Synthesis of biphenylyltetrazole derivatives of 1-aminopyrroles as angiotensin II antagonists

Article abstract of DOI:10.1016/S0014-827X(98)00100-1

Based on preliminary molecular modelling study, the synthesis of two different classes of biphenylyltetrazole derivatives of 1-aminopyrroles, as potentially active non-peptide angiotensin II (AII) antagonists, is reported. Some NH-Boc protected 1-aminopyr

Full text of DOI:10.1016/S0014-827X(98)00100-1

Il Farmaco 54 (1999) 64–76
Synthesis of biphenylyltetrazole derivatives of 1-aminopyrroles as
angiotensin II antagonists
Orazio A. Attanasi a,*, Spartaco M. Colombani b, Lucia De Crescentini a,
Raffaello Giorgi b,1, Susanna Monti b, Ada Perrone a, Francesca R. Perrulli a,
Anna Rita Renzetti c, Stefania Santeusanio a
a Institute of Organic Chemistry, Faculty of Science, Urbino Uni6ersity, Piazza della Repubblica 13, I-61029 Urbino, Italy
b Departments of Medicinal Chemistry and Pharmacology, Laboratori Guidotti S.p.A., Via Li6ornese 897, 56122 S. Piero a Grado, Pisa, Italy
c Departments of Medicinal Chemistry and Pharmacology, Menarini Ricerche S.p.A., Via Sette Santi 3, I-50131 Florence, Italy
Received 1 September 1998; accepted 30 November 1998
Abstract
Based on preliminary molecular modelling study, the synthesis of two different classes of biphenylyltetrazole derivatives of
1-aminopyrroles, as potentially active non-peptide angiotensin II (AII) antagonists, is reported. Some NH-Boc protected
1-aminopyrroles were deprotected, N-acylated, N-alkylated with 5-[4%-bromomethyl-1,1%-biphenyl-2-yl]-1-triphenylmethyl-1H-te-
trazole, and then detritylated to give the first class of title compounds. Other 1-NH-Boc protected 1,2-diaminopyrroles were
regioselectively subjected to the 1-alkylation with 5-[4%-bromomethyl-1,1%-biphenyl-2-yl]-1-triphenylmethyl-1H-tetrazole, to the
acylation of the amino group at 2-position of the pyrrole ring, and then to the detritylation process to yield the second class of
title compounds. © 1999 Elsevier Science S.A. All rights reserved.
Keywords: Pyrrole; Biphenylyltetrazole; Deprotection; N-Acylation; N-Alkylation
1. Introduction
initial lead reported by Takeda 1a (see Fig. 1) [5],
researchers at DuPont discovered Losartan 1b (see Fig.
1) [6], the first orally active nonpeptide AII antagonist
which was marketed for the treatment of hypertension
(1994, Cozaar) [7]. Weinstock et al. [8] showed that the
replacement, in Losartan, of the biphenylyltetrazole
(BPT) moiety, which is linked to an imidazole ring via
The renin–angiotensin system (RAS) is known to
play an important role in the regulation of blood
pressure and electrolyte balance [1,2]. The blocking of
the RAS with angiotensin-converting enzyme (ACE)
inhibitors has been shown to be effective in the control
of hypertension and congestive heart failure also if
there are some side effects such as dry cough and
angioedema caused by the non specific action of ACE
[3,4].
On the basis of the effectiveness of ACE inhibitors in
cardiovascular control, there has been intense activity
regarding the discovery of oral angiotensin II (AII)
antagonists as a means of inhibiting the RAS, with the
hope of obtaining greater pharmacological selectivity
than observed with ACE inhibitors. Starting from the
* Corresponding author. Fax: +39-722-2907.
E-mail address: attanasi@fis.uniurb.it (O.A. Attanasi)
1 Deceased on 1st August 1998.
Fig. 1.
