RO2468

This is overall an excellent chemical probe against MDM2, based on in vitro biochemical binding affinity, cellular activity and selectivity, and an impressive in vivo/xenografts model efficacy. It is important, though, to discuss the RO2468 compound in relation to two of its analogues that possess similar activities both in vitro and in vivo: RO5353 (which is reported alongside of RO2468 in the same study by Zhang and co-workers, ACS Letters 2014) and RO8994 (which is the parental compound described by the same team in Bioorg. Med. Chem. 2014 and herein). The compound binding affinity (IC50 = 6 nM) is almost identical to RO5353 (7 nM) and RO2468 (5 nM). In cancer cells, the antiproliferative potency (IC50 = 0,015) and selectivity (742) values of RO2468 are comparable to/in the same range of other two compounds, although RO5353 shows the best potency (IC50 = 0.007) and selectivity (1023) in cell cultures. With regard the efficacy in in vivo, the three compound show comparable activities, exhibiting tumour regression between 6 to 10 mg/kg in SJSA1 osteosarcomas xenografts in nude mice with chronic dosing over 2-4 weeks, with no significant weight loss. They also possess very similar/comparable profiles for their in vivo pharmacokinetics characteristics. Although for none of these three analogues selectivity studies have been yet carried out against other proteins of the same family - their structural complexity, robust SAR and low nanomolar binding affinities can be overall a good indicator of a low probability of off-target effects. Furthermore, Zhang and co-workers have reported that RO8994 has acceptable toxicity profiles in both rodent and non-rodent dose range finding studies – although data are not reported (Bioorg. Med. Chem. 2014). While this information has not been provided for the other two compounds – based on their close structural characteristics, they could show the same profile.

It should be noted that both RO8994 and RO5353 show a much better oral bioavailability than RO2468 (F% 92 vs. 46) – and that RO8994 has a t1/2 more than double compare to both RO2468 (and RO5353).

The synthesis of compound RO8994 has been optimized for large scale activities, which offers a practical advantage. Although the same synthetic approach has been applied to prepare the RO2468 and RO5353, this was not specifically optimised for prepare large amounts. Nonetheless, these two compounds have been used enantiomerically pure compare to RO8994, which is used as racemic mixture. As the use of an enantiomerically pure compound is always best, and based on the overall profile for the in vitro and in vivo data, R05353 is likely the best probe out the three analogues and in general one of the best among the currently-available small molecules against MDM2.