Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid: Rational design, synthesis and biological evaluation

Article abstract of DOI:10.1007/s00726-010-0651-y

A series of o-, m- and p-benzyl tetrazole derivatives 11a-c has been designed, synthesized and evaluated as potential Angiotensin II AT1 receptor antagonists, based on urocanic acid. Compound 11b with tetrazole moiety at the m-position showed moderate, however, higher activity compared to the o- and p-counterpart analogues. Molecular modelling techniques were performed in order to extract their putative bioactive conformations and explore their binding modes.

Full text of DOI:10.1007/s00726-010-0651-y

Amino Acids (2011) 40:411–420
DOI 10.1007/s00726-010-0651-y
ORIGINAL ARTICLE
Towards non-peptide ANG II AT1 receptor antagonists
based on urocanic acid: rational design, synthesis
and biological evaluation
•
George Agelis Amalia Resvani Minos-Timotheos Matsoukas Theodore Tselios
•
•
•
•
Konstantinos Kelaidonis Dimitra Kalavrizioti Demetrios Vlahakos John Matsoukas
•
•
Received: 19 April 2010 / Accepted: 2 June 2010 / Published online: 6 July 2010
Ó Springer-Verlag 2010
Abstract A series of o-, m- and p-benzyl tetrazole
derivatives 11a–c has been designed, synthesized and
evaluated as potential Angiotensin II AT1 receptor antag-
onists, based on urocanic acid. Compound 11b with tetra-
zole moiety at the m-position showed moderate, however,
higher activity compared to the o- and p-counterpart ana-
logues. Molecular modelling techniques were performed in
order to extract their putative bioactive conformations and
explore their binding modes.
inactivation by an ACE inhibitor (Wyvratt and Patchett
1985) (Captopril, Enalapril etc.), a non-peptide ANG II
AT1 receptor blocker (Carini et al. 1991) (Losartan, Irbe-
sartan, Candesartan etc.) or a renin inhibitor (Aliskiren)
(Lam and Choy 2007), has been established as a successful
therapeutic approach for the treatment of hypertension and
other cardiovascular diseases (Dzau 1994). Experimental
evidence for ANG II, the primary effector component of
RAS, has supported a bioactive conformation characterized
by a charge relay system between Tyr hydroxyl, His
imidazole and Phe carboxylate (Matsoukas et al. 1994),
as well as spatial proximity of the three key amino acids
Tyr-His-Phe which seems to be responsible for agonistic
activity (Matsoukas et al. 2000; Turner et al. 1990). Thus,
conformational analysis using 2 D NMR techniques in
receptor-simulating environments has shown proximity of
the three key amino acid side chains and the formation of
tyrosinate anion has been demonstrated by nanosecond
time-resolved fluorescence studies (Turner et al. 1991;
Matsoukas et al. 1995).
Keywords AT1 receptor Á Urocanic acid Á
Pharmacophore Á Molecular modelling
Introduction
Renin–angiotensin System (RAS) is a well-known cascade
playing a pivotal role in cardiovascular homeostasis and its
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-010-0651-y) contains supplementary
material, which is available to authorized users.
The DuPont group was the first to develop Losartan
(DuP 753) (Chiu et al. 1990), an orally effective ANG II
receptor blocker, which is metabolized in vivo to the
more potent full antagonist EXP3174 (Wong et al. 1990).
Structure–activity relationships (SAR) studies have illus-
trated the importance of the tetrazole and phenyl groups of
Losartan (Carini et al. 1991; Duncia et al. 1990) for binding
to the AT1 receptor. Alkyl lipophilic substituents, such as
biphenyl group at the 1-position and a linear alkyl group at
the 2-position of the imidazole ring gave rise to compounds
of higher affinity due to a tight fit into the hydrophobic
pocket. On the other hand, remarkable binding affinity
emerges in the presence of acidic groups such as carboxyl
or its isoster tetrazole group which interacts with the
positive charge of the receptor binding site. Furthermore, a
G. Agelis (&) Á A. Resvani Á M.-T. Matsoukas Á T. Tselios Á
K. Kelaidonis Á J. Matsoukas (&)
Department of Chemistry, University of Patras,
26500 Patras, Greece
e-mail: aggelisgeorge@hotmail.com
J. Matsoukas
e-mail: imats@chemistry.upatras.gr
D. Kalavrizioti
Department of Pharmacology, School of Medicine,
University of Patras, 26500 Patras, Greece
D. Vlahakos
Department of Internal Medicine, ‘ATTIKON’ University
Hospital, Athens, Greece
123

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G. Agelis et al.
nitrogen atom at the 3-position enhances the activity through
hydrogen bonding to the receptor (Thomas et al. 1992).
On the basis of these findings, we designed, synthesized
and evaluated a series of benzyl tetrazole compounds 11a–c
based on urocanic acid as potential ANG II receptor antago-
nists. The reason we used urocanic acid as a template was to
replace the imidazole butyl group with an ester group similar
tothe oneinOlmesartan(Yanagisawa etal. 1996) in whichthe
aliphatic moiety is the bulky ester group (5-methyl-2-oxo-1,2-
dioxol-4-yl)methyl which in vivo is metabolized to the car-
boxyl moiety. In particular, replacement of the butyl group of
Losartanwithpropanoicmethylestermayenhance theaffinity
and act as a prodrug (Kubo et al. 1993). The biphenyl tetrazole
functionality of Losartan was replaced by the phenyl tetrazole
group, positioning tetrazole moiety at the o-, m- and p-posi-
tion. The reason for the shortening of the biphenyl group was
to evaluate the ability of tetrazole group to trigger activity
located on a phenyl group instead of a biphenyl moiety and on
the other hand the ability of a single phenyl group to interact
with the receptor.
