Enabling HTS Hit follow up via Chemo informatics, File Enrichment,

and Outsourcing

Enabling HTS Hit follow up via Chemo informatics, File Enrichment,

and OutsourcingGraham F Smith

Sandwich Chemistry

Pfizer Global Research and Development

Graham F Smith

Sandwich Chemistry

Pfizer Global Research and Development

OverviewOverview

File Enrichment

Chemo informatics

High Throughput Chemistry

HTS

Enabled Hit Follow Up &Productive lead Discovery

Presentation OverviewPresentation Overview

HTS at Pfizer 10 years ago

File Enrichment

Chemo-informatics

Outsourcing hit follow up

HTS at Pfizer now.

HTS at Pfizer 10 years agoHTS at Pfizer 10 years ago

A 500,000 screening file• Of mixed quality and purity

Early days of parallel chemistry and automation in chemistry andpurification• Synthesis capacity > purification capacity > chemo informatics ability

Typical HTS gave 10% of lead matter being parallel chemistry friendlyCrude informatics tools for HTS follow up• Mostly Excel

The parallel chemistry friendly hits given more effort and therefore typically start more projects• Singleton hits - higher chemistry cost and risk• Parallel chemistry hits already have SAR, starting materials and high

speed method

File Enrichment – our early thoughtsFile Enrichment – our early thoughts

Drug discovery fails more often than it succeeds

Some very difficult drug targets

Fewer targets than before

Some targets in the past, the industry has succeeded but Pfizer hasn’t

One possible cause• Industry file said to be approx

3,000,000 in 1999• Pfizer’s was approx 500,000• Narrow range of structural types

0

0.5

1

1.5

2

2.5

3

3.5

01 02 03 04 05 06

Year

File

Siz

e in

Milli

ons

George M Milne Jr (Pfizer), Annual Reports in Medicinal Chemistry, 2003, 38, 383-396

rationale for file enrichmentrationale for file enrichment

70% of PGRD project leads originate as HTS hits

Quality of drug candidate is reflected in quality of the lead matter

One significant source of Discovery and Development attrition is chemical in nature

Screening file attributes will drive downstream processes, efficiency and overall success in Medicinal Chemistry

Taking attrition early is good BUT suffering attrition on approaches early can be costly, • Therefore having more series options critical• Testing the mechanism is the keyGunther Wess (Aventis), Drug Discovery Today, 7 (10), 2002, 533-535.

file enrichment strategy: consider attrition from outsetfile enrichment strategy: consider attrition from outset

We know what chemotypes are more likely to fail in development

We know clinical candidates are similar to leads

Build this knowledge into library design• make and screen drug-like or lead-

like compounds

hithit

leadlead

candidatecandidate

drugdrug

Beautiful compound conceptBeautifulBeautiful compound concept

parallel chemistry

no no toxicophorestoxicophores

rule of 5rule of 5compliantcompliant

pure and stablepure and stable

PfizerPfizerexclusiveexclusive

Lipinski, C.A. Chris Lipinski Discusses Life and Chemistry afterthe Rule of Five. Drug Discovery Today 2003, 8, 12-16.

File Enrichment InitiativeFile Enrichment Initiative

Aim to make a 3,000,000 compound pure compound file by compound design and acquisition

Hits enabled by having established parallel chemistry protocols

The largest screening file of any pharmaceutical company

More hits on difficult targets – better choices on easier targets

Faster / cheaper lead discovery in Pfizer enabled for the long term

File Enrichment 1 – “the big four”File Enrichment 1 – “the big four”

Neurogen• Informatics based design and synthesis technology• Predicts the next // chemistry compound to make

ArQule• Broad experience of high throughput parallel chemistry• Delivering ~500,000 compounds and associated

protocols

Evotec• Ultra High Throughput Screening uHTS

Aurora• Ultra High Throughput Screening uHTS

The Evotec ultra high throughputscreening system

The Evotec ultra high throughputThe Evotec ultra high throughputscreening systemscreening system

• Capacity to analyse >100,000 compounds per day• Assay volume of 1 μl - saving in reagent and compound usage• 2080 wells on a single assay plate

File Enrichment 2002-2006File Enrichment 2002-2006

ArQule• 800,000• Developing ~200 new chemistry protocols

Tripos • 440,000 compounds• 12,000 privileged monomers

Chembridge• 500,000 compounds• 4,000 privileged monomers

Chem RX (Discovery partners )• 500,000 compounds

Evotec Technology• Developed in collaboration• Created uHTS centres of emphasis

1 mergers and 1 acquisition with associated legacy compound fileacquisition• Warner Lambert• PharmaciaAlan Procter (Pfizer), Drug Discovery and Development, December, 2003See various collaboration press releases

