Drew Residential Schoolon Medicinal Chemistry

Chemical Diversity

Generation and Use in Drug Discovery

Philip F. Hughes

InnovaSyn, LLC

Chapel Hill, NC

Chemical Diversity Generation and Use in Drug Discovery

OverviewReasons, History, Economics, Definitions

Combinatorial Chemistry/ Parallel SynthesisSynthesis Methods

split/mix, array

solid phase, solution phase

Equipment

Purification Methods

Analytical Methods

Conclusions

Why Chemical Diversity?

ReasonsThe biggest reason for continued interest in

Chemical Diversity is the recent ability of scientists to evaluate very large numbers of molecules in biological systems.

i.e.

High Throughput Screening

High Throughput Screening

Biotechnology

Genomics

Computers

Robotics

Chemistry

synergy

Current Screeningcapacities of

2000-100,000Samples/Day

in multiple assays

Where will the Samples come from?

History

1990:

A medicinal chemists made

2-6 compounds / month

at $2,500-$10,000 / compound

Compounds were tested once in a single assay.

Leftover compound sent for storage

Old Molecular Diversity

Company Chemical Storage20,000-400,000 compounds, many similar

classes, some >100 yrs. old

Natural Productslarge number, not clean,

test as “mixtures”

Classical Medicinal Chemistrytoo slow or too expensive

New Requirements

We need to increase the compound synthesis rate by

20 to 1000 foldThis is less than the increase in

screening capacity because we’re now willing to test each compound in numerous assays

Going Faster

4 Ways to go Faster1. Use Combinations

• Reuse

2. Do many things at the same time• Parallel processing

3. Speed up the process

4. Get someone else to do it• Automation• Outsourcing

The Answer

Combinatorial Chemistry

Combinatorial chemistry is a technology through which large numbers of structurally distinct molecules may be synthesized in a time and resource-effective manner, and then be efficiently used for a variety of applications

Nick Terrett

From the Tetnet page on Elsevier.com

Two Major Approaches

Split & Mix“Real Combinatorial Chemistry”

Array Synthesis“Parallel Synthesis”

“Spatially-Addressable Synthesis”

“Matrix Array Synthesis”

Split & Mix

Originated in peptide synthesisSimple efficient chemistry (amides)

Long linear sequence of reactions

Solid Phase approaches known

H2NNH

HN

NH

HN

OH

R1-i

O R1-j

O R1-k

O R1-l

O R1-m

O

# of reagents = 10# of reactions = steps ● reagents; 5 ● 10 = 50# of products = reagentssteps; 105 = 100,000

Split & Mix

# of reagents = 3# of reactions =3+ 3 + 3 = 9# of products = 3 x 3 x 3 = 33 = 27

A Big Mixture

A

n

A

C

B B

C

AB

BB

CB

Pool

AA

BA

CA

AC

BC

CC

AAB

BAB

CAB

AAA

BAA

CAA

AAC

BAC

CAC

ABB

BBB

CBB

ABA

BBA

CBA

ABC

BBC

CBC

ACB

BCB

CCB

ACA

BCA

CCA

ACC

BCC

CCC

A

C

B Pool

A

C

B

n/3

n/9n/27

Dealing with Mixtures

Options• Test as a mixture

Encoded Libraries• Tags

• Nucleotide• Chemical

• Labeled reactors

Big Mixture Testing

Deconvolution generally requires repeated synthesis of smaller and smaller mixtures followed by retesting.

This only made sense back when screening capacity was limited.

www.mixturesciences.com - positional scanning

1024

512

512

256

256

128

128

64

64

32

32

16

16

8

etc.

Nucleotide Tags

Beads selected based on binding to target

Nucleotide “code” can be defined for natural or unnatural monomers

Nucleotide sequence can be amplified by PCR

1. S. Brenner, R. A. Lerner, Proc. Natl. Acad. Sci. USA, 89, 5381-5383 (1992)

2.. M. C. Needels, d. G. Jones, E. H. Tate, G. L. Jeinkel, L. M. Kochersperger, W. J. Dower, R. W. Barrett, M. A. Gallop. Proc. Natl. Acad. Sci. USA, 90, 10700-10704 (1995)

Nucleotide

Peptide

Chemical Tags - Pharmacopeia

Example: Arylsulfonamide inhibitors of Carbonic Anhydrase

• 7 X 31 X 31 library: 6727 members (R1-R2-R3)• Each reagent encoded by a unique combination of 3-5 tags based on a binary

code: coding 2n-1 members requires n tags• Tag incorporated by Rh-catalyzed carbene insertion into polymer C-H• Tags released from oxidatively labile linker with (NH4)2Ce(NO3)2, followed by

Electron Capture GC (silylated tags)

H2NO2S

HN

O

NCO2H

O

R3 R2R1

ON2

O

OCH3

(CH2)nOAr

Cl Cl

Cl

ClCl

Cl

Cl

Cl

Ar =

n = 3-12 n = 4-6 13 tags total

Chemical Tags - Pharmacopeia

M.H.J. Ohlmeyer, R.N. Swanson, L. W. Dillard, J.C. Reader, G. Aronline, R. Koabyashi, M. Wigler, W. C. Still, Proc. Natl.Acad. Sci. USA, 90, 10922-10926 (1993).

