Losartan-antioxidant hybrids: Novel molecules for the prevention of hypertension-induced cardiovascular damage

Article abstract of DOI:10.1021/jm9003957

We report the first examples of a new series of antioxidant-sartan hybrids (AO-sartans), which were made by adding an antioxidant fragment to the hydroxymethyl side chain of losartan. Experiments performed in cultured cells demonstrate that these new hybr

Full text of DOI:10.1021/jm9003957

7220 J. Med. Chem. 2009, 52, 7220–7227
DOI: 10.1021/jm9003957
Losartan-Antioxidant Hybrids: Novel Molecules for the Prevention of Hypertension-Induced
Cardiovascular Damage
†
Gonzalo Garcıa, Manuel Rodrıguez-Puyol, Ramon Alajarın, Isabel Serrano, Patricia Sanchez-Alonso, Mercedes Griera,
‡
†
‡
†
‡
ꢀ
ꢀ
§
†
,‡
Juan J. Vaquero,*,† Diego Rodrıguez-Puyol, Julio Alvarez-Builla, and Marıa L. Dıez-Marques*
ꢀ
ꢀ
†
‡
Departamento de Quımica Organica, Departamento de Fisiologıa and Hospital Prıncipe de Asturias, Universidad de Alcala, Campus
§
´
Universitario, 28871-Alcalaꢀ de Henares, Madrid, Spain
ꢀ
´
´
ꢀ
Received March 27, 2009
We report the first examples of a new series of antioxidant-sartan hybrids (AO-sartans), which were
made by adding an antioxidant fragment to the hydroxymethyl side chain of losartan. Experiments
performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin
II effect with increased antioxidant ability. In hypertensive rats, these compounds show properties that
suggest they may be more useful than losartan for controlling hypertension and preventing hyperten-
sion-induced cardiovascular damage.
Introduction
antihypertensive potency, but mainly to increase their ability
to prevent tissue damage in the cardiovascular system.3
Oxidative stress is a well-known mechanism that is respon-
sible for the development of vascular damage.4 Different
pathogenic stimuli involved in cardiovascular diseases, such
as activated macrophages, hyperglycaemia, oxidized low den-
sity lipoprotein (LDL), and even angiotensin II, exert their
harmful effects, at least partially, through an increased local
synthesis of reactive oxygen species.5-8 These active metabo-
lites induce a significant endothelial dysfunction9 and are able
to modify the normal balance among proliferation, apoptosis,
and extracellular matrix synthesis in heart and arterial
walls.10-12 Some beneficial effects of antihypertensive mole-
cules have been attributed, at least partially, to their antiox-
idant ability.13-15
The above-mentioned considerationsprompted us to devel-
op a new class of sartan derivatives by adding an antioxidant
fragment to these drugs. We hypothesized that the adminis-
trationof thesehybridcompoundswould bea morespecific or
efficient means to target antihypertensive or antioxidant
molecules to cardiovascular cells. Herein, we report our
strategy of incorporating a variety of antioxidant moieties
onto the primary alcohol group of losartan through an ester
linkage to generate new losartan-antioxidant hybrids, which
retain the ability to block the angiotensin II effect, with
increased antioxidant ability.
Although it is believed that lowering blood pressure is the
main protective mechanism of antihypertensive treatments,
other possibilities to explain the beneficial effects of these
drugs have been proposed. It is a well-known fact that local
changes that take place at the vascular level, such as the
increased oxidative stress, may also be a relevant mechanism
ofcardiovascular disease progressionin hypertensive patients.
Thus, treatments that interfere with this oxidative stress could
be useful tools for management of these individuals.
Among antihypertensive agents, angiotensin converting
enzyme (ACEa) inhibitors have been widely used in the
treatment of hypertension. However, in the past two decades,
research focused on drugs that can replace ACE inhibitors in
hypertension therapy, without their side effects (mainly
coughing), has led to the discovery of sartans (Chart 1).1
Drugs of this class are antagonists of angiotensin II at the AT1
receptor and block the action of angiotensin II in a potentially
more complete and specific way than ACE inhibitors.
Lowering blood pressure is not the only beneficial effect of
angiotensin IIblockade. Angiotensin IIplays a significantrole
in the progression of tissue damage in cardiovascular dis-
eases,2 and the beneficial effects of these drugs in the preven-
tion of cardiovascular morbidity and mortality cannot be
solely explained by their antihypertensive action. In this
context, some chemical interventions have been performed
on sartan molecules in order to modify, where possible, their
Chemistry
*To whom correspondence should be addressed. Phone þ34-91-
8854761; fax þ34-91-8854686; e-mail juanjose.vaquero@uah.es.
a Abbreviations: ABTS, 2,20-azino-di-(3-ethyl benzothiazoline-6-sul-
fonic acid); ACE, angiotensin converting enzyme; AO, antioxidant; Bn,
benzyl; EDTA, ethylenediamine tetraacetic acid; DMEM, Dulbecco0s
Modified Eagle Medium; HNE, 4-hydroxy-2-nonenal; HPLC-MS/MS,
high-performance liquid chromatography-tandem mass spectrometry;
LDH, lactate dehydrogenase; LDL, low density lipoprotein; L-NAME,
Nω-Nitro-L-arginine methyl ester hydrochloride; PCSA, planar cell
surface area; QqQ, triple-quadrupole; TBAF, tetra n-butyl ammonium
fluoride; TBDMS, tert-butyldimethylsilane; THF, tetrahydrofuran;
VSMC, vascular smooth muscle cells.
Among the sartans shown in Chart 1, losartan16 was chosen
as the appropriate candidate for chemical manipulation on
the basis of its high activity as an angiotensin II receptor
blocker, along with the presence in its structure of a hydro-
xymethyl moiety, which made it suitable for attaching the
antioxidant fragment in a simple synthetic chemical process.
