Deprotonation mechanism and log P values of new antihypertensive thiomorpholinylmethylphenols: A combined experimental and theoretical study

Article abstract of DOI:10.1021/je100470g

Four new antihypertensive thiomorpholinylmethylphenol compounds were synthesized by its potential antihypertensive and antiarhythmic properties. The pK_a values were determined experimentally, with the aid of program SQUAD, by Capillary Zone Electrophoresis (CZE) (T = 310.15 K and I = 0.05 mol·dm^-3) and by UV spectrophotometry at pseudop ysiological conditions (T = 310.15 K and I = 0.15 mol·dm^-3), obtaining good agreement between the values determined with both techniques. A theoretical study was followed to propose a deprotonation mechanism for each compound. The log P values were also determined between n-octanol and water and compared with the values of other similar compounds to relate it with its possible biological activity.

Full text of DOI:10.1021/je100470g

J. Chem. Eng. Data 2010, 55, 4323–4331
4323
Deprotonation Mechanism and log P Values of New Antihypertensive
Thiomorpholinylmethylphenols: A Combined Experimental and Theoretical
Study†
‡
§
‡
§
§
´
Karla Sanpedro-Montoya, Beatriz Mart´ınez-Pe´rez, Annia Galano, Enrique Angeles, V´ıctor H. Abrego,
Mar´ıa Teresa Ram´ırez-Silva,‡ and Alberto Rojas-Herna´ndez*,‡
´
Universidad Auto´noma Metropolitana-Iztapalapa, Departamento de Qu´ımica, Area de Qu´ımica Anal´ıtica, Ap. Postal 55-534,
San Rafael Atlixco 186, Col. Vicentina, 09340 Me´xico, D.F. Me´xico, and Universidad Nacional Auto´noma de Me´xico,
FES-Cuautitla´n, Campo 1, Departamento de Ciencias Qu´ımicas, Laboratorio de Qu´ımica Medicinal, Av. 1ero de Mayo S/N,
Col. Sta. Mar´ıa las Torres, 54740 Cuautitla´n Izcalli, Edo. de Me´xico, Me´xico
Four new antihypertensive thiomorpholinylmethylphenol compounds were synthesized by its potential
antihypertensive and antiarhythmic properties. The pKa values were determined experimentally, with the
aid of program SQUAD, by Capillary Zone Electrophoresis (CZE) (T ) 310.15 K and I ) 0.05 mol · dm-3)
and by UV spectrophotometry at pseudophysiological conditions (T ) 310.15 K and I ) 0.15 mol · dm-3),
obtaining good agreement between the values determined with both techniques. A theoretical study was
followed to propose a deprotonation mechanism for each compound. The log P values were also determined
between n-octanol and water and compared with the values of other similar compounds to relate it with its
possible biological activity.
deprotonation mechanisms for the LQMs studied, the deproto-
nation paths shown in Scheme 1 were taken into account in
this work.
Introduction
The most common treatment for fighting hypertension is
through antihypertensive drugs. Nowadays there are a large
number of such substances available. Unfortunately, most of
them have adverse secondary effects. Therefore, the develop-
ment and detailed study of new potential antihypertensive
substances are of current and vital interest.
Experimental Section
Reagents. NaOH (0.99, Merck) and HCl (0.37, Merck) were
used to prepare acidic and basic solutions for spectrophotometric
experiments and to adjust the pH of buffers in CZE. Methanol
(spectroscopic grade, Baker), NaH2PO4 and Na2HPO4 (0.99,
Fluka), Na3PO4 (0.96, Aldrich), and H3PO4 (0.854, Baker) were
employed to prepare the buffers for CZE experiments or stock
solutions of the LQMs synthesized. All aqueous solutions were
prepared with deionized water (0.182 MΩ·m), purified with
Millipore Milli-Q Gradient equipment.
General Synthesis Procedure. An appropriate substituted
phenol (1 equiv), formaldehyde (2 equiv), and thiomorpholine
(1 equiv) (solvent free) were mixed in a round flask fitted with
a condenser. The mixture was irradiated with infrared light using
a medicinal infrared lamp (250 W), and the reaction was
monitored by thin-layer chromatography. The mixture was
separated by column chromatography on silica gel using a
hexane/ethyl acetate gradient solvent.1
4-Nitro-2-(thiomorpholin-1-ylmethyl)phenol (LQM324). Mp
) (112 to 114) °C. Yield: 0.7. Reaction time: 25 min. 1H NMR
(200 MHz; CDCl3; Me4Si, δH): 9.44 (1H, s, OH), 8.10 (1H,
m), 7.95 (1H, m), 6.86 (1H, m), 3.81 (2H, s), 2.83 (4H, m),
2.78, (4H, m). 13C NMR (δC): 164.16, 140.19, 125.40, 124.85,
120.70, 116.52, 61.54, 54.30, 27.69. FAB-MS (M+1) 255
(1.00), 254 (0.60), 154 (0.30), 136 (0.25). Calculated for
C11H14N2O3S: C(0.5195), H(0.0550), N(0.1102), O(0.1887),
S(0.1261).
