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Drew Residential School on Medicinal Chemistry Chemical Diversity Generation and Use in Drug Discovery Drew Residential School on Medicinal Chemistry Chemical Diversity Generation and Use in Drug Discovery Philip F. Hughes Innova. Syn, LLC Chapel Hill, NC

Chemical Diversity Generation and Use in Drug Discovery Overview Reasons, History, Economics, Definitions Combinatorial Chemical Diversity Generation and Use in Drug Discovery Overview Reasons, History, Economics, Definitions Combinatorial Chemistry/ Parallel Synthesis Methods split/mix, array solid phase, solution phase Equipment Purification Methods Analytical Methods Conclusions

Why Chemical Diversity? Reasons The biggest reason for continued interest in Chemical Diversity is 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. i. e. High Throughput Screening

High Throughput Screening Biotechnology Genomics Computers Robotics Chemistry synergy Current Screening capacities of 2000 High Throughput Screening Biotechnology Genomics Computers Robotics Chemistry synergy Current Screening capacities of 2000 -100, 000 Samples/Day in multiple assays Where will the Samples come from?

History 1990: A medicinal chemists made 2 -6 compounds / month at $2, 500 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 Storage 20, 000 -400, 000 compounds, many similar classes, 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 We need to increase the compound synthesis rate by 20 to 1000 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 4 Ways to go Faster 1. Use Combinations • Reuse 2. Do Going Faster 4 Ways to go Faster 1. 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 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 Two Major Approaches Split & Mix “Real Combinatorial Chemistry” Array Synthesis “Parallel Synthesis” “Spatially-Addressable Synthesis” “Matrix Array Synthesis”

Split & Mix Originated in peptide synthesis Simple efficient chemistry (amides) Long linear sequence 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 & Mix # of reagents = 3 # of reactions =3+ 3 = Split & Mix # of reagents = 3 # of reactions =3+ 3 = 9 # of products = 3 x 3 = 33 = 27 A Big Mixture

Dealing with Mixtures Options • Test as a mixture Encoded Libraries • Tags • 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 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 Beads selected based on binding to target Nucleotide “code” can be defined 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 Example: Arylsulfonamide inhibitors of Carbonic Anhydrase • 7 X 31 Chemical Tags - Pharmacopeia Example: Arylsulfonamide inhibitors of Carbonic Anhydrase • 7 X 31 library: 6727 members (R 1 -R 2 -R 3) • Each reagent encoded by a unique combination of 3 -5 tags based on a binary code: coding 2 n-1 members requires n tags • Tag incorporated by Rh-catalyzed carbene insertion into polymer C-H • Tags released from oxidatively labile linker with (NH 4)2 Ce(NO 3)2, followed by Electron Capture GC (silylated tags)

Chemical Tags - Pharmacopeia M. H. J. Ohlmeyer, R. N. Swanson, L. W. Dillard, 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 ECLi. PS™ encoding technology ICCB at Harvard iccb. med. harvard. edu/

Chemical Tags - Pharmacopeia 1. Clip off compounds for testing 2. Clip off tags Chemical Tags - Pharmacopeia 1. Clip off compounds for testing 2. Clip off tags for analysis (23 -1) • (25 -1) = 7 • 31 = 6727 compounds 3 + 5 = 13 tags 7+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 www. irori. com Discovery Partners International

Labeled Reactors Radio Encoded Tags - Irori Similar to resin split and mix except 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 Split and Mix Synthesis Points Large diversity requires but can also utilize a longer synthetic sequence Generally makes a smaller amount (p. M to n. M) 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 Use parallel synthesis in a matrix format (8 x 12 array) - 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 Larger numbers of compounds are available from one scaffold or reaction 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 Reagents 8 X 12 Plates

Array Synthesis Points • Large diversity requires but can also utilize the large diversity 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 (u. M to m. M) of fewer compounds