Among the antioxidant candidates to be incorporated in the
losartan structure, a series of phenols were selected as the best
choices because of their well-known redox properties.
r
pubs.acs.org/jmc
Published on Web 10/28/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7221
Chart 1. Sartans, a Family of Angiotensin II Receptor Antagonists
whereas the formation of 6c required the removal of the silyl
protecting group with tetrabutyl ammonium fluoride (TBAF)
in tetrahydrofuran (THF) followed by hydrogenolysis. The
attempted deprotection of 5a under these conditions needed
higher amounts of the catalyst in order to complete its
conversion, thus giving 7a as the main product, and only
traces of 6a were obtained.
Chart 2. Antioxidant Phenolic Derivatives
In the attempts to remove the trityl group from 5d using
palladium on carbon, we detected the formation of the
derivative 7d as the main product, which results from the
deprotection and dehalogenation of 5d. This unexpected
result was convenient to establish the role of the halogen in
the target compounds. Derivatives 7a-c were also prepared
under the same conditions used for 7d. The series of losartan-
antioxidant hybrids obtained are shown in Chart 3.
A selection of the most potent antioxidant phenols are
compounds 2a-d17 (Chart 2).
Although there are different possibilities for the linkage
between losartan and the antioxidant, the simplest approach
involves an esterification reaction, and this was the initial
choice from a trityl-protected losartan prepared according to
the literature procedure.16 Attempts to carry out the esterifi-
cation reaction using some unprotected phenols failed or gave
very low yields of the corresponding ester. As a result, the
hydroxygroupsin phenols2a,b wereprotected withthe benzyl
group(Bn) and 2c wasprotectedwithtert-butyldimethylsilane
(TBDMS). The hydroxy group in phenol 2d was difficult to
protect, probably due to the steric hindrance of the two vicinal
tert-butyl groups, and this compound was used without
protection.
The esterification reaction between the protected losartan 3
and 2d and protected phenols 4a-c was attempted using
different conditions and reagents for the activation of the
carboxylic acid and the primary alcohol. The best results were
obtained using Mitsunobu conditions18 with the correspond-
ing esters 5a-d being obtained in moderate yields (Scheme 1).
Deprotection of losartan derivatives 5a-d to give the desired
esters was carried out under different conditions depending on
the protecting group. In all cases, deprotection of the tetrazole
ring was required, so compound 5d was used as a model to
establish the best conditions for removing the trityl group.
Hydrogenolysis in the presence of Pt/C (5%) afforded the best
yield for the ester 6d. Under these conditions, compound 5b
was transformed into the corresponding derivative 6b by
removal of both the benzyl and trityl protecting groups,
Results and Discussion
After the syntheses of 6 and 7, the antioxidant properties
were assessed.17 Losartan displayed minimal antioxidant
ability, which increased 4-8-fold in the synthesized com-
pounds, with 6b, 7a, and 7b showing the best antioxidant
ability (Table 1). At concentrations of 1 mmol/L, these
products display the same antioxidant ability as 1 mmol/L
of compound 2b (0.36 ( 0.03 mmol/L).
To test the ability of the synthesized compounds to block
angiotensin II in vitro, two kinds of experiments were per-
formed in cultured mesangial cells. These human cells express
angiotensin II receptors and show a contractile response in the
presence of this peptide.19 First, the binding of radiolabeled
angiotensin II was tested in the presence of the different
compounds.20 All of them inhibited the binding of the labeled
peptide. The maximum inhibition was observed with losartan
(47%), followed by 6b (41%) and 7b (40%), although the
differences observed between the different compounds were
not statistically significant (Table 1).
Second, the ability of angiotensin II toreduce theplanar cell
surface area (PCSA) in cells pretreated with the same com-
poundswas analyzed. ChangesinPCSAareconsidered tobe a
consequence of cell contraction and are due to the interaction
of angiotensin II with its receptor.20 The different compounds
tested inhibited the angiotensin II-induced PCSA reduction.
This inhibition ranged between 62% with losartan and 77%

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Garcıa et al.
7222 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Scheme 1. Synthesis of Losartan-Antioxidant Hybrids
Chart 3. Losartan-Antioxidant Hybrids Prepared
Table 1. Antioxidant Ability and Angiotensin II Binding of Losartan
Derivatives 6 and 7a
compound
antioxidant ability
Ang II binding
Losartan
0.04 ( 0.01
0.31 ( 0.03b
0.25 ( 0.03b
0.16 ( 0.02b
0.31 ( 0.04b
0.31 ( 0.04b
0.27 ( 0.03b
0.24 ( 0.02b
53 ( 8%
59 ( 7%
68 ( 7%
65 ( 10%
66 ( 9%
60 ( 6%
65 ( 11%
77 ( 11%
6b
6c
6d
7a
7b
7c
7d
a Antioxidant ability is expressed as antioxidant equivalent concen-
tration, in mmol/L, per 1 mmol/L of product. Ang II binding is
expressed as percentage of the binding of labeled angiotensin II
(100%) in the presence of the same concentration of the products
(1 μmol/L). Results are the mean ( SEM of 8 independent experiments.
b p < 0.05 vs losartan.