J. J. Baldwin, J. J. Burbaum, I. Henderson, M. H. J. Ohlmeyer, J. Am. Chem. Soc., 117 5588-5589 (1995).

Pharmacopeia’s web site www.pcop.com ECLiPS™ encoding technologyICCB at Harvard iccb.med.harvard.edu/

T1T3

T2

T3

T1

T2

T2

T2

T1

AA

C

B B

C

AB

BB

AA

BA

AC

BC

A

C

B

T2

T1

T4

T2

T3

T1

CB

CC

CAT1

T3

T4

T3

T3T4

n

Pool

T1

T2

T1T2

T3

T4

T3T4

Chemical Tags - Pharmacopeia

1. Clip off compounds for testing

2. Clip off tags for analysis

1. 2.

T1

T3

T2

T3

T3

T2

AB

AA CB

CC

T1

T3

AA

T2

T3

AB

T4

T2

CB T3

T2

T1

CCT3

T4

T4

T1

T2

T3T4

(23-1)•(25-1)•(25-1) = 7•31•31 = 6727 compounds3 + 5 + 5 = 13 tags7+31+31=69 reagents, 69 x 2 = 138 reactions

Labeled Reactors Radio Encoded Tags - Irori

www.irori.com Discovery Partners International

Labeled Reactors Radio Encoded Tags - Irori

Similar to resin split and mix except that each reactor can is tracked throughout the synthesis. Each product is made once and each can contains only one product. Irori calls this “directed sorting”, which has been automated

A similar package is available from Mimotopes

www.mimotopes.com Now owned by Fisher Scientific

Split and Mix Synthesis Points

Large diversity requires but can also utilize a longer synthetic sequence

Generally makes a smaller amount (pM to nM) of a greater number of compounds

Efficiency requires multiple sites (3 or more) of diversity

Data handling and analysis can be complex

Generally applicable to only solid phase synthetic approaches

A

n

A

C

B B

C

AB

BB

CB

Pool

AA

BA

CA

AC

BC

CC

AAB

BAB

CAB

AAA

BAA

CAA

AAC

BAC

CAC

ABB

BBB

CBB

ABA

BBA

CBA

ABC

BBC

CBC

ACB

BCB

CCB

ACA

BCA

CCA

ACC

BCC

CCC

A

C

B Pool

A

C

B

n/3

n/9n/27

Array Synthesis

Use parallel synthesis in a matrix format (8 x 12 array) - 20 reagents with 1 or 2 reactions gives 96 products

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

HO

N

NHRA--H

O

R1-12

OH

RA--H NH

R1-12

O

RA--H NH

R1-12

Large Array Synthesis

Larger numbers of compounds are available from one scaffold or reaction scheme

Lay out a Super Grid• 72 X 72 reagents or wells

• 9 X 6 plates = 54 plates

• 5184 compounds

• Chemists make multiple plates at a time

• Need 72 + 72 reagents

A1 A2

B1 B2

C1 C2

Reagents

8 X 12 Plates

Array Synthesis Points

• Large diversity requires but can also utilize the large diversity of commercially available reagents

• More efficient when an array of reactions is treated as a unit – parallel processing

• Efficiency requires at least 2 sites of diversity

• Data handling simpler - one site, one compound

• Applicable to both solid and solution phase synthetic approaches

• With micro-titer plate format, one can borrow equipment from biologists (a first)

• Efficiencies gained in matrix format make this a combinatorial technique

• Make greater quantities (uM to mM) of fewer compounds

1 2 3 4 5 6 7 8 9 10 11 12

A

B

C

D

E

F

G

H

Solution and Solid Phase Organic Chemistry

Definitions for the sake of discussion:

• Solution Phase Organic Chemistry is chemistry like it used to be (pre 1990).

• Solid Phase Organic Chemistry (SPOC) is chemistry where some part of the target molecule is covalently attached to an insoluble support somewhere during the synthetic sequence.

