Synthesis, binding studies and in vivo biological evaluation of novel non-peptide antihypertensive analogues

Article abstract of DOI:10.1016/j.bmc.2006.02.044

AT₁ antagonists (SARTANs) constitute the last generation of drugs for the treatment of hypertension, designed and synthesized to mimic the C-terminal segment of the vasoconstrictive hormone angiotensin II (AngII). They exert their action by blocking the binding of AngII on the AT₁ receptor. Up to date eight AT₁ antagonists have been approved for the regulation of high blood pressure. Although these molecules share common structural features and are designed to act under the same mechanism, they have differences in their pharmacological profiles and antihypertensive efficacy. Thus, there is still a need for novel analogues with better pharmacological and financial profiles. An example of a novel synthetic non peptide AT₁ antagonist which devoids the classical template of SARTANs is MM1. In vivo studies showed that MMK molecules, which fall in the same class of MM1, had a significant antihypertensive (40-80% compared to the drug losartan) activity. However, in vitro affinity studies showed that losartan has considerably higher affinity. The theoretical docking studies showed that MM1 acts on the same site of the receptor as losartan. They exert hydrophobic interactions with amino acid Val108 of the third helix of the AT₁ receptor and other hydrophobic amino acids in spatial vicinity. In addition, losartan favours multiple hydrogen bondings between its tetrazole group with Lys199. These additional interactions may in part explain its higher in vitro binding affinity.

Full text of DOI:10.1016/j.bmc.2006.02.044

Bioorganic & Medicinal Chemistry 14 (2006) 4353–4360
Synthesis, binding studies, and in vivo biological evaluation of
novel non-peptide antihypertensive analogues
b,
b
b
T. Mavromoustakos,a,* P. Moutevelis-Minakakis, C. G. Kokotos, P. Kontogianni,
A. Politi,a P. Zoumpoulakis,a J. Findlay,c A. Cox,c A. Balmforth,d
A. Zogae and E. Iliodromitise
*
aInstitute of Organic and Pharmaceutical Chemistry, National Hellenic Research Foundation, Athens, Greece
bDepartment of Chemistry, University of Athens, Athens 15784, Greece
cSchool of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK
dSchool of Biomedical Sciences, University of Leeds, Leeds, LS2 9JT, UK
eOnassis Cardiac Surgery Center, 356 Sygrou Ave., Athens, Greece
Received 21 November 2005; revised 23 January 2006; accepted 24 February 2006
Available online 20 March 2006
Abstract—AT1 antagonists (SARTANs) constitute the last generation of drugs for the treatment of hypertension, designed and syn-
thesized to mimic the C-terminal segment of the vasoconstrictive hormone angiotensin II (AngII). They exert their action by block-
ing the binding of AngII on the AT1 receptor. Up to date eight AT1 antagonists have been approved for the regulation of high blood
pressure. Although these molecules share common structural features and are designed to act under the same mechanism, they have
differences in their pharmacological profiles and antihypertensive efficacy. Thus, there is still a need for novel analogues with better
pharmacological and financial profiles. An example of a novel synthetic non-peptide AT1 antagonist which devoids the classical tem-
plate of SARTANs is MM1. In vivo studies showed that MMK molecules, which fall in the same class of MM1, had a significant
antihypertensive (40–80% compared to the drug losartan) activity. However, in vitro affinity studies showed that losartan has con-
siderably higher affinity. The theoretical docking studies showed that MM1 acts on the same site of the receptor as losartan. They
exert hydrophobic interactions with amino acid Val108 of the third helix of the AT1 receptor and other hydrophobic amino acids in
spatial vicinity. In addition, losartan favors multiple hydrogen bondings between its tetrazole group with Lys199. These additional
interactions may in part explain its higher in vitro binding affinity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
class of drugs called SARTANS.14–24 To comprehend
the stereoelectronic requirements for receptor binding,
the stereochemical features of angiotensin II, its peptide
antagonists sarmesin and sarilesin, synthetic peptide
analogues, and AT1 non-peptide antagonists (commer-
cially available and novel compounds) were explored.
