Advanced MedicinalAdvanced MedicinalChemistryChemistry

AstraZeneca R&D Charnwood

Lecture 10:Lecture 10:

Case History – Inducible Nitric Oxide Synthase (iNOS)

inhibitors

TargetIdentification

HTS

Hit-to-Lead(HtL)

New Lead OptimisationProjects (LO)

CandidateDrug (CD)

Active-to-Hit(AtH)

3 months to2 years!

3-4 months

3 months

6-9 months

2 years

The Drug Discovery Process

Nitric Oxide Synthase – Biological Mechanism

NO

2O2 , NADPH, NADP

FMN/FAD/H4B

L-Arginine L-Citrulline

+NH N

H

NH2

NH2

COOHO N

H

NH2

NH2

COOH

• iNOS - induced NOS• induced, constitutively active, Ca2+ independent

• over expression causes inflammation & pain

• nNOS - neuronal NOS• constitutively expressed, Ca2+ dependent

• long term memory, GI motility, stroke

• eNOS - endothelial NOS

• constitutively expressed, Ca2+ dependent

• vasodilatation - inhibition causes increased blood pressure!

Early Compounds based on arginine

N

NH2F

iNOS 0.6 Msel. vs eNOS x 160

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

NNH

N

NH2

O

OEt

iNOS 0.7 Msel. vs eNOS x 60

Best Compound in the series

NNH

N

NH2

O

N CN

F

F

iNOS 0.035 M (isolated enzyme)cell 1.1 M (DLD-1 cell)

sel. vs eNOS > 1000sel. vs. nNOS x 22

Rat PKCl = 57 ml/min/kg

t1/2 2.4 hoursBioavailability 75%

•AZ10896372 - a potent and selective inhibitor

Other ‘Amidine-like’ Series

• We pursued many other series, for example

Cell 4 uM

O

CN

N

NH

N

OMe

Cell 1.6 uM

N NH

N

O

Cell 1.5 uM

S

O

N

NH2

• 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

NH2

NH

NO2

OHO

Me

N+

O O

NH

N

iNOS 9 M

NH

O

Cl

O

iNOS 5 MiNOS 11 MJMC, 1998, 41(14), 2636

But how do they bind to iNOS?

Finding a New Lead

• Decided to explore ‘Abbott’ compound further

NH2

NH

NO2

OHO

Me iNOS 11 MJMC, 1998, 41(14), 2636

iNOS ~50% at 1 Monly 1 well active

Me

NO2

NH

CONH2

OH

Me

F

NO2

R1-80NH2

‘tyrosine amide’

Finding a New Lead

• Decided to explore ‘Abbott’ compound further

NH2

NH

NO2

OHO

Me iNOS 11 MJMC, 1998, 41(14), 2636

Me

F

NO2

R1-80NH2

‘tyrosine amide’

Me

NO2

O

NH2

CONH2

iNOS 1.2 Msel. vs eNOS x 3

Check Structure!

N

Me

NH

Glu371

Using Crystal Structure data

Salt bridge

Me

NO2

O

NH2

CONH2

Glu371

Using Crystal Structure data

Residue moved

N

Me

NH

Me

NO2

O

NH2

CONH2

Gln257

Glu371

Combining data

N

Me

NH

Me

NO2

O

NH2

CONH2

Gln257

Glu371

Haem acids

Using Crystal Structure to Design a New Series

iNOS 0.9 Msel. vs eNOS x100

(racemic)

Me

NO2

ONH2 O

NH2

Cl

Cl

iNOS 2 Msel. vs eNOS >50sel. vs nNOS >50

(racemic)

• Move Gln257 and add amine to bind haem acids

Me

NO2

O

NH2

CONH2

N

Me

NH

+

Gln257

Glu371

ONH2

Cl

Cl

iNOS 2 Msel. vs eNOS >50sel. vs nNOS >50

(racemic)

Improving Potency

O

Cl

NH2

CN

F

iNOS 0.006 Mcell 0.7 M

sel. vs eNOS >13,000sel. vs. nNOS x35

active in vivoCl = 35 ml/min/kg

t1/2 7 hours

ONH2

Cl

R

R = CliNOS 2 M

sel. vs eNOS > x 50 (racemic)

