Chemical and biological evaluation of dipeptidylboronic acids as warheads for pro-, pro-soft, and 'ultra-smart' cancer therapeutics

Dipeptidylboronic acids are potent inhibitors of therapeutically important enzymes such as DPP-IV, FAP, and the proteasome, often achieving Ki values in the picomolar range. Xaa-boroPro dipeptides are the best-characterized of these compounds, and several Xaa-boroPro derivatives are currently being tested in clinical trials for the treatment of diabetes, cancer, and thrombosis. These compounds have been found to undergo a pH-dependent intramolecular cyclization reaction that is correlated with attenuation in their enzymatic inhibition potency. Because of this attenuation in potency, this phenomenon can potentially be exploited to provide the benefits of a soft-drug (a compound that becomes inactivated away from the intended target through metabolic of chemical processes). This intrinsic chemical feature of dipeptidylboronic acids when coupled with a pro-drug strategy (a compound that is delivered in an inactive form and later activated by specific metabolism or chemistry), render these compounds rich with possibilities in the development of pro-soft drugs and "ultra-smart" drugs (compounds that are reversibly activated at the intended target and inactivated away from the intended target) that are target-site specific.;Aminoacyl-leucineboronic acids (Xaa-boroLeu) are chemically and structurally similar to the Xaa-boroPro dipeptides and have potential in the design of anti-cancer agents due to their structural similarity to the FDA-approved VelcadeRTM. Here, dipeptides of boroLeucine with a variety of natural and non-natural P2 amino acids have been examined for their proteasome inhibitory potency in vitro and in cells against the proteasome, as well as for their cytotoxic potential. In spite of the large variety in P2 side chains, the majority of the compounds were no more than 10-fold less potent than VelcadeRTM in vitro. These compounds also exhibited a pH-dependent attenuation in their inhibition of the proteasome. One- and two-dimensional NMR experiments definitively showed that the Xaa-boroLeu dipeptides reversibly cyclize in a pH-dependent manner like the related Xaa-boroPros. Interestingly, thionamide derivatives of Phe-boroLeu, Ala-boroLeu, and Gly-boroLeu exhibited an irreversible pH-dependent attenuation in their potency that was demonstrated to be the result of deboronation.;Despite the excellent potency of the majority of these compounds in vitro, most showed a much-reduced ability to inhibit the proteasome in cells. The presence of the free amino group was thought to render the Xaa-boroLeu dipeptides susceptible to degradation by cellular aminopeptidases and reduce the cell permeability of the compounds relative to VelcadeRTM. Aminopeptidase degradation was ruled out as the cause for the reduction in in-cell median inhibitory concentration (ICIC50) against the proteasome, as dipeptidylboronic acids showed no degradation from cell lysate or intact cells. Consistent with the hypothesis that cell permeability was the cause of the reduced ICIC50 values, hydrophobic P2 amino acids offered improved in-cell inhibition and cytotoxicity.;Since the cyclization phenomenon is a characteristic phenomenon of dipeptidylboronic acids, factors that contribute to or influence it were examined. Model compounds were used to determine the features of dipeptidylboronic acids that contribute to the pKa values of the boronic acid and amino groups. The amide was found to have an acid-strengthening effect of nearly 1.5 units on the boronic acid, and the amino and boronic acid groups had significant effects on one another's pKa values. Microscopic and macroscopic equilibrium constants were determined from NMR titration data. These data revealed that the differences in pKa values for Gly-boroPro, Ala-boroAla, and Phe-boroLeu do not account for the dramatic differences in the extent of cyclization and stability of the cyclic species in these compounds. Instead this difference is likely due to differences in conformational preferences and flexibility.;The deboronation of Phe-boroLeu and its thionamide analog were also examined. Deboronation occurs in both compounds. However, the rate of deboronation is much faster in the thionamide than in the amide. Furthermore, the thionamide is not observed to cyclize.;Finally, pro-drug candidates were assayed for their ability to release dipeptidylboronic acid warheads in the presence of FAP. LC/MS-based cleavage assays demonstrated that pro-drugs containing FAP cleavage sequences branched from the P2 amino acid side chain released active warhead at a slower rate than pro-drugs with the activation sequence attached to the N-terminus of the warhead. Pro-drugs possessing the iso-peptide linked FAP cleavage sequence exhibited a difference in both the in-cell proteasome inhibition and cytotoxicity in the presence or absence of FAP. The one pro-drug that was tested in an FAP-expressing cell line showed specific activation in this cell line over the non-FAP-expressing cell line.;The work described herein aimed to understand the chemistry and biochemistry of Xaa-boroLeu dipeptidylboronic acids. Specifically, it was desired to obtain an understanding of the factors that govern the potency, stability, and cytotoxicity of these compounds in order to facilitate the development of pro-, soft, and pro-soft drugs that are safer and more effective.