~$1 BillionFE

investment

File Enrichment – managing diversityFile Enrichment – managing diversity

La Jolla Groton Ann Arbor Sandwich

Generation of ideas for library protocols

Global Ideas overlapresolution

La Jolla Groton Ann Arbor SandwichPrioritizationof ideas fordevelopment

Library chemistry development

Library idea Management Tool

La JollaLibrary production and purification

Enhancing Chemo-informaticsEnhancing Chemo-informatics

Enable designs• Monomers• Protocols• Chemical Knowledge• Enumeration• Filtering• Prioritise lead series

Share Designs• Visualise large libraries• Use common desktop tools which aid collaboration

The Pfizer Global Virtual Library -PGVLThe Pfizer Global Virtual Library -PGVL

All resources connected in one application• All Pfizer Monomers stored in one location• All Pfizer // synthesis reactions and protocols

– Stored in one location– With enumeration and structure checking– With knowledge of prior parallel chemistry reactions

• All Pfizer in silico properties available• Filtering, clustering tools and visualisation

All parallel chemistry HTS Hit Follow up work comes from within this tool

• A research sharing and visualisation tool• Links to Excel, Spotfire etc.

Enables choice from ~10,000,000,000,000 potential products• This virtual space grows with each new chemistry protocol and

monomer discovered• PGVL ~ (PGRL)2

HTS Hit triage protocol – Pipeline PilotHTS Hit triage protocol – Pipeline Pilot

Raw data• Compound Name• % inhibition

• Structures• Physical properties (Mw, clogP, etc)• Potential false positives/negatives (Bayesian statistics)

• Library protocol if any• Custom substructures (known series etc)• Clustering of actives by Murcko frame• Known aggregators, reactive groups• Ligand efficiency

M Murcko (Vertex), J. Med. Chem. 1999, 42, 5095-5099A L Hopkins (Pfizer), Drug Discovery Today. 2004 May 15;9(10):430-1

Pipeline Pilot treePipeline Pilot tree

HTS Actives

HTS Hits

HTS library outputHTS library output

multiplelead options

for most targets

Global Protocol databaseGlobal Protocol database

intranet access

All Parallel Chemistry searchable in one place

All 9 PGRD site use this tool

All File enrichment research captured in the global database

A link from compound to parallel synthesis protocol

Architecture based on Formal ReactionsArchitecture based on Formal Reactions

Protocols are associated into Formal Reactions

VRXN-2-00001Reductive amination

as a family of protocols

aldehydes

nu

cleo

phili

cam

ines

Two protocolsof broadest scope

cover thelargest range of potential

----thus potential

for re-use:Capturing these iswhat really builds

the asset inglobal

Chemical Knowledge Bases

VRXN-2-00001 isdefined by the Formal Reaction scheme and spans the space covered byall suitable amines & aldehydesin the linked inventory db

R1 H

O

NH

HR2

HR1

H

N

H

HR2

+

Formal Reactions as Families of ProtocolsFormal Reactions as Families of Protocols

Name of Formal Reaction

(broad scope rxns)

# of explicit protocols within reaction family

Source (each color represents one of the Pfizer R&D sites)

VRXN-2-00001 13VRXN-2-00002 11VRXN-2-00004 4VRXN-2-00005 16VRXN-2-00007 9VRXN-2-00009 12VRXN-2-00010 39VRXN-2-00011 15VRXN-2-00013 8VRXN-2-00016 9VRXN-2-00017 7VRXN-2-00018 3VRXN-2-00081 2VRXN-3-00007 4VRXN-3-00029 21

Chemist’s DesktopChemist’s Desktop

Access to 543 (and growing) Formal Reactions

Scope & Limitations for the thousands of Protocols under those Formal Reactions is scientific basis for • Round-the-clock automated monomer mining • PGVL server-side automated search engine system uses

thousands of stored query components running on Pipeline Pilot

• Linked to global inventory• Search engine defines the precise VL for each protocol and

(by concatenation) the overall VL for each Formal Reaction

PGVL exceeds 10 trillion compounds 1013 +

PGVL Hub, an internally developed application

PGVL Hub on the Chemist’s DesktopPGVL Hub on the Chemist’s Desktop

Access to virtual libraries from the desktopAccess to virtual libraries from the desktop

