An efficient synthesis of a rationally designed 1,5 disubstituted imidazole AT1 Angiotensin II receptor antagonist: Reorientation of imidazole pharmacophore groups in losartan reserves high receptor affinity and confirms docking studies

Article abstract of DOI:10.1007/s10822-010-9371-3

A new 1,5 disubstituted imidazole AT₁ Angiotensin II (AII) receptor antagonist related to losartan with reversion of butyl and hydroxymethyl groups at the 2-, 5-positions of the imidazole ring was synthesized and evaluated for its antagonist activity (V8). In vitro results indicated that the reorientation of butyl and hydroxymethyl groups on the imidazole template of losartan retained high binding affinity to the AT ₁ receptor concluding that the spacing of the substituents at the 2,5- positions is of primary importance. The docking studies are confirmed by binding assay results which clearly show a comparable binding score of the designed compound V8 with that of the prototype losartan. An efficient, regioselective and cost effective synthesis renders the new compound as an attractive candidate for advanced toxicological evaluation and a drug against hypertension.

Full text of DOI:10.1007/s10822-010-9371-3

J Comput Aided Mol Des (2010) 24:749–758
DOI 10.1007/s10822-010-9371-3
An efficient synthesis of a rationally designed 1,5 disubstituted
imidazole AT1 Angiotensin II receptor antagonist: reorientation
of imidazole pharmacophore groups in losartan reserves high
receptor affinity and confirms docking studies
•
George Agelis Panagiota Roumelioti Amalia Resvani
•
•
•
•
Serdar Durdagi Maria-Eleni Androutsou Konstantinos Kelaidonis
•
•
Demetrios Vlahakos Thomas Mavromoustakos
•
John Matsoukas
Received: 16 February 2010 / Accepted: 17 June 2010 / Published online: 10 July 2010
ꢀ Springer Science+Business Media B.V. 2010
Abstract
A
new 1,5 disubstituted imidazole AT1
primary importance. The docking studies are confirmed by
binding assay results which clearly show a comparable
binding score of the designed compound V8 with that of
the prototype losartan. An efficient, regioselective and cost
effective synthesis renders the new compound as an
attractive candidate for advanced toxicological evaluation
and a drug against hypertension.
Angiotensin II (AII) receptor antagonist related to losartan
with reversion of butyl and hydroxymethyl groups at the 2-,
5-positions of the imidazole ring was synthesized and
evaluated for its antagonist activity (V8). In vitro results
indicated that the reorientation of butyl and hydroxymethyl
groups on the imidazole template of losartan retained high
binding affinity to the AT1 receptor concluding that the
spacing of the substituents at the 2,5- positions is of
Keywords Angiotensin II receptor antagonist ꢀ
Losartan ꢀ Molecular modeling ꢀ
Docking theoretical calculations ꢀ NMR spectroscopy
Docking Studies: Serdar Durdagi, Thomas Mavromoustakos
Introduction
Electronic supplementary material The online version of this
article (doi:10.1007/s10822-010-9371-3) contains supplementary
material, which is available to authorized users.
The Renin-Angiotensin System (RAS) plays a key role in
regulating cardiovascular homeostasis and electrolyte/fluid
balance in normotensive and hypertensive subjects [1]. The
octapeptide AII is formed within the RAS from Angio-
tensin I by Angiotensin-Converting Enzyme (ACE) and is
one of the most powerful vasoconstrictors. AII is also
found to be a growth factor implicated in cardiac, vascular
hypertrophy and the ventricular remodeling following
myocardial infarction. Consequently, the RAS has been a
prime target for the therapy of cardiovascular diseases [2].
Reducing the levels of AII by inhibition of ACE, is con-
sidered a legitimate approach for treating hypertension.
Although the resulted ACE inhibitors are successful drugs,
they suffer from side effects such as dry cough and angi-
oedema, because they may increase the levels of bradyki-
nin [3–5]. A new and more specific approach to block the
RAS is considered the antagonism of AII at its receptor site
[6, 7]. Peptide analogues of AII such as sarilesin and
sarmesin inhibit the action of AII by competitive binding to
G. Agelis (&) ꢀ P. Roumelioti ꢀ A. Resvani ꢀ
M.-E. Androutsou ꢀ K. Kelaidonis ꢀ J. Matsoukas
Department of Chemistry, University of Patras,
Patras 26500, Greece
e-mail: aggelisgeorge@hotmail.com
S. Durdagi
University of Calgary, Department of Biological Sciences,
Institute for Biocomplexity and Informatics, Calgary, Alberta,
Canada
T. Mavromoustakos
Department of Chemistry, University of Athens, Athens, Greece
D. Vlahakos
Department of Internal Medicine, ‘ATTIKON’ University
Hospital, Athens, Greece
J. Matsoukas (&)
Eldrug S.A., Patras, Greece
e-mail: eldrug@eldrug.gr
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J Comput Aided Mol Des (2010) 24:749–758
receptor is not crystallized and its 3D putative bioactive
conformation is based solely on homology models.
Materials and methods
Protein structure
The 3D model of the AT1 receptor used in our docking
studies was kindly provided by Tuccinardi et al. [31]. The
construction of this model is based on X-ray bovine rho-
dopsin structure, molecular procedure and available site-
directed mutagenesis data [32].