These results show that the attachment of an antioxidant
moiety to losartan is able to increase its antioxidant ability
without modifying its basic properties as an angiotensin II
receptor blocker. However, these in vitro results should be
confirmed by an in vivo approach. For this purpose, 6b or/and
7b were selected as representative hybrids. Wistar rats were
treated for 8 weeks with an inhibitor of the synthesis of nitric
oxide, Nω-nitro-L-arginine methyl ester hydrochloride
(L-NAME), to induce hypertension, and received losartan,
6b or 7b, for the last 4 weeks. Two additional groups of
with 7b, but no statistically significant differences were ob-
served in the inhibition of the contraction showed by the
different compounds.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7223
Table 2. Summary of the Main Biochemical Parameters for the Different Groups of Animalsa
C
L-NAME
LOS
AO (2b)
LOSþAO
6b
7b
glucose (mg/dL)
cholest (mg/dL)
triglyc (mg/dL)
Ca (mg/dL)
144 ( 5
49 ( 3
165 ( 10b
57 ( 6
149 ( 8
48 ( 1
170 ( 14b
58 ( 6
70 ( 7
8.8 ( 0.2
5.2 ( 0.4
591 ( 33
77 ( 12
24 ( 1
168 ( 14b
54 ( 3
154 ( 10
52 ( 4
149 ( 11
55 ( 2
52 ( 14
8.5 ( 0.3
4.5 ( 0.3
588 ( 20
85 ( 9
84 ( 15b
8.5 ( 0.3
4.5 ( 0.5
643 ( 4b
93 ( 12
65 ( 13
8.3 ( 0.4
5 ( 0.3
622 ( 26
91 ( 4
69 ( 11
8.5 ( 0.3
4 ( 0.4
610 ( 7
93 ( 10
22 ( 4
52 ( 12
8.6 ( 0.4
4.5 ( 0.5
561 ( 49
83 ( 4
60 ( 17
8.6 ( 0.3
4.5 ( 0.47
600 ( 12
85 ( 7
protein (mg/dL)
LDH (UI/L)
AST (UI/L)
ALT (UI/L)
28 ( 3
32 ( 5
24 ( 1
24 ( 2
25 ( 2
creatinine (mg/dL)
urea (mg/dL)
Naþ (mmol/L)
0.42 ( 0.02
25 ( 3
146 ( 3
4.4 ( 0.1
0.44 ( 0.01
29 ( 4
151 ( 4
0.42 ( 0.02
24 ( 3
151 ( 3
4.7 ( 1
0.47 ( 0.02
27 ( 1
148 ( 7
4.1 ( 1.3
0.44 ( 0.01
24 ( 3
148 ( 4
4.2 ( 0.2
0.42 ( 0.01
24 ( 5
151 ( 4
4.6 ( 0.2
0.43 ( 0.01
24 ( 4
151 ( 3
4.4 ( 0.2
K
þ (mmol/L)
4.5 ( 0.2
a C, Control; L-NAME, Nω-Nitro-L-arginine methyl ester hydrochloride; LOS, losartan; AO, Antioxidant (2b); LOSþAO, losartanþantioxidant;
Ca, calcium; LDH, lactic dehydrogenase; AST, aspartate transaminase; ALT, alanine transaminase. Results are the mean ( SEM of 10 rats. b p < 0.05 vs
control group.
animals subjected to an 8 week treatment with L-NAME were
also included, some rats that received just the antioxidant
compound 2b, and another group of rats that were cotreated
with losartan and 2b, simultaneously but separately. In these
two groups, the treatments were also administered for the last
4 weeks. All compounds were administered in equimolar
concentrations. The main biochemical parameters of the
different groups of animals are given in Table 2. Although
the nature of the molecules under investigation (i.e., losartan
and 2b) did not suggest the potential appearance of adverse
effects, this analysis was performed to evaluate the possible
toxic effect of drug administration. In short, metabolic,
hepatic, and renal parameters were studied. An 8 week
treatment with L-NAME induced a slight but statistically
significant increase in serum glucose, triglycerides, and LDH
concentrations. Similar serum glucose changes were observed
in rats treated with L-NAME plus 2b, with or without
losartan, whereas the biochemical parameters of the other
animals, those treatedwithL-NAMEpluslosartan,6band7b,
were comparable to those of control animals. These results
enabled us to rule out a toxic effect of the newly synthesized
hybrids, at least on the basis of the parameters studied.
Losartan decreased systolic blood pressure in rats treated
with L-NAME, but the values observed after 4 weeks of
treatment did not reach the control values. Similar results
were observed when the antioxidant molecule was adminis-
tered at the same time as losartan. Treatment with 6b and 7b
not only diminished systolic blood pressure but normalized it
(Figure 1A). Two macroscopic characteristics were selected to
evaluate the consequences of these changes in blood pressure,
heart weight, and aorta thickness. In the case of heart weight
(Figure 1B), losartan and 6b and 7b were able to normalize the
increased heart weight induced by L-NAME, whereas in
terms of aorta thickness (Figure 1C), the three compounds
prevented the effect of L-NAME, albeit only partially. The
administration of the antioxidant molecule did not modify
either blood pressure or aorta thickness, and only minimally
decreased the L-NAME-induced cardiac hypertrophy
(Figure 1).
differences are subtle and, in the case of heart damage, not
statistically significant.
For this reason, a more sophisticated analysis of vascular
damage was performed: the vascular content of two extra-
cellular matrix proteins, fibronectin and collagen type I, as
well as the local protein oxidative damage, measured as the
accumulation of 4-hydroxy-2-nonenal-lysine (HNE-lysine),
were evaluated in the different groups of animals. L-NAME
administration induced an increased accumulation of fibro-
nectin (Figure 2A) and collagen I (Figure 2B) as well as
increased oxidative damage (Figure 2C). Losartan slightly
but significantly decreased the collagen I content in vessel
walls, and when administered together with 2b, it also de-
creased the fibronectin content and the HNE-lysine accumu-
lation without reaching the values observed in the control
animals. The compounds 6b and 7b completely normalized
the changes induced by L-NAME in vascular walls (Figure 2).
These results, together with the changes in blood pressure,
suggest that long-term treatment with these new compounds
may prevent more efficiently the cardiovascular damage
induced by hypertension than with the use of standard
angiotensin II blockers. In addition, the ability of the new
compounds to prevent the accumulation of extracellular
matrix proteins suggests that, on a long-term basis, it is
possible that these drugs minimize significantly the structural
damage induced by hypertension in vascular walls.