0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(98)00100-1

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
65
a methylene group, implies poor activity while the
imidazole moiety can be successfully replaced by other
cyclic and acyclic structures 1c (see Fig. 1).
Therefore, the imidazole heterocycle has been postu-
lated as useful mainly to link the required functionali-
ties. In fact, Alvarez-Builla and co-workers have
confirmed recently that imidazole ring in Losartan ref-
erence can be replaced by 2-n-butylbenzimidazole or
1H-naphthol[2,3-d][1,2,3]triazole conferring promising
activities to the molecules prepared. Indeed they have
shown that even an arylthienyl bridge can be used
instead of a biphenyl without a significant decrease in
activity [9].
Ellingboe et al. [10b] prepared a series of pyrido-
[2,3-d]pyrimidine by using the common structural simi-
larities among some of the reported AII antagonists.
They demonstrated the importance of the electronic
and steric character of the substituents on the
pyrido[2,3-d]pyrimidine ring system.
A substituent capable of participation in a hydrogen
bond was not beneficial at positions near the nitrogen
atoms of the pyrimidine ring (positions 2, 4) while the
introduction of alkyl groups in these positions increased
binding affinity; on the other hand a hydrogen-bond
acceptor near the nitrogen atom of the saturated ring
(position 7) appeared important for binding to the AT1
receptor.
Fig. 2.
Based on our previous results, we were interested in
exploring a series of biphenylyltetrazole derivatives of
1-aminopyrroles (see Fig. 2). In fact, in the previous
heteroaromatic rings and in Winn et al.’s report [10a]
the presence of a nitrogen atom outside the ring could
favour an increased binding affinity of the molecules
with the receptor because of an enlargement of the zone
of negative potential around these nitrogen atoms, in-
creasing the potency of the AII antagonists.
2. Molecular modelling
The alkyl groups may fit into a lipophilic pocket in
the AT1 receptor and hydrogen-bond acceptor adjacent
to the point of attachment of the BPT group in the
heterocyclic portion may increase drug–receptor inter-
actions and binding affinity.
Thus, a target compound should incorporate a car-
boxylic acid, an alkyl group and a tetrazole substituted
biphenyl moiety, each attached to a central heterocycle.
However, the exact location of these groups and the
optimum heterocycle ring still had to be determined
exactly [10a].
In a previous work [2], we modified the imidazole
system with the substitution of the chlorine in DUP 753
with some electron-deficient heteroaromatic moieties
leading to the conjugated bis-heteroaryl system. This
replacement produced compounds with potent AII an-
tagonist activity.
We chose to compare the MEP distributions and
structures of compounds 5a, 5b, 5e and 9a (see Fig. 2)
with those of DUP 753 (Losartan) [11] (see Fig. 3) and
A 81988 [10a] (see Fig. 4) considered as reference
systems.
Since the X-ray coordinates of DUP 753 and A
81988 were not available to us, several minimum energy
structures have been generated using the SYBYL molecu-
lar modelling software [12] starting from model built
arrangements with randomly chosen but reasonable
dihedral angles. The partial charges for the electrostatic
contribution in SYBYL have been computed with the
Gasteiger–Hu¨ckel (GH) method [13–15] and the
We verified, by molecular modelling studies, that the
substituents in heterocycle rings are able to modify the
charge distribution in such a way as to influence the
activity. In order to correlate the results of the binding
data with molecular structure we examined the molecu-
lar electrostatic potential (MEP) of our compounds.
The MEP is a very important index of reactivity and
recognition in that its magnitude and shape can give
useful information on the site and geometry of likely
attack. The MEP is a measurable quantity that depends
on the three-dimensional (3D) structure and charge
distribution of the molecule.
Fig. 3. Minimum energy structure of DUP 753 chosen as reference
geometry.

