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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
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 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.
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)
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