Introduction

In the discovery of biologically active small molecules, for example via high-throughput screening, carboxylic acids may be found among the "hit" compounds. With a pK_A of ∼5, acids are ionized at physiological pH and therefore are not expected to penetrate cell membranes readily. The activity of such a molecule in a cell-based or in vivo screen could be explained in several ways. It could act on an extracellular target (such as an integral membrane receptor), it could have very high potency on an intracellular target that is moderated by the low intracellular concentrations (much lower than the concentration applied), or it could exploit a native uptake pathway to attain sufficient intracellular concentrations to act on an intracellular target. One strategy to distinguish these possibilities is the preparation of an ester and examination of its activity. The idea here is that as an uncharged molecule, the ester is capable of passive diffusion through the cell membrane and once inside the cell, the ester may be hydrolyzed to the acid by nonspecific esterases. When the biological target is intracellular, the higher intracellular concentrations of the active form of the compound that could thereby be achieved would be reflected in a higher measured potency. Ester formation can also be applied to pharmaceutical agents to modify their pharmacokinetics or ease of administration as another application of this "pro-drug" strategy. It has been applied to many known drugs (1,2,3,4) such as ramipril/ramiprilat (5,6) and melagatran/ximelagatran (7,8), and to the compounds studied here, aspirin and indomethacin (9,10,11,12,13).

Broadly, the preparation of a methyl ester from an acid is a straight-forward, even trivial chemical transformation. However, essentially all known methods would require at least a solvent partitioning extraction, and likely some form of chromatography, to obtain a pure product. Thus, obtaining the ester needed for the experiments described above is easy when multidisciplinary teams that include chemists work together. As high-throughput screening of compound libraries becomes more widely used in the field of chemical genetics, those who identify carboxylic acid hits from such screens may not have an established working relationship with chemists who could prepare the needed ester. The described method was developed to address this circumstance and permit a laboratory with little chemical background, experience, or equipment to effectively prepare esters for experiments addressing the site of action of a newly discovered agent.

The tactics described here ((Figure 1)) are adapted from a method to prepare esters using a solid-phase reagent (14). A strong base quaternary ammonium anion exchange resin is used to deprotonate the carboxylic acid. The resulting carboxylate is sequestered on the resin by electrostatic interactions. Then, the alkylating agent methyl iodide is added, and the methyl ester is formed via a nucleophilic substitution reaction. The resulting ester, as a neutral compound, has little affinity for the charged resin and dissociates from it. Any unreacted acid and other products of the reaction remain on the resin, meaning that the ester and the volatile reactant methyl iodide should be the only compounds present in the solution eluted from the resin. Little attention need be paid to the stoichiometric relationship of the acid to the reagents because they are all removed to phases different from the ester product. No analytical balance is required because the chosen reaction scale permits reasonably accurate determination of reactant weight using an electronic balance. No aqueous/organic partitioning is needed for purification of the reaction mixture because solid/liquid partitioning takes its place. No further purification of the product (by chromatographic methods, for example) should be necessary because the methyl ester is the only compound remaining upon evaporation of the eluent from the resin. The method as presented (mostly) gives high chemical yields, but even if its efficiency were much lower, the described scale of the reaction should still provide ample quantities of esters for further biological testing and, because of the solid-liquid-gas phase partitioning strategy, should still provide a very pure ester. Evidence that the biological target is intracellular comes, as in the example described here, when the methyl ester proves more potent than the parent carboxylic acid. A sensitive assay (such as mass spectrometry or possibly HPLC) for these compounds would be needed to prove the thesis that the ester is transported into the cell and then hydrolyzed to the active substance.