Antihypertensive and antiarrhythmic properties of a para-hydroxy[bis(ortho- morpholinylmethyl)]phenyl-1,4-DHP compound: Comparison with other compounds of the same kind and relationship with logP values

Article abstract of DOI:10.1016/j.ejmech.2010.07.027

A new para-hydroxy[bis(ortho-morpholinylmethyl)]phenyl-1,4-DHP substituted compound, (4-(4-hydroxy-3,5-bis(morpholin-4-ylmethyl)phenyl)-2,6-dimethyl-1,4- dihydropyridine-3,5-dicarboxylic acid diethyl ester, LQM300), with antihypertensive and antiarrhythmic properties, has been synthesized. Four pKa values of this compound have been determined with the aid of the program SQUAD, at pseudo-physiological conditions (T = 37 °C and I = 0.15 M) by UV spectrophotometry and at T = 25 °C and I = 0.05 M by Capillary Zone Electrophoresis (CZE). The logP = 2.7 ± 0.2 between n-octanol and water, has been estimated by UV spectrophotometry. The antihypertensive and antiarrhythmic efficacies as well as the logP values have been compared with other compounds of the same kind and related with their structure.

Full text of DOI:10.1016/j.ejmech.2010.07.027

European Journal of Medicinal Chemistry 45 (2010) 4622e4630
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antihypertensive and antiarrhythmic properties of a para-hydroxy[bis(ortho-
morpholinylmethyl)]phenyl-1,4-DHP compound: Comparison with other
compounds of the same kind and relationship with logP values
,
b
Victor H. Abrego a, Beatriz Martínez-Pérez a, Luis A. Torres a, Enrique Ángeles a , Luisa Martínez ,
*
J. Lorena Marroquín-Pascual c, Rosario Moya-Hernández c, Héctor Adrián Amaro-Recillas c,
Juan Carlos Rueda-Jackson c, Damaris Rodríguez-Barrientos d, Alberto Rojas-Hernández d
,
*
a Universidad Nacional Autónoma de México, FES-Cuautitlán, Laboratorio de Química Medicinal, Campo 1. Av. 1ero de mayo S/N. 54714 Estado de México, México
b Universidad Nacional Autónoma de México, FES-Cuautitlán, Laboratorio de Farmacología del Miocardio, Campo 1. Av. 1ero de mayo S/N. 54714 Estado de México,
México
c Universidad Nacional Autónoma de México, FES-Cuautitlán, UIM, Laboratorio de Fisicoquímica Analítica, Campo 4. Km 2.5 Carretera Cuautitlán Teoloyucan, 54740
Estado de México, México
d Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Química, Área de Química Analítica, Apdo, Postal 55-534, 09340 México, D. F. México
a r t i c l e i n f o
a b s t r a c t
Article history:
A
new para-hydroxy[bis(ortho-morpholinylmethyl)]phenyl-1,4-DHP substituted compound, (4-(4-
Received 24 February 2010
Received in revised form
13 July 2010
Accepted 16 July 2010
Available online 23 July 2010
hydroxy-3,5-bis(morpholin-4-ylmethyl)phenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid
diethyl ester, LQM300), with antihypertensive and antiarrhythmic properties, has been synthesized. Four
pKa values of this compound have been determined with the aid of the program SQUAD, at pseudo-
physiological conditions (T ¼ 37 ꢀC and I ¼ 0.15 M) by UV spectrophotometry and at T ¼ 25 ꢀC and
I ¼ 0.05 M by Capillary Zone Electrophoresis (CZE). The logP ¼ 2.7 ꢁ 0.2 between n-octanol and water,
has been estimated by UV spectrophotometry. The antihypertensive and antiarrhythmic efficacies as well
as the logP values have been compared with other compounds of the same kind and related with their
structure.
Keywords:
Cardiovascular effects
Antihypertensives
Bis(morpholinylmethyl)phenols
DHP
ꢀ 2010 Elsevier Masson SAS. All rights reserved.
pKa values
logP
1. Introduction
properties of some methyltiomorpholinylphenols have been de-
monstrated [12].
