Title: Drew Residential School on Medicinal Chemistry
1Drew Residential Schoolon Medicinal Chemistry
- Chemical Diversity
- Generation and Use in Drug Discovery
- Philip F. Hughes
- InnovaSyn, LLC
- Chapel Hill, NC
2Chemical 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
- Equipment
- Purification Methods
- Analytical Methods
- Conclusions
3Why 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.
i.e. High Throughput Screening
4High Throughput Screening
Current Screening capacities of 2000-100,000 Sampl
es/Day in multiple assays
Biotechnology Genomics Computers Robotics Chemistr
y
synergy
Where will the Samples come from?
5History
- 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
6Old Molecular Diversity
- Company Chemical Storage
- 20,000-400,000 compounds, many similar classes,
some gt100 yrs. old - Natural Products
- large number, not clean,
- test as mixtures
- Classical Medicinal Chemistry
- too slow or too expensive
7New Requirements
- We need to increase the compound synthesis rate
by - 20 to 1000 fold
- This is less than the increase in screening
capacity because were now willing to test each
compound in numerous assays
8Going 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
9The 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
10Two Major Approaches
- Split Mix
- Real Combinatorial Chemistry
- Array Synthesis
- Parallel Synthesis
- Spatially-Addressable Synthesis
- Matrix Array Synthesis
11Split 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
12Split Mix
of reagents 3 of reactions 3 3 3 9
of products 3 x 3 x 3 33 27
A Big Mixture
13Dealing with Mixtures
- Options
- Test as a mixture
- Encoded Libraries
- Tags
- Nucleotide
- Chemical
- Labeled reactors
14Big 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
15Nucleotide 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)
16Chemical 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)
17Chemical 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). - Pharmacopeias web site www.pcop.com
ECLiPS encoding technology - ICCB at Harvard iccb.med.harvard.edu/
18Chemical Tags - Pharmacopeia
1.
2.
1. Clip off compounds for testing
2. Clip off tags for analysis
(23-1)(25-1)(25-1) 73131 6727 compounds 3
5 5 13 tags 7313169 reagents, 69 x 2
138 reactions
19Labeled Reactors Radio Encoded Tags - Irori
www.irori.com Discovery Partners International
20Labeled 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
21Split 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
22Array Synthesis
- Use parallel synthesis in a matrix format (8 x 12
array) - 20 reagents with 1 or 2 reactions gives
96 products
23Large 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
Reagents
8 X 12 Plates
24Array 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
25Solution 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 (SPRs) may be made
using SPOC. They (SPRs) have also made solution
phase combinatorial chemistry easier.
26Solid 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 youre interested in
doing chemistry on and releasing at the end of
the synthesis
27Linkers
28An 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)
29Reaction Path
30Solid 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)
- Cant 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)
31Solution 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
32Solution 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).
33Solution Phase Organic Chemistry
3072 Compounds Single isomer gt 95
IC50 420 nM FTase Competitive Inhibitor
iterate
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
34Solution 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 (SPRs) is possible
- No linkers required, less excluded chemistry
- Requires development of parallel workup and
purification methods - Called Parallel Synthesis or Rapid Parallel
Synthesis (RPS)
35Trends over the Last Decade
Sld P SM
Sld P Array
10,000
Solu P Array 2004
of Compounds
1000
Solu P Array 1996
Classical Organic Synthesis
0
time
Solution Phase Array or Parallel Synthesis now
dominates
Dev. times for solid phase
36Equipment for Solid Phase Organic Chemistry
- Split Mix
- Standard labware with gentle stirring
- Array
- Geyson Pin Approach
- Bottom filtration
- Top filtration
- Little stuff
- Big stuff
37Geysen 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 www.mimotopes.com
38Equipment for Solid Phase Organic Chemistry
Problem How do you put 24-96 of these together?
Bottom Filter
Top Filter
39Original 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 Diversity 1995, 1, 13-20
40Commercial 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
FlexChem www.robsci.com www.scigene.com
MiniBlock www.bohdan.com www.Autochem.com
Polyfiltronics/Whatman www.whatman.com
Charybdis Technologies www.spike.cc
41Parallel 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
42Equipment for Parallel Solution Phase Organic
Synthesis
- One at a time Synthesis
- Parallel Synthesis
43Equipment for Parallel Solution Phase Organic
Synthesis
Generic Reactor Block
44Equipment for Solution Phase Organic Synthesis
45Equipment for Solution Phase Organic Synthesis
Biotage http//www.personalchemistry.com/
http//cemsynthesis.com
46Solution/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
47Solid 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
48Purification Methods
- Solid Phase
- Wash exhaustively
- product dependent cleavage
- Solution Phase - Parallel Purification
- Extraction
- liquid-liquid, acid/base
- SPE, scavenging resins
- Fluorous Synthesis
- Chromatography
49Scavenging Resins
S. W. Kaldor, J. E. Fritz, J. Tang, E. R.
McKinney, Biorganic Med. Chem. Lett..,
6,3041-3044 (1996).
50Fluorous 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
51Liquid Handling RobotsA Primer
52Purification 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
53Filtration
- Salt Removal
- Covalent and Ionic Scavenging Resin Removal
Robot Tip
Filter plate
Source plate
Destination plate
54Extractions
- Liquid-Liquid
- 1. Positional Heavy Solvent Extraction
- 2. Positional Light Solvent Extraction
- 3. Liquid Detection Light Solvent Extraction
55Chromatography 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
56Chromatography Example
- Cyclic Urea Plate, wells 1-48, Before and After
Filtration through Silica gel
57Commercial 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
58Commercial 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
59High Through-PutPrep HPLC
- Systems based on UV and/or ELSD
- Biotage
- Gilson
- Argonaut
- Isco
- Systems based on Mass Spect
- MicroMass, PE Sciex, Shimadzu, Agilent
60Analytical 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.
61Robotic TLC Plate Spotting
62Example 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
63Mass 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 lt 1 min. each
- LC/MS becoming much more routine (5 min. each)
64Analytical Data AnalysisLC/MS
MicroMass Diversity Browser
Lilly RTP Analytical Viewer
65Analytical Data AnalysisNMR
ACDs SpectView
SLAVA
SLAVA
66Trends
- 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.
67Conclusions
- 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.
68Further Information
www.combichem.net www.combichemlab.com www.5z.com
www.combinatorial.com www.netsci.org www.innovasyn
.com www.google.com
69Archiving 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