Combinatorial Chemistry At Sphinx/Lilly

1. Combinatorial ChemistryAt Sphinx/Lilly • Why do Combinatorial Chemistry? • Speed • Economics

2. Screening Speed • Current High Efficiency Screening • 2000 compounds screened per day per assay (125,000 tot.) • Multiple assays run concurrently • 10-30 screens per year projected to increase 5 to 10-fold by the year 2000

3. Combinatorial Economics • The classical cost/compound $2500-$10,000 each. • (5 assays x 2000 compounds x $10,000) = $100,000,000.00/day • To take advantage of the screening capacity, we need to make compounds faster and cheaper.

4. New Requirements • We needed to increase the compound synthesis rate by 50 to 1000 fold • How? • Old Engineering Maxim “good, fast, cheap - pick two”

5. Ground Rules • Drug-like molecules • Single compounds • 20 µmol each. • Purity priorities • Flexible synthesis methods • Automation as needed

6. How Do We Do It? • Use multiple parallel synthesis in a matrix format - 20 reagents with 2 reactions gives 96 products

7. How Do We Do It? • Take as much technology from High Throughput Screening (HTS) as possible. • pros • Experience with parallel formats • Experience with robotics • cons • Materials compatibility issues

8. How Do We Do It? • Use simple, disposable equipment • Take some simple chemistry and start scaling it up until it hurts • Identify the bottlenecks and work to open them up until some other part of the process becomes the slow part

9. Simple Chemistry • Suitable Test Chemistry-A Bisamide Library

10. Simple Equipment • Solid Phase Chemistry Reactor • Beckman 96 deep-well titer plate

11. Simple Equipment • Solid Phase Chemistry Reactor Plate in a Plate Clamp

12. Reaction Path

13. Plate Layout R2 Scaffold R1

14. Library Synthesis Planning • Lay out a Super Grid • 72 X 72 reagents or wells • 9 X 6 plates • 5184 compounds • Make reagents • 72 1 M acylating agents solutions • 180 g of resin-scaffold • 20 mg/well (1 mmol/g) Reagents 8 X 12 Plates

15. Reagent Addition • You need • a device that will take up a large amount of solution and easily deliver smaller quantities • compatibility with all organic materials • disposable • cheap?

16. Repeater Pipette • Takes up large volume and quickly and accurately dispenses smaller quantities • Disposable polypropylene liquid holder • Dispenses in 1µL to 5 mL per shot • Adaptable to leur fittings • Compatible with slurries

17. Reaction Path

18. Resin to Plate Addition • Isopycnic Slurry • Mix solvents until the resin neither sinks nor floats while tracking the solvent ratio • Dilute with the solvent ratio to get desired resin/vol ratio • Using a modified Eppendorf Repeater Pipette 50 mL tip, add resin to plates

19. First Acylation • Add a CH2 Cl2 solution of DMAP and pyridine to the entire plate • Add 8 unique acylating agents to each row • Cap and tumble

20. Tumbling • Plates are attached to a square bar which slowly rotates. Mixing is effected by the up and down motion of an air bubble. • This device is known with affection as the “Rotissarie”

21. Washing resins • To wash the resins, the plates are removed from the clamp and placed into a trough • Solvent is then delivered to the wells via an 8-way manifold from a pump • A 6-way valve allows selection from a variety of solvents • The resins are washed using a solvent sequence and allowed to drain • This process has been automated essentially as shown

22. Nitro Reduction • Add a DMF solution of SnCl2•H2O to the entire plate • Cap, tumble and wash

23. Second Acylation • Add a CH2 Cl2 solution of DMAP and pyridine to the entire plate • Add 12 unique acylating agents to each column • Cap and tumble and wash

24. Product Cleavage • Plate now contains 96 different molecules • Add cleavage agent, cap and tumble

25. Product Collection • 1. Remove the plate from the clamp upside-down • 2. Place under a 2 mL plate • 3. Invert and remove the caps • 4. Wash resins 4 2 3 1

26. Reaction Path

27. Product Analysis • On each Plate • 1H-NMRs, 4 random samples • Mass Spects initially, 4 random samples FAB or IS Now, all wells • TLC, all wells • Weight, entire plate (well average)

28. Robotic TLC Plate Spotting • The TECAN 5052 • Spots 2-96 well titer plate to 4-10 X 20 TLC plates, 48 spots per TLC plate 1A-H, 2 A-H A1-12, B1-12

29. 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

30. C B D A Example TLC Plate • Some Pertinent Points • Analyze an entire plate 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 linerarity of detection • No structural information

31. Purification Methods • Filtration • Salt Removal • Covalent and Ionic Scavenging Resin Removal • Extractions • Liquid-Liquid • SPE - Solid Phase Extraction • Chromatography • Silica • C18 Based on using our reactor as a 96 position chromatography column/filter

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

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

34. Extractions • SPE - Solid Phase Extraction 1. Add Sulphonic acid resin to grab amine products 2. Transfer to Filter Plate and wash away contaminents 3. Elute clean products off with 1 N HCl in Methanol

35. Chromatography • Silica Gel • C18 1. Dissolve Samples in a suitable solvent 2. Transfer to little chromatography columns 3. Elute clean products and/or collect fractions

36. Chromatography Example • Cyclic Urea Plate, wells 1-48, Before and After Filtration through Silica gel

37. Diamino Alcohol SuperLibrary

38. Bis-Amide Libraries

39. Other Chemistries

40. Other Chemistries

41. Summary • Fast • Capacity for 100,000 compounds/year • Cheap • Inexpensive, flexible and often disposable equipment • 1 robot ($50 G) for 20 people • Good • Good Enough • < µM Leads in CNS, cardiovascular and cancer screens

42. Acknowledgements • The Sphinx Durham Chemistry Group SeanHollinshead JeanDefauw • The Sphinx Cambridge Chemistry Group Hal Meyers • The Kaldor Group at Lilly in Indianapolis