AT1 antagonists are designed to mimic the C-terminal
part of Ang II.25–28,45 Based on superimposition studies
of losartan with the model proposed for sarmesin, a new
avenue was explored in an attempt to design and synthe-
size novel AT1 antagonists. Thus, few years ago we have
briefly reported MM1 synthesis which possesses pyrro-
lidinone instead of biphenyltetrazole template. Its ste-
reoelectronic properties were also analyzed and
compared with those of losartan.29 MM1 was designed
to mimic conformational characteristics of His6-Pro7-
Phe8 and constitutes the first lead compound. Two other
derivative compounds were also synthesized namely
MMK2 and MMK3. Detailed synthesis of the three
Angiotensin II (AII) is a vasoconstrictive peptide hor-
mone formed within the renin–angiotensin system
(RAS) which plays an important role in regulating car-
diovascular homeostasis.1–3 Research efforts for the con-
trol of hypertension have focused on competing Ang II
binding to AT1 receptors. The research efforts of Du-
Pont were crowned by the discovery of the first potent
and orally active non-peptide AII antagonist losartan
(COZAAR).4–14 New molecules similar in structure
appeared in the pharmaceutical market defining a new
Keywords: Angiotensin II; AT1 antagonists; MM1; MMK2; MMK3;
FlexX; Docking.
*
Corresponding authors. Fax: +30 2107273872 (T.M.); fax: +30
2107274761 (P.M.); e-mail addresses: tmavro@eie.gr;
pminakak@chem.uoa.gr
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.02.044

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T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
N
O
N
N
O
N
N
N
X
OMe
X = H (MM1)
X = OMe (MMK2)
MMK3
Figure 1. Chemical structures of MM1–MMK3.
compounds is reported in this research activity. The
structures of losartan, MM1, MMK2, and MMK3 are
shown in Figure 1. In addition, comparative in vitro
binding studies with AT1 and AT2 receptors are per-
formed, as well as in vivo experiments with adult nor-
motensive male New Zealand White rabbits.
Moreover, theoretical (docking) studies were applied to
investigate possible interactions between the prototype
of these series MM1 with binding amino acids. Such
studies may aid to provide a plausible explanation for
the in vivo and in vitro results strengthening the rational
design of novel analogs with improved binding affinities.
Figure 2. Synthesis of (5S)-1-benzyl-(1H-imidazol-1-yl-methyl)-2-pyr-
rolidinone (MM1), (5S)-1-(3-methoxybenzyl)-5-(1H-imidazol-1-yl-
methyl)-2-pyrrolidinone (MMK2), and (5S)-1-(3-methoxybenzyl)-5-
(1H-benzimidazol-1-ylmethyl)-2-pyrrolidinone (MMK3).
2. Results
2.1. Synthesis of MM1, MMK2, and MMK3
The synthesis of MM1, MMK2 and MMK3 is shown in
Figure 2 and is presented in detail in the Experimental
Part. Briefly, S-pyroglutamic acid (1) was converted to
methyl (S)-pyroglutamate (2) by treatment with SOCl2
in methanol. This ester was subsequently dissolved in
dry THF and benzyl bromide (or m-methoxy-benzyl
bromide) was added. After cooling to 0 ꢁC, NaH was
added portionwise to gain compounds 3a and 3b,
respectively. Reduction of 3a and 3b with LiBH4 led
to the alcohols 4a and 4b. After tosylation, the activated
tosyl esters 5a and 5b were converted to the target com-
pounds MM1 (6a) and MMK2 (6b) by treatment with
freshly prepared lithium imidazolide and MMK3 (7)
by treatment with lithium benzimidazolide, freshly pre-
pared as well.
Figure 3. In vitro competition binding studies with membranes
containing human AT1 receptor.
2.2. Biological studies
showed high antihypertensive activity (72% and 80%
relatively to losartan defined as 100%). MMK3
showed smaller antihypertensive activity (48%).
The experiments in membranes containing AT1 recep-
tors and cell cultures were repeated on triplicates. The
mean values of the obtained results were identical.