In 2006 Vela´zquez et al.1 revisited the previous research by
Stout et al.2,3 and synthesized a new series of thiomorpholinyl-
methylphenol compounds. Some of them are shown in Table
1. They are intended to show antiarrhythmic and antihyperten-
sive activity and low toxicity since their physicochemical
properties and structure are similar to those of changrolin and
its derivatives. However, there are no reported data on the acid
constants of these thiomorpholinylmethylphenol compounds.
Thus, it is the main aim of the present work to estimate them
for the substances shown in Table 1, under pseudophysiological
conditions of temperature and ionic strength, as well as to
determine the log P values of these compounds between
n-octanol and water, to compare them with those of similar
compounds with demonstrated antihypertensive properties. It
is also our purpose to propose a probable deprotonation
mechanism for each of the studied compounds, i.e., to establish
the relative order in which the deprotonation processes take
place, following the same methodology described elsewhere.4
It provides information on the prevailing structure at each pH
interval, which is very important since different structures
interact in different ways with the surrounding species present
in biological environments. To establish the more plausible
4-Hydroxy-3-(thiomorpholin-1-ylmethyl)benzonitrile (LQM330).
Mp ) (168 to 170) °C. Yield: 0.85. Reaction time: 15 min. 1H
NMR (200 MHz; CDCl3; Me4Si, δH): 8.22 (1H, s, OH), 7.47
(1H, m), 7.28 (1H, m), 6.84 (1H, m), 3.74 (2H, s), 2.85 (4H,
† Part of the “Sir John S. Rowlinson Festschrift”.
* Corresponding author. E-mail: suemi918@xanum.uam.mx.
‡ Universidad Auto´noma Metropolitana-Iztapalapa.
§ Universidad Nacional Auto´noma de Me´xico.
10.1021/je100470g  2010 American Chemical Society
Published on Web 08/03/2010

4324 Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010
Table 1. Thiomorpholinylmethylphenol Ions of the Compounds Studied in This Work
Scheme 1. (R ) H, Br, CN, NO2)
(200 MHz; CDCl3; Me4Si, δH): 10.66 (1H, s, OH), 7.20 (1H,
m), 6.95 (1H, m), 6.79(1H, m), 3.71 (2H, s), 2.82 (4H, m), 2.71,
(4H, m). 13C NMR (δC): 157.58, 128.94, 128.76, 120.79,
119.27, 116.12, 62.27, 54.42, 27.93. FAB-MS (M+1) 288
(0.40), 184 (1.00). Calculated for C11H14NOS: C(0.4584),
H(0.0490), Br(0.2772), N(0.0486), O(0.0555), S(0.1113).
In the precedent paragraphs, yield, relative strength of MS
signals, and composition of compounds in their elements have
been reported with the corresponding fractions, not percentages.
Capillary Zone Electrophoresis (CZE) Study. Conditions
for the Electropherograms Acquisition. CZE experiments were
achieved with a Beckman P/ACE MDQ with DAD equipment.
A fused silica capillary with 50 µm of internal diameter, a total
length (Lt) of 30.2 cm, and an effective length from injection
to detector (Ld) of 20.0 cm on the long side or 10.2 cm on the
short side was used. Sample injection was hydrodynamic using
6.89·10-3 MPa (1 psi) by 10 s. Temperature was imposed at
310.15 K and voltage (V) at 20 kV (using reverse polarity only
for pH < 3.1). Acetone was used as an electroosmotic flow
marker and internal standard. pH of buffers was measured with
a pH/Ion Analyzer (MettlerToledo) potentiometer and a com-
bined pH electrode. Calibration of the potentiometer was
achieved with three buffer standard solutions of 4.00, 7.00, and
10.00 (Radiometer Analytical).
m), 2.76, (4H, m). 13C NMR (δC): 162.04, 133.43, 132.70,
121.73, 119.31, 117.22, 102.38, 61.51, 54.33, 27.75. FAB-MS
(M+1) 235 (0.70), 154 (1.00). Calculated for C12H14N2OS:
C(0.6151), H(0.0602), N(0.1196), O(0.0683), S(0.1368).
