Discovery of 5-(2-chloro-4′-(1H-imidazol-1-yl)-[1,1′-biphenyl]-4-yl)-1H-tetrazole as potent and orally efficacious S-nitrosoglutathione reductase (GSNOR) inhibitors for the potential treatment of COPD

Article abstract of DOI:10.1016/j.bmcl.2018.10.012

Endogenous nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO) serve as reservoir for bioavailable nitric oxide (NO) and mediate NO-based signaling, inflammatory status and smooth muscle function in the lung. GSNOR inhibition increases pulmonary GS

Full text of DOI:10.1016/j.bmcl.2018.10.012

Accepted Manuscript
Discovery of 5-(2-Chloro-4'-(1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-1H-tet-
razole as potent and orally efficacious S-Nitrosoglutathione reductase (GSNOR)
inhibitors for the potential treatment of COPD
Nagarajan Muthukaman, Sanjay Deshmukh, Shital Tondlekar, Macchindra
Tambe, Dnyandeo Pisal, Neelam Sarode, Siddharth Mhatre, Samitabh
Chakraborti, Daisy Shah, Vikram M. Bhosale, Abhay Kulkarni, Mahamad
Yunnus A. Mahat, Satyawan B. Jadhav, Girish S. Gudi, Neelima Khairatkar-
Joshi, Laxmikant A. Gharat
PII:
S0960-894X(18)30802-3
https://doi.org/10.1016/j.bmcl.2018.10.012
BMCL 26071
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 July 2018
28 September 2018
10 October 2018
Please cite this article as: Muthukaman, N., Deshmukh, S., Tondlekar, S., Tambe, M., Pisal, D., Sarode, N., Mhatre,
S., Chakraborti, S., Shah, D., Bhosale, V.M., Kulkarni, A., Mahat, M.Y.A., Jadhav, S.B., Gudi, G.S., Khairatkar-
Joshi, N., Gharat, L.A., Discovery of 5-(2-Chloro-4'-(1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-1H-tetrazole as
potent and orally efficacious S-Nitrosoglutathione reductase (GSNOR) inhibitors for the potential treatment of
COPD, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.012
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Discovery of 5-(2-Chloro-4'-(1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-1H-
tetrazole as potent and orally efficacious S-Nitrosoglutathione reductase
(GSNOR) inhibitors for the potential treatment of COPD
Nagarajan Muthukamana, Sanjay Deshmukha, Shital Tondlekara, Macchindra Tambea, Dnyandeo
Pisala, Neelam Sarodea, Siddharth Mhatrea, Samitabh Chakrabortib, Daisy Shahb, Vikram M.
Bhosaleb, Abhay Kulkarnib, Mahamad Yunnus A. Mahatc, Satyawan B. Jadhavc, Girish S. Gudic,
Neelima Khairatkar-Joshib, Laxmikant A. Gharata,*
aChemical Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra
400709, India
bBiological Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra
400709, India
cDrug Metabolism and Pharmacokinetics, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi
Mumbai, Maharashtra 400709, India
Corresponding author. Tel.: +91 022 6772 0000x3208.
E-mail address: Laxmikant.gharat@glenmarkpharma.com (L.A. Gharat).
Abstracts:
Endogenous nitrosothiols (SNOs) including S-nitrosoglutathione (GSNO) serve as
reservoir for bioavailable nitric oxide (NO) and mediate NO-based signaling, inflammatory status
and smooth muscle function in the lung. GSNOR inhibition increases pulmonary GSNO and
induces bronchodilation while reducing inflammation in lung diseases. In this letter, design,
synthesis and structure–activity relationships (SAR) of novel imidazole-biaryl-tetrazole based
GSNOR inhibitors are described. Many potent inhibitors (30, 39, 41, 42, 44, 45 and 58) were
identified with low nanomolar activity (IC50s: < 15 nM) along with adequate metabolic stability.
Lead compounds 30 and 58 exhibited good exposure and oral bioavailability in mouse
pharmacokinetic (PK) study. Compound 30 was selected for further profiling and revealed
comparable mouse and rat GSNOR potency, high selectivity against alcohol dehydrogenase
(ADH) and carbonyl reductase (CBR1) family of enzymes, low efflux ratio and permeability in
PAMPA, a high permeability in CALU-3 assay, significantly low hERG activity and minimal off-
target activity. Further, an in vivo efficacy of compound 30 is disclosed in cigarette smoke (CS)
induced mouse model for COPD.
Key Words: Bronchodilation; S-nitrosoglutathione (GSNO); glutathione (GSH); GSNOR
inhibitor; reductase; bioavailability; cigarette smoke (CS).
S-Nitrosoglutathione reductase (GSNOR) also identified as glutathione-dependent
formaldehyde dehydrogenase (FDH), is a zinc-dependent dehydrogenase.1 It is a member of the