Electrospray Platform LC instrument HRMS experiments
were carried out in a micrOTOF-II (University of Notre
Dame, Indianapolis, USA) mass spectrometer equipped
with an ion spray ionization source. Microanalyses were
performed on a Carlo Erba EA 1108 CHNS elemental
analyser in the Laboratory of Instrumental Analysis of the
University of Patras. All reactions were carried out in dry
solvents. TLC was performed on silica gel 60 F254 plates
(Merck, Germany). Purification of final products was per-
formed with Waters Preparative HPLC (Waters Prep LC
Controller) using SunFire Prep C18 column (50 9 100 mm)
with 5 lm packing material. Separation was performed with
stepped linear gradient from 5% acetonitrile to 90% acetoni-
trile in 50 min with a flow rate of 9 mL/min. Supplementary
data are also available.
General synthesis of alkyl bromides 5a–c
A solution of 1a (7.8 mL, 66.24 mmol), NaN3 (4.31 g
66.24 mmol), Sn(n-Bu)3Cl (19.76 mL, 72.86 mmol) in dry
Tol (35 mL) was refluxed for 4 days. After completion of the
reaction, the solvent was evaporated resulting in a colourless
oil 2a used in the next reaction without further purification.
Compound 2a (20.86 g, 46.37 mmol) was dissolved in a
mixture of THF (60 mL), H2O (20 mL), 2 N HCl (26 mL,
52 mmol) and stirred at RT. After 16 h, the solvents were
concentrated and the residue was diluted in H2O (80 mL),
extracted with EtOAc (3 9 100 mL) andthe organic layer was
washed with Brine, dried andconcentrated. The oily residue 3a
(0.77 g, 44.05 mmol) was dissolved in DCM (110 mL), TEA
(15.50 mL, 110.12 mmol), TrtCl (13.47 g, 48.45 mmol) were
added and stirred at RT for 1 h. The mixture was diluted with
DCM, washed successively with 5% w/v NaHCO3 (93), H2O
(92), dried over Na2SO4 and concentrated. Recrystallization
from diisopropyl ether (DIE) afforded 4a as a white solid.
A mixture of 4a (14.80 g, 37.0 mmol), MBS (6.58 g,
37.0 mmol), DBP (0.89 g, 3.7 mmol) in CCl4 (100 mL) was
refluxed for 8 h. The mixture was allowed to cool at 40°C, the
yellow solid was filtered off and the filtrate was concentrated in
vacuo. The oily residue was recrystallized from DIE to afford
5a as a pure white solid (13.85 g, 78%). Compounds 5b–c
were prepared by a similar procedure.
Molecular modelling techniques were applied in order to
extract the possible bioactive conformations of the
designed compounds. The putative energy minimum con-
formations of analogues 11a–c (Fig. 1) were superimposed
with the proposed bioactive conformation of EXP3174
(Fig. 5) in order to gain information on the orientation of
their pharmacophore groups and were docked to the AT1
receptor, to understand their binding properties. The syn-
thesis of the target compounds 11a–c included a facile few-
step method in high yields. In vitro and in vivo experiments
were performed in order to evaluate the antihypertensive
activity of these synthesized analogues. Compound 11b
with benzyl tetrazole moiety at the m-position showed
moderate, however, higher activity compared to the o- and
p-counterpart analogues (Fig. 4).
Materials and methods
General remarks
Starting materials were purchased by Aldrich and used as
received. Melting points were determined with a Stuart
Data for compound 4a: Yield: 84%; mp 140–142°C; ESI–
1
1
SMP 10 apparatus and are uncorrected. H NMR and 13C
MS (m/z): 160.37 (M ? H?-Trt), 243.23 (Trt); H MMR
NMR spectra were recorded on a Bruker Avance DPX
spectrometer at 400.13 and 100.62 MHz, respectively.
