Relative Role of Halogen Bonds and Hydrophobic Interactions in

Jul 23, 2010 - ... and Hydrophobic Interactions in Inhibition of Human Protein Kinase ... of human CK2α by 4,5,6,7-tetrabromobenzotriazole (TBBt), we...
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J. Phys. Chem. B 2010, 114, 10601–10611

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Relative Role of Halogen Bonds and Hydrophobic Interactions in Inhibition of Human Protein Kinase CK2r by Tetrabromobenzotriazole and Some C(5)-Substituted Analogues Romualda Wa˛sik,† Maja Łebska,† Krzysztof Felczak,†,§ Jarosław Poznan´ski,† and David Shugar*,†,‡ Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawin´skiego 5a, 02-106 Warszawa, Poland, and DiVision of Biophysics, Institute of Experimental Physics, UniVersity of Warsaw, 93 Z˙wirki i Wigury St., 02-089 Warszawa, Poland ReceiVed: March 30, 2010; ReVised Manuscript ReceiVed: June 28, 2010

To examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2R 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 CK2R 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 CK2R, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand. Introduction Protein kinase CK2 (previously known as casein kinase 2, albeit casein is not an in vivo substrate), the most pleiotropic of known protein kinases, is a Ser/Thr kinase, also known to phosphorylate Tyr residues. It is constitutively active, but attempts to clarify its mode of regulation remain ongoing.1 It plays a key role as an antiapoptopic factor and is, consequently, a highly promising drug target, particularly since its level is markedly elevated in all human cancers and experimental tumors.1,2 This underlines the importance of development of permeable low molecular weight selective inhibitors of this enzyme, as well as its intrinsically catalytically active subunits CK2R and CK2R′.2 The first reported promising low molecular weight inhibitor, 4,5,6,7-tetrabromotriazole, now known as TBBt (or TBB), and early on shown to be cell-permeable,3 was shortly thereafter followed by the finding that the corresponding 4,5,6,7-tetrabromo-1H-benzimidazole (TBBz) is a comparably good inhibitor of CK2.4 Although many more potent inhibitors of CK2 have since been reported,5 TBBt and TBBz continue to be widely applied. Screening of TBBt against a small panel of over 30 different kinases demonstrated that it is reasonably selective for CK2.6 Subsequent screening of a number of analogues of TBBt and TBBz versus a larger panel of 80 kinases revealed three major * Corresponding author: Phone: +48 22 592 3511. Fax: +48 22 592 2190. E-mail: [email protected]. † Polish Academy of Sciences. ‡ University of Warsaw. § Present address: Center for Drug Design, University of Minnesota, Minneapolis, Minnesota 55455.

off-targets for TBBz and also showed that TBBt is more selective toward CK2R.5 A characteristic feature of the crystal structures of complexes of CK2R (and one other kinase; see below) with TBBt and some of its analogues substituted on the triazole ring is the presence of what are now widely known as halogen bonds, that is, noncovalent electrostatic interactions between a classical hydrogen bond acceptor (electronegative O, N, or S) and a polarizable, partially electropositive halogen atom (Cl, Br, I). Statistical analysis of crystal structures of many proteins complexed with halogenated ligands7 demonstrated that such halogen bonds exhibit a geometry similar to that of hydrogen bonds,includingadirectionalpreference,butwithadonor-acceptor distance significantly shorter than the sum of their van der Waals radii. Fluorine, because of its high electronegativity, is a very poor halogen bond acceptor but is a good acceptor from standard hydrogen bond donors.8 However, despite the clear evidence of short halogen bonding contacts in protein structures, their energetic contribution is still under active debate.9 Thus, analysis of the effect of a halogen bond on the isomerization of a four-stranded DNA junction led to estimation of the halogen bond contribution as 2-5 kcal/ mol stronger than a hydrogen bond in an analogous environment.10 However, it should be noted that the energy contribution was calculated on the assumption that the observed conformational switch is not accompanied by a change in DNA hydration entropy. From a design perspective, it would appear that one may substitute a halogen bond for a hydrogen bond in the active site of an enzyme to increase both affinity and/or specificity. A recent example of such an approach, involving the enzyme ketosteroid isomerase, was based on replacement of the tyrosine

10.1021/jp102848y  2010 American Chemical Society Published on Web 07/23/2010

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hydrogen bond donor in the oxyanion hole of the enzyme by semisynthetic enzymes containg para-halogenated phenylalanine derivatives in place of the tyrosine hydrogen bond donor. All of the halogenated enzymes exhibited activity comparable to that of the phenylalanine mutant, leading to the conclusion that a halogen bond could not, in this system, functionally replace a hydrogen bond in its active site.9 Furthermore, analysis of structures of T4 lysozyme binding various halogenated benzene derivatives demonstrated that (with the exception of an iodine substituent) pentafluorobenzene and its bromo and chloro congeners display heterogeneity of binding sites, strongly suggesting predominance of van der Waals interactions rather than halogen bonding per se. Moreover, comparison of association constants led to a rough estimate of halogen bond strength as 0.5-0.7 kcal/mol, hence much less than that for a canonical hydrogen bond.11 The most important factor enabling appropriate estimation of halogen bond strength is the decomposition of the observed change in free energy to the accompanying changes in entropy and enthalpy. As demonstrated for halogenated anesthetic compounds either by van’t Hoff analysis of the temperature dependence of binding halothane to BSA12 or by calorimetric studies on binding of halothane and isoflurane to the four-helix bundle motif of Ferritin,13 both entropic and enthalpic effects contribute comparably to binding. Similarly, binding of chlorohydroxyphenyl acetal by CprK protein, monitored by isothermal titration calorimetry, was reported to be largely entropically driven.14 A systematic study of binding of rationally designed halogenated peptides by a small modular domain of a mammalian neuronal protein demonstrated that, for all compounds studied (with the exception of fluorinated peptides), contributions of enthalpic and entropic (e.g., hydrophobic) effects were virtually equal.15 With a view to better understanding the selectivity, affinity, and mode of binding of TBBt to CK2, we have synthesized a series of analogues in which one of the Br atoms is replaced by other substituents and examined the activities of these ligands as inhibitors relative to that of TBBt. In some respects, this approach is similar to that adopted by Kraut et al.9 (see above). The bromine atom selected for replacement is that at C(5), which was found to be involved in halogen bonding in the crystal structure of TBBt bound to maize CK2R [pdb1j91]16 and human pCDK2 [pdb1P5E],17 as well as in other TBBt analogues bound to maize CK2R [pdb2oxd, pdb2oxx, pdb2oxy]18 [pdb1zoe, pdb1zog].19 It should be noted that the TBBt molecule in the two complexes differs by its prototropic state. In the complex with CK2R, the ligand molecule is asymmetric with C4a-C7a, C7a-N1, N1-N2, N2-N3, and N3-C4a bond lengths of 1.38, 1.40, 1.24, 1.42, and 1.38 Å, respectively, suggesting that, in contrast to the monoanionic form highly predominant in aqueous medium, the TBBt molecule is in the neutral N(1)-H/N(3)-H form (see Scheme 4). In the complex with pCDK2, TBBt is symmetric, and C4a-C7a, C7a-N1, N1-N2, N2-N3, and N3-C4a bond lengths are 1.45, 1.38, 1.43, 1.43, and 1.38 Å, respectively. This indicates that N1-N2, N2-N3 (1.43 Å), and C4a-C7a (1.45 Å) are single chemical bonds, indicative of TBBt in the neutral N(2)-H form. Note that, for the symmetrical anionic form, the expected length of the nitrogen-nitrogen bond is 1.33 Å, which is an average value of single (1.43 Å) and double (1.24 Å) chemical bonds. On the other hand, QM/MM calculations suggested that TBBt analogues bind efficiently to CK2R in the monoanionic form,20 and thus both hydrophobic forces and electrostatic interactions together drive inhibition of CK2R activity.

