Combinatorial libraries: strategies and methods for …...Discovery chemistry: stage 1 • High Throughput Screening (HTS): – Rapid, automated screening of compounds for specific

Combinatorial libraries: strategies and methods for ‘lead’ discovery

Alan SpiveyDepartment of Chemistry

University of Sheffield

Key sources of information• WWW:

– Diversity information pages [http://www.5z.com/divinfo/]– J.Combinatorial Chem. [http://acsinfo.acs.org/journals/jcchff/index.html]– Combi. Chem. H.T.S. [http://www.bscipubl.demon.co.uk/cchts/index.html]

• Books– Combinatorial peptide and nonpeptide libraries-a handbook, Ed. G.Jung,

VCH, Weinheim, 1996.– Combinatorial chemistry-synthesis and application, S.R.Wilson, A.W.Czarnik,

Wiley, New York, 1997.

• Reviews– ‘Combinatorial chemistry’, Chem. Rev. 1997, 97(2), special issue.– ‘Combinatorial chemistry’, Curr. Opin. Chem. Biol. 1998, 2(3) & 1999, 3(3).– ‘Combinatorial chemistry’, S. Borman, Chem & Eng. News 1997, Feb24, 43.

Format and scope of lecture• What is combinatorial chemistry?• The drug discovery process• Approaches to combinatorial library

synthesis:– mix and split synthesis– parallel synthesis– encoded tagging

• Library types:– oligomeric libraries– template based libraries

• Combinatorial drug discovery!

What is combinatorial chemistry?

Combinatorial chemistry is a useful tool for rapidly optimizing molecular properties, particularly ones that are difficult to design a priori…

Nature uses a combinatorial approach to generate diverse functional macromolecules such as antibodies to recognize a vast array of antigens.

The drug discovery process

• The total cost of bringing a new drug to market is typically ~£250m (i.e. EXPENSIVE!)

• Of this, £170m is spent on DISCOVERY RESEARCH.

• This reflects the large amount of TIME involved in synthesising new compounds.

• A typical chemist can synthesise ~100 compounds a year using traditional techniques.

• SOLID PHASE ORGANIC SYNTHESIS (SPOS) and COMBINATORIAL CHEMISTRY are beginning to revolutionise this situation.

DiscoveryResearch

Pre-clinicaltrials

Clinicaltrials

Registration and launch

2-20 years8000-10000compounds

2-3 years20-30

compounds

3-5 years4-5

compounds

2-3 years1 compound

Pre-clinical candidates

Regulatory/ethical clearance

Registrationwith healthauthorities

Marketed Drug

Idea

Discovery chemistry: stage 1

• High Throughput Screening (HTS):– Rapid, automated screening of

compounds for specific biological activity.

• Role of combinatorial chemistry:– Very large libraries.– Maximum diversity libraries.– Mix and split libraries (& parallel

synthesis).– Mixtures of compounds (& single

compounds).

DiscoveryBiology

Automation ofassay

Biological assay established

Assay adapted forHigh Throughput Screening (HTS)

Lead structure

Pre-clinical candidate

Therapeutic area identified

MedicinalChemistry

CompoundHTS

Combinatorial Chemistry

CombinatorialChemistry

Discovery chemistry: stage 2

• Medicinal chemistry:– Systematic optimisation of molecular

and physicochemical properties of lead compound

• Role of combinatorial chemistry:– Small libraries.– ‘Targeted/focussed’ libraries.– Parallel synthesis libraries.– Single compounds.

DiscoveryBiology

Automation ofassay

Biological assay established

Assay adapted forHigh Throughput Screening (HTS)

Lead structure

Pre-clinical candidate

Therapeutic area identified

MedicinalChemistry

CompoundHTS

Combinatorial Chemistry

CombinatorialChemistry

Traditional vs. combinatorial• Traditional synthesis:

• Combinatorial synthesis:

A + B AB

compounds prepared one at a time, characterised and screened

reaction of 3 reagents Ax with 3 reagents By provides a library of 32 (i.e. 9) compounds AxBy

introduction of a third set of 3 reagents Cz increases the library size to 33 (i.e. 27) compounds AxByCz

A1-3 B1-3+A1B1

A2B1A3B1

A1B2

A2B2A3B2

A1B3

A2B3A3B3

Approaches to ‘combinatorial’ library synthesis

• In vivo - biological methods:– Phage display, plasmids, polysomes etc.

