| Combinatorial Chemistry At Sphinx/Lilly : Combinatorial Chemistry At Sphinx/Lilly Why do Combinatorial Chemistry?
Speed
Economics |
| Screening Speed : 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 |
| Combinatorial Economics : 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. |
| New Requirements : New Requirements We needed to increase the compound synthesis rate by 50 to 1000 fold
How?
Old Engineering Maxim
“good, fast, cheap - pick two” |
| Ground Rules : Ground Rules Drug-like molecules
Single compounds
20 µmol each.
Purity priorities
Flexible synthesis methods
Automation as needed |
| How Do We Do It? : How Do We Do It? Use multiple parallel synthesis in a matrix format - 20 reagents with 2 reactions gives 96 products |
| How Do We Do It? : 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 |
| How Do We Do It? : 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 |
| Simple Chemistry : Suitable Test Chemistry-A Bisamide Library Simple Chemistry |
| Simple Equipment : Solid Phase Chemistry Reactor
Beckman 96 deep-well titer plate Simple Equipment |
| Simple Equipment : Solid Phase Chemistry Reactor
Plate in a Plate Clamp Simple Equipment |
| Reaction Path : Reaction Path |
| Plate Layout : Plate Layout Scaffold R2 R1 |
| Library Synthesis Planning : 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 |
| 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? Reagent Addition |
| Repeater Pipette : 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 |
| Reaction Path : Reaction Path |
| Resin to Plate Addition : 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 |
| First Acylation : 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 |
| Tumbling : 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” |
| Washing resins : 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 |
| Nitro Reduction : Nitro Reduction Add a DMF solution of SnCl2•H2O to the entire plate
Cap, tumble and wash |
| Second Acylation : 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 |
| Product Cleavage : Product Cleavage Plate now contains 96 different molecules
Add cleavage agent, cap and tumble |
| Product Collection : 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 |
| Reaction Path : Reaction Path |
| Product Analysis : 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) |
| Robotic TLC Plate Spotting : 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 A1-12, B1-12 1A-H, 2 A-H |
| Archiving TLC Plates : 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 |
| Example TLC Plate : 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 |
| Purification Methods : 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 |
| Filtration : Filtration Salt Removal
Covalent and Ionic Scavenging Resin Removal Source plate Robot Tip Destination plate Filter plate |
| Extractions : Extractions Liquid-Liquid
1. Positional Heavy Solvent Extraction
2. Positional Light Solvent Extraction
3. Liquid Detection Light Solvent Extraction
|
| Extractions : 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 |
| Chromatography : 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 |
| Chromatography Example : Chromatography Example Cyclic Urea Plate, wells 1-48, Before and After Filtration through Silica gel |
| Diamino Alcohol SuperLibrary : Diamino Alcohol SuperLibrary |
| Bis-Amide Libraries : Bis-Amide Libraries |
| Other Chemistries : Other Chemistries |
| Other Chemistries : Other Chemistries |
| Summary : 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 |
| Acknowledgements : Acknowledgements The Sphinx Durham Chemistry Group
SeanHollinshead
JeanDefauw
The Sphinx Cambridge Chemistry Group
Hal Meyers
The Kaldor Group at Lilly in Indianapolis
|