• Solid Phase Reagents (SPR) are insoluble reagents used in solution phase chemistry (like 10% Pd/C or polyvinyl pyridine). They (SPR’s) may be made using SPOC. They (SPR’s) have also made solution phase combinatorial chemistry easier.

Solid Phase Organic Chemistry

•Core is usually 1% crosslinked polystyrene•Spacer, if present, is usually a polyethylene glycol

TentaGelTM, or ArgoGelTM (www.argotech.com)Give more solution-like reactivity with lower resin loading

•Linker, if present, provides an orthogonal method for releasing the scaffold

•Scaffold is the part that you’re interested in doing chemistry on and releasing at the end of the synthesis

OO

O

O

S

Br

NO2

Core Spacer Linker Scaffold

n

Linkers

CL

Merrifield

O

OH

Wang

O

OCH3

H3CO

(N or O)

O

R

Rink amideRink acid

NH

O

NO2

O O or R

O

Greenbergphotolinker

SiN

N

R3

O

R1

R2

H3CCH3

Ellman

O

N

NH

O

RA--HO

R1-12

O

O

NH2

NH

O

RA--HO

O

NO2

NH

O

RA--HO

O

NO2

NH2

OCO 2H

HO

NO2

NH2

RA--HCl

O

HO

N

NHRA--H

O

R1-12

O

DMF

DMF

DIC, pyridine, DMAP

SnCl2. (H2O)2

1) n-BuLi, TMEDA

cyclohexane

2) CO 2

NaOCH3

4:1 THF/MeOH

pyridine, DMAP CH2Cl2

R1-12Cl

O

pyridine, DMAP CH2Cl2

HH

An Example

H. V. Meyers, G. J. Dilley, T. l. Durgin, T. S. Powers. N. A. Winssinger, H. Zhu, M. R. Pavia, Molecular Diversity,1,13020 (1995)

Reaction Path

Acylation Cleavage

Expose

Wash

Wash

Wash

Collection &

purificationSubmit

Analysis

1,3

2

496-well reactor

Solid Phase Organic Chemistry

Products are insoluble• Easier to manipulate physically

• Easier to clean up, can wash exhaustively

• Can use excess reagents to drive reactions to completion

• No bimolecular reactions (infinite dilution)

• Can’t use Solid Phase Reagents (SPR)

• Modified kinetics (generally slower, greater rate distribution, all sites not equal)

• Requires new analytical methods

• Requires linking chemistry (limits reaction conditions, constrains product structure)

Solution Phase Organic Chemistry

More compounds means less time per compound

This requires:• Good generalized procedures

• Short synthetic sequences

• High yield reactions

• Stoichiometric addition of reactants

• Parallel or high throughput purification methods

Solution Phase Organic Chemistry

Multiple Component Condensation Reactions

Armstrong, R.W., Combs, A.P., Tempest, P.A., Brown, S.D., & Keating, T.A. Account. Chem. Res., 29, 123-131 (1996).

H2N NH2

O

R2

O

O

O

R3+ R1CHO +

NH

NH

O

R2

R1

O

OR3

R1COOH + R2NH2 + + R5NC

O

R3 R4

R1 NR2

O

HN

O

R5

R3 R4

NH2

Biginelli

Ugi

+ R1CHO +

R2R4

R5

R6

R3

NH

R2

R3 R4R5

R6

R1GriecoKobayashi

R1

N

R2R3

Solution Phase Organic Chemistry

N O

O2N

Br

OH

N

O2N

R2

CO2Me

NR1 O

O2N

R2

R3

R2CO2Me

(CH3O)2

P

O

CO2CH3

R3-NH3 AcOH

EtOH, refluxH3C-NO2

R2CHO

R1CHO

DBUKOMe

6 Nitrobutyrates

3072Compounds

Single isomer> 95%

Shinji Nara, Rieko Tanaka, Jun Eishima, Mitsunobu Hara, Yuichi Takahashi, Shizuo Otaki, Robert J. Foglesong, Philip F. Hughes, Shelley Turkington, and Yutaka Kanda. J. Med. Chem.; 2003, 46, 2467-2473

IC50 = 420 nM FTaseCompetitive Inhibitor

N

O2N

OH

N

OH

Br

IC50 = 1.9 nM FTasefor enantiomer shown

iterate

Solution Phase Organic Chemistry

Products are soluble• Byproducts and excess reagents are also

soluble and accumulated with each step• Direct analysis is much easier (tlc, nmr, ms,

hplc, lc/ms)• Kinetics are uniform and familiar• Use of solid phase reagents (SPR’s) is possible• No linkers required, less excluded chemistry• Requires development of parallel workup and

purification methods

Called Parallel Synthesis or Rapid Parallel Synthesis (RPS)