MM1 as well as MMK2 and MMK3 showed no consid-
erable activity compared to prototype losartan (IC50 val-
ue ꢀ10ꢁ9 M) for the AT1 receptor (Fig. 3). Preliminary
experiments indicate that the three molecules, like losar-
tan, do not compete for [125I]Sar1,Ile8-angiotensin II
binding at AT2 receptors. Figure 4 summarizes the anti-
hypertensive effect and the degree of potency of the three
compounds in a preparation of anesthetized rabbits
made hypertensive by AII infusion. MM1 and MMK2
2.3. Theoretical docking studies
The early models have been constructed based on 3D
structure of bacteriorhodopsin (BR) but it is now com-
monly accepted that BR is not a good template for mod-
eling GPCRs, since it has very low homology to this
family30 and the relative orientation of transmembrane
segments is different. The more recent models are based
on the 3D structure of rhodopsin (Rh) that was revealed

T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
4355
200
180
160
140
120
100
80
60
40
20
0
Normal BP
Start AII
Addition of
Antagonist
Stop AII
MM1 MMK2 MMK3 Losartan
Antagonist % BP reduction
Potency (% losartan)
Losartan
MM1
100
72
-25 2
-18 4
-20 3
-12 1
MMK2
MMK3
80
48
Figure 4. In vivo biological data for compounds MM1–MMK3. The
adult normotensive male New Zeland White Rabbits were first become
hypertensive with the infusion of angiotensin II. The antihypertensive
activity of novel non-peptide molecules was compared with the control
AT1 antagonist losartan. All concentration 5 lg/ml provided with
0.8 lg/min. Final concentrations of antagonists were 2 mg/ml.
by electronic microscopy31 or, in much more detail, by
recent X-ray crystallography.32
For the definition of the active site, several site-directed
mutagenesis studies have been performed in order to
examine the residues involved either in ligand binding
(agonists or antagonists) or in signal transduction.33–43
These studies are some times controversial and do not
establish unequivocally the active site of the AT1 recep-
tor.33,35,39,40,44 The key amino acids for binding of non-
peptide AT1 antagonists are Val108, Asn111, Ala163,
Arg167, Lys199, Ser252, His256, Asn295, Leu300, and
Phe301. These amino acids have been reported for spe-
cific interactions with non-peptide antagonists of the
class of SARTANs. Other amino acids mostly in the
third and seventh helices are important for the mainte-
nance of the integrity of the pocket. These reported ami-
no acids cover a wide area of the receptor thus cannot
determine a unique binding pocket. For this reason the-
oretical calculations have been applied to identify re-
gions of the receptor, capable of binding.49 SiteID
module of Sybyl6.8 showed that losartan and MM1
can accommodate in thirteen pockets of different sizes
(Fig. 5). The pocket selected (Table 1) for further dock-
ing studies was the one which included three of the most
important amino acids (Val108, Lys199, and His256) for
antagonist binding. This pocket is located near the polar
head groups and exceeds down to the hydrophobic core
of the phospholipid bilayers of the membrane in which
the receptor is embedded. This observation is important
if we consider that AT1 antagonists have been found to
locate themselves near the mesophase of the phospho-
lipid bilayers. Thus, the specific pocket may be easily
accessible by the ligands after their diffusion which con-
firms the proposed two-step mechanism of action for
AT1 antagonists.
Figure 5. Thirteen resulted theoretical pockets found after applying
SiteID software on the model of AT1 receptor.
putative active site. This algorithm places a base
fragment of the ligand in the pocket and then constructs
fragment by fragment the remainder while searching for
favorable interactions between the ligand and the
protein trying to avoid steric overlaps at each step.
During this procedure, the conformation of the protein
is kept stable. The docking and subsequent scoring were
Table 1. amino acids of the AT1 receptor which constitute the binding
pocket, as found with SiteID module of SYBYL6.8 software
Aminoacid
TM
Val108
Leu112
Val116
Phe170
Phe182
Lys199
Asn200
Gly203
Phe204
Pro207
Phe208
Phe249
Trp253
His256
Gln257
Ile288
3
3
3
4 (extracellular)
4 (extracellular)
5
5
5
5
5
5
6
6
6
6
7
7
7
Ala291
Tyr292
Application of the FlexX algorithm (Sybyl6.8—TRI-
POS) showed that MM1 could fit spontaneously in the

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T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
Figure 6. The incorporation of MM1 (left) in the putative pocket of AT1 receptor (right) as it is revealed using theoretical and experimental
data.
performed using the formal charges and the default
parameters of the FlexX program.