In order to analyze the mechanisms responsible for the
differences observed between the different groups of animals,
we measured the blood levels of losartan, its main metabolite
EXP-3174, 2b and 6b, after chronic exposure to losartan, 2b,
losartan þ 2b, and 6b. We wanted to gain insight into the
stabilityofthe hybridtoesterases andensurethattheobserved
pharmacological effects are associated with the presence of
circulating levelsof the hybrid rather than hydrolysis products
or even the carboxy-metabolite of losartan EXP-3174 (more
potent than losartan at the AT1 receptor level). We also
wanted to compare the steady-state circulating levels of
losartan, 2b, and the hybrid after chronic administration, to
analyze the possibility that significant differences in circulat-
ing levels could explain, at least partially, the observed effects.
Before performing the analysis of plasma, we carried out a
stability study of the 6b-containing drinking water solution.
After 2 days, 6b remains mainly nonhydrolyzed (95.9%) in the
drinking water, and only a low amount of losartan was
detected (4.1%).
The analysis of the changes in blood pressure, as well as the
evaluation of heart weight and aorta thickness, suggest that6b
and 7b are at least as potent as losartan in the control of
hypertension and in the prevention of cardiovascular damage.
Moreover, a morecareful analysis of the datasuggests thatthe
two newly synthesized compounds may have an increased
antihypertensive effect and, in the case of 6b, a more marked
protective effect in heart damage than losartan. However,
Wistar rats were treated with losartan, 2b, losartan plus 2b,
and 6b for 4 weeks, plasma was obtained at sacrifice, and the

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Garcıa et al.
7224 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Figure 2. Analysis of the vascular content of two extracellular
matrix proteins: fibronectin (FN) (A) and Col I (collagen type I)
(B) and the local protein oxidative damage, measured as HNE
(accumulation of HNE-lysine) (C). Wistar rats were treated with
Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) for 8
weeks and losartan (LOS), LOSþAO, 6b, and 7b were administered
for the last 4 weeks. Representative Western blots and densitometric
analysis from 10 separate experiments are shown. Results are
expressed as percent of the basal value, and they are the mean
SEM of the densiometric values. *p < 0.05 vs control. **p < 0.05 vs
L-NAME.
Figure 1. Changes in systolic blood pressure (A); heart weight (B);
aorta thickness (C). Wistar rats were treated with Nω-nitro-L-
arginine methyl ester hydrochloride (L-NAME) for 8 weeks and
Losartan (LOS), the antioxidant compound 2b (AO), LOSþAO, 6b,
and 7b were administered for the last 4 weeks. In the cases of heart
weight, values were corrected to the animal weight. Results are
mean ( SEM of 10 rats. * p < 0.05 vs control. **p < 0.05 vs vehicle.
levels of losartan, EXP-3174, 2b, and 6b were measured. Two
different plasma samples from each animal were analyzed
with basically identical results. A significant amount of 6b
(90.3-93.5%) was detected in plasma, with only minimal
amounts of losartan (3.1-3.3%) and EXP-3174 (3.3-6.6%)
(see Supporting Information), thus supporting the high meta-
bolic stability of the ester linkage in 6b and the low levels of
circulating losartan and EXP-3174 after the oral exposure of
Wistar rats to 6b. Moreover, total plasma concentrations of
antihypertensive molecules, including losartan, EXP-3174,
and 6b, were higher in the animals receiving the 6b compound
than in the other groups (Table 3). No significant amounts of
2b were detected in plasma samples.
The high concentration of angiotensin II blocking mole-
cules detected after 6b administration was probably respon-
sible for the complete normalization of blood pressure in these
animals. Even in these conditions, no significantly hypoten-
sive effect was demonstrated. Moreover, these high levels of
antihypertensive molecules, togetherwiththe presenceof6bin

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7225
Table 3. Concentration (nM) of Losartan, EXP-3174 and 6b in Rat
Plasma Samples from the Different Groups of Ratsa
residue was dissolved in MeOH (0.35 M in relation to starting
material) and treated with 2.3 M NaOH (1.2 equiv), and the
mixture was heated at reflux for 1 h. Solvents were evaporated
under reduced pressure, and the residue was dissolved in
water and extracted with Et2O. Aqueous phase was made acidic
(pH 1-2) with 1 M HCl, extracted with EtOAc, and the organic
extracts dried over MgSO4. After filtering and removing solvent,
4a-c were obtained as white solids.
General Procedure for the Synthesis of 5a-d. A solution of 3
(0.025 M) and PPh3 (0.025 M) in Et2O was added dropwise over
a solution of 4a-d (0.025 M) in Et2O under an argon atmo-
sphere. The mixture was stirred at room temperature for 12 h,
OPPh3 was filtered off and the solvent was evaporated under
reduced pressure. Residue was chromatographed on silica gel
using hexane/EtOAc 1:1 to give 5a-d as yellowish oils.
General Procedure for the Catalytic Hydrogenation of 5a,b,d
and 8c. Catalyst was added over a solution of 5a,b,d or 8c in the
corresponding solvent, and the reactor was flushed with argon
for 10 min. Then, reaction mixture was kept under a hydrogen
atmosphere at 1 atm and stirred at room temperature for the
time indicated in each case. Mixture was filtered through Celite,
and solvent was evaporated under reduced pressure. Residue
was chromatographed on silica gel using the more convenient
eluent in each case to give 6b-d or 7a-d.
groups
losartan (n = 4) losartan þ 2b (n = 4)
6b (n = 6)
losartan
EXP-3174
6b
24.4 ( 2.6
39.0 ( 5.2
---;
11.1 ( 1.4
19.4 ( 2.3
---;
8.0 ( 1.1
13.0 ( 1.3
171.0 ( 16.4
192.0 ( 20.1
total
63.4 ( 5.3
30.5 ( 4.2
a Each data represents the mean ( SEM.
the circulation, a substance with antioxidant properties, could
explain the decreased vascular content in extracelular matrix
proteins and HNE-lysine observed in the 6b-treated animals.
