Combinatorial Chemistry
At Sphinx/Lilly
Why do Combinatorial Chemistry?
• Speed
• Economics
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 10fold by the year 2000
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
We needed to increase the
compound synthesis rate by
50 to 1000 fold
How?
Old Engineering Maxim
“good, fast, cheap - pick two”
Ground Rules
•
•
•
•
•
•
Drug-like molecules
Single compounds
20 µmol each.
Purity priorities
Flexible synthesis methods
Automation as needed
How Do We Do It?
Use multiple parallel synthesis in a
matrix format - 20 reagents with 2
reactions gives 96 products
1
A
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
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?
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
NO 2
1) n-BuLi, TMEDA
NH 2
HO
cyclohexane
NO 2
O
CO 2H
DIC, pyridine, DMAP
2) CO 2
NH 2
O
DMF
O
Cl
O
R A--H
NO 2
O
NH
O
pyridine, DMAP
CH 2Cl 2
O
RA--H
O
O
H
H
O
N
R1-12
NH
O
RA--H
N
R1-12
NaOCH 3
4:1 THF/MeOH
HO
NH
O
Cl
R 1-12
NH
O
DMF
O
O
NH 2
O
SnCl 2. (H 2O) 2
RA--H
RA--H pyridine, DMAP
CH 2Cl2
Simple Equipment
Solid Phase Chemistry Reactor
1
2
3
4
5
6
7
8
9
10
11
12
A
B
C
D
E
20 µM
Polyethylene frit
F
G
H
Small hole drilled
into the bottom of each
well
Beckman 96 deep-well titer plate
Polypropylene
Simple Equipment
Solid Phase Chemistry Reactor
Plate in a Plate Clamp
Reaction Path
Analysis
Expose
2
Wash
Acylation
96-well
reactor
1,3
Wash
Wash
Cleavage
4
Collection
&
transfer
Submit
Plate Layout
R2
Cl
Scaffold
H 3CO
Cl
CN
N
N
N
C
C
C
O
O
O
Cl
Cl
OCH 3
Cl
O
NHR 2
HO
O
NHR 1
Cl
Cl
O
H 3CO
Cl
O
Cl
O
H 3CO
R1
Cl
O
O
Cl
Cl
O
Cl
Cl
O
Cl
Cl
O
Cl
O
Cl
O
Cl
O
O
Cl
Cl
O
O
Cl
O
Cl
O
Cl
Cl
O
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
A1
A2
B1
B2
C1
C2
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?
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
Analysis
Expose
2
Wash
Acylation
96-well
reactor
1,3
Wash
Wash
Cleavage
4
Collection
&
transfer
Submit
Resin to Plate Addition
add 1mL per well
Isopycnic Slurry
1
3
4
5
• 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
2
eppendorf
2:1 methylene
chloride: DMF
to get
30 mg/mL
First Acylation
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
2
1
5
1
A
O
NO 2
3
4
Add a CH2 Cl2 solution of
DMAP and pyridine to
the entire plate
Add 8 unique acylating
agents to each row
Cap and tumble
ep pen
do rf
O
NH 2
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”
1
Plate
Reactions
High torque
Ov erhead
Stirrer
Motor
3
3/8 "
Square
Steel Rod
2
Rubber
coupling
4
24.5
1
Ov erhead
Stirrer
Controller
Standard
Laboratory Hood
Racks
Ball Bearings
held with lab
clamps
Washing resins
Solvent Bottles
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
6-way valve
Pump
Pump Controller
To Waste
Teflon Coated Aluminum Washing Trough
Nitro Reduction
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
2
1
O
5
1
A
3
4
Add a DMF solution of
SnCl2•H2O to the entire
plate
Cap, tumble and wash
O
ep pen
NO 2
NH
do rf
O
RA--H
Second Acylation
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
2
1
5
1
A
O
3
NH 2
4
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
ep pen
do rf
O
NH
O
RA--H
Product Cleavage
2
O
B
C
D
E
F
G
H
2
3
4
5
6
7
8
9
10
11
12
1
5
1
A
4
Plate now contains 96
different molecules
Add cleavage agent, cap
and tumble
H
3
ep pen
do rf
O
O
N
R1-12
NH
O
RA--H
Product Collection
1. Remove the plate from the clamp upsidedown
2. Place under a 2 mL plate
3. Invert and remove the caps
4. Wash resins
Resin
2
Hole
Frit
1
Resin
3
Frit
Hole
4
Reaction Path
Analysis
Expose
2
Wash
Acylation
96-well
reactor
1,3
Wash
Wash
Cleavage
4
Collection
&
transfer
Submit
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
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
1
A1-12, B1-12
2
3
4
5
6
7
8
9
10
11 12
A
B
1
C
D
E
48
F
G
1
96
49
H
1
2
3
4
A
5
6
7
8
9
10
11 12
1
2
3
4
B
C
D
E
F
2
G
H
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
Camera
Computer
Scanner
UV light box
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
A
B
C
D
Purification Methods
Filtration
Based on using our reactor as
a 96 position chromatography
column/filter
• Salt Removal
• Covalent and Ionic
Scavenging Resin Removal
Extractions
• Liquid-Liquid
• SPE - Solid Phase Extraction
Chromatography
20 µM
Polyethylene frit
Small hole drilled
into the bottom of each
well
Polypropylene
• Silica
• C18
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 Detection
Light Solvent
Extraction
1
2
1
3-1 3-2 3-3
2
3
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
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
Cyclic Urea Plate, wells
1-48, Before and After
Filtration through Silica
gel
Diamino Alcohol SuperLibrary
Bis-Amide Libraries
NHR 1
H
NR1
HO
NHR 1
R2 HN
HO
OH
NR2
H
Bisamide phenol
55 plates
NHR 2
Indane
50 plates
CH3
Cyclopentane
58 plates
Other Chemistries
R
O 2N
H
N
O
X
OCH 3
S
Nuc
S
O
R1 R2 NH(10-15 eq)
O 2N
O
Br
O
DM F, RT
S
O
N
E
N
R1
O
N
R2
DM F, RT
S
O
H
H
+
E , Py, DM AP
CH2 Cl2
R1
R2
O
SnCl2 •H2 O(10 eq)
O 2N
S
O
1. NaOH, M eOH, THF
2. HCl, M eOH
3. SiO 2
E
N
R1
OCH 3
N
R2
S
O
Other Chemistries
O
O
ArCHO
O
N
Ph
O
CO 2Me
Ar
CH 3
LiBr, DBU, THF
NaOM e/M eOH/THF
OH
O
Ar
TFA/CH2 Cl2
O
O
Ar
MeO 2C
Ph
N
H
MeO 2C
N
H
Ph
OH
O
RNCO, Py
DM AP, CH2 Cl2
O
O
Ar
Ar
TFA/CH2 Cl2
O
N
N
R
O
N
Ph
N
R
O
O
Ph
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
The Sphinx Durham Chemistry Group
SeanHollinshead
JeanDefauw
The Sphinx Cambridge Chemistry Group
Hal Meyers
The Kaldor Group at Lilly in Indianapolis