alcohol dehydrogenase (ADH) family and is called class III alcohol dehydrogenase that regulates
the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO).1 The
importance of GSNOR in controlling the levels of both intracellular GSNO and nitrosated proteins
was established in knockout models of yeast and the nomenclature “GSNO reductase” was
introduced to reflect this important physiological role.1b GSNOR enzyme that catalyzes the
reduction of S-nitrosoglutathione has been recognized as a potential therapeutic target and it
products GSNO and SNOs are implicated in many diseases2 including respiratory,3 cardiovascular
(pulmonary hypertension, thrombosis and altered platelet function),4 gastrointestinal systems
(inflammatory bowel diseases),5 cerebral ischemia and compromised host defense.6 GSNOR is a
highly conserved enzyme and can be found in both prokaryotic and eukaryotic organisms.
Although GSNOR is expressed in all tissues, its activity levels are highest in the liver followed by
kidney, heart, lung, spleen and thymus.7.
Cellular GSNO is one of the major source of biologically stable nitric oxide (NO), thus
forms adducts with cysteine containing peptides and proteins.8 Therefore, inhibition of GSNOR
prevents GSNO metabolism and allows accumulation of GSNO, which leads to protein S-
nitrosylation. It is also thought to influence the levels of NO and nitrite (NO2-) that form via
reactions of GSNO with glutathione (GSH) or various reactive oxygen or nitrogen species.9 The
role of GSNOR in NO metabolism is further established by studies in GSNOR knockout mice.7,10
Moreover, an increase of GSNO in an OVA-sensitized and OVA-challenged mice ameliorated
airway hyper reactivity (AHR).11 Overall, these studies suggest that a therapeutic approach in
which airway GSNO levels are increased by treatment with GSNOR inhibitors could provide a
novel therapeutic approach for reducing allergic inflammation, AHR in asthma, COPD and other
related lung diseases. The therapeutic utility of GSNOR inhibitors were well established in animal
models of asthma12 and COPD,3c which provided a rational for inhibiting GSNOR as a drug target
for respiratory indication
O
COOH
Cl
COOH
H2N
COOH
O
O
N
N
HO
CF3
HO
2, N91115 (Nivalis Pharma)
Ph-II for Cystic Fibrosis
h IC50 = 16 nM
3, (Nivalis Pharma)
h IC50 = 36.8 nM
N
1, N6022 (Nivalis Pharma)
Ph-I for Asthma
h IC50 = 20 nM
N
Figure 1. Representative GSNOR inhibitors.

Removed benzene ring
to get novelty
A N
COOH
B
C
Cl
COOH
N
N
4
5
Introduced
bioisostere
HO
2
N
N
N
A
C
B
D
N
N
N
Introduced 5-member
heterocycle to get novelty
H
Figure 2. Scaffold hopping of 2 led to novel GSNOR inhibitors 4 and 5.
The campaign to identify a potent and selective GSNOR inhibitor attained a significant
milestone with the discovery of N6022 (1) and N91115 (2).13-17 N6022 is a potent and selective
GSNOR inhibitor (human GSNOR IC50: 20 nM) and has > 1000-fold selective over other human
ADH enzymes. N6022 did not inhibit hERG channel and was negative in Ames, but it has poor
oral bioavailability (%F: < 10%) and significant CYP liability.13,14 N6022 has completed Ph-I
clinical trial as i.v agent to treat acute asthma.15 N91115 (2) is also a potent GSNOR inhibitor
(GSNOR hIC50: 16 nM) which has good oral bioavailability and has completed Ph-II clinical trials
recently for cystic fibrosis.16 Likewise, another GSNOR inhibitor 3 is also disclosed by Nivalis
Pharma having human GSNOR potency of 37 nM IC50, respectively. As part of our on-going drug
discovery program in the respiratory indication, we identified N91115 (2)16c as a potential starting
point to design novel GSNOR inhibitor. Applying scaffold hopping strategy led to novel GSNOR
inhibitor 4 which was expected to retain GSNOR potency similar to 2 as exemplified in Figure 2.
The bioisosteric replacement of the carboxylic acid in compound 4 with tetrazole led to compound
5, which was projected to offer enhanced GSNOR potency and favorable pharmacokinetic
properties.17
In this letter, we describe the synthesis and pharmacological activity of 4'-(1H-imidazol-1-
yl)-[1,1'-biphenyl]-4-carboxylic acid derivatives 4 and 5-(4'-(1H-imidazol-1-yl)-[1,1'-biphenyl]-4-
yl)-1H-tetrazole derivatives 5 as depicted in Schemes 1-3 and Tables 1-3. Commercially available
4'-bromo-[1,1'-biphenyl]-4-carbonitrile (6) was reacted with imidazole in the presence of CuI to
afford compound 7.18 The nitrile in compound 7 was hydrolyzed independently to amide 8 and
carboxylic acid 4 as shown in Scheme 1. Further, the biaryl nitrile 7 was converted to tetrazole 5
using NaN3 in DMF at 100 °C.19 Next, the reaction of 7 with hydroxylamine hydrochloride to
afford biphenyl carboximidamide 9, followed by CDI reaction in THF at 70 °C afforded 1,2,4-
oxadiazol-5(2H)-one derivative 10 in 55% yield.19 Further, compounds 12 and 13 were prepared
by reacting commercially available 1,1'-biphenyl-4,4'-dicarbonitrile 11 with one equivalent of

NaN3 and as well as excess NaN3 in the presence of Et3NHCl in DMF to afford in 30% and 70%
yield (Scheme 1). Compounds 18 and 21 shown in Table 1 were prepared as described in Scheme
2. Commercially available 4-amino-3-chlorobenzonitrile 14c was subjected to Sandmeyer reaction
to afford iodo compound 15c, followed by Suzuki coupling with 4-bromophenylboronic acid and
4-aminophenylboronic acid independently to afford compounds 16c and 19, respectively.
Compound 16c was reacted with 1,2,4-triazole in the presence of CuI and K2CO3 to provide
compound 17, followed by tetrazole formation using NaN3 to give compound 18 in 60% yield.
Similarly, N-tetrazole compound 21 was prepared by reaction of 19 with NaN3 and trimethyl
orthoformate in acetic acid,20 followed by tetrazole formation afforded N- and C-linked tetrazole
in 55% yield.
O
NH
N
a
e
N
N
N
f
Br
CN
N
N
X
N
O
NHOH
N
H
6
9
10
7:
8:
4:
5:
X = CN
b
X = CONH2
X = COOH
X = C-tetrazole
c
d
N
N
N
N
N
g
d
N
N
N
NC
NC
CN
N
N
H
N
H
N
H
12
11
13
Scheme 1. Reagents and conditions: (a) CuI (0.5 eqv.), K2CO3 (2.4 eqv.), imidazole, 1,10-phenanthroline (0.5 eqv.),
100 °C, 12 h, 70%; (b) K2CO3 (0.7 eqv), 50% H2O2, DMSO, rt, 3 h, 60%; (c) NaOH, EtOH-water (4:1), 80 °C, 70%;
(d) NaN3 (6 eqv.), Et3NHCl (6 eqv.), DMF, 80-90 °C; 10 h, 80%; (e) NH2OH.HCl (2 eqv.), Et3N, EtOH, 80 °C, 4 h;
(f) CDI, Et3N, THF, 70 °C, 6 h, 55%; (g) NaN3 (1.1 eqv.), Et3NHCl (2 eqv.), DMF, 70 °C, 15 h, 30%.
Table 1
SAR on biphenyl scaffold
R1
R2
R3
Compd R1
R2
R3 GSNOR IC50 in nMa
or % inhi. @10 µMb
4a
4
OH
COOH
COOH
H
H
25%
N
62 nM
N
N
N
N
N
N
N
N
N
5
7
8
H
H
H
22 nM
10%
NH
N
CN
CONH2
60%