Today there are many compounds used as antihypertensives
[1e9]. Despite the great number of antiarrhythmic drugs known
today, a satisfactory treatment does not exist and it has been
necessary to develop long-lasting, oral route agents with minor
indirect effects [6]. In Mexico arterial hypertension is a serious
public health issue, so it is important to develop new compounds
with antihypertensive activity, but less adverse effects and at more
accessible costs. Recently the synthesis of some morpholinyl-
methylphenols, as well as methyltiomorpholinylphenols, (LQM
compounds, based on the Region 2 of changrolin molecule [10]) has
been reported [11] and the antihypertensive and antiarrhythmic
Due to the great potential observed for the LQM compounds,
1e3, shown in Fig. 1, having antihypertensive and antiarrhythmic
properties [12], it was decided to synthesize LQM300 (compound 4,
Fig. 2) that merges in the same molecule two fragments with
antihypertensive properties: a 1,4-DHP moiety and a changrolin-
Region 2-like moiety [10] (Figs. 1 and 2). The second fragment has
also antiarrhythmic properties. With these structural changes it
was expected that the new molecule had an ambivalent antihy-
pertensive and antiarrhythmic action: blockage of Ca2þ channels
(associated to 1,4-DHP moiety) and binding to Naþ channels (sug-
gested for the changrolin-Region 2 moiety [13]).
The aim of the present work is to determine the antihyperten-
sive and antiarrhythmic properties of the compound 4 (LQM300)
and to determine its logP value and of the compounds 1e3,
between water and n-octanol, in order to compare it with those of
similar molecules and trying to relate these values with the
* Corresponding authors. Tel.: þ52 55 58044670; fax: þ52 55 58044666.
E-mail addresses: angeles@unam.mx (E. Ángeles), suemi918@xanum.uam.mx
(A. Rojas-Hernández).
0223-5234/$ e see front matter ꢀ 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.027

V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
4623
Fig. 2. Chemicalstructuresofcompound 4(LQM300). a)Neutralspecies H(LQM300)¼ HL
compound showing the1,4-DHP and changrolin-like moieties. b) Fully protonated species
H4(LQM300)3þ ¼ H4L3þ
.
4-(4-Hydroxy-3,5-bis(morpholin-4-ylmethyl)phenyl)-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester (compound 4,
Fig. 2) was prepared as described elsewhere [11] from 4-hydrox-
yphenyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylic acid
diethyl ester and thiomorpholine and formaldehyde (37%) were
mixed in a round flask fitted with a condenser. The mixture was
irradiated with infrared light for 10 min using a medicinal infrared
lamp (250 Watts) and the reaction was monitored by TLC. Yield:
85%. The mixture was passed through a silica gel chromatography
column using gradient hexane/ethyl acetate, than afforded one
crystalline product.
Fig. 1. Changrolin and some LQMs previously studied [12].
antihypertensive efficacy of LQM compounds. It was decided to
obtain the logP of compound 4 by determining its extracted fraction
as a function of pH and its pKa values using Spectrophotometric
(UV) and Capillary Zone Electrophoresis (CZE) studies through data
processing by the program SQUAD [14e17].
4-(4-Hydroxy-3,5-bis(morpholin-4-ylmethyl)phenyl)-2,6-dimethyl-
1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester. Physico chem-
ical properties. Mp 145e147 ꢀC. IR (cmꢂ1; CHCl3 film) 3450, 3540,
3056, 2932,1691. 1H NMR (300 MHz; CDCl3; Me4Si, dH): 6.93 (2H, s),
5.6 (1H, s, OH), 4.87 (1H, s, NH), 4.1 (4H,q, J ¼ 7.0 Hz), 3.71 (8H, m),
3.51 (2H, s), 2.49, (8H, m), 2.35 (6H, s) 1.22 (6H, t, J ¼ 14.0 Hz). 13C
2. Chemistry
2.1. Synthesis and properties of the LQM compounds
2.1.1. Synthesis of LQM300 (compound 4)
4-Hydroxyphenyl-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarbox-
ylic acid diethyl ester was prepared from 4-hydroxybenzaldehyde
with ethyl acetoacetate in ammonia gave the 1,4-dihydropyridine
resulting from the normal Hantzsch reaction, Yield, 80%. Mp
230e232 ꢀC. FT-IR (KBr): 3331, 1689, 1656, 1490, 1244, 1126,
NMR (d CDCl3): 167.7; 154.2; 143.3; 138.2; 128.7; 121.1; 104.4; 66.9;
59.5; 53.2; 38.7; 19.6; 14.3. FAB-MS (M þ 1) 544 (20%), 456 (96%),
252 (100%). Calculated for C29H41N3O7, C 64.07%; H 7.60%; N 7.73%;
O 20.60%.
720 cmꢂ1, 1H NMR (300 MHz, CDCl3):
d
¼ 1.25 (t, J ¼ 7.1 Hz, 6H), 2.34
(s, 6H), 4.12 (q, J ¼ 7.1 Hz, 4H), 5.32 (s,1H), 5.77 (s,1H), 7.24e7.41 (m,
4H). 13C NMR (CDCl3):
¼ 167.8, 148.5, 142.3, 130.4, 130.9, 127.9,
103.9, 59.8, 39.4, 19.3, 14.1.
