Synthesis and Physico-Chemical Properties in Aqueous Medium of All Possible Isomeric Bromo Analogues of Benzo-1H-Triazole, Potential Inhibitors of Protein Kinases.

In ongoing studies on the role of the individual bromine atoms of 4,5,6,7-tetrabromobenzotriazole (TBBt) in its relatively selective inhibition of protein kinase CK2α, we have prepared all the possible two mono-, four di-, and two tri- bromobenzotriazoles, and determined their physico-chemical properties in aqueous medium. They exhibited a general trend of a decrease in solubility with an increase in the number of bromines on the benzene ring, significantly modulated by the pattern of substitution. For a given number of attached bromines, this was directly related to the electronic effects resulting from different sites of substitution, leading to marked variations of pKa values for dissociation of the triazole proton. Experimental data (pKa, solubility) and ab initio calculations demonstrated that hydration of halogenated benzotriazoles is driven by a subtle balance of hydrophobic and polar interactions. The combination of QM-derived free energies for solvation and proton dissociations was found to be a reasonably good predictor of inhibitory activity of halogenated benzotriazoles vs CK2α. Since the pattern of halogenation of the benzene ring of benzotriazole has also been shown to be one of the determinants of inhibitory potency vs some viruses and viral enzymes, the present comprehensive description of their physico-chemical properties should prove helpful in efforts to elucidate reaction mechanisms, including possible halogen bonding, and the search for more selective and potent inhibitors

Isomeric Mono-, Di-, and Tri-Bromobenzo-1H-Triazoles as Inhibitors of Human Protein Kinase CK2α.

To further clarify the role of the individual bromine atoms of 4,5,6,7-tetrabromotriazole (TBBt), a relatively selective inhibitor of protein kinase CK2, we have examined the inhibition (IC(50)) of human CK2α by the two mono-, the four di-, and the two tri- bromobenzotriazoles relative to that of TBBt. Halogenation of the central vicinal C(5)/C(6) atoms proved to be a key factor in enhancing inhibitory activity, in that 5,6-di-Br(2)Bt and 4,5,6-Br(3)Bt were almost as effective inhibitors as TBBt, notwithstanding their marked differences in pK(a) for dissociation of the triazole proton. The decrease in pK(a) on halogenation of the peripheral C(4)/C(7) atoms virtually nullifies the gain due to hydrophobic interactions, and does not lead to a decrease in IC(50). Molecular modeling of structures of complexes of the ligands with the enzyme, as well as QSAR analysis, pointed to a balance of hydrophobic and electrostatic interactions as a discriminator of inhibitory activity. The role of halogen bonding remains debatable, as originally noted for the crystal structure of TBBt with CK2α (pdb1j91). Finally we direct attention to the promising applicability of our series of well-defined halogenated benzotriazoles to studies on inhibition of kinases other than CK2

Relative role of halogen bonds and hydrophobic interactions in inhibition of human protein kinase CK2alpha by tetrabromobenzotriazole and some C(5)-substituted analogues.

To examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2alpha by 4,5,6,7-tetrabromobenzotriazole (TBBt), we have synthesized a series of 5-substituted benzotriazoles (Bt) and the corresponding 5-substituted 4,6,7-tribromobenzotriazoles (Br3Bt) and examined their inhibition of human CK2alpha relative to that of TBBt. The various C(5) substituents differ in size (H and CH3), electronegativity (NH2 and NO2), and hydrophobicity (COOH and Cl). Some substituents were halogen bond donors (Cl, Br), while others were fluorine bond donors (F and CF3). Most of the 5-substituted analogues of Br3Bt (with the exception of COOH and NH2) exhibited inhibitory activity comparable to that of TBBt, whereas the 5-substituted analogues of the parent Bt were only weakly active (Br, Cl, NO2, CF3) or inactive. The observed effect of the volume of a ligand molecule pointed to its predominant role in inhibitory activity, indicating that presumed halogen bonding, identified in crystal structures and by molecular modeling, is dominated by hydrophobic interactions. Extended QSAR analysis additionally pointed to the monoanion and a preference for the N(1)-H protomer of the neutral ligand as parameters crucial for prediction of inhibitory activity. This suggests that the monoanions of TBBt and its congeners are the active forms that efficiently bind to CK2alpha, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand

Inhibitory activities (IC₅₀) of brominated Bt derivatives predicted on the basis of: (A) V_mol and experimental pK_a; (B) <i>ab initio</i> derived ΔG_solv(anion) and ΔG_diss free energies; and (C) autodock-derived free energy of binding (ΔG_bind).