H
N
a N6022 (1) was used as a positive control in
this experiment and in-house IC50 of N6022
(1) is 20 nM. For the
N
10
12
H
H
152 nM
27%
N
O
N
O
N
NC-
N
literature reported potency of N6022 (1), see
Ref. 13d.
NH
N
N
b IC50 values are derived from graphs plotted
with data from a minimum of two experiment
in duplicates.
N
N
13
H
3279 nM
N
NH
N
N
N
b % Inhibition values are means of two
experiments in duplicates.
H
N
N
N
18
21
Cl
Cl
593 nM
36%
Initially,
the
synthesized
N
N
N
NH
N
N
compounds from Scheme 1 were
evaluated for human GSNOR potency
before generating additional SAR in
N
N
N
N
NH
N
order to understand scaffold feasibility and required functionality as illustrated in Table 1.
Commercially available 4'-hydroxy-[1,1'-biphenyl]-4-carboxylic acid 4a having similar terminal
functional groups as in N91115 (2) was tested for human GSNOR potency, which unveiled low
GSNOR inhibition at 10 µM concentration.21 Next, imidazole derived carboxylic acid analog 4
designed based on scaffold hopping strategy, afforded IC50 of 62 nM GSNOR potency which is
roughly 4-fold lesser in comparison to clinical candidate N91115 (2). Encouraged by this result,
compound 5 having tetrazole as a bioisostere replacement of carboxylic acid analog 4 provided 3-
fold enhanced GSNOR potency (IC50: 22 nM) and is comparable to N91115 (2). Tetrazole in
compound 5 was bioisosterically replaced with other groups, such as CN (7), CONH2 (8) and 1,2,4-
oxadiazol-5(2H)-one (10) derived analogs unveiled substantial loss of potency. Similarly,
bioisosteric replacement of imidazole in compound 5 with CN (12), C-tetrazole (13), 1,3,4-triazole
(18) and N-tetrazole (21) derived analogs displayed inferior GSNOR potency (Table 1).21

: R1 = F; R3 = H
: R1 = Me; R3 = H
: R1 = Cl; R3 = H
: R1 = CF3; R3 = H
R3
N
23
24
25
26
O
N
OH
R1
d
R3
N
R3
NH2
R1
I
N
a
b
c
N
e
N
N
N
CN
N
Br
CN
B(OH)2
N
NC
NC
R1
H
R1
R1
R1
: R1 = H
14
: R1 = H
15
: R1 = H; R3 = Me
: R1 = H
: R1 = F
: R1 = Me
: R1 = Cl
: R1 = CF3
27
: R1 = H; R3 = Me
: R1 = F; R3 = H
: R1 = Me; R3 = H
: R1 = Cl; R3 = H
: R1 = Cl; R3 = Me
: R1 = CF3; R3 = H
22
16
: R1 = F
: R1 = Me
: R1 = Cl
15a
15b
15c
15d
: R1 = F
: R1 = Me
: R1 = Cl
: R1 = F; R3 = H
: R1 = Me; R3 = H
: R1 = Cl; R3 = H
: R1 = Cl; R3 = Me
: R1 = CF3; R3 = H
Br
14a
14b
14c
14d
22a
22b
22c
22d
22e
16a
16b
16c
16d
28
29
30
31
32
: R1 = CF3
: R1 = CF3
f
NH2
b
N
N
N
N
e
N
N
N
N
CN
N
N
B(OH)2
H
Cl
Cl
17
18
N
g
N
N
N
N
e
N
N
H2N
CN
N
N
CN
N
N
H
N
Cl
Cl
21
Cl
20
19
R3
N
R2
O
1 = H; R2 = Cl; R3 = H
N
38:
39
R
: R1 = H; R2 = Me; R3 = H
OH
R1
d
R3
N
R3
R2
R2
R2
NH2
R2
I
N
N
e
N
N
a
b
c
CN
N
Br
CN
N
N
B(OH)2
Br
Br
R2
H
R1
R1
1 = H; R2 = Cl; R3 = H
R1
R1
R
1 = H; R2 = Cl
36:
: R2 = Cl
33
: R2 = Cl
40:
41
R
1 = H; R2 = Cl; R3 = H
34
37:
R
: R1 = H; R2 = Me; R3 = H
: R1 = H; R2 = Et; R3 = H
: R1 = H; R2 = Et; R3 = Me
: R1 = Cl; R2 = Me; R3 = H
: R1 = F; R2 = Et; R3 = H
: R2 = Me
: R2 = Et
: R2 = Me
: R2 = Et
: R1 = H; R2 = Me
: R1 = H; R2 = Et
: R1 = Cl; R2 = Me
: R1 = F; R2 = Et
CN
: R1 = H; R2 = Me; R3 = H
: R1 = H; R2 = Et; R3 = H
: R1 = H; R2 = Et; R3 = Me
: R1 = Cl; R2 = Me; R3 = H
: R1 = F; R2 = Et; R3 = H
34a
34b
36a
36b
36c
36d
33a
33b
37a
37b
37c
37d
37e
: R1 = H
35
42
43
44
45
: R1 = Cl
: R1 = F
35a
35b
Scheme 2. Reagents and conditions: (a) PTSA (2 eqv.), NaNO2 (1.5 eqv.), KI (1.5 eqv.), CH3CN, 0 °C to rt, 2 h, 60-
80%; (b) Pd(PPh3)4 (50 mol%), Na2CO3 (2 eqv.), THF or 1,4-dioxane, 90-100 °C, 10-14 h, 50-70%; (c) CuI (0.5 eqv.),
K2CO3 (2.4 eqv.), imidazole and or 2-methylimidazole, 1,10-phenanthroline (0.5 eqv.), 100 °C, 12 h, 50-70%; (d)
NaOH, EtOH-water (4:1), 80 °C, 60-70%; (e) NaN3 (6 eqv.), Et3NHCl (6 eqv.), DMF, 80-90 °C; 10-14 h, 60-80%; (f)
CuI (0.5 eqv.), K2CO3 (2.4 eqv.), 1,2,4-triazole, 1,10-phenanthroline (0.5 eqv.), 90 °C, 12 h, 60%; (g) NaN3,
(Et3O)3CH, AcOH, 100 °C, 12 h, 70%.

