Streamlining the purification of drug-like compounds “dry-loaded” onto silica.

By Dr Nigel Vicker, Group Leader, Discovery Chemistry,Dept of Pharmacy &

Pharmacology, University of Bath Solvent removal using centrifugal vacuum evaporation is an established means to eradicate the drying bottleneck for chemists engaged in parallel synthesis and purification. It has been proven that during centrifugal vacuum evaporation the structural integrity of drug-like compounds remains intact in typical reaction solvents such as DMF. The removal of high boiling solvents by the application of centrifugal vacuum evaporation and the control of unwanted ‘bumping’ in such cases, and with solvent mixtures is also a proven benefit to the drug discovery chemist. In this study we extend these advantageous uses of centrifugal evaporation to the adsorption of drug-like molecules as an automated means of improving parallel purification.

Figure 1 The new Genevac DD-4X evaporator

When purifying compounds either by classical chromatography on a single column or more commonly nowadays in a parallel fashion using automation, it is common to adsorb a mixture of compounds of varied structure and polarity on a small amount of silica and ‘dry-load’ the mixture onto the column prior to chromatography. Classically compounds are adsorbed typically on silica in a round-bottomed flask followed by solvent removal using a traditional rotary evaporator. This method although usually efficient at adsorption has a number of limitations such as bumping which may result in loss of compound up the vapour duct (this can also occur with adsorbed compound when releasing the vacuum) and removal from the flask as it is common for some compound or silica to stick to the sides of the flask. With the parallel paradigm now an established part of drug discovery, possibly the biggest limitation is the slow throughput and lack of automation of the above process.

Here we compare the adsorption of drug-like molecules on silica using a traditional rotary evaporator and a Genevac DD-4 centrifugal evaporation system and evaluate the separation and recovery of mixtures of these drug-like molecules. The compounds chosen for the study are depicted below; these include the steroid estradiol, a heterocyclic compound caffeine and the tubulin binding drug colchicine. These compounds were chosen due to their structural diversity and their different chromatographic properties on silica, with the least polar compound (estradiol) chromatographically mobile in ethyl acetate/hexane mixtures and the most polar (colchicine) requiring ethyl acetate/methanol for chromatography.

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Estradiol ColchicineCaffeine Materials and Methods. Estradiol and caffeine were purchased from the Aldrich Chemical Co. (Gillingham, UK) and colchicine was obtained from Avocado (Heysham, UK). All organic solvents of A.R. grade were supplied by Fisher Scientific (Loughborough, UK), the solvents used for dissolving the compounds were dichloromethane and methanol and the solvents used for chromatography were hexane, ethyl acetate and methanol. Compounds were adsorbed on Sorbsil C60 silica gel from Merck and chromatography was performed on 10g Isolute® pre-packed Flash Si columns from Argonaut Technologies.

Figure 2: A dried silica-adsorped sample is removed from the evaporator

Experimental Protocol. Each compound (100 mg) was dissolved in solvent (5 mL), for solubility reasons and also to examine the effects of removal of solvent mixtures estradiol was dissolved in methanol and caffeine and colchicine were dissolved in dichloromethane to give a combined volume of 15 mL. Silica (3g) was added to the mixed compounds in solution in either a 100 mL round bottomed flask or a 12-station Radleys Carousel reaction station. The solvents were removed either on a rotary evaporator (100 mL flask) using a water pump and water bath temperature of 40° C or a Genevac DD-4 centrifugal vacuum evaporator using the AquaspeedTM setting at 30° C for 45 min (Carousel tube). The adsorbed compounds were then ‘dry-loaded’ onto 10g pre-packed Argonaut columns and chromatography was performed using an Argonaut Flashmaster II in an automated fashion using a gradient elution developed for these compounds.

Results and Discussion. Adsorption of the compounds onto silica in both cases gave a good mobile stationary phase. In the case of adsorption in the centrifugal evaporator as the solvent containing the compound mixture was much higher than the level of silica some compound appeared to stick to the sides of the tube. This was overcome by using approximately the same volume of solvent mixture as silica (~ 6 mL) and this gave complete adsorption and a mobile silica plug at the bottom of the tube. Each component of the mixture was separated to baseline resolution and recovery of each component was the same for each method. This clearly shows that the use of centrifugal vacuum evaporation is an attractive automated alternative for the adsorption of compound mixtures prior to chromatography.

Figure 4: Method screen from the flash Chromatograph

Conclusions Centrifugal evaporation has been demonstrated to be an attractive alternative of dry-loading samples prior to chromatography, and after work up of parallel synthesis reactions offers a combinatorial method of dry-loading to streamline the parallel purification process. As the Genevac DD-4 has been designed to hold reaction blocks directly from the Radleys Greenhouse synthesiser and tubes from the Radleys Carousel reaction station, an extension of this paradigm is the automated parallel adsorption of reaction mixtures directly onto silica, using centrifugal evaporation, prior to automated parallel purification leading to a new and exciting extension of this technology. For more information on this application of centrifugal evaporation please contact: Genevac Ltd Farthing Rd Ipswich Suffolk IP1 5AP 01473 240000 www.genevac.co.uk