Advanced MedicinalAdvanced MedicinalChemistryChemistry

Dr Jeff Stonehouse

AstraZeneca R&D Charnwood

Lecture 2:Lecture 2:

Finding a Lead

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

Lead Compounds from a Variety of Sources

4. Natural Ligands

5. Existing Drugs

6. High Throughput Screening (HTS)

N

S

O

NHR

O

OOH

penicillins

O

OHOO

O

O

O

OOH

O

O

O

OH

NHO

H

taxol

NH

NN

N

O

O

SN

O O

N

Viagra

1. Chance Discovery

2. Natural Products

3. Clinical Observation

Natural Ligands

OH

OH

OHNH

RR=H adrenaline

R=Me noradrenaline

Catechol bioisostere

(toxicity)Increased size

(selectivity and duration)

Catechol bioisostere

(toxicity)

Increased size (selectivity and duration)

NH

OH

OHNH

O

O

H

Formoterol AstraZeneca

OH

OHNH

OH

Salbutamol GlaxoSmithKline

N

O

O

NNH

OO

Cialis

Eli Lilly

NH

NN

N

O

O

SN

O O

N

Levitra

Bayer

Existing Drugs

Also known as the “Me-Too” or “Me-Better” Approach

Issues: short duration

Multiple side effects and incompatibility with other drugs

NH

NN

N

O

O

SN

O O

N

Viagra

Pfizer

Fewer side effects and incompatibility with other drugs

36h duration (“the weekend pill”)

BEWARE: Patent Issues!!

High Throughput Screening (HTS)

• validated, tractable targets

• target selection for HTS

• industrialised process

• HTS assay technologies and automation

• chemical diversity

• sample selection for HTS

How?

“An industrialised process which brings together validated, tractable targets and chemical diversity to rapidly identify novel

lead compounds for early phase drug discovery”

50-70% of new drug projects originate from a HTS

Establishing a HTS

OH

N

Cl

O

O

chemical space

compoundcollection

compoundselection

human & pathogen genomes

validated/tractabletargets

targetID

HT ScreenDevelopment

Microtitre Plates – the HTS test tube

9mm9mm

96300-100300-100ll9mm pitch9mm pitch

384LV25-525-5ll4.5mm pitch4.5mm pitch

384100-25100-25ll4.5mm pitch4.5mm pitch

153610-110-1ll2.25mm pitch2.25mm pitch

For 200K data points:

125 x 1536 well plates

2000 x 96 well plates

500 x 384 well plates

Charnwood HTS Technologies; 1995-2001

3%

16%

2%4%

1%30%

1%

19%

24%

SPA FLIPR Filter Fluorescence Reporter Yeast TR-FRET Alphascreen FP

•Screening can utilise numeroustechnologies e.g radioactivity,fluorescence, luminescence

•None are universally applicable, eachwith advantages and disadvantages

High throughput radioligand binding assays

Scintillation Proximity Assay – the first true homogeneous HTS screening technology

Nothing bound

bead not activated,

no light

Antibody/receptor

Molecule too far away to activate bead

Molecule cannot bind

Bound molecule

bead activated

light produced

I125

I125

Molecule binds

I125

I125

Suitable for I125, 3H, 33P

SPA (Scintillation Proximity Assay)

• First true homogeneous HTS technology• Allows throughput of ~30K compounds/day in

384 format• Easy to automate, no significant volume of

aqueous waste

BUT:

•Radioactive (safety headaches)

•Long read times (>30min/plate)

•Susceptible to quench artefacts

•Not applicable to all targets

FLIPR – a high throughput fluorimeter

Fluorescent Imaging Plate ReaderReal-time simultaneous imaging of 96- & 384-well platesUsed for HTS Ca2+ flux assays and ion channel screening

PCPC

Cooled CCD CameraCooled CCD Camera

96/384-Tip Pipettor96/384-Tip Pipettor

Drawer HoldingDrawer Holding5 Microplates5 Microplates

6 W Argon Ion Laser6 W Argon Ion Laser

• Cells loaded with fluorescent dye sensitive to Ca2+ (fluo-3)

• CCD camera images base of microtitre plate

• Addition of receptor agonist stimulates Ca2+ release, resulting in fluorescence increase