R2
2 = H; R1 = H
49:
50
R
O
: R2 = Cl; R1 = H
: R2 = Me; R1 = H
: R2 = Me; R1 = F
N
N
51
52
N
OH
R1
d
c
R2
R2
R2
R2
N
N
N
N
N
a
b
N
Br
CN
N
CN
Br
Br
(HO)2B
N
H
N
N
N
N
CN
R1
R1
2 = H; R1 = H
R1
2 = H; R1 = H
R1
: R2 = H
46
47:
R
48:
R
53:
R
2 = H; R1 = H
: R2 = Cl
: R1 = H
35
: R2 = H; R1 = Me
: R2 = H; R1 = Cl
: R2 = Cl; R1 = H
: R2 = Cl; R1 = F
: R2 = Cl; R1 = Me
: R2 = Me; R1 = H
: R2 = Me; R1 = F
: R2 = NO2; R1 = H
: R2 = H; R1 = Me
: R2 = H; R1 = Cl
: R2 = H; R1 = Me
: R2 = H; R1 = Cl
: R2 = Cl; R1 = H
: R2 = Me; R1 = H
: R2 = Me; R1 = F
: R2 = Cl; R1 = F
: R2 = Cl; R1 = Me
46a
46b
46c
47a
47b
47c
47d
47e
47f
48a
54
55
56
57
58
59
60
: R2 = Me
35a
: R1 = Cl
: R1 = F
: R1 = Me
48b
: R2 = NO2 35b
48c: R2 = Cl; R1 = H
: R2 = Cl; R1 = F
: R2 = Cl; R1 = Me
: R2 = Me; R1 = H
: R2 = Me; R1 = F
: R2 = NO2; R1 = H
: R2 = NO2; R1 = F
35c
48d
48e
48f
47g
48g
47h
47i
48h
: R2 = NO2; R1 = F
48i
e
I
NH2
g
N
N
N
N
N
h
f
N
CN
N
CN
CN
N
CN
N
N
N
N
R1
R1
R1
R1
1 = H
61a
64:
63:
R
1 = H
: R1 = F
R
1 = H
: R1 = F
1 = H
62:
R
61:
R
: R1 = F
: R1 = F
64a
63a
62a
B(OH)2
a
d
N
N
N
d
N
N
N
N
N
N
CN
N
N
N
N
H
N
N
N
N
1 = H
: R1 = F
H
R1
68
67
65:
66
R
Scheme 3. Reagents and conditions: (a) Pd(PPh3)4 (50 mol%), Na2CO3 (2 eqv.), THF and or 1,4-dioxane, 90-100 °C,
8-14 h, 40-60%; (b) CuI (0.5 eqv.), K2CO3 (2.4 eqv.), imidazole, 1,10-phenanthroline (0.5 eqv.), 100 °C, 12 h, 40-
70%; (c) NaOH, EtOH-water (4:1), 80 °C, 60-70%; (d) NaN3 (6 eqv.), Et3NHCl (6 eqv.), DMF, 80-90 °C; 10-14 h,
60-80%; (e) Fe/HCl. methanol, rt, 2h, 60%; (f) PTSA (2 eqv.), NaNO2 (1.5 eqv.), KI (1.5 eqv.), CH3CN, 0 °C to rt,
2h, 60-70%; (g) (i) Ethynyltrimethylsilane (1.5 eqv.), CuI (20 mol %), TEA (2 eqv.), DMSO, rt, 6h, 60%; (ii) TBAF,
45 °C, 6h, 70%; (h) Pd-C/H2, pyridine (cat.), ethanol, rt, 16 h.
Having identified optimum terminal A-ring (imidazole) and D-ring (tetrazole and or
carboxylic acid) in the scaffold, additional compounds were synthesized by introducing various
substituents and an insertion of nitrogen in B-, and C-rings with the purpose of improving GSNOR
potency and physicochemical properties. Therefore, compounds 23-32, 38-45, 49-60, 65-66 and
68 were synthesized and tested for GSNOR potency as shown in Scheme 2, 3 and Tables 2, 3.
Commercially available and meta-substituted 4-aminobenzonitriles 14 and 14a-d were subjected
to diazotization in the presence of NaNO2 and KI (Sandmeyer reaction) to afford 4-
iodobenzonitrile derivatives 15 and 15a-d, and further Suzuki coupling with 4-
bromophenylboronic acid provided biphenyl derivatives 16 and 16a-d in 50-70% yield. Copper

