 160 M) was observed.
l
Compounds 8d, 8f, and 8h were tested in mouse pharmacoki-
netic studies. Oral bioavailability of these compounds was 0.7%,
1.6%, and 0.49%, respectively, compared to 4.4% for N6022 reported
earlier.20 The plasma clearance (CL) after intravenous (IV) adminis-
tration was 54.1, 31.6, and 16.8 ml/min/kg for 8d, 8f, and 8h,
respectively, compared to 37.7 ml/min/kg for N6022.20
N
N
N
Compound 8d was tested in a 5-day mouse toxicity study with
intravenous QD dosing at 1, 10, or 50 mg/kg. The results of this
study suggest that treatment of male CD-1 mice with 8d for 5 days
had no adverse effects. This study resulted in a no observable ad-
verse effect level (NOAEL) of 50 mg/kg for IV treatment. Compound
8f was also tested in a 5-day mouse toxicity study with IV BID dos-
ing at 5, 25, or 50 mg/kg. To our surprise, the treatment of male CD-
1 mice with 8f for 5 days resulted in significant adverse effects on
numerous study endpoints. In particular, histological findings
demonstrated toxicity to the liver, spleen, and thymus of treated
animals. The NOAEL for 8f could not be established from the study
and was considered to be <10 mg/kg/day.
Table 2
Imidazole replacement with six-membered rings
Compd
R1
GSNOR IC50
9.8
(lM)
N
6l
N
6m
6n
23.92
19.03
H
N
O
The efficacy of GSNOR inhibitors was assessed in an animal
model of asthma, a disease influenced by dysregulated GSNOR
and altered function of NO, GSNO, and SNOs. In a mouse model
of ovalbumin-induced asthma,29 compound 8d attenuated metha-
choline-induced bronchoconstriction (airway hyper-responsive-
ness) and eosinophil infiltration into the lungs following a single
IV dose administered 24 h prior to the methacholine challenge.
Efficacy was observed at doses P0.01 mg/kg compound 8d. In a
similar study, compound 8f also attenuated methacholine-induced
airway hyper-responsiveness and eosinophil infiltration into the
lungs following a single IV dose of 1 mg/kg administered 24 h prior
to the methacholine challenge.
O
6o
6p
6q
6s
8.17
43.94
8.78
6.16
N
HN
N
N
In conclusion, based on the crystal structure of GSNOR inhibi-
tors and crystal structure of ketoconazole with CYP 3A4, substitu-
tion of the imidazole at 2-position with small alkyl group and
replacement of the phenyl ring with thiophene led to potent
GSNOR inhibitors that demonstrated significantly reduced CYP
inhibition. These findings validate the initial hypothesis of the
imidazole as a key pharmacophore for the binding of GSNOR inhib-
itors to cytochrome enzymes causing CYP inhibition. However, de-
spite the improved in vitro profile of GSNOR inhibitor 8f, this
compound exhibited a less attractive pharmacological profile than
N6022 due to the surprise in vivo toxicity observed in the 5-day
exploratory toxicity evaluation in mice. The studies described in
this paper provided more insight into the understanding of GSNOR
inhibition, the inhibitor–enzyme interaction, and structure–in vi-
tro toxicity relationship of GSNOR inhibitors.
However, the GSNOR inhibition activity was also reduced by sub-
stitution, particularly with a larger group such as cyclopropyl
(17) and isopropyl (8c). Hydroxymethyl (18) and trifluoromethyl
(19) substitution at 2-postion of imidazole diminished the GSNOR
inhibitory activity.
In order to maintain or improve the GSNOR inhibition activity
and remove CYP inhibition, the phenyl ring connecting to the
imidazole was replaced with a variety of heterocycles (Table 3).
Replacement of phenyl ring on N6022 with thiophene (8d, 8e)
maintained the GSNOR inhibition activity, but the CYP inhibition
issue remained (8d). Substitution at 2-position of the imidazole
with a methyl group (8f) not only maintained the GSNOR inhibi-
tion activity, also reduced the CYP inhibition to <30% as compared
to >50% inhibition for N6022 (Table 3). The thiophene regioisom-
er of 8f (8h) also achieved a similar result. Heterocyclic analogs
other than thiophene, such as furan (20), thiazole (21), pyridines
(22, 23) and pyrimidine (24) lost significant GSNOR inhibitory
activity.
Acknowledgments
The authors are grateful to Dr. J. Singh and his team at deCODE
chemistry for the synthetic chemistry support. We also thank Dr.
Zhaojun Zhang and his colleagues at Chempartner, Shanghai for
the synthesis of some of the GSNOR inhibitors and Dr. Larry Ross
at SRI for screening GSNOR inhibitors in the enzyme assay.
Selected GSNOR inhibitors were assessed for potential off-target
activity with a panel of 54 transmembrane and soluble receptors,
ion channels, and monoamine transporters. Off-target effects were
estimated from the percent inhibition of receptor radio-ligand

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X. Sun et al. / Bioorg. Med. Chem. Lett. 21 (2011) 5849–5853
Table 3
SAR and cytochrome P450 activities of imidazole analogs
Compd
R2
Ar
GSNOR IC50
(l
M)
% Of CYP inhibition at 10
2C19
lM
1A2
85
2C9
81
2D6
89
3A4
60
N6022
8a
H
0.020
0.075
0.13
95a
55
25
—
Me
Et
11
6
20
15
—
11
20
—
31
27
—
8b
17
cPr
6.96
—
—
—
—
8c
iPr
Inactive
2.31
—
—
—
—
18
HOCH2
CF3
—
—
—
—
19
Inactive
—
—
—
—
8d
8e
20
H
H
H
0.022
0.026
0.90
77
—
92
—
83
—
96
—
91
—
S
S
—
—
—
—
—
O
N
21
22
23
H
H
H
1.57
0.36
2.30
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
S
N
N
N
24
8f
H
0.15
—
—
—
—
—
N
S
Me
0.017
À8
15
25
10
27
8g
8h
Et
0.048
0.021
0.048
—
—
—
—
—
S
Me
Me
À11
—
13
—
11
—
12
—
18
—
S
S
8i
a
IC50 = 0.77 l
M.20
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