2
A. Szymaniec-Rutkowska et al. / Journal of Molecular Liquids 293 (2019) 111527
proximal network of water-water H-bonds much more ordered than
that in the bulky water [30,31], while an increased population of
water molecules strongly bound to the solute is observed for proteins
purchased from Fluorochem (Hadfield, UK). All bromo-substituted
benzotriazoles were synthesized according to the previously used
methods [45,46]. 4,5,6,7-Tetrachloro-1H-benzotriazole was synthesized
according to literature procedure [47].
[32] or amino acids [33]. All in all, it indicates that hydrophobic effect,
i.e. solute-induced changes in the organization of the solvation shell, in-
cluding both static and dynamic properties, gather the thermodynamics
of the molecule in an aqueous solvent.
Melting points (uncorrected) were determined in open capillary
tubes, using a Büchi B504 apparatus. The reaction progress was moni-
tored with the use of thin-layer chromatography (TLC) analysis using
silica gel plates (Kieselgel, 60F254, E. Merck, Darmstadt, Germany). Col-
Hydrophobic effect is of extreme importance in a drug design ap-
proach, especially for highly hydrophobic ligands, the binding affinity
of which may be predominated by the unfavorable interactions of the
free solute with an aqueous solvent. The effective inhibitory activity de-
pends on the apparent free energy of protein-ligand interactions, the
deconvolution of which to the contributions of direct protein-ligand in-
teractions and the solvent-driven effect of desolvation (both for ligand
and the protein) is difficult, and in most cases remains unresolved.
Leo, Hansh and Elkins successfully used partition coefficients in octan-
umn chromatography was performed on Silica Gel 60
M
(0.040–0.063 mm, E. Merck, Darmstadt, Germany). High-resolution
mass spectra were recorded on an LTQ Orbitrap Velos instrument
(Thermo Scientific). NMR spectra were recorded in DMSO d ; signal as-
6
signment for the chlorinated benzotriazoles based on the data already
published for bromo-benzotriazoles [45,46,48]. The purity of all studied
compounds was controlled with the use of HPLC twice: in isocratic con-
ditions (2: 1 methanol: 20 mM ammonium formate, (AF) pH 6.5), and
with the linear gradient of 20 mM AF pH 6.5 × 65–95% aq. MeOH over
30 min at a flow rate of 0.7 mL/min.
1
-ol/water system (LogP) as a measure of solute hydrophobicity
[
34,35]. This approach has soon become fundamental, and since then
LogP data routinely support drug design procedures. However, aqueous
solubility (LogS) and pH-dependent distribution coefficient (LogD) are
also commonly used. Partition coefficients usually correlates with li-
gand binding affinities [36,37], however, the dependency of ligand affin-
ity on LogP is generally nonlinear, thus indicating an optimal drug
hydrophobicity [2].
A solution of 10 mmol of the appropriate benzene-1,2-diamine in
3.5 mL of acetic acid (AcOH) and 1 mL of water was cooled to 0–5 °C,
followed by the addition of 15 mmol of sodium nitrite in 2 mL of
water. The mixture was stirred for 2 h at room temperature. After
the completion of the reaction, a solvent was evaporated and the res-
idue was co-evaporated with toluene (3 × 20 mL). The crude product
was partitioned between water (20 mL) and ethyl acetate (20 mL),
the organic phase was washed with the saturated solution of sodium
In the case of halogenated ligands, the exact contributions of halogen
bonding, hydrophobic effect and halogen-induced changes in the solute
electronic properties to the free energy of ligand binding cannot be ex-
tracted directly from the experimental thermodynamic data. It should
be noted that, although the enthalpy of solute-solvent interaction can
be determined calorimetrically by the combination of the enthalpy of
solution and the enthalpy of sublimation, there is still no experimental
method to estimate directly the entropic contribution to the solute-
solvent interactions. It should also be stressed that, in order to improve
the preliminary steps of drug design procedures, there is a strong need
for at least a semi-quantitative method to assess these interactions.