Fig. 1 Compound V8 and losartan, the first non-peptide AII receptor
antagonist
the receptor. However, their application as clinical agents
is limited due to short duration of action, poor bioavail-
ability and partial agonist activity. These AII type I and
type II antagonists aided in the identification of pharma-
cophore groups and for the design of AII non-peptide mi-
metics [8, 9]. The discovery by DuPont of the first potent
and orally active non-peptide AII antagonist losartan [10]
has stimulated extensive research interest in this area.
Several patents and publications have appeared over the
past few years [11, 12] describing new AII receptor
antagonists including candesartan, irbesartan, valsartan,
telmisartan, tasosartan and eprosartan, which have been
proven safe and effective in the treatment of hypertension
and other cardiovascular disorders [13–21].
In silico docking studies
Molecular Docking studies were performed using GOLD
(v.4.1.1) [33] and Glide extra precision (XP) implemented
Induced Fit Docking (IFD) protocol (v5.0) [34–36] docking
programs under the Linux operating system.
(i) GOLD: The binding interactions were derived using
the genetic algorithm. Genetic algorithm is a stochastic
technique used in GOLD docking program [33, 37–39]. In
the genetic algorithm, the following steps are applied: (i) A
population of potential binding poses at defined binding
pocket is set up at random; (ii) Each member of the popu-
lation is encoded as a chromosome, which contains infor-
mation about the mapping of protein–ligand interactions;
(iii) Each chromosome is assigned a fitness score based on its
predicted binding affinity and the chromosomes within the
population are ranked according to fitness; (iv) The popu-
lation of chromosomes is iteratively optimized. In this work,
the default genetic algorithm parameters were used (popu-
lations size 100, selection pressure 1.1, number of islands 5,
migrate 10, mutate 95, crossover 95, niche size 2, and
number of operation 107000).
Our work has been focused in recent years on the study
of the conformational analysis of the peptide hormone AII,
the competitive antagonist sarmesin, as well as other cyclic
peptide derivatives. Moreover, comparative studies of
sarmesin with losartan using a combination of NMR
spectroscopy and computational chemistry, proposed
important conformational, steric and electronic properties
for antagonist activity [22–28].
Herein, we report the synthesis, the in vitro biological
evaluation and the molecular docking results of the
designed non-peptide AT1 AII receptor antagonist V8 [29].
In this study, we optimized the synthetic strategies leading
to key 1,5 imidazole scaffolds as drug leads, in contrast to
our past efforts which involved a multi-step, poorly effi-
cient synthetic strategy [30]. A general alkylation protocol
has been developed in this research which facilitates the
synthesis of a 1,5 disubstituted imidazole derivative by
selective alkylation of N-3 nitrogen of 4(5)-butylimidazole
with biphenyl tetrazole moiety where the N-1 nitrogen is
temporarily protected by the trityl group. More impor-
tantly, the designed and synthesized compound V8 is
evaluated for in vitro antihypertensive activity which
showed similar binding affinity for AT1 receptor compared
to losartan (Fig. 1) in accordance with the molecular
docking theoretical calculations. Thus, our results con-
firmed that rational design can be of aid in systems where
The GOLD fitness function is made up of following
components: protein–ligand hydrogen bond energy; pro-
tein–ligand van der Waals (vdw) energy; ligand internal
vdw energy and ligand torsional strain energy (definitions
have been taken from the GOLD user manual [40]):
!
X X
Aij Bij
di8j di4j
Etotal
¼
ꢁ
protein ligand
X X
þ
½ðEda þ EwwÞ ꢁ ðEdw þ EawÞꢂ
protein ligand
(
)
ꢀ
ꢁ
X
X
Cij Dij
12
1
n
þ
ꢁ
þ
di6j
V 1 þ cosðjnjwÞ
:
ligand 2
jnj
d
ij
ligand
Block functions of two docking scores (GOLD fitness
score and ChemScore) with default values were used. A
block function has the following form [40]:
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751
8
9
=
A full list of the parameters used for GOLD docking
studies is provided in the supporting information.
1
if x ꢃ xideal
<
xꢁxideal
Bðx; xideal; xmaxÞ ¼ 1:0 ꢁ x
if xideal ꢃ x ꢃ xmax
if x [ xmax
:
maxꢁxideal
:
;
(ii) Glide/IFD: The receptor was mapped with grid-based
calculations. In grid-based calculation procedure the target
protein is embedded in a three-dimensional grid. Then, a
probe atom is sequentially located at each grid point, the
interaction energy between the probe atom and the target
atoms is computed, and the value is stored in the grid. The
energy of interaction of this single atom with the protein is
assigned to the grid point. [41] The active site was defined
0
In the GOLD implementation of ChemScore, the block
function can be also convoluted with a Gaussian function:
þ1
R
Bðx ꢁ u; xideal; xmaxÞgðu; rÞdu
B0 ðx; xideal; xmax; rÞ ¼
ꢁ1
þ1
R
gðu; rÞdu
˚
by 20 and 46 A inner and outer cubic grid boxes, centered
ꢁ1
ꢁu2=2r2
on the point that is the center of mass of residues K199 and
H256. The IFD protocol under the Schrodinger molecular
modeling package [35, 36] was used in order to eliminate
clashes between receptor and ligand atoms and for the
receptor to gain partial flexibility to the receptor. Before the
docking simulations, the complexes were submitted to the
protein preparation module of Schrodinger. Ligands were
constructed using the Schrodinger’s Maestro module and
then geometry optimization was performed for these
gðu; rÞ ¼ e
:
For example, hydrogen bonding is computed as the sum
over all possible donor–acceptor pairs, such that one atom
belongs to the protein and other belongs to the ligand. Each
term in the summation is the product of three Gaussian-
smoothed block functions. The purpose of the block
functions is to reduce the contribution of a hydrogen
bond according to how much its geometry deviates from:
(i) ideal H…A distance (ii) ideal D-H…A angle and (iii)
ligands using Polak-Ribiere conjugate gradient (PRCG)
-1
minimization (0.0001 kJA mol-1, convergence criteria).