66
O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
The SYBYL force field with the inclusion of the elec-
trostatic contribution in the calculation (GH charges,
m=R) was able to satisfactorily reproduce our ab initio
model structure, thus conformational searches for com-
pounds 5a, 5b, 5e and 9a have been performed with
SYBYL. The threshold used was a maximum energy
difference of 10 kcal/mol with respect to the most stable
conformer.
We compared the MEP produced by the GH charges
of some low energy conformers of the molecules 5a, 5b,
5e and 9a with the MEP of the molecules DUP 753
(better reference) and A 81988 (see Figs. 6 and 7).
Fig. 4. Minimum energy structure of A 81988 chosen as reference
geometry.
dielectric constant has been set equal to the value of R,
the separation between a pair of atoms (m=R).
In order to reduce the number of possible conforma-
tions we compared the structures obtained with sim-
plified models optimised with ab initio calculations
carried out at the SCF level with a 3-21G basis set
[2,16], and with the X-ray resolved structures of some
other AII receptor antagonists [17–21] contained in the
Cambridge Structural Database [22].
By following this procedure, we found suitable refer-
ence geometries of the two compounds DUP 753 and A
81988 (see Fig. 5).
In order to determine the 3D structure of the N-acyl-
1-aminopyrrole, a model system, in which the R1, R2,
R3, R4, R6 chains were replaced by hydrogen atoms and
the R5 chain was replaced by a methyl group (see Fig.
2), was optimised by ab initio calculations [23] carried
out at the SCF level with a 6-31G* basis set [24,25].
The final structure showed a perpendicular arrange-
ment of the pyrrole ring with respect to the NCO plane.
Fig. 6. MEP of A 81988 and DUP 753 produced by Gasteiger–
Hu¨ckel partial charges (black, light grey solid surfaces= −5 and +5
kcal/mol, respectively; black, grey, light grey grid surfaces= −1 and
+1 kcal/mol, respectively).
˚
The optimised value of the N–N distance (1.37 A) was
slightly longer than a standard double bond between N
atoms but decidedly shorter than standard single
bonds.
Fig. 7. MEP of simplified models of A 81988 and DUP 753, in which
the BPT segment has been replaced by a methyl group, produced by
Gasteiger–Hu¨ckel partial charges (black, light grey solid surfaces=
−5 and +5 kcal/mol, respectively; black, grey, light grey grid
surfaces= −1 and +1 kcal/mol, respectively).
Fig. 5. Superimposition between DUP 753 and A 81988.

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
67
The isopotential surfaces at +5 and −5 kcal/mol
and at +1 and −1 kcal/mol of the complete structures
and of the simplified models, in which the BPT segment
has been replaced by a methyl group (see Figs. 7, 9, 11,
13 and 15) were examined. By comparison of the
isopotential surfaces of the complete structures with the
partial structures we noted that the BPT segment gave
a positive contribution in the positive part and a nega-
tive contribution in the negative part, increasing the
extension of both zones (see Figs. 6, 8, 10, 12 and 14).
The overall shape of the isopotential surfaces at +1
and −1 kcal/mol of some low energy conformers of
the new molecules were similar to that of the reference
structures, even if greatly enlarged.
The molecules examined showed the negative and
positive isopotential surfaces oriented in a similar way
to that of the reference molecules.
Molecular mechanics (SYBYL) and quantum mechan-
ics (Gaussian94) [23] calculations, geometries and MEP
visualisations have been performed on the IRIS/4D-
420-GTXB workstation.
Fig. 8. MEP of DUP 753 and 5a produced by Gasteiger–Hu¨ckel
partial charges (black, light grey solid surfaces= −5 and +5 kcal/
mol, respectively; black, grey, light grey grid surfaces= −1 and +1
kcal/mol, respectively).
Fig. 10. MEP of DUP 753 and 5b produced by Gasteiger–Hu¨ckel
partial charges (black, light grey solid surfaces= −5 and +5 kcal/
mol, respectively; black, grey, light grey grid surfaces= −1 and +1
kcal/mol, respectively).