Wa˛sik et al. SCHEME 1

SCHEME 2

Results and Discussion Synthetic Procedures. The starting compound, 4-Br-1,2phenylenediamine (1b, see Scheme 1) was obtained in good yield, according to a previously published procedure,21 by reduction of 4-Br-2-nitroaniline22 with SnCl2. The monosubstituted 5-F- (2a), 5-Br- (2b), 5-CF3- (2c), and 5-NO2- (2d) benzotriazoles (Bt) were prepared from the appropriate ophenylenediamines by nitrous-acid-promoted cyclization23 (see Scheme 1), while 5-NH2-benzotriazole (2i) was obtained in 60% yield by HI reduction24 of the parent 5-nitro analogue (2d) or with the use of SnCl2 in HCl (see Scheme 2). To our knowledge, there are no reports on direct bromination of monosubstituted benzotriazoles, with the exception of 5-ClBt (2f) to 5-Cl-Br3Bt (3f).23 The 5-F- and 5-CF3-, 5-CH3-, 4,6,7tribromobenzotriazoles (Br3Bt) (3a, 3c, 3e) were prepared from the 5-monosubstituted benzotriazoles by direct bromination in concentrated nitric acid.23 However, direct bromination of 5-COOH- and 5-NO2- benzotriazoles (2g, 2d) led to a complex mixture of dibrominated products and a low yield of the required tribromo analogues. To circumvent difficult separations and low yields of the desired tribromo analogues, we have developed new straightforward procedures leading to only one product. Oxidation of 5-CH3-Br3Bt (3e) with the use of KMnO4 led to 5-COOH-Br3Bt (3g) in 72% yield and high purity (see Scheme 3). Then 5-NO2Br3Bt (3d) was obtained in 70% yield by nitration of Br3Bt (3h), prepared by decarboxylation in quinoline of the parent 5-COOHBr3Bt in 68% yield. In attempts to synthesize 5-NH2-Br3Bt (3i), reduction of 5-NO2-Br3Bt (3d) by SnCl2 led to partial debromination, with dibromo isomers as main products. This drew our attention to a recent report on the use of HI for selective reduction of aromatic nitro compounds to amines and claimed to proceed with excellent chemoselectivity, without affecting other functional groups, including halides.24 In our hands, its application to reduction of 5-NO2-Br3Bt (3d) led to a mixture of monobromo and bromo-iodo analogues of 5-NH2-Bt (2i), as determined by high-resolution mass spectroscopy (see Scheme 3). We have, however, successfully applied this procedure to preparation of 5-NH2-Bt (2i) from its 5-nitro congener (2d). The foregoing difficulties also prompted us to attempt direct reduction of 5-NO2-Br3Bt (3d) with use of Fe powder in acetic acid25 and resulted in the desired 5-NH2 congener (3i, see Scheme 3) in 74% yield.

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SCHEME 3

TABLE 1: Inhibition (IC50, and Standard Error in Parentheses, in µM) of Human CK2r by 5-Substituted Benzotriazoles and 5-Substituted-4,6,7-Tribromobenzotriazoles

* Roughly estimated since titration was performed for ligand concentrations in the range of 0-100:M.

Inhibitory Activity against Human CK2r. The inhibitory activities of the various analogues against CK2R are presented in Table 1. The 5-substituted 4,6,7-tribromobenzotriazoles with a hydrophobic group in the 5-position (H, Me, Cl, Br, NO2) exhibit similar inhibitory activities, with IC50 values in the low micromolar range, but with the 5-Br significantly more active. For the analogues with hydrophilic groups in the 5-position, COOH (dissociated in neutral aqueous medium) and NH2 activity is substantially decreased. With a 5-fluorine substituent, which may play a role in hydrogen bonding, the appropriately substituted analogue has activity similar to the parent Br3Bt. All of the experimental data leading to the determination of IC50 are presented in Supporting Information Figure 1. Physicochemical Properties of TBBt Derivatives. Unlike the neutral form of TBBt which, because of symmetry, may exist as two prototropic tautomers, the 5-substituted analogues may exist as three prototropic forms (see Scheme 4). Moreover, the TBBt analogues in aqueous solution at pH close to neutral may display a protonation equilibrium. Comparison of the UV-vis spectra recorded in aqueous solution at three pH values (2, 7, 12) demonstrated that, at neutral pH, C(5)-substituted

SCHEME 4: Prototropy of the Neutral Forms of 5-Substituted Benzotriazoles (X ) H) and Their 4,6,7-Tribromo Analogues (X ) Br)

Br3Bt analogues are largely dissociated, whereas C(5)-substituted Bt analogues are generally neutral. The fraction of the anionic form in neutral solution was estimated assuming that the spectra recorded at pH 7 are a superposition of those recorded for neutral (pH 2) and anionic (pH 12) forms. This approach was successful for most of the compounds, and the estimated populations of neutral forms at physiological pH are collected in Table 2. Ab initio calculations further demonstrated the predominance of these two forms, while the third possible, the symmetrical N(2)-H, is barely detectable, if at all. The above method failed for 5-NH2-Bt (2i), indicating that this compound experiences an additional ionic equilibriumsmost likely a cationic form at low pH. The spectra of TBBt, 5-ClBr3Bt, and 5-CH3-Br3Bt (3f, 3e) in acidic medium display a long-wavelength tail arising from light scattering of aggregated particles. This indicates that these three compounds, which are monoanions at pH 7, are poorly soluble in their neutral forms. In parallel, standard UV-monitored titrations versus pH were performed to determine the pKa values for dissociation of the triazole ring proton (Table 3). The prototropic equilibrium of the neutral forms, monitored with the aid of 13C NMR spectroscopy in anhydrous DMSO, demonstrated existence of a relatively slow exchange between at least two prototropic forms, resulting in significant broadening of 13C resonance lines observed for most of the compounds (Figure 1).

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TABLE 2: Structural Parameters of the Benzotriazole Derivativesa dipole [D] ligand

rmsd #X #H ASA ∆ASA ASAap ∆ASAap H(N1) H(N2) H(N3) ∆Gsolv ∆G1 ∆G2 ∆G3 ∆Gdiss [Å2] bonds bonds [Å2] [Å2] [Å2] [Å2] [kcal mol-1] [kcal mol-1] [kcal mol-1] [kcal mol-1] [kcal mol-1]

Bt 5-NH2-Bt 5-F-Bt 5-CH3-Bt 5-Cl-Bt 5-NO2-Bt 5-COOH-Bt 5-Br-Bt 5-CF3-Bt Br3Bt 5-NH2-Br3Bt 5-F-Br3Bt 5-CH3-Br3Bt 5-Cl-Br3Bt 5-NO2-Br3Bt 5-COOH-Br3Bt TBBt 5-CF3-Br3Bt