˚
ideal directionality with respect to the acceptor atom [40].
P
DGHꢁbond
¼
B0 ðDr; Drideal; Drmax; rrÞꢀ
Protonation states of ligands and residues were created
using LigPrep and Protein Preparation modules under the
Schrodinger package at neutral pH. IFD uses the Glide
docking program to account the ligand flexibility and the
refinement module and the Prime (v.1.6) program [35, 36]
to account for flexibility of the receptor. Schrodinger’s IFD
protocol model uses the following steps (the description
below is taken from the IFD user manual [35, 36]): (i)
Constrained minimization of the receptor with an RMSD
alldonorꢁacceptor pairs
B0 ð Da; Daideal; Damax; raÞ ꢀ B0 ꢄ ðDb; Dbideal; Dbmax; rbÞ
(r in the above equation defines the Gaussian smearing
sigma associated with each term).
Protein binding site can be mapped in several ways in
GOLD (i.e. if r is the radius, the binding site will be defined
˚
as all atoms lie within r A of the specified point (this can be
done in several ways, e.g. by specifying the approximate
centre of the binding site and taking all atoms that lie within
a specified radius of this point or from an atom in receptor’s
residues). In this study, binding site was constructed using
˚
cutoff of 0.18 A. (ii) Initial Glide docking of each ligand
using soft potentials (0.5 van der Waals radii scaling of non-
polar atoms of ligands and receptor using partial charge
cutoff of 0.15). (iii) Derived docking poses were refined
using the Prime Induced Fit module of Schrodinger. Resi-
˚
r = 15 A from a well known active site residue K199
(from its terminal N atom). In GOLD docking algorithm
one or more side chains of residues at the active site can be
treated as flexible. Thus, active site residues S109, F182,
Y184, K199, N200, W253, H256, Q257, T287 and I288 are
treated to be flexible. Each flexible side chain will be
allowed to undergo torsional rotation around one or more of
its acyclic bonds during docking. Once a side chain has
been selected, it must be required to define one or more
allowed rotamers. Each rotamer specifies the torsion angles
that are permitted to vary, and the allowed values or ranges
of values for those torsion angles. In this study, ten crucial
amino acids in binding site were selected and treated their
side chains as flexible. Throughout the docking, the defined
residues side chains are rotated with 10ꢁ increment and
scanning by 360ꢁ and aimed to find the optimized inter-
actions between docked ligand and receptor residue. By this
way, the optimized side chain conformations of residues are
determined.
˚
dues within 5.0 A of ligand poses were minimized in order
to form suitable conformations of poses at the active site of
the receptor. (iv) Glide re-docking of each protein–ligand
complex. Full list of used parameters for Glide docking
studies were provided in the supporting information.
Prediction of pharmacokinetic properties
QikProp module (v.3.1) under Schrodinger molecular
modeling package was used to produce predicted phar-
macokinetic properties of new compound V8.
In vitro binding experiments
The binding buffer solution was comprised of 20 nM Tris–
HCl, 100 mM NaCl, 5 mM MgCl2 and was adjusted to
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pH = 7.4. For the binding studies, 0.1% BSA was added in
the buffer. The buffers were stored at 4 ꢁC between the
experiments. Five drug concentrations for the experiments,
ranging between 10-5 and 10-9 in ascending powers of
ten, were used and ran at triplicates. All drugs were DMSO
soluble and when diluted, the DMSO concentration did not
exceed 1% v/v.
iodine. Flash chromatography was performed using silica
gel (230–400 mesh, Merck). Purity of the intermediate and
the final products was determined by analytical RP-HPLC,
followed by UV detection at 230, 254 nm and identified by
1
ESI–MS and H NMR.
Synthesis of the alkylating agents (3), (4) [42–44]
The radioligand used for the experiments was [125I]
Sar1,Ile8-AII, a non specific peptide appropriate for both
AT1 and AT2 receptors. A constant concentration of radi-
oligand of 0.1 nM (&40,000 cpm) was maintained
throughout. The total binding is defined as the binding in
the absence of competitive compounds. The non-specific
binding in the presence of 10-5 M losartan was about
300 cpm.
M-(Trityl)-5-[40-(bromomethyl)-biphenyl-2-yl]tetrazole (3)
1
Rf = 0.40 (Hex/EtOAc, 85:15); mp 135–137 ꢁC; H NMR
(CDCl3): d 7.83–6.82 (m, 23 H), 4.62 (s, 2 H); ESI–MS m/z
[L ? H-Tr]? 315.48, [Tr] 242.95.
