1. Combinatorial Chemistry At 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. Suitable Test Chemistry-A Bisamide Library Simple Chemistry

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

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

12. Reaction Path

13. Plate Layout Scaffold R2R2 R1R1

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. 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? Reagent Addition

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 CH 2 Cl 2 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 SnCl 2 H 2 O to the entire plate Cap, tumble and wash

23. Second Acylation Add a CH 2 Cl 2 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 3 4 2 1

26. Reaction Path

27. Product Analysis On each Plate 1 H-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 A 1-12, B 1-12 1 A-H, 2 A-H

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. 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 B D C A

31. Purification Methods Filtration Salt Removal Covalent and Ionic Scavenging Resin Removal Extractions Liquid-Liquid SPE - Solid Phase Extraction Chromatography Silica C 18 Based on using our reactor as a 96 position chromatography column/filter

32. Filtration Salt Removal Covalent and Ionic Scavenging Resin Removal Source plate Robot Tip Destination plate Filter 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 C 18 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

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