It would be expected that the inhibition in the HNE-lysine
content, the selected index of oxidative tissue damage, would
be similar when comparing administration of losartan þ 2b
with 6b or 7b treatments, but this was not the case. As
previously mentioned, the circulating levels of angiotensin II
blocking molecules were higher in the 6b treated animals than
intheratsreceivinglosartanþ 2b, andangiotensinIIblockade
may diminish oxidative stress.21 Moreover, the circulating
levels of 2b were undetectable after oral administration,
probably as a consequence of its rapid removal from the
circulation,22 and this fact may also contribute to a lower
antioxidant effect. Finally, the observed differences in the
antioxidant effect could be also explained by the fact that
HNE lysinewas only analyzed invascularwalls, a tissuewitha
higherAngIIreceptor content, towhichthe newlysynthesized
molecules could gain access more specifically.
3-(3,4-Dihydroxy-phenyl)propionic Acid 2-butyl-5-chloro-
3-[20-(2H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazol-4-yl-
methyl ester (6b). Pt/C (5%) (1.1 equiv/protecting group), 5b
(1.3 ꢀ 10-2 M; MeOH/EtOAc 4:3), 48 min, eluent EtOAc/
MeOH/AcOH (60:20:5), Rf
0.63, oil,
EtOAc/MeOH/AcOH60:20:5
yield 10%. IR (KBr) [cm-1] 2957, 2361, 1732, 1604, 1528,
1460, 1352, 1259, 1146, 1115, 956, 816, 759, 667, 634. 1H
NMR (200 MHz, CD3OD) δ 7.72-7.36 (m, 4H), 7.11 (d, J =
8.2 Hz, 2H), 6.87 (d, J = 8.5 Hz, 2H), 6.71-6.53 (m, 3H), 5.08 (s,
2H), 5.00 (s, 2H), 2.78 (t, J = 7.2 Hz, 2H), 2.47 (t, J = 7.8 Hz,
2H), 2.34 (t, J = 7.7 Hz, 2H), 1.58 (quin, 2H, J = 7.7 Hz), 1.39
(hex, 2H, J = 7.2 Hz), 0.90 (t, J = 7.2 Hz, 3H). 13C NMR (50
MHz, CD3OD) δ 175.1, 157.0, 150.7, 147.1, 147.0, 142.6, 140.5,
136.5, 132.2, 131.6, 131.5, 130.7, 129.9, 128.9, 126.9, 124.8,
124.4, 123.1, 122.1, 121.9, 118.0, 78.4, 73.4, 62.2, 55.7, 32.0,
30.9, 27.3, 26.0, 23.3, 22.0, 14.0, 12.9, 12.1, 11.9. ES-MS m/z: 587
(Mþ1). Anal. Calcd. for C55H53N6O4Cl: C (63.42), H (5.32), N
(10.90); found C (63.53), H (5.21), N (11.04).
In summary, the results provide in vitro evidence that these
losartan/antioxidant hybrids retain the ability to block
the angiotensin II effect, with increased antioxidant ability.
In hypertensive rats, the compounds show properties that
suggest they may be more useful than losartan to control
hypertension and to prevent the hypertension-induced cardi-
ovascular damage.
Experimental Section
3-(3,4-Dihydroxy-phenyl)-propionic Acid 2-Butyl-3-[20-(2H-
tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazol-4-ylmethyl Es-
ter (7b). Pd/C (30%) (0.76 equiv/protecting group), 5b (3.3 ꢀ
10-2 M; MeOH/CHCl3 2:1), 4 h, eluent MeOH/EtOAc 1:5, Rf
General Chemical Methods. Melting points were determined
with a Gallenkamp apparatus and are uncorrected. H NMR
1
and 13C NMR spectra were recorded at 200 and 50 MHz,
respectively, on a Varian Gemini 200. Chemical shifts are given
in ppm relative to solvent. Signals are quoted as singlet (s), broad
singlet (bs), doublet (d), triplet (t), quartet (q), quintet (quin),
hexaplet (hex), and multiplet (m). Coupling constants (J) are
given in hertz (Hz). MS were recorded on a Hewlett-Packard
5988A or a Hewlett-Packard 1100MSD mass spectrometers.
Elemental analyses were performed on a Heraeus CHN rapid
analyzer. FTIR spectra were recorded on a Perkin-Elmer FTIR
1725X spectrophotometer. Reagents and solvents were ob-
tained from commercial sources and used without further
purification. TLC was carried out on Alugram Sil G/UV254
silica gel plates. Preparative gravity column chromatography
was performed on Merck silica gel. Reported yields are not
optimized. Purities of the hybrids and new compounds were
determined by elemental analysis, which indicated >95% pur-
ity of each product (see Supporting Information).
0.3, oil, yield 46%. IR (KBr) [cm-1] 3441, 3137,
EtOAc/MeOH5:1
3058, 2958, 2931, 2871, 2505, 1941, 1736, 1603, 1519, 1458, 1354,
1283, 1143, 1114, 1005, 966, 819, 784, 759, 635. 1H NMR (200
MHz, CD3OD) δ 7.58-7.31 (m, 4H), 7.10 (d, J = 7.1 Hz, 3H),
6.80 (d, J = 8.2 Hz, 3H), 6.71-6.59 (m, 2H), 6.40 (dd, J = 2.0
Hz, J = 8.2 Hz, 1H), 5.08 (s, 2H), 5.00 (s, 2H), 2.66 (t, J = 7.4
Hz, 2H), 2.64 (t, J = 7.4 Hz, 2H), 2.34 (t, 2H, J = 7.3 Hz), 1.58
(quin, 2H, J = 7.5 Hz), 1.31 (hex, 2H, J = 7.2 Hz), 0.89 (t, 3H,
J = 7.1 Hz). 13C NMR (50 MHz, CD3OD) δ 173.9, 162.0, 151.8,
146.0, 144.4, 142.4, 141.8, 135.9, 133.0, 131.6, 131.1, 130.6,
130.2, 130.1, 128.4, 128.1, 127.8, 126.5, 120.3, 116.3, 116.2,
56.1, 36.7, 31.2, 30.7, 27.2, 23.3, 14.0 . ES-MS m/z: 553
(Mþ1). Anal. Calcd. for C31H32N6O4: C (67.37), H (5.83), N
(15.20); found C (67.42), H (5.61), N (15.06).
