New losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual drugs: Ester, amide and amine linkers

Article abstract of DOI:10.1016/j.ejmech.2012.01.043

We report new examples of a series of losartan-hydrocaffeic hybrids that bear novel ester, amide and amine linkers. These compounds were made by linking hydrocaffeic acid to the side chain of losartan at the C-5 position of the imidazole ring through different strategies. Experiments performed in cultured cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect and have increased antioxidant ability. Most of them reduced arterial pressure in rats better or as much as losartan.

Full text of DOI:10.1016/j.ejmech.2012.01.043

European Journal of Medicinal Chemistry 50 (2012) 90e101
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
New losartan-hydrocaffeic acid hybrids as antihypertensive-antioxidant dual
drugs: Ester, amide and amine linkers
,
,d
Gonzalo García a, Isabel Serrano b d, Patricia Sánchez-Alonso a, Manuel Rodríguez-Puyol b
,
Ramón Alajarín a , Mercedes Griera d, Juan J. Vaquero a , Diego Rodríguez-Puyol
**
,
,
b,
,
c,d
*
*
Julio Álvarez-Builla a, María L. Díez-Marqués b
,d,
a Departamento de Química Orgánica, Facultad de Química, Universidad de Alcalá, Campus Universitario, 28871-Alcalá de Henares, Madrid, Spain
b Departamento de Fisiología, Facultad de Medicina, Universidad de Alcalá, Campus Universitario, 28871-Alcalá de Henares, Madrid, Spain
c Hospital Universitario Príncipe de Asturias, Campus Universitario, 28871-Alcalá de Henares, Madrid, Spain
d Instituto Reina Sofía de Investigación Nefrológica, Madrid, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
We report new examples of a series of losartanehydrocaffeic hybrids that bear novel ester, amide and
amine linkers. These compounds were made by linking hydrocaffeic acid to the side chain of losartan at
the C-5 position of the imidazole ring through different strategies. Experiments performed in cultured
cells demonstrate that these new hybrids retain the ability to block the angiotensin II effect and have
increased antioxidant ability. Most of them reduced arterial pressure in rats better or as much as losartan.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Received 3 June 2011
Received in revised form
5 December 2011
Accepted 22 January 2012
Available online 30 January 2012
Keywords:
Antihypertensive
Antioxidant
Hybrid drug
Hydrocaffeic acid
Losartan
1. Introduction
Angiotensin converting enzyme (ACE) inhibitors have been
widely used in hypertension therapy. However, in the past two
Drug hybridization strategies are valuable tools in the discovery of
new drugs with either improved affinity for one bioreceptor or dual
action on more than one bioreceptor related with a multifactorial
disease [1,2]. The hybridization approach has been applied, for
instance, to combat drug-resistant bacteria [3,4] or parasites [5] and in
the developmentof hybrid anticancer [6] orcardiovascular [7,8] drugs.
Antihypertensive drugs exert their main protective mechanism
through lowering blood pressure, although the beneficial effects of
these drugs can also be explained in conjunction with other possi-
bilities. A relevant mechanism of cardiovascular disease progression
in hypertensive patients may also be increased oxidative stress at
the vascular level. Thus, treatments that interfere with this oxidative
stress could be useful tools for management of these individuals.
decades, ACE inhibitors have been partially displaced by antago-
nists of angiotensin II, mainly because the latter are free of the side
effects (mainly coughing) associated with ACE inhibitors. The
angiotensin II antagonists (sartans), of which losartan (Fig. 1) [9] is
the first marketed drug of this type, exert their antihypertensive
effect by blocking the angiotensin II AT1 receptor [10].
Lowering blood pressure is not the only beneficial effect of
angiotensin II blockade-based antihypertensives in the prevention
of cardiovascular morbidity and mortality. Angiotensin II plays
a significant role in the progression of tissue damage in cardio-
vascular diseases [11]. In this context, some chemical interventions
have been performed on sartans in order to increase their ability to
prevent tissue damage in the cardiovascular system while retaining
their antihypertensive potency [12].
It is well established that oxidative stress is a mechanism that
leads to the development of vascular damage [13]. The harmful
effects of the increased local synthesis of reactive oxygen species is
a consequence, at least partially, of different pathogenic stimuli
involved in cardiovascular diseases, such as activated macrophages,
hyperglycaemia, oxidized low density lipoprotein (LDL), and even
* Corresponding authors. Departamento de Química Orgánica, Edificio de Farm-
acia, Universidad de Alcalá, Campus Universitario, 28871-Alcalá de Henares,
Madrid, Spain. Tel.: þ34918854622; fax: þ34918854686.
** Corresponding author. Departamento de Fisiología, Facultad de Medicina,
Universidad de Alcalá, Campus Universitario, 28871-Alcalá de Henares, Madrid, Spain.
Tel.: þ34918854551; fax: þ34918854590.
E-mail address: ramon.alajarin@uah.es (R. Alajarín).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.043

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
91
OH
OH
Cl
OH
OH
OH
N
HO
OH
OH
OH
Bu
R
N
N
N
NH
N
HO
HO
HO
O
R
4b
4c
O
4a
O
N
O
Bu
N
O
H2O
H2O2
OH
N
N
NH
R = CH2OH; Losartan (1)
R = CO2H; EXP-3173 (5)
N
OMe
O
.
Ferryl myoglobin[Fe4+ =O]
O
H-Metmyoglobin-Fe3+
4d
HO
OH
2: R = Cl
3: R = H
CMe3
OH
HO
Hydrocaffeic acid (4a)
.-
-
ABTS2
ABTS
(blue/green)
O
(colorless)
HO
HO
CMe3
Fig. 1. Dual antihypertensive-antioxidant hybrids 2 and 3.
4e
.
+
AO
AO-H
angiotensin II [14e17]. These oxygen-active metabolites induce
significant endothelial dysfunction [18e20] and are able to modify
the normal balance between proliferation, apoptosis, and extra-
cellular matrix synthesis in heart and arterial walls [21e23]. Some
beneficial effects of antihypertensive molecules have been attrib-
uted, at least in part, to their antioxidant ability [24e26].
Me
OH
CMe3
OMe
OH
OH
HO
HO
O
Me
CMe3
OMe
Me
O
Me
O
O
4f
4g
4h
We recently reported a hybridization approach to dual drugs by
linking the antihypertensive losartan (1) to antioxidant phenols
[27]. The novel losartan-antioxidant hybrids 2 and 3 (Fig. 1), which
were made by adding hydrocaffeic acid (4a) to the hydroxymethyl
side chain of 1, retain the ability to block the angiotensin II effect
and have increased antioxidant ability. With this strategy, we tried
to block the angiotensin II receptor and to deliver significant
amounts of antioxidant molecules to the vascular walls, simulta-
neously. In hypertensive rats, compound 2 showed properties that
suggest it may be more useful than 1 for controlling hypertension
and cardiovascular damage. In addition, it prevented oxidative
vascular damage more effectively than an equimolar mixture of 1
and the antioxidant fragment 4a. These beneficial effects were due
to the presence of significant amounts of compound 2 in plasma
(90.3e93.5% after oral administration), since it was stable to plas-
matic hydrolases after a 4-week treatment.
Fig. 2. Metmyoglobin/ABTS antioxidant assay (AO-H ¼ antioxidant) and structures of
phenols tested.
intensity decreases proportionally. The antioxidant ability is
expressed as TEAC (Trolox Equivalent Antioxidant Capacity), that is,
the antioxidant equivalent concentration in mM that gives the
same percentage change of absorbance of the ABTS.- as that of
1
mM of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-
carboxylic acid, 4h). Antioxidant capacity may also be expressed
as ABTS inhibition percentage [31].
A heriarchical antioxidant potency order could be established
for the phenols tested (Table 1): 4a
> 4g z 4e z 4h
(reference) > 4b z 4d > 4f z 4c z 1. Antioxidant capacity of some
of these compounds was previously reported using a method
slightly different [32]. Antioxidant capacity test showed that the
best phenols were hydrocaffeic acid (4a), syringic acid (4g), and
3,5-di-tert-butyl-4-hydroxyphenyl acetic acid (4e). Phenols tested
were 6e7-fold more potent than losartan (1), being 4a the most
potent antioxidant phenol assayed. Then, 4a was selected to be
linked to losartan through different linkers and was compared to 1
and our previously reported hybrid 2. For each of the hybrids the
number of atoms in the spacer between the phenolic and imidazole
rings was the same than that of 2.
Following on from our studies on the role of the linker on the
activity, herein we report different strategies for the incorporation
of new ester, amide and amine linkers between the losartan and the
antioxidant moiety to generate new hybrids. These new losartan
analogues have been evaluated by their ability to block the angio-
tensin II effect and their antioxidant and antihypertensive activities.
2. Results and discussion
2.1.1. Structureeactivity relationship (SAR) studies
2.1. Antioxidant activity of phenols
Some correlations are evident between the antioxidant activity
measured and the type of spacer linking the aromatic ring to the
carboxylic acid group or the type of substituent on ortho-position to
The antioxidant ability of an array of 8 phenols was performed
using the ABTS method and compared with losartan as we reported
previously (Fig. 2) [27]. This is an electron transfer-based antioxi-
dant capacity assay [28] first reported by Miller and Rice-Evans [29]
and later improved [30]. The principle of this antioxidant assay is
the formation of a ferryl myoglobin radical from metmyoglobin and
hydrogen peroxide. The ferryl myoglobin radical can oxidize
2,20-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to
generate a radical anion ABTS.- (since sulfonic acid groups are fully
deprotonated), a soluble chromogen that is blue/green in colour
and can be determined spectrophotometrically at 600 nm. Anti-
oxidant suppress this reaction by electron donation radical scav-
enging and inhibit the formation of the coloured radical anion
Table 1
Antioxidant capacity assay for phenols 4a-g and losartan.
Compound
TEAC (mM, n ¼ 3)a
4a
4b
4c
4d
4e
4f
4g
1
1.274 ꢂ 0.069
0.200 ꢂ 0.099
0.153 ꢂ 0.085
0.202 ꢂ 0.014
1.058 ꢂ 0.115
0.181 ꢂ 0.059
1.067 ꢂ 0.141
0.179 ꢂ 0.013
ꢁ
a
ABTSꢀ in a concentration dependent manner and the colour
Trolox (4h) as reference standard (see Experimental).