Trends over the Last Decade

0

1000+

# of Compounds

timeDev. times for

solid phase

Sld P Array

Sld P S&M

Solu P Array2004

Classical Organic

Synthesis

Solu P Array1996

10,000+

Solution Phase Array or Parallel Synthesis now dominates

Equipment for Solid Phase Organic Chemistry

Split & MixStandard labware with gentle stirring

ArrayGeyson Pin Approach

Bottom filtration

Top filtration

Little stuff

Big stuff

Geysen Pin Method

Resins attached to pins in an 8 x 12 array format

Reagents or wash solvents in a 96 deep-well plate

Drop it in to run reactions or wash resins

Kits available from Mimotopeswww.mimotopes.com

Equipment for Solid Phase Organic Chemistry

Problem: How do you put 24-96 of these together?

Bottom Filter Top Filter

Solid Phase Chemistry Reactor

Plate in a Plate Clamp

Original Sphinx Reactor

Plate Bottom acts as a 96-way valve

Strip Caps used to seal reaction after reagent addition

Plate removed from clamp for resin washing

H.V. Meyers, G.J. Dilley, T.L. Durgin, et al Molecular Diversity 1995, 1, 13-20

Commercial Apparatus for Solid Phase

FlexChemwww.robsci.com

www.scigene.com

Charybdis Technologieswww.spike.cc

Polyfiltronics/Whatmanwww.whatman.com

MiniBlockwww.bohdan.com

www.Autochem.com

ArgonautQuest 210Nautilus 2400Trident

Bohdan Ram

Tecan Combitec

Advanced Chemtech 496

Myriad Core

All Discontinued Big stuff is a bad idea.

Little Stuff

Big Stuff

Parallel Solution Phase Organic Synthesis

Equipment – An Array of Vessels• Heating and cooling• Mixing• Inert Atmosphere• Access for addition and sampling

Methods• Reactants and reagents added as

solutions or slurries• Run at equimolar scale• Separate the reaction from the workup

Equipment for Parallel Solution Phase Organic Synthesis

One at a time Synthesis

Parallel Synthesis

Equipment for Parallel Solution Phase Organic Synthesis

Generic Reactor Block

Equipment for Solution Phase Organic Synthesis

Reactor Blocks

Equipment for Solution Phase Organic Synthesis

MicroWave

http://cemsynthesis.comBiotagehttp://www.personalchemistry.com/

Solution/Slurry Addition

1

5

4

32

eppendorf

Eppendorf Repeater Pipette• Good for Repeated Additions of one

Solution• Disposable Polypropylene Syringe

Barrels• Easily adaptable to Leur fittings

(needles)• Can deliver from 0.5 uL to 5 mL• Inexpensive and Fast (better than

robots)• Can Deliver Slurries with Modifications

Solid Addition

Solid addition plates/Vacuum systems

Solid as a slurry• 10% Pd on Carbon in Ethanol

• NaHB(OAc)3 in Dichlorethane

• Resins as isopycnic slurries

Purification Methods

Solid Phase• Wash exhaustively

• product dependent cleavage

Solution Phase - Parallel Purification• Extraction

• liquid-liquid, acid/base

• SPE, scavenging resins

• Fluorous Synthesis

• Chromatography

Scavenging Resins

N

N

NH

O

NH

O

BuO

O

S. W. Kaldor, J. E. Fritz, J. Tang, E. R. McKinney, Biorganic & Med. Chem. Lett.., 6,3041-3044 (1996).

NH2

R1 N C O

R2 NH2

+CHCl3, RT, 3h

NH

NH

O

R2R1

NH

NH

R1

O

1 eq.

1.5 eq.

Fluorous Synthesis

Fluorous (C6F12) Phase

Aqueous Phase

Halocarbon (CH2Cl2) Phase

D. P. Curran, M. Hoshino, J. Org. Chem., 1996, 61, 6480-6481.D. P. Curran and Z. Luo, Fluorous Synthesis with Fewer Fluorines (Light Fluorous Synthesis): Separation of Tagged from Untagged Products by Solid-Phase Extraction with Fluorous Reverse Phase Silica Gel, J. Am. Chem. Soc., 1999, 121, 9069. http://fluorous.com