Suitable monomers retrieved for each reaction protocol

Properties calculated

Inventory and location searched

Clustering and filtering pre and post enumeration

Spotfire Design analysisSpotfire Design analysis

Hit Follow Up (2006…)Hit Follow Up (2006…)

A strategic modification of File Enrichment contracts• Hit Follow-up – (HF)• Med Chem Support

Emphasis on Pfizer design• Pfizer monomers and protocols• Custom synthesis of key monomers and templates• Development and optimisation of // chemistry

Fast turnaround a contractual priorityCompound supplied enough for project screening and Pfizer global HTS activitiesSupported by local Pfizer Parallel Chemistry• Core expertise

HTS at Pfizer nowHTS at Pfizer now

uHTS capacity with Evotec and other platformsAutomated triage process based on • Clustering of actives and analysis of near neighbours• Hot monomer analysis• Parallel chemistry protocols• In Silico predicted physicochemical and ADME properties

Several libraries initiated on priority series• Several series / chemotypes progressed to reduce risk of

project attritionMonomer and template synthesis initiated where neededProtocol development where needed

HTS at Pfizer now – cont.HTS at Pfizer now – cont.

Iterations of library design are synthesised to address series issues versus project criteria

Designs include global and project specific in silico models

Measures of success: SandwichMeasures of success: Sandwich

96% of projects using parallel synthesis• 138 libraries of >96 compounds each across the

Sandwich discovery portfolio in 2003

Can identify multiple series to pursue from most HTS

Several leads from HTS & parallel synthesis currently in lead to CAN phase and early development

33% of clinical candidates resulted from FE enabled hits• applying multiple libraries (including structure based drug

design) & traditional medicinal chemistry synthesis

IKK2 inhibitors from HTS to multiple leadsIKK2 inhibitors from HTS to multiple leads

Weak HTS hits from early FE library• 2 active custom

templates• 5.6 billion potential

analogues• 4880 inactives in RL

6 rounds of library synthesisPotent IKK2 inhibition in acidic, basic and neutral series found

N

N

NH

NH

N

NNH

N

NH

NN NH

SO2NH2

O

N

NH

NN NN

NH

N

N N

N

NH

NH

NH

NMe

OH

O

N N

N

NH

N

NH

NMe

Me

UK-379,421IKK2 IC50 9.4 µM

UK-387,618IKK2 IC50 = 820 nMPBMC IC50 1.5 µM

1st loop693 compounds

UK-411,930IKK2 IC50 1.58 µM

UK-436,303IKK Ki 129 nM Kinase X IC50 1250 nMPBMC IC50 690 nMClean at 10 µM against HERG

UK-426,075IKK2 Ki 5 nM Kinase X Ki 184 nMPBMC IC50 >40 mM

N

N

Cl

Cl N

N

NR'2

NR"2

R R

1) R'2NH

2) R"2NH

Crystal Structure of UK-411,930 bound to CDK2 - SBLDCrystal Structure of UK-411,930 bound to CDK2 - SBLD

2.3A resolution data SRS, Daresbury.Key pyrazole Donor Acceptor Donor interaction

0

10

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40

50

60

0 1 2 3 4 5&6

Closed-loop iteration

% o

f co

mpo

unds

act

ive

at 1

0μM

1

10

100

1000

10000 activity of most potent com

pound (nM)

Overview of Closed-loops for IKKOverview of ClosedOverview of Closed--loops for IKKloops for IKK

Activity goes up with library iteration

Number of actives increases with library iteration

novel low MWt PDE-5 inhibitors from closed loop chemistry on HTS hitsnovel low MWt PDE-5 inhibitors from closed loop chemistry on HTS hits

Closed loop (CL) involves iterative design, synthesis, purification, sample logistics and screening loops

Combining singleton and exploratory library synthesis demonstrated that series was not flawed

Closed-loop synthesis identified 2-aminopyridine as novel PDE5 pharmacophore

2 closed loops from CP-X (HTS hit)• eliminated toxicophore, reduced MWt by over 200 Da, raised ligand efficiency, good

selectivity over 1, 9 and 6• found 5 other active templates in addition to UK-C

Aim: novel potent, selective, low clogP templates for o.d. PDE-5 i

Y

XO2N NHR1

NR2 R3

Y

XO2N NHR4

NR2 R3

Y

X NHR4

NHR5

Y

X NR6

NR8 R9

R7

193 compound library

264 compound library

MedChemSBDD?