• Whole plate is read simultaneously, allowing kinetic analysis

• ‘Functional’ screen (i.e.whole cell) – greater relevance than simpler screening methods

• Throughput is 1000x greater than cuvette-based fluorimeter assay

FLIPR – how it works

Establishing a HTS

OH

N

Cl

O

O

chemical space

compoundcollection

compoundselection

human & pathogen genomes

validated/tractabletargets

targetID

HT ScreenDevelopment

The AstraZeneca Compound CollectionThe AstraZeneca Compound Collection

19931993ASTRA ARCUSASTRA ARCUS ASTRA PAIN CONTROLASTRA PAIN CONTROL

19941994

19991999

Not a recipe for an optimal screening bankNot a recipe for an optimal screening bank

Ca 9% compound overlapCa 9% compound overlap

Compound Collection Enhancement

• AZ global initiative to boost screening collection

– Target: ensure viable Hits from 75% of AZ HTS

• Five-year initial lifespan. Two concurrent themes…

AcquisitionAcquisition

300K from 107 available

Stringent filters

Big Medchem input

Accept IP risks

SynthesisSynthesis

Nominal 500K over 5 years

Target-class focus

Aligned to Research Areas

Early Bioscience input

CCE Structure

• Chemistry deliberately embedded in Research Areas

– Not centralised

– Benefit of Project exposure

– Feeds parallel synthesis skill back into projects

CompoundManagement

AP

Channel

Södertälje

KinaseAlderley

Park

CentralBioscience

Cheminformatics

GPCR

Charnwood

ProteaseMölndal

HTSCharnwood

HTSAP

HTSUS

HTSMölndal

~60 Scientists Med ChemBioscience

Comp ChemInformatics

Types of reactions

amide coupling

sulphonamide formation

reductive amination

Boronic acid coupling

Multicomponent reaction (3 variants so far)

Sulphonamide arylation

Ester hydrolysis

Acyl sulphonamide formation

Urea formation

Epoxide opening

Anhydride opening

Condensation to form benzamidazoles

Mitsunobu

N-, O- and S-Alkylation

Sulfonylurea formation

benzoxazinone formation

Pyridone formation

tetrazole formation

Boc or t-butyl deprotection

cyclization to heterocycles (21 types - see list)

Nucleophilic aromatic substitutions (2 types)

aminopyrazoles

imidazopyridines

imidazothiazoles

imidazopyrimidines

aminothiazoles

aminooxadiazoles

triazolopyrimidines

aminotriazoles

aminobenzimidazoles

triazolopyridines

pyrazolopyrimidine

3-aminoquinolines

triazolopyridazines

triazolopyrazines

thiazolidin-4-one

3-amino-1,2,4-triazoles

pyrimidin-2-ones

triazolo[1,5-c]quinazoline

imidazolidin-2-one

quinazolinone

1,2,4-oxadiazole

CCE – Library Chemistry

3 most commonly used reactions-

Amide coupling

Reductive amination

Sulphonamide formation

CCE – Common Combinatorial Reactions

• Amide Coupling

R3OH

O

N

R2

HR1

N

R2

R1

R3

O

+

HATU, Et3N

NMP

• Sulphonamide Formation

N

R2

HR1

N

R2

SR1

R3

O O

SCl R3

O O

+

Et3N

NMP

• Reductive Amination

N

R2

HR1

N

R2

R1

R3H R3

O

+AcOH, NMP

Na(AcO)3BH

N NN

N

O

N

N+

PF6-

NO

HATU

NMP

Mechanism

Amide

Coupling R

3OH

OO

NN

+

NN

N

N

PF6-

Sulphonamide

Formation N

R2

HR1

SCl R3

O O

Reductive

Amination

N

R2

HR1H R3

O

H+

N

R2

R1OH

R3-H+

H+

N+

R2

R1

R3-H2O

B

H

O

O

O

OO

O

Na+

N

R2

R1

R3

N

R2

SR1

R3

O O

N+

H Cl

-H+

+

R3

O

ON+

N

PF6-

+H+

-H+

N

R2

HR1

N

R2

R1

R3

O+H+

-H+