Pharmacology. Experimental in Vitro and in Vivo Methods.
Measurement of the Total Antioxidant Ability. The antioxidant
ability of the different compounds was evaluated with a com-
mercial kit (Total Antioxidant Status Assay Kit, Calbiochem,
La Jolla, CA).17 The assay relies on the ability of antioxidants
to inhibit the oxidation of a chromogen (ABTS) by metmyoglo-
bin and hydrogen peroxide. Oxidation is monitored by reading
the absorbance at 600 nm. The antioxidant ability is propor-
tional to the reduction of the absorbance, and it is expressed
as mM.
Chemistry. General Procedure for the Synthesis of 4a-b.
Benzyl bromide (1.5 equiv/hydroxy or carboxy group) and
K2CO3 (2.0 equiv/hydroxy or carboxy group) were added over
a solution of 2a-c (0.55 M) in DMF under an argon atmosphere
and heated at 60 °C for 20 h. The reaction mixture was cooled at
room temperature and filtered, and the solvent was removed
under reduced pressure. Residue was dissolved in EtOAc and
washed with 1 M HCl and brine and dried over MgSO4. After
filtering, solvent was removed under reduced pressure, the

´
Garcıa et al.
7226 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22
Experimental Studies in Cells. Vascular smooth muscle cells
(VSMC) were obtained from thoracic aortas of Wistar rats by
methods described previously.20 Briefly, thoracic aortas from
Wistar rats were removed, cleaned, dissected into small strips,
and incubated in DMEM/Ham0s F-12 medium (Biowhittaker,
Walkersville, MD) with collagenase type IV at 37 °C for 45 min.
The digested strips were seeded onto dishes and maintained in
10 mL of DMEM/Ham0s F-12 medium with 10% fetal calf
serum (Biowhittaker), at 37 °C, in a humidified atmosphere of
5% CO2. Culture media were changed every 3 days. The primary
cultures on days 20 to 22 were passaged by trypsinization
(trypsin-EDTA). The cells were used between the third and fifth
passages.
Angiotensin II binding experiments were performed to assess
the ability of the different compounds to block the angiotensin
II receptor. For this purpose, VSMC were gently removed from
the dishes and resuspended in assay buffer (20 mM Tris-HCl,
5 mM glucose, 130 mM NaCl, 5 mM KCl, 10 mM sodium
acetate, pH 7.4), at a protein concentration of 0.5 μg/μL.
Binding experiments were performed by incubating 0.15 nM
125I-Angiotensin II (Amersham International, Buckingham-
shire, UK) with 125 μg of cell protein, in the presence of losartan
or the different synthesized compounds, for 45 min. The free
radioactivity was separated from the bound radioactivity by
centrifugation at 11 000 g for 2 min, and the resultant pellet was
washed three times with ice-cold 0.15 M NaCl. The radioactivity
was counted in a Kontron gamma counter (Kontron Instru-
ments AG, Zurich, Switzerland). Nonspecific binding was con-
trolled by incubating some samples with an excess of angiotensin
II (10-4 M).20
The ability of the different compounds to block angiotensin II-
induced cell contraction was also tested. This analysis was
performed by studying the changes in planar cell surface area
(PCSA) under different experimental conditions.20 In every
experiment, cells were washed and placed in Tris-glucose buffer
(20 mM Tris, 130 mM NaCl, 5 mM KCl, 10 mM sodium acetate,
and 5 mMglucose, pH 7.45) containing 2.5mMCaCl2. Cells were
incubated with 1 μM angiotensin II, in the presence of losartan or
the different synthesized compounds, for 30 min. Photographs
(TMS-F Photomicroscope, Nikon, Tokyo, Japan, magnification
150ꢀ) of the same cells were taken at times 0 and 30 min. PCSA
was determined by computer-aided planimetric techniques.
Animal Studies. Two-month-old male Wistar rats were
treated with Nω-nitro-L-arginine methyl ester hydrochloride
(L-NAME) in the drinking water (20 mg/kg/day). Four weeks
after starting L-NAME, some rats were treated with losartan
(20 mg/kg/day), 2b (10 mg/kg/day), losartan plus 2b (20 mg/kg/
day plus 10 mg/kg/day), 6b (28 mg/kg/day), and 7b (30 mg/kg/
day). These treatments were administered daily, for a controlled
period of time (12.00-18.00 h), also in drinking water. Ten
animals were included in each experimental group. Arterial
pressure was monitored in conscious animals with a tail cuff
sphygmomanometer (LE 5001 Pressure Meter, Letica Scientific
Instruments, Hospitalet, Spain).23 After 8 weeks, animals were
sacrificed under halothane anesthesia, between 9.00 and 10.00 h.
Blood samples were taken, and heart and aorta removed. Blood
was centrifuged and serum stored until analysis. Heart was
weighed. A portion of descending thoracic aorta was transferred
to ice-cold Hank0s, cleaned and flushed with 4% formaldehyde
solution. Then, it was cut into 2-3 mm rings, fixed on slides, and
photographed. Media thickness was measured by computer-
aided planimetric techniques.