mediated coupling of 16 and 16a-d with imidazole and 2-methylimidazole yielded imidazole
derived biphenyls 22 and 22a-e in 30-60% yield.18 Nitrile in compounds 22a-c and 22e were
hydrolyzed using aqueous sodium hydroxide to afford biphenylcarboxylic acid derivatives 23-26
in 60-80% yield. Further, nitrile compounds 22 and 22a-e were converted to tetrazole derivatives
27-32 using NaN3 in DMF at 100 °C (Scheme 2).19 Similarly, ortho-substituted 4-bromoanilines
33 and 33a-b were exposed to diazotization with NaNO2/KI gave iodo derivatives 34 and 34a-b,
which was further subjected to Suzuki coupling with arylboronic acids 35, 35a-b to afford biphenyl
derivatives 36 and 36a-d in 40-70% yield. Compounds 36 and 36a-d were reacted with imidazole
and 2-methylimidazole in the presence of CuI to afford 37 and 37a-e, followed by tetrazole
formation using NaN3 to give biphenyltetrazole derivatives 40-45 in >50% yield. Besides,
biphenylcarboxylic acids 38 and 39 were prepared from the corresponding nitriles 37 and 37a
using aqueous sodium hydroxide in 70-80% yield (Scheme 2).
Subsequently, we also explored the synthesis of imidazole-biaryl-tetrazole derivatives
having nitrogen incorporated in the B-ring in order to further improve potency and
physicochemical properties as described in Scheme 3. Commercially available 3-substituted 2,5-
dibromopyridines 46 and 46a-b were coupled with various arylboronic acids 35 and 35a-c in the
presence of Pd(PPh3)4 to afford 2-phenylpyridine derivatives 47 and 47a-i in 40-70% yield. Copper
catalyzed imidazole coupling of 47 and 47a-i afforded compounds 48 and 48a-i in 40-70% yield.
Compounds 48 and 48a-g were further converted to tetrazole derivatives 53-60 using
NaN3/Et3NHCl in DMF to provide 60-80% yield. The carboxylic acid derivatives 49-52 were
synthesized from the corresponding nitrile derivatives 48, 48c and 48f-g using aqueous sodium
hydroxide in ethanol at 80 °C. Furthermore, nitro reduction of 48h-i gave amines 61 and 61a,
followed by diazotization provided iodo-derivatives 62 and 62a in > 50% yield over two steps.
Sonogashira coupling of 62 and 62a with ethynyltrimethylsilane, followed by TMS-cleavage to
afford 63 and 63a and further hydrogenation with Pd/C-H2 afforded compounds 64 and 64a.
Reaction of nitrile derivatives 64 and 64a with NaN3/Et3N-HCl in DMF yielded tetrazole analogs
65 and 66 in 60% yield. Compound 67 was prepared by Suzuki coupling of iodo derivative 62
with cyclopropylboronic acid, followed by tetrazole formation with NaN3 afforded compound 68
in 65% yield.
Table 2
SAR on A, B and C-ring modification

R2
O
N
N
A
B
C
X
OH
R1
R2
a N6022 (1) was used as a positive control in this experiment and in-
house IC50 of N6022 (1) is 20 nM. For the
literature reported potency of N6022 (1), see Ref. 13d.
b IC50 values are derived from graphs plotted with data from a
minimum of two experiment in duplicates.
Having identified initial leads 4 and 5 (Table 1),
further lead optimization was initiated on B- and C-rings
to afford compounds with improved potency. Initial set of
Compd R1
X
GSNOR
IC50 (nM)b
23
24
25
26
38
39
49
50
51
52
F
H
H
H
H
Cl
Me
H
Cl
C
C
C
C
C
C
N
N
N
N
29
29
30
38
59
20
214
89
41
36
Me
Cl
CF3
H
H
H
H
H
F
substituted
imidazole-biphenyl-carboxylic
acid
Me
Me
derivatives 23-26 and 39 (Table 2) were evaluated for
GSNOR potency.21 Among this, fluoro, methyl, chloro
and CF3 substituted C-ring carboxylic acid analogs 23-26 revealed only marginal improvement in
GSNOR potency (IC50: 29-38 nM range), whereas compound 39 (IC50: 20 nM) having methyl
substituent in the B-ring unveiled 3-fold improvement in potency compared to initial lead 4 (IC50:
62 nM). Next, among the substituted pyridine derived biaryl carboxylic acids 49-52 assessed for
GSNOR potency, only methyl substituted pyridine biaryl compounds 51 and 52 exhibited GSNOR
potency comparable to non-pyridine derivatives 23-26, whereas other pyridine derived analogs 49
and 50 showed substantial loss of potency. Though few GSNOR inhibitors with acid functionality
(Table 2) were obtained in comparable GSNOR potency to N6022 (1) and N91115 (2), these
compounds were not considered for further development as oral drug, due to potential formation
of acyl glucuronide through phase-II metabolism.22, 23
Table 3
SAR on A-, B- and C-ring modification
R3
R2
N
N
N
N
N
A
B
D
C
N
X
H
R1
R2 R3
Compd R1
X
GSNOR
IC50 (nM)b
27
28
29
30
31
H
F
Me
Cl
Cl
H
H
H
H
H
Me
H
H
H
C
C
C
C
C
97
20
26
9.2
37
Me

a N6022 (1) was used as a positive control in this
experiment and in-house IC50 of N6022 (1) is 20 nM. For
the
literature reported potency of N6022 (1), see Ref. 13d.
b IC50 values are derived from graphs plotted with data from
a minimum of two experiment in duplicates.
Hence, the focus was shifted to various
tetrazole analogs 27-32, 40-45, 53-60, 65-66 and
68, which are bioisosteric replacement of
carboxylic acid in which expected to offer
enhanced potency and improved pharmacokinetic
(PK) properties (Table 3). Though, initial tetrazole
lead 5 (IC50: 22 nM) has comparable GSNOR
potency to clinical candidates [N6022 (1) and
N91115 (2)], it had high inhibition against major
32
40
41
42
43
44
45
53
54
55
56
57
58
59
60
65
66
68
CF3
H
H
H
H
Cl
F
H
Me
Cl
H
H
F
F
Me
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
C
C
C
C
C
C
C
N
N
N
N
N
N
N
N
N
N
N
39
22
14
6.1
24
12
11
32
44
308
20
17
14
27
935
66
62
74
Cl
Me
Et
Et
Me
Et
H
H
H
Cl
Me
Me
Cl
Cl
Et
F
H
Et
Cy
CYP isoforms tested (1A2, 2D6, 3A4, 2C9 and 2C19) and low liver microsomal stability in mice
(~ 30% remaining after 1h incubation).21 Therefore, it was presumed that diverse substitution and
nitrogen insertion on the biaryl ring would probably deliver enhanced potency, reduced CYP
liability, adequate metabolic stability and an improved physicochemical properties. In order to
address CYP liability on compound 5, 2-methylimidazole as A-ring was introduced and that led to
compound 27 (IC50: 97 nM), which revealed 5-fold lower GSNOR potency in comparison to
compound 5. However, the CYP inhibition of compound 27 was significantly low (< 33% @ 10µM
to major CYP isoforms tested, Table 4) in addition to high metabolic stability in human, rat and
mouse liver microsomes. Next, we focused our attention on the B- and C-ring modification of
compound 5 to afford improved GSNOR potency. Among the many C-ring substituted compounds
(28-32) tested for GSNOR potency, ortho-substituted chloro biphenyl derivative 30 (IC50: 9.2 nM)
provided improvement in potency compared to initial lead 5. On the other hand, bulky CF3-
substituted C-ring compound 32 (IC50: 39 nM) gave one-fold lower GSNOR potency and all other
compounds exhibited marginally lower potency (Table 3) compared to compound 5. Further,
substitution on B- and B-C ring yielded compounds 40-45 revealed enhanced GSNOR potency
compared to compound 5. Particularly, ethyl substituted B-ring compound 42 (IC50: 6.1 nM)
showed greater than 3-fold higher potency and its corresponding 2-methylimidazole A-ring analog
43 (IC50: 24 nM) exhibited comparable potency to lead 5 and noticeably reduced potency than 42.
The lower potency of 2-methylimidazole derived A-ring analogs 27, 31 and 43 further confirmed