2.1.2. Synthesis of LQM301, LQM302 and LQM303 (compounds
1e3)
These compounds (Fig. 1) have been synthesized as described
elsewhere [11,12].
d

4624
V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
Fig. 3. Doseseeffect curve (mean ꢁ SD, n ¼ 5) of the LQM300 compound for a) systolic
pressure (SP), b) diastolic pressure (DP) and c) heart rate (HR), using IAP model in male
Wistar rats anesthetized. The hypotensor effect of the LQM300 compound on the SP
and DP such as HR diminution was found for every dose used.
Fig. 4. Effect on a) SP, b) DP and c) HR of LQM300 after oral administration, using SHR
model. In these cases a dose of 0.1 mg/kg was used. Results are given on mean ꢁ SD,
n ¼ 5.
2.2. Chemicals
Compound 4 was administrated using an intravenous physio-
logical saline solution for the invasive model and an oral glucose
solution (5%) in the spontaneous hypertenses rat model.
The studies of noninvasive arterial pressure model were carried
out in a SPAM apparatus. The software used to collect data was
SIEVART 1. This equipment is at the Instituto Nacional de Car-
diología “Ignacio Chávez” from Mexico City.
All chemicals were analytical grade including H3PO4, Na2HPO4,
Na3PO4$12H2O (Química Meyer, Mexico), NaH2PO4$H2O (Técnica
Química, Mexico), sodium acetate (Merck), NaCl and boric acid
(Productos Químicos Monterrey, Mexico).
3. Antihypertensive and antiarrhythmic properties of the
LQM300 compound
3.1. Biological material
3.2. Biological activity
9-month old Male Wistar rats and male spontaneously
hypertensive rats (SHR) were obtained from the Centro de
Investigación y de Estudios Avanzados (CINVESTAV-IPN). All
animal procedures were conducted according to the Federal
Regulations for Animals Experimentation and Care (SAGARPA,
NOM-062-ZOO-1999, Mexico).
3.2.1. Invasive arterial pressure model (IAP)
Rats were anesthetized by intra-peritoneal injection of
sodium pentobarbital (40 mg/kg). Once the rat had reached
a sufficient depth of anesthesia, carotid arteria was cannulated
using a catheter model PE50, which was connected to a pressure
transducer. Then, femoral vein was dissected using this route to

V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
4625
introduce in the rat different doses of compound
a catheter PE10.
4 using
Basal heart rate, systolic and diastolic pressure were monitored,
the tested doses of compound 4 in this work were 0.0001, 0.001,
0.01, 0.1 and 1.0 mg/kg. Data were collected using a Digi-Med
pressure analyzer equipped with DMSI-200_1 software.
3.2.2. Non invasive arterial pressure model or spontaneous
hypertenses rats (SHR)
Five spontaneously hypertensive rats were used. Heart rate,
systolic and diastolic pressure were collected at the beginning and
every 10 min for 2 h. After that the compound 4 was orally
administrated (0.1 mg/kg) and heart rate, systolic and diastolic
pressures were monitored in the same way.
3.3. Antihypertensive and antiarrhythmic properties
In order to obtain fast and reliable data about the activity of the
compound 4, the IAP Model was used, and it provided us a fast
manner to know if this compound really decreases the arterial
tension. Fig. 3 represents the curves dose-response for systolic and
diastolic pressure (a and b, respectively) and heart rate (c) for the
IAP model. The dose which showed more hypotensive activity was
0.1 mg/kg even on heart rate.
Fig. 5. Effective mobility of compound 4 (LQM300) as a function of the aqueous
solution pH. Markers represent experimental values (the average of three replicates)
while solid line represents fitting achieved with a four acidebase equilibria model and
data of Table 1. Vertical lines signal the position of the pKa values determined at
T ¼ 25 ꢀC and I ¼ 0.05 M and in the top of the figure the predominance-zone diagram
[18] of the species of compound 4 has been drawn.
The SHR model was applied to evaluate the pharmacologic
effect on the diastolic and systolic pressure and for heart rate too.
The systolic and diastolic pressure reduction practically was initi-
ated after oral administration, and reached its maximum effect
after 50 min of the beginning of the experiment, and it remained
until the end of the experiment (120 min) Figs. 4a and b. The effect
on heart rate was also observed at the beginning until the end of
the experiment, and it reached a maximum level after 40 min
Fig. 4c.
phenol group. In this way, the neutral species HL could be molec-
ular and non-zwitterionic.
