1 / 69

Drew Residential School on Medicinal Chemistry

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

Solomon
Download Presentation

Drew Residential School on Medicinal Chemistry

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Drew Residential Schoolon Medicinal Chemistry • Chemical Diversity • Generation and Use in Drug Discovery • Philip F. Hughes • InnovaSyn, LLC • Chapel Hill, NC

  2. 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 Equipment Purification Methods Analytical Methods • Conclusions

  3. 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

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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. Two Major Approaches • Split & Mix “Real Combinatorial Chemistry” • Array Synthesis “Parallel Synthesis” “Spatially-Addressable Synthesis” “Matrix Array Synthesis”

  11. 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

  12. Split & Mix # of reagents = 3 # of reactions =3+ 3 + 3 = 9 # of products = 3 x 3 x 3 = 33 = 27 A Big Mixture

  13. Dealing with Mixtures • Options • Test as a mixture • Encoded Libraries • Tags • Nucleotide • Chemical • Labeled reactors

  14. 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

  15. 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)

  16. 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)

  17. 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/

  18. Chemical Tags - Pharmacopeia 1. 2. 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

  19. Labeled Reactors Radio Encoded Tags - Irori www.irori.com Discovery Partners International

  20. 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

  21. 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

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

  23. 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

  24. 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

  25. 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.

  26. 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

  27. Linkers

  28. 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)

  29. Reaction Path

  30. 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)

  31. 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

  32. 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).

  33. Solution Phase Organic Chemistry 3072 Compounds Single isomer > 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

  34. 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)

  35. 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 time Solution Phase Array or Parallel Synthesis now dominates Dev. times for solid phase

  36. 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

  37. 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

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

  39. 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

  40. 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 FlexChem www.robsci.com www.scigene.com MiniBlock www.bohdan.com www.Autochem.com Polyfiltronics/Whatman www.whatman.com Charybdis Technologies www.spike.cc

  41. 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

  42. Equipment for Parallel Solution Phase Organic Synthesis • One at a time Synthesis • Parallel Synthesis

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

  44. Equipment for Solution Phase Organic Synthesis • Reactor Blocks

  45. Equipment for Solution Phase Organic Synthesis • MicroWave Biotage http://www.personalchemistry.com/ http://cemsynthesis.com

  46. eppendorf 4 3 5 2 1 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

  47. 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

  48. Purification Methods • Solid Phase • Wash exhaustively • product dependent cleavage • Solution Phase - Parallel Purification • Extraction • liquid-liquid, acid/base • SPE, scavenging resins • Fluorous Synthesis • Chromatography

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

  50. 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

More Related