that only imidazole is well tolerated in the A-ring to hold onto GSNOR potency (Table 1).
Compounds 44 and 45 (IC50s: 12 nM and 11 nM) having substitution on both B- and C-rings
afforded enhanced potency than compound 5 and as comparable to compound 30 (IC50: 9.2 nM),
respectively.
Next set of compounds 53-60, 68-69 and 71 having nitrogen incorporated in the B-ring
along with various substituents either in B-ring or C-ring and or B-C ring were evaluated for
GSNOR potency.21 Compounds 53-55 having nitrogen in the B-ring and methyl, chloro-
substituents in C-ring exhibited decreased enzyme potency (Table 3). Specifically, compound 55
(IC50: 308 nM), which is a nitrogen inserted B-ring analog of compound 30 (IC50: 9.2 nM) revealed
35-fold drop in GSNOR potency. Further, compounds 56 and 57 having Cl, and CH3-substituents
as well as nitrogen in the B-ring provided potency similar to lead 5. Among the di-substituted B-
C ring compounds including nitrogen in the B-ring (58-60) tested for GSNOR potency, compound
60 having chloro- and methyl in the ortho-position of biphenyl B-C ring revealed greater than 100-
fold diminished potency. Other two di-substituted pyridine derived biaryl compounds (58, 59)
having smaller fluorine in the ortho-position of biaryl C-ring, and methyl, chlorine in the ortho-
position of nitrogen inserted B-ring unveiled potency similar to lead 5. The probable reason for
inferior potency of compound 60 would be due to non-planar geometry of the molecule because
of bis-ortho substitution on the B-C ring of biaryl. Further, nitrogen inserted B-ring compounds
(68, 69 and 71) having substitution on the B-ring (ethyl and cyclopropyl) showed reduced potency
(IC50: > 60 nM) and precisely pyridine biaryl analog 68 (IC50: 66 nM) revealed 10-fold diminished
GSNOR potency compared to structurally similar analog 42 (IC50: 6.1 nM). Overall, most of the
pyridine derived biaryl analogs exhibited decreased GSNOR potency in comparison to simple
biphenyl derivatives except few (56-58) which retained potency similar to lead 5 (Table 3).
Table 4
Metabolic stability and CYP inhibition data of selected GSNOR compounds
Compd Metabolic
% CYP inhibition @ 10 µM
stability in liver concentrationsb
microsomesa
H / R / M
1A2 2D6 3A4 2C9 2C19
5
66 / 58 / 31
63 / 82 / 92
100 / 83 / 43
100 / 64 / 48
99 / 68 / 44
100 / 71 / 58
5 / 99 / 87
74
10
32
22
39
56
13
73
37
52
14
44
37
n.i
54
-2
55
4
77
18
86
37
86
94
48
69
33
86
18
84
86
67
27
30
39
41
42
43
26
33
18

--
44
45
56
57
58
59
47
41
20
1
63
52
11
9
43
30
1
83
78
64
61
60
50
76
88
42
69
45
42
-- : not determined; n.i: no inhibition.
a Percentage of test compound remaining after
60 min incubation with liver microsomes
(human, rat and mouse) at
37 °C. MS experiment was conducted in
triplicates [see supporting info (SI) for details].
b Cytochrome P450 (CYP) % inhibition as
compared to control (no inhibitor) and
97 / 80 / 78
83 / 39 / 19
94 / 50 / 17
100 / 53 / 42
--
17
19
n.i
-15
n.i
12
n.i
conducted in triplicates (see SI for
details).
Table 5
Mouse oral PK data of GSNOR compounds 30 and 58.
Parameter a,b,c
30
58
a Cmax, AUC0-24, Tmax, and T1/2 were determined
based on oral dosing and clearance (CL), volume of
distribution
Dose: i.v / PO (mg/kg)
3 / 30
3 / 30
CL (mL/min/Kg)
Vz (L/Kg)
T1/2 (h), i.v
1.4
0.41
3.4
17
2.8
1.9
(Vz) were determined based on i.v dosing.
Bioavailability (%F) was determined based on both
oral and i.v dosing.
Cmax (ng/mL)
AUC (ng.h/mL)
Tmax (h)
T1/2 (h), oral
% F
8963 ± 1667
69851 ± 14737 14170 ± 942
2
6
21
0.38
2565 ± 417
b Vehicle for Oral dosing-2.5µL/mL Tween 80+
0.5% MC Solution; Vehicle for i.v dosing- PEG200:
water premix
0.5
5.3
49
(3:2) - 90% + Water (q.s). Male C57 mice were
used.
c See supporting info (SI) for the experimental
details.
Lung/plasma ratio (L/P)
0.62
After establishing improved GSNOR potency, some of the potent inhibitors were further
evaluated for microsomal stability and CYP inhibition studies (Table 4).21 Majority of the tested
analogs revealed high metabolic stability in human and rat liver microsomes, and moderate to low
stability in mouse liver microsomes except few analogs (Table 4). CYP inhibition study of
tetrazole analogs 5 and 30 revealed higher CYP inhibition (≥50% @ 10µM) for all five major CYP
isoforms (1A2, 2D6, 3A4, 2C9 and 2C19) tested. On the other hand, carboxylic acid analog 39
unveiled minimal CYP inhibition to all five CYP isoforms evaluated. Likewise, 2-
methylimidazole analog 27, which is structurally similar to lead 5 provided gratifyingly low CYP
inhibition for all the isoforms tested. In addition, substituted biphenyl derivatives 42, 44 and 45
exhibited higher CYP2C9 and CYP2C19 liability. Conversely, 2-methylimidazole analog 43,
which is structurally similar to the potent analog 42 still displayed CYP2C19 liability. Besides,
moderate CYP2C9 and CYP2C19 liability were observed to pyridine derived biaryl compounds
(56, 57 and 58). Overall, compounds from this series still revealed either CYP2C9 or 2C19 and or
both CYP isoform liabilities, which is undesirable.
A single dose pharmacokinetic (PK) study of compounds 30 and 58 were carried out after
intravenous (i.v) and oral administration on male C57 mice.21 As shown in Table 5, compound 30
displayed low plasma clearance (CL) and volume of distribution (Vz) after 3 mg/kg i.v dose in