4.2. UV spectrophotometry
The absorption spectra obtained in this work for [HL] ¼
8.83 ꢃ 10ꢂ5 M aqueous solutions (compound 4) at different pH
values for pseudo-physiological conditions (T ¼ 37 ꢀC and 0.15 M
ionic strength (I)) are shown in Fig. 6.
4. Results and discussion
4.1. CZE experiments
It is remarkable that at this concentration the compound forms
a solid in the 6.7 < pH < 10.4 range. Then, absorbance values of 23
absorption spectra ecorresponding to aqueous solutions of
8.83 ꢃ 10ꢂ5 M of the compound 4 in the 1.5 ꢄ pH ꢄ 6.8 and
The molecular structure of compound 4 (Fig. 2) permits to
expect until four acid functions for the totally protonated species
(H4L3þ): the phenol and the three nitrogen atoms that might be
protonated in an acid aqueous medium.
The effective mobility of compound 4 as a function of pH is
shown in Fig. 5. The experimental values were fitted to the equation
(1) using the program SQUAD, as explained before [16,17], with
a chemical model involving four global formation equilibria.
10.4 ꢄ pH ꢄ 13.1 pH ranges and in the 230 nm ꢄ ꢄ 425 nm range
l
(with increments of Dl ¼ 5 nm) were introduced to program
SQUAD, in order to refine global formation constants and molar
absorptivity coefficients for several models. The best refinement is
presented in Table 1. The molar absorptivities obtained in the same
refinement are shown in Fig. 7.
ueff ¼ uLQM300 ¼ uLfL þ uHL HL
f
þ . þ uH L H L
f
þ . þ uH L H L
f
j
j
n n
z
2þ l
z
3þ l
H4L
H4L3þ
H3L
H3L2þ
zH Lþ lH Lþ
z
LlL
2
2
¼
fL þ
fH Lþ
þ
fH L2þ
þ
fH L3þ
2
3
4
F
F
F
F
ꢀ
ꢁ
lLQM300
lLQM300
þ
þ
z zLfL þ zH L fH L þ zH L2þ f
þ zH L3þ f
¼ zLQM
(1)
H3L2þ
H4L3þ
2
2
3
4
F
F
where fHjL (j ˛ {0, 1, 2, 3, 4}) are the fractions of the compound 4
(LQM300) species (that contain L in the system). [19], Table 1.
Analyzing the information obtained by CZE for compound 4 it
seems that pKa1 ¼ 1.15 may be related to the nitrogen atom of the
1,4-DHP moiety [1] and, by analogy with the behavior of some
pyperidinylmethylphenols with bulky non-polar substituents in
para position, previously studied [17], the pKa2 ¼ 4.07 and
pKa3 ¼ 6.75 values could be associated to the nitrogen atoms of
morpholines. Then, the pKa4 ¼ 11.28 should be linked then to the
4.3. logP determinations
4.3.1. Distribution ratio, logP0 ¼ f(pH), between n-octanol and
aqueous solutions for compound 4 (LQM300)
The absorption spectra obtained to quantify compound
4
(LQM300) concentration in n-octanol ([LQM300]o) is shown in
Fig. 8a, for the concentrations of the calibration curve. A comparison
of the molar absorptivities of the neutral HL ¼ HLQM300 species
(Fig. 8b), in aqueous and n-octanol phases, indicates that the

4626
V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
Table 1
mobility curves. The wavelength selected to work for analysis of
samples of extraction systems was 360 nm and linear regression of
pKa values (pKai) of compound 4 (LQM300) obtained with the aid of program
SQUAD, from CZE and spectrophotometric data, shown in Figs. 5 and 6.
the calibration curve gives: A(360) ¼ (7370 ꢁ 340)[LQM300]o
(0.0037 ꢁ 0.0034), when concentrations are given in molarity.
þ
Acidity equilibria
pKai ꢁ sa
Absorbance values of the n-octanol phase have been recorded
and interpolated in the calibration curve shown in Fig. 8c, after
phase separation for the liquideliquid extraction experiments, in
order to obtain [LQM300]o; the concentration of compound 4 on
aqueous phase [LQM300]aq has been obtained by difference of total
LQM300 and LQM300 in n-octanol and, then the fraction of
CZE
Spectrophotometry
T ¼ 25 ꢀC and bI ¼ 0.05 M T ¼ 37 ꢀC and
bI ¼ 0.15 M
H4L3þ%H3L2þ þ Hþ
H3L2þ%H2Lþ þ Hþ
H2Lþ%HL þ Hþ
HL%Lꢂ þ Hþ
1.153 ꢁ 0.24
4.076 ꢁ 0.084
6.748 ꢁ 0.039
11.323 ꢁ 0.050
1.5c
4.118 ꢁ 0.021
7.172 ꢁ 0.023
11.2798 ꢁ 0.0087
compound 4 in the octanol phase ðELQM300 Þ in the extraction may
o
be calculated by definition (equation (2)).