Solution and Solid Phase Organic Chemistry Definitions for the sake of discussion: • Solution 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 Solid Phase Organic Chemistry • Core is usually 1% crosslinked polystyrene • Spacer, if present, is usually a polyethylene glycol Tenta. Gel. TM, or Argo. Gel. TM (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

Linkers Linkers

An Example H. V. Meyers, G. J. Dilley, T. l. Durgin, T. S. Powers. 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 Reaction Path

Solid Phase Organic Chemistry Products are insoluble • Easier to manipulate physically • Easier 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: • 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. 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 Chemistry 3072 Compounds Single isomer > 95% IC 50 = 420 Solution Phase Organic Chemistry 3072 Compounds Single isomer > 95% IC 50 = 420 n. M FTase Competitive Inhibitor iterate IC 50 = 1. 9 n. M 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 Chemistry Products are soluble • Byproducts and excess reagents are also 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 Sld P S&M Sld P Array 10, 000+ Solu Trends over the Last Decade Sld P S&M Sld P Array 10, 000+ Solu P Array 2004 # of Compounds 1000+ Solu P Array 1996 Classical Organic Synthesis 0 Solution Phase Array or Parallel Synthesis now dominates time Dev. times for solid phase

Equipment for Solid Phase Organic Chemistry Split & Mix Standard labware with gentle stirring 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 Resins attached to pins in an 8 x 12 array format 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 www. mimotopes. com

Equipment for Solid Phase Organic Chemistry Problem: How do you put 24 -96 of Equipment for Solid Phase Organic Chemistry Problem: How do you put 24 -96 of these together? Bottom Filter Top Filter

Original Sphinx Reactor Solid Phase Chemistry Reactor Plate in a Plate Clamp Strip Caps 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 Diversity 1995, 1, 13 -20

Commercial Apparatus for Solid Phase Argonaut Quest 210 Nautilus 2400 Trident Little Stuff Flex. Commercial Apparatus for Solid Phase Argonaut Quest 210 Nautilus 2400 Trident Little Stuff Flex. Chem www. robsci. com www. scigene. com Big Stuff Mini. Block www. bohdan. com www. Autochem. com Bohdan Ram Tecan Combitec Advanced Chemtech 496 Myriad Core Polyfiltronics/Whatman www. whatman. com Charybdis Technologies www. spike. cc All Discontinued Big stuff is a bad idea.

Parallel Solution Phase Organic Synthesis Equipment – An Array of Vessels • • Heating 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 One at a time Synthesis Parallel Synthesis

Equipment for Parallel Solution Phase Organic Synthesis Generic Reactor Block Equipment for Parallel Solution Phase Organic Synthesis Generic Reactor Block

Equipment for Solution Phase Organic Synthesis Reactor Blocks Equipment for Solution Phase Organic Synthesis Reactor Blocks

Equipment for Solution Phase Organic Synthesis Micro. Wave Biotage http: //www. personalchemistry. com/ http: Equipment for Solution Phase Organic Synthesis Micro. Wave Biotage http: //www. personalchemistry. com/ http: //cemsynthesis. com

Solution/Slurry Addition 1 5 • Good for Repeated Additions of one Solution • Disposable Solution/Slurry Addition 1 5 • Good for Repeated Additions of one Solution • Disposable Polypropylene Syringe Barrels • Easily adaptable to Leur fittings (needles) • Can deliver from 0. 5 u. L to 5 m. L • Inexpensive and Fast (better than robots) • Can Deliver Slurries with Modifications 3 2 4 Eppendorf Repeater Pipette eppendorf

Solid Addition Solid addition plates/Vacuum systems Solid as a slurry • 10% Pd on Solid Addition Solid addition plates/Vacuum systems Solid as a slurry • 10% Pd on Carbon in Ethanol • Na. HB(OAc)3 in Dichlorethane • Resins as isopycnic slurries

Purification Methods Solid Phase • Wash exhaustively • product dependent cleavage Solution Phase - 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 S. W. Kaldor, J. E. Fritz, J. Tang, E. R. Mc. Kinney, Scavenging Resins S. W. Kaldor, J. E. Fritz, J. Tang, E. R. Mc. Kinney, Biorganic & Med. Chem. Lett. . , 6, 30413044 (1996).