1.2 1.3 1.3 0.9 1.5 1.5 1.7 1.5 0.8 1.3 1.3 1.2 0.9 1.6 1.9 1.6 1.6 1.3

0 0 0 0 0.5 0 0 0.3 0 1.7 1.5 1.8 1.8 1.9 1.2 1.5 2.3 2.6

4.6 3.4 3.8 2.5 3.5 3.4 3.1 3.8 3.8 3.4 3.6 3.7 3.0 3.1 3.3 4.2 3.5 3.3

226 249 240 252 259 279 277 282 282 362 371 366 372 379 378 381 391 389

193 205 199 209 223 227 246 243 232 310 311 317 325 310 312 330 322 329

131 101 111 157 165 94 101 188 97 286 264 272 297 302 246 254 313 249

102 79 87 118 131 74 89 152 79 237 213 230 251 239 198 216 250 210

5.6 6.8 6.0 5.8 5.8 7.9 16.1 5.8 6.1 3.7 4.5 4.8 4.1 4.5 7.0 16.5 4.4 4.9

0.7 2.5 2.0 1.2 2.4 6.3 15.9 2.5 3.4 2.0 1.1 3.5 1.3 3.5 6.7 15.7 3.4 1.1

5.7 6.9 3.4 6.2 3.2 3.2 11.9 3.2 2.9 5.3 6.3 4.0 5.8 4.3 4.9 11.1 4.4 6.3

-3.3 -5.7 -2.7 -1.0 -1.0 -6.2 -63.3 -1.0 -3.8 4.3 3.2 3.9 5.9 5.8 1.7 -55.4 5.3 2.7

0.0 2.8 0.4 0.5 0.3 0.0 0.0 0.0 2.2 0.6 2.7 0.6 1.2 0.0 0.0 3.0 0.0 1.0

5.1 5.9 5.4 3.6 5.6 7.0 6.7 5.4 6.1 3.4 4.8 3.6 5.3 4.1 4.4 5.1 5.5 5.2

0.0 0.0 0.0 0.0 0.0 2.4 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.4 0.7 0.0 0.0 0.0

59.5 62.5 57.8 59.9 56.7 54.3 53.1* 57.4 58.4 51.4 53.9 51.1 52.8 49.7 45.8 48.3* 49.7 50.4

a The rmsd is a measure of the average displacement of a ligand in the structure of the complex; #X bonds, # Hbonds are the average number of halogen and hydrogen bonds, estimated from the calculated structures of the complexes; ASA is the average solvent-accessible area, ∆ASA its change upon complex formation; and ASAap and ∆ASAap are the values calculated only for the apolar atoms (e.g., excluding N, O, HN, and HO). All of these parameters were estimated as average values calculated for the 20 lowest energy structures of the complexes. The population of the major protomeric forms of the neutral ligands, P, and their dipole moments, D, were estimated with the aid of quantum-mechanical calculations, using the PCM model of aqueous solvent; solvation free energy, ∆Gsolv was calculated ab initio for the lowest energy protomeric form of the neutral ligand; ∆G1, ∆G2, and ∆G3 are the QM-derived relative free energies of the three possible protomeric forms, and ∆Gdiss is the estimated free energy of dissociation of the triazole proton (cf. eq 3 for details). *Values calculated for the nondissociated carboxyl group.

TABLE 3: Parameters Employed for Prediction of Inhibitory Activities of the Various Ligandsa IC50 [µM ] ligand Bt 5-NH2-Bt 5-F-Bt 5-CH3-Bt 5-Cl-Bt 5-NO2-Bt 5-COOH-Bt 5-Br-Bt 5-CF3-Bt Br3Bt 5-NH2-Br3Bt 5-F-Br3Bt 5-CH3-Br3Bt 5-Cl-Br3Bt 5-NO2-Br3Bt 5-COOH-Br3Bt TBBt 5-CF3-Br3Bt

pKa 2i 2a 2e 2f 2d 2g 2b 2c 3h 3i 3a 3e 3f 3d 3g 3c

8.56 9.23 7.96 8.79 7.62 6.31 8.08 7.55 7.23 5.38 5.95 4.77 6.20 5.20 4.49 5.19 5.10 4.58

(0.04) (0.04) (0.05) (0.05) (0.08) (0.07) (0.06) (0.07) (0.05) (0.04) (0.05) (0.04) (0.30) (0.30) (0.07) (0.05) (0.30) (0.04)

Xma 0.03 0.01 0.10 0.02 0.19 0.83 0.92* 0.22 0.37 0.98 0.92 0.99 0.86 0.98 1.00 0.02* 0.99 1.00

log P 1.21 0.52 1.49 1.62 1.93 1.20 0.40 2.03 2.09 3.39 2.71 3.55 3.78 4.09 3.28 2.32 4.24 4.22

(0.17) (0.40) (0.35) (0.21) (0.37) (0.33) (1.15) (0.37) (0.23) (0.31) (0.23) (0.36) (0.35) (0.31) (0.29) (0.96) (0.32) (0.46)

log S

Vmol [Å3]

#F

experimental

-1.38 -1.52 -1.44 -1.53 -1.96 -1.66 -1.38 -2.14 -2.08 -3.95 -3.78 -4.06 -4.33 -4.58 -4.05 -3.87 -4.80 -4.42

103 115 106 120 119 125 128 125 129 168 179 171 185 182 189 192 188 192

0 0 1 0 0 0 0 0 3 0 0 1 0 0 0 0 0 3

nonactive nonactive 180 (30) 440 (140) 72 (10) 54 (8) 190 (80) 51 (7) 109 (21) 6.6 (0.7) 73 (21) 3.51 (0.17) 2.49 (0.43) 1.20 (0.09) 2.26 (0.18) nonactive 0.61 (0.09) 2.28 (0.21)

R (Vmol)

QSAR

R(log D#)

R(log S)

228 113 191 84 90 62 55 62 52 4.9 2.6 4.3 1.9 2.2 1.5 1.2 1.5 1.3

170 120 170 110 75 68 68 48 110 3.7 2.5 3.5 2.3 1.5 1.6 1.5 1.0 2.3

456 2.8 × 105 178 476 66 42 175 38 171 4.2 72 3.3 4.3 1.0 2.6 1815 0.9 1.9

220 176 200 173 88 141 220 66 72 3.7 4.9 3.1 2.0 1.4 3.2 4.2 1.0 1.8

a

Experimental pKa; Xma, population of monoanionic form at neutral pH; log P and log S are consensus values of calculated partition and solubility coefficients; Vmol, calculated molecular volume; IC50, experimental values and those estimated from the four best single-parameter nonlinear regression models, and, finally, by QSAR. *Corrected for ionization of carboxyl group.

Preliminary QM calculations pointed to the N(1)-H and N(3)-H neutral forms as the main interexchanging states (cf. ∆G1, ∆G2, and ∆G3 in Table 2). The calculated free energy of dissociation of the triazole proton (∆Gdiss in Table 2) was found to be dependent on the nature of the 5-substituent, being lowest for the neutral carboxyl and highest for the amino derivatives. This is in line with the resonance patterns observed in the 13C NMR spectra, where 5-NH2-Br3Bt (3i) exhibits two separate sets of slightly broadened resonance lines, corresponding to the N(1)-H and N(3)-H forms, while 5-COOH-Br3Bt (3g) displays a set of seven narrow resonance lines. For the other compounds, with C(5) substituents displaying intermediate electronic properties, the patterns of the resonance lines fall in the medium

exchange/coalescence regime, leading to extremely broad lines with a width exceeding 1 kHz. Addition of a small amount of water (∼1% v/v) led to significant broadening of the resonance lines, pointing to an increase in proton transfer rates via the more efficient water-mediated one. An analogous effect has been reported for benzotriazole derivatives symmetrically substituted on the benzene ring.26 The appropriate structure-related data are summarized in Tables 2 and 3. Molecular Modeling of Inhibitor Binding to Human CK2r. Two Modes of Binding TBBt by CK2r. For each of the Bt and Br3Bt analogues, a set of 10 lowest energy structures, selected from 150 models of their complexes with human CK2R

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Figure 2. Lowest energy structure of the modeled complex of human CK2R with 5-NO2-Br3Bt. The protein backbone is represented as a ribbon model, the benzotriazole derivative by thick lines, and its volume by a transparent yellow surface. The side chains of the residues found interacting with the ligand in most of the low-energy structures are identified; their apolar bonds are in black, while those with oxygen and nitrogen are in red and blue, respectively. Backbone carbonyl groups of V45, G46, and S51, as well as side chain oxygen atoms of S51, E114, N118, and D175, make relatively highly populated halogen bond-like contacts. The 5-NO2 substituent (denoted by an arrow) is located in the peripheral region of the enzyme binding pocket and is therefore partially accessible to solvent.

