Drug transporter assays

Drug transporter proteins can be a very important consideration faced by drug discovery scientists. They are transmembrane proteins which facilitate transport of molecules both in and out of the cell. Therapeutic molecules can be inhibitors or substrates for these transporters. This can have a huge impact on the ADME (absorption, distribution, metabolism and excretion) properties of a therapeutic compound. The most widely studied drug transporter is P-gp (P-glycoprotein), but there are a variety of others expressed within the body.
Driven by the requirement to identify inhibitors of transporter proteins at an earlier stage, a group at Bristol –Myers Squibb have published a 384 well based assay panel in this article J Biomolecular Screening 2013, 19 (9), 1072-1083 http://dx.doi.org/10.1177/1087057113494807 The authors outline a range of assay formats they developed for a variety of different drug transporters using both fluorescence and radioactive methods in 384 well format, for speed and to support higher compound numbers. An example of one assay format developed was with the use of calcein-AM as a fluorescent reporter to measure P-gp inhibition. Calcein-AM can freely pass through the cell membrane and is converted via intracellular esterases into calcein (which does not pass freely). Uninhibited P-gp transporter can efflux the calcein-AM to the external environment before this esterase activity can occur. If the P-gp transporter is inhibited the calcein-AM will remain in the cell to be converted, and can be monitored using cellular imaging techniques. The authors made a comparison of the values obtained in the new 384 format against the current standard 96 well format (3H digoxin) and the correlation between the two methods was acceptable. This 384 well method offers a much faster response to generate data than the earlier 96 well methodology.
Using these assays the authors were able to detect inhibitors of P-gp and other transporters, however currently they are not able to determine if a compound is a substrate for these transporters. If a method could be produced in the same manner and plate format that accomplished this, it would be another step forward for drug transporter assays.

Clean and Economic Synthesis of Alkanesulfonyl Chlorides from S-Alkyl Isothiourea Salts via Bleach Oxidative Chlorosulfonation


A current eFirst paper by Jiaxi Xu et al. published in Synthesis DOI: 10.1055/s-0033-1338743 describes a simple and synthetically practical method to produce alkylsulfonyl chlorides from an alkyl chloride, bromide or mesylate.

Many of the previous methods for the formation of sulfonyl chlorides, such as the Reed reaction, have been referenced by Xu in his paper, but from these methods it is generally accepted that S-alkyl isothiourea salts are considered as easily accessible, inexpensive, and green starting materials.

The oxidative chlorosulfonation transformation of S-alkyl isothiourea salts into sulfonyl chlorides can be achieved by using many different reagents such as chlorine gas, H2O2–HCl, KMnO4/HCl, HCl-treated silica gel with PhIO and NaClO3/HCl. Xu has previously published the use of N-chlorosuccinimides and NaClO2  as the oxidative chlorinating reagents under acidic conditions but it was thought that the use of house-hold bleach would simplify product purification and be more atom efficient (scheme 1).



Table 1 describes some of the early optimisation of the oxidative chlorosulfonation step. Xu suggest that to achieve the best results the bleach should be titrated to know the actual concentration of NaClO.



S-alkyl isothiourea salt 2a was not entirely soluble in water and so would contaminate the product 3a when it was isolated by filtration.  By adding diethyl ether to the reaction highly pure 3a could be isolated and the amount of undesired hydrolysed product was reduced. With these newly optimised reaction conditions Xu synthesised a range of alkyl and benzyl sulfonyl chlorides in very good yields (Table 2).



To show that this chemistry was scalable Xu preformed a reaction on a 50 mmol scale and achieved a 96% yield over the 2 steps (scheme 2).

scheme 4


A mechanism similar to that proposed by Xu for his NCS/HCl oxidative chlorosulfonation has been postulated (scheme 3). In mild acidic aqueous solutions a chlorinium ion will be formed which is able to transform the S-alkyl

isothiourea salt into alkylsulfonyl methanimidamide salt 4 by 2 chlorination oxidation steps. 4 is then hydrolysed to sulfinic acid 5 which is subsequently oxidised to sulfonyl chloride 3.



This synthesis of alkylsulfonyl chlorides presented by Xu represents a cheap, simple and scalable method. With the use of bleach as the oxidant and without the need for any chromatography this method is one of the more environmentally friendly processes to produce alkylsulfonyl chlorides.