• In vitro - synthetic methods:– Mix and split using Solid Phase Organic Synthesis (SPOS).

• Cleavage from the solid support following ‘mix and split’ results in complex mixtures (pools) of compounds. Screening of these mixtures yields ‘hits’ whose identity must be determined by ‘deconvolution’.

• If screening can be performed ‘on-bead’ (i.e. ‘one-bead one-compound’ libraries) then deconvolution can be avoided.

– Parallel synthesis using Solid Phase Organic Synthesis (SPOS).• Spatially separate synthesis of single compounds whose identity is

uniquely defined by their location.

Mix & split synthesis: libraries of mixtures of compounds in solution

• Screening complex mixtures of compounds in solution can give false ‘hits’ due to synergistic effects.

• Identification of a compound within the mixture responsible for the ‘hit’ requires iterative deconvolution.

• Houghton Nature, 1991, 354, 84.

Deconvolution

Identity of hit

A1A2

A3

A1 A2 A3

A1B1

A2B1A3B1

A1B2

A2B2A3B2

A1B3

A2B3A3B3

A1B3C1

A2B3C1A3B3C1

A1B3C2

A2B3C2A3B3C2

A1B3C3

A2B3C3A3B3C3

A1B2C1

A2B2C1A3B2C1

A1B2C2

A2B2C2A3B2C2

A1B2C3

A2B2C3A3B2C3

A1B1C1

A2B1C1A3B1C1

A1B1C2

A2B1C2A3B1C2

A1B1C3

A2B1C3A3B1C3

B1 B2B3

C1 C2C3

Mix & Split

Mix & Split

Split

31=3

32=9

33=27

Mix & Cleavefromresin

'Pool' of allcompoundsin solution 'Hit'

Screenmixture

Mix & split synthesis: ‘one-bead one-compound’ libraries

• Requires a very sensitive screening protocol which can accommodate resin bound compounds.

• Identification of a ‘hit’ compound on (or from) a single bead (~100pm) by: – analytical methods e.g. Edman

sequencing of peptides, MALDI-TOFMS, single bead NMR…

– reading ‘encoding tags’ on beads.

Identity of hit

Analyticalidentificationof compound

A1A2

A3

A1 A2 A3

A1B1

A2B1A3B1

A1B2

A2B2A3B2

A1B3

A2B3A3B3

A1B3C1

A2B3C1A3B3C1

A1B3C2

A2B3C2A3B3C2

A1B3C3

A2B3C3A3B3C3

A1B2C1

A2B2C1A3B2C1

A1B2C2

A2B2C2A3B2C2

A1B2C3

A2B2C3A3B2C3

A1B1C1

A2B1C1A3B1C1

A1B1C2

A2B1C2A3B1C2

A1B1C3

A2B1C3A3B1C3

B1 B2B3

C1 C2C3

Mix & Split

Mix & Split

Split

31=3

32=9

33=27

Mix &screen

'on-bead'Isolate

'hit'bead

Clark Still’s encoded tagging protocol• Still Acc. Chem. Res. 1996, 29, 155.

• Each ‘monomer’ used in the library synthesis has an associated encoded tag.

• The tags are chlorinated aromatic compound which can be analysed at sub-picomolar levels by Electron Capture Gas Chromatography (ECGC).

• Allows for hit identification at one-bead fidelity for any type of library

A1 + 1%T1

A1

B1 + 1% T4 Mix & Split

Split

Identity of hit

Mix &screen

'on-bead'

Isolate'hit'

beadscleave tag by oxidation

and 'read' by ECGC

A3 + 1%T3

A2 + 1%T2T1

A2

T2

A3

T3

B2 + 1% T5

B3 + 1% T6

A1B1

T1

A2B1

T2

A3B1

T3

T4

T4

T4

A1B2

T1

A2B2

T2

A3B2

T3

T5

T5

T5

A1B3

T1

A2B3

T2

A3B3

T3

T6

T6

T6

Mechanism of Clark Still encoded tags• Still J. Org. Chem. 1994, 59, 4723.