CP-Xmwt 545clogP 5.47IC50 PDE5 33nMIC50 PDE6 30,000nM

UK-Amwt 439clogP 3.87IC50 PDE5 77nMIC50 PDE6 620nM

UK-Bmwt 309clogP 3.53IC50 PDE5 255nMIC50 PDE6 755nM

UK-Cmwt 335clogP 3.08IC50 PDE5 16nMIC50 PDE6 75nM

Structure Based Library DesignStructure Based Library Design

Templates

Favoured monomers e.g. 2-aminopyridines

Pendant ionisable centrefor solubility and SAR probe

Library design incorporated the positive structural filtersNegative filters of cLogP of 4 and MW of 400 were applied264/576 targeted compounds made.

N

NR'

R Amine 2

Amine1

NN

N

n PrN

NH N

N H

M e

NM e

Criteria UK-469413 Lead Target

PDE5 IC50 71nM32v6, 5v10, 5v113671.9, 3.318, ER<112-24 hours

≤ 50nMSelectivity >10x vs PDE’sMWt <400LogD, LogP 1-2, <3Caco abs. >10, ER<1Pred. Hu T1/2 >12 hours

PDE5 Library lead: UK-469,413PDE5 Library lead: UK-469,413

PDE5 2nd generation seriesPDE5 2nd generation series

UK-469,413

←Gln775 close to C7-aminopyridine methyl

NN

N

nPrN

NH

N

NH

Me

NMe 7

Gln817 H-bonds to C7-aminopyridine

Mike Palmer et al, Current Topics in Medicinal Chemistry, 2006, in press.

Profiles of selective PDE5 leadsProfiles of selective PDE5 leads

CriteriaPDE5 IC50PDE6 Sel.PDE11Caco2TPSAN,O count

Lead 20.80nM

42x 10x

18, ER 1939

NN

N

N

NH

N

N

EtO

ONHMe

Me

NH

Lead 30.50nM

104x 160x

2, ER>1012211

Great pharmacology, but… efflux problems.

NN

N

N

NH

N

N

EtO

Me

NH

Oxytocin hit–to-lead chemistryOxytocin hit–to-lead chemistry

HTS hits from FE project• Open ring by product of Ugi Benzdiazepinedione library• 2-Aminonicotinic acids yield non-cyclised materials

Library synthesis yielded 284 compounds

Most potent compounds confirmed as functional antagonists

O

NNH

N

Cl

ONH2 OO

O

NNH2

N

Cl

ONH2N

NH2

O N

O

CONH2

N

NH2

O NN

CONH2

UK-366045IC50 (n=2, Liquid): 470 nMIC50 (n=2, Solid): 377 nM

UK-366207IC50 (n=2, Liquid): 1860 nMIC50 (n=2, Solid): 1265 nM

HTS Actives

Rac-UK-458564IC50 (n=2): 49 nM

Rac-UK-458567IC50 (n=2): 86 nM

Round 1

Keating, T.A. & Armstrong, R.W. (1995) "Molecular diversity via a convertible isocyanidein the Ugi four-component condensation," J. Am. Chem. Soc. 117: 7842-7843

Resolution of Oxytocin library activesResolution of Oxytocin library actives

Resolution of library actives provided compounds meeting the target potency and selectivity criteria

Series accepted as leads for further development.

N

NH2

O N

O

CONH2

UK-469137IC50 7 nM (n=13) pA2 8.85MW 423 cLogP 3.1 CaCo2 flux 21

Racemic-UK-458564IC50 (n=2): 49 nM

Resolution

Round 1 actives:

UK-469138IC50 65 nM

Chiral HPLCResolution

Long Acting Beta 2 agonistsLong Acting Beta 2 agonists

Beta2 0.74 nM EC50 HTS hitParallel synthesis friendly project strategyEnabled rapid progress of the projectPartnering TA with PMC group

• Protected head groups

Optimised for potency and durationSalmeterol like potency and DOA foundExciting potential for long DOA in humans

OH

NH

OH

NH

O

N

OH

R3

R4

head Linker Tail

G 0.049

Compound NR3R4 Beta-2 EC50(nM)

NH

NH

MeO

H 0.020

INH

MeS

0.038

J NH

F

F

0.053

K NH

OH

0.164

L NH

SO2NH2

0.122

M 0.038

Compound NR3R4 Beta-2 EC50(nM)

NH

N

NH

S

N

N 0.031

O NH 0.058

P 0.022

QNH

OMe

0.254

R NH 0.051

N

Me

Maraviroc - Faster to CAN with parallel synthesisMaraviroc - Faster to CAN with parallel synthesis