MM1 showed a good fit into the hydrophobic core of the
pocket. Starting with the four low energy conformations
of MM1 as found elsewhere with NMR studies and
molecular modeling,29 the resulting docked conforma-
tion was specific and is presented in (Fig. 6). The interac-
tions between the amino acids located in the cavity of AT1
receptor and pharmacophore segments of MM1 are
mainly hydrophobic in nature and involve phenyl ring
of MM1 with Phe204 of AT1 receptor and pyrrolidinone
with amino acids Trp253 and His256 (Fig. 7). MM1 did
not show any strong electrostatic interaction or hydrogen
bond with the amino acids of the binding site.
3. Discussion
The use of SITE ID algorithm determined putative
pockets (active site of AT1 receptor) for MM1 and losar-
tan molecules. Among those revealed by this algorithm
Phe204
one contained amino acids reported by mutational stud-
His256
ies. This pocket, which was supported by experimental
and theoretical calculations, was considered for further
studies. The application of FlexX algorithm on this
pocket showed that MM1 could fit spontaneously in
the putative active site. MM1 is docked mainly because
of the hydrophobic interactions between its lipophilic
segments and hydrophobic amino acids in the core of
the pocket. A major difference with losartan is that
losartan is proposed to stabilize itself in the pocket with
electrostatic interactions and hydrogen bonds.49 These
bonds may be of biological significance and must be
considered for the rational design of new AT1 antago-
nists using pyrrolidinone template as a scaffold. Docking
experiments supported our rational design for MM1.
Trp253
N
O
N
Lys199
N
Val108
Figure 7. Docking of MM1 into putative pocket of AT1 receptor.

T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
4357
As it is mentioned the in vitro experiments showed that
MM1 has considerably lower in vitro activity. The
in vivo experiments, as explained in the introduction,
show that MM1 is almost equipotent with losartan.
Two plausible explanations can be given for this obser-
vation: (a) The diffusion of both molecules into the
receptor site may be facilitated (i.e., proteins); (b) the
receptor site adopts different conformation. It should
be pointed out that in the in vivo experiments, the drug
is given after rabbits have become hypertensive, while in
the in vitro studies receptors were in a ‘rest state’.
(6.53 mL, 0.055 mol) was added under stirring in one
portion. The mixture was cooled to 0 ꢁC and NaH46
(60% in paraffin oil, 3 g, 0.075 mol) was added portion-
wise. After stirring for 30 min at 0 ꢁC, the mixture was
stirred for 30 min at rt. The product was washed twice
successively with a saturated aqueous NH4Cl solution
and H2O, and the aqueous layer was extracted with
Et2O. The combined organic layers were washed with
brine, dried with Na2SO4, and evaporated. The residue
was purified by column chromatography (Silica gel 60)
using a petroleum ether/Et2O (3:7–1:9) mixture and
finally only diethyl ether as eluent.
25
D
4. Conclusion
Product was obtained as an oil. Yield 3.6 g (45%) ½aꢂ
+3.5 (c 1, MeOH).
Our studies confirm our rational design for the proto-
type MM1 and its analogues MMK2 and MMK3. The
docking theoretical study will guide the second step in
our rational design which includes the synthesis of deriv-
ative molecules of MM1 which must contain an acidic
group (i.e., carboxylate or an isosteric tetrazole) in order
to form electrostatic interactions or hydrogen bonds be-
tween MMK derivatives and the AT1 receptor pocket.
This will be a strict requirement for improving the anti-
hypertensive activity not only because it will provide
multiple contacts with the receptor but also because it
may optimize its partitioning in membrane bilayers.
1H NMR (CDCl3): d = 7.29–7.15 (m, 5H, aromatic), 5.0
(d, J = 14.6 Hz, 1H, CHHPh), 4.0 (d, J = 14.6 Hz, 1H,
CHHPh), 3.98 (m, 1H, CH), 3.63 (s, 3H, OCH3),
2.63–2.27 (m, 4H, 2· CH2). 13C NMR (CDCl3):
d = 175.2, 172.4, 135.9, 128.9, 128.7, 127.9, 58.9, 52.5,
45.8, 29.7, 23.0.
Compounds (5S)-1-benzyl-5-hydroxymethyl-2-pyrrolid-
inone (4a)47 and (5S)-1-benzyl-5-hydroxymethyl-2-pyr-
rolidinone tosyl ester (5a)48 were prepared according
to the literature.