comparison to compound 58. As well, after 30 mg/kg PO administration, compound 30 unveiled
roughly 4-fold higher Cmax and AUC0-24 h in comparison to compound 58 despite having similar
oral half-life (T1/2). The oral bioavailability of both compounds were found to be low to moderate
(%F: 21 and 49). As GSNOR is expressed in lung, and COPD efficacy evaluation requires
adequate lung distribution of orally available compounds, lung partitioning of 30 and 58 was
evaluated and found to be comparable as disclosed in Table 5.21
Table 6
Summary of in vitro data of compound 30
N
N
N
N
N
N
H
Cl
a Human, rat and mouse IC50 values are derived
from graphs plotted with data from a minimum of
two experiment
Property
30
GSNOR hIC50 (ADH5)
GSNOR mIC50 (ADH5)
GSNOR rIC50 (ADH5)
ADH1B IC50
9.2 nM
7.1 nM
in duplicates.
b See Table 4 foot notes for details.
c Plasma protein binding (PPB) was determined
using equilibrium dialysis method. 10 µM
concentrations were used
9.1 nM
54 µM
CBR1
24% inhi. @10 µM
100 / 83 / 43
Met.stability (% remaining,
H, R, M)
(see SI for details) and the data provided are %
bound.
d PAMPA permeability assay was carried out using
2% lecithin dodecane mixture to determine the
passive
CYP % inhi. @ 10µM;
1A2 / 2D6 / 3A4 / 2C9 / 2C19
PPB (% bound, H / M)
PAMPA (Peff x 10-6 cm/sec)
CALU-3 (Papp x 10-6 cm/sec)
32 /52 / 55 / 86 / 86
>99.5 / 98.5
0.12 (low)
8.23 (high)
0.8
permeability and the study was conducted in
triplicates.
e
CALU-3 assay was performed at Syngene
International Ltd, Bangalore, India.
Efflux ratio [Papp(B-A) / Papp(A-
B)]
f For hERG assay details (patch clamp), see Ref.
25.
hERG inhibition (Patch clamp)
POR (CEREP data):
11% @10 µM
g POR data presented here for 30, which showed >
50% inhibition at 10 µM test concentration against
receptors, ion channels and enzyme targets. MT3-
Melatonin receptor. See Ref. 26 for details.
MT3: 100%;
P2Y: 59%;
BZD: 50%;
LTB4 (h): 64%;
Cl- channel: 50%;
I2 (antagonist): 88%;
PDE6: 60%
Subsequently, compound 30 was
chosen for further profiling after having
encouraging PK, metabolic stability and
human GSNOR potency comparable to
clinical lead compounds N6022 (1) and
N91115 (2). The mouse and rat potency of
compound 30 (mIC50: 7.1 nM and rIC50: 9.1
nM) were comparable to human GSNOR
potency and further demonstrated good
Solubility data:
Solubility in DMSO
20 mg/mL
Solubility at FaSSIF (pH 6.2)
Solubility at FeSSIF (pH 5.2)
3.5 mcg/mL
0.6 mcg/mL
1.1 mcg/mL
Solubility in acetate buffer (pH
4.5)
Solubility in phosphate buffer (pH 7 mcg/mL
6.8)
0.1N HCl
20 mcg/mL

selectivity against alcohol dehydrogenase (ADH) and carbonyl reductase (CBR1) family of
enzymes (Table 6). Compound 30 appeared to be highly protein bound in human and mouse, and
revealed low permeability in PAMPA assay and the corresponding efflux ratio was found to be
low (0.8). However, CALU-3 (human airway epithelial cells) permeability24 of compound 30 was
found to be higher than PAMPA. The hERG channel activity25 of compound 30 was found to be
low (11% inhibition @10µM in patch clamp) and further exhibited minimal off-target liability
(few hits in CEREP panel data, which is not significantly relevant to GSNOR inhibitor).26
Solubility data of 30 is also presented in Table 6 both in Fed-state and fasted state and has low
aqueous solubility.
Figure 3. Effect of 30 on cigarette smoke (CS) induced pulmonary inflammation.
Results are shown as Mean ± S.E.M. (n = 7/group).
Consequently, compound 30 was further evaluated for in vivo efficacy in cigarette smoke
(CS) induced pulmonary inflammation for COPD in mice (widely used model for efficacy
study).21,16c Male C57BL/6 mice were exposed to Kentucky Research Grade cigarettes (3R4F)
twice daily in a whole body inhalation exposure chamber (SIU24, ProMech Lab Holding AB,
Sweden) for seven days, while GSNOR inhibitor 30 was dosed orally for seven days, one hour
prior to 1st smoke exposure. The PDE4 inhibitor, roflumilast (approved oral drug) was used as a
positive control, naive mice exposed to air (vehicle) was served as negative control. On day-8 after
the last smoke exposure, animals were euthanized and subjected to bronchoalveolar lavage (BAL),
and then fluid was collected. Total and differential BAL cell counts were performed from the BAL
fluid. Data was analyzed using Graph Pad Prism software and statistical analysis was conducted
using one way ANOVA followed by Dunnett’s multiple comparison test. Study results revealed
significant dose dependent inhibition of cellular infiltration in BALF of the animals exposed to

