Inducible Nitric Oxide Synthase (iNOS) inhibitors
AstraZeneca R&D Charnwood

Target
Identification
3 months to
2 years!
HTS
3-4 months
Active-to-Hit
(AtH) 3 months
Hit-to-Lead
(HtL)
6-9 months
New Lead
Optimisation
Projects (LO)
2 years
Candidate
Drug (CD)

Nitric Oxide Synthase – Biological Mechanism
H N
H
N
NH
2
NH
2
L-Arginine
COOH
2O
2
, NADPH, NADP
FMN/FAD/H
4
B
O
NH
2
H
N
NH
2
L-Citrulline
COOH +
NO
• iNOS - induced NOS
• induced, constitutively active, Ca 2+ independent
• over expression causes inflammation & pain
• nNOS - neuronal NOS
• constitutively expressed, Ca 2+ dependent
• long term memory, GI motility, stroke
• eNOS - endothelial NOS
• constitutively expressed, Ca 2+ dependent
• vasodilatation - inhibition causes increased blood pressure!

Early Compounds based on arginine
Simple 1-isoquinolinamines were potent iNOS inhibitors, although prone to aromatisation.
BMCL, 2001, 11(11) 1023.
Stability was markedly improved by making the spirocyclic quinazolines
JMC, 2003, 46(6), 913-916
N
F NH
2 iNOS 0.6 m M sel. vs eNOS x 160
O
H
N
N OEt
N
NH
2 iNOS 0.7 m M sel. vs eNOS x 60

Best Compound in the series
• AZ10896372 - a potent and selective inhibitor
F
F
H
N
N
NH
2
N
O
N CN iNOS 0.035 m
M (isolated enzyme) cell 1.1 m
M (DLD-1 cell) sel. vs eNOS > 1000 sel. vs. nNOS x 22
Rat PK
Cl = 57 ml/min/kg t
1/2
2.4 hours
Bioavailability 75%

Other ‘Amidine-like’ Series
• We pursued many other series, for example
OMe
N
O
N
N
H
Cell 4 uM
CN
O N NH
N
Cell 1.6 uM
O
S
H
2
N
N
Cell 1.5 uM
• all had low cell potency and/or sub-optimal pharmacokinetics
Move away from the amidine isosteres!

Non-amidine inhibitors – Literature Leads
• Some weak non-amidine inhibitors were known
Me
O OH
H
N
NH
2
NO
2 iNOS 11 m
M
JMC, 1998, 41(14),
2636
N
+
O O
N
H
N iNOS 9 m M
Cl
O
N
H
O iNOS 5 m
M
But how do they bind to iNOS?

Finding a New Lead
O OH
Me
H
N
NH
2 iNOS 11 m
M
JMC, 1998, 41(14),
2636
NO
2
Me
NO
2
F
R
1-80
NH
2
‘tyrosine amide’
Me
NO
2
N
H
CONH
2
OH iNOS ~50% at 1 m
M only 1 well active

Finding a New Lead
O OH
Me
H
N
NH
2 iNOS 11 m
M
JMC, 1998, 41(14),
2636
NO
2
Me
NO
2
F
R
1-80
NH
2
‘tyrosine amide’
Me
NH
2
CONH
2
NO
2
O
Check Structure!
iNOS 1.2 m M sel. vs eNOS x 3

Using Crystal Structure data
Salt bridge
Glu 371
Me
N N
H

Me
NO
2
O
NH
2
CONH
2
Using Crystal Structure data
Residue moved Glu 371

Me
NO
2
O
Me
N N
H
NH
2
CONH
2
Combining data
Gln 257
Glu 371

Me
NO
2
O
Me
N N
H
NH
2
CONH
2
Gln 257
Haem acids
Glu 371

Using Crystal Structure to Design a New Series
• Move Gln 257 and add amine to bind haem acids
Me
NO
2
O
Me
+
NH
2
CONH
2
N N
H
Me
NO
2
O
NH
2 iNOS 0.9 m M sel. vs eNOS x100
(racemic)
Cl
NH
2
Cl
O iNOS 2 m
M sel. vs eNOS >50 sel. vs nNOS >50
(racemic)

Cl
Cl
O
NH
2 iNOS 2 m
M sel. vs eNOS >50 sel. vs nNOS >50
(racemic)
Gln 257
Glu 371