<p>All relations point to predominance of hydrophobic interactions (V_mol or ΔG_solv(anion)), accompanied by protonation of the anionic form of the ligand upon binding to CK2α (pK_a or ΔG_diss) (see text for details).</p

Structures of all possible halogenated derivatives of benzotriazole.

<p>Structures of all possible halogenated derivatives of benzotriazole.</p

Distribution of short halogen-acceptor (O, N, S, π system) contacts identified in 21 accessible structures of complexes of CK2α with halogenated ligands.

<p>The Gaussian cumulative distribution was fitted to the crystallographic data for halogen to donor distance (solid line in panel A) and, according to the Anderson-Darling test, experimental data up to 3.7 Å agrees with a normal distribution (panel B). However, pairs separated by more than 3.7 Å are overrepresented, clearly limiting the maximal distance for eventual halogen-bonding interactions to the sum of donor and acceptor VdW radii. Note that an isolated water molecule (red triangles in panels A, C) is an equally favorable acceptor to the protein (O, N, S, π-electrons). The cumulative distribution of the experimental data is visibly better represented by a bi-normal distribution (panel C), in which the contribution of an additional narrow peak represents putative halogen-bonding (panel D). This is additionally supported by the distribution of angles X…Acc-C and C-X…Acc, which, for short halogen-acceptor distances, are substantially restricted to the regions favoring halogen bond formation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898.s002" target="_blank">Figure S2</a>).</p

Schematic representation of the effects of stepwise bromination of the benzene ring of benzotriazole (Bt), including interdependence of the molecular volume (A) and pK_a (B) of the products with their IC₅₀ for inhibition of protein kinase CK2α: (a) Green arrows follow sequential bromination of the central vicinal C(5)/C(6) atoms, leading to a moderate decrease of pK_a and a large decrease in IC₅₀; (b) Red arrows illustrate the lesser effects of bromination of the peripheral C(4)/C(7) atoms, resulting in a significant decrease of pK_a, with virtually no gain in inhibitory activity; (c) Black lines link products with the same number of bromine atoms, but substantially differing in pK_a and IC₅₀, culminating in the di- and tri-brominated derivatives with inhibitory activities comparable to that of TBBt.

<p>Note that the parent Bt (pKa 8.56), which is not an inhibitor (IC₅₀>2 mM), is located outside the diagrams.</p

Location of all nine halogenated Bt derivatives in complex with CK2α.

<p>For each ligand the average location determined from the 3 ns trace of Molecular Dynamics performed in the presence of explicit aqueous solvent is presented in relation to X-ray structure of CK2α (pdbj91). All ligands were found to bind in the same orientation (see panel A), in the position almost identical to that found for TBBt in the crystal structure of the complex with CK2α (see panel B, TBBt, from PDB, in magenta and putative location of Bt in green). The lowest-energy structures identified in 3 ns MD traces are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898.s003" target="_blank">Figure S3</a>.</p

Experimental values for triazole proton dissociation (pK_a), aqueous solubility (C_w), and inhibitory activity (IC₅₀) against CK2α compared with molecular volumes of brominated Bt derivatives, <i>ab initio</i> derived free energies of proton dissociation (ΔG_diss), free energies of solvation of the anionic forms, free energy of binding to CK2α estimated with the aid of Autodock for ligands both in neutral and monoanionic state (ΔG_bind), average ligand movement upon 3 ns Molecular Dynamics of the complex with CK2α in aqueous solution (RMFS), and the displacement between average ligand location estimated from 3 ns MD simulations in aqueous medium from the location of TBBt in the crystal structure with CK2α (RMSD).

a<p>Experimental data determined for all compounds under identical conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898-Wsik1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898-Wsik2" target="_blank">[22]</a>.</p>b<p>This work.</p>c<p>Values calculated previously using the same approach for all compounds <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898-Wsik1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048898#pone.0048898-Wsik2" target="_blank">[22]</a>.</p