N2

O

O

OMe

OClmn

10 different α,ω-diols (n = 3-12)4 different chlorophenols (m = 2-5)

40 different tags

[Rh]O

O

OMe

OClmn

OR

Rh(O2CCF3)3CH2Cl2, Δ

O

OO

MeOClm

n

ORCH2Cl2

Buchnerreaction

1) ceric ammoniumnitrate (CAN)

2)

Me

OTMS

NTMS

OTMSOClm

n

'read' usingElectron Capture Gas Chromatography

Parallel synthesis: spatial separation gives single compounds

• Suitable for the synthesis of relatively small libraries as each compound requires its own reaction ‘well’.

• Each reaction ‘well’ may be anything from a small flask to a radio-frequency tagged ‘tea bag’ to an etched region on a silicon chip!

• Once screening has identified a hit no further work is required to deduce the identity of the active compound although it is routine practice to independently verify the structure.

Identity of hit

Spatial locationof hit defines

its identity

'Hit'

Screeneach

'position'

A1A2

A3

A1 A2 A3

A1B1

A2B1

A3B1

A1B2

A2B2

A3B2

A1B3

A2B3

A3B3

A1B1C1 A1B1C2 A1B1C3

B1 B2 B3

Split

Split

C1 C2 C3

etc (x27)...

Split± cleaveagefrom resin

A1B1C1 A1B1C2 A1B1C3

spatially separated libraries

etc (x27)...

or

Keeping track of ‘tea-bag’ parallel synthesis: Irori radio-frequency tagging

• http://www.irori.com/

Library types• There are essentially two strategically distinct types of library at the

molecular level:

Buildingblock 1

Buildingblock 2

Buildingblock 3

Buildingblock 4

Oligomeric

peptides/peptoidsoligonucleotidesoligosaccharidesunnatural oligomerspolyaromatics

Template

Buildingblock 1

Buildingblock 2

Buildingblock 3

Buildingblock 4

Template based

drug-like moleculesnatural product-like moleculesheterocycle based molecules

Balasubramanian’s peptide library• Protein tyrosine phosphatase substrate library (oligomeric).• Balasubramanian J. Am. Chem. Soc. 1997, 119, 9568.• Library synthesis:

NH2

1) Fmoc-Aaa-OH, PyBOP,HOBt, DIPEA2) piperidine

ncycles

K

amino methylfunctionalisedKieselguhr

N-G-Q-Q-P-I-L--Xaa-Xaa-A-Xaa-Fmoc

OPO3H

mix and splitwith encoding

Tx

K

one-bead one-peptideencoded libraryencoding via dummy peptide sequence incorporating

glycine in place of phosphotyrosine

Balasubramanian’s peptide library• Protein tyrosine phosphatase substrate library (oligomeric).• Balasubramanian J. Am. Chem. Soc. 1997, 119, 9568.• Library screening:

1) leukocyte antigen receptor protein tyrosine phosphatase (PTP)2) α-chymotrypsin3) fluorescent labelling of N-terminus with carboxyfluorescein:

N-G-Q-Q-P-I-LK

F

F

Tx

Hits

sequenceencoding tag peptide

Identity of PTPsubstrate sequences =

N-G-Q-Q-P-I-L--X3aa-X2aa-A-X1aa-Fmoc

OPO3H

Tx

K

one-bead one-peptideencoded library

"preference for at least two acidicresidues in variable positions anda glutamic acid residue at X1aa"

Beck-Sickinger’s cyclic peptide library• Neuropeptide Y analogue library (oligomeric).• Beck-Sickinger J. Org. Chem. 1999, 64, 4353.• Library synthesis:

wOH

NHFmoc

CO2Me

HO

PPh3, DEADMitsunobu

wO

NHFmoc

CO2Me

1) piperidine2) Fmoc-Aaa-OH, DIC, HOBt3) piperidine

ncycles

wO

NH-R-Q-R-Xaa-K-S-P-Y-Xaa-H

CO2Me

1) LiOH2) TBTU, HOBt, DIPEA

wO

NH-R-Q-R-Xaa-K-S-P-Y-Xaa

O

TFAXaa-Y-P-S-K-Xaa-R-Q-R

parallel synthesis

spatially separatedsolution library

Wang resin

• Neuropeptide Y analogue library (oligomeric).• Beck-Sickinger J. Org. Chem. 1999, 64, 4353.• Library screening:

Beck-Sickinger’s cyclic peptide library

Xaa-Y-P-S-K-Xaa-R-Q-R competitive binding assay in solutionwith radiolabelled neuropeptide Y Hits

spatially separatedsolution library

identity defined byspatial location

"weak competitive binding

at μM level by range of derivatives"

Oligonucleotide libraries

• These oligomeric libraries are generally prepared using in-vivo‘biological’ methods and screened using Systematic Evolution of Ligands by Exponential enrichment (SELEX) procedures.