Fluorous Synthesis Fluorous (C 6 F 12) Phase Aqueous Phase Halocarbon (CH 2 Cl Fluorous Synthesis Fluorous (C 6 F 12) Phase Aqueous Phase Halocarbon (CH 2 Cl 2) 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 m. L Loop Tees Robot Arm 6 -Way Liquid Handling Robots A Primer 10 m. L Loop Tees Robot Arm 6 -Way Valve X Tip Y Z Syringes System Solvent

Purification Methods Filtration Use Parallel Filtration and a Liquid Handling Robot • Salt Removal Purification Methods Filtration Use Parallel Filtration and a Liquid Handling Robot • Salt Removal • Covalent and Ionic Scavenging Resin Removal Extractions • Liquid-Liquid • SPE - Solid Phase Extraction Chromatography • Silica • C 18 • Fluorous Silica

Filtration Salt Removal Covalent and Ionic Scavenging Resin Removal Robot Tip Filter plate Source Filtration Salt Removal Covalent and Ionic Scavenging Resin Removal Robot Tip Filter plate Source plate Destination plate

Extractions Liquid-Liquid 1. Positional Heavy Solvent Extraction 2. Positional Light Solvent Extraction 3. Liquid Extractions Liquid-Liquid 1. Positional Heavy Solvent Extraction 2. Positional Light Solvent Extraction 3. Liquid Detection Light Solvent Extraction

Chromatography and SPE Silica Gel Fluorous Silica Gel C 18 Ion Exchange 1. Dissolve Chromatography and SPE Silica Gel Fluorous Silica Gel C 18 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 148, Before and After Filtration through Silica gel Chromatography Example Cyclic Urea Plate, wells 148, Before and After Filtration through Silica gel

Commercial 24 & 96 -well Filter Plates Varian Oros technologies Robbins Scientific Polyfiltronics Whatman Commercial 24 & 96 -well Filter Plates Varian Oros technologies Robbins Scientific Polyfiltronics Whatman Spike International http: //www. varianinc. com http: //www. oroflex. com http: //www. robsci. com http: //www. polyfiltronics. com http: //www. whatman. com http: //www. spike. cc

Commercial Robotics Robots • TECAN http: //www. tecan-us. com • Hamilton http: //www. hamiltoncomp. 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 • Auto. Chem http: //www. mtautochem. com • weighing, extraction, transfers • Innova. Syn http: //www. innovasyn. com • extraction, transfers, TLC spotting • J-KEM http: //www. jkem. com

High Through-Put Prep HPLC Systems based on UV and/or ELSD Biotage Gilson Argonaut Isco High Through-Put Prep HPLC Systems based on UV and/or ELSD Biotage Gilson Argonaut Isco Systems based on Mass Spect Micro. Mass, PE Sciex, Shimadzu, Agilent

Analytical methods Solid Phase - few high throughput methods • • NMR - gel 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 Robotic TLC Plate Spotting

Example TLC Plate Some Pertinent Points • Analyze an entire plate (96 compounds) at 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 A B C D

Mass Spectroscopy Mass Spectrometers used in Combinatorial Labs • Use an Ion Spray technique 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 Micro. Mass Diversity Browser Lilly RTP Analytical Viewer Analytical Data Analysis LC/MS Micro. Mass Diversity Browser Lilly RTP Analytical Viewer

Analytical Data Analysis NMR ACD’s Spect. View SLAVA Analytical Data Analysis NMR ACD’s Spect. View SLAVA

Trends 1. With higher screening throughput there is a trend away from making or 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 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. 5 z. com www. combinatorial. Further Information www. combichem. net www. combichemlab. com www. 5 z. 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 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