Fig. 11. MEP of simplified models of DUP 753 and 5b produced by
Gasteiger–Hu¨ckel partial charges (black, light grey solid surfaces=
−5 and +5 kcal/mol, respectively; black, grey, light grey grid
surfaces= −1 and +1 kcal/mol, respectively).
Fig. 9. MEP of simplified models of DUP 753 and 5a produced by
Gasteiger–Hu¨ckel partial charges (black, light grey solid surfaces=
−5 and +5 kcal/mol, respectively; black, grey, light grey grid
surfaces= −1 and +1 kcal/mol, respectively).

68
O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
Fig. 12. MEP of DUP 753 and 5e produced by Gasteiger–Hu¨ckel
partial charges (black, light grey solid surfaces= −5 and +5 kcal/
mol, respectively; black, grey, light grey grid surfaces= −1 and +1
kcal/mol, respectively).
Fig. 14. MEP of DUP 753 and 9a produced by Gasteiger–Hu¨ckel
partial charges (black, light grey solid surfaces= −5 and +5 kcal/
mol, respectively; black, grey, light grey grid surfaces= −1 and +1
kcal/mol, respectively).
Fig. 13. MEP of simplified models of DUP 753 and 5e produced by
Gasteiger–Hu¨ckel partial charges (black, light grey solid surfaces=
−5 and +5 kcal/mol, respectively; black, grey, light grey grid
surfaces= −1 and +1 kcal/mol, respectively).
3. Chemistry
Fig. 15. MEP of simplified models of DUP 753 and 9a produced by
Gasteiger–Hu¨ckel partial charges (black, light grey solid surfaces=
−5 and +5 kcal/mol, respectively; black, grey, light grey grid
surfaces= −1 and +1 kcal/mol, respectively).
A series of N-acyl-1-aminopyrrole compounds [26]
containing the BPT moiety linked to the N-amino
group by a methylene group (5a–e), was obtained as
shown in Scheme 1. The removal of the Boc protect-
ing group (CO2CMe3) from NH-Boc protected 1-
aminopyrrole derivatives 1a–e was carried out by
heating at 170°C. The resulting deprotected 1-
aminopyrrole derivatives 2a–e (69.5–91.3%), dissolved
in anhydrous THF, were at first treated with valeryl
chloride in the presence of anhydrous pyridine, to
yield the NH-acylated compounds 3a–e (80.4–94.5%).
The subsequent N-alkylation of the derivatives 3a–e
with 5-[4%-bromomethyl-1,1%-biphenyl-2-yl]-1-triphenyl-
methyl-1H-tetrazole was achieved in dichloromethane
at room temperature (r.t.) in the presence of sodium
hydroxide providing the derivatives 4a–e (59.2–
88.7%). Finally, the removal of the trityl protecting
group (Ph3C) from the tetrazole ring was performed
by treatment of the compounds 4a–e under

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
69
Scheme 1.
reflux with methyl or ethyl alcohol, giving the final
products 5a–e in good yields (86.2–93.4%).
these new compounds unfortunately were found inac-
tive (pKi55).
The reaction of 1-NH-Boc protected 1,2-diamino-
pyrroles 6a–b (see Scheme 2) with 5-[4%-bromomethyl-
1,1%-biphenyl-2-yl]-1-triphenylmethyl-1H-tetrazole in di-
chloromethane at r.t. in the presence of sodium hydrox-
ide led exclusively to the N-alkylation of the amino
group linked to the nitrogen heteroatom, producing the
compounds 7a–b (57–74.8%). Acylation of the amino
group at 2-position of the pyrrole ring of these com-
pounds with valeryl chloride at r.t. furnished the
derivatives 8a–b (63.8–64%). The target products 9a–b
(79.9–95.1%) were obtained by detritylation of the
compounds 8a–b with methyl or ethyl alcohol under
reflux.