Here, we test our former model of hydrophobic solvation, in which
the observed excess volume (β) that represents the difference between
the experimentally measured partial molar volume and the estimated in
silico molecular volume was attributed directly to the effect of the reor-
ganization of water molecules in the solvation shell [38–42]. This exten-
sive thermodynamic parameter was proposed as a measure of the free
energy of the reorganization of water molecules surrounding the solute
molecule. The application of the proposed model is now tested for five
rationally selected bromo-benzotriazoles and their five chloro-
analogues. The hydrophobicity of solutes was independently estimated
from RP-HPLC retention times, the values of which were proven to cor-
relate with LogP data [43]. We have also analyzed other physicochemi-
cal parameters that are routinely determined upon the early steps of
4
hydrogen carbonate and dried over magnesium sulfate (MgSO ). The
products were purified by crystallization from nitromethane and/or
by column chromatography on silica gel using a chloroform – meth-
anol 97:3–95:5 v/v mixture as eluent. Reaction products were ana-
lyzed by use of mass-spectrometry (Waters Q-TOF Premier Mass
Spectrometer) and NMR spectroscopy (Varian INOVA 500 Spectrom-
eter, see Supp. Fig. 1). Purity of the products was assessed using in-
ternal standard quantitative NMR method (qNMR) [49]. It should
be noted that the formal qNMR-derived purity determined for 4-
2 2
BrBt, 4-ClBt, 5,6-Cl Bt and 5,6-Br Bt increased significantly upon ad-
dition a small amount of water to the DMSO solution, which in-
creased proton exchange rates. This observation indicates that the
nuclear relaxation process accompanying protomeric equilibrium
(generally N1-H and N3-H forms predominates) significantly con-
tribute to the obtained NMR spectra, the best proof of which is the
strong broadening of the H-7 resonance line in 4-BrBt and 4-ClBt
(Supp. Fig. 1A,B).
4-Chloro-1H-benzotriazole (4-ClBt): yield 550 mg (24%);
mp. 170.6–172.1 °C (lit. 168.5–169.5 °C) [50]; HRMS (ESI): m/z [M
+
+ H] calc. For C
6
H
5
ClN
3
: 154.01665, 156.01370 found: 154.01665,
1
156.01362; H NMR 500 MHz (DMSO d
6
) δ [ppm]: 7.87 (bs, 1H, H-7);
7.50–7.57 (m, 2H, H-5,H-6: 7.56; assigned as ad, 1H, H-5, J = 7.4 Hz;
7.51 and at, 1H, H-6, J = 7.4 Hz); qNMR purity (PqNMR): 89.3%; (Supp.
Fig. 1B).
a w
drug design procedure (pK , LogC , LogP, LogS).
All tested ligands, which are the derivatives of the first-reported
low-mass ATP-competitive inhibitor of protein kinase CK2, 4,5,6,7-
tetrabromo-1H-benzotriazole [44], are expected to bind at the ATP-
binding site of the catalytic domain of protein kinase CK2 (CK2α), so
in this way we have assessed the applicability of various methods
confronting the thermodynamic parameters determined for free ligands
with their binding affinities deduced from the thermal shift assay.
5-Chloro-1H-benzotriazole (5-ClBt): yield 400 mg (32%);
mp. 158.7–159.8 °C (lit. 156–157 °C) [46,51]; HRMS (ESI): m/z [M
+
+ H] calc. for C
6
H
5
ClN
3
: 154.01665, 156.01370 found: 154.01665,
1
156.01363; H NMR 500 MHz (DMSO d
6
) δ [ppm]: 8.05 (s, 1H, H-4);
1 2
7.99 (d, 1H, H-7, J = 8.8 Hz); 7.50 (dd, 1H, H-6, J = 7.5 Hz, J =
1.9 Hz); PqNMR: 93.9%; (Supp. Fig. 1D).
4
,6-Dichloro-1H-benzotriazole (4,6-Cl
2
Bt): yield 773 mg (49%); mp.
+
2
. Material and methods
246–247.7 °C (lit. 245.5–246.5 °C) [52]; HRMS (ESI): m/z [M + H] calc.
1
for C
6
H
4
Cl
2
N
3
: 187.97768; 189.97473 found: 187.97748; 189.97446; H
) δ [ppm]: 8.01 (s, 1H, H-7); 7.67 (d, 1H, H-5,
2
.1. General procedure for synthesis chloro-benzotriazoles
NMR 500 MHz (DMSO d
6
J = 1.4 Hz); PqNMR: 95.3%; (Supp. Fig. 1F).
Commercially available chemicals were of reagent grade and used as
5,6-Dichloro-1H-benzotriazole (5,6-Cl
2
Bt): yield 690 mg (32%);
received. 3-chlorobenzene-1,2-diamine (95%) was purchased from abcr
Karlsruhe, Germany), 4-chlorobenzene-1,2-diamine (97%) and 3,5-di-
chlorobenzene-1,2-diamine (97%) were purchased from Sigma Aldrich
Munich, Germany), 4,5-dichlorobenzene-1,2-diamine (95%) was
mp. 263.5–266.5 °C (lit. 264–266 °C); HRMS (ESI): [48,53] m/z [M
+
(
6 4 2 3
+ H] calc. for C H Cl N : 187.97768; 189.97473 found: 187.97752;
1
6
189.97451; H NMR 500 MHz (DMSO d ) δ [ppm]: 8.31 (s, 2H, H-4,H-
(
7); PqNMR: 84.0%; (Supp. Fig. 1H).