92
G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
the hydroxyl group responsible for the activity (on the para-position
to the spacer). The presence of a methylene group linked to the
phenol ring seems to enhance the radical scavenging properties
against ABST.- (4a vs 4b and 4c; 4e vs 4f). Also, a double-bond
resulted more active than if there is not a spacer (4b vs 4c).
Regarding to the type of substituent, a methoxy group is slightly
better than a hydroxyl group (4d vs 4c) and much better than
a t-butyl group (4g vs 4f).
bromide and the product was reduced with LAH to give alcohol 6
(56%). On the other hand, aldehyde 7 was converted to ester 8 (86%)
in a one-pot three-step process involving cyanohydrin formation,
oxidation and alcoholysis [33]. Ester 8 was then hydrolyzed in
a basic medium to give acid 9 (78%). Alcohol 6 and acid 9 were
linked in a Mitsunobu reaction to yield ester 10 (68%) [34].
Compound 10 was alkylated under phase-transfer conditions with
bromide 11, which was prepared as reported previously [9]. An
equimolar regioisomeric mixture of esters 12 (25%) and 13 (24%)
was obtained and separated by chromatography. Both 12 and 13
were hydrogenated at atmospheric pressure to remove trityl and
benzyl groups to give hybrids 14 (70%) and 15 (69%), respectively. It
was observed that the hydrogenation time is dependent on the
batch of catalyst used. It was critical to monitor the reaction by TLC
in order to minimize reduction of the CeCl bond.
2.2. Chemistry
We first planned to prepare a new hybrid bearing an ester linker
between losartan and the antioxidant moieties but with the reverse
position of these fragments around the ester group in 2 (Scheme 1).
Thus, hydrocaffeic acid (4a) was treated with excess benzyl
1) Benzyl bromide
K2CO3, DMF
20 h, 60 ºC
BnO
BnO
OH
HO
CO2H
HO
2) LAH, Et2O, THF
2.5 h, rt, 56%
6
4
Cl
Cl
Cl
NaCN, AcOH
N
N
N
MnO2, EtOH
NaOH, EtOH
OEt
OH
Bu
Bu
CHO
N
H
Bu
N
H
N
H
60 ºC, 20 h
86%
reflux, 2.5 h
78%
O
O
9
8
7
CH2Br
BnO
CPh3
OBn
N
N
Cl
N
N
N
6
DEAD, Ph3P
Et2O, 12 h, rt
11
O
+
Bu
N
H
(n-Bu)4PBr, NaOH
H2O, CH2Cl2
24 h, rt
9
O
10 (68%)
BnO
OBn
Cl
N
O
BnO
OBn
Bu
N
N
N
N
Tr
O
N
N
N
N
Cl
Tr
+
N
N
O
Bu
N
O
12 (25%)
13 (24%)
H2 (1 atm)
H2 (1 atm)
HCCl3
MeOH
HCCl3
MeOH
30% Pd-C
100 min, rt
70%
30% Pd-C
75 min, rt
69%
HO
OH
Cl
N
HO
O
OH
N
Bu
N
N
H
O
N
N
N
N
N
Cl
H
N
N
O
Bu
15
N
O
14
Scheme 1. Synthesis of esters 14 and 15.

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
93
A hybrid bearing an amide linker was prepared next. In this case
the losartan moiety bears the amine functional group and the
antioxidant moiety bears the carboxylic acid group (Scheme 2).
Hydrocaffeic acid (4a) was protected as the dibenzyl ether 16 as
reported previously [27]. Reaction of 4a with excess benzyl
bromide and subsequent basic hydrolysis gave carboxylic acid 16
(69%). Aldehyde 17 was prepared as previously reported [9] and
was then reacted with hydroxylamine to give oxime 18 quantita-
tively with simultaneous deprotection of tetrazole ring. The
reduction of 18 was tested with LAH in Et2O or THF, but the reaction
did not take place. We then protected 18 with trityl chloride since
oxime 18 proved to be highly insoluble in both polar and non polar
solvents. Reaction of 18 with excess trityl chloride in the presence
of TBAI yielded the fully protected compound 19 (48%). Subsequent
reduction of the O-trityl-oxime 19 was performed with LAH to give
amine 20 in modest yield (43%). The amidation reaction between 16
and 20 was carried out using CDI to yield amide 21 (64%). Finally,
complete removal of the benzyl and trityl protecting groups was
carried out by catalytic hydrogenation to give the hybrid 22 (72%).
We also explored the synthesis of an amide hybrid bearing both
losartan and antioxidant moieties in reverse positions (Scheme 3).
Firstly, alkylation of phthalimide with alcohol 6 was performed
under Mitsunobu conditions to give phthalimide 23 (94%), which
was then reacted with hydrazine to yield amine 24 (65%). Aldehyde
17 was used as the starting material for the preparation of the
carboxylic acid 5 (EXP-1371), the active metabolite of losartan [35].
1) Benzyl bromide, K2CO3
DMF, 20 h, 60 ºC
BnO
BnO
HO
CO2H
CO2H
HO
2) NaOH, H2O, MeOH
2 h, reflux, 69%
16
4
Cl
CHO
Cl
N
N
NOH
Bu
Bu
N
N
Tr
N
N
NH2OH.HCl
N
N
NH
N
N
N
EtOH, 3 h, reflux
99%
18
17
Cl
Cl
N
N
NOTr
NH2
Bu
Bu
N
N
Tr
N
Tr
N
TrCl, K2CO3
N
N
N
N
LAH, THF
N
N
TBAI, MeCN
3 h, reflux
48%
3,5 h, rt
43%
19
20
BnO
OBn
Cl
N
H
N
1) CDI, THF
20 h, 60 ºC
Bu
N
H2 (1 atm), 30% Pd-C
HCCl3, MeOH
O
16
N
Tr
2) 20, THF
4 h, 60 ºC, 64%
N
N
N
45 min, rt
68%
21
HO
OH
Cl
N
H
N
Bu
N
O
N
NH
N
N
22
Scheme 2. Synthesis of amide 22.

94
G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
OBn Cl
CHO
OBn
Phthalimide
DEAD, Ph3P
N
O
BnO
BnO
Bu
N
N
OH
THF, 20 h , rt
94%
Tr
CH2Cl2, MgSO4
17 h, rt
N
N
N
24
+
N
23
O
6
OBn
BnO
17
NH2NH2, EtOH
Cl
N
NH2
OEt
10 h, reflux
65%
Bu
OBn
OBn
Cl
N
N
Cl
24
O
N
N
NH
N
N
N
CHO
Bu
N
Bu
N
Cl
N
Tr
Tr
N
N
N
N
N
N
+
N
N
NaCN, AcOH
CHO
Bu
N
MnO2, EtOH
Tr
25
N
N
N
+
(85:15)
N
21 h, reflux
(68:32)
Cl
30
17
N
CHO
Bu
N
OBn
OBn
17
Cl
Cl
N
NH
N
N
N
H
N
N
OH
Bu
Bu
N
N
NaBH4
CH2Cl2, MeOH
Tr
Tr
N
N
N
N
N
N
N
+
N
Cl
4 h, rt
26
N
OH
Bu
N
O
29 (34%)
N
31 (40%)
NaOH, EtOH
reflux, 3 h
NH
N
26 (20%)
N
+
OH
OH
R
N
H
N
5 (78%)
H2 (1 atm)
30% Pd-C
Bu
N
N
NH
OBn
OBn
29
N
N
HCCl3, MeOH
160 min, rt
Cl
N
H
N
32 (R = Cl, 40%)
33 (R = H, 15%)
1) CDI, THF
20 h, 60 ºC
Bu
N
O
N
5
NH
N
N
2) 24,THF, 4 h, 60 ºC
Scheme 4. Synthesis of amine 32.
26%
30:17 (68:32) as determined by 1H NMR analysis. This mixture was
further reacted with sodium borohydride to give amine 29 (34%)
and trityl-protected losartan 31 (40%) [9]. Amine 29 was subse-
quently deprotected by catalytic hydrogenation for 2,5 h to give the
amine 32 (40%) and dehalogenated product 33 (15%). In order to
avoid reduction of CeCl bond, the process was carried out in
shorter times but conversion of starting material was not complete
and yield could not be improved.
27
OH
OH
Cl
N
H
N
H2 (1 atm)
30% Pd-C
Bu
N
O
N
NH
N
MeOH, 1 h, rt
65%
N
2.3. Spectroscopic data
28
1H and 13C NMR spectra were recorded for the synthesized
hybrids using standard experiments in CD3OD. Data were collected
and compared between either the hybrids or between them and
losartan (see Fig. 3). As expected no significant differences were
found for chemical shifts of the protons and carbons at the butyl
Scheme 3. Synthesis of amide 28.
Aldehyde 17 was treated under similar conditions as for 7 to give an
inseparable mixture of ester 25 and aldehyde 26 (85:15). The
isolated mixture was then hydrolyzed under basic conditons to give
acid 5 (78%) and aldehyde 26 [9] (20%) which were then separated.
Subsequent amidation between 5 and 24 was carried out using
similar conditions as for 16 to yield amide 27 in low yield (26%),
probably as a consequence of the lower reactivity of the imidazole-
carboxylic acid 5. Finally, the protecting groups of 27 were removed
by catalytic hydrogenation to give the hybrid 28 (65%).
Another linker of interest was the amino group (Scheme 4). The
link between the losartan and antioxidant units was formed by
reductive amination. Reaction of amine 24 and aldehyde 17,
followed by addition of sodium borohydride gave amine 29. Imine
formation was not complete after 17 h and resulted in a mixture of
chain. The mean values for dH1 (0.90
ꢂ
0.02 ppm), dH2
(1.35 ꢂ 0.04 ppm), dH3 (1.59 ꢂ 0.03 ppm) and dH4 (2.54 ꢂ 0.10 ppm)
were calculated. These values match with the chemical shifts of the
corresponding protons of losartan (0.89, 1.33, 1.56, 2.59 ppm
respectively). For dH5, slightly higher but still not significant devi-
ations were found for ester 14 (5.59 ppm) and amide 28 (5.46 ppm)
in comparison with losartan (5.30 ppm). These protons are shifted
to lower field probably due to both electronic and anisotropic
effects of the carbonyl group in position C-6. For hybrids 2
(5.08 ppm), 15 (5.30 ppm), 22 (5.02 ppm) and 32 (5.27 ppm), dH5 is
slightly lower or the same that than of losartan (5.30 ppm). For
hybrids 2, 22 and 32, dH6 (5.00, 4.28 and 3.73 ppm) correlates with
the presence of carbonyloxy, carbonylamino and amino groups