cigarette smoke upon compound 30 treatment, and the efficacy dose of 100 mg was comparable
to Roflumilast. Thus, lead compound 30 (oral GSNOR inhibitor) demonstrated significant anti-
inflammatory effects in cigarette smoke induced pulmonary inflammation in mice.16c
In summary, we have successfully identified a series of potent and selective GSNOR
inhibitors having imidazole and tetrazole functional groups in both end of the core structure and
many compounds have been identified with IC50: < 25 nM and the best two compounds 30 and 42
in this series were having IC50: < 10 nM potency. Most of the selected GSNOR inhibitors were
metabolically stable across species (human, rat and mouse) and majority of them had few CYP
isoform liabilities (2D6, 2C9 and 2C19). Selected lead compound 30 demonstrated favorable in
vitro properties, low hERG activity, minimal off-target (POR) activity and reasonable oral
pharmacokinetics (PK) including moderate oral bioavailability. Further, compound 30
demonstrated ability to attenuate pulmonary inflammation (in vivo
efficacy), which supports the therapeutic potential of GSNOR
inhibitor for the treatment of COPD. Since most of the disclosed GSNOR
inhibitors has CYP liability, further SAR optimization of this series to afford minimal CYP liable
compounds along with intranasal PK and efficacy study (inhalation route) will be the subject of
future publication.
Acknowledgements
We would like to thank Dr. Sanjib Das for the review of this manuscript, and Mr. Dayanidhi Behera
for in vitro metabolic stability, CYP inhibition data to few selected compounds, and scientists from
the analytical support group for their help in compound characterization. We also thank Dr. Pravin
S Iyer for his support and encouragement.
Supplementary data
Supplementary data associated with this article can be found in the online version, at
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(b) Gallant, M.; Beaulieu, C.; Berthelette, C.; Colucci, J.; Crackower, M. A et al., Bioorg. Med.
Chem. Lett. 2011, 21, 288;
(c) Wang, J. L.; Carter, J.; Kiefer, J. R.; Kurumbail, R. G.; Pawlitz, J. L. et al., Bioorg. Med. Chem.
Lett. 2010, 20, 7155.
23. Although, there are many carboxylic acid-containing drugs have been marketed worldwide, some
of them have been withdrawn from the market due to idiosyncratic drug toxicities arising from the
metabolism of the carboxylic acid moiety. For related references, see: (a) Lassila, T.; Hokkanen,
J.; Aatsinki, S-M.; Mattila, S.; Turpeinen, M.; Tolonen, A. Chem Res Toxicol. 2015, 28, 2292;
(b) Fung, M.; Thornton, A.; Mybeck, K. Drug Inf J. 1992, 26, 1-15.
24. CALU-3 assay study was carried out at Syngene International Ltd, Bangalore, India.
25. hERG channel assay study was conducted at Eurofins Lab-USA (http://www.eurofinsus.com).
26. POR study (CEREP panel screen) was conducted at Eurofins lab-France (previously known as
Cerep SA, France; http://www.eurofins.com).
Highlights:
 41 imidazole-biaryl-tetrazole derivatives had been synthesized, 7 exhibiting low nM
GSNOR potency.
 Lead compounds 30 and 58 demonstrated good oral exposure and bioavailability in
mouse PK study.
 Advanced lead 30 displayed comparable rodent potency, good selectivity and minimal
off-target activity.
 Lead compound 30 was efficacious in cigarette smoke induced mouse model for COPD.

Discovery of 5-(2-Chloro-4'-(1H-imidazol-1-yl)-[1,1'-biphenyl]-4-yl)-1H-
tetrazole as potent and orally efficacious S-Nitrosoglutathione reductase
(GSNOR) inhibitors for the potential treatment of COPD
Nagarajan Muthukamana, Sanjay Deshmukha, Shital Tondlekara, Macchindra Tambea, Dnyandeo
Pisala, Neelam Sarodea, Siddharth Mhatrea, Samitabh Chakrabortib, Daisy Shahb, Vikram M.
Bhosaleb, Abhay Kulkarnib, Mahamad Yunnus A. Mahatc, Satyawan B. Jadhavc, Girish S. Gudic,
Neelima Khairatkar-Joshib, Laxmikant A. Gharata,*
aChemical Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra
400709, India
bBiological Research, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi Mumbai, Maharashtra
400709, India
cDrug Metabolism and Pharmacokinetics, Glenmark Pharmaceuticals Limited, Glenmark Research Center, Navi
Mumbai, Maharashtra 400709, India
Corresponding author. Tel.: +91 022 6772 0000x3208.
E-mail address: Laxmikant.gharat@glenmarkpharma.com (L.A. Gharat).
N
N
N
N
N
N
H
Cl
30
(Lead compound)
GSNOR hIC50 (ADH5): 9.2 nM
GSNOR mIC50 (ADH5): 7.1 nM
GSNOR rIC50 (ADH5): 9.1 nM
ADH1B IC50: 54 µM; CBR1: 24% inhi. @10µM
hERG (patch clamp): 11% @10µM
Mouse %F (30 mg/kg): 21
COPD efficacy (CS model): efficacious
Effect of 30 on cigarette smoke (CS) induced pulmonary inflammation