Electrical and optical
Ionic effective
Absorptivities at
properties of the five
mobilities of species
l
¼ 360 nm
species involved obtained (uHz jL
ꢁ
s
) ꢃ 108/m2 Vꢂ1 sꢂ1 (˛Hz jL
ꢁ
s)/L
with the aid of program
SQUAD
molꢂ1 cmꢂ1
vo½LQM300ꢅo
ELQM300 ¼ 100%
(2)
o
sreg ¼ 0.05825d
sreg ¼ 0.0063d
n
LQM300T
H4L3þ
3.000c
5788 ꢁ 170
5413 ꢁ 40
5548 ꢁ 27
7900 ꢁ 64
9030 ꢁ 34
H3L2þ
H2Lþ
HL
2.091 ꢁ 0.094
where vo represents the volume of n-octanol phase while n
represents the total amount of substance of compound 4 in the
extraction system.
On the other hand, the fraction of compound 4 in the n-octanol
phase may be obtained through equation (3), for a system with
buffering in pH and phosphates [20]:
LQM300T
1.000c
0.000c
Lꢂ
ꢂ1.048 ꢁ 0.054
a
s
represents the standard deviation of the statistical estimator.
b
c
I represents the ionic strength of the solution.
Fixed during refining.
sreg represents the standard deviation of the regression, fitting mobilities or
d
absorbances.
rP0
ELQM300
¼
(3)
o
1 þ rP0
predominant species at pseudo-physiological conditions is practi-
cally the same (due to the equality of both molar absorptivities). This
gives evidence to associate to the neutral species the molecular non-
zwitterionic character, as explained before by the electrophoretic
where r is the volume ratio of n-octanol to aqueous phase while P0 is
the conditional partition coefficient (or distribution ratio, DR) of
compound 4, given by the equation (4).
Fig. 7. Molar absorptivity coefficients (˛) of the compound
4 (LQM300) species
Fig. 6. Representative absorption spectra of compound 4. [LQM300] ¼ 8.83 ꢃ 10ꢂ5
M
obtained by absorbance data refined with program SQUAD (Table 1). Error bars are
only appreciated for the (more uncertain) coefficient of H4L3þ species. a) Short UV
wavelengths. b) Long UV wavelengths.
solutions. a) Experimental behavior in acidic media. b) experimental behavior in basic
media.

V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
4627
½HLꢅo
aq
P0 ¼
Lꢂ þ½HLꢅaqþ HLþ þ H L2þ þ H L3þ
½
ꢅ
½
ꢅ
½
ꢅ
½
ꢅ
2
3
aq
aq
aq
P
b
Hþ
½
ꢅ
1
¼
¼ PfHL
ð4Þ
3
1þ
b
Hþ
þ
b
Hþ 2þ
b
Hþ 3þ
b
Hþ
½
ꢅ
½
ꢅ
½
ꢅ
½
ꢅ
1
2
3
4
By combining equations (3) and (4) and remembering that for this
compound r ¼ 1, equation (5) was obtained.
Fig. 9. Fraction of compound 4 (LQM300) in the n-octanol phase. Markers represent
experimental points and solid line is the fitting obtained with equation (5) for
a logP ¼ 2.7 ꢁ 0.2.
ꢂ
ꢃ
1
ELQM300 ¼ 100%
(5)
o
1 þ 1=ðPfHL
Þ
The fitting of the curve shown in Fig. 9 has been obtained by
substituting the global formation constants given for pseudo-
physiological conditions (Table 1) and a value of P ¼ 102.7 in
equation (5). Furthermore, from Fig. 9 it can be seen that at pseudo-
physiological conditions P0 ¼ P ¼ 102.7
.