5. Conclusions
Unfortunately the examined compounds did not pos-
sess remarkable affinity for AT1 receptor. The reduced
binding affinity shown by these central 1-aminopyrrole
compounds might be due to the fact that the conforma-
tional behaviour of these molecules is not in agreement
with the geometrical activity model derived from pub-
lished data and theoretical calculations. The almost
planar disposition of the 1-aminopyrrole moiety could
allow the structure to exist in conformations not opti-
mal for receptor binding. Nevertheless, there is a con-
ceptual difference between the computational results
and the way the compounds act in the biological
experiments.
4. Pharmacology
The series of compounds (5a, 5b, 5e, and 9a) were
measured for their interaction with the receptor by way
of their inhibition of [3H]AII binding to rat adrenal
cortex membrane preparations (AT1 receptors). In spite
of the promising chemical and theoretical assumptions,
The calculations deal with molecules in the gas
phase, no outside influences are present at all, whereas
compounds in a biological environment are surrounded
by a complicated liquid phase where they interact with
many other molecules in polar and apolar circum-

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O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
Scheme 2.
6. Experimental
stances. This might cause discrepancies in any compari-
son with experimental results and does not facilitate
our search for a relationship between antagonist activ-
ity and calculated properties.
6.1. Chemistry
The energy differences among the rotational isomers
of our compounds are small enough to allow the sub-
stituents large rotational freedom. Interactions with
solvent molecules might determine the actual conforma-
tion of the compounds rather than the intramolecular
forces that dictate the molecular mechanic equilibrium
geometries.
More populated low-energy conformers, which are
incompatible with the hypothesised receptor-bound
structure, might be present. Thus the important func-
tional groups could assume spatial positions that give
rise to unfavourable interactions inside the receptor.
However, it should be emphasised that this obviously
is pure speculation and is based on possible interpreta-
tions of the negative results and that this is not a model
directly supported by the presented data.
Commercially available solvents were used without
further purification, except for THF, which was dis-
tilled on sodium hydroxide and stored under 4A molec-
˚
ular sieves. Valeryl chloride was commercial material
(Janssen Chimica) and was used without further puri-
fication. 5-[4%-Bromomethyl-1,1%-biphenyl-2-yl]-1-tri-
phenyl-methyl-1H-tetrazole was prepared as reported
[27]. 1-NH-Boc protected aminopyrroles 1a–b are
known [28], 1c–e are new compounds and were pre-
pared by analogous methodology; 6a was previously
obtained [29], 6b was synthesised according to the same
procedure. Characterisation and spectroscopic data of
1c–e and 6b are reported below. Melting points were
determined in open capillary tubes with a Gallenkamp
apparatus and are uncorrected. All yields referred to
pure isolated products. All FT-IR spectra were per-

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
71
6.1.1. Synthesis of the 1-aminopyrrole deri6ati6es
(2a–e)
formed in KBr for Nujol mulls and were obtained
using a Nicolet Impact 400 spectrophotometer. MS
spectra were performed with Hewlett–Packard
a
The NH-Boc protected 1-aminopyrrole derivatives
1a–e (1 mmol) were heated in an oil bath at 170°C
until the starting compounds disappeared (20–40 min,
monitored by TLC). The resulting dark residues were
purified by chromatography on a silica gel column by
elution with cyclohexane–ethyl acetate mixtures to
give the relevant 1-aminopyrrole derivatives 2a–e.
2a: 69.5% yield as yellowish powder from
dichloromethane–light petroleum (b.p. 40–60°C), m.p.
104–106°C. 1H NMR l: 1.20 (t, 3H, J=6.6 Hz,
OCH2CH3), 2.24 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.65
(s, 2H, CH2CO2CH2CH3), 4.09 (q, 2H, J=6.6 Hz,
OCH2CH3), 5.50 (s, 2H, NH2), 6.26 (s, 1H, CH). IR
(KBr) cm−1: 3335, 1707, 1657. MS m/z (rel. int.%):
224 (M+, 30), 151 (100). Anal. Calc. for C11H16N2O3:
C, 58.91; H, 7.19; N, 12.49. Found: C, 58.79; H, 7.30;
N, 12.65%.