M-(Benzyl)-5-[40-(bromomethyl)-biphenyl-2-yl]
tetrazole (4)
Two kinds of membranes were used for the binding
experiments. Membranes containing human AT1 receptor
purchased from PerkinElmer Life Sciences, Inc., Boston,
MA, USA and membranes containing either AT1 or AT2
receptors kindly provided by Prof. A. Balmforth, Bio-
medical Sciences, University of Leeds. 23.5 lg of mem-
brane protein was used in each binding assay. Binding
assay comprised of: 25 lL radioligand, 25 lL test com-
pound or buffer and 50 lL membrane sample. Incubations
were carried out at room temperature for at least 1.5 h. The
samples were harvested using a Brandel Cell Harvester on
GF/B filters pre-soaked in 1% v/v polyethylimine and
washed with chilled binding. The radioactivity retained on
the filters was determined on a Packard RiasStar 5405
gamma counter. Binding assays with intact cells were
carried out in a similar manner to that given above except
that the buffer used was made isotonic by incubation of
150 mM NaCl. 105 cells in 50 lL buffer replaced the use
of membranes. Washing of filters was also carried out
using isotonic buffer.
1
Rf = 0.47 (Hex/EtOAc, 1:1); mp 135–138 ꢁC; H NMR
(CDCl3): d 7.69–6.77 (m, 13 H), 4.85 (s, 2 H), 4.47 (s, 2
H); ESI–MS m/z [L ? H]? 406.29.
4-Butyl-1-(trityl)-imidazole (6)
To a solution of the imidazole derivative 5 (80.5 mmol) in
dry dichloromethane (DCM, 240 mL), triethylamine (TEA,
27.8 mL, 201.25 mmol), trityl chloride (TrCl, 2.47 g,
88.5 mmol) were added and stirred at room temperature for
1 h. The mixture was diluted in H2O, extracted with DCM
and the organic phase was washed with 5% w/v NaHCO3,
H2O, dried over Na2SO4, filtered and concentrated.
Recrystallization from diisopropylethyl ether (DIE) affor-
ded 6. Yield 88%; Rf = 0.45 (Hex/EtOAc, 20:80); mp 97-
100 ꢁC; 1H NMR (CDCl3) d 8.25 (s, 1H), 7.49–7.10 (m, 15
H), 6.69 (s, 1 H), 2.75 (t, 2 H, J = 7 Hz), 1.62 (quint. 2 H,
J = 7 Hz), 1.36 (sext, 2 H J = 7 Hz), 0.92 (t, 3 H,
J = 7 Hz); ESI–MS m/z [L ? H]? 367.30; Anal. Calcd
for C26H26N2: C, 85.21; H, 7.15; N, 7.64; found: C, 85.14;
H, 6.92; N, 7.48.
Chemistry
Starting materials were purchased by Aldrich and used as
received. Melting points were determined with an Elec-
trothermal 9100 melting point apparatus and are uncor-
rected. Infrared spectra were recorded on a Perkin-Elmer
General procedure for alkylation and subsequent
detritylation
1
16PC spectrophotometer. H NMR spectra were obtained
To a solution of 6 (9.95 g, 27.2 mmol) in dry DCM
(80 mL), 3 (16.6 g, 30 mmol) was added and the resulting
mixture was stirred at room temperature for 24 h under
nitrogen. The solution was concentrated and precipitation
from diethyl ether (DEE) afforded 7. Then, 7 was added to
a solution (70 mL) of 40% trifluoroacetic acid (TFA) in
DCM, triethylsilane (TES, 8.8 mL, 54.4 mmol) was added
dropwise and the mixture was stirred for 1 h at ambient
temperature. The solvent was concentrated and recrystal-
lization from DEE afforded 8. Compound 12 was prepared
by a similar procedure.
in CDCl3, CD3OD and DMSO-d6 on a Bruker Avance
DPX-400 spectrometer. Chemical shifts are given in d
values (ppm) using tetramethylsilane (TMS) as the internal
standard, and coupling constants (J) are given in Hertz
(Hz). Electrospray-ionization mass spectra (ESI–MS) were
obtained on a Electrospray Platform LC instrument.
Microanalyses were performed on a Carlo Erba EA 1108
CHNS elemental analyzer. Analytical thin layer chroma-
tography (TLC) was performed on silica gel 60 F254 plates
from Merck reagents and visualized by UV irradiation and
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753
Trifluoroacetate salt of 5-butyl-1-[[20-(2H-tetrazol-
5-yl)biphenyl-4-yl]methyl]imidazole (8)
modification of the synthetic procedure (37% aq. HCHO,
DMF, 140 ꢁC, 8 h).
5-Butyl-2-hydroxymethyl-1-[[20-[[N-(2-chlorotrityl)]
tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole (10)
Tield 63%; Rf = 0.50 (1-BuOH/AcOH/H2O, 4:1:1); mp
68–70 ꢁC; 1H NMR (CDCl3) d 8.91 (s, 1 H), 7.71–7.23 (m,
9 H), 5.45 (s, 2 H), 2.60 (t, 2 H, J = 7 Hz), 1.66 (quint, 2
H, J = 7 Hz), 1.41 (sext, 2 H, J = 7 Hz), 0.95 (t, 3 H,
J = 7 Hz); ESI–MS m/z [L ? H]? 359.30; Anal. Calcd
for C23H23F3N6O2: C, 58.47; H, 4.91; N, 17.79; found: C,
58.34; H, 4.78; N, 17.92.