5.1.2. (5S)-1-Benzyl-5-(1H-imidazol-1-ylmethyl)-2-pyrro-
lidinone (6a). Freshly prepared lithium imidazolide
(0.22 g, 3 mmol) was added in a solution of the tosyl es-
ter 5a (0.54 g, 1.5 mmol) in DMSO (4 mL) at room tem-
perature. The resulting mixture was stirred for 24 h at
60 ꢁC under argon atmosphere. After addition of water
(2 mL), the mixture was extracted with ethyl acetate.
The organic layer was washed with brine to neutral
pH, dried with Na2SO4, and evaporated under reduced
pressure and the residue was purified by column chro-
matography (silica gel, elution system: AcOEt/MeOH
9:1). The product (0.24 g) was obtained as an oil. Yield
62%.
5. Experimental
5.1. Synthesis of MM1, MMK2, and MMK3
Mps were determined on an mp apparatus (Buchi) and
¨
are uncorrected. Specific rotations were measured at
25 ꢁC on an electronic polarimeter Perkin-Elmer 343
using a 10 cm cell. NMR spectra were recorded in
CDCl3 on a 200 MHz spectrometer. S-Pyroglutamic
acid, and 3-methoxy-benzyl bromide were purchased
from Aldrich and benzyl bromide from Fluka. TLC
plates (silica gel 60 F254) and silica gel 60 (70–230 or
230–400 mesh) for column chromatography were
purchased from Merck. THF was dried by standard
procedures and stored over molecular sieves. DMSO
was purchased from Merck and stored also over molec-
ular sieves. All other solvents and chemicals were of re-
agent grade and used without further purification. Fast
atom bombardment (FAB) mass spectra were recorded
using a VG analytical ZAB-SE instrument. Electron
spray ionization (ESI) mass spectra were recorded on
a Finnigan, Surveyor MSQ Plus spectrometer.
25
½aꢂ +8.3 (c 1, MeOH) Rf = 0.58 (CHCl3–MeOH 7:3).
D
1H NMR (CDCl3): d = 7.41–7.11 (m, 7H, aromatic),
6.80 (s, 1H, aromatic), 5.10 (d, J = 15.1 Hz, 1H,
CHHPh), 4.05 (d, 2H, CH2N), 3.96 (d, J = 15.1 Hz,
1H, CHHPh), 3.78 (m, 1H, CH), 2.35–1.78 (m, 4H,
2· CH2). 13C NMR (CDCl3): d = 175.0, 137.5, 135.9,
130.2, 128.9, 128.0, 119.2, 56.8, 48.7, 44.9, 29.1, 22.4.
Anal. Calcd for C15H17N3O: C, 70.56; H, 6.71; N, 16.46.
Found: C, 70.42; H, 6.88; N, 16.40. MS (ESI): m/z = 278
(17%) (M+Na)+, 256 (100%) (M+H)+.
Lithium imidazolide and lithium benzimidazolide were
prepared as follows: Imidazole (250 mg, 3.6 mmol) or
benzimidazole (425 mg, 3.6 mmol) was dissolved in
10 mL dry toluene and 1.6 mL (2.5 M) of nBuLi was
added at 0 ꢁC. The mixture was stirred for 2 h at room
temperature and evaporated. The resulted solid in both
cases was dried under vacuum overnight.
5.1.3. (5S)-1-(3-Methoxybenzyl)-5-carbomethoxy-2-pyr-
rolidinone (3b). Methyl (S)-pyroglutamate (7.2 g,
0.05 mol) was reacted with m-methoxybenzyl bromide
(11.06 g, 0.055 mol) under the same conditions as for
compound 3a, described above. The residue was
purified by column chromatography (Silica gel 60)
using a petroleum ether–AcOEt (3:7 to 1:9) mixture
as eluent.
5.1.1. (5S)-1-Benzyl-5-carbomethoxy-2-pyrrolidinone
(3a). Methyl (S)-pyroglutamate (7.2 g, 0.05 mol) was
dissolved in dry THF (250 mL) and benzyl bromide

4358
T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
The product is obtained as an oil. Yield: 5.2 g (40%).