Improving Potency
Cl
R
O
NH
2
R = Cl iNOS 2 m
M sel. vs eNOS > x 50
(racemic)
R = CN iNOS 0.9 m
M sel. vs eNOS x 110
(racemic)
Cl
CN
O NHMe iNOS 0.009 m
M cell 0.7 m
M sel. vs eNOS >10000 sel. vs. nNOS x150 not active in vivo
Cl = 94 ml/min/kg t
1/2
1.3 hours
F
Cl
CN
O NH
2 iNOS 0.006 m M cell 0.7 m M sel. vs eNOS >13,000 sel. vs. nNOS x35 active in vivo
Cl = 35 ml/min/kg t
1/2
7 hours

A New Series of iNOS Inhibitors
– pKa = 9.6, logD = 0.8
– stable in in vitro in rat, dog & human microsomes and hepatocytes.
– dog t
1/2
= 11 hours, F = 70%
– radiolabelled study shows no glutathione displacement of F
– not nNOS selective enough < 50 fold
– unacceptable CYP 2D6 (0.3 m
M)
– Other activities (5-HT, NA uptake)
F
Cl
CN
O NH
2 iNOS 0.006 m M cell 0.7 m M sel. vs eNOS >10,000 sel. vs. nNOS x35 active in vivo
Cl = 35 ml/min/kg t
1/2
7 hours

Series Optimisation - Requirements
• Selectivity versus eNOS
– All compounds, selectivity of >1000 fold!
• Cellular potency need < 1 m
M
– Dose to Man, off-target selectivity
• Selectivity versus nNOS > 50 fold
• Metabolic stability

O
O
S
N
Me
S
N
F
S
F iNOS Potency – Overview of SAR
Cl
Me
Me
N N
O
N
N
S
N S
N
S
N
S
N
N
N
R
L
W
Y
NR
CN
S
CN
F
O
F
N
S
N
H or F
Cl or Me
N
O or S
Me
N
S
N
N
OCHF
2
S
N

S N
R iNOS Potency – Overview of SAR
N
S
CN
L
S
N
W
Y
NR
CH
3
CH
3
N N
N
O
O N
Me
O
N
S
N
F

iNOS Potency – Overview of SAR
• O,S iNOS 
• N,C iNOS 
2,
2
• iNOS 
2
2
• iNOS 
• iNOS 
R
CN
L
W
Y
NR

nNOS Selectivity
• Selectivity vs. nNOS improves;
– R1 = NH
2
, NHMe, NMe
2
– R1 = H & R2 = F, Cl, OMe
• but iNOS potency falls away!
– L = ‘S’ vs. ‘O’
– Y = -C(CH
2
OH)-
• and iNOS potency increases!
R2
• Selectivity vs. nNOS decreases;
– R1 = Br, CF
3
, CH
2
F, CHF
2 or R2 = F
• but iNOS potency increases!
R1
CN
L
Y
NH
2

Best Compound of the Series
N
OMe
S
N
OH
NH
2 logD = 1.2, pKa = 8.2
iNOS 0.002 m
M, cell 0.1 m
M sel. vs. eNOS > 10,000 sel. vs. nNOS x 50
Cyp2D6 = 0.6 m
M, 5HT = 4 m
M
& NA > 10 m
M, hERG = 16 m
M
Rat PK - poor

Crystal Structure
Trp 366
N
OMe
S
N
OH
NH
2
Gln 257
Met 368
Glu 371

Synthesis
BnO
O
O
O
O
N
O
NHtBOC
(MeO)
2
C(Me)
2
CSA toluene (90g, 100%)
NaBH
4
, THF-H
2
0, O
°C
(85g, 100%)
H
2
Pd/C EtOH
(86g, 83%)
H O
O
BnO
O
OH
NHtBOC
O
N tBOC
MeNHOMe.HCl EDCI
25 °C DCM (60g, 97%)
PhMgBr, THF O °C
(64g, 95%)
O
O
N tBOC
Borane, THF, -10
°C
(R)-Me-CBS catalyst d.r. = 4:1
37g (58%)
H O
O
N tBOC

H O
Completion of Synthesis
O
N tBOC
1) PPh
3
, DIAD, 0 °C, THF
2) PhCOSH
Ph
O
S
O
N tBOC
OMe
+
N
CN
SO
2
Me
1) NH
3
, MeOH
2) Cs
2
CO
3
, DMF (89%)
3) MeOH, HCl (87%)
OMe
OH
N
S
CN
29.4g 99.6% pure
NH
2