Yield 52%; Rf = 0.27 (CHCl3/MeOH, 97:3); mp 191–
193 ꢁC; IR (KBr) 3,366, 3,134, 2,940, 2,926, 2,854 cm-1
;
1H NMR (CDCl3) d 7.97–6.74 (m, 23 H), 4.95 (s, 2 H),
4.45 (s. 2 H), 2.30 (t, 2 H, J = 7 Hz), 1.50 (quint, 2 H,
J = 7 Hz), 1.27 (sext, 2 H, J = 7 Hz), 0.85 (t, 3 H,
J = 7 Hz); ESI–MS m/z [L ? H]? 666.81; Anal. Calcd
for C41H37ClN6O: C, 74.03; H, 5.61; N, 12.63; found: C,
74.12; H, 5.55; N, 12.53.
5-Butyl-1-[[20-[(N-benzyl)tetrazol-5-yl]biphenyl-
4-yl]methyl]imidazole (12)
Yield 61%; Rf = 0.34 (CHCl3/MeOH, 95:5); mp 69–71 ꢁC;
1H NMR (CDCl3) d 7.65–6.78 (m, 15 H), 5.04 (s, 2 H), 4.84
(s, 2 H), 2.37 (t, 2 H, J = 7 Hz), 1.54 (quint, 2 H, J = 7 Hz),
1.35 (sext, 2 H, J = 7 Hz), 0.89 (t, 3 H, J = 7 Hz); ESI–MS
m/z [L ? H]? 449.38; Anal. Calcd for C30H29F3N6O2: C,
64.05; H, 5.20; N, 14.94; found: C, 64.19; H, 4.92; N, 14.78.
5-Butyl-2-hydroxymethyl-1-[[20-[(N-benzyl)tetrazol-
5-yl]biphenyl-4-yl]methyl]imidazole (13)
Yield 70%; Rf = 0.32 (CHCl3/MeOH, 96:4); IR (KBr)
3,358, 3,128, 2,944, 2,926, 2,852 cm-1; 1H NMR (DMSO-
d6) d 7.73–6.84 (m, 13 H), 6.18 (s, OH), 5.39 (s, 2 H), 5.12
(s. 2 H), 4.72 (s. 2 H), 2.38 (t, 2 H, J = 7 Hz), 1.40 (quint,
2 H, J = 7 Hz), 1.24 (sext, 2 H, J = 7 Hz), 0.80 (t, 3 H,
J = 7 Hz); ESI–MS m/z [L ? H]? 479.50; Anal. Calcd
for C29H30N6O: C, 72.78; H, 6.32; N, 17.56; found: C,
72.49; H, 6.28; N, 17.40.
5-Butyl-1-[[20-[[N-(2-chlorotrityl)]tetrazol-5-yl]biphenyl-
4-yl]methyl]imidazole (9)
To a solution of 8 (8.1 mmol) in dry DCM (25 mL), N,N-
diisopropylethylamine (DIPEA, 4.2 mL, 24.3 mmol) was
added and the mixture was cooled to -10 ꢁC under
nitrogen. Then, 2-chlorotrityl chloride (ClTrCl) (2.8 g,
8.9 mmol) was added in three portions and the mixture was
allowed to warm at room temperature. After 1 h, the
mixture was diluted in H2O, extracted with DCM and the
organic phase was washed successively with 5% w/v citric
acid, H2O, dried over Na2SO4 and concentrated. Purifica-
tion by flash chromatography (CHCl3/MeOH, 97:3) affor-
ded 9. Yield 65%; Rf = 0.43 (CHCl3/MeOH, 97:3); mp
Trifluoroacetate salt of 5-butyl-2-hydroxymethyl-
1-[[20-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole
(V8)
10 (0.3 mmol) was added to a solution (1 mL) of 20% TFA
in DCM, TES (0.048 mL, 0.3 mmol), was added dropwise
and stirred for 1 h at ambient temperature. The reaction
mixture was concentrated and recrystallization from DEE
afforded V8. Yield 80%; Rf = 0.53 (1-BuOH/AcOH/H2O,
4:1:1); mp 109–111 ꢁC; IR (KBr) 3,366, 3,126, 2,954, 2,926,
2,862, 1,670 cm-1; 1H NMR (CD3OD) d 7.73–7.13 (m, 9 H),
5.45 (s, 2 H), 4.83 (s, 2 H), 2.56 (t, 2 H, J = 7 Hz), 1.64
(quint, 2 H, J = 7 Hz), 1.41 (sext, 2 H, J = 7 Hz), 0.95 (t, 3
H, J = 7 Hz); ESI–MS m/z [L ? H]? 389.13; Anal. Calcd
for C24H25F3N6O3: C, 57.37; H, 5.01; N, 16.72; found: C,
57.53; H, 5.17 N, 16.61.
1
87–88 ꢁC; H NMR (CDCl3) d 7.97–6.74 (m, 24 H), 4.95
(s, 2 H), 2.61 (t, 2 H, J = 7), 1.54 (quint, 2 H, J = 7), 1.33
(sext, 2 H, J = 7), 0.89 (t, 3 H, J = 7); ESI–MS m/z
[L ? H]? 636.72; Anal. Calcd for C40H35ClN6: C, 75.63;
H, 5.55; N, 13.23; found: C, 75.42; H, 5.33; N, 13.18.