2b: 84.7% yield as white powder from ethyl ether,
m.p. 79.5–81.5°C. 1H NMR l: 1.19 (t, 3H, J=7.1
Hz, OCH2CH3), 2.48 (s, 3H, CH3), 3.68 (s, 2H, CH2-
CO2CH2CH3), 4.12 (q, 2H, J=7.1 Hz, OCH2CH3),
5.61 (s, 2H, NH2), 6.04 (s, 1H, CH), 7.47–7.55 (m,
3H, arom.), 7.62–7.66 (m, 2H, arom.). IR (KBr)
cm−1: 3352, 3272, 1722, 1612, 1564. MS m/z (rel.
int.%): 286 (M+, 80), 213 (100). Anal. Calc. for
C16H18N2O3: C, 67.12; H, 6.34; N, 9.78. Found: C,
67.00; H, 6.45; N, 9.66%.
1
5995C spectrometer. All H NMR spectra were mea-
sured using a Bruker AC-200 (200 MHz) Fourier
transform spectrometer equipped with cryomagnet, in
DMSO-d6 solution unless otherwise stated. All the
spectra were measured at 298 K. Chemical shifts (lH)
are reported in ppm downfield from internal Me4Si.
J-Values are given in Hz. The abbreviations used are
as follows: s, singlet; d, doublet; t, triplet; q, quartet;
m, multiplet; br s, broad singlet. The assignments of
NH and NH2 signals were confirmed by the disappear-
ance of the signals after addition of deuterium oxide.
Macherey–Nagel precoated silica gel SIL G-25UV254
plates (0.25 mm thick) were used for analytical TLC
˚
and silica gel Amicon LC 60A (35–70 m) for column
chromatography.
1c: 55.4% yield as colourless crystals from ether,
1
m.p. 103–104°C. H NMR l: 1.45 (s, 9H, OBut), 2.29
(s, 6H, 2CH3), 3.50–3.63 (m, 5H, CH2CO2CH3 and
OCH3), 6.39 (s, 1H, CH), 10.30 (br s, 1H, NH). IR
(KBr) cm−1: 3270, 1740, 1660, 1640, 1580. MS m/z
(rel. int.%): 310 (M+, 29), 254 (44), 195 (100). Anal.
Calc. for C15H22N2O5: C, 58.05; H, 7.15; N, 9.03.
Found: C, 57.98; H, 7.24; N, 8.93%.
1d: 72.2% yield as colourless crystals from ether,
m.p. 152–154°C (dec.). 1H NMR l: 1.46 (s, 9H,
OBut), 2.29 (s, 3H, CH3), 3.52–3.67 (m, 5H,
CH2CO2CH3 and OCH3), 6.16 (s, 1H, CH), 7.47–7.68
(m, 5H, arom.), 10.38 (br s, 1H, NH). IR (KBr)
cm−1: 3195, 1740, 1730, 1620, 1600, 1570. MS m/z
(rel. int.%): 372 (M+, 91), 316 (100), 271 (45), 256
(59). Anal. Calc. for C20H24N2O5: C, 64.50; H, 6.50;
N, 7.52. Found: C, 64.36; H, 6.62; N, 7.36%.
1e: 62% yield as white powder from dichlo-
romethane–light petroleum (b.p. 40–60°C), m.p. 137–
139°C. 1H NMR l: 1.28 (t, 3H, J=7.1 Hz,
OCH2CH3), 1.45 (s, 9H, OBut), 1.94 (s, 3H, CH3),
2.01 (s, 3H, CH3), 2.17 (s, 3H, CH3), 4.30 (q, 2H,
J=7.1 Hz, OCH2CH3), 10.36 (br s, 1H, NH). IR
(KBr) cm−1: 3316, 1742, 1716, 1638, 1601. MS m/z
(rel. int.%): 324 (M+, 51), 251 (69), 151 (100). Anal.