R = CNiNOS 0.9 M

sel. vs eNOS x 110 (racemic)

O

Cl

NHMe

CN

iNOS 0.009 Mcell 0.7 M

sel. vs eNOS >10000sel. vs. nNOS x150

not active in vivoCl = 94 ml/min/kg

t1/2 1.3 hours

A New Series of iNOS Inhibitors

O

Cl

NH2

CN

F– pKa = 9.6, logD = 0.8

– stable in in vitro in rat, dog & human microsomes and hepatocytes.

– dog t1/2 = 11 hours, F = 70%

– radiolabelled study shows no glutathione displacement of F

– not nNOS selective enough < 50 fold – unacceptable CYP 2D6 (0.3 M)– Other activities (5-HT, NA uptake)

iNOS 0.006 Mcell 0.7 M

sel. vs eNOS >10,000sel. vs. nNOS x35

active in vivoCl = 35 ml/min/kg

t1/2 7 hours

Series Optimisation - Requirements

• Selectivity versus eNOS – All compounds, selectivity of >1000 fold!

• Cellular potency need < 1 M– Dose to Man, off-target selectivity

• Selectivity versus nNOS > 50 fold

• Metabolic stability

iNOS Potency – Overview of SAR

LY

NR

CN

WR

N N

N

S

Me

N

N

N

S

Me

N

S

N

O

N

O or S

Cl or Me

H or FN

N

S

MeN

S

OO

S

CN

S

MeN

N

S

OCHF2

N

N

S

O

F F

N

S

F

FN

N

S

Cl

iNOS Potency – Overview of SAR

NS

NN

S

F

N

O

O

N

NO

Me

LY

NR

CN

WR

S

S

NN N

CH3

CH3

iNOS Potency – Overview of SAR

LY

NR

CN

WR

• Linker L• O,S iNOS • N,C iNOS

• Y = CH2, W = CH2OH• iNOS

• Y = bond or -CH2CH2-• iNOS

• any R, except R = Me• iNOS

nNOS Selectivity

• Selectivity vs. nNOS improves;– R1 = NH2, NHMe, NMe2

– R1 = H & R2 = F, Cl, OMe• but iNOS potency falls away!

– L = ‘S’ vs. ‘O’

– Y = -C(CH2OH)-

• and iNOS potency increases!

• Selectivity vs. nNOS decreases;– R1 = Br, CF3, CH2F, CHF2 or R2 = F

• but iNOS potency increases!

LY

NH2

CN

R1

R2

Best Compound of the Series

N

N

S NH2

OH

OMe

logD = 1.2, pKa = 8.2

iNOS 0.002 M, cell 0.1 M

sel. vs. eNOS > 10,000sel. vs. nNOS x 50

Cyp2D6 = 0.6 M, 5HT = 4 M& NA > 10 M, hERG = 16M

Rat PK - poor

Glu371

Met368

Trp366

Gln257

N

N

S NH2

OH

OMe

Crystal Structure

OOO

BnO NHtBOC

N

O

O

OOH

BnO NHtBOC

NaBH4, THF-H20, O°C

(85g, 100%)

Synthesis

O

N

O

tBOC N

O

OHtBOC

37g (58%)

Borane, THF, -10 °C

(R)-Me-CBS catalyst d.r. = 4:1

PhMgBr, THF O°C

(64g, 95%)

O

OH N

O

tBOC

H2 Pd/C EtOH

(86g, 83%)

MeNHOMe.HCl EDCI

25°C DCM (60g, 97%)

(MeO)2C(Me)2

CSA toluene (90g, 100%)

N

O

OHtBOC

1) NH3, MeOH2) Cs2CO3, DMF (89%)3) MeOH, HCl (87%)

29.4g 99.6% pure

NH2

OH

S

N

CN

OMe

N

O

S

O

PhtBOC

N

OMe

CN

SO2Me+

1) PPh3, DIAD, 0°C, THF2) PhCOSH

Completion of Synthesis