General procedure for hydroxymethylation
In a sealed glass tube a mixture of 9 (0.94 mmol), 37%
aqueous formaldehyde solution (aq. HCHO, 0.24 mL,
8.46 mmol), DIPEA (1.15 mL, 6.58 mmol) and N,N-
dimethylformamide (DMF, 2 mL) was heated for 16 h at
85 ꢁC. The reaction mixture was diluted in H2O and
extracted with EtOAc. The organic phase was washed
successively with 5% w/v citric acid, H2O, dried over
Na2SO4 and filtered and concentrated. Purification by flash
chromatography (CHCl3/MeOH, 97:3) afforded 10. Com-
pound 13 was prepared by a similar procedure, with a slight
Trifluoroacetate salt of 5-butyl-2-hydroxymethyl-
1-[[20-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole
(V8)
To a solution of 13 (0.42 mmol) in AcOH (3 mL), 10% Pd/
C (0.06 g) was added as catalyst and the mixture was stirred
under H2 at room temperature. After 24 h, the reaction
mixture was filtered through a pad of Celite to remove the
catalyst and the filtrate was concentrated. Recrystallization
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from DEE afforded V8. Yield 75%. The analytical data
were given above.
Results and discussion
Computational analysis studies
Docking results
Experimental and predicted IC50 values of compound V8
and losartan are tabulated at Table 1. Both docking algo-
rithms have confirmed that compound V8 has high binding
affinity at the AT1 receptor. Although ChemScore under
estimates and Glide/Induced Fit Docking (IFD) binding
score overestimates the compounds’ binding energies at
AT1 receptor, they are able to show that compound V8 has
about 3–4 fold lower binding affinity than losartan. GOLD/
ChemScore and Glide/IFD docking algorithms have shown
that compound V8 has 2.7 and 3.9 fold lower affinity than
that of losartan, respectively. These values are in good
agreement with experimental result (3.3 fold). GOLD fit-
ness scores for both compounds are similar which is rea-
sonable since both ligands have very similar topology. It
must be noted that used described docking programs are
not applicable to predict absolute DG or IC50 values
attributed to the lack in conformational sampling for
degrees of freedom in solute and receptor in docking
studies, the oversimplified treatment of environmental
effects and the lack of well-defined strategy for computa-
tions of the entropic components [45].
Fig. 2 a Glide docking poses: Binding interactions of compound V8.
b Losartan at the active site of AT1. c Superimposition of binding
poses of losartan and compound V8 (light colored bonds show
compound V8 and dark bonds show losartan. High ranking docking
poses of losartan and V8 ligands were used in superimposition.
Derived xyz coordinates of ligands at the binding site were extracted
from complex structures and superimposed)
Figures 2a and b illustrate the binding interactions of
best binding pose of compound V8 and losartan at the
active site of AT1 receptor produced by Glide/IFD docking
algorithm. Compound V8 forms H-bonds with T260, Q257,
Y113, G203; it has close contacts with amino acid residues
G196, K199, T260, W253 and G203; whereas losartan
forms H-bonds with Q257, Y113 and H183 and it has close
contacts with T260, N200, H256, A181 and Y113. Derived
binding poses have been superimposed at Fig. 2c. (first
ranking docking poses of losartan and V8 ligands were
used in superimposition. Derived xyz coordinates of
ligands at the binding site were extracted from complex
structures and are superimposed). Both ligands show
similar orientation at the active site of the receptor except
the tetrazole and phenyl (bound to tetrazole) pharmaco-
phores. These pharmacophores in compound V8 and lo-
sartan orient in opposite directions at the active site. This is
expected since syn and anti conformations, as the combi-
nation of NMR studies with Molecular Modeling calcula-
tions showed, have similar low energy values reflecting the
flexibility of the tetrazole ring [28]. This flexibility is not
surprising to exist when V8 approaches the active site.
Table 1 Experimental vs. predicted IC50 values and docking scores of losartan and compound V8
GOLD SCORE
CHEM SCORE
GLIDE/IFDa
EXP. IC50 (nM)
DG (kcal/mol)
Predicted IC50 (nM)b
DG (kcal/mol)
Predicted IC50 (nM)
LOSARTAN
78.29
81.41
-9.81
-9.22
b
71.3
-12.30
-11.49
1.09
4.26
16.4 1.6
53.8 6.4
V8
191.9
a
Induced Fit Docking/XP module were used. Predicted IC50 values were calculated using the DG = RTlnIC50 equation, (T = 300 K)
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J Comput Aided Mol Des (2010) 24:749–758
755
Fig. 4 Binding studies of target derivative V8 using cell cultures.
Compound V8 has similar activity compared to losartan
Predicted pharmacokinetic properties of compound V8
Pharmacokinetic properties were predicted using QikProp
module of Schrodinger molecular modeling package. Qik-
Prop module has shown that all predicted pharmacokinetic
properties of V8 are inside the 95% range of available drug
database (*2,000 known drugs), (see supporting informa-
tion). Thus, the molecule can be a potential drug and more
extensive pharmacological studies are in progress.