• e.g. The discovery of very high-affinity RNA and DNA ligands to human IgE which inhibit binding to the Fcε receptor I.

• Wiegand J. Immunology 1996, 157, 221 (and references therein).

Unnatural backbone oligomer libraries

NH

OHN

O

O

R2O

R1

Backbone

oligocarbamate

Monomers

FmocNH

O OPNP

O

R

OO

PO

OP

R2

R1

oligo-phosphodiester

O O

O O

NH

SHN S

R2

R1

vinylogous sulfonamidopeptide

BocNH

SO2Cl

R

O O

O O

N N N

vinylogous sulfonamidopeptide

R1

NCO

PO

ODMTrRNPr2

O O O

R3R2Cl

O+ R-NH2

Schultz’s purine library• Kinase inhibitor library (template based).• Schultz Science 1998, 281, 533.• Library synthesis:

OOH

Mitsunobureaction

O N

NN

N F

Cl

N

NN

N F

Cl

HR1NH2 2

6

O N

NN

N F

NH

Nucleophilicsubstitution

(C-2)

R2NH2

R1

O N

NN

N NH

NHR1

parallel synthesis

R2

N

NN

N NH

NHR1

R2

OH

TFA

spatially separatedsolution library

PS resin with acid labile alcohol linkerpre-functionalised with hydroxyethyl

group

Schultz’s purine library• Kinase inhibitor library (template based).• Schultz Science 1998, 281, 533.• Library screening:

Screen each positionfor inhibition of

h-CDK2-cyclin A kinase complexHits identity defined by

spatial location

N

NN

N NH

NHR1

R2

OH

spatially separatedsolution library

N

NN

N NH

HN Cl

OH

6

92

R3

3 & 4 substituted anilines

& benzylamines

R4

Ala, Val or Ilederived amino

alcohols

small alkyle.g. Me

Schreiber’s ‘natural product’ library• Protein epitope binding library (template based). • Schreiber J. Am. Chem. Soc. 1998, 120, 8565.• Library synthesis:

NH2PyBOP, DIPEA

NMP, rt.

Me

O

O

OH

Me

O

O

NH

HO

ONO

HATU, DIPEADMAP, rt

ON

O

O

O

HO

HN

H

H

II

R1

Pd(II)/Cu(I)

ON

O

O

O

HO

HN

H

H

R1

ON

HO

OO

HN

H

R1O

R2HNR2NH2R3CO2H

DCC, DMAP

ON

O

OO

HN

H

R1O

R2HN

R3

O

mix and splitwith Clark Still

encoding

one-bead one-compoundencoded library

T

TT T

PS withphotolabile

amide linker

Schreiber’s ‘natural product’ library• Protein epitope binding library (template based). • Schreiber J. Am. Chem. Soc. 1998, 120, 8565.• Library screening:

ON

O

OO

HN

H

R1O

R2HN

R3

O

one-bead one-compoundencoded library

hv

ON

O

OO

NH2H

R1O

R2HN

R3

O

compound from singlebead in 'nano-droplet'

T

T

screen forprotein binding

Hits

'read' usingElectron Capture Gas Chromatography

Identity of hit

"...several members of thislibrary activate a reportergene in mink lung cells!"

Nicolaou’s sarcodictyin library • Tubulin-microtubule disruptant library (template based). • Nicolaou J. Am. Chem. Soc. 1998, 120, 10814.• Library synthesis:

O

Me

Me Me

H

H

Me

OH

OAc

OTIPSO

OH3 PPTS, CH2Cl2

O

Me

Me Me

H

H

Me

O

OAc

OTIPS

parallel synthesis

1) NaOMe2) R1-LG O

Me

Me Me

H

H

Me

O

OR1

OTIPS1) TBAF2) Dess-Martin3) NaClO24) R2-LG

O

Me

Me Me

H

H

Me

O

OR1

O OR2

PPTS, R3-OHO

Me

Me Me

H

H

Me

O

OR1

O OR2

R3

spatially separatedlibrary in IRORI

'tea bags'