4.3.2. logP between n-octanol and water for LQM301, LQM302 and
LQM303 (compounds 1e3)
The experimentation followed for compound 4 (LQM300) has
demonstrated that the complete set of pKa values might explain its
extraction behavior. But, as this experimentation is very long, it was
decided to determine only the logP value of compounds 1e3
(LQM301, LQM302 and LQM303) between n-octanol and water, in
order to compare it with that obtained for compound 4. The volume
of the aqueous solutionwas raised to obtain better precision for these
three estimators. The linear regression equations of the calibration
curves obtained were A(286) ¼ (10.49 ꢁ 0.08) ꢃ 10ꢂ3[LQM301]o
þ
(ꢂ0.0306 ꢁ 0.0050), A(286) ¼ (12.12 ꢁ 0.07) ꢃ 10ꢂ3[LQM302]o
þ
(0.1383 ꢁ 0.0029), and A(280) ¼ (10.04 ꢁ 0.11) ꢃ 10ꢂ3[LQM303]o
þ (0.1280 ꢁ 0.0058); when concentrations are given in parts per
million (ppm). The extracted fractions of LQM301, LQM302 and
LQM303 were obtained from equation (6) and then its P values from
equation (7).
½LQM30Xꢅo
ELQM30X
¼
(6)
(7)
o
½LQM30Xꢅo; initial
ꢂ
ꢃ
E
1
r
LQM30Xo
PLQM30X
¼
1 ꢂ ELQM30X
o
Table 2 shows the comparison of logP values of compound 4
(LQM300) with other LQMs and other antihypertensive substances.
Table 2
logP values between n-octanol and water for several antihypertensive substances.
Compound
logP
Reference
1 (LQM301)
2 (LQM302)
3 (LQM303)
4 (LQM300)
>3.048
This work
This work
This work
This work
Fig. 8. Results for the calibration curve of the quantification of [LQM300]o. a) Absorption
spectra of HL in n-octanol. b) Molar absorptivity coefficients of aqueous neutral
(HLQM300)and anionic (HLQM300ꢂ) species, and octanolic neutralspecies (HLQM300ꢀ)
of compound 4. It is relevant the similarity of the absorptivity coefficients of the neutral
species in both phases. c) Graphic representation of regression curve. The error bars are
represented over the experimental points in the absorbance direction; the solid line
represents the maximum verisimilitude line, the dashed lines represent the slope
confidence limits at 95% significance level and the curve lines (hyperboles) represent the
confidence limits of the absorbances at the same significance level.
1.96 ꢁ 0.03
2.03 ꢁ 0.08
2.7 ꢁ 0.2
Nifedipine
Felodipine
2.40
3.86
[1]
[21]
Captopril
Losartan
1.02
4.01
[1]
[21]

4628
V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
higher logP values than for the other substituents. On the other
hand, the order observed for logP (2 ꢄ 3 < 4) may be explained by
the size of the substituent in the para position of the changrolin-
Region 2-like moiety of the molecules, as expected: higher the size
of the substituent, higher the logP value.
4.3.3. Comparison of antihypertensive and antiarrhythmic effects of
compound 4 (LQM300) with captopril, losartan, and 1e3
compounds (LQM301, LQM302 and LQM303) and relationship with
log P values
The antihypertensive and antiarrhythmic effects of compound 4
(LQM300) and other substances are presented in Fig. 10. The curves
of the effect percentage as a function of the dose, for the Invasive
Arterial Pressure (IAP) model in male Wistar rats anesthetized, are
given for antihypertensive (Figs. 10aec) and antiarrhythmic
(Fig. 10d) properties. In general and for a dose of 1 mg/kg the
antihypertensive effect of the compound 4 is placed between
compounds 3 and 2 or 1. This may be due to the bulky substituent
1,4-DHP in the para position of the bis(morpolinylmethyl)phenol of
compound 4 with respect to the tert-butyl substituent in the para
position of the bis(tiomorpholinylmethyl)phenol of compound 3.
Then, it seems that it is important to maintain the double nitrogen-
ring substitution (morpholines or tiomorpholines ortho to the
hydroxyl group of the phenol) in the structure, to have good anti-
hypertensive properties. The antiarrhythmic effect of compound 4
is poorer than for the others LQMs at a dose lower or equal to
0.1 mg/kg. In general the antihypertensive and antiarrhythmic
effects of compound 4 are similar to those of losartan.
5. Conclusions
The four pKa values of a compound 4 (LQM300) have been
determined by UV spectrophotometry (at pseudo-physiological
conditions) and CZE (at 25 ꢀC and 0.05 M of ionic strength). These
values and extraction of the compound 4 (LQM300), between
n-octanol and water, have been used to determine the logP value.
The order observed of logP values of LQM compounds determined
in this work follows the order: 2 ꢄ 3 < 4 < 1.
