In Silico Prediction of Off Target Activities of Chemical Probes

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Chemical probes are frequently described as important tools in the validation of new drug targets, yet many of them are of little value due a number of important factors frequently including poor selectivity. This issue was highlighted by Frye in 2010 (Frye, Nature Chemical Biology 2010, 6(3), 159-161) where he illustrated the problem with the case of staurosporine, a promiscuous kinase inhibitor and the subject of 8,000 publications, many of which make conclusions which are not valid due to the compounds promiscuity. Frye defines a set of criteria which a probe should have in order for it to be a valuable tool to the drug discovery scientist. One key criterion is selectivity and he states that it is important that ‘a quality chemical probe has sufficient in vitro potency and selectivity data to confidently associate its in vitro profile to its cellular or in vivo profile,’ Which is valid suggestion.
Determination of selectivity is difficult as the majority of groups including pharmaceutical companies only have access to a limited range of selectivity assays and establishing selectivity against targets related to the protein of interest is relatively straightforward; however assessing broader selectivity is an issue both with respect to choosing targets to screen against and accessing suitable assays.
A recent publication highlights the importance of assessing broad selectivity and describes an in silico approach capable of predicting off target liabilities (Antolín and Mestres, ACS Chemical Biology 2014, ahead of print). The authors apply their software to the full set of chemical probes from the NIH Molecular Libraries Program (MLP) with some very interesting findings, illustrated by the examples below.
Table 1 shows the activity at the canonical (original target) of four probes in the collection together with off target activity identified through in silico testing and subsequently demonstrated in a biological assay. In each case the probes are more potent at the newly discovered target than the original published target.

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ML006 was published as a weak probe for the S1P3 shingosine receptor but has micromolar potency for mTOR kinase. ML124 and ML204 were originally identified as probes for TRP channels, TRPML3/2 and TRPC4/5 respectively but show off target activity as acetylcholinesterase inhibitors (ML204) and sigma receptor ligands (ML124). ML141 was identified as a highly potent carbonic anhydrase inhibitor having originally been described as a selective inhibitor of Cdc42 GTPase. The latter discovery regarding ML141 should possibly have been identified earlier as the template in question is a well documented carbonic anhydrase inhibitor chemotype (Weber et al J. Med. Chem. 2004, 47(3),550-7). These results thus pose significant questions as to the usefulness of this set of compounds as probes.
The publication from Antolin et al not only highlights the caution that workers need to exercise when using chemical probes but also offer a potential strategy for checking the validity of the probe before drawing conclusions regarding experiments in which the probes have been used.

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

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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).

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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.

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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).

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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).

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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.

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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.