Figure 1. 13C spectra of 5-substituted derivatives of (A) benzotriazole and (B) 4,6,7-tribromobenzotriazole in anhydrous DMSO, and (C) after addition of 1% (v/v) water to the DMSO solutions of B.

obtained from SA simulations, were subjected to further analysis. Initially, location of a potential inhibitor with respect to the CK2R binding site was tested in terms of the rmsd values estimated for the heavy atoms of the heterocyclic ring. All compounds were found flexibly located in the protein pocket, which readily adapts to small inhibitor movement. The rmsd of their average locations varied from 1 Å (5-CF3-Bt (2c), 5-CH3-Bt (2e), 5-CH3-Br3Bt (3e)) up to 2 Å (5-NO2-Br3Bt (3d), 5-COOH-Bt (2g), 5-NH2-Br3Bt (3i)). Comparison of the ensembles of lowest energy structures obtained for the various ligands points to a weak, but noticeable, ligand-specific preference in its location inside the CK2R pocket. Both observations clearly indicate that the CK2R binding site is flexible enough to bind a broad class of heteroaromatic compounds. In particular, for TBBt, two alternative binding modes were found by the SA procedure (see Experimental Section), both closely corresponding to those found in the crystal structures of TBBt

complexed with either maize CK2R [pdb1j91] or human pCDK2 [pdb1p5e] (see Supporting Information Figure 2). Common Pattern of Interactions. For each TBBt analogue, the close contacts with CK2R residues found in at least 7 of the 10 lowest energy structures of the complexes were analyzed. This pointed to a common pattern of intermolecular interactions stabilizing location of Bt and Br3Bt congeners in the CK2R binding pocket. Thus the side chain carboxyl of Asp175 recognizes the N(2)-N(1)-H triazole ring configuration, and this interaction was found for all of the ligands. Additionally, the Ser51 hydroxyl is hydrogen bonded to the triazole N(2). Halogen atoms of the Bt benzene ring make numerous contacts with aliphatic side chains of the residues Val45, Val53, Ile66, Phe113, Met163, and Ile174. This stabilizes the complex by protecting the hydrophobic halogen atoms from solvent accessibility. Additionally, some intermolecular contacts between ligand bromine atoms and CK2R backbone (Val45, Gly46, Ser51) and side chain (Ser51, Asn118, Asp175) oxygen atoms are observed. These may be identified as halogen bond interactions that stabilize the complex (see Figure 2). The pattern of interactions involving Val45, Ser51, Glu114, and Asp175 was

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found to be common to all of the complexes modeled with the tribromobenzotriazole ligands. Substituents on the Benzene Ring Modify Stability of the Complex. The flexible location of the Bt analogues in the CK2R pocket permits a C(5) substituent to be either partially solventexposed or fully protected from solvent accessibility, so that both polar and apolar C(5)-substituted derivatives could be efficiently bound by the enzyme. Both theoretical (QM calculations) and experimental (13C NMR and UV titration) data demonstrate that a C(5) substituent on the benzene ring modulates the distribution of electron density on the triazole ring, hence affecting both the relative protomer populations and the pKa, which may result in modulation of the crucial interaction with the Asp175 side chain; for example, 5-NO2-Br3Bt (3d) activity is close to that of the parent TBBt or 5-Cl-Br3Bt (3f), whereas 5-CH3-Br3Bt (3e) and Br3Bt (3h) are somewhat less active. Structure-Activity Relationships. Correlation of Inhibitor ActiWity with Its Structure. For each TBBt congener, its molar volume (Vmol), surface area (Smol), as well as the solventaccessible area (ASA) and its apolar part (ASAap) were estimated (see Experimental Section). The populations of the three neutral prototropic forms obtained by the ab initio method, the disorder of a ligand location in the CK2R pocket estimated from molecular modeling simulations, and the experimentally derived population of neutral and charged forms at pH 7 were also considered as potential factors influencing ligand activity. Analysis was restricted to active ligands which exhibited IC50 values below 100 µM. The best single-parameter correlation was obtained for ln(IC50) versus Vmol, which, according to LCW theory,27 is an exact descriptor of hydrophobic interactions (see Table 3 and Figure 3A). This strongly points to hydrophobic interactions as the dominant factor influencing inhibitory activity. Correlation of Inhibitor ActiWity with Structure of the Complex. The 10 lowest energy structures of the complexes were then subjected to more detailed analysis. For each complex, the average number of identified halogen, hydrogen, and fluorine bonds was estimated. Reduction of the solvent-accessible area and its apolar part, upon complex formation, was calculated for each structure of the complex and then averaged. The mean movement of a ligand in the complex (rmsd) was also estimated. Following the concept of atomic solvation parameterization (ASP),28 intermolecular hydrophobic interactions may also be evaluated in terms of changes in the solvent-accessible surface upon complex formation. Reduction of the apolar part of the solvent-accessible surface area of a ligand upon complex formation is also significantly correlated with its inhibitory activity (not shown). In general, there is no correlation between inhibitor volume, or its activity, with its flexibility in the bound state, estimated as the rmsd describing the average displacement within the complex. This again points to the large flexibity of the CK2R pocket, enabling it to accommodate a wide class of compounds of various sizes. The effect of electron density on a C(5) substituent in direct electrostatic interactions requires further analysis. QSAR Analysis. All physical and structural parameters for the compounds collected in Tables 2 and 3 were subjected to predictions of inhibitory activity, and in accord with the QSAR approach,29 we assumed that ln(IC50) could be expressed as a linear combination of the analyzed parameters. Following the method of principal component analysis (PCA),30,31 we stepwise iteratively reduced the number of parameters required for

Wa˛sik et al.