CHHPh), 3.62 (m, 1H, CH), 2.42–1.94 (m, 7H, CH3,
2· CH2).
25
½aꢂ +4.70 (c 1.5, MeOH), Rf = 0.45 (AcOEt/petroleum
D
ether 4:1).
13C NMR (CDCl3): d = 174.9, 159.9, 145.3, 137.5, 132.2,
129.6, 129.7, 127.8, 120.0, 113.5, 113.1, 68.6, 55.3, 55.1,
44.3, 29.6, 21.6, 21.0.
1HNMR(CDCl3):7.23(m,1H,aromatic),6.79(m,3H,aro-
matic), 5.01 (d, J = 14.8 Hz, 1H, CHHPh), 4.06 (m, 1H,
CH), 3.94 (d, J = 14.8 Hz, 1H, CHHPh), 3.78 (s, 3H,
OCH3),3.70(s,3H,COOCH3),2.62–2.00(m,4H,2·CH2).
5.1.6. (5S)-1-(3-Methoxybenzyl)-5-(1H-imidazol-1-ylm-
ethyl)-2-pyrrolidinone (6b). Lithium imidazolide (0.22 g,
3 mmol) was added in a solution of the tosyl ester 5b
(0.58 g, 1.5 mmol) in dimethylsulfoxide, under the same
conditions as for compound 6a, described above. The
residue was purified by column chromatography (Silica
gel 60, elution system: AcOEt/MeOH 1:1). The product
(0.23 g) was obtained as an oil. Yield 55%.
13C NMR (CDCl3): 175.0, 172.2, 159.7, 137.2, 129.6,
120.6, 113.8, 113.1, 58.5, 55.1, 52.3, 45.4, 29.4, 22.7.
5.1.4. (5S)-1-(3-Methoxybenzyl)-5-hydroxymethyl-2-pyr-
rolidinone (4b). LiBH4 (0.1 g, 5 mmol) was added in dry
tetrahydrofuran (THF) (2 mL) under argon atmosphere.
Afterwards, a solution of the aforementioned substance
3 b (1.1 g, 4.55 mmol) in dry THF (2.5 mL) was added
dropwise at room temperature. The reaction was
quenched 45 min later, with the addition of an aqueous
acetic acid solution (20%) at 0 ꢁC. Then the mixture was
neutralized with solid Na2CO3. The mixture was extract-
ed with ethyl acetate and the organic layer was washed
with water. After drying with sodium sulfate and evap-
oration, the product was obtained as a pure oil.
25
½aꢂ +10.9 (c 0.78, MeOH), Rf = 0.39 (AcOEt/MeOH
D
1:1).
1H NMR (CDCl3): d = 7.39–6.81 (m, 7H, aromatic),
5.01 (d, J = 15.2 Hz, 1H, CHHPh), 4.02 (d, 2H,
CH2N), 3.87 (d, J = 15.2 Hz, 1H, CHHPh), 3.80 (s,
3H, OCH3), 3.75 (m, 1H, CH), 2.14–1.78 (m, 4H,
2· CH2).
13C NMR (CDCl3): d = 175.0, 160.0, 137.5, 130.2, 130.0,
120.2, 119.2, 113.8, 113.2, 56.9, 55.3, 48.7, 44.9, 29.1,
22.4.
Yield 0.94 g (97%).
25
½aꢂ ꢁ81.2 (c 1.1, MeOH), Rf = 0.51 (CHCl3/MeOH
D
9:1).
Anal. Calcd for C16H19N3O2: C, 67.35; H, 6.71; N,
14.73. Found: C, 67.29; H, 6.79; N, 14.68. MS (FAB)
m/z: 286 (100%) (M+H)+, 204 (10%).
1H NMR (CDCl3): d = 7.23 (m, 1H, aromatic), 6.81 (m,
3H, aromatic), 4.82 (d, J = 15.2 Hz, 1H, CHHPh), 4.16
(d, J = 15.2 Hz, 1H, CHHPh), 3.77 (s, 3H, OCH3), 3.76
(m, 1H, CH), 3.53 (m, 2H, CH2OH), 2.80 (b, 1H, OH),
2.42–2.01 (m, 4H, 2· CH2).
