1	Compound Solubility and HTS Screening Christopher A. Lipinski Adjunct Senior Research Fellow Pfizer Global R&D, Groton Labs Christopher_A_Lipinski@Groton.Pfizer.Com
2	The symptoms to be concerned about Erratic HTS screening results that seem to bear little or no relationship to the biology and maybe even to the chemistry erratic rates of primary hits in HTS screens erratic re-confirm rates on primary HTS actives differences among screening sites differences among compound collections
3	Minimum Acceptable Solubility in ug/mL Bars shows the minimum solubility for low, medium and high permeability (Ka) at a clinical dose. The critical range for aqueous and DMSO solubility is around 10 uM
4	14.2 Percent of 1597 phase II compounds have low aqueous solubility (<20 ug/mL)
5	31.2 Percent of 2246 commercial compounds have low aqueous solubility (<= 20 ug/mL)
6	39.8 Percent of 33093 medicinal chemistry compounds have low solubility (<= 20 ug/mL)
7	Evidence for 35-40% poor aqueous solubility 70,000 compounds screened at Pfizer, Groton Capsugel Symposium June 2003 Tokyo solubility formulation, customer feedback Collaborator feedback to Matrical “Sonicman” HTS plate based sonicator
8	Dimension of the solubility problem 40% of compounds have poor aqueous solubility Half the problem due to size / lipophilicity Half the problem due to crystal packing DMSO no better than water for compounds insoluble due to crystal packing Suggests an upper limit of 20% compounds insoluble in dry DMSO at 10uM Will this limit ever be achieved? Depends on whether a nucleation event occurs Depends on number of freeze thaw cycles
9	Compounds differ in aqueous and DMSO solubility based on crystalline form Ostwalds “rule of stages” Sequence of compound batch isolation proceeds towards thermodynamically most stable form 1 - amorphous - highest energy solid form 2 - highest energy crystalline polymorph 3 - lowest energy crystalline polymorph Amorphous is the highest energy form most soluble in water and DMSO
10	Early discovery compound purity Pressure on chemistry to increase output crystallization has disappeared Combinatorial compounds are now being purified by automated procedures 90% pure by evaporative light scattering 80% pure by UV detection Compounds “appear” more soluble amorphous state impurities enhance solubility
11	Fraction of Pfizer Groton compounds having melting point field information
12	Consequences of amorphous compounds Amorphous DMSO solubility is always higher than when compound is crystalline Amorphous compounds from combichem or medchem initially easily dissolve in DMSO allows preparation of DMSO stocks Sets stage for later precipitation problems
13	Thermodynamic aqueous solubility
14	Metastable supersaturated zone
15	Unstable supersaturated zone
16	Water and DMSO solubility Large, lipophilic compound aqueous insoluble DMSO greatly helps aqueous solubility Very crystalline compound may show no computational problem no “rule of 5” violation aqueous insoluble high melting point strong intermolecular crystal lattice DMSO does not help aqueous solubility
17	DMSO really helps aqueous solvation problems when the problem is size / lipophilicity Compound has to make a “hole” in DMSO to dissolve easier to do this in DMSO than in water no H-bond donor / acceptor networks to disrupt DMSO has a high dielectric constant solvates compound dipoles almost all drugs have dipoles
18	Sample storage in DMSO
19	Sample in DMSO lifetime Compound disappears from DMSO solution What is the explanation? Chemical integrity in DMSO keep cold and frozen avoid oxygen keep dry Compound solubility in DMSO cold and / or frozen is the worst choice possible avoid freeze thaw cycles
20	DMSO – water phase diagram
21	Timing factor in compound DMSO solubility Once a compound crystallizes from DMSO it will not easily re-dissolve crystallized compound is in a lower energy, higher melting point, less DMSO soluble form Narrow working window (time window) for keeping most compounds dissolved in DMSO 1 to 2 days at room temperature explains why compounds are active when freshly made but not when stored Freeze thaw cycles increase the probability of crystallization
22	Summary Crystalline state is important to aqueous solubility Crystalline state is important to DMSO solubility be alert for compound precipitation from DMSO do not store liquid DMSO stocks in the refrigerator minimize time once DMSO stocks are diluted expect erratic HTS screening results dependent on minor compound handling differences minimize freeze thaw cycles Poor DMSO solubility is here to stay
23	Recent Developments in DMSO solubility New Software DMSO solubility prediction software Pharma Algorithms http://ap-algorithms.com/dmso.html Chemical Diversity Labs http://www.currentdrugdiscovery.com/pdf/2003/500632.pdf New Hardware Matrical “SonicMan” plate based sonicator http://www.matrical.com/Literature/SonicManFlier.pdf
24	Acknowledgements The generous support of Pfizer Global R&D, Groton Labs in my post retirement activities is gratefully acknowledged