Chemical shifts are reported in units, parts per million
(ppm) downfield from tetramethylsilane (TMS) and cou-
pling constants (J) are given in Hertz (Hz). HPLC analysis
was performed on a Alliance Waters 2695 equipped with a
Waters 2996 Photodiode Array Detector UV–vis, using a
XBridge C18 column (4.6 9 150 mm, 3.5 lm). Electro-
spray ionization mass spectra (ESI–MS) were obtained on a
(400 MHz, CDCl3): d (ppm) 8.05 (d, 1H, J = 7.1 Hz), 7.33–
7.14 (m, 18H), 2.48 (s, 3H); 13C NMR (100 MHz, CDCl3): d
(ppm) 164.13, 140.71, 139.24, 137.58, 136.33, 131.88,
129.91, 129.50, 128.40, 127.96, 125.59, 126.52, 125.94,
81.98, 21.74; Anal. Calcd for C27H22N4: C, 80.57, H, 5.51, N,
13.92. Found: C, 80.66, H, 5.63, N, 14.05.
Data for compound 4b: Yield: 85%; mp 139–141°C;
ESI–MS (m/z): 160.33 (M ? H?-Trt), 242.85 (Trt); 1H
NMR (400 MHz, CDCl3): d (ppm) 7.97–7.93 (m, 2H),
123

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid
413
Fig. 1 Energy minima of
EXP3174 and compounds
11a–c after molecular
modelling simulations which
consisted of Energy
Minimization, Molecular
Dynamics and Energy
Minimization runs sequentially
for a total time of 1 ns
7.37–7.16 (m, 17H), 2.41 (s, 3H); 13C NMR (100 MHz,
CDCl3): d (ppm) 164.55, 141.79, 138.91, 131.40, 130.68,
129.78, 129.48, 129.06, 128.68, 128.39, 128.15, 127.96,
127.79, 127.63, 127.17, 124.59, 83.44, 21.73; Anal. Calcd
for C27H22N4: C, 80.57, H, 5.51, N, 13.92. Found: C,
80.43, H, 5.41, N, 13.78.
141.65, 139.94, 131.35, 130.66, 129.78, 128.77, 128.44,
128.30, 128.21, 128.17, 127.83, 127.64, 127.47, 124.78,
83.66, 33.19; Anal. Calcd for C27H21BrN4: C, 67.37, H,
4.40, N, 11.64. Found: C, 67.43, H, 4.31, N, 11.54.
Data for compound 5c: Yield: 78%; mp 136–138°C; ESI–
MS (m/z): 239.17 (M ? H?-Trt), 243.10 (Trt); HRMS-ESI
(m/z): [M ? Na]? calcd for C27H21BrN4Na, 503.0847; found,
503.08336;1HNMR(400 MHz,CDCl3):d(ppm)8.13(d, 2H,
J = 8.3 Hz) 7.49–7.16 (m, 17H), 4.52 (s, 2H); 13C NMR
(100 MHz, CDCl3): d (ppm) 163.53, 146.86, 141.32, 139.88,
132.45, 130.31, 130.09, 129.51, 129.30, 128.39, 127.94,
127.82, 127.50, 127.34, 127.28, 127.10, 83.26, 32.85; Anal.
Calcd for C27H21BrN4: C, 67.37, H, 4.40, N, 11.64. Found: C,
67.28, H, 4.62, N, 11.49.
Data for compound 4c: Yield: 87%; mp 146–148°C; ESI–
1
MS (m/z): 160.34 (M ? H?-Trt), 242.75 (Trt); H NMR
(400 MHz, CDCl3) d (ppm) 8.03 (d, 2H, J = 8.0 Hz), 7.35–
7.16 (m, 17H), 2.40 (s, 3H); 13C NMR (100 MHz, CDCl3): d
(ppm) 164.14, 141.45, 140.42, 130.34, 129.48, 128.32,
127.95, 127.78, 127.33, 127.00, 124.76, 83.04, 21.53; Anal.
Calcd for C27H22N4: C, 80.57, H, 5.51, N, 13.92. Found: C,
80.72, H, 5.69, N, 13.82.
Data for compound 5a: Yield: 78%; mp 135–137°C;
ESI–MS (m/z): 239.17 (M ? H?-Trt), 243.23 (Trt);
HRMS-ESI (m/z): [M ? Na]? calcd for C27H21BrN4Na,
Methyl urocanate 7
1
503.0847; found, 503.0835; H NMR (400 MHz, CDCl3):
A mixture of urocanic acid 6 (1 g, 7.24 mmol), anhydrous
Na2SO4 (0.14 g, 1 mmol) and conc. H2SO4 (1 mL) in dry
MeOH (15 mL) was refluxed for 24 h. After completion of
the reaction the solid Na2SO4 was filtered off, the solvent
was removed in vacuo and the residue was diluted in
EtOAc, washed successively with saturated NaHCO3/H2O
(93) and Brine (91), dried and concentrated. Precipitation
with petroleum ether (40–70°C) afforded pure 7 as a white
solid (0.99 g, 90%).
d (ppm) 8.19–8.17 (m, 1H), 7.49–7.19 (m, 18H), 4.92 (s,
2H); 13C NMR (100 MHz, CDCl3): d (ppm) 162.90,
141.28, 136.74, 131.54, 130.44, 130.36, 130.15, 128.94,
128.41, 127.94, 127.85, 127.29, 126.34, 83.44, 32.42;
Anal. Calcd for C27H21BrN4: C, 67.37, H, 4.40, N, 11.64.
Found: C, 67.58, H, 4.56, N, 11.78.
Data for compound 5b: Yield: 79%; mp 137–139°C;
ESI–MS (m/z): 239.07 (M ? H?-Trt), 243.16 (Trt);
HRMS-ESI (m/z): [M ? Na]? calcd for C27H21BrN4Na,
Data for compound 7: mp 92–94°C; ESI–MS (m/z):
153.22 (M ? H?); 1H MMR (400 MHz; CD3OD): d (ppm)
7.78 (s, 1H), 7.61 (d, 1H, J = 16.0 Hz), 7.43 (s, 1H), 6.44
(d, 1H, J = 16.0 Hz), 4.85 (s, 3H); 13C NMR (100 MHz,
1
503.0847; found, 503.0833; H NMR (400 MHz, CDCl3):
d (ppm) 8.10–8.08 (m, 1H), 7.50–7.16 (m, 18H), 4.54 (s,
2H); 13C NMR (100 MHz, CDCl3): d (ppm) 163.91,
123

414
G. Agelis et al.
CD3OD): d (ppm) 168.14, 137.43, 135.25, 134.35, 124.0,
114.35, 50.61. All data were consistent with literature
(Pirrung and Pei 2000).
furnished pure 11a as a white foam (0.49 g, 80%). Com-
pounds 11b–c were prepared by a similar procedure.