In vitro studies
Binding experiments using two different sources of mem-
brane preparations containing the AT1 receptor, one
membrane preparation containing AT2 receptors and cell
cultures were performed in order to study the specificity of
V8. The experiments in membranes containing AT1
receptors and cell cultures were repeated on three occa-
sions and using triplicate determinations showed similar
results. Compound V8 was found to have high affinity for
the AT1 receptor and only *3-fold lower affinity than that
of losartan (IC50 values of 53.8 6.4 and 16.4 1.6 nM,
respectively, Fig. 4). In order to confirm that V8 similar to
losartan has no binding affinity to AT2 receptor, experi-
ments were performed in membrane bilayers containing
solely this receptor. The experiments indicated that V8 did
not have any competition for [125I]Sar1,Ile8-AII binding at
AT2 receptors. Therefore, compound V8 has similar
binding and biological properties as the prototype losartan.
Fig. 3 a GOLD docking poses: Binding interactions of compound
V8. b Losartan at the active site of AT1. c Superimposition of binding
poses of losartan and compound V8 (light colored bonds show
compound V8 and dark bonds show losartan. Top docking poses of
losartan and V8 ligands were used in superimposition. Derived xyz
coordinates of ligands at the binding site were extracted from
complex structures and superimposed
Figures 3a and b illustrate the binding interactions of the
best binding pose of compound V8 and losartan at the
binding site of the AT1 receptor produced by GOLD.
Compound V8 forms H-bonds with S109 and G203 and has
close contacts with K199, N200, G196, H256, Y184, A181,
V108, F182 and H183. Losartan forms multiple H-bonds
with K199, and it has close contacts with following amino
acids H183, N200, G196, Y184 and F182. GOLD docking
poses for compound V8 and losartan at the active site have
different orientations; losartan’s butyl side chain orients
inward to the receptor, whereas butyl side chain of com-
pound V8 orients outward (the same superimposition pro-
tocol explained for Glide was performed, Fig. 3c).
Different orientations of ligands’ binding poses at the
active site of the receptor show that binding site of AT1
may be able to form different conformations with similar
binding energy.
Synthesis of target compound V8
This research has focused on the design and synthesis of an
analogue that differs in the substitution pattern around the
imidazole ring compared to losartan. Thus, the alkyl chain
and hydroxymethyl group possess different topographical
position in an attempt to optimize the mimicry of lipophilic
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superimposition of the butyl chain with isopropyl group of
Ile5 in AII and to probe the significance of the position of
hydroxymethyl group. An established procedure was
employed in the preparation of biphenyl intermediate 3 and
4 (Scheme 1) [42, 43].
diisopropylethylamine (DIPEA) and selective chlorotrity-
lation of the tetrazole moiety with 2-chlorotrityl chloride
(Cl-TrCl) at -10 ꢁC led to 9. Hydroxymethylation at C-2
of the imidazole ring with 37% aqueous formaldehyde (aq.
HCHO) solution in a sealed glass tube at 85 ꢁC afforded 10
in 52% yield. Finally, deprotection of the 2-chlorotrityl
group with 20% TFA furnished the final compound V8.
We used 2-chlorotrityl instead of Tr group as tetrazole
protection because the former is less stable as protecting
group in nitrogen bearing moieties (primary and secondary
amines, imidazole, tetrazole, etc.) compared to the Tr
group [46–50]. Introduction of chlorine atom in the phenyl
ring of Tr group produces more stable carbocation through
hyperconjugation, compared to Tr carbocation. Therefore,
this group is more acid labile compared to Tr group and
more easily removed to afford the target compound V8.
A second more efficient synthetic route for compound
V8 was achieved, utilizing the brominated biphenyl benzyl
tetrazole derivative 4, through debenzylation of 13 with
hydrogenolysis via the shorter route depicted at Scheme 2.
In particular, alkylation of 6, removal of the Tr group and
hydroxymethylation of 12 led to 13. Benzyl (Bn) protection
of the tetrazole group permitted us to employ higher tem-
peratures (140 ꢁC) in the hydroxymethylation reaction
(yield 70%), due to the thermal stability compared to the 2-
chlorotrityl group. The latter began to cleave at tempera-
tures above 90 ꢁC at which equilibrium of optimum
hydroxymethylation/minimum detritylation was achieved.