2-(Thiomorpholin-1-ylmethyl)phenol (LQM331). Mp ) (120
1
to 121) °C. Yield: 0.82. Reaction time: 23 min. H NMR (200
MHz; CDCl3; Me4Si, δH): 10.66 (1H, s, OH), 7.20 (1H, m),
6.95 (1H, m), 6.79 (1H, m), 3.71 (2H, s), 2.82 (4H, m), 2.71,
(4H, m). 13C NMR (δC): 157.58, 128.94, 128.76, 120.79,
119.27, 116.12, 62.27, 54.42, 27.93. FAB-MS (M+1) 210
(0.40), 106 (1.00). Calculated for C11H15NOS: C(0.6312),
H(0.0722), N(0.0669), O(0.0764), S(0.1532).
Capillary Conditioning and Storing. At the beginning of the
day, as usual, the capillary was washed with 0.1 mol ·dm-3
NaOH during at least 5 min at 0.138 MPa, followed by another
5 min, at 0.138 MPa, wash with deionized water and a last 15
min, 20 psi, phosphate buffer wash (pH 7). Each run was started
by a 1 min, 0.034 MPa, wash with 0.1 mol ·dm-3 NaOH,
4-Bromo-2-(thiomorpholin-1-ylmethyl)phenol (LQM334). Mp
) (119 to 121) °C. Yield: 0.76. Reaction time: 18 min. 1H NMR

Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010 4325
followed by a 1 min, 0.034 MPa, deionized water wash, then a
2 min, 0.034 MPa, phosphate buffer wash of the desired pH.
The capillary was stored full of deionized water.
Determination of log P of LQM Compounds between
n-Octanol and Water. Presaturation of Phases. n-Octanol
(Baker) and deionized water obtained from a Milli-Q equipment
of Millipore (0.182 MΩ·m) were presaturated to maintain the
volume ratio constant during liquid-liquid extraction processes.
Calibration CurWes. For each LQM compound, a calibration
curve was prepared, by solving adequate amounts of each LQM
in n-octanol presaturated with water.
Buffers Employed and Stock Solutions of LQMs. Buffer pH
solutions, 50 mmol ·dm-3 in phosphates, were prepared by
mixing adequate volumes of stock 0.50 mmol · dm-3 H3PO4,
H2PO4-, HPO42-, or PO43- aqueous solutions, adjusting finally
the pH of each solution with 0.1 mol ·dm-3 NaOH or HCl as
required, before the filling of the 25.00 cm3 volumetric flask to
the mark. Individual solutions of LQM324, LQM330, LQM331,
and LQM334 were prepared solving around 5 mg of each
compound in the minimum amount of methanol before filling
to the mark with deionized water the 50.00 cm3 volumetric flask.
Sample injected was added with acetone (as needed to have a
5 % acetone solution v/v).
Procedure to Obtain EffectiWe Electrophoretic Mobility. At
least three independent acquisitions of electropherogram were
achieved for each pH value and compound. Effective electro-
phoretic mobilities (ueff) were determined as the difference
between the apparent (observed) mobility (uapp ) uLQM′)srelated
with the reciprocal of its migration time (tLQM′)sand the mobility
of the electroosmotic flow marker (ueof)srelated with the
reciprocal of its migration time (teof)-in agreement with the
equation4,5
An amount of 1 cm3 of a LQM stock solution in n-octanol,
with a concentration sited around the middle of the calibration
curve, was mixed with 9 cm3 of deionized water, presaturated
with n-octanol. Vigorous stirring was undertaken during 5 min,
and the phases were allowed to separate. A similar mixture in
each case was used as a blank for absorbance measurements,
but changing n-octanol presaturated of deionized water in place
of the corresponding LQM stock solution. The UV absorption
spectra for the n-octanol phase were recorded in the Perkin-
Elmer Lambda 20 or 950 spectrophotometers, using quartz cells
with an optical path length of 1 cm. The concentration of the
corresponding LQM compound in the n-octanol phase was
obtained by interpolation of the absorbance measured on the
calibration curve, and this determination was repeated three
times.
The linear regression equations of the calibration curves
obtained were
LtLd
V
1
tLQM′
1
teof
A(315) ) (55.49 ( 1.34)·10-3([LQM324]o/mg ·dm-3) +
(-0.018 ( 0.024) with r2 ) 0.9989 for LQM324
ueff ) uapp - ueof ) uLQM′
)
-
(
)
Spectrophotometric Study. Solution Preparation. An acidic
solution, at an adequate concentration for spectrophotometric
measurements, was prepared by solving a convenient mass of
the LQM desired on a volume of 0.01 mol ·dm-3 HCl with 0.14
mol·dm-3 NaCl aqueous mixture. Also, a basic solution was
prepared by solving a convenient mass of the LQM desired on
a volume of 0.01 mol ·dm-3 NaOH with 0.14 mol ·dm-3 NaCl
aqueous mixture. Then, for each LQM, the acidic solution was
titrated with the basic solution (and vice versa), to maintain the
ionic strength (I) equal to 0.15 mol ·dm-3 during titration. The
temperature of the titration cell was maintained at 37 °C with
a Cole-Parmer recirculator. Atmosphere over the solution was
kept inert over passing a dry nitrogen flow.