UK-107,543 (HTS hit)400 nM CCR5 blocker

no antiviral activity CYP450 inhibitor

UK-427,857antiviral IC90 1 nM

selectiveno CYP450 inhibition

jun00dec97

N

N

N

N

Me

NNNH

O

NN

Me

Me Me

F

F965 analogues

Dorr et al, Antimicrob Agents Chemother., 2005, 49, 11 Armour & Wood, Progress in Medicinal Chemistry, 2005, 43, 239WO2003084954

CCR5 antagonist - MaravirocCCR5 antagonist - Maraviroc

Most final products made by HSA

Maraviroc first made in a filter tube with solid phase reagent• Blood rotator used for

agitation

Crude products purified by prep HPLC

N NN

NNH

R

Cpd

R AV IC90

(nM) HLM K+

channel LogD Polar

Surface Area (A2)

34 O

8 55 30% @ 300 nM

1.6 75.5

40 O

2 21 18% @ 300 nM

2.1 59.9

41 O

CF3

14 >120 14% @

300 nM 1.8 62.4

42 O

FF

0.5 51 0% @ 300 nM

2.1 77.3

NNNH2

NN

Me

Me Me

NNNH

NN

Me

Me Me

R

OCDI

RCO2HDCM RT o/n

NEP Parallel Chemistry StrategyNEP Parallel Chemistry Strategy

O

OH NH

OR

O

OH NH

OR

O

OH NH

OR

O

OH NH

OR

OMe

O

OH NH

O

O

R

OMe

O

O OH

O

R1

O

OH NH

O

R1

R2R2NH2

MWt. <400, LogD Between –0.5 and +1

+i) WSCDI, HOBtii) TFA

Initially retain a small R1(S1‘) based on known CandoxatrilatSAR

– start with nPr, for expediency and start racemic

Rapid exploration of R2 using parallel chemistry– diverse set of amines based on Desired drug properties

As SAR emerges, incorporate chiral and diverse R1 S1'

NEP inhibitor SARNEP inhibitor SAR

Successful and rapid SAR exploration

UK-447,841 discovered

Short half life prn dose regimen required T1/2 7-10h

High absorption (F)

O

OH NH

O

Cl

OMe

UK-447,841

R2 dNEP (nM) MWt. LogD Caco-2

24 339 0.5 9/14

R1

6 393 -0.2 -

100 375 1.0 6/10

nPrN N

SMe

O

OMe

OMe

F

Cl

OMeMe

(CH2)2OMe

(CH2)2OMe

(CH2)2OMe

(CH2)2OMe

4 333 0.8 21/21

3 379 0.5 12/17

0.5 396 1.0 12/14

David C. Pryde et al, J. Med. Chem. 2005WO2002002513

OverviewOverview

File Enrichment

Chemo informatics

High Throughput Chemistry

HTS

Enabled Hit Follow Up &Productive lead Discovery

ConclusionsConclusions

HTS hit follow up is supported by huge global resources

FE created a 3MM compound pure parallel chemistry rich file

Global Chemo informatics resources able to quickly mine 1 trillion potential molecules

Contract research to support early stage lead optimisation and SAR generation

AcknowledgementsAcknowledgements

Everyone!

Additional SlidesAdditional Slides

Pfizer Overview

Industry pressures

Pfizer OverviewPfizer Overview

World’s leading healthcare company• Biggest investor in R&D worldwide

– 2004 R&D investment: $7.5 billion• The largest foreign owned R&D investor in the UK

122,000 employees across 60 sites in 31 countries• sales in over 150 countries:• 12,000 R&D employees worldwide

– 2,700 people in R&D in UK• 6900 Pfizer UK employees• Biggest supplier of medicines to the NHS

INDUSTRY AVERAGES:

• 100 Screens Produce 12 Candidates• 1 in 12 survival rate during development • Only 1 of 4 products shows significant profit• 12 years from idea to market• 12-13 years of market exclusivity• Up to $800 million investment per product

Pre-Clin Phase I Phase II Phase III RegistrationMARKET

Animal Toxicity, Chemical Stability,Superior Compound

Human PK, Toleration,Formulation

Efficacy, SafetyDifferentiation, Dose

Long-term SafetyApproval?

The Attrition CurveThe Attrition Curve

11 22 33 44 55 >12 years>12 years

60%60%

25%25%

IdeasIdeas Leads Leads Dev. Candidates Dev. Candidates PhIIaPhIIa ClinClin. . ProductsProducts

25%25%

100 Projects 1 Product

% P

roje

cts

% P

roje

cts

25%25%