Jolla, CA) or rabbit anti HNE-lysine polyclonal antibody
(Chemicon). Membranes were washed, incubated with the
secondary antibody conjugated with horseradish peroxide
(goat antirabbit IgG; Bio-Rad Laboratories, Richmond, CA)
for 30 min, and washed extensively. Blots were developed by
chemiluminescence using the ECL Western blotting system
(Pharmacia-Biotech, Uppsala, Sweden). Densitometric analysis
was performed with appropriate software (NIH Image 1.55;
Bethesda). Blots were stripped and reblotted with an antibody
against β-tubulin, as a control of charge.24
Blood Level Determination of Losartan, Losartan Metabolites,
2b, and 6b after Chronic Exposure to Drugs. Additional groups
of animals were treated for 4 weeks with losartan (n = 4), 2b
(n = 4), losartan plus 2b (n = 4), and 6b (n = 6), sacrificed, and
blood was obtained. The administration of the treatments and
the sacrifice were performed as previously reported. Plasma
circulating levels of the drugs were analyzed using HPLC-MS/
MS (QqQ). For the blood level determination of losartan, EXP-
3174 and 6b, SPE-C18 cartridges were conditioned with MeOH
and equilibrated with 2% formic acid. A plasma aliquot (100 μL)
was diluted up to 1 mL with 2% formic acid and applied on
the cartridge, washed with MeOH/water 1:1 (1 mL) and eluted
with MeOH (1 mL). Aliquots (2 μL) from the eluting fraction
were taken and injected in a HPLC-MS-QqQ instrument
(HPLC model 1200 coupled through an orthogonal electrospray
interface model G1607A to a triple quadrupole model 6410 all of
them from Agilent Technologies) using an Ascentis Express
column (25 mm ꢀ 2.1 mm i.d.; 2.7 μm from Supelco) and an
isocratic mobile phase (25% MeOH with 0.1% v/v formic acid
plus 45% water with 0.1% v/v formic acid; 0.5 mL/min) at 40 °C.
For the blood level determination of 2b and its metabolites, a
plasma aliquot (100 μL) was diluted with 0.1 mM sodium
acetate buffer (pH = 5, 100 μL) and 200 mM HCl/MeOH
(200 μL). The mixture was vortexed and ultrasonicated (5 min).
Then, the homogenized sample was extracted with EtOAc (6 ꢀ
600 μL). For each extraction, the sample was shaken for 7 min,
centrifuged (6200g) for 5 min, and phases were separated.
Organic extracts were pooled and dried under N2. Dried extracts
were diluted with 15% MeCN/H2O (100 μL). Aliquots (5 μL)
were taken and injected in a HPLC-MS-QqQ instrument
(see above) using a C18 Tripple End-capping LUNA column
(250 mm ꢀ 4.6 mm i.d.; 5 μm Phenomenex) and a gradient
(A, water with 0.1% v/v formic acid; B, MeOH with 0.1%
v/v formic acid; starting mobile phase, 75% A plus 25% B;
0.5 mL/min) at 25 °C.
Statistical Analysis. In every case, data shown are the mean (
SEM of a variable number of experiments (see figure legends),
and in some cases, they are expressed as percentages of the
control values. Because the number of data points in each
distribution was never over 10, nonparametric statistics was
selected to compare the different groups of results. p values <
0.05 were considered statistically significant.
Acknowledgment. This work was supported by grants
ꢀ
from the Ministerio de Educacion y Ciencia (projects
CTQ2008/04313 and SAFF 2007-623471), Instituto de Salud
ꢀ
Carlos III (Red de Investigacion Renal, (REDinREN),
RD06/0016/0016 and PI 070695)) and IRSIN.
Supporting Information Available: Chemical experimental
procedures for the synthesis and fully characterization of com-
pounds 4-7. HPLC chromatograms from 6b-containing dink-
ing water and plasma samples of rats treated with 6b, losartan
and losartan þ 2b. This material is available free of charge via
the Internet at http://pubs.acs.org.
Serum biochemical parameters were measured with an auto-
matic analyzer (Hitachi 717, Boehringer Mannheim, Man-
nheim, Germany). Vascular samples were lysed in protein lysis
buffer, and total extract proteins (μg/lane) were size-fractio-
nated by SDS-PAGE and transferred to polyvinylidene difluor-
ide membranes. After blocking these membranes with 5%
nonfat milk for 1 h, they were incubated with rabbit anticollagen
type I or antifibronectin monoclonal antibodies (Chemicon, La
References
(1) Wexler, R. R.; Greenlee, W. J.; Irvin, J. D.; Goldberg, M. R.;
Prendergast, K.; Smith, R. D.; Timmermans, P. B. M. W. M.

Article
Nonpeptide Angiotensin II Receptor Antagonists: The Next
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 22 7227
(13) Bartosz, M.; Kedziora, J.; Bartosz, G. Antioxidant and Prooxidant
Properties of Captopril and Enalapril. Free Radic. Biol. Med. 1997,
23, 729–735.
Generation in Antihypertensive Therapy. J. Med. Chem. 1996,
39, 625–656.
(2) Daugherty, A.; Cassis, L. Angiotensin II-Mediated Development
of Vascular Diseases. Trends Cardiovasc. Med. 2004, 14, 117–120.
(3) For a recent example, see: Breschi, M. C.; Calderone, V.;
Digiacomo, M.; Macchia, M.; Martelli, A.; Martinotti, E.;
Minutolo, F.; Rapposelli, S.; Rossello, A.; Testai, L.; Balsano, A.
New NO-Releasing Pharmacodynamic Hybrids of Losartan and
Its Active Metabolite: Design, Synthesis, and Biopharmacological
Properties. J. Med. Chem. 2006, 49, 2628–2639.
(14) Aslam, S.; Santha, T.; Leone, A.; Wilcox, C. Effects of Amlodipine
and Valsartan on Oxidative Stress and Plasma Methylarginines in
End-Stage Renal Disease Patients on Hemodialysis. Kidney Int.
2006, 70, 2109–21015.
(15) Wu, T. C.; Chen, Y. H.; Leu, H. B.; Chen, Y. L.; Lin, F. Y.; Lin, S. J.;
Chen, J. W. Carvedilol, a pharmacological Antioxidant, Inhi-
bits Neointimal Matrix Metalloproteinase-2 and -9 in Experimen-
tal Atherosclerosis. Free Radic. Biol. Med. 2007, 43, 1508–15022.
(16) Carini, D. J.; Duncia, J. V.; Aldrich, P. E.; Chiu, A. T.; Johnson, A.
