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Drew Residential School on 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. Overview Reasons, History, Economics, Definitions

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drew residential school on medicinal chemistry
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
Chemical Diversity Generation and Use in Drug Discovery
  • Overview

Reasons, History, Economics, Definitions

  • Combinatorial Chemistry/ Parallel Synthesis

Synthesis Methods

split/mix, array

solid phase, solution phase


Purification Methods

Analytical Methods

  • Conclusions
why chemical diversity
Why Chemical Diversity?
  • Reasons

The biggest reason for continued interest in Chemical Diversity is the recent ability of scientists to evaluate very large numbers of molecules in biological systems.


High Throughput Screening

high throughput screening
High Throughput Screening

Current Screening

capacities of



in multiple assays







Where will the Samples come from?

  • 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
Old Molecular Diversity
  • Company Chemical Storage

20,000-400,000 compounds, many similar classes, some >100 yrs. old

  • Natural Products

large number, not clean,

test as “mixtures”

  • Classical Medicinal Chemistry

too slow or too expensive

new requirements
New Requirements
  • We need to increase the compound synthesis rate by
  • 20 to 1000 fold
  • This is less than the increase in screening capacity because we’re now willing to test each compound in numerous assays
going faster
Going Faster
  • 4 Ways to go Faster
    • Use Combinations
      • Reuse
    • Do many things at the same time
      • Parallel processing
    • Speed up the process
    • Get someone else to do it
      • Automation
      • Outsourcing
the answer
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
Two Major Approaches
  • Split & Mix

“Real Combinatorial Chemistry”

  • Array Synthesis

“Parallel Synthesis”

“Spatially-Addressable Synthesis”

“Matrix Array Synthesis”

split mix
Split & Mix
  • Originated in peptide synthesis

Simple efficient chemistry (amides)

Long linear sequence of reactions

Solid Phase approaches known

# of reagents = 10

# of reactions = steps ● reagents; 5 ● 10 = 50

# of products = reagentssteps; 105 = 100,000

split mix12
Split & Mix

# of reagents = 3

# of reactions =3+ 3 + 3 = 9

# of products = 3 x 3 x 3 = 33 = 27

A Big Mixture

dealing with mixtures
Dealing with Mixtures
  • Options
    • Test as a mixture
  • Encoded Libraries
    • Tags
      • Nucleotide
      • Chemical
    • Labeled reactors
big mixture testing
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
nucleotide tags
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)
chemical tags pharmacopeia
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)
chemical tags pharmacopeia17
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 technology
  • ICCB at Harvard iccb.med.harvard.edu/
chemical tags pharmacopeia18
Chemical Tags - Pharmacopeia



1. Clip off compounds for testing

2. Clip off tags for analysis

(23-1)•(25-1)•(25-1) = 7•31•31 = 6727 compounds

3 + 5 + 5 = 13 tags

7+31+31=69 reagents, 69 x 2 = 138 reactions

labeled reactors radio encoded tags irori
Labeled Reactors Radio Encoded Tags - Irori

www.irori.com Discovery Partners International

labeled reactors radio encoded tags irori20
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
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
array synthesis
Array Synthesis
  • Use parallel synthesis in a matrix format (8 x 12 array) - 20 reagents with 1 or 2 reactions gives 96 products
large array synthesis
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


8 X 12 Plates

array synthesis points
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
solution and solid phase organic chemistry
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
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
an example
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)

solid phase organic chemistry30
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
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 chemistry32
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).

solution phase organic chemistry33
Solution Phase Organic Chemistry



Single isomer

> 95%

IC50 = 420 nM FTase

Competitive Inhibitor


IC50 = 1.9 nM FTase

for enantiomer shown

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

solution phase organic chemistry34
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
Trends over the Last Decade

Sld P S&M

Sld P Array


Solu P Array


# of



Solu P Array


Classical Organic Synthesis



Solution Phase Array or Parallel Synthesis now dominates

Dev. times for solid phase

equipment for solid phase organic chemistry
Equipment for Solid Phase Organic Chemistry
  • Split & Mix

Standard labware with gentle stirring

  • Array

Geyson Pin Approach

Bottom filtration

Top filtration

Little stuff

Big stuff

geysen pin method
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 Mimotopes


equipment for solid phase organic chemistry38
Equipment for Solid Phase Organic Chemistry

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

Bottom Filter

Top Filter

original sphinx reactor
Original Sphinx Reactor
  • Solid Phase Chemistry Reactor

Plate in a Plate Clamp

Strip Caps used to seal reaction after reagent addition

Plate removed from clamp for resin washing

Plate Bottom acts as a 96-way valve

H.V. Meyers, G.J. Dilley, T.L. Durgin, et al Molecular Diversity1995, 1, 13-20

commercial apparatus for solid phase
Commercial Apparatus for Solid Phase

Big Stuff

  • Argonaut
    • Quest 210
    • Nautilus 2400
    • Trident
  • Bohdan Ram
  • Tecan Combitec
  • Advanced Chemtech 496
  • Myriad Core
  • All Discontinued
  • Big stuff is a bad idea.

Little Stuff









Charybdis Technologies


parallel solution phase organic synthesis
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
Equipment for Parallel Solution Phase Organic Synthesis
  • One at a time Synthesis
  • Parallel Synthesis
equipment for solution phase organic synthesis45
Equipment for Solution Phase Organic Synthesis
  • MicroWave




solution slurry addition







Solution/Slurry Addition
  • 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
  • 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
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
Scavenging Resins

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

fluorous synthesis
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

liquid handling robots a primer

10 mL Loop




6-Way Valve



System Solvent




Liquid Handling RobotsA Primer
purification methods52
Purification Methods
  • Filtration
    • Salt Removal
    • Covalent and Ionic Scavenging Resin Removal
  • Extractions
    • Liquid-Liquid
    • SPE - Solid Phase Extraction
  • Chromatography
    • Silica
    • C18
    • Fluorous Silica

Use Parallel Filtration

and a Liquid Handling Robot

  • Salt Removal
  • Covalent and Ionic Scavenging Resin Removal

Robot Tip

Filter plate

Source plate

Destination plate

  • Liquid-Liquid

1. Positional Heavy Solvent Extraction

2. Positional Light Solvent Extraction

3. Liquid Detection Light Solvent Extraction

chromatography and spe
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
Chromatography Example
  • Cyclic Urea Plate, wells 1-48, Before and After Filtration through Silica gel
commercial 24 96 well filter plates
Commercial 24 & 96-wellFilter Plates
  • Varian http://www.varianinc.com
  • Oros technologies http://www.oroflex.com
  • Robbins Scientific http://www.robsci.com
  • Polyfiltronics http://www.polyfiltronics.com
  • Whatman http://www.whatman.com
  • Spike International http://www.spike.cc
commercial robotics
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 put prep hplc
High Through-PutPrep HPLC
  • Systems based on UV and/or ELSD





  • Systems based on Mass Spect
  • MicroMass, PE Sciex, Shimadzu, Agilent
analytical methods
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.
example tlc plate





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
mass spectroscopy
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 analysis lc ms
Analytical Data AnalysisLC/MS

MicroMass Diversity Browser

Lilly RTP Analytical Viewer

analytical data analysis nmr
Analytical Data AnalysisNMR

ACD’s SpectView



  • 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.
  • 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
Further Information








archiving tlc plates
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