5.1.7. (5S)-1-(3-Methoxybenzyl)-5-(1H-benzimidazol-1-
ylmethyl)-2-pyrrolidinone (7). Lithium benzimidazolide
(0.372 g, 3 mmol) was added in a solution of the tosyl es-
ter 5b (0.58 g, 1.5 mmol) in dimethylsulfoxide, under the
same conditions as for compound 6a, described above.
The residue was purified by column chromatography
(Silica gel 60, elution system: CHCl3/MeOH 9:1). The
product (0.38 g) was obtained as an oil, which was solid-
ified during storage at 4 ꢁC. Yield 75%.
13C NMR (CDCl3): d = 176.1, 159.7, 138.1, 129.7, 120.0,
113.5, 112.7, 61.9, 58.6, 55.1, 44.2, 30.5, 20.8.
MS (FAB) m/z: 236 (100%) (M+H)+, 204 (39%).
5.1.5. (5S)-1-(3-Methoxybenzyl)-5-hydroxymethyl-2-pyr-
rolidinone tosyl ester (5b). A solution of alcohol 4b
(0.76 g, 3.3 mmol) in methylene chloride (10 mL) was
cooled at 0 ꢁC. Triethylamine (0.51 mL) and tosyl chlo-
ride (0.685 g, 3.3 mmol) were added in three portions,
successively, under cooling. The solution was stirred at
room temperature overnight. The mixture was washed
with a cooled 1 N HCl solution (2 mL), a saturated
NaHCO3 solution (1.5 mL), and brine. Then it was
dried with Na2SO4 and evaporated. The residue was
purified by column chromatography (Silica gel 60) using
AcOEt/petroleum ether (9:1) as eluent. Product was ob-
tained as an oil. Yield 0.83 g (65%).
25
½aꢂ +22.3 (c 0.7, CHCl3), Rf = 0.92 (AcOEt/MeOH
D
1:1).
1H NMR (CDCl3): d = 7.74–6.60 (m, 9H, aromatic),
4.98 (d, J = 15.2 Hz, 1H, CHHPh), 4.01 (d, 2H,
CH2N), 3.80 (s, 3H, OCH3), 3.75 (d, J = 15.2 Hz, 1H,
CHHPh), 3.62 (m, 1H, CH), 2.45–1.80 (m, 4H, 2· CH2).
13C NMR (CDCl3): d = 175.0, 159.9, 145.3, 137.5, 130.0,
129.8, 127.9, 120.2, 113.5, 113.2, 68.9, 55.3, 55.2, 44.4,
29.7, 21.7.
Anal. Calcd for C20H21N3O2: C, 71.62; H, 6.31; N,
12.53. Found: C, 71.47; H, 6.35; N, 12.48. MS (ESI):
m/z = 358 (20%) (M+Na)+, 336 (100%) (M+H)+.
25
½aꢂ +23.7 (c 1, CHCl3), Rf = 0.49 (AcOEt/petroleum
D
ether 9:1).
1H NMR (CDCl3): d = 7.70 (m, 2H, aromatic), 7.38 (m,
2H, aromatic), 7.21 (m, 1H, aromatic), 6.71 (m, 3H, aro-
matic), 4.82 (d, J = 15.2 Hz, 1H, CHHPh), 3.98 (m, 2H,
CH2OS), 3.80 (s, 3H, OCH3), 3.78 (d, J = 15.2 Hz, 1H,
5.2. In vitro binding studies
Membranes containing human AT1 receptors were
purchased from PerkinElmer Life Sciences, Inc., Boston,

T. Mavromoustakos et al. / Bioorg. Med. Chem. 14 (2006) 4353–4360
4359
MA, USA, and membranes containing either AT1 or
AT2 receptors were kindly provided by Prof. A. Balm-
forth, Biomedical Sciences, University of Leeds. (3-
hypertension was induced by infusing angiotensin II
via a syringe pump (Harvard Apparatus Pump 22, Har-
vard Apparatus, Natick. MA, USA) at a constant rate
of 0.2 mL/min (1 lg/min). Each compound including
losartan was initially dissolved with 0.05 mL DMSO
and then diluted in dextrose 5% at a final concentration
of 2 mg/mL. Five minutes after the establishment of
hypertension, three equal boluses of each compound
were given via an ear vein 20 min apart. The blood pres-
sure was monitored for 20 min after the third bolus and
then angiotensin infusion was halted and blood pressure
was recorded for an additional period of 20 min until the
end of the experiment. Boluses of the same dose of losar-
tan, as positive control, were used in the same experi-
mental model.