Data for compound 11a: ESI–MS (m/z): 313.30
(M ? H?); HRMS-ESI (m/z): [M ? H]? calcd for
C15H17N6O2, 313.1413; found, 313.1401; 1H MMR
(400 MHz, CD3OD): d 8.54 (s, 1H), 7.99–7.97 (m, 1H),
7.66–7.61 (m, 2H), 7.40–7.38 (m, 1H), 7.35 (s, 1H), 5.80 (s,
2H), 3.65 (s, 3H), 2.93 (t, 2H, J = 7.2 Hz), 2.69 (t, 2H,
J = 7.2 Hz); 13C NMR (100 MHz, CD3OD): d (ppm)
172.72, 157.78, 135.35, 134.87, 132.17, 131.30, 130.54,
130.26, 129.97, 126.19, 117.24, 51.37, 49.24, 31.26, 18.88;
Anal. Calcd for C17H17F3N6O4: C, 47.89, H, 4.02, N, 19.71.
Found: C, 47.96, H, 4.11, N, 19.62.
Methyl 3-(imidazol-4-yl)propionate 8
A solution of 7 (1 g, 6.58 mmol) in MeOH (10 mL) was
introduced in a hydrogenation flask and 0.10 g of Pd–C
(10%) was added as catalyst. The mixture was stirred under
H2 for 8 h at RT and after completion of the reaction, the
catalyst was filtered through a pad of Celite and the filtrate
was concentrated in vacuo to furnish a white solid 8 (0.96 g,
95%) which showed adequate purity for the next step.
Data for compound 8: mp 95–97°C; ESI–MS (m/z):
155.41 (M ? H?); 1H MMR (400 MHz, CD3OD): d (ppm)
7.58 (s, 1H), 6.81 (s, 1H), 3.66 (s, 3H), 2.88 (t, 2H,
J = 7.2 Hz), 2.66 (t, 2H, J = 7.2 Hz); 13C NMR (100 MHz,
CD3OD): d (ppm) 173.59, 135.96, 134.53, 115.75, 50.67,
33.27, 21.76. All data were consistent with literature (Pir-
rung and Pei 2000).
Data for compound 11b: ESI–MS (m/z): 313.30
(M ? H?); HRMS-ESI (m/z): [M ? H]? calcd for
C15H17N6O2, 313.1413; found, 313.1403; 1H MMR
(400 MHz, CD3OD): d (ppm) 8.99 (s, 1H), 8.04 (d, 1H,
J = 7.8 Hz), 7.94 (s, 1H), 7.64 (t, 1H, J = 7.8 Hz), 7.50 (d,
1H, J = 7.8 Hz), 7.42 (s, 1H), 5.60 (s, 2H), 3.60 (s, 3H), 2.93
(t, 2H, J = 7.2 Hz), 2.64 (t, 2H, J = 7.2 Hz); 13C NMR
(100 MHz, CD3OD): d(ppm) 172.70, 157.65, 135.90, 135.60,
134.65, 130.44, 129.98, 127.46, 127.13, 126.09, 117.95,
51.34, 49.88, 31.32, 18.84; Anal. Calcd for C17H17F3N6O4: C,
47.89, H, 4.02, N, 19.71. Found: C, 47.81, H, 4.19, N, 19.57.
Data for compound 11c: ESI–MS (m/z): 313.45 (M ? H?);
HRMS-ESI (m/z): [M ? H]? calcd for C15H17N6O2, 313.1413;
found, 313.1406; 1H MMR (400 MHz, CD3OD): d (ppm) 8.98
(s, 1H), 8.09 (d, 2H, J = 8.2 Hz), 7.48 (d, 2H, J = 8.2 Hz),
7.42 (s, 1H), 5.59 (s, 2H), 3.63 (s, 3H), 2.91 (t, 2H, J = 7.2 Hz),
2.66 (t, 2H, J = 7.2 Hz); 13C NMR (100 MHz, CD3OD): d
(ppm) 172.32, 157.67, 136.68, 135.49, 134.27,128.17, 127.62,
125.72, 117.47, 50.97, 49.42, 30.86, 18.42; Anal. Calcd for
C17H17F3N6O4: C, 47.89, H, 4.02, N, 19.71. Found: C, 47.92,
H, 3.95, N, 19.86.
Methyl 3-(1-tritylimidazol-4-yl)propionate 9
To a solution of compound 8 (1.0 g, 6.53 mmol) in DCM
(7 mL) were added TEA (1.80 mL, 13.06 mmol), TrtCl
(2.0 g, 7.18 mmol) and the resulting solution was stirred at
RT for 2 h. The mixture was diluted with DCM, washed
successively with 5% w/v NaHCO3 (93), H2O (92), dried
over Na2SO4 and concentrated. Recrystallization from DIE
afforded 9 (2.25 g, 87%) as a white solid.