A(254) ) (76.85 ( 4.25)·10-3([LQM330]o/mg ·dm-3) +
(-0.015 ( 0.045) with r2 ) 0.9963 for LQM330
A(275) ) (12.95 ( 0.10)·10-3([LQM331]o/mg ·dm-3) +
(0.0068 ( 0.0061) with r2 ) 0.9999 for LQM331
and
A(287) ) (0.72 ( 0.05)·10-3([LQM334]o/mg ·dm-3) +
(0.007 ( 0.011) with r2 ) 0.9917 for LQM334
pH Measurements. Solution pH, with a resolution of ∆pH
) ( 0.001, was measured with a Tacussel LHP 430T
potentiometer equipped with a Radiometer Analytical combined
pH electrode F-69627. The pH of the solution was corrected
by cell efficiency6,7 with the equation4
where [LQM3XX]o represents the concentration of correspond-
ing LQM in the octanol phase.
Extracted Fractions and log P Values. The extracted
fractions of the corresponding LQM were obtained from the
equation
pHcalibration - pHobserved
[LQM3XX o
]
pHcorrected ) pHobserved
+
Ef
[
]
pHcalibration
ELQM3XX
)
o
[LQM3XX o,initial
]
Ef is an empirical parameter related to potentiometric cell
efficiency that tends to zero while cell efficiency tends to 100
%. The results obtained with this equation are practically the
same as those for the methods proposed by Wescott6 or Bates.7
Conditions for the Absorption Spectra Acquisition. The
absorption spectrum for each solution was achieved with a
Perkin-Elmer Lambda 950 spectrophotometer, using quartz cells
of 1 cm path length and a temperature of 310.15 K.
Equilibrium Constants Determination. The SQUAD program
was used to obtain the formation constants by fitting UV
absorption spectra8,9 or effective electrophoretic mobilities4,5
with pH and chemical composition of the solutions.
and then its P values from the equation
E
LQM3XXo
1
log PLQM3XX ) log
(
)
[
r 1 - ELQM3XX
]
o
Computational Details. All the electronic calculations were
performed with the Gaussian 0310 package of programs. The
stationary points were first modeled in the gas phase (vacuum),
and solvent effects were included a posteriori by single-point
calculations using the polarizable continuum model, specifically

4326 Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010
Table 2. Deviation of the Calculated pKa Values from the Experimental Data for the LQM331 System
∆Ggas
CBS-QB3
CBS-QB3
CBS-QB3
CBS-QB3
∆Gs
pKa1 (SE)a
pKa2 (SE)a
MUEb
PCM/HF/6-31+G(d,p)
10.28 (1.39)
10.19 (1.30)
10.38 (1.49)
10.28 (1.39)
9.84 (0.95)
10.49 (1.60)
11.57 (1.18)
11.51 (1.12)
11.50 (1.11)
11.44 (1.05)
14.91 (4.52)
10.54 (0.15)
1.29
1.21
1.30
1.22
2.74
0.88
PCM/HF/6-311++G(d,p)
CPCM/HF/6-31+G(d,p)
CPCM/HF/6-311++G(d,p)
CPCM/B3LYP/6-311++G(d,p)
CPCM/HF/6-311++G(d,p)
CBS-QB3
B3LYP/6-311++G(d,p)
a Signed error. b Mean unsigned error.
the integral-equation-formalism (IEF-PCM).11-14 In PCM cal-
culations, the choice of the solute cavity is important because
the computed energies and properties strongly depend on the
cavity size. In the present study, the cavity has been systemati-
cally built by the united atom model for the Hartree-Fock
(UAHF) method15 which is the recommended method for
predicting free energies of solvation according to the Gaussian
03 User’s Reference.16
Different levels of theory have been tested, within this
scheme, for LQM331 (R ) H). The results are shown in Table
2. To choose a level of theory for computing ∆Gs, the ∆Ggas
was calculated as accurately as possible (CBS-QB3). As this
table shows, when the solvation energies are obtained using the
B3LYP functional instead of HF a large error arises for the
second pKa. This is a logical finding since the UAHF method
has been optimized for HF/6-31G(d).15 Due to the size of the
systems studied in the present work, an alternative, less
computationally expensive method was tested for computing
∆Ggas: B3LYP/6-311++G(d,p). Even though the error in the
first pKa is larger than when calculating ∆Ggas with CBS-QB3,
the error in the second one is smaller, leading to the lowest
average error among all the tested methods.
Relative Gibbs free energies in solution have been computed
using the Hess law and thermodynamic cycles, explicitly
including solvation free energies (∆Gs) From this cycle, the
On the basis of these results, the pKa values for all the other
systems were calculated using the last approach; i.e., geometry
optimizations and frequency calculations were performed at the
B3LYP/6-311++G(d,p) level of theory, followed by CPCM
single-point calculations at the HF/6-311++G(d,p) level to
obtain the ∆Gs values.