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
95
OH
OH
OH
Cl
OH
9
7
Y
N
1
H3C
5
4
Cl
3
9
7
z
8
10
N
1
X
3
6
O
N
5
10
H3C
4
6
2
N
8
2
O
2, 14, 22, 28, 32
15
X = CH2, CO; Y = O, CO, NH;
z = CO, CH2, NH
Fig. 3. General structures for NMR data.
respectively and with the hydroxy group in losartan (4.50 ppm). For
ability is expressed as antioxidant equivalent concentration, in mM
per 1 mM of product. The results are the mean of 8 independent
experiments and SEM are in the range between ꢂ0.01 and ꢂ0.04
with p < 0.05 vs 1. All the new hybrids elicited lower antioxidant
ability than ester hybrid 2 (0.31 ꢂ 0.03 mM). At concentrations of
1 mM, this product displays the same antioxidant ability as 1 mM of
compound 4a (0.36 ꢂ 0.03 mM).
protons at C8eC10 positions
13C NMR spectra there are not significant differences between
hybrids and losartan for dC4 Dd ¼ 1.5 ppm).
Dd ¼ 0.4 ppm) and dC5
13C NMR data showed that the carbonyl group of both ester 2
dCO ¼ 175.1 ppm) and amide 22 (dCO ¼ 174.9 ppm) correlates with
an alkanoate ester and an alkanamide respectively. For 14
dCO ¼ 162.0 ppm), 15 (dCO ¼ 163.3 ppm) and 28 (dCO ¼ 162.2 ppm)
d match with calculated values. In the
(
(
(
(
the carbonyl group correlates with a conjugated ester or amide.
According to proton and carbon chemical shift values for hybrids
and losartan, the small differences observed do not suggest
conformational preferences other than that of losartan and for this
reason NOE experiments were not performed. As reported previ-
ously by Mavromoustakos [36,37] and Yoo [38] for losartan and
other non peptide angiotensin II AT1 antagonists, protons at C-4 and
C-5 positions are pointing out each other. The slight downfield shift
observed for H5 in 14 and 28 supports this idea. This downfield shift
should be stronger if protons at C-5 position were pointing out
towards the carbonyl group at C-6. Hybrids 2, 15, 22 and 32, which
lack of this group at C-6, show the same dH5 value than losartan or
slightly shifted to upper field. Thus, we can conclude that, in
methanol, these hybrids keep a conformation close to that reported
for losartan.
2.4.1.1. Structureeactivity relationship (SAR) studies. A hierarchical
antioxidant potency order could be established for the hybrids
prepared and it was compared to hydrocaffeic acid (4a), losartan (1)
and its metabolite 5: 4a > 2 > 22 z 15 > 28 > 32 > 14 > 1 z 5.
Then, to obtain a good antioxidant activity, losartan must be the
alcohol or amine moiety of both ester (2 vs 14) and amide (22 vs 28)
hybrids. However, there is not a strong preference between an ester
or amide for the antioxidant activity (2 vs 22 and 14 vs 28). Hybrid
15, where losartan is the carboxylic acid moiety of the ester linker,
also gives a clear antioxidant activity. This means that a reverse
ester group is better accepted when the bifenilmethyl residue is
linked to the nitrogen N-1 atom of the imidazole ring (15 vs 14). The
compound 15 is an exception by its structure, among all the series
indicated, and should be further explored in future papers.
2.4.2. Analysis of the ability to block angiotensin II-dependent cell
contraction
2.4. Biological activities
The ability of angiotensin II to reduce the planar cell surface area
(PCSA) in cells pre-treated with the different compounds was
analyzed. Changes in PCSA are considered to be a consequence of
cell contraction, and are due to the interaction of angiotensin II with
its receptor [42]. The analysis was performed in cultured smooth
muscle cells, incubated with angiotensin II, in the presence of 1 or
the different synthesized compounds, for 30 min (the concentra-
2.4.1. Antioxidant activity of hybrids
The antioxidant properties of the different compounds (Fig. 4)
was evaluated by the same method used for the phenols 4 and our
previous reported ester hybrids (Total antioxidant status assay kit,
Calbiochem, La Jolla, CA, U.S.A) [29]. Losartan (1) or its metabolite 5
displayed minimal antioxidant ability (0.03 ꢂ 0.01 mM) and this
increased 3e9 fold in the synthesized compounds, with 15 and 22
showing the best antioxidant ability (Table 2) [39e41]. Antioxidant
tion of angiotensin II and the different compounds were 1 mM).
Photographs (TMS-F Photomicroscope, Nikon, Tokyo, Japan,
magnification 150ꢃ) of the same cells were taken at times 0 and
30 min. PCSA was determined by computer-aided planimetric
techniques.
Results are included in the Table 2. The different compounds
tested inhibited the angiotensin II-induced PCSA reduction. This
inhibition ranged between 95% with 15 and 88% with 14. However,
statistically significant differences were not observed in the inhi-
bition of the contraction shown by the different newly synthesized
compounds, with respect to 1 and 2, except for compound 14. These
results show that the attachment of hydrocaffeic acid (4a) to 1 is
able to increase the antioxidant ability without markedly modi-
fying its basic properties as an angiotensin II receptor blocker.
Table 2
Antioxidant ability and inhibition of cellular contraction of the new hybrids
prepared and reference compounds.
Compound
1a
2
TEAC (mM; n ¼ 8)
Cell contraction inhibition (%; n ¼ 5)
0.03 ꢂ 0.01
0.31 ꢂ 0.03
0.36 ꢂ 0.03
0.03 ꢂ 0.01
0.10 ꢂ 0.02
0.24 ꢂ 0.03
0.27 ꢂ 0.04
0.17 ꢂ 0.02
0.13 ꢂ 0.02
e
93
94
85
e
4a
5b
14
15
22
28
32
Ang II
88
95
90
93
92
82
2.4.3. Antihypertensive activity
a
Losartan.
Wistar rats were treated for 1 week with an inhibitor of the
b
2-Butyl-5-chloro-3-[20-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazole-
4-carboxylic acid (EXP-3147). It is the major active metabolite of losartan in the
liver, and primarily responsible for the therapeutic response (see Ref. [39]). EXP3174
is 10e40 fold more potent than 1 and has a longer duration of action (see Ref. [40]
and [41]).
synthesis of nitric oxide, N
chloride ( -NAME), to induce hypertension, and received losartan or
the different hybrids for 3 days. Arterial pressure was measured
before and after the treatment with -NAME and after the
u-nitro-L-arginine methyl ester hydro-
L
L

96
G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
OH
OH
OH
OH
Cl
Cl
N
N
N
O
O
N
N
N
Bu
Bu
N
O
O
N
OH
OH
H
Cl
N
N
N
O
N
N
Bu
Bu
N
NH
NH
N
N
O
15
14
2
OH
OH
OH
OH
OH
OH
Cl
Cl
Cl
H
N
N
N
H
N
N
H
N
N
Bu
Bu
N
O
N
O
N
N
N
N
N
N
NH
NH
N
NH
N
N
22
32
28
Fig. 4. Structures of hybrids prepared.
treatment with the antihypertensive drug. Then the pressure drop
was calculated. Results are shown in Table 3. The more potent
antihypertensive was 2 which produced a pressure drop of 2-fold
than that of losartan. Ester 15 and amide 22 were antihyperten-
sives as potent as losartan. Ester 14 was slightly less potent than
losartan. Unexpectedly amide 28 didn’t show any antihypertensive
effect. Hybrid amine 32 was not tested.
antioxidant moieties have been prepared. These new hybrids
showed an antioxidant ability ranging from 3 to 9 times than that of
1 or its active metabolite 5, and were as potent as 1 in inhibiting
cellular contraction with values ranging from 88 to 95% inhibition.
Most of them showed to be antihypertensives as good as losartan.
Biological activities tested showed that 2 was the best hybrid, since
it combines both the higher antihypertensive and antioxidant
activities. It is better antihypertensive than losartan and may give
an extra tissue protection through its antioxidant properties.
Hybrids 15 and 22 are also promising candidates as antihyperten-
sive drugs since they produce a drop of arterial pressure as good as
losartan and they keep enough antioxidant capacity. Thus, 15 and
22 represent potential hits for further studies. No evidence have
been found that any of the products behave as prodrugs, meta-
bolically dissociating into its components.
Structureeactivity relationship studies showed that to have
a good losartaneantioxidant hybrid the antioxidant phenol must
bear at least to hydroxy groups, one of them in para-position with
respect to the spacer chain. The spacer must have at least a meth-
ylene group between the phenol and the linker functional group.
The linker must be preferably an ester, the better being losartan the
alcohol moiety. Also, an amide is well accepted if losartan provides
the amine moiety. Also an ester linker which bears the imidazole
ring linked to the carbonyl group is well accepted, but in this case
the biphenylmethyl moiety must be linked to the nitrogen N-1 of
the imidazole ring.
2.4.3.1.. Structureeactivity relationship (SAR) studies. The hierar-
chical antihypertensive potency order found for the hybrids tested
andlosartanwas:2>15 z22z1>14 > 28(inactive).Whenlosartan
is the alcohol moiety of theesterlinker(2 vs 14) thehybrid behaves as
a better antihypertensive. The same situation also occurs when los-
artan is the amine moiety of the amide linker (22 vs 28). Indepen-
dently of the position of losartan around the linker, an ester is better
accepted than an amide (2 vs 14 and 22 vs 28). The position of
the biphenylmethyl side chain also affects the potency (14 vs 15). If
the biphenylmethyl group is linked to the nitrogen N-1 atom (15) of
the imidazole ring the activity is enhanced over the position N-3 (14).
3. Conclusion
In summary, novel losartanehydrocaffeic acid hybrids bearing
ester, amide and amine linkers between the antihypertensive and
Table 3
4. Experimental
Antihypertensive effect of the new hybrids prepared and reference compounds.
Compound
P0(mm Hg)a
P1 (mm Hg)b
P2 (mm Hg)c
P2ꢁP1 (mm Hg)
4.1. Chemistry
1
2
14
15
22
28
126 ꢂ 5
139 ꢂ 6
127 ꢂ 5
136 ꢂ 5
131 ꢂ 3
128 ꢂ 4
171 ꢂ 3d
175 ꢂ 5d
175 ꢂ 3d
177 ꢂ 5d
175 ꢂ 2d
171 ꢂ 6d
157 ꢂ 6d,e
13 ꢂ 1
29 ꢂ 4f
9 ꢂ 2f
14 ꢂ 2
13 ꢂ 1
1 ꢂ 1f
145 ꢂ 2e
167 ꢂ 3d,e
163 ꢂ 4d,e
162 ꢂ 5d,e
170 ꢂ 5d
Melting points were determined with a GALLENKAMP appa-
ratus and are uncorrected. 1H NMR and 13C NMR spectra were
recorded at 200 MHz 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), sextet (sext) and multiplet (m). Coupling
constants (J) are given in Hertz (Hz). MS were recorded on
HewlettePackard 5988A or HewlettePackard 1100MSD mass
spectrometers. Elemental analyses were performed on a Heraeus
a
Basal systolic arterial pressure.
b
c
d
e
f
Systolic arterial pressure after 1-week treatment with L-NAME.
Systolic arterial pressure after 3-day treatment with hybrid drug.
p < 0.05 vs basal.
p < 0.05 vs
L-NAME.
p < 0.05 vs losartan.