Data for compound 9: mp 142–143°C; ESI–MS (m/z):
1
397.33 (M ? H?); H MMR (400 MHz, CDCl3): d (ppm)
8.02 (s, 1H), 7.42–7.08 (m, 15H), 6.75 (s, 1H), 3.62 (s, 3H),
3.07 (t, 2H, J = 7.2 Hz), 2.85 (t, 2H, J = 7.2 Hz); 13C
NMR (100 MHz, CDCl3): d (ppm) 173.05, 141.10, 136.45,
129.94, 129.16, 128.86, 128.86, 128.29, 84.33, 51.97,
33.42, 22.06. All data of compounds were consistent with
literature (Pirrung and Pei 2000).
Molecular modelling
Molecular dynamics and minimization
General synthesis of alkylated compounds 11a–c
All computer calculations were performed on a Pentium IV
workstation equipped with a 2.14 GHz processor using
Discovery Studio version 2.0 molecular modelling systems
by Accelrys Software Inc. (San Diego, CA, USA). The
molecular modelling procedure included Energy Minimi-
zation (using the algorithms Steepest Descents for 9000
iterations and Conjugate Gradient for 9000 iterations)
using the CHARMm force field. Dielectric constant was set
to 45 for better simulation of the receptor environment
during all the experiments as well as the SHAKE algorithm
To a solution of 9 (1.0 g, 2.53 mmol) in dry DCM (7 mL),
alkyl bromide 5a (1.46 g, 3.04 mmol) was added under N2
atmosphere and the reaction was allowed to proceed for
48 h at RT. The mixture was concentrated in vacuo and
trituration with DEE afforded 10a as a yellow solid
(1.32 g, 60%), used as obtained for the next step. A solu-
tion of 10a (1.0 g, 1.14 mmol) in 2 mL TFA/DCM (1:1) in
the presence of TES (0.15 mL, 1 mmol) was stirred at RT
and after 1 h the solvent was concentrated. Trituration with
DEE afforded a yellow solid, followed by purification with
preparative RP-HPLC (5% acetonitrile to 90% acetonitrile
in 50 min with a flow rate of 9 mL/min) and lyophilization
˚
and RMSD 0.001 A as energy convergence criterion. MD
runs of EXP3174 and analogues 11a–c, were performed
using the CHARMm force field as follows: Heating, from
0–300 K gradually, and Equilibration were set with a time
123

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid
415
step of 0.002 ps for a total time of 0.1 ns while the time
step of Production was 0.002 ps for a total time of 1 ns.
Parameters on saving results frequencies were set in such a
way in order to extract 300 conformations for each mole-
cule. Energy Minimization runs were performed (using the
algorithms Steepest Descents for 9000 iterations and
Conjugate Gradient for 9000 iterations) on the final 300
conformations, respectively. Analogues 11a–c were
superimposed with the proposed bioactive conformation of
EXP3174 in order to compare the groups of our com-
pounds. The conformations of the two rings (imidazole and
phenyl) of the studied compounds and EXP3174 were used
in the superimpositions in order to determine the orienta-
tion of the tetrazole group, which is critical for the antag-
onistic activity.
from previous mutagenesis studies (Clement et al. 2005),
were located in the binding site of our model. EXP3174 was
manually docked in the binding pocket which consists of
these residues as the input control ligand in order to define the
interaction area. A Monte-Carlo conformation generation
was performed for the 11a–c and EXP3174 compounds in
order to obtain a set of various conformations to be docked
into the defined binding site. In that stage, the ligands were
energy minimized in terms of the Smart Minimizer method,
which begins with the Steepest Descent method, followed by
the Conjugate Gradient method for faster convergence
towards a local minimum in the presence of a fixed or par-
tially flexible receptor. Finally, various scoring functions
(PLP1, PLP2, Jain, and PMF) were applied to the ligand–
receptor interactions recorded. According to the calculated
scores, the best docked poses of each compound were ana-
lysed for interaction with the receptor in order to give a
detailed explanation of the experimental results.
Docking studies
The molecular docking techniques provide useful infor-
mation of drug affinity for the receptor, and various models
and studies have been performed on the AT1 receptor and
non-peptide antagonists (Tuccinardi et al. 2006; Patny et al.
2006). Our study includes the binding modes of com-
pounds 11a–c as ANG II antagonists and EXP3174. The
AT1 receptor model was generated using the recent human
beta-2 adrenergic receptor crystal structure determined at
Biological activity
In vitro studies
Radioligand binding assay We used membrane suspen-
sions containing cloned human ANG II receptor subtype 1
in sf9 cells (AT1 receptor, Perkin Elmer 6110121). In a
final volume of 170, 100 lL membrane suspensions
(40 lg/lL) were incubated for 120 min at 37°C under mild
stirring in assay buffer (20 mM Tris–HCl pH 7.4, 5 mM
MgCl2, 1 mM EDTA, 0.1% BSA). The AT1 receptor
concentration (Bmax = 2.9 pmol/mg protein) and the
affinity of radioligand (Kd = 0.74 nM) are available from
the manufacturer. In the displacement experiments, the
membranes were incubated with 0.5 nM [125I]-Sar1-Ile8-
ANG II (specific activity 1846 lCi/lg) and investigating
ligands at final concentration of 10-5 M. Unlabelled ANG
II at a concentration of 10 lM was used to estimate non-
specific binding. The bound fraction was separated from the
free radioligand by rapid vacuum filtration through Millipore
GF/C filters. The filters were washed immediately nine times
with 500 lL of ice-cold 50 mM Tris–HCl pH 7.4 and the
retained radioactivity was measured in gamma counter
(Packard Ria Star 5405). Preincubation of GF/C filters with
0.3% polyethylinimine was used to minimize non-specific
absorption of the radioligand to filters. All determinations
were performed at least three times. Non-specific binding was
defined as the amount of radioligand bound in the presence of
cold ANG II and specific binding was defined as the total
radioligand bound minus non-specific binding.