α,ω-diol functionalisedhydroxymethyl-PS

Nicolaou’s sarcodictyin library • Tubulin-microtubule disruptant library (template based). • Nicolaou J. Am. Chem. Soc. 1998, 120, 10814.• Library screening:

O

Me

Me Me

H

H

Me

O

OR1

O OR2

R3

spatially separatedlibrary in IRORI

'tea bags'

screen for induction of tubulin polymerisationand

cytotoxicity with ovarian cancer cells Hits identity defined byspatial location

O

Me

Me Me

H

H

Me

OH

O OMe

o

N

NMe

side chain crucial

both N'simportant

ketals tolerated

esters prefered over amides; reduction to alcohol not tolerated

DeWitt’s quinolone library• Ciprofloxazin analogue library (template based). • DeWitt Tet. Lett. 1996, 37, 48115, and patent: WO 94/08711, 1994.• Library synthesis:

O

O O

N

F

FF

1)2) R1-NH23) Δ

MeO OMe

NMe2

THFO

O O

N

F

FNR1

R2-NH2NMP, Δ

O

O O

N

F

NH

NR1

R2

TFAHO

O O

N

F

NH

NR1

R2

W W

W

parallel synthesis

spatially separatedlibrary in DIVERSOMER

reaction tubes

Wang resin

OHW

DMAPtolueneEt-ester

DeWitt’s quinolone library• Ciprofloxazin analogue library (template based). • DeWitt Tet. Lett. 1996, 37, 48115, and patent: WO 94/08711, 1994.• Library screening:

HO

O O

N

F

NNR1

R2

spatially separatedlibrary in DIVERSOMER

reaction tubes

Screen for gyrase inhibition in solution Hits identity defined byspatial location

HO

O O

N

F

NN

R3

NH

ciprofloxazin

Gallop’s mercaptoacyl proline library

O

O

O

O

T T

mix and split

TentaGelresin

OHTR1

NHFmoc N Ar

R1

1) 20% piperidine, DMF2) ArCHO

3) CH(OMe)34) Ac2O, DIPEA

DCC-DMAP

ZAgNO3, Et3NMeCN

O

O

THN Ar

ZR1

AcS R2 COCl1)

2) TFA3) ethylenediamine

HO

ON Ar

ZR1

O R2 SH

480 member poolof compounds

• Angiotensin Converting Enzyme (ACE) inhibitor library (template based). • Gallop J. Am. Chem. Soc. 1995, 117, 7029.• Library synthesis:

Gallop’s mercaptoacyl proline library• Angiotensin Converting Enzyme (ACE) inhibitor library (template based). • Gallop J. Am. Chem. Soc. 1995, 117, 7029.• Library screening:

HO

ON Ar

ZR1

O R2 SH

480 member poolof compounds

Screened forACE inhibition

DECONVOLUTIONVIA FOUR ITERATIONS OF

SUB-LIBRARY RE-SYNTHESIS AND SCREENING

N

O

HS

CO2Me

HO2CMe

Ki ~ 160pM3 x more potent than captopril

Ellman’s benzodiazepine library• Benzodiazepine library (template based). • Ellman J. Org. Chem. 1997, 62, 2885.• Library synthesis:

NH2

aminomethyl-PS

NHBpoc

SnMe3SiMe Me

parallel synthesis

1) R1COCl, Pd(0)2) TFA

NH2

SiMe Me

O

R1

FOC NHFmoc

R21)

2) 20% piperidine DMF3) 5% AcOH, Δ

SiMe Me

N

HN

O

R2

R1

1) RLi2) R3-Br, DMF

SiMe Me

N

NO

R2

R1

R3

HFN

NO

R2

R1

R3

H

spatially separatedsolution library

Ellman’s benzodiazepine library• Benzodiaxepine library (template based). • Ellman J. Org. Chem. 1997, 62, 2885.• Library screening:

SiMe Me

N

NO

R2

EWG

R3N

NO

R2

R1

R3

H

spatially separatedsolution library

screening of this library was not reported because it pertained that some of the compounds in the library still

contained silicon due to an anomolous cleavage mechanism which was particularly troublesome when R1

was an electron withdrawing substituent

F

Summary• What is combinatorial chemistry?• The drug discovery process• Approaches to combinatorial library synthesis:

– mix and split synthesis– parallel synthesis– encoded tagging

• Library types:– oligomeric libraries– template based libraries

• Combinatorial drug discovery.