L.; Pierce, M. E.; Price, W. A.; Santella, J. B., III; Wells, G. J.;
Wexler, R. R.; Wong, P. C.; Yoo, S.-E.; Timmermans, P. B. M.W.
M. Nonpeptide Angiotensin II Receptor Antagonists: The
Discovery of a Series of N-(Biphenylylmethyl)Imidazoles as Po-
tent, Orally Active Antihypertensives. J. Med. Chem. 1991, 34,
2525–2547.
(4) Stocker, R.; Keaney, J. J. Role of Oxidative Modifications in
Atherosclerosis. Physiol. Rev. 2004, 84, 1381–478.
(5) Rajagopalan, S.; Meng, X. P.; Ramasamy, S.; Harrison, D. G.;
Galis, Z. S. Reactive Oxygen Species Produced by Macrophage-
Derived Foam Cells Regulate the Activity of Vascular Matrix
Metalloproteinases in Vitro. Implications for Atherosclerotic Pla-
que Stability. J. Clin. Invest. 1996, 98, 2572–2579.
(6) Ushio-Fukai, M.; Alexander, R. W.; Akers, M.; Yin, Q.; Fujio, Y.;
(17) The antioxidant activity was carried out by using the kit “Total
Antioxidant Status Assay”, Miller, N. J.; Rice-Evans, C.; Davies,
M. J.; Gopinathan, V.; Milner, A. A Novel Method for Measuring
Antioxidant Capacity and Its Application to Monitoring the
Antioxidant Status in Premature Neonates. Clin. Sci. 1993, 84,
407–412.
(18) Mitsunobu, O. The Use of Diethyl Azodicarboxylate and Triphe-
nylphosphine in Synthesis and Transformation of Natural Pro-
ducts. Synthesis 1981, 1–28.
Walsh, K.; Griendling, K. K. Reactive Oxygen Species Mediate
the Activation of Akt/Protein Iinase B by Angiotensin II
in Vascular Smooth Muscle Cells. J. Biol. Chem. 1999, 274,
22699–22704.
(7) Uemura, S.; Matsushita, H.; Li, W.; Glassford, A. J.; Asagami, T.;
Lee, K. H.; Harrison, D. G.; Tsao, P. S. Diabetes Mellitus
Enhances Vascular Matrix Metalloproteinase Activity: Role of
Oxidative Stress. Circ. Res. 2001, 88, 1291–1298.
(8) Zmijewski, J. W.; Moellering, D. R.; Le Goffe, C.; Landar, A.;
Ramachandran, A.; Darley-Usmar, V. M. Oxidized LDL Induces
Mitochondrially Associated Reactive Oxygen/Nitrogen Species
Formation in Endothelial Cells. Am. Physiol. Heart Circ. Physiol.
2005, 2, 852–861.
(9) Cai, H.; Harrison, D. G. Endothelial Dysfunction in Cardiovas-
cular Diseases: The Role of Oxidant Stress. Circ. Res. 2000, 87,
840–844.
(10) Li, P. F.; Dietz, R.; von Harsdorf, R. Differential Effect of
Hydrogen Peroxide and Superoxide Anion on Apoptosis and
Proliferation of Vascular Smooth Muscle Cells. Circulation 1997,
96, 3602–3609.
(11) Locher, R.; Brandes, R. P.; Vetter, W.; Barton, M. Native LDL
Induces Proliferation of Human Vascular Smooth Muscle Cells via
Redox-Mediated Activation of ERK 1/2 Mitogen-Activated Pro-
tein Kinases. Hypertension 2002, 39, 645–650.
(19) Ardaillou, R.; Chansel, D.; Chatziantoniou, C.; Dussaule, J. C.
Mesangial AT1 Receptors: Expression, Signaling, and Regulation.
J. Am. Soc. Nephrol. 1999, 11, S40–46.
ꢀ
(20) Torrecillas, G.; Boyano-Adanez, M. C.; Medina, J.; Parra, T.;
ꢀ
Griera, M.; Lopez Ongil, S.; Arilla, E.; Rodrıguez Puyol, M.;
Rodrıguez Puyol, D. Role of Hydrogen Peroxide in the Contractile
Response to Angiotensin II. Mol. Pharmacol. 2001, 59, 104–112.
(21) Schmieder, R. E.; Hilgers, K. F.; Schlaich, M. P.; Schmidt, B. M.
Renin-Angiotensin System and Cardiovascular Risk. Lancet 2007,
369, 1208–1219.
(22) Poquet, L.; Clifford, M. N.; Williamson, G. Investigation of the
Metabolic Fate of Dihydrocaffeic Acid. Biochem. Pharmacol. 2008,
75, 1218–1229.
ꢀ
(23) Perez-Rivero, G.; Ruiz-Torres, M. P.; Rivas-Elena, J. V.; Jerkic,
ꢀ
M.; Dıez-Marques, M. L.; Lopez-Novoa, J. M.; Blasco, M. A.;
Rodrıguez-Puyol, D. Mice Deficient in Telomerase Activity
Develop Hypertension Because of an Excess of Endothelin
Production. Circulation 2006, 114, 309–317.
(12) Okada, H.; Woodcock-Mitchell, J.; Mitchell, J.; Sakamoto, T.;
Marutsuka, K.; Sobel, B. E.; Fujii, S. Induction of Plasminogen
Activator Inhibitor Type 1 and Type 1 Collagen Expression in Rat
Cardiac Microvascular Endothelial Cells by Interleukin-1 and Its
Dependence on Oxygen-Centered Free Radicals. Circulation 1998,
97, 2175–2182.
(24) Cruz, C. I.; Ruiz-Torres, P.; del Moral, R. G.; Rodrıguez-Puyol,
M.; Rodrıguez-Puyol, D. Age-Related Progressive Renal Fibrosis
in Rats and Its Prevention with ACE Inhibitors and Taurine. Am.
J. Physiol. Renal Physiol. 2000, 278, F122–129.