A comparison of logP values of compound 4 with respect to
other LQM compounds, as well as the percentage of the effect as
a function of dose for antihypertensive properties, suggests that
there is a maximum effect for intermediate values of logP e around
2.0 e between n-octanol and water when there is a double ortho
substitution in the phenol ring (of LQM compounds) and may be
a molecular non-zwitterionic neutral species. The antiarrhythmic
properties seem not to be so dependent of these remarks, because
compound 4 has good antiarrhythmic effect for low doses.
6. Experimental protocols
6.1. UV spectrophotometry
6.1.1. Equipment conditions
UV absorption spectra were recorded with a PerkineElmer
Lambda 18 spectrophotometer, using quartz cells of 1 cm optic path
length, during titration of basic solution of compound 4 (LQM300)
with acid solution of 4, and vice versa.
Fig. 10. Comparison of the doseeeffect percentage curves of the compound
4
(LQM300) and other compounds using IAP model in male Wistar rats anesthetized. a)
Effect on the systolic pressure. b) Effect on the diastolic pressure. c) Effect on the mean
pressure. d) Effect on heart rate. The effect of compound 4 is intermediate to other
LQM compounds and similar to the effect of losartan. Data taken from Figs. 3 and 4 for
compound 4, and from Ref. [12] for all the other compounds.
6.1.2. Samples preparation
A stock aqueous solution of 4 was prepared by solving 5 mg at
the minimum amount of HCl 0.1 M required in a 50 ml volumetric
flask and filling to the mark with deionized water.12 ml of the stock
aqueous solution of 4 were mixed with necessary amounts of NaOH
and NaCl in a 50 ml volumetric flask and filling to the mark with
deionized water, in order to obtain the basic solution of 4 (pH z 13
The values obtained in the present work show good compatibility
with those reported for other antihypertensives. In fact, it seems
that a chloride substituent in the LQM compounds originate higher
solubility and affinity to the n-octanol phase, as it occurs with the
other antihypertensive compounds (losartan and felodipine), given

V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
4629
and 0.15 M of ionic strength). In a similar way the acid solution of 4
was prepared (pH z 1 and 0.15 M of ionic strength), but changing
NaOH by HCl.
values. Effective mobility (ueff) of LQM300 was calculated as the
difference of measured mobility (or apparent mobility, uapp) and
electroosmotic mobility (ueof), by applying the equation (8):
!
6.1.3. Spectrophotometric titrations
LtLd
V
1
1
ueff ¼ uLQM ¼ uapp ꢂ ueof
¼
ꢂ
(8)
A volume of 25 ml of basic (acid) solution of 4 was placed on
a glass cell and titrated with the acid (basic) solution of 4, in order
to decrease (increase) the pH by 0.25 pH units. The temperature of
the cell was maintained at 37 ꢁ 0.1 ꢀC with a PolyScience 9105
water recirculator bath and the inert atmosphere was achieved
passing a nitrogen flow over the solution. The titrated solution was
stirred magnetically with a Corning PC-353 device. The pH of the
solution was measured with a Mettler Toledo MA235 pH meter
equipped with a combined electrode INLAB 413. The pH-meter was
calibrated previously with commercial 4, 7 and 10 buffer solutions.
tLQM teof
where Lt represents the total length of the capillary, Ld accounts for
length from injection to detector and V the applied voltage for
electro-migration
6.3. logP determination
In order to determine the logarithmic value of the partition
coefficient (logP) between n-octanol and water, experiments have
been designed to determine the n-octanol fraction of compound 4
as a function of pH. For compounds 1e3 experiments have been
designed to determine its corresponding fraction at neutral pH.
6.2. Capillary zone electrophoresis (CZE)
6.2.1. Equipment conditions
CZE experiments were carried out with a PACE-MDQ Capillary
Electrophoresis System (Beckman-Coulter, Fullerton CA, US) equip-
ped with a diode array detector and temperature control cartridges.
Instrument control and data processing were made with 32 KARAT
5.0 Software. The wavelength detection was set at 254 nm. New and
untreated fused silica capillary (from Polymicro Technologies,
Phoenix USA) of 60 cm (10 cm to detector in the short side and 50 cm
6.3.1. Solutions for the calibration curves of compounds 1e4
A convenient mass of compounds 1e4 was solved in n-octanol
in order to obtain a stock solution of concentration 5.00 ꢃ 10ꢂ4 M,
4.00 ꢃ 10ꢂ4 M, 3.00 ꢃ 10ꢂ4 M, and 1.47 ꢃ 10ꢂ4 M, respectively. The
solutions for each calibration curve were prepared by taking
5e25 ml with volumetric pipettes and diluting with n-octanol to
25.00 ml in a volumetric flask.
in the long side) and 75 mm of internal diameter was used to carryout
the electropherograms. The voltage applied was set at 20 kV. The
sample was injected hydrodynamically at 5 psi for 0.5 s.