Gibbs free energy of reaction in solution (∆Gsol) can be obtained
as the sum of the Gibbs free energy of reaction in vacuum
(∆Ggas) and the difference in solvation free energies (∆∆Gs)
Results and Discussion
∆Gsol ) ∆Ggas + ∆∆Gs
where ∆∆Gs and ∆Ggas are calculated as
∆∆Gs ) ∆Gs(A-) + ∆Gs(H+) - ∆Gs(HA)
(1)
Stability of LQM Compounds. The stability studies on the
four studied compounds were carried out by spectrophotometric
analysis of their aqueous solutions that were followed in function
of time for acid, basic, and neutral solutions. The cyano and
nitro compounds (LQM330 and LQM324) showed higher
stability than the other two (LQM331 and LQM334). The last
compounds start to slowly precipitate after a few hours. On the
basis of these results, we have used fresh solutions, prepared
as diluted as possible.
(2)
(3)
∆Ggas ) Ggas(A-) + Ggas(H+) - ∆Ggas(HA)
Capillary Zone Electrophoresis (CZE) at T ) 310.15 K
and I ) 0.05 mol ·dm-3. After the stability tests, the following
step was the determination of the pKa values of each LQM
compound, at T ) 310.15 K and I ) 0.05 mol ·dm-3 by capillary
zone electrophoresis. The mobility curves as a function of pH
are shown in Figure 1, and the parameters of their fitting with
the program SQUAD, as explained elsewhere,4,5 are shown in
Table 3. An equation of the effective mobility as a function of
pH for LQM species is presented in eq 5
with ∆Gs representing the free energies of solvation. In all the
cases, the reference state is 1M. For the cycles that involve
protons, the ∆Ggas(H+) and ∆Gs(H+) values have been derived
from experiments. We have used ∆Ggas(H+) ) -6.28
kcal·mol-1 ) -26.28 kJ·mol-1 and ∆Gs(H+) ) -264.61 kcal/
mol ) -1107.13 kJ ·mol-1. This procedure has been previously
used in calculation of pKa values with successful results.17-21
The aqueous Gibbs free energies for the deprotonation
reactions are in turn used to compute the acid equilibrium
constant (Ka), according to
uL′ ) u f + uHL HL + u +f
f
-
-
+
L
L
H2L H2L
a1+pKa2-2pH)
uL + uH L+[10(pK
]
sol/RT
Ka ) e-∆G
(4)
-
2
)
(6)
1 + 10(pK
+ 10(pK
a2-pH)
a1+pKa2-2pH)
Then, using the pKa definition
pKa ) -log(Ka)
-
+
where fL , fHL, and fH L are the molar fractions of the distribution
2
(5)
diagram23,24 of the LQM344 species; uL , uHL (that is equal to
0 because it is electrically neutral), and uH L are their corre-
-
+
2
This approach is based on the knowledge that computational
methods poorly reproduce the solvation energies of protons, and
it has been successfully used before by different authors.21,22
sponding ionic mobilities; and pKa1 and pKa2 are the minus
logarithmic values of the acidity constants of H2L+ and HL
species, respectively.

Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010 4327
Figure 1. Effective mobility curves (ueff) as function of pH, obtained at T ) 310.15 K and I ) 0.05 mol·dm-3. The markers represent the experimental
results, with error bars calculated as the standard deviation of three replicates, and the solid lines the fittings obtained with the aid of the program SQUAD.
In the top of each figure, the corresponding predominance-zone diagram of species23,24 is represented. (a) LQM324. (b) LQM330. (c) LQM331. (d) LQM334.
Table 3. pKa Values for Acidity Equilibria and Effective Ionic
corresponds to a monoanion (L- ) (lqm3XX)-). Nevertheless,
Mobilities of Each Species uH (lqm,3XX)z, Refined with SQUAD from
j
Effective Electrophoretic Mobilities (ueff, Figure 1) at T ) 310.15 K
it is noteworthy that on one hand the monoprotonated electrically
neutral species (HL ) H(lqm324) and H(lqm334)), for the nitro
and bromo derivatives, respectively, reaches a maximum fraction
(fHL ≈ 0.95) in its corresponding pH (6.8 and 7.9); on the other
hand, this neutral species (HL ) H(lqm330) and H(lqm331))
only reaches a maximum value around 0.82 in its corresponding
pH (8.5 and 9.6) for the cyano and hydrogen derivatives. In
other words, at physiological pH the neutral species predomi-
nates for the nitro and bromo derivatives, but the cation is the
predominant species at these conditions for cyano and hydrogen
derivatives.