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
97
CHN Rapid analyzer. FTIR spectra were recorded on a PerkineElmer
FTIR 1725X spectrophotometer. Reagents and solvents were
obtained 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. Purity of the
hybrids and new compounds was determined by elemental anal-
ysis (accuracy of within ꢂ0.4%) and HPLC, which indicated a purity
>98% for each product.
4.1.4. 2-Butyl-5-chloro-3H-imidazole-4-carboxylic acid (9)
To a solution of 8 (0.57 g, 2.49 mmol) in EtOH (20 mL) was added
10 N NaOH (1 mL) and the mixture was heated under reflux for
2.5 h. The solvent was evaporated and the residue was dissolved in
H2O (10 mL) and extracted with Et2O (10 mL). The aqueous phase
was treated with 1 M HCl to give pH ¼ 3e4, extracted with AcOEt
(3 ꢃ 10 mL) and dried (MgSO4). The desiccant was filtered off and
evaporated under reduced pressure to give 9 (0.39 g, 78%) as a dark
brown oil: Rf ¼ 0.37 (AcOEt/MeOH 2:1); mp: 192.2e193.0 ꢄC; 1H
NMR (300 MHz, CD3OD):
J ¼ 7.6 Hz, 2H),1.38 (sext, J ¼ 7.4 Hz, 2H), 0.97 ppm (t, J ¼ 7.4 Hz, 3H).
13C NMR (300 MHz, CD3OD):
¼ 161.5, 152.0, 134.7, 118.6, 31.5, 28.7,
23.1, 14.0 ppm. IR (KBr):
d
¼ 2.69 (t, J ¼ 7.6 Hz, 2H), 1.71 (quin,
4.1.1. General hydrogenation method
In a round-bottomed flask the compound to be hydrogenated
was dissolved in MeOH or CHCl3/MeOH and 30% PdeC was added.
The flask was capped with a septum and flushed with argon. A
balloon containing hydrogen was connected to the flask through
a needle and a syringe. The reaction mixture was stirred at room
temperature until TLC showed complete conversion of starting
material. The mixture was filtered through a short pad of Celite,
washed with MeOH and evaporated under reduced pressure. The
residue was purified by column chromatography on silica gel.
d
n
¼ 3202, 2959, 2874, 2439, 1942, 1670,
1569, 1518, 1326, 1279, 1238, 1087, 824, 776, 760, 686 cmꢁ1. MS (EI,
70 eV) m/z (%): 202 (8) [M þ H]þ, 160 (76) [M ꢁ 42]þ, 142 (100)
[M ꢁ 60]þ. Anal. Calcd for C8H11ClN2O2: C 47.41, H 5.47, N 13.82,
found: C 47.22, H 5.48, N 13.95.
4.1.5. 2-Butyl-5-chloro-3H-imidazole-4-carboxylic acid 3-(3,4-bis-
benzyloxy-phenyl)-propyl ester (10)
A solution of 6 (2.00 g, 5.74 mmol) and Ph3P (1.50 g, 5.74 mmol)
in anh. Et2O (75 mL) was added dropwise to a solution of 9 (1.16 g,
5.74 mmol) and DEAD (0.99 g, 5.74 mmol) in anh. Et2O/THF 3:1
(100 mL). The reaction mixture was stirred for 12 h at rt. The
mixture was filtered and the solvents were removed under reduced
pressure. The residue was chromatographed on silica gel using
hexane/AcOEt 1:1 to give 10 (2.08 g, 68%) as a yellowish oil:
4.1.2. 3-(3,4-Bis-benzyloxy-phenyl)-propan-1-ol (6)
A solution of 4 (0.50 g; 2.74 mmol) and benzyl bromide (2.11 g;
12.34 mmol) in anh. DMF (5 mL) was treated with anh. K2CO3
(2.27 g; 16.46 mmol) for 20 h at 60 ꢄC under an argon atmosphere.
The reaction mixture was cooled to rt, filtered and evaporated under
reduced pressure. The residue was dissolved in AcOEt (10 mL),
washed with 1 M HCl (3 ꢃ 5 mL), satd. NaCl (5 ꢃ 5 mL) and dried
(MgSO4). The desiccant was filtered off and the solvent was evap-
orated under reduced pressure. The residue was dissolved in anh.
Et2O/THF 1:1 (18 mL), added to a slurry of LiAlH4 (0.10 g; 2.71 mmol)
in anh. Et2O at 0 ꢄC and the mixture was stirred at room temperature
for 2.5 h. 20% HCl was added to give pH ¼ 1 and the mixture was
extracted with AcOEt/Et2O 1:1 (4 ꢃ 10 mL). The organic phase was
washed with satd. NaCl and dried (Na2SO4). The desiccant was
filtered off and the solvent was evaporated under reduced pressure.
The residue was chromatographed on silica gel using hexane/AcOEt
1:1. Compound 6 (0.68 g; 56%) was obtained as a colourless oil:
Rf ¼ 0.52 (hexane/AcOEt 1:1); 1H NMR (200 MHz, CDCl3):
d
¼ 9.46
(bs, 1H), 7.43e7.24 (m, 10H), 6.86e6.67 (m, 3H), 5.11 (s, 2H), 5.10 (s,
2H), 4.23 (t, J ¼ 6.2 Hz, 2H), 2.67 (t, J ¼ 7.7 Hz, 4H), 1.97 (quin,
J ¼ 6.6 Hz, 2H), 1.69 (quin, J ¼ 7.5 Hz, 2H), 1.34 (sext, J ¼ 7.5 Hz, 2H),
0.90 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CDCl3):
d
¼ 159.9,
151.1, 148.7, 147.2, 137.2, 137.1, 134.3, 128.3, 127.7, 127.2, 121.2, 116.7,
115.6, 115.2, 71.4, 71.3, 64.2, 31.4, 30.1, 29.9, 28.3, 22.2, 13.6 ppm; IR
(KBr):
n
¼ 3261, 3018, 2961, 2874, 1680, 1558,1510,1454, 1424,1405,
1327, 1215, 1136, 1073, 1024, 755, 696, 696 cmꢁ1. MS (EI, 70 eV) m/z
(%): 555 (100) [M þ Na]þ; Anal. Calcd for C31H33ClN2O4: C 69.84, H
6.24, N 15.25, found: C 70.00, H 6.36, N 15.42.
Rf
¼ 7.38e7.21 (m, 10H), 6.78 (d, J ¼ 8.2 Hz, 1H), 6.72 (s, 1H), 6.61 (d,
J ¼ 8.2 Hz, 1H), 5.06 (s, 2H), 5.04 (s, 2H), 3.48 (t, J ¼ 6.2 Hz, 2H), 2.51
¼
0.31 (hexane/AcOEt 1/1); 1H NMR (200 MHz, CDCl3):
d
4.1.6. 2-Butyl-5-chloro-3-[20-(2-trityl-2H-tetrazol-5-yl)-biphenyl-
4-ylmethyl]-3H-imidazole-4-carboxylic acid 3-(3,4-bis-benzyloxy-
phenyl)-propyl ester (12) and 2-Butyl-5-chloro-1-[20-(2-trityl-2H-
tetrazol-5-yl)-biphenyl-4-ylmethyl]-1H-imidazole-4-carboxylic acid
3-(3,4-bis-benzyloxy-phenyl)-propyl ester (13)
(t, J ¼ 7.6 Hz, 2H), 1.70 (quin, 2H) ppm; IR (CHCl3):
n
¼ 3390, 3031,
2937, 1588, 1511, 1453, 1424, 1380, 1262, 1217, 1135, 1023, 909, 849,
808, 755, 696, 666 cmꢁ1; MS (EI, 70 eV) m/z (%) 348 (55) [M þ H]þ;
Anal. calcd for C23H24O3: C 79.28, H 6.94, found: C 79.00, H 6.99.
To a mixture of 10 (1.88 g, 3.53 mmol), tetrabutylphosphonium
bromide (0.120 g, 0.353 mmol), 10 N NaOH (0.8 mL), H2O (2.8 mL)
and CH2Cl2 (11 mL) was added a solution of 11 (1.96 g, 3.53 mmol)
in CH2Cl2 (17 mL). The reaction mixture was stirred for 24 h at rt.
H2O (15 mL) was added, the organic phase was separated, washed
with H2O (2 ꢃ 15 mL) and dried (MgSO4). The mixture was filtered
and the solvent was evaporated under reduced pressure. The
residue was chromatographed on silica gel using hexane/AcOEt 2:1.
Compound 12. Colourless oil (0.88 g, 25%): Rf ¼ 0.4 (hexane/AcOEt
4.1.3. 2-Butyl-5-chloro-3H-imidazole-4-carboxylic acid ethyl ester
(8)
To a solution of 7 (3.00 g, 16.07 mmol) in EtOH (120 mL) was
added NaCN (4.17 g, 85.2 mmol), AcOH (1.54 g, 25.71 mmol) and
MnO2 (29.34 g, 337.47 mmol). The mixture was stirred for 20 h at
60 ꢄC. After cooling, the mixture was filtered and the solvent was
evaporated under reduced pressure. The residue was dissolved in
Et2O (60 mL) and washed with H2O (30 mL). The organic phase was
dried (MgSO4), the desiccant was filtered off and the solvent was
evaporated under reduced pressure to give 8 as a dark brown oil
(3.20 g, 86%): Rf ¼ 0.21 (hexane/AcOEt/MeOH 4:1:0.05); 1H NMR
2:1); 1H NMR (300 MHz, CDCl3):
d
¼ 7.91e7.88 (m, 1H), 7.48e7.21
(m, 22H), 7.08 (d, J ¼ 7.8 Hz, 2H), 6.93e6.65 (m, 11H), 5.44 (s, 2H),
5.11 (s, 2H), 5.10 (s, 2H), 4.12 (t, J ¼ 6.1 Hz, 2H), 2.63 (t, J ¼ 7.4 Hz,
2H), 2.50 (t, J ¼ 7.7 Hz, 2H), 1.90 (quin, J ¼ 6.8 Hz, 2H), 1.64 (quin,
J ¼ 7.9 Hz, 2H),1.27 (sext, J ¼ 7.3 Hz, 2H), 0.85 ppm (t, J ¼ 7.3 Hz, 3H);
(200 MHz, CDCl3):
d
¼ 10.71 (bs, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 2.70 (t,
J ¼ 7.6 Hz, 2H), 1.70 (quin, J ¼ 7.5 Hz, 2H), 1.43e1.22 (m, 5H),
13C NMR (300 MHz, CDCl3):
141.3, 141.1, 140.6, 137.4, 137.2, 136.6, 134.6, 134.5, 130.7, 130.2, 130.1,
129.9, 129.7, 128.3, 128.1, 127.6, 127.5, 127.3, 127.2, 126.1, 125.3, 121.2,
117.1, 115.7, 115.3, 82.8, 71.5, 71.3, 63.8, 48.2, 31.5, 30.2, 29.4, 26.8,
d
¼ 163.9, 159.4, 152.3, 148.8, 147.1,
0.89 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CD3OD):
d
¼ 160.3,
152.5, 134.9, 118.3, 61.8, 31.4, 28.8, 23.1, 14.6, 13.9 ppm; IR (CHCl3):
n
¼ 3617, 3270, 3019, 2965, 2400, 1682, 1508, 1423, 1215, 1073, 757,
668 cmꢁ1; MS (EI, 70 eV) m/z (%): 230 (8) [M þ H]þ, 188 (91)
[M ꢁ 42]þ, 142 (100) [M ꢁ 88]þ; Anal. Calcd for C10H15ClN2O2: C
52.06, H 6.55, N 12.14, found: C 51.99, H 6.59, N 12.23.
22.3, 13.7 ppm; IR (CHCl3):
n
¼ 3423, 3059, 3030, 2955, 2929, 2869,
1701, 1603, 1511, 1491, 1451, 1424, 1392, 1374, 1264, 1224, 1138, 1103,
1025, 1005, 903, 880, 747, 696 cmꢁ1; MS (APCI) m/z (%): 1008 (100)