O S

NO2

Br

O

(CF2)n O S

NO2

Br

O

CF3

Replace resin with fluorous handle

Liquid Handling RobotsA Primer

6-Way Valve

Syringes

Tip

10 mL Loop

XY

Z

Tees

RobotArm

System Solvent

Purification Methods

Filtration• Salt Removal

• Covalent and Ionic Scavenging Resin Removal

Extractions• Liquid-Liquid

• SPE - Solid Phase Extraction

Chromatography• Silica

• C18

• Fluorous SilicaSmall hole drilled

into the bottom of each well

20 µM Polyethylene frit

Polypropylene

Use Parallel Filtration and a Liquid Handling Robot

Filtration

Salt Removal

Covalent and Ionic Scavenging Resin Removal

Source plate

Robot Tip

Destination plate

Filter plate

Extractions

Liquid-Liquid1. Positional Heavy

Solvent Extraction

2. Positional Light Solvent Extraction

3. Liquid Detection Light Solvent Extraction

1

2

1 2

3-1 3-2 3-3

3

Chromatography and SPE

Silica Gel

Fluorous Silica Gel

C18

Ion Exchange

1. Dissolve Samples in a suitable solvent

2. Transfer to little chromatography columns

3. Elute clean products and/or collect fractions

Chromatography Example

Cyclic Urea Plate, wells 1-48, Before and After Filtration through Silica gel

Commercial 24 & 96-wellFilter Plates

Varian http://www.varianinc.comOros technologies http://www.oroflex.comRobbins Scientific http://www.robsci.comPolyfiltronics http://www.polyfiltronics.comWhatman http://www.whatman.comSpike International http://www.spike.cc

Commercial Robotics

Robots• TECAN http://www.tecan-us.com• Hamilton http://www.hamiltoncomp.com• Gilson http://www.gilson.com

Custom solutions• Chemspeed http://www.chemspeed.com

• Complete reaction stations

• AutoChem http://www.mtautochem.com• weighing, extraction, transfers

• InnovaSyn http://www.innovasyn.com• extraction, transfers, TLC spotting

• J-KEM http://www.jkem.com

High Through-PutPrep HPLC

Systems based on UV and/or ELSDBiotage

Gilson

Argonaut

Isco

Systems based on Mass SpectMicroMass, PE Sciex, Shimadzu, Agilent

Analytical methods

Solid Phase - few high throughput methods• NMR - gel phase, MAS

• IR - works well

• MS - laser assisted removal and ionization

• elemental analysis - must analyze starting materials

Solution Phase - some high throughput methods• TLC - ideal for parallel analysis

• MS - ion spray, 45 sec./sample, reports at 2 sec./sample

• NMR - high throughput with flow probes 2 min./sample

• HPLC, LC-MS 5 min./sample

The challenge is not so much to collect the data as to analyze it.

Robotic TLC Plate Spotting

Example TLC Plate

Some Pertinent Points• Analyze an entire plate (96

compounds) at once

• Trends are easy to spot• Note similar impact of substituent

change

• Common impurities

• Common by-products

• Can Spot Across or Down to See Trends

• Non linearity of detection

• No structural information

B DCA

Mass Spectroscopy

Mass Spectrometers used in Combinatorial Labs

• Use an Ion Spray technique (ES or APCI) to allow flow injection analysis (FIA)

• Auto Samplers sample from multiple 96 well plates

• Use quadrapoles for mass filters

• Have data analysis and reduction packages for matrix analysis

• Can run samples at < 1 min. each

• LC/MS becoming much more routine (5 min. each)

Analytical Data AnalysisLC/MS

MicroMass Diversity Browser

Lilly RTP Analytical Viewer

Analytical Data AnalysisNMR

SLAVASLAVA

ACD’s SpectView

Trends

1. With higher screening throughput there is a trend away from making or testing mixtures.

2. With better purification methods, SPOC no longer dominates combinatorial chemistry.

3. Everyone is demanding purer products and more material with better characterization.

4. Equipment complexity is dropping as we learn to be clever rather than over-engineer. There are more commercial options though big machines are going away.

5. The methods of Parallel Synthesis are slowly finding their way into all aspects of synthetic chemistry.

6. Handling data (registration, analysis, results) remains a major challenge.

7. Combinatorial Chemistry/ Parallel Synthesis is here to stay.

Conclusions

By application of robotics, computers, clever engineering and chemistry, the methodology now exists to synthesize, with reasonable purity and yield, medicinally relevant organic molecules at 100 to 10,000 times the rate possible just 10 years ago. The field of Combinatorial Chemistry/ Parallel synthesis is evolving and melding with classical Medicinal Chemistry.

Further Information

www.combichem.net

www.combichemlab.com

www.5z.com

www.combinatorial.com

www.netsci.org

www.innovasyn.com

www.google.com

Archiving TLC Plates

UV Images • Captured using a UV Light Box with a Visible

Camera

Visible Images• Captured using a Scanner

All Images Stored on Disk and Printed for Notebook storage

Computer UV light box

Camera

Scanner