Removal of the Bn group was performed by catalytic hy-
drogenolysis using 10% palladium in carbon (Pd/C) to
yield the final V8. Although the two experimental
The preparation of the target compound V8 is depicted
at Scheme 2. Firstly, selective tritylation [46–50] of N-1
nitrogen of compound 5 by trityl chloride (TrCl) in the
presence of triethylamnie (TEA) led to 6. Subsequent
alkylation of the N-3 nitrogen was carried out using the
brominated biphenyl trityl tetrazole derivative 3 as alkyl-
ating agent, afforded 7 in good yield (65–70%). The rela-
tive decrease in the yield was owed to the tendency of the
trityl group (Tr) to be eliminated during the alkylation,
giving a mixture of Np-substituted and Ns, Np-disubstituted
products [51]. Simultaneous removal of the Tr groups
using 40% trifluoroacetic acid (TFA) in dichloromethane
(DCM) in the presence of triethylsilane (TES) as scavenger
provided 8 as TFA salt. Neutralization with N,N-
Br
i
N
N
N
N
NH
NR
CN
N
N
2
R=Tr, 3
R=Bn, 4
1
Scheme 1 Reagents and conditions: (i) 1. TrCl or BnBr, TEA, DCM,
rt, 1 h; 2. N-bromosuccinimide, dibenzoyl peroxide, CCl4, reflux, 8 h
Bu
Bu
Bu
Bu
Bu
Bu
e1
b1
a
d1
c
N
N
N
N
N
N
N
N
HN
N
N
N
Tr
Tr
Br
HO
5
6
N
N
N
N
N
N
N
N
HN
N
N
N
Tr
N
ClTr
ClTr
N
N
N
b2
f
7
8
9
10
Bu
Bu
Bu
Bu
N
N
N
N
N
N
N
N
Tr
d2
e2
c
Br
HO
HO
N
N
N
N
N
N
N
N
HN
N
N
N
Bn
Bn
Bn
N
N
N
N
11
12
13
V8
Scheme 2 Reagents and conditions: (a) TrCl, TEA, DCM, rt, 1 h;
(b1). 3, DCM, rt, 24 h; (b2) 4, DCM, rt, 24 h; (c) 40% TFA in DCM,
TES, rt, 1 h; (d1) ClTrCl, DIPEA, DCM, -10 ꢁC, 1 h; (d2) 37% aq.
HCHO, DMF, 140 ꢁC, 8 h; (e1) 37% aq. HCHO, DIPEA, DMF
85 ꢁC, 16 h; (e2) 10% Pd/C, AcOH, H2, rt, 24 h; (f) 20% TFA in
DCM, TES, rt, 1 h
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J Comput Aided Mol Des (2010) 24:749–758
757
procedures of V8 are quite similar, the second approach is
more attractive because it leads to higher overall yield
through efficient shorter synthetic route.
the imidazole ring of the prototype drug losartan without
affecting significantly the bioactivity; (b) this tolerance of
the receptor can trigger the interest of medicinal chemists
to synthesize more active molecules based on modifica-
tions of imidazole ring; (c) the derived parallel results
between in vitro data and docking theoretical calculations
make this task reasonable.
Conclusions
In 1994, a model of AII based on NMR studies using 2D
ROESY techniques in receptor simulating environments
has been developed which involves an aromatic ring cluster
and consequently a charge relay system formed from the
triad of amino acids Tyr4-His6-Phe8 was suggested [52].
Another theoretical study showed that these three amino
acids which are a strict requirement for AII to exert its
agonist activity govern its conformation [28]. Comparative
nuclear magnetic resonance studies of the backbone
structure between peptide agonists and antagonists have
shown that agonists display ring clustering and form a
charge relay system [53] Such clustering is also present to
the competitive antagonist [Tyr(OMe)4]AII (sarmesin)
which lacks the potential of the charge relay system and the
form of the tyrosinate anion which is a strict requirement
for agonist activity in the proposed model [54, 55]. In
addition, the proposed conformation of AII and sarmesin
overlay the non-peptide AII receptor antagonist losartan
and its analogues when molecular modeling techniques and
superimposition studies are applied [28]. Furthermore, the
ring cluster conformation is supported by the design and
synthesis of novel constrained AII cyclic analogues
[Sar1,Lys3,Glu5]AII, [Sar1,Asp3,Glu5]AII, which possess
agonist activities when tested in the rat uterus assay and in
anesthetized rabbits [56, 57]. These potent cyclic analogues
were designed to have a major molecular feature, the
integrity of the ring cluster [58–60]. This model has
enabled us to further explore spatial characteristics of AII
pharmacophore groups and to design and synthesize non-
peptide losartan analogues. An important feature for
activity is the presence of at least a negative charge pro-
vided by a carboxylate group. Indeed, the antihypertensive
activity of losartan is largely due to a long-acting metab-
olite (EXP 3174), which is produced in vivo as a result of
the conversion of hydroxymethyl to carboxylate, a
molecular feature also met in AT1 antagonists of eprosartan
and valsartan [61].
In conclusion, this study shows a new, general method for
the synthesis of key 1,5 disubstituted imidazole intermedi-
ates, which could be used for the facile synthesis of imid-
azole based AII receptor antagonists. Reorientation of butyl
and hydroxymethyl substituents in losartan led to potent AT1
AII receptor antagonist. The synthesis was regioselective,
facile and high yielding, rendering it a cost effective process.
This convenient method allows the introduction of alkylating
agents bearing desired pharmacophore groups and this may
be a useful tool for the design and synthesis of potent sub-
stances for diverse pharmaceutical uses. This study indicates
that the hydroxymethyl group as well as the butyl chain is
essential for triggering activity.
Acknowledgments This work was supported by grants from the
Ministry of Development of Greece, General Secretariat of Research
and Technology, EPET II, 115/PENED, 1999/EPAN, 114 from the
Ministry of Education (postgraduate program ‘‘Medicinal Chemistry’’
EPEAEK). We also acknowledge NATO (Linkage Grant 974548). We
also acknowledge Prof. J. Findlay and Dr. Alan Cox from School of
Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT,
UK who kindly provided the laboratory facilities to Prof. T. Mavro-
moustakos a recipient of Royal Society scholarship. We also
acknowledge A. Suarez for her linguistic amendment of the manuscript.
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