98
G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
[M þ H]þ; Anal. Calcd for C64H57ClN6O4: C 76.13, H 5.69, N 8.32,
found: C 76.02, H 5.72, N 8.45. Compound 13. Yellowish oil (0.85 g,
24%): Rf ¼ 0.16 (hexane/AcOEt 2:1); 1H NMR (300 MHz, CDCl3):
reduced pressure and the residue was triturated with AcOEt
(100 mL) to give 18 (7.69 g, 99%) as a white solid: Rf ¼ 0.06 (hexane/
AcOEt 2:1); mp: 215e217 ꢄC; 1H NMR (200 MHz, CD3OD):
d
¼ 8.11 (s,
d
¼ 7.96e7.93 (m, 1H), 7.52e7.21 (m, 22H), 7.11 (d, J ¼ 8.2 Hz, 2H),
1H), 7.75e7.43 (m, 4H), 7.18 (s, 4H), 5.82 (s, 2H), 2.95 (t, J ¼ 7.6 Hz,
6.92e6.69 (m, 11H), 5.12 (s, 2H), 5.11 (s, 2H), 5.03 (s, 2H), 4.33
(t, J ¼ 6.6 Hz, 2H), 2.66 (t, J ¼ 7.6 Hz, 2H), 2.55 (t, J ¼ 7.9 Hz, 2H), 2.04
(quin, J ¼ 7.2 Hz, 2H), 1.61 (quin, J ¼ 7.9 Hz, 2H), 1.27 (sext, J ¼ 7.3 Hz,
2H), 0.83 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CDCl3):
2H), 1.61 (quin, J ¼ 7.6 Hz, 2H), 1.38 (sext, J ¼ 7.6 Hz, 2H), 0.92 ppm
(t, J ¼ 7.1 Hz, 3H); 13C NMR (200 MHz, CD3OD):
d
¼ 156.5, 151.6,
142.6, 140.7, 136.9, 135.4, 132.5, 131.7, 131.5, 130.7, 129.1, 127.7, 124.2,
123.9,122.6, 50.8, 29.8, 26.2,13.1, 13.8 ppm; IR (KBr):
n
¼ 2999, 1800,
d
¼ 163.8, 161.9, 149.0, 148.8, 147.2, 141.2, 141.15, 141.1, 137.4, 137.3,
1568, 1476, 1418, 1189, 1152, 1058, 994, 874, 778, 763, 703, 610 cmꢁ1
;
134.8, 133.1, 130.6, 130.1, 129.9, 129.8, 128.3, 128.2, 127.7, 127.68,
127.64, 127.5, 127.3, 127.2, 126.8, 126.1, 125.4, 123.4, 121.2, 115.5,
115.3, 82.8, 71.5, 71.3. 63.9. 46.9, 31.6, 30.3, 29.7, 27.6, 22.3,
MS (APCI) m/z (%): 436 (100) [M þ H]þ; Anal. Calcd for C22H22ClN7O:
C 60.61, H 5.08, N 22.49, found: C 60.78, H 5.15, N 22.58.
13.6 ppm; IR (CHCl3):
n
¼ 3422, 3059, 3030, 2954, 2868, 1715, 1603,
4.1.10. 2-Butyl-5-chloro-3-[20-(2-trityl-2H-tetrazol-5-yl)-biphenyl-
4-ylmethyl]-3H-imidazole-4-carbaldehyde O-trityl-oxime (19)
A mixture of 18 (4.00 g, 9.17 mmol), TBAI (1.016 g, 2.75 mmol),
anh. K2CO3 (2.78 g, 20.17 mmol) and Ph3CCl (7.67 g, 20.17 mmol) in
anh. MeCN (220 mL) was heated for 3 h under reflux. The reaction
mixture was filtered and the solvent was evaporated. The residue
was dissolved in CHCl3 (100 mL), washed with satd. NaCl
(2 ꢃ 100 mL), H2O (2 ꢃ 100 mL) and dried (Na2SO4). The mixture was
filtered and the solvent was evaporated to give a yellowish oil, which
was chromatographed on silica gel using hexane/AcOEt 3:1. The
resulting oil was triturated with Et2O to give 19 (2.98 g, 48%) as
a white solid: Rf ¼ 0.43 (hexane/AcOEt 3:1); mp: 208e210 ꢄC; 1H
1534, 1511, 1449, 1427, 1324, 1262, 1190, 1155, 1136, 1081, 1037, 1005,
903, 880, 847, 817, 783, 747, 697, 677 cmꢁ1. MS (APCI) m/z (%): 1009
(100) [M þ H]þ; Anal. Calcd for C64H57ClN6O4: C 76.13, H 5.69, N
8.32, found: C 76.23, H 5.87, N 8.45.
4.1.7. 2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazole-4-carboxylic acid 3-(3,4-dihydroxy-
phenyl)-propyl ester (14)
Following the general hydrogenation method, 12 (0.55 g,
0.54 mmol) in MeOH/CHCl3 7:3 (14 mL) was treated with 30% PdeC
(0.35 g) for 100 min. The residue was triturated with Et2O and
chromatographed using AcOEt/MeOH 2:1 to give 14 (0.22 g, 70%) as
a white solid: Rf ¼ 0.55 (AcOEt/MeOH 2:1); mp: 150e151 ꢄC; 1H
NMR (200 MHz, CDCl3):
d
¼ 8.38 (s,1H), 7.97 (dd, J ¼ 1.4 Hz, J ¼ 7.3 Hz,
1H), 7.65e7.53 (m, 2H), 7.46e7.21 (m, 25H), 7.05e7.00 (m, 8H), 6.49
(d, J ¼ 8.2 Hz, 2H), 4.99 (s, 2H), 2.32 (t, J ¼ 7.6 Hz, 2H), 1.61 (quin,
J ¼ 7.3 Hz, 2H), 1.23 (sext, J ¼ 7.6 Hz, 2H), 0.86 ppm (t, J ¼ 7.3 Hz, 3H);
NMR (300 MHz, CD3OD):
d
¼ 7.57e7.40 (m, 4H), 7.1 (d, J ¼ 8.2 Hz,
2H), 6.9 (d, J ¼ 8.2 Hz, 2H), 6.68e6.64 (m, 2H), 6.51e6.48 (m, 1H),
5.59 (s, 2H), 4.22 (t, J ¼ 6.2 Hz, 2H), 2.68 (t, J ¼ 7.6 Hz, 2H), 2.59
(t, J ¼ 7.4 Hz, 2H), 1.93 (quin, J ¼ 7.1 Hz, 2H), 1.61 (quin, J ¼ 7.1 Hz,
2H), 1.35 (sext, J ¼ 7.3 Hz, 2H), 0.91 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR
13C NMR (200 MHz, CDCl3):
140.3, 136.5, 134.9, 132.8, 132.5, 132.1, 131.9, 131.4, 131.2, 130.9, 130.2,
129.8, 129.6, 129.4, 129.2, 129.0, 128.4, 128.2, 127.2, 121.3, 93.3, 84.8,
d
¼ 166.0,153.3,146.0,143.5,143.2,141.9,
(300 MHz, CD3OD):
d
¼ 162.0, 160.6, 154.0, 146.2, 144.4, 142.7, 141.8,