Figure 3. Experimental IC50 values as a function of (A) Vmol, molecular volumes of the ligands; (B) log[Pcons], calculated coefficients of ligand partition between polar and apolar phases; (C) log[S], calculated solubility coefficients. The high correlation coefficients of 0.94 (0.99 for TBBt, 3a, 3f, 3h: dashed line), 0.89, and 0.96, respectively, indicate that the mechanism of inhibition by benzotriazole derivatives substituted on the benzene ring is largely driven by hydrophobic interactions. According to the presented analysis, the IC50 for the hitherto unknown TIBt (o) was estimated as 0.12 ÷ 0.5 µM. All models overestimate activity of 2e and 3i.

prediction of activity. The final, optimal model underlines the role of the molecular volume of the ligand, the number of fluorine atoms attached, and the fraction of the neutral form estimated at pH 7. The average number of halogen bonds was found to be virtually nonsignificant at the 10% significance level. The model points to predominance of the volume effect of the ligand, which, located deeply within the enzyme binding site, is thus protected from solvent accessibility. Polar substituents (COOH, NH2) attached to the benzene ring destabilize the

5-Substituted TBBt Inhibitors of CK2R

Figure 4. Optimal model that can be used for prediction of inhibitory activities of benzotriazole and its analogues substituted on the benzene ring. IC50 is the experimentally measured value, log P is the predicted polarity; pKa values were determined experimentally, and XN(1)-H is the population of the N(1)-H protomer, estimated by QM calculations. This model is the only one which properly predicts the activities of 2e and 3i and points to Bt, 2i, and 3g as inactive compounds (cf. Table 3 for the details).

complexes, shifting the equilibrium toward the dissociated, free state of the ligand. Contact of a fluorine atom on the benzene ring with a proximal protein carbonyl/carboxyl oxygen increases IC50 1.5- to 5-fold, due to unfavorable electrostatic interactions between negatively charged centers. The final results of regression analysis (Figure 4) are summarized in Table 4. Bearing in mind that QSAR analysis pointed to hydrophobic interactions involving the monoanionic form of a ligand as the dominant driving factor in its inhibitory activity, we tested the combinations of descriptors related to the hydrophobic properties of the ligand. The best results were obtained for the log P and log S coefficients, which estimate partition of the molecule in an octanol-water two-phase system and its solubility in water, respectively (Figure 3B,C). Since the QSAR method pointed to the populations of the monoanion and the QM-derived populations of the neutral N(1)-H protomer as important for prediction of ligand inhibitory activity, we introduce, following the method for correction of the partition coefficient of ionizable compounds,32 the corrections of log P as follows:

The final model, which accurately predicts IC50 values of all the active compounds, with an R2 value of 0.98, is presented in Figure 4. This model, based on the log D# descriptor appears to be the best because it not only predicts the inhibitory activity of all TBBt derivatives displaying IC50 < 500 µM but also points to Bt, 5-COOH-Br3Bt (3g), and 5NH2-Bt (2a) as inactive compounds. In order to judge the statistical significance of all the parameters in the proposed model, an additional procedure was carried out to test independently the significance of log P, log(Xma), and log(XN(1)-H) parameters used in eq 1. The p statistics values of 9 × 10-10, 1.5 × 10-5, and 5.9 × 10-6 obtained for log P, log(Xma), and log(XN(1)-H), respectively, clearly indicate that, despite the ligand polarity being the dominant factor, both protomeric and dissociation equilibria almost equally drive its binding to CK2R.

J. Phys. Chem. B, Vol. 114, No. 32, 2010 10607 Effects of Fluorine and Halogen Bonding. According to the final two-parameter QSAR model, a fluorine atom attached to the benzene ring of a Bt derivative increased the IC50 by a factor ∼1.4 (see Table 4). This is largely due to the unfavorable change in free energy upon transfer of a fluorine atom from the polar solvent to the apolar environment of the protein pocket. Consequently, derivatives with a fluorine atom on the benzene ring are weaker inhibitors than expected according to their molecular volume, Vmol. Less clear is the role of halogen bonds. In contrast to literature data,33 a halogen bond (defined as the close contact between a halogen and O or N electron-rich atoms) was not found to be significant in stabilization of the ligand in the binding pocket. This conclusion must be judged bearing in mind the predominance of hydrophobic interactions in stabilization of the protein-ligand complexes, which are directly correlated with the number of halogen atoms in the ligand. Analysis of the parameters of the models that include the effect of halogen bonding indicates that the contribution of hydrophobic interactions to the final free energy of complex formation (∼-5 kcal/mol) is an order of magnitude larger than the average energy estimated for a putative halogen bond (∼0.2 kcal/mol). This may be readily verified independently by examination of ligand aqueous solubility. Solubilities in neutral aqueous medium of benzotriazole and some mono-, di-, tri-, and tetrabromobenzotriazoles are shown in Figure 5. Hydrophobic effects are responsible for the observed decrease of solubility with increasing molecular volume of the compounds. Since each water molecule exposes an oxygen atom capable of participating in a halogen bond, it appears that the eventual halogen bonding effect is masked by existence of dominant hydrophobic solvation. Hence, the observed halogen bond interaction may orientate the ligand molecule in the apolar protein cavity, thus slightly decreasing the free energy of binding, but the ligand-binding process is generally driven by hydrophobic interactions that are represented as the molecular volume, or log D, as shown in Figure 3A,C. This is also strongly supported by the reported 20-fold decrease of inhibitory activity of TBBt against a CK2RV66A/I174A double mutant.2 We are presently examining possible extension of the structure-activity relationship described above to predict inhibitory properties of other structurally related analogues of TBBt, for example, those in which all four bromine atoms are replaced by other substituents, with preliminary promising results. For example, tetramethylbenzotriazole (TMBt) is predicted to exhibit very low activity (consensus log P ) 2.04 ( 0.57; pKa ) 8.85;26 IC50,calcd ) 140 µM), in agreement with early experimental results.4 Tetrachlorobenzotriazole (TCBt), with consensus log P ) 3.71 ( 0.27; pKa ) 4.57;26 IC50,calcd ) 2.6 µM, is predicted as more than 2-fold less active than TBBt (IC50,calcd ) 1.2 µM), again in agreement with experimental results.4 The hitherto unknown tetraiodobenzotriazole (TIBt; consensus log P ) 4.77 ( 1.12; pKa < 4.5; IC50,calcd ) 0.6 µM) was estimated to be at least two times more active than TBBt. This is in accord with analysis of a series of 4,5,6,7-tetrahalogeno-1H-isoindole-1,3(2H)-dione inhibitors,34 where replacement of the four bromines on the benzene ring by iodines decreased IC50 by a factor of 3-10, depending on the nature of the substituent on N(2). It is also consistent with the report5 that tetraiodobenzimidazole is up to 10-fold more active than tetrabromobenzimidazole. It should be noted that, whereas the hydrophobicity of the monoanionic forms of the ligands is the dominant factor describing their inhibitory activity, the propensity for protonation of the N(1) nitrogen is also significant.

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TABLE 4: Parameters of the Four Best Models Describing the Inhibitory Activities of a Series of Benzotriazoles Substituted on the Benzene Ring, According to the Equation ln(IC50) ) ∑wi · xi, Where the Corresponding Structural Parameters are Listed in Table 3a weight, wi model R(log P) R(log D#) R(log S) R (Vmol) QSAR

value t-stat P value t-stat P value t-stat P value t-stat P value t-stat P

intercept (SE) (SE) (SE) (SE) (SE)

7.2 (0.6) 12.7 1.7 × 10-7 5.2 (0.1) 45.0 1.2 × 10-15 7.6 (0.4) 20.2 4.9 × 10-10 11.5 (0.8) 15.2 3.1 × 10-8 11.3 (0.6) 17.6 2.8 × 10-8

log P/log D -1.68 (0.18) 9.2 3.3 × 10-6 -1.35 (0.05) 27.0 8.3 × 10-13 1.59 (0.11) 14.3 1.8 × 10-8

Vmol

#F

-0.059 (0.005) 12.4 2.1 × 10-7 -0.059 (0.004) 14.7 1.3 × 10-7

0.26 (0.11) 2.3 4.7 × 10-2

R2 (df)

F (R)

rank -6

0.89 (11)

85.1 (3.3 × 10 )

5

0.98 (13)

730 (8.3 × 10-13)

1

0.95 (11)

206 (1.8 × 10-8)

2

0.94 (11)

154 (2.1 × 10-7)

4

0.96 (11)

112 (4.3 × 10-7)

3

a

For each model, the value of R2, the number of degrees of freedom (df), Snedoker statistics (F), and the corresponding significance level (R) are presented. The models were ranked according to their significance level, R.