a
and I ) 0.05 mol ·dm-3
buH (lqm3XX)z ·108 λH (lqm3XX)z ·104
c
j
j
compounds and
related equilibria
pKai
m2 ·V-1 ·s-1
m2 ·S·eq-1
d
d
d
d
LQM324, σreg ) 0.032
H2L+ a HL + H+
HL a L- + H+
H2L+
5.296 ( 0.018
8.329 ( 0.129
1.367 ( 0.015 13.19 ( 0.14
-1.250 ( 0.115 12.06 ( 1.1
L-
LQM330, σreg ) 0.019
H2L+ a HL + H+
HL a L- + H+
H2L+
7.530 ( 0.002
9.529 ( 0.019
1.750 ( 0.001 14.96 ( 0.01
-1.737 ( 0.006 16.76 ( 0.06
L-
UV Spectrophotometry at Pseudophysiological Conditions
(T ) 310.15 K and I ) 0.15 M). Due to the potential medical
applications of the studied compounds, the UV spectrophoto-
metric study was carried out under pseudophysiological condi-
tions: T ) 310.15 K and I ) 0.15 mol ·dm-3. The most
representative spectra, in acidic and basic media, are shown in
Figure 2. Several isosbestic points, corresponding to acid-base
equilibra, are observed. This information, together with the data
gathered from CZE for number and nature of species in aqueous
solutions, allowed us to refine the same models with the program
SQUAD.8,9 The refined pKa results are reported in Table 4, while
the refined molar absorptivity coefficients are shown in Figure
3.
LQM331, σreg ) 0.008
H2L+ a HL + H+
HL a L- + H+
H2L+
8.597 ( 0.006
10.516 ( 0.003
0.831 ( 0.014
8.02 ( 0.13
L-
-1.203 ( 0.002 11.60 ( 0.02
LQM334, σreg ) 0.013
H2L+ a HL + H+
HL a L- + H+
H2L+
6.195 ( 0.009
9.592 ( 0.001
2.305 ( 0.014 22.24 ( 0.13
L-
-1.919 ( 0.115
18.5 ( 1.1
a Equivalent conductivities of each species λH (lqm3XX)z have been
j
calculated from uH (lqm3XX)z
.
b The mobility of neutral species has been
j
fixed to 0.000 m2 ·V-1 ·s-1 during refining. c λ values are calculated with
equation |z|λH (lqm3XX)z ) FuH (lqm3XX)z, where F accounts for the constant
j
j
of Faraday.4,5,25 d σreg represents the standard deviation of the regression.
It is interesting to remark the similarity of the set of molar
absorptivity coefficients of LQM331 and LQM334 species,
while for LQM324 and LQM330 there are more differences.
These could be attributed to the electro-attracting capacity of
the substituent in the para position to the phenol group, greater
All the compounds show the expected behavior (Figure 1).
The double-protonated species correspond to a monocation
(H2L+ ) H2(lqm3XX)+), while the fully deprotonated one

4328 Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010
Figure 2. UV absorption spectra of (absorbance, A, as a function of wavelength, λ) LQM compounds studied in this work. (a) [LQM324] ) 2.44·10-5
mol·dm-3. (b) [LQM330] ) 4.61·10-5 mol·dm-3. (c) [LQM331] ) 4.78·10-5 mol·dm-3. (d) [LQM334] ) 3.85·10-5 mol·dm-3
.
Table 4. pKa Values Obtained under Pseudophysiological
Syracuse Research Corporation (SRC), from USA.26-29 As it
can be seen in Table 5, there is great dispersion of the calculated
values. In fact, ACDLogP and ALOGPS 2.1 programs fail for
more than 0.15 log P units in two of the four LQMs studied,
while the SRC program fails by the same amount in three of
the four cases. The miLogP program fails for more than 0.15
log P units in all the LQMs studied. In other words, the different
predictive methods render significantly different results for the
same compound.
Conditions (T ) 310.15 K and I ) 0.15 mol ·dm-3), Refined with
SQUAD from Spectrophotometric Data (Figure 2)
pKa1 ( σ
pKa2 ( σ
σ
reg, Ua
LQM324
LQM330
LQM331
LQM334
5.450 ( 0.001
7.437 ( 0.008
8.89 ( 0.11
8.412 ( 0.001
9.380 ( 0.004
10.391 ( 0.075
9.261 ( 0.009
0.006, 0.038
0.002, 0.008
0.007, 0.032
0.009, 0.019
6.421 ( 0.032
a σreg represents the standard deviation of the regression, while U
takes into account the sum of squares of the residuals.