50.2, 31.3, 28.4, 24.2, 15.7 ppm; IR (KBr):
n
¼ 3059, 3010, 2960, 2872,
136.4, 134.1, 131.8, 131.3, 130.7, 130.3, 128.2, 126.7, 120.7, 116.5, 116.3,
65.3, 49.4, 32.4, 31.5, 30.5, 27.5, 23.2, 14.0 ppm; IR (KBr):
2957, 2870, 1701, 1604, 1527, 1486, 1459, 1425, 1397, 1361, 1267,
1208, 1148, 1119, 813, 760, 702, 669 cmꢁ1; MS (APCI) m/z (%): 587
(100) [M þ H]þ; Anal. Calcd for C31H31ClN6O4: C 63.42, H 5.32, N
14.31, found: C 63.25, H 5.45, N 14.54.
1613, 1520, 1491, 1448, 1410, 1264, 1216, 1187, 1156, 1033, 1003, 979,
962, 925, 879, 755, 699, 667, 633 cmꢁ1; MS (APCI) m/z (%): 921 (3)
[M þ H]þ, 865 (14) [M ꢁ 55 þ H]þ; Anal. Calcd for C60H50ClN7O: C
78.28, H 5.47, N 10.65, found: C 78.54, H 5.57, N 10.85.
n
¼ 3179,
4.1.11. {2-Butyl-5-chloro-3-[20-(2-trityl-2H-tetrazol-5-yl)-
biphenyl-4-ylmethyl]-3H-imidazol-4-yl}-methylamine (20)
4.1.8. 2-Butyl-5-chloro-1-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-1H-imidazole-4-carboxylic acid 3-(3,4-dihydroxy-
phenyl)-propyl ester (15)
Following the general hydrogenation method, 13 (0.71 g,
0.70 mmol) in MeOH/CHCl3 7:3 (18 mL) was treated with 30% PdeC
(0.45 g) for 75 min. The residue was triturated with Et2O and chro-
matographed using AcOEt/MeOH 2:1 to give 15 (0.29 g, 69%) as
a white solid: Rf ¼ 0.47 (AcOEt/MeOH 2:1); mp: 177.5e177.7 ꢄC; 1H
A solution of 19 (1.93 g, 2.1 mmol) in anh. THF (40 mL) was added
to a slurry of LiAlH4 (1.19 g, 31.5 mmol) in anh. THF (40 mL) at 0 ꢄC
under an argon atmosphere. The reaction mixture was stirred for
3.5 h at rt. Satd. NH4Cl was added and the mixture was extracted
with AcOEt (2 ꢃ 40 mL) and dried (Na2SO4). The desiccant was
filtered off and the solvent was evaporated under reduced pressure.
A yellowish oil was obtained and this was chromatographed on
silica gel using first hexane/AcOEt 1:1 and then AcOEt/MeOH 5:1 to
give 20 (0.60 g, 43%) as a white foam: Rf ¼ 0.43 (AcOEt/MeOH 5:1);
NMR (200 MHz, CD3OD):
d
¼ 7.61e7.49 (m, 4H), 7.16 (d, J ¼ 8.0 Hz,
2H), 6.99 (d, J ¼ 7.8 Hz, 2H), 6.69 (d, J ¼ 8.0 Hz, 2H), 6.55 (d, J ¼ 8.0 Hz,
1H), 5.3 (s, 2H), 4.30 (t, J ¼ 6.3 Hz, 2H), 2.72e2.60 (m, 4H), 2.03 (quin,
J ¼ 6.7 Hz, 2H), 1.59 (quin, J ¼ 7.7 Hz, 2H), 1.35 (sext, J ¼ 7.5 Hz, 2H),
1H NMR (200 MHz, CDCl3):
d
¼ 7.93e7.89 (m, 1H), 7.51e7.38 (m, 2H),
7.36e7.20 (m, 10H), 7.09 (d, J ¼ 7.9 Hz, 2H), 6.92e6.87 (m, 6H), 6.72
(d, J ¼ 7.9 Hz, 2H), 5.12 (s, 2H), 3.49 (s, 2H), 2.47 (t, J ¼ 7.7 Hz, 2H),
1.63 (quin, J ¼ 7.9 Hz, 2H), 1.43 (bs, 2H), 1.26 (sext, J ¼ 7.6 Hz, 2H),
0.90 ppm (t, J ¼ 7.5 Hz, 3H); 13C NMR (300 MHz, CD3OD):
¼ 163.3,
d
160.9, 151.3, 146.1, 144.3, 142.5, 141.9, 135.2, 134.2, 131.7, 131.3. 130.9.
130.7, 128.5, 127.8, 127.0, 120.7, 125.0, 120.7, 116.5, 116.3, 65.1, 48.0,
0.83 ppm (t, J ¼ 7.6 Hz, 3H); 13C NMR (200 MHz, CDCl3):
d
¼ 163.8,
147.5, 141.2, 140.8, 134.8, 130.6, 130.2, 130.1, 129.9, 129.8, 128.2, 127.6,
127.6, 126.2, 126.1, 125.7, 125.0, 82.8, 46.9, 34.6, 29.8, 22.4, 13.8 ppm;
32.4, 31.7, 30.6, 28.1, 23.2, 14.0 ppm; IR (KBr):
n
¼ 3137, 3057, 2957,
2870, 2712,1699,1651,1603,1531,1456,1406,1360,1284,1257,1210,
1155, 1117, 1088, 1044, 1013, 1006, 865, 842, 816, 783, 758 cmꢁ1; MS
(APCI) m/z (%): 587 (100) [M þ H]þ; Anal. Calcd for C31H31ClN6O4: C
63.42, H 5.32, N 14.31, found: C 63.21, H 5.32, N 14.02.
IR (CHCl3):
n
¼ 3060, 3010, 2958, 2932, 2871, 1600, 1577, 1493, 1447,
1425, 1410, 1356, 1250, 1216, 1157, 1029, 1005, 932, 880, 754,
698 cmꢁ1. MS (ESþ) m/z (%): 664 (22) [M þ H]þ; Anal. calcd for:
C41H38ClN7: C 74.13, H 5.76, N 14.76, found: C 74.02, H 5.24, N 14.59.
4.1.9. 2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazole-4-carbaldehyde oxime (18)
To a solution of 17 (11.83 g, 17.84 mmol) in EtOH (280 mL),
NH2OH.HCl (2.72 g, 39.25 mmol) was added. The reaction mixture
was heated for 3 h under reflux. The solvent was evaporated under
4.1.12. 3-(3,4-Bis-benzyloxy-phenyl)-N-{2-butyl-5-chloro-3-[20-(2-
trityl-2H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazol-4-
ylmethyl}-propionamide (21)
A solution of CDI (0.118 g, 0.72 mmol) in anh. THF (3 mL) was
added to a slurry of 16 (0.12 g, 0.33 mmol) in anh. THF (3 mL) under