Figure 5. Correlation of experimentally measured solubilities (cx) of benzotriazole and its mono-, di-, tri-, and tetrabromo analogues with their molecular volumes (Vmol). Bearing in mind the correlation between IC50 and Vmol (0.92, cf. Table 4 and Figure 3A), the high correlation between Vmol and cx (0.998) supports the mechanism of inhibition via hydrophobic interactions.

Experimental Section Chemical Methods. Melting points (uncorrected) were determined in open capillary tubes, using a Bu¨chi apparatus B504. UV absorption spectra were recorded on a Cary 300 instrument. All compounds were checked by thin-layer chromatography (TLC) on 0.2 mm Merck silica gel 60 F254 plates. Preparative separations were carried out by column chromatography, using Merck silica gel (230-400 mesh) or on preparative glass plates (2 mm, Merck silica gel 60 F254), using the following solvent systems: (A) CHCl3/MeOH (9:1), (B) EtOAc/n-heptan/AcOH (2:2:0.2). Mass spectrometry was performed with a Micromass ESI Q-TOF spectrometer. 1H and 13C NMR spectra were recorded in DMSO-d6 on a Varian 500 or Varian 400 (5-bromobenzotriazole) instrument. 13C spectra of 5-substituted 4,6,7-tribromobenzotriazoles and 4,6,7-tribromobenzotriazole were recorded in aqueous medium.26 Spectra were analyzed with the aid of the MestRe-C (version 2.3a) program (Cobas, Cruces Mestre-C 2.3a). Chemical shifts are in parts per million relative to tetramethylsilane (Me4Si, δ ) 0). Starting materials: 5-F- and 5-NO2-1,2-phenylenediamines

(1a, 1d), benzotriazole, 5-CH3-, 5-COOH-, and 5-Cl-benzotriazoles (2e, 2g, 2f) were products of Aldrich or Lancaster (USA), and 4,5,6,7-tetrabromobenzotriazole (TBBt) was synthesized as previously reported.23 Synthesis. 5-Fluoro-1H-benzotriazole (2a). To a cooled (0 °C) and stirred solution of 1a (126 mg, 1 mmol) in 2 mL of AcOH and 0.8 mL of H2O was added 114 mg (1.65 mmol) of NaNO2 dissolved in 0.7 mL of H2O. Stirring was continued at room temperature for 20-30 min; 0.5 mL of HCl was then added, and the resulting solution was poured into a mixture of EtOAc and brine (1:1 v/v, 50 mL). Organic layers were separated, dried over Na2SO4, filtered, and excess was solvent removed in vacuo. The residue was dissolved in EtOH/H2O, treated with activated charcoal, filtered, and evaporated to give 116 mg (85% yield) of 2a; mp 148-149 °C, lit. 144-145.8 °C,36 Rf (A) 0.63, (B) 0.58. UV λmax (ε): (pH 2) 258.5 nm (5780), 273.5 (5595); (pH 7) 260 nm (5960), 274 nm (6130); (pH 12) 275 nm (9690). 1H NMR δ: 8.03 (dd, 1H, J ) 4.7 Hz, J ) 8.9 Hz), 7.73 (dd, 1H, J ) 2.3 Hz, J ) 8.8 Hz), 7.35 (td, 1H, J ) 2.3 Hz, J ) 9.2 Hz). 13C NMR δ: 160.31 (J ) 241.9 Hz), 137.61 (J ) 13.3 Hz), 137.36, 117.63 (J ) 10.7 Hz), 114.63 (J ) 27.5 Hz), 99.04 (J ) 26.7 Hz). MS for C6H5FN3 [M + H]+: found 137.9920, calcd 138.0468. 5-Bromo-1H-benzotriazole (2b). To a cooled (0 °C) and stirred solution of the dihydrochloride of 1b (260 mg, 1 mmol) in 6.5 mL of H2O was added 0.26 mL of concentrated HCl to a solution of NaNO2 (114 mg, 1.65 mmol) in 0.7 mL of H2O. Stirring was continued at room temperature for 30 min. The solid product was filtered and washed with H2O. Recrystallization from EtOH/H2O gave 163 mg (83% yield) of 2b; mp 152.2-153.9 °C, lit. 150 °C,37 Rf (A) 0.61, (B) 0.6. UV λmax (ε): (pH 2) 265 nm (5372), 282.5 nm (5525); (pH 7) 282.5 nm (6190); (pH 12) 282 nm (6967). 1H NMR δ: 8.17 (dd, 1H, J ) 0.6 Hz, J ) 1.7 Hz), 7.89 (dd, 1H, J ) 0.6 Hz, J ) 8.7 Hz), 7.5 (dd, 1H, J ) 1.7 Hz, J ) 8.7 Hz). 13C NMR δ: 137.86, 133.38, 131.7, 128.48, 117.46, 116.95. MS for C6H4Br1N3 [M + H]+: found 199.9303, calcd 199.9646. 5-Trifluoromethyl-1H-benzotriazole (2c). To a cooled (0 °C) and stirred solution of 1c (176 mg, 1 mmol) in 2 mL of AcOH and 0.8 mL of H2O was added 114 mg (1.65 mmol) of NaNO2 dissolved in 0.7 mL of H2O. Stirring was continued at room temperature for 20-30 min; 0.5 mL of HCl was then added,