6.3.2. Absorbance as a function of 1e4 compounds concentration
The absorption UV spectrum of each LQM solution was recorded
in a PerkineElmer Lambda 18 spectrophotometer, using quartz
cells with an optical path length of 1 cm.
6.2.2. Capillary conditioning
Before use the capillary was flushed with 1.0 M NaOH for 10 min
and then with deionized water for 10 min. In order to achieve
repeatable migration times, the capillary was rinsed with water
3 min, buffer running 5 min before each run.
6.3.3. Preparation of pH phosphate buffers
Several buffer solutions of phosphates were prepared in
a similar way that those used for CZE experiments, but a 0.1 M
phosphates concentration, but without fixing the ionic strength.
The pH of each solution was adjusted using a Mettler Toledo MA235
pH-meter equipped with a combined electrode INLAB 413. The pH-
meter was calibrated previously with commercial 4, 7 and 10 buffer
solutions.
6.2.3. Preparation of buffer solutions for CZE experiments
Since thestudied compound is a complex molecule, it was studied
in a wide range of pH (2e12.5). The electrolyte solutions were
prepared using a worksheet designed in our Lab to adjust the ionic
strength, considering the quantity of ions produced at each pH value;
this worksheet shows the quantity of NaCl to add (if necessary) in
order to obtain the required ionic strength, 0.05 M in this case. In
order to obtain the pH requested we used the following ionic pairs.
6.3.4. Extraction procedure of compound 4 and interpolation on the
calibration curve
5 ml of stock solution of compound 4 were mixed with 5 ml of
the pH buffer desired in an extraction flask. Vigorous stirring was
undertaken during 2 min and the phases were allowed to separate.
As a blank for absorbance measurements was used a similar
mixture in each case, but changing n-octanol in place of the stock
solution of 4. The UV absorption spectra for the n-octanol phase
were recorded in the PerkineElmer Lambda 18 spectrophotometer.
The concentration of compound 4 in the n-octanol phase was
obtained by interpolation of the absorbance measured on the
calibration curve.
pH range
Reagents used
2.0 e 3.5
3.5 e 5.7
6.0 e 8.2
8.2 e 10.7
10.9 e 12.5
H3PO4/H2POꢂ4
CH3COOH/CH3COOꢂ
H2POꢂ4 /HPO24ꢂ
H3BO3/H2BOꢂ3
HPO24ꢂ/PO34ꢂ
The pH of each buffer solution was measured by a Beckman
pH-meter and a Futura Gel-Filled combined electrode.
F 310
6.2.4. Sample preparation
6.3.5. Extraction procedure of compounds 1e3 and interpolation on
the calibration curve
A stock methanol solution of 450
mg of compound 4 was
prepared. An aliquot of 100 L of this solution was added to 1.7 ml
m
10 ml of 1, 2 or 3 stock solution were mixed with 90 ml of
deionized water Type I. Vigorous stirring was undertaken during
5 min and the phases were allowed to separate. As a blank for
absorbance measurements was used a similar mixture in each case,
but changing n-octanol in place of the LQM300 stock solution. The
UV absorption spectra for the n-octanol phase were recorded in the
PerkineElmer Lambda 18 spectrophotometer. The concentration of
1, 2 or 3 compound in the n-octanol phase was obtained by
of buffer solution at the pH required in order to obtain an adequate
concentration of the compound. Sample was injected hydrody-
namically to the capillary from the last solution.
6.2.5. Effective electrophoretic mobility determination (ueff
)
Migration times of 4 (tm ¼ tLQM300) and acetone (to ¼ teof, used as
electroosmotic flow (eof) marker) were obtained for different pH

4630
V.H. Abrego et al. / European Journal of Medicinal Chemistry 45 (2010) 4622e4630
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Acknowledgments
The authors wish to acknowledge to PAPIIT/UNAMProjects No
IN203609, IN211108, IN218408 and CONACYT Project No 82473 by
partially support for this work. We would like to thank to C. Barajas,
F. Sotres, M. Hernández, P. García and D. Jiménez for their skillful
technical assistance and DGSCA-UNAM for their support. VHA, as
a part of the project Cátedra de Química Medicinal of FESC-UNAM,
and DR-B, as member of Research Group of Analytical Chemistry
CA-UAM-I-33 of PROMEP, acknowledge to CONACYT by grant
support and AR-H for support to sabbatical leave in FESC-UNAM
during 2009.
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