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
99
an argon atmosphere. The reaction mixture was heated for 20 h at
60 ꢄC. A solution of 20 (0.22 g, 0.33 mmol) in anh. THF (3 mL) was
added and the reaction mixture was stirred for 4 h at 60 ꢄC. The
solvent was evaporated under reduced pressure and the residue
was dissolved in CHCl3 (5 mL). The organic phase was washed with
satd. NaCl (5 mL) and the aqueous phase was extracted with CHCl3
(3 ꢃ 5 mL). The combined organic extracts were dried (MgSO4),
filtered and evaporated under reduced pressure. The residual oil
was chromatographed on silica gel using hexane/AcOEt 1:2 to give
21 (0.19 g, 58%) as a yellowish oil: Rf ¼ 0.47 (hexane/AcOEt 1:2); 1H
1006, 969, 944, 914, 886, 855, 791, 766, 733, 708, 698, 605 cmꢁ1; MS
(APCI) m/z (%): 500 (100) [M þ Na]þ; Anal. calcd. for: C31H27NO4: C
77.96, H 5.69, N 2.93, found: C 78.00; H 5.71, N 3.00.
4.1.15. 3-(3,4-Bis-benzyloxy-phenyl)-propylamine (24)
To a solution of 23 (3.12 g, 6.54 mmol) in EtOH (30 mL) was
added hydrazine monohydrate (0.32 g, 6.54 mmol) and the mixture
was heated for 10 h under reflux. The solvent was removed under
reduced pressure and the residue was triturated with CHCl3. The
solid was filtered off and the filtrate was evaporated under reduced
pressure. The residual oil was chromatograped on silica gel using
MeOH to give 24 (1.47 g, 65%) as a yellowish oil: Rf ¼ 0.17 (MeOH);
NMR (200 MHz, CDCl3):
d
¼ 7.87e7.83 (m, 1H), 7.39e7.14 (m, 22H),
7.03 (d, J ¼ 8.2 Hz, 2H), 6.91e6.87 (m, 6H), 6.74e6.61 (m, 4H),
6.54e6.49 (m, 1H), 5.23 (t, J ¼ 5.7 Hz, 1H), 5.06 (s, 2H), 5.01 (s, 2H),
5.00 (s, 2H), 4.1 (d, J ¼ 5.7 Hz, 2H), 2.63 (t, J ¼ 7.6 Hz, 2H), 2.40 (t,
J ¼ 7.7 Hz, 2H), 2.02 (t, J ¼ 7.6 Hz, 2H), 1.0 (quin, J ¼ 7.6 Hz, 2H), 1.20
(sext, J ¼ 7.3 Hz, 2H), 0.79 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR
1H NMR (300 MHz, CDCl3):
d
¼ 7.48e7.25 (m, 10H), 6.89e6.80 (m,
2H), 6.70 (dd, J ¼ 1.8 Hz, J ¼ 8.0 Hz,1H), 5.15 (s, 2H), 5.12 (s, 2H), 2.65
(t, J ¼ 7.1 Hz, 2H), 2.55 (t, J ¼ 7.7 Hz, 2H), 1.69 (quin, J ¼ 7.3 Hz, 2H),
1.34 ppm (bs, 2H); 13C NMR (300 MHz, CDCl3):
d
¼ 148.7, 147.1,
(200 MHz, CDCl3):
d
¼ 171.6, 163.8, 148.7, 148.0, 147.3, 141.2, 141.1,
137.5, 137.3, 135.6, 128.3, 127.6, 127.6, 127.29, 127.2, 121.1, 115.6,
115.3, 71.5, 71.3, 41.6, 35.3, 32.6 ppm; IR (CHCl3):
2858, 1587, 1511, 1453, 1424, 1380, 1263, 1223, 1137, 1079, 1023, 852,
806, 735, 696 cmꢁ1; MS (APCI) m/z (%): 348 (100) [M þ H]þ; Anal.
calcd for: C23H25NO2: C 79.50, H 7.25, N 4.03, found: C 79.25, H 7.52,
N 4.08.
140.7, 137.3, 137.2, 134.8, 133.8, 130.6, 130.2, 130.1, 129.9, 129.7,
128.3, 128.2, 127.6, 127.2, 127.2, 126.1, 124.8, 122.4, 120.9, 115.3,
115.2, 82.8, 71.3, 71.2, 46.8, 37.8, 31.5, 30.7, 29.7, 26.7, 22.3,13.7 ppm;
n
¼ 3031, 2929,
IR (CHCl3):
n
¼ 3293, 3062, 3010, 2958, 2931, 2871, 1662, 1511, 1450,
1424, 1257, 1216, 1016, 1005, 755, 697 cmꢁ1; MS (APCI) m/z (%):
1008 (100) [M þ H]þ; Anal. calcd for: C64H58ClN7O3: C 76.21, H 5.79,
N 9.72, found: C 76.05, H 5.70, N 9.65.
4.1.16. 2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazole-4-carboxylic acid (5) and 2-butyl-5-chloro-
3-[20-(2H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazole-4-
carbaldehyde (26)
4.1.13. N-{2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazol-4-ylmethyl}-3-(3,4-dihydroxy-phenyl)-
propionamide (22)
Following the general hydrogenation method, 21 (0.44 g,
0.44 mmol) in MeOH/CHCl3 7:3 (9 mL) and 30% PdeC (0.33 g) were
stirred for 45 min at rt. The residue was chromatographed using
AcOEt/MeOH 5:1 to give 22 (0.18 g, 68%) as a white solid; Rf: 0.31
(AcOEt/MeOH 5:1); mp: 178.4e178.6 ꢄC; 1H NMR (300 MHz,
To a solution of 17 (2.00 g, 3.01 mmol) in EtOH (23 mL) was
added NaCN (0.78 g, 16 mmol), AcOH (0.29 g, 4.81 mmol) and MnO2
(5.50 g, 63.21 mmol) and the mixture was stirred for 21 h under
reflux. The reaction mixture was filtered and the solvent was
removed under reduced pressure. The residue was dissolved in
Et2O (12 mL), washed with H2O (12 mL) and dried (MgSO4). The
desiccant was filtered off and the solvent was removed under
reduced pressure to give a dark brown oil. The oil was dissolved in
EtOH (45 mL), 10 N NaOH (1 mL) was added and the mixture was
heated for 3 h under reflux. The solvent was removed under
reduced pressure, the residue was dissolved in H2O (10 mL) and 1 M
HCl was added to give pH ¼ 3e4. The mixture was extracted with
AcOEt (3 ꢃ 10 mL) and the combined organic extracts were dried
(MgSO4). The mixture was filtered and the solvent was evaporated
under reduced pressure to give a dark brown oil, which was
chromatographed on silica gel using AcOEt/MeOH 2:1. Compound 5:
yellowish solid (1.02 g, 78%); Rf ¼ 0.28 (AcOEt/MeOH 2:1); mp:
177e179 ꢄC (Lit [35]: 176e178 ꢄC); 1H NMR (200 MHz, MeOD):
CD3OD):
d
¼ 7.58 (dd, J ¼ 1.5 Hz, J ¼ 7.1 Hz 1H), 7.54e7.44 (m, 2H),
(dd, J ¼ 1.4 Hz, J ¼ 7.7 Hz 1H), 7.1 (d, J ¼ 8.2 Hz, 2H), 6.8 (d, J ¼ 8.0 Hz,
2H), 6.64e6.60 (m, 2H), 6.44 (dd, J ¼ 2.1 Hz, J ¼ 7.8 Hz 1H), 5.02 (s,
2H), 4.28 (s, 2H), 2.62 (t, J ¼ 7.5 Hz, 2H), 2.55 (t, J ¼ 7.6 Hz, 2H), 2.16
(t, J ¼ 7.5 Hz, 2H), 1.56 (quin, J ¼ 7.5 Hz, 2H), 1.32 (sext, J ¼ 7.5 Hz,
2H), 0.89 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CD3OD):
d
¼ 174.9, 160.7, 149.9, 146.1, 144.5, 142.6, 141.4, 136.5, 133.4, 131.7,
131.4, 130.8, 130.7, 128.7, 128.4, 127.8, 126.5, 124.2, 120.5, 116.5,
116.2, 47.9, 38.4, 32.3, 31.8, 30.8, 27.3, 23.2, 14.0 ppm; IR (KBr):
n
¼ 3252, 3059, 2957, 2870, 2715,1650, 1604,1527,1459, 1424,1360,
1258, 1209, 1152, 1116, 1075, 1005, 819, 778, 759 cmꢁ1; MS (APCI)
m/z (%): 586 [M þ H]þ; Anal. calcd for: C31H32ClN7O3: C 63.52, H
5.50, N 16.72, found: C 63.25, H 5.48, N 16.62.
d
¼ 7.60e7.40 (m, 4H), 7.07 (d, J ¼ 8.0 Hz, 2H), 6.89 (d, J ¼ 8.0 Hz,
2H), 5.72 (s, 2H), 2.62 (t, J ¼ 7.5 Hz, 2H), 1.56 (quin, 2H), 1.32 (sext,
4.1.14. 2-[3-(3,4-Bis-benzyloxy-phenyl)-propyl]-isoindole-1,3-dione
(23)
2H), 0.88 ppm (t, J ¼ 7.1 Hz, 3H); IR (KBr):
n
¼ 3365, 2957, 1599,
1458, 1407, 1359, 1261, 1173, 1108, 1054, 1006, 976, 894, 816, 760,
668 cmꢁ1; MS (ESþ) m/z (%): 437 (100) [M þ H]þ; Anal. calcd for:
C22H21ClN6O2: C 60.48, H 4.84, N 19.23, found: C 60.52, H 4.95, N
19.25. Compound 26: white solid (0.25 g, 20%); mp: 155e157 ꢄC
(Lit [35]: 154e155 ꢄC).
To a solution of 6 (2.58 g, 7.41 mmol), Ph3P (1.94 g, 7.41 mmol)
and phthalimide (1.09 g, 7.41 mmol) in anh. THF (60 mL) was added
DEAD (0.174 g, 7.41 mmol) under an argon atmosphere. The reac-
tion mixture was stirred for 20 h at rt. The solvent was evaporated
under reduced pressure and the residue was chromatographed on
silica gel using hexane/AcOEt 1:1 to give an oil, which was tritu-
rated in Et2O to yield 23 (0.22 g; 94%) as a white solid: Rf ¼ 0.72
(AcOEt/MeOH 1:1); mp: 91e93 ꢄC; 1H NMR (300 MHz, CDCl3):
4.1.17. 2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazole-4-carboxylic acid [3-(3,4-bis-benzyloxy-
phenyl)-propyl]-amide (27)
d
¼ 7.81e7.70 (m, 2H), 7.69e7.66 (m, 2H), 7.45e7.26 (m, 10H),
A solution of CDI (0.41 g, 2.5 mmol) in anh. THF (15 mL) was
added to a slurry of 5 (0.5 g, 1.14 mmol) in anh. THF (40 mL) under
an argon atmosphere. The mixture was heated for 20 h at 60 ꢄC. A
solution of 24 (0.87 g, 2.50 mmol) in anh. THF (15 mL) was added
and the mixture was heated for 4 h at 60 ꢄC. The solvent was
evaporated under reduced pressure and the residue was dissolved
in CHCl3 (40 mL) and washed with satd. NaCl (40 mL). The aqueous
phase was extracted with CHCl3 (3 ꢃ 40 mL) and the combined
6.81e6.78 (m, 2H), 6.69e6.66 (m, 1H), 5.12 (s, 2H), 5.05 (s, 2H), 3.68
(t, J ¼ 7.0 Hz, 2H), 2.57 (t, J ¼ 7.6 Hz, 2H), 1.95 ppm (quin, J ¼ 7.3 Hz,
2H); 13C NMR (75 MHz, CDCl3):
d
¼ 168.3, 148.8, 147.1, 137.4, 137.3,
134.5, 133.8, 132.0, 128.3, 127.6, 127.3, 127.2, 123.0, 121.0, 115.3, 71.4,
71.1, 37.6, 32.6, 29.7 ppm; IR (CHCl3):
2863,1779,1769, 1713,1607, 1587, 1517, 1465, 1454,1436, 1427, 1398,
1380, 1365, 1331, 1266, 1245, 1224, 1188, 1136, 1106, 1083, 1027,
n
¼ 3461, 3061, 3030, 2934,

