Drug Discovery The use of solid phase combinatorial chemistry and parallel synthesis

Problems with Traditional Synthesis 1 chemist 1 molecule –Can only make one molecule at a time Each synthesis very time consuming –Multistep syntheses have loss at each step –Purification of products very time-consuming between steps. Yields can be low and produces very few molecules at a time for testing

Solid Phase Synthesis Technology adapted from Biochemistry and peptide synthesis Addresses problem of purification steps Typically purifications involve such processes as –Recrystallization –Distillation –Extraction All very time consuming and have losses that lower yields

Solid Phase Synthesis First step is to attach the starting molecule to an inert solid. –Typically inert polymers or resins are used. –These are commercially available

Solid Phase Synthesis Since the molecule is attached to a solid, any other chemicals added or products can be removed by filtration. –Typically reactions undertaken in vessels with frits at the bottom (for filtration) –“Tea bags” also used Here, solids are dipped into solutions containing subsequent reagents. Next, they can be removed from the “dip” and placed into a wash solution

Solid Phase Synthesis After all reactions are done the product is still attached to the insoluble bead. –Can be washed as in previous slide or, in the case of a reaction well, excess solvent is washed through Finally, the product is cleaved from the bead and isolated.

Advantages of Solid Phase Synthesis Purification of each product can be achieved in one step. –Only purification technique is filtration –Washing is simple Because all we do is change solution or move a “tea bag” from solution to solution, this process can be easily automated.

Disadvantages to Solid Phase Synthesis Not all syntheses can be done solid phase –Some molecules don’t attach well to beads –Some chemistry just doesn’t work in this fashion –Removal of product from bead, can be damaging to product if not careful Typically, kinetics not the same –Reaction rates can be slower

Parallel Synthesis Method for making many molecules at the same time. Reactions done “in parallel” –Have to be analogous reactions Same basic chemistry Same conditions of temp, solvents, time etc. Reactions done at same time. –Not all will give same yields as analogous is NOT identical

Next Several Slides The slides below are an example or how we can make several compounds at one time using parallel synthesis and solid phase combinatorial chemistry. Please look in the examples for the principles of solid phase and parallel synthesis. Note that the number of reactions is significantly fewer than the number of compounds. –Number of reactions is additive –Number of compounds multiplicative

12-well reaction block

12 well reaction block Again, this is an example This is simply a teflon block with wells drilled in it. –All of the reactions done in the block are done at the same time. If the block is heated, all wells are heated etc Same solvents in each well etc.

Cross section of a well on the reaction block Each well in the reaction block has a frit on the bottom If the wholc block is placed on a vacuum, liquids will go through the frit Solids stay behind Can purify substances by filtration Frit. Plate on vacuum

Scaffold A scaffold is a molecule on which the structure of the final molecules is built Has a basic structure May have several reactive sites –These are called points of diversity –This is where other molecules are added

Example The molecule to the right has 2 reactive sites It is attached to a bead for solid phase synthesis For the next slides, we’ll call this scaffold, S

Parallel Synthesis The Scaffold is now added to each well In this example, there are twelve wells, so presently there are twelve copies of the same molecule.

Add Scaffold to each well SS S S S S S S S S S S

Well after addition of scaffold Each well on the plate has the scaffold.

Diversity Groups For lack of a better term, these are the things that will be reacted with the reactive centers on the scaffold Remember “R” means the “rest” of the molecule –R groups can be methyl, ethyl, propyl, isopropyl, benzyl etc –The reaction takes place at the other end

Diversity Groups for our example = A - D = 1 - 3

Reaction of First Reactant with Scaffold Excess reagent and water can be filtered out, These R’s are A - D

Addition of First Reactant to Scaffold Each reaction proceeds in the same fashion They are analogous –Same chemistry –Same other product (in this case, water) –Same time Only difference will be the R groups –Remember, in this example they are A - D

Well after first reaction, filtration and washing Each Column on the plate has a different R group (A – D). The other products were removed by filtration.

Wells after addition of first reagent There are now 4 different products There have been 4 different reactions –However, though we added four different reagents, all of the reactions were undertaken simultaneously.

Wells after Addition of first reagent SBSA SB SC SD

Addition of Second Reactant to Scaffold Each reaction proceeds in the same fashion They are analogous –Same chemistry –Same other product (in this case, HCl) –Same time Only difference will be the R groups –Remember, in this example they are 1 - 3

Reaction of Second Reactant with Scaffold + HCl Excess reagent filtered through along with HCl ions.

Wells after second reaction and washing Each Row on the plate has a different R group. Excess reagent and HCl removed by filtration

Wells after addition of second reagent There are now 12 different products There have been 7 different reactions –However, though we added seven different reagents, all of the reactions were undertaken simultaneously.

There are now twelve different products SB1SA1 SA2 SA3 SB2 SB3 SC2 SC1 SC3 SD1 SD2 SD3

Combinatorial Advantage Number of reactions is additive –4 + 3 = 7 reactions Number of products is multiplicative –4 X 3 = 12 products In this example, not a big deal, but imagine if we did more steps

Isolation of Product Product is cleaved from the bead Solubilized product is filtered through well into a small test tube.

Contents of Test Tube (Isolated compound) Remember there are 12 different ones

Summary This example illustrates a couple of things –Parallel synthesis Though there were 7 reactions, there were only 2 sets of reactions. We had to add 7 different reagents, but there were only 2 events where we did the reactions –Solid phase synthesis Original scaffold on a bead so excess reagents and other products removed by filtration Intermediate products easily washed –Combinatorial advantage Only did 7 reactions, but got 12 products Would be more pronounced if more steps