˚
2.4 A (2RH1) (Cherezov et al. 2007) as the template.
Alignment was performed manually taking into account the
highly conserved amino acid residues (Mirzadegan et al.
2003) N1.50, the LA-AD (L2.46, A2.47, A2.49, and
D2.50), DRY (D3.49, R3.50 and Y3.51) motif, the tryp-
tophan in the fourth transmembrane helix W4.50, the two
prolines P4.59 and P6.50, and the NP–Y motif in TM7
(N7.49, P7.50 and Y7.53). The construction of the receptor
was performed by means of the Modeller programme
taking into consideration the two disulfide bonds between
C18 and C274 as well as between C101 and C180 formed
in the extracellular domain. Using the Modeller loop
refinement tool, the loops were refined according to the
DOPE SCORE (discrete optimized protein energy) assess
method, and the receptor was subjected to a molecular
dynamics simulation for a time of 2 ns in a vacuum envi-
ronment, with the backbone of the seven transmembrane
helices harmonically restrained in order to allow them
some freedom near a particular position in space.
All docking experiments were performed using the
CHARMm force field in terms of the Ligandfit method
which docks ligands into the active site using a shape filter
and Monte-Carlo ligand conformation generation. The
important amino acids A3.28(104), N3.35(111), L3.36
(112), Y3.37(113), K5.42(199), W6.48(253), H6.51(256),
T6.55(260), A7.42(291), N7.45(294), N7.46(295), C7.47
(296), and L7.48(297), known to effect binding affinity
Intracellular Ca2? ([Ca2?]i) mobilization assay Primary
rat aortic smooth muscle cells (RASMCs) were cultured
in Dulbecco’s modified Eagle’s medium (DMEM)
123

416
G. Agelis et al.
supplemented with 1 mM L-glutamine, 20 mM NaHCO3,
1% streptomycin–penicillin and 10% foetal bovine serum
(FBS). Confluent RASMCs were collected by trypsiniza-
tion (0.05% trypsin, 0.02% EDTA). The cells were washed
in loading buffer (145 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1 mM CaCl2, 5.6 mM glucose, 5 mM HEPES, pH
7.4) and then were resuspended in loading buffer plus 0.1%
BSA to a final concentration of 4 9 105 cells/mL. Cell
suspensions were then incubated with 2.5 lM Fura-2/AM
in DMSO for 30 min at 37°C in the dark. After incubation
cells were washed extensively with loading buffer, were
adjusted to a concentration of 1.5 9 105 cells/mL and were
stored at 37°C for 30 min or at RT for 60 min in order
hydrolysis of intracellular Fura-2/AM took place. ANG II
10-6 l was added directly to promote Ca2? mobilization.
RASMCs were pretreated with the test substances at a
concentration of 10-5 M for 300 s. Calcium transients
were measured with a PERKIN ELMER LS45 spectroflu-
orometer at RT to minimize compartmentalization and cell
extrusion of the fluorescent dye (excitation at 340 and
380 nm, emission at 505 nm). The level of Ca2? mobili-
zation was determined from the excitation ratio of 340 and
380 nm (R = F340/F380).
multichannel recorder. ANG II and test antagonist were
diluted in 5% dextrose.
Results and discussion
Chemistry
The synthetic procedure for titled compounds 11a–c was
divided in two steps. The first step (Fig. 2) involved the
synthesis of the o-, m- and p-bromomethylphenyltetrazoles
5a–c by conversion of the tolunitriles 1a–c to the corre-
sponding organostannanes 2a–c with tributyltin choride
(Sn(n-Bu)3Cl) and sodium azide (NaN3) in 70% yield
(Luitjen et al. 1963; Sisido et al. 1971). Acid hydrolysis
with 2 N HCl in H2O/THF (1:3), followed by tritylation of
tetrazole using trityl chloride (TrtCl) furnished 1H-tetrazole
derivatives 4a–c in high yield (85%). Bromo-derivatives
5a–c were prepared by bromination with (N-bromosuccini-
mide) NBS and dibenzoyl peroxide (DBP) as initiator in 78%
1
yield. The H NMR spectra of 5a–c were similar, showing
singlet peaks at d 4.92, 4.54, 4.52, respectively, due to
the methylene protons, whereas the phenyl protons were
observed at the aromatic region as multiplet peaks.