On the other hand, it seems that a bromine substituent in the
LQM compounds originates higher solubility and affinity to the
n-octanol phase, as it occurs with antihypertensive compounds
with chloride substituents (e.g., losartan and felodipine), giving
higher log P values than for the other substituents.
for -NO2 than for -CN than for -Br. There is a bathochromic
shift for all the wavelengths of maximum absorption for
LQM324, as expected. Nevertheless, the differences in magni-
tude of molar absorptivity coefficients are difficult to explain
because they are also related with the energy differences of the
molecular orbitals involved in the electronic transition, in each
case (and in each case they could be different).
In addition, we believe that the experimental pKa values
obtained spectrophotometrically are more accurate than those
determined by electrophoresis because the imposition of ionic
strength is better by the experimental task achieved (with NaCl
in the first case and phosphate buffers in the latter). In fact, the
differences observed in the pKa values (up to 0.33) between
the two methods should be attributed mainly to the different
ionic strength values imposed in both cases.
Theoretical Study of the Deprotonation Mechanisms. The
LUMO density surfaces obtained for the H2L+ species are shown
in Figure 4. According to this criterion, the site with the highest
acid character depends on the para substituent in the phenol
moiety. For those species with nitro and cyano substituents, the
phenolic OH shows larger contribution to the LUMO density
than the amino sites; i.e., the phenolic H is expected to be the
most acid one and therefore the one involved in the first pKa.
For all the H and Br para substituents, the amino groups
contribute to the LUMO densities in a larger proportion than
the phenolic OH; i.e., the most acid sites are expected to be
those in the thiomorpholine group. However, this is only a
qualitative criterion, and a more detailed study based on Gibbs
free energies is needed to establish, with a higher degree of
certainty, the deprotonation mechanism.
Log P Values Obtained at Ambiance Temperature. Table 5
shows the comparison of log P values of LQM compounds
studied in the present work with those calculated with the
programs: ACDLogP of Adavanced Chemistry Development
Laboratories, from Canada; ALOGPS 2.1 of VCC Laboratories,
from Germany; miLogP of Molinspiration and KowWin of
Gibbs free energies for all possible deprotonation processes
have been computed at 298.15 K. Table 6 shows the ∆Gsol

Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010 4329
Figure 3. Absorptivity coefficients ε of the species of LQM compounds studied in this work, determined by the program SQUAD. (a) LQM324. (b) LQM330.
(c) LQM331. (d) LQM334. Solid line (-) represents the absorptivity of L, dotted line represents the absorptivity of HL (- -) and segmented line (- - -)
represents the absorptivity of H2L+.
Table 5. Experimental log P Values between n-Octanol and Water
for LQM Compounds Studied in the Present Work and Calculated
log P Values Calculated with Different Programs
log P
log P calcda
ACD ALOGPS
KowWin
SRC
substance
exptl
LogP
2.1
miLogP
LQM324
LQM330
LQM331
LQM334
1.591 ( 0.016
1.631 ( 0.009
1.743 ( 0.038
2.126 ( 0.017
1.53
1.56
1.45
2.45
1.77
1.67
1.78
2.58
2.00
1.80
1.07
2.85
2.13
1.83
1.73
2.62
a The uncertainties for the calculated values are 0.5 or greater.
values corresponding to the different pathways considered for
the first deprotonation. These energy values are in good
agreement with the predictions based on the LUMO density
distribution. For the species with NO2 and CN groups at the
para sites of phenols, the most feasible process is the one
involving the OH in the phenol group; i.e., the first pKa should
be associated with the acidity of this site. For all the other
substituents, the pathways involving amino groups lead to more
favorable processes, i.e., less endergonic.
Figure 4. LUMO density surfaces, computed with isodensity value of 0.02
au.
Table 6. Gibbs Free Energies of Reaction in kJ ·mol-1 for All
Possible First Deprotonation Pathways at T ) 298.15 K
acid sites
system
-OH
|-NH+|
R ) H
87.32
84.52
50.33
39.71
59.87
41.30
51.46
51.38
R ) Br
R ) CN
R ) NO2
Once the most viable path for the first deprotonation has been
identified for H2L+ systems, the deprotonation routes are
completely specified since only two deprotonation processes are
considered. Accordingly, path A is proposed as the most viable
one if R ) CN or NO2, and path B if R ) H or Br.
The pKa values calculated according to Scheme 2 are reported
in Table 7. They are in good agreement with those obtained
experimentally for pKa2 values and in some agreement for pKa1
values (Tables 3 and 4), probably due to the results obtained in
the calibration procedure of the calculation method (see Section
2). A correlation between calculated and experimental pKa
values is shown in Figure 5 (without considering the pKa1 value

4330 Journal of Chemical & Engineering Data, Vol. 55, No. 10, 2010
Table 8. Mean Signed Errors MSE, Mean Unsigned Errors MUE,
and Root-Mean-Square Errors RMSE, for Calculated pKa Values
with Respect to Those Estimated by Spectrophotometric and
Electrophoresis Techniques, Without Considering the pKa1 Value of
the LQM331 Compound
Scheme 2
spectrophotometry
electrophoresis
MSE
MUE
RMSE
0.607
0.607
0.676
0.578
0.610
0.721
calculations reproduce the experimental values. This agreement
permits us to have some confidence in the used methodology,
as well as in the theoretical predictions, and supports the
elucidation of the deprotonation paths proposed above.