5-Substituted TBBt Inhibitors of CK2R and the resulting solution was poured into a mixture of EtOAc and brine (1:1 v/v, 50 mL). Organic layers were separated, dried over Na2SO4, filtered, and excess solvent was removed in vacuo. The residue was dissolved in EtOH/H2O, treated with activated charcoal, filtered, and evaporated to give 182 mg (97% yield) of 2c; mp 125.2-127.1 °C, lit. 132 °C.38 UV λmax (ε): (pH 2) 259.5 nm (5001); (pH 7) 274.5 nm (5236); (pH 12) 276 nm (7226). 1H NMR δ: 8.4 (s, 1H,), 8.09 (d, 1H, J ) 8.5 Hz), 7.72 (d, 1H, J ) 8.5 Hz). 13C NMR δ: 140.74, 139.65, 125.87 (q, J ) 32.2 Hz), 125.14 (q, J ) 272.5 Hz), 122.54 (q, J ) 3.9 Hz), 116.2, 115.4 (q, J ) 3.9 Hz). MS for C7H5F3N3 [M + H]+: found 188.0430, calcd 188.0436. 5-Nitro-1H-benzotriazole (2d). To a mixture of 1d (153 mg, 1 mmol) in 2 mL of AcOH and 0.8 mL of water at 0 °C was added a solution of NaNO2 (114 mg, 1.65 mmol) in 0.7 mL of water. The solid was collected by filtration and washed with H2O to afford 149 mg (91% yield) of pure light tan 2d; mp 212.4-213.7 °C, lit. 215-216 °C,39 Rf (B) 0.48. UV λmax (ε): (pH 2) 290 nm (7527); (pH 7) 307.5 nm (6905); (pH 12) 313 nm (7471). 1H NMR δ: 8.93 (d, 1H, J ) 2.0 Hz), 8.28 (dd, 1H, J ) 2.0 Hz, J ) 9.0 Hz), 8.10 (d, 1H, J ) 9.0 Hz). 13C NMR δ: 144.60, 140.55, 138.69, 121.01, 114.25, 114.12. MS for C6H5N4O2 [M + H]+: found 165.0361, calcd 165.0413. 5-Amino-1H-benzotriazole (2i). (a) To a solution of 2d (164 mg, 1 mmol) in 1 mL of concentrated HCl was added dropwise a solution of SnCl2 (569 mg, 3 mmol) in 2 mL of concentrated HCl. The resulting mixture was stirred for 1 h at room temperature. The pale blue product was filtered off, washed with EtOAc, and dried to afford 162 mg (95% yield) of the hydrochloride 2i. (b) Reduction with HI was as follows: A suspension of 2d (164 mg, 1 mmol) in 3 mL of aqueous HI (57%, unstabilized) was heated at 90 °C for 3 h. After cooling to room temperature, it was diluted with EtOAc (50 mL) and washed with saturated Na2S2O3 until all formed iodine was removed, as indicated by disappearance of the dark purple color. The reaction mixture was brought to neutrality with saturated NaHCO3, and the organic layer was dried over Na2SO4 and evaporated. The residue was dissolved in n-hexane/EtOH (2:1) and concentreated HCl added. The resulting precipitate of the hydrochloride of 2i was filtered off and washed with n-hexane to give 102 mg (60% yield). Rf (A): 0.22. UV λmax (ε): (pH 2) 258.5 nm (3550), 274.5 nm (2970); (pH 7) 302 nm (3910); (pH 12) 276.5 nm (3270), 303.5 nm (3400). 1H NMR δ: 10.0 (br s, NH2), 8.02 (dd, 1H, J ) 0.5, J ) 8.8 Hz), 7.89 (s, 1H), 7.39 (dd, 1H, J ) 1.9, J ) 8.8). 13 C NMR δ: 138.06, 131.04, 120.69, 116.81, 107.71 MS for C6H7N4 [M + H]+: found 135.0768, calcd 135.0671. 5-Fluoro-4,6,7-tribromo-1H-benzotriazole (3a). To a solution of 2a (137 mg, 1 mmol) in 1.6 mL of 70% HNO3 was added 0.221 mL (4.3 mmol) of Br2, and the solution was heated at 130 °C with stirring for 15 h. The resulting crystalline product was collected on a filter and washed with HNO3 and H2O. Recrystallization from EtOH give 82 mg (22% yield) of 3a: mp 239.3-242.5 °C, Rf (A) 0.41, (B) 0.73. UV λmax (ε): (pH 2) 280.5 nm (12840); (pH 7) 291 nm (13585); (pH 12) 292 nm (13570). 13C NMR δ: 154.22 (J ) 243.62 Hz), 138.16, 137.77, 112.6 (J ) 27.6 Hz), 112.28, 93.53 (J ) 28.9 Hz). MS for C6H2Br3N3F [M + H]+: found 373.7695, calcd 373.7762. 5-Trifluoromethyl-4,6,7-tribromo-1H-benzotriazole (3c). To a solution of 2c (187 mg, 1 mmol) in 7 mL of 70% HNO3 was added 0.410 mL (8 mmol) of Br2, and the solution was heated under reflux with stirring for 64 h. The solution was cooled to room temperature, and the resulting crystalline product collected on a filter and washed with HNO3 and H2O. Purification by

J. Phys. Chem. B, Vol. 114, No. 32, 2010 10609 silica gel column chromatography (0-15% methanol in chloroform) gave 85 mg (20% yield) of pure 3c: mp >300 °C (dec.), Rf (B) 0.65. UV λmax (ε): (pH 2) 279.5 nm (2630); (pH 7) 290.5 nm (2580), 303 nm (2530); (pH 12) 290.5 nm (2640), 302.5 nm (2590). 13C NMR δ: 144.74, 143.52, 123.14 (q, J ) 275.9 Hz), 118.14 (q, J ) 28.8 Hz), 115.186, 112.03, 110.21 (q, J ) 2.44 Hz). MS for C7H2Br3F3N3 [M + H]+: found 423.7738, calcd 423.7730. 5-Nitro-4,6,7-tribromo-1H-benzotriazole (3d). 4,6,7-tribromobenzotriazole (3h) (356 mg, 1 mmol) was dissolved in concentrated H2SO4 (1.75 mL) and stirred magnetically at room temperature. NaNO3 (850 mg, 10 mmol) was added stepwise over a period of 10 min. The resulting suspension was heated with stirring at 70 °C until the reaction was complete (∼4 h); the cooled mixture was poured into a mixture of ice and water and the resulting precipitate filtered off, washed with H2O, and dried. Recrystallization from AcOH and H2O gave 277 mg (69% yield) pure 3d: mp 242.5-245.5 °C, Rf (B) 0.52. UV λmax (ε): (pH 2) 277.5 nm (8770); (pH 7) 289.5 nm (9640); (pH 12) 289 nm (9920). 13C NMR δ: 149.23, 139.96, 139.80, 113.55, 112.78, 102.72. MS for C6H2Br3N4O2 [M + H]+: found 400.7722, calcd 400.7707. 5-Methyl-4,6,7-tribromo-1H-benzotriazole (3e). This compound was prepared in 76% yield (282 mg) starting from 2e (133 mg, 1 mmol) as described for 3a, but with more prolonged stirring (24 h): mp 238.2-241.1 °C, Rf (A) 0.68, (B) 0.77. UV λmax (ε): (pH 7) 292.5 nm (10460); (pH 12) 292.5 nm (10980); (MeOH) 281 nm (10120), 294.5 nm (8900). 1H NMR δ: 2.64 (s, 3H). 13C NMR δ: 139.51, 138.6, 136.53, 111.34, 125.31, 108.25, 25.32.MS for C7H5Br3N3 [M + H]+: found 369.7946, calcd 369.8013. 5-Chloro-4,6,7-tribromo-1H-benzotriazole (3f). This compound was prepared in 70% yield from 2f as described for 3e, but with more prolonged stirring (41 h). Repeated recrystallization from MeOH gave 272 mg of pure 3f: mp 250.2-252.7 °C, lit. 249-251 °C,23 Rf (A) 0.39, (B) 0.73. UV λmax (ε): (pH 7) 291.5 nm (10440), 299.5 nm (10670); (pH 12) 291 nm (10610), 299.5 nm (10850) (MeOH) 286 nm (8920), 300 nm (8420), 310.5 nm (6040). 13C NMR δ: 139.30, 132.40, 123.37, 112.15, 108.90. MS for C6H2Br3N3 [M + H]+: found 389.7514, calcd 389.7467. 5-Carboxy-4,6,7-tribromo-1H-benzotriazole (3g). A suspension of 3e (370 mg, 1 mmol) in 3.32 mL of 0.3 M KOH was stirred and heated to ∼90 °C. KMnO4 was added in three portions (207, 146, and 67 mg) over 3-4 h. Stirring and heating was continued for another 2-3 h at 100 °C, and the resulting precipitate of MnO2 was filtered off with the aid of Celite Hyflo and washed with hot water. The clear colorless solution was acidified with concentrated HCl, and the resulting precipitate was collected by filtration and washed with H2O to afford 260 mg (72%) pure 3g: mp 297.4-300.5 °C, Rf (B) 0.14. UV λmax (ε): (pH 2) 283.5 nm (10480); (pH 7) 296 nm (14890); (pH 12) 296 nm (13480). 13C NMR δ: 168.17, 140.03, 139.56, 138.30, 119.30, 111.78, 105.38. MS for C7H3Br3N3O2 [M + H]+: found 399.7423, calcd 399.7755. 4,6,7-Tribromo-1H-benzotriazole (3h). A suspension of 3g (400 mg, 1 mmol) in 0.666 mL (5.6 mmol) of quinoline was heated until the temperature reached 210-220 °C and kept at this temperature for 10-15 min, cooled, and 30 mL of hexane was added. The resulting precipitate was collected and washed several times with hexane to remove residual quinoline. Recrystallization from EtOH/H2O gave 242 mg (68%) of pure 3h: mp 239.2-241.9 °C, Rf (A) 0.55, (B) 0.75. UV λmax (ε): (pH 2) 279 nm (11000); (pH 7) 289 nm (12730); (pH 12) 289 nm