[
125I]iodotyrosyl4) Sar1 Ile8, Angiotensin II was pur-
chased from Amersham Biosciences, Little Chalfont,
Bucks, UK, and all other general reagents were from
Sigma, Poole, Dorset, UK.
5.3. In vitro binding experiments
The binding buffer solution comprised 20 mM Tris–
HCl, 100 mM NaCl, 5 mM MgCl2, and 0.1% (w/v)
BSA, and was adjusted to pH 7.4. Competitor drug
concentrations ranged between 10ꢁ5 and 10ꢁ9 M and
triplicate determinations made. All drugs were DMSO
soluble and when diluted the DMSO concentration did
not exceed 1% (v/v). The radioligand used for the
experiments was [125I]Sar1,Ile8-angiotensin II, a non-
specific peptide appropriate for both AT1 and AT2
receptors. A constant concentration of radioligand of
0.1 nM (ꢃ40,000 cpm) was maintained in all samples.
The total binding is defined as the binding in the ab-
sence of competitive compounds. Non-specific binding,
in the presence of 10ꢁ5 M losartan, was about 300 cpm.
Two kinds of membranes were used for the binding
experiments containing human AT1 or AT2 receptors.
23.5 lg of membrane protein was used in each binding
assay. Binding assay comprised: 25 lL radioligand,
25 lL test compound or buffer, and 50 lL membrane
sample. Incubations were carried out at room temper-
ature 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 buffer. The radioactivity retained on
the filters was determined on a Packard RiasStar
5405 gamma counter.
5.5. Theoretical calculations for AT1 binding
A homology model based on the X-ray structure of rho-
dopsin human AT1 receptor developed by James Ellis
and Dan Donnelly (University of Leeds, UK) was used
for these studies. Determination of the active site: SiteID
module of the SYBYL6.8 (TRIPOS) was used to scan
the protein and provide the theoretical binding sites
located in the seven helices of AT1 receptor. Default
parameters of the SiteID program were used.
Docking. FlexX algorithm of SYBYL6.8 (TRIPOS) was
conducted for the docking analysis between the ligand
(MM1) and the selected site of the receptor.
Ligand and receptor preparation. The structure of the
ligand was prepared in MOL2 format using the 2D
sketcher module of Sybyl6.8 and Gasteiger–Huckel
charges were assigned to the ligand atoms. The ligand
was subjected to minimization until converged to a max-
ꢁ1. MM1 conforma-
˚
A
imum derivative of 0.001 kcalꢁ1
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 inclusion of 150 mM NaCl.
One hundred and five cells in 50 ll buffer replaced the
use of membranes. Washing of filters was also carried
out using isotonic buffer.
tion derived from NMR studies and molecular
modelling was used as the initial reference structure,29
after the removal of the hydrogen atoms and the atom
charges. Possible bound waters and ligands were re-
moved from the protein and hydrogen atoms were add-
ed subsequently.
5.4. In vivo experiments
Acknowledgments
Adult normotensive male New Zealand White rabbits
weighing between 2.5 and 3.3 kg were used in the study
following previously described methods.50,51 In brief,
animals were anesthetized by pentobarbitone (30 mg/
kg), intubated, and mechanically ventilated with 100%
oxygen using a respirator for small animals (MD Indus-
tries, Mobile, Al, USA). The tidal volume was 15 mL
and the rate was adjusted to keep blood gases within
normal rage. Two polyethylene catheters were inserted,
one in the carotid artery for continuous blood pressure
monitoring via a transducer attached to a multichannel
recorder (Nihon-Kohden, Model 6000, Japan) and the
other one in the jugular vein for the administrations of
solution made by diluting angiotensin II (AII) (Hyper-
tensin, CIBA) in 5% dextrose at a final concentration
of 5 lg/mL. Based on previous testing with this rabbit
preparation submaximal angiotensin II-dependent
The work was funded by the GSRT under the EPAN 4.5
Hercules 2005-2007. Financial support from the
EPEAEK program ‘Organic Synthesis and Applications
in Chemical Industry’ is highly appreciated.
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