In summary, from the theoretical calculations, it can be
proposed that the nature of the para substituent in the phenol
group influences not only the acidity of the different protons
(phenolic and thiomorpholinic) in H2L+ but also the route of
the consecutive deprotonation processes.
Table 7. Calculated pKa Values, According to Pathways in Scheme 2
pKa1
pKa2
Finally, the nature of the zwitterionic character of the neutral
species could also be related to the differences in magnitude of
molar absorptivity coefficients. The set of molar absorptivities
of LQM331 is very similar to LQM344, and the neutral species
seems to be non-zwitterionic in both cases. The differences are
greater with the set of molar absorptivities of the species of
LQM330 and LQM324, where the neutral species seems to be
zwitterionic.
R ) H
10.49
7.24
8.82
6.96
10.54
10.05
9.40
R ) Br
R ) CN
R ) NO2
8.60
of LQM331). The theoretical calculations reproduce the ex-
perimental trends, although with a systematic error of about 1
unit for pKa1 value, which are also in line with chemical
intuition. For instance, the presence of the nitro group at the
para site of phenol significantly increases the acidity of the
phenolic proton, which is explained by the high electro-
withdrawing character of this substituent. Moreover, both pKa
values decrease when this substituent is present, which suggests
that it also influences the acidity of the protons in the N atoms
of thiomorpholine groups, despite the presence of a methylene
group between thiomorpholine and the phenolic ring.
Conclusions
Stability tests of the studied compounds were found to safely
choose the work concentrations and the titration conditions.
The pKa values for the four antihypertensive studied com-
pounds were determined by UV spectrophotometry at T )
310.15 K and I ) 0.15 mol ·dm-3 and CZE at T ) 310.15 K
and I ) 0.05 mol ·dm-3
.
These experimental results permit us to predict a better
physiological behavior of LQM324 (nitro) and LQM334
(bromo) by the main predominance of its electrically neutral
species in these conditions, even though the four LQMs studied
in the present work follow at least the majority of the Lipinsky
rules, for the development of drugs.30
The deprotonation mechanisms were elucidated using theo-
retical calculations. From the theoretical results, it can also be
proposed that the nature of the para substituent in the phenol
group influences not only the acidity of the different protons
(phenolic and thiomorpholinic) but also the route of the
consecutive deprotonation processes. The theoretical results
permit us to propose that the neutral species of the Br derivative
might confer lower solubility of these species with respect to
the other derivatives, at physiological conditions, and this
behavior was observed with the log P value determined
experimentally.
The deviations of the calculated pKa values from those
obtained by spectrophotometric and electrophoresis techniques
are reported in Table 8 (without considering the pKa1 value of
LQM331), in terms of mean signed errors (MSE), mean
unsigned errors (MUE), and root-mean-square errors (RMSE).
Since the theoretical chemical accuracy is generally accepted
to be around 1 kcal·mol-1 ) 4.184 kJ·mol-1, for the highest
levels of theory, and this would imply an error of about 0.73
units of pKa, at T ) 298.15 K, it can be stated that the present
Acknowledgment
A.G., M.T.R.-S., and A.R.-H. thank Laboratorio de Visualizacio´n
y Co´mputo Paralelo at UAM-Iztapalapa for the access to its
computer facilities. E.A. also wants to acknowledge DGSCA for
access to the KAMBALM supercomputer and to thank C. Barajas,
F. Sotres, M. Herna´ndez, R. Valadez, and D. Jime´nez for their
skillful technical assistance.
Figure 5. Correlation between calculated pKa values, pKa(calc), and
experimental pKa values, pKa(exp), without considering the pKa1 value of
LQM331. [, points that use pKa values obtained by spectrophotometry; 9,
points that use those obtained by CZE. Solid line (s) fits the equation
pKa(calc) ) 0.732pKa(exp) + 2.867 for spectrophotometric data, while the
dotted line (---) fits the equation pKa(calc) ) 0.683pKa(exp) + 3.241 for
electrophoretic data.
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Received for review May 4, 2010. Accepted July 19, 2010. We are
indebted to UNAM through PAPIIT-IN218408, IN203609, and
CONACyT 46124 and 82473 projects for partial financial support.
K.S.-M. and A.R.-H. want to acknowledge CONACyT for a scholarship
to follow postgraduate studies and support for sabbatical leave on
FESC-UNAM during 2009, respectively.
JE100470G