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(12760). 1H NMR δ: 8.06 (s, 1H). 13C NMR δ: 140.61, 139.61, 132.72, 122.89, 110.09, 108.84. MS for C6H2Br3N3 [M + H]+: found 355.8037, calcd 355.7857. 5-Amino-4,6,7-tribromo-1H-benzotriazole (3i). A mixture of 3d (401 mg, 1 mmol) and Fe powder (298 mg, 5.34 mmol) in AcOH (20 mL) was stirred for 6 h at room temperature, then for 18 h at 110 °C, cooled to room temperature, and 279 mg (5 mmol) of Fe powder was added, and stirring was continued for 18 h at 110 °C. The reaction mixture was cooled, diluted with 40 mL of EtOAc, filtered, and the resulting solid was washed with EtOAc. The filtrate and washings were combined and washed with H2O. The organic layer was separated, dried over Na2SO4, filtered, and volatiles were removed in vacuo. Purification of the crude product by silica gel column chromatography (0-15% MeOH/CHCl3) gave 274 mg (74%) of 3i: mp (dec.) 249.6-251.9 °C, Rf (B) 0.62. UV λmax (ε): (pH 2) 290 nm (5860), 320 nm (6440); (pH 7) 294 nm (7430), 320 (6350); (pH 12) 294 nm (8190), 320 nm (6620). 13C NMR δ: 142.93, 137.04, 136.87, 113.56, 111.65, 84.61. MS for C6H4Br3N4 [M + H]+: found 370.8145, calcd 370.7966. Enzyme and Activity Measurements. The cDNA of CK2R was a kind gift of Prof. Lorenzo A. Pinna (Padua University). Protein was expressed in Escherichia coli and then purified as described elsewhere.35 Protein kinase activity was determined in a medium (50 µL total volume) containing CK2R (0,1 µg), casein (75 µg), γ-[32P]ATP (10 µM icpm/pmol), Tris buffer pH 7.5 (50 mM), MgCl2 (10 mM), and appropriate concentrations of inhibitor in 1 µL of DMSO. After 20 min incubation at 30 °C, the reaction was terminated by spotting 40 µL of the reaction mixture onto a 3MM filter strip and washing five times for 10 min with 5% TCA. Incorporation of 32P was measured in an LKB Flexi-Vial scintillation counter (activities are expressed against controls with DMSO). CK2R activity was measured for the pure enzyme and in the presence of 0.5, 1, 2, 5, 10, 20, 30, 40, and 50 µM ligand solution. The additional ligand concentrations were tested for less active compounds (75 and 100 µM for 5COOH-Br3Bt (3g), 5CF3-Bt (2c), 5F-Bt (2a), and 5NH2-Br3Bt (3i)), and the most active TBBt was also tested in 0.2, 0.4, 0.6, and 0.8 µM concentration. The dose-response data were analyzed according to the three-parameter Hill-Slope logistic model in the form

A0

A(x) ) 1+

( ) x IC50

z

(2)

where the activity of the enzyme, A0, and ligand IC50 were optimized, and the slope coefficient, z, was constrained to 1, reproducing the expected form of a 1:1 complex. Independent series of experiments were analyzed together, assuming for each an individual enzyme activity, A0, and the same value of the IC50 parameter. Molecular Modeling of Inhibitor Binding. Ab Initio Calculations. The initial coordinates of Bt and TBBt were adopted from the crystal structure of TBBt complexed with maize CK2R,16 and their C(5) derivatives were built with the aid of homemade software. This was followed by ab initio analysis with the aid of the GAMESS 6.0 program.40 The DFT calculations were performed with the B3LYP functional,41 using the 6-31G(d,p) basis set,42 previously found applicable in the analysis of symmetrical Bt systems.26 Geometry preoptimizations were done in vacuo, and ESP charges were calculated to mimic the electrostatic properties of the molecules. Correction for the

solute-solvent interaction was estimated using the polarizable continuum model (PCM) approach,43 including the electrostatic partition of solute interaction with the apparent surface charge distribution (Gelec), dispersion (Gdisp) and repulsion (Grep) contributions to the solvation free energy,44 and the ClaveriePierotti cavitation energy45 (Ecav, cf. ref 46 for the most recent review). The zero-point energies (Ezp) and their thermal corrections for translational (Gtrans), rotational (Grot), and vibrational (Gvib), calculated using ideal gas rigid rotor and harmonic normal mode approximations, were used to convert the internal energies to the Gibbs free energies at 298.15 K. In principle, changes of free energy were analyzed for three different processes: (a) the protomeric equilibria, ∆G1, ∆G2, ∆G3, respectively, for the forms presented in Scheme 4, (b) dissociation of the triazole N-H proton (∆Gdiss), and (c) solvation (∆Gsolv) according to the following formulas: Gsolv ) Eelec + Grep + Gdisp + Ecav + Ezp + Gtrans + Grot + Gvib ∆Gi ) Gsolv(state i) - min (Gsolv(state j)) j)1,2,3

∆Gdiss ) Gsolv(monoanion) - Gsolv(neutral) + Ghydr(H+) ∆Gsolv ) Gsolv - G0

(3) where G0 is the QM-derived Gibbs free energy in vacuo. It should be noted that both carboxy derivatives carry the COOgroup, hence ∆Gdiss was calculated as the difference in free energy of the doubly ionized molecule and its monoanionic form. The proton hydration free energy, Ghydr(H+), was taken as -262.3 kcal/mol.47 The molecular surface, its contribution from polar and apolar atoms, their changes upon binding to CK2R, and the volume for each compound were calculated according to the algorithm of tessellation implemented in the GEPOL package.48 Simulated Annealing Procedure. The 141 residue fragment (Tyr39-Ala179) of the CK2R subunit of the human CK2 holoenzyme [pdb1JWH],49 comprising all of the amino acid residues proximal to the postulated binding site for TBBt and its derivatives, was used as template of the protein target. Structural calculations were performed with the aid of the simulated annealing (SA) protocol implemented in X-PLOR.50 The original parallhdg force-field parametrization was extended for the various derivatives, based on ab initio calculated geometries and ESP charges. Coordinates for Bt derivatives in the complex were also adopted from the structure of TBBt bound to maize CK2R [pdb1j91],16 and the initial structures were relaxed upon 1000 steps of energy minimization, with all protein backbone atoms kept fixed, while other protein atoms were allowed to move. This was followed by the SA procedure, comprising 6 ps evolution at 3000 K, 3 ps cooling down to 300 K, and final energy minimization. To preserve the general protein fold, all backbone residues located outside a 6 Å sphere around the TBBt binding site were fixed in high-temperature cycles and released upon final minimization. Finally, for each derivative, 150 random SA calculations were performed, and the 10 lowest energy structures were subjected to further analysis. Structural analysis was performed with the aid of MolMol.51 The affinity of an inhibitor for CK2R was judged in terms of preferred complex organization, defined as the rmsd of a derivative, calculated when the protein backbone atoms were superimposed. Additionally, according the concept of atomic solvation parameters (ASP),52 the average solvent-accessible area and its reduction upon binding to enzyme were estimated for

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