In vivo studies
The second step included esterification, reduction,
tritylation and selective Np alkylation of urocanic acid
(Fig. 3). Thus, urocanic methyl ester 7 resulted from
esterification with dry MeOH in the presence of concen-
trated H2SO4 and Na2SO4 in 90% yield (Pirrung and Pei
Adult normotensive male New Zealand white rabbits,
weighing between 2.5 and 3.3 kg, were anaesthetized by
pentobarbitone (30 mg/kg) via an ear vein, intubated and
mechanically ventilated with 100% oxygen using a respi-
rator for small animals. The tidal volume was 15 mL and
the rate was adjusted to keep blood gases within normal
range. ANG II dependent hypertension was induced by
infusing ANG II via a syringe pump through a pre-inserted
polyethylene catheter at a constant rate of 0.2 mL/min
(1 lg/min). After the establishment of hypertension, three
boluses of each compound 11a–c of 0.5, 1 and 2 mg were
administrated via the jugular vein and the changes of blood
pressure were recorded through a transducer attached to a
1
2000). H NMR data of 7 showed two singlet peaks at d
7.78 and 7.43 corresponding to the H-2 and H-5 of the
imidazole ring. Moreover, the two vinylic protons appeared
as doublets at d 6.44 and 7.61 (J = 16.0 Hz), respectively,
while methyl protons were located at d 4.85 as a singlet
peak. The resulting ester was subjected to hydrogenation in
the presence of catalyst 10% Pd–C in MeOH to afford 8 in
1
95% yield. The H NMR spectra of 8 showed two triplet
peaks at d 2.88 and 2.66 (J = 7.2 Hz), assigned to the
methylene protons.
Fig. 2 Reagents and
conditions. a Sn(n-Bu)3Cl,
NaN3, Tol, 110°C, 96 h; b 2 N
HCl in THF/H2O (3:1), RT,
16 h; c TrtCl, TEA, DCM, RT,
1 h; d NBS, 10% DBP, CCl4,
reflux, 8 h
123

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid
417
Fig. 3 Reagents and
conditions. a MeOH, conc.
H2SO4, Na2SO4, reflux, 24 h;
b H2, 10% Pd–C, MeOH, RT,
24 h; c TrtCl, TEA, DCM, RT,
1 h; d 5a–c, DCM, RT, 48 h;
e TFA/DCM (1:1), TES, RT,
1 h
Protection of the Ns-position was achieved by the ste-
rically demanding trityl blocking group. Thus, tritylation of
8 with TrtCl and triethylamine (TEA) in DCM afforded
displacement of [125I]-Sar1-Ile8-ANG II from the AT1
receptors, whereas Losartan at the same conditions caused
100% displacement. In case of intracellular Ca2? mobili-
zation assay, the stimulation of AT1 receptors with ANG II
in concentrations of 10-6 M causes the mobilization of
intracellular Ca2? from endoplasmic reticulum to cyto-
plasm and is detected using dye Fura-2/AM. The test
substance 11b was found to induce a 37% inhibition of the
ANG II action in contrast with the reference AT1 antago-
nist Losartan which caused a complete inhibition.
1
only the Ns- substituted analogue 9 in 87% yield. The H
NMR spectra showed two singlet peaks at d 8.02 and 6.75
corresponding to the H-2 and H-5 of the imidazole ring and
a multiplet peak at d 7.42–7.08 due to the 15 phenyl pro-
tons. Selective Np alkylation of 9 with 5a–c in dry DCM
for 2 days afforded the fully protected 10a–c in 60% yield
(Lauth-de Viguerie et al. 1994). Moderate yield was owed
to the tendency of the trityl group to be eliminated during
the alkylation, giving a mixture of Np-substituted and Ns,
Np-disubstituted products (Lauth-de Viguerie et al. 1994;
Anthony et al. 1999). Deprotection of the Trt group with
trifluoroacetic acid (TFA)/DCM (1:1) in the presence of
triethylsilane (TES) as scavenger provided the final target
compounds 11a–c after purification in 80% yield as TFA
Moreover, compounds 11a–c were evaluated for their in
vivo ANG II antagonistic activities. The in vivo activity
was determined by the inhibition of the pressor response
induced by infusion of ANG II in anaesthetized normo-
tensive male rabbits. The in vivo experiments showed that
compounds 11a and 11c did not significantly decrease
mean arterial blood pressure, whereas 11b with phenyl
tetrazole moiety at the m-position showed moderate,
however, comparatively better activity compared to the
11a and 11c (Fig. 4).
1
salts. The H NMR spectra of 11a–c showed singlet peaks
at d 5.80, 5.60, 5.59, respectively, due to the methylene
protons of the alkylating moiety. Purification of 11a–c was
performed by preparative RP-HPLC using C18 column as
1
stationary phase and identification by ESI–MS, H NMR
and 13C MNR.
Biological activity
The new synthesized compounds 11a–c were initially
evaluated in radioligand binding and intracellular Ca2?
mobilization assay (in vitro) at a final concentration of
10-5 l. Although the used concentration was high enough,
there were indications for mild to moderate activity of
substance 11b which required further study. Competitive
binding experiments revealed that 11b caused 25%
Fig. 4 Percentage (%) drop in mean blood pressure following three
boluses of 0.5, 1 and 2 mg of each compound in anaesthetized rabbits
made hypertensive by constant infusion of ANG II
123

418
G. Agelis et al.
Structure–activity relatioships
Moreover, the binding experiment results indicate a
different orientation of the pharmacophores of the ligands.