100
G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
organic extracts were dried (MgSO4). The desiccant was filtered off,
the solvent was evaporated under reduced pressure and the residue
was chromatographed on silica gel using AcOEt/MeOH 2:1 to give
27 (0.23 g, 26%) as a yellow solid that was crystallized from hexane/
AcOEt 1:1: Rf ¼ 0.73 (AcOEt/MeOH 1:1); mp: 134e136 ꢄC; 1H NMR
1510, 1450, 1379, 1248, 1157, 1136, 1026, 930, 904, 880, 747,
696 cmꢁ1; MS (APCI) m/z (%): 995 (100) [M þ H]þ; Anal. calcd for
C64H60ClN7O2: C 77.28, H 6.08, N 9.85, found: C 77.12, H 6.14, N 9.99.
Compound 31: white solid (0.36 g, 40%); mp: 165e167 ꢄC (Lit [9]:
167e169 ꢄC).
(300 MHz, CD3OD):
d
¼ 7.58e7.26 (m, 14H), 7.08 (d, J ¼ 8.2 Hz, 2H),
6.95 (d, J ¼ 8.2 Hz, 2H), 6.93e6.88 (m, 2H), 6.72e6.69 (m, 1H), 5.47
(s, 2H), 5.10 (s, 2H), 5.08 (s, 2H), 3.27 (t, J ¼ 6.8 Hz, 2H), 2.68
(t, J ¼ 7.7 Hz, 2H), 2.55 (t, J ¼ 7.6 Hz, 2H), 1.77 (quin, J ¼ 7.3 Hz, 2H),
1.61 (quin, J ¼ 7.5 Hz, 2H), 1.37 (sext, J ¼ 7.3 Hz, 2H), 0.92 ppm
4.1.20. 4-[3-({2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-
4-ylmethyl]-3H-imidazol-4-ylmethyl}-amino)-propyl]-benzene-1,2-
diol (32) and 4-[3-({2-Butyl-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazol-4-ylmethyl}-amino)-propyl]-benzene-1,2-
diol (33)
Following the general hydrogenation method, 29 (0.19 g,
0.21 mmol) in MeOH/CHCl3 77:23 (13 mL), and 30% PdeC (0.17 g) were
stirred for 160 min. The residue was chromatographed using AcOEt/
MeOH/TEA (2:1:0.2). Compound 32: Orange solid (0.05 g, 40%):
Rf ¼ 0.36 (AcOEt/MeOH/TEA 2:1:0.2); mp: 97e99 ꢄC; 1H NMR
(t, J ¼ 7.3 Hz, 3H); IR (CHCl3):
n
¼ 3123, 3032, 2931, 2860, 2609,
1958, 1648, 1511, 1258, 1135, 1063, 1013, 930, 852, 751, 696,
663 cmꢁ1; MS (ESþ) m/z (%): 766 (100) [M þ H]þ; Anal. calcd for
C45H44ClN7O3: C 70.52, H 5.78, N 12.79, found: C 69.72, H 5.74, N
12.25.
4.1.18. 2-Butyl-5-chloro-3-[20-(2H-tetrazol-5-yl)-biphenyl-4-
ylmethyl]-3H-imidazole-4-carboxylic acid [3-(3,4-dihydroxy-
phenyl)-propyl]-amide (28)
Following the general hydrogenation method, 27 (0.20 g,
0.26 mmol) in MeOH (5 mL) and 30% PdeC (0.94 g) were stirred for
1 h. The residue was chromatographed using AcOEt/MeOH 2:1 to
give 28 (0.10 g, 65%) as a yellowish solid: Rf ¼ 0.49 (AcOEt/MeOH
2:1); mp: 180.2e181.6 ꢄC; 1H NMR (300 MHz, CD3OD):
(300 MHz, CD3OD):
d
¼ 7.59e7.41 (m, 4H), 7.13 (d, J ¼ 8.2 Hz, 2H), 6.86
(d, J ¼ 6.8 Hz, 2H), 6.68e6.62 (m, 2H), 6.48e6.45 (m, 1H), 5.27 (s, 2H),
3.73 (s, 2H), 2.68e2.63 (m, 4H), 2.47 (t, J ¼ 7.5 Hz, 2H), 1.71 (quin,
J ¼ 7.3 Hz, 2H), 1.62 (quin, J ¼ 7.3 Hz, 2H), 1.35 (sext, J ¼ 7.0 Hz, 2H),
0.91 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CD3OD):
d
¼ 162.6,
150.3, 146.1, 144.3, 142.4, 135.9, 134.3, 131.7, 131.1, 131.0, 130.0, 128.9,
128.2, 126.4, 123.3, 120.6, 116.4, 116.2, 48.8, 48.2, 41.3, 33.6, 31.4, 30.9,
27.4, 23.2, 14.0 ppm; IR (KBr):
n
¼ 3381, 2925, 2139, 1636, 1457, 1357,
d
¼ 7.56e7.32 (m, 4H), 7.09 (d, J ¼ 8.2 Hz, 2H), 6.95 (d, J ¼ 8.2 Hz,
1257, 1207, 1150, 1119, 1013, 897, 812, 785, 759 cmꢁ1; MS (ES) m/z (%):
572 (100) [M þ H]þ; Anal. calcd for C31H34ClN7O2: C 65.08, H 5.99, N
17.13,found:C65.12, H6.02, N17.25.Compound 33. Yellowsolid(0.02 g,
15%): Rf ¼ 0.12 (AcOEt/MeOH/TEA 2:1:0.2); mp: 100e101 ꢄC; 1H NMR
2H), 6.68e6.64 (m, 2H), 6.51e6.48 (m, 1H), 5.46 (s, 2H), 3.29
(t, J ¼ 6.8 Hz, 2H), 2.69 (t, J ¼ 7.6 Hz, 2H), 2.49 (t, J ¼ 7.7 Hz, 2H), 1.76
(quin, J ¼ 7.3 Hz, 2H), 1.61 (quin, J ¼ 7.5 Hz, 2H),1.36 (sext, J ¼ 7.5 Hz,
2H), 0.92 ppm (t, J ¼ 7.3 Hz, 3H); 13C NMR (300 MHz, CD3OD):
(300 MHz, CD3OD):
d
¼ 7.63e7.43 (m, 4H), 7.14e7.11 (m, 3H), 6.82 (d,
d
¼ 162.2, 161.3, 152.0, 146.1, 144.3, 142.6, 142.1, 136.2, 134.5, 131.8,
J ¼ 8.0 Hz, 2H), 6.71e6.65 (m, 2H), 6.53e6.50 (m,1H), 5.27 (s, 2H), 3.99
(s, 2H), 2.94 (t, J ¼ 7.7 Hz, 2H), 2.74 (t, J ¼ 7.6 Hz, 2H), 2.57 (t, J ¼ 7.4 Hz,
2H), 1.90 (quin, J ¼ 7.7 Hz, 2H), 1.64 (quin, J ¼ 7.0 Hz, 2H), 1.37 (sext,
J¼ 7.4 Hz, 2H), 0.92 ppm (t, J¼ 7.3 Hz, 3H); 13C NMR(300 MHz, CD3OD):
131.2, 130.7, 130.5, 130.2, 129.8, 128.2, 127.2, 123.0, 120.6, 116.5,
116.2, 40.1, 33.5, 32.3, 30.7, 27.3, 23.2, 14.0 ppm; IR (KBr):
2957, 2870, 1646, 1515, 1455, 1361, 1263, 1197, 1114, 1006, 814, 777,
760 cmꢁ1. MS (ESþ) m/z (%): 586 (100) [M þ H]þ; Anal. calcd for
C31H32ClN7O3: C 63.52, H 5.50, N 16.72, found: C 63.75, H 5.74, N
16.45.
n
¼ 3061,
d
¼ 162.6,152.5,146.4,144.8,142.6,142.4,135.7,133.1,131.7,131.1,130.9,
130.1, 129.3, 128.4, 126.4, 120.5, 116.5, 116.4, 47.7, 41.8, 32.9, 31.0, 29.4,
27.5, 23.3, 14.0 ppm; IR (KBr):
n
¼ 3418, 2957, 2869, 2349, 2297, 1603,
1519, 1459, 1407, 1358, 1282, 1151, 1118, 1011, 955, 889, 820, 786,
760 cmꢁ1. MS (API-ESþ) m/z (%): 538 (100) [M þ H]þ; Anal. calcd for:
C31H35N7O2: C 69.25, H 6.56, N 17.13, found: C 69.42, H 6.75, N 17.21.
4.1.19. [3-(3,4-Bis-benzyloxy-phenyl)-propyl]-{2-butyl-5-chloro-3-
[20-(2-trityl-2H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazol-
4-ylmethyl}-amine (29) and {2-Butyl-5-chloro-3-[20-(2-trityl-2H-
tetrazol-5-yl)-biphenyl-4-ylmethyl]-3H-imidazol-4-yl}-methanol
(31)
4.2. Antioxidant activity of phenols and hybrids
To a solution of 17 (0.90 g, 1.35 mmol) and 24 (0.471 g,
1.35 mmol) in CH2Cl2 (36 mL) was added anh. MgSO4 (0.16 g) and
the mixture was stirred for 17 h at rt under an argon atmosphere.
The mixture was filtered and the solvent was removed under
reduced pressure. The residue was dissolved in anh. MeOH/CH2Cl2
2:1 (36 mL) under an argon atmosphere, NaBH4 (0.10 g, 2.71 mmol)
was added slowly and the mixture was stirred for 4 h at rt. The
solvent was evaporated under reduced pressure and the residue
was dissolved in CH2Cl2 (18 mL), washed with satd. NaCl
(2 ꢃ 10 mL) and dried (MgSO4). The desiccant was filtered off and
the solvent was removed under reduced pressure. The residue was
chromatographed on silica gel using hexane/AcOEt 1:2. Compound
29: colourless oil (0.46 g, 34%); Rf ¼ 0.25 (hexane/AcOEt 1:2); 1H
The antioxidant ability of the different compounds was evalu-
ated with a commercial kit (Total antioxidant status assay kit)
purchased from Calbiochem (La Jolla, CA, USA) [29]. The assay relies
on the ability of antioxidants to inhibit the oxidation of a chro-
mogen (ABTS) by metmyoglobin and hydrogen peroxide. Oxidation
is monitored by reading the absorbance at 600 nm. The antioxidant
ability is proportional to the reduction of the absorbance, and it is
expressed as mM. Experimental protocol was modified slightly. A
96-well plate was used instead of a cuvette. Lower volume of
chromogen (metmyoglobin and ABTS; 250
m
L), water (5
m
L), stan-
dard (Trolox, 5
m
L) and sample solutions (10ꢁ1 M in PBS, 5
mL) were
used. Phenols stock solutions were prepared dissolving 5 mg of
sample in 1 equivalent volume of 1.622 M K2CO3 and then diluted
up to assay concentrations. Two wells were used for each sample.
Initial absorbance (A0) was red after mixing solutions. Then,
substrate solution (200 mL) was added, solutions were mixed and
absorbance (A) was red after exactly 3 min. The mean of 3 (phenols)
NMR (300 MHz, CDCl3):
d
¼ 7.95e7.92 (m, 1H), 7.47e7.22 (m, 22H),
7.09 (d, J ¼ 8.2 Hz, 2H), 6.94e6.91 (m, 6H), 6.83 (d, J ¼ 8.0 Hz 2H),
6.73 (d, J ¼ 7.8 Hz 2H), 6.62 (dd, J ¼ 1.9 Hz, J ¼ 8.2 Hz 1H), 5.14
(s, 2H), 5.11 (s, 4H), 3.44 (s, 2H), 2.46 (t, J ¼ 7.7 Hz, 6H), 1.63 (sext,
J ¼ 7.7 Hz, 4H), 1.28 (sext, J ¼ 7.4 Hz, 2H), 1.06 (bs, 1H), 0.85 ppm
or 8 (hybrids) assays was used to calculate TEAC as in Eq. (1)
(t, J ¼ 7.3 Hz, 3H); 13C NMR (75 MHz, CDCl3):
¼ 163.9, 148.8, 147.6,
d
(D
¼ AꢁA0).
TEACðmMÞ ¼ ½Standardꢅð
ꢃ ð ABlank ꢁ
147.1, 141.28, 141.2, 140.7, 137.4, 137.3, 135.5, 134.9, 130.6, 130.2,
130.1, 129.9, 129.7, 128.3, 128.2, 127.7, 127.6, 127.3, 127.2, 127.1, 126.1,
125.0, 123.6, 121.1, 115.6, 115.2, 82.8, 71.5, 71.3, 48.3, 46.8, 41.8, 32.9,
D
ABlank ꢁ
D
D
ASampleÞ=
D
AStandardÞ
(1)
31.5, 29.7, 26.7, 22.4, 13.7 ppm; IR (CHCl3):
n
¼ 3029, 2930, 1586,

G. García et al. / European Journal of Medicinal Chemistry 50 (2012) 90e101
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This work was supported by grants from the Ministerio de
Ciencia e Innovación (project CTQ2008-04313/BQU), Instituto de
Salud Carlos III (Red de Investigación Renal, REDinREN, RD6/0016/
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