All docked poses for 11a–c and EXP3174 were similar,
with close RMSD values, indicating a stable ligand–
receptor interaction model. EXP3174 also has the advan-
tage of a more extended conformation, interacting with all
the helices contributing amino acids to the binding site
(Fig. 6). The main amino acids participating in hydrogen
bond interactions were S3.33(109), L(191), N5.43(200)
and Y7.43(292). Hydrophobic contacts stated the higher
affinity of the m- against the o- and p-compounds. All
conformers presented interactions with the critical residues
of V3.32(108), L3.36(112), Y3.37(113), L(191), K5.42
(199), G5.46(203), F5.47(204), W6.48(253) and H6.51
(256) (Fig. 7).
In vitro and in vivo results showed moderate, but higher
activity of 11b, rather than the o- and p-counterparts, in
agreement with molecular modelling studies. These studies
supported that the global minimum conformation of the
11b analogue bearing the three pharmacophore groups,
imidazole, phenyl and tetrazole distanced identically to the
conformation of EXP3174, in contrast with 11a, 11c.
The conformations of the two rings (imidazole and
phenyl) of the synthesized compounds and EXP3174 were
tethered in several superimpositions (Fig. 5) in order to
determine the orientation of the essential for receptor–
substrate interaction tetrazole group, which is in evident
spatial proximity between 11b conformer and EXP3174.
Ultimately, even though the tetrazole group seemed to be
appropriately positioned, the shortening of the biphenyl
group, the presence of an electron-withdrawing ester group
and the lack of an aliphatic electron-donating group as in all
Sartans essential for higher affinity and activity, could justify
the moderate biological activity of 11b. Consequently, the
proper pharmacophore spacing is of primary importance in
non-peptide antagonists for high binding affinity.
Specifically, the best docked pose for each of the
four compounds was further examined in order to give an
elaborate visual explanation to the in vivo experiments.
EXP3174 fits in the binding site with the two phenyl
rings interacting with the TM3 and TM5 residues and
especially with K5.42(199) of the binding pocket. The
imidazole group is in spatial proximity with the residues
I7.39(288) Y7.43(292) of TM7 and the important tetrazole
Fig. 5 Superimposition between EXP 3174 and 11a–c compounds, respectively, illustrating spatial pharmacophore proximity
Fig. 6 Overview of EXP3174
in the binding site, formed
between the TM3, TM5, TM6
and TM7 helices. The molecular
surface is colour-coded by
interpolated partial atom charge
of nearest neighbours to the
surface. The extracellular loops
are not present for better
visualization
123

Towards non-peptide ANG II AT1 receptor antagonists based on urocanic acid
419
Fig. 7 Binding modes of the tested compounds, compared to EXP3174. Transmembrane helices 1, 2 and 4 are not visible for better clarity.
Compounds 11a–c (a–c) and EXP3174 (d) are presented
˚
group in radius less than 3 A, with N5.43(200) of TM5
and H6.51(256), Q6.52(257) of TM6. On the contrary, the
11a–c poses with the highest affinity bind in a different
manner, due to their smaller volume than EXP3174 of
*70% according to their van der Waals surface area
(Fig. 7). 11a has a totally different orientation than 11b and
11c, placing phenyl group far from the lipophilic pocket of
the interaction site. Conformers 11b and 11c share a sim-
ilar orientation but with one essential difference. Even
though their phenyl and carbonyl groups interact with
K5.42(199), N5.43(200) and S3.33(109), respectively
(helices 3 and 5), their main contradiction is the m- con-
former’s methyl ester group which interacts with the side
chain of W6.48(253). This proximity to the TM6 helix can
interpret the increased affinity of 11b to the AT1 receptor.
The m-substituted tetrazole in 11b results in the accom-
modation of the imidazole and tetrazole groups towards
TM3 and TM5 helices, as well as the proximity of the
methyl ester group to W6.48(253) and TM6. Lower
activity of 11b compared to Losartan was due to (1) the
lack of a lipophilic butyl pharmacophore group in the
imidazole moiety necessary for binding in AT1 receptor
antagonists (2) the smaller molecular weight and volume of
our compounds than EXP3174, which eliminate their
interaction with the important TM7 region of the binding
site and (3) the shorter length of the phenyl template
compared to the biphenyl moiety, necessary for high
affinity to the receptor’s lipophilic pocket, as well as proper
spacing of pharmacophore groups and activity.
Acknowledgments This research project was supported by the
Ministry of Development, General Secretariat of Research and
Technology of Greece [EPAN: ‘‘IRAKLIS’’ YB 84 (B321/2005)]. We
thank Dr Andreas Papapetropoulos for providing primary rat aortic
smooth muscle cells (RASMC). Also, we would like to thank Dimitris
Vahliotis (Instrumental Analysis Laboratory, University of Patras) for
recording the NMR spectra.
Conclusion
This study describes the synthesis and biological evalua-
tion of a series of o-, m- and p-benzyl tetrazole derivatives
11a–c as potential ANG II AT1 receptor antagonists, based
on urocanic acid. The synthetic strategy of the target
compounds 11a–c included a facile few step method with
high yields. Compound 11b with tetrazole moiety at the m-
position showed moderate, however, higher activity com-
pared to the o- and p-compounds 11a and 11c, respectively.
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