Calc. for C16H24N2O5: C, 59.24; H, 7.46; N, 8.64.
Found: C, 59.36; H, 7.32; N, 8.78%.
2c: 75.9% yield as yellowish powder from
dichloromethane–light petroleum (b.p. 40–60°C), m.p.
93–93.5°C. 1H NMR l: 2.23 (s, 3H, CH3), 2.43 (s,
3H, CH3), 3.62 (s, 3H, OCH3), 3.66 (s, 2H,
CH2CO2CH3), 5.50 (s, 2H, NH2), 6.26 (s, 1H, CH). IR
(KBr) cm−1: 3340, 3280, 1710, 1660. MS m/z (rel.
int.%): 210 (M+, 55), 195 (22), 151 (100). Anal. Calc.
for C10H14N2O3: C, 58.05; H, 7.15; N, 9.03. Found: C,
57.96; H, 7.23; N, 9.03%.
2d: 83.1% yield as yellowish crystals from
dichloromethane–light petroleum (b.p. 40–60°C), m.p.
1
103–104°C. H NMR l: 2.47 (s, 3H, CH3), 3.62 (s,
3H, OCH3), 3.70 (s, 2H, CH2CO2CH3), 5.60 (s, 2H,
NH2), 6.03 (s, 1H, CH), 7.47–7.65 (m, 5H, arom.). IR
(KBr) cm−1: 3359, 3270, 1733, 1720, 1613, 1563. MS
m/z (rel. int.%): 272 (M+, 95), 213 (100). Anal. Calc.
for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29. Found:
C, 66.31; H, 5.78; N, 10.43%.
6b: 60.02% yield as beige powder from ether–light
1
petroleum (b.p. 40–60°C), m.p. 165–168°C (dec.). H
NMR l: 1.43–1.54 (m, 15H, cyclic CH2 and OBut),
2.16 (s, 3H, CH3), 3.62 (s, 3H, OCH3), 3.32–3.40 (m,
5H, cyclic CH2), 4.71 (s, 2H, NH2), 9.90 (s, 1H, NH).
IR (KBr) cm−1: 3440, 3360, 3220, 1725, 1700, 1670.
MS m/z (rel. int.%): 380 (M+, 44), 324 (57), 280 (56),
195 (61), 84 (100). Anal. Calc. for C18H28N4O5: C,
56.83; H, 7.42; N, 14.73. Found: C, 56.95; H, 7.42; N,
14.60%.
1
2e: 91.3% yield as yellow oil. H NMR (CDCl3) l:
1.37 (t, 3H, J=7.1 Hz, OCH2CH3), 2.08 (s, 3H, CH3),
2.12 (s, 3H, CH3), 2.43 (s, 3H, CH3), 4.30–4.41 (m,
4H, NH2 and OCH2CH3). IR (KBr) cm−1: 3354,
3220, 1735, 1627. MS m/z (rel. int.%): 224 (M+, 18),
151 (100). Anal. Calc. for C11H16N2O3: C, 58.91; H,
7.19; N, 12.49. Found: C, 58.92; H, 7.31; N, 12.34%.

72
O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
1
6.1.2. Synthesis of the N-acyl-1-aminopyrrole
3d: 91.7% yield as yellow oil. H NMR (CDCl3) l:
deri6ati6es (3a–e)
0.94 (t, 3H, J=7.2 Hz, COCH2CH2CH2CH3), 1.31–
1.49 (m, 2H, COCH2CH2CH2CH3), 1.63–1.78 (m, 2H,
COCH2CH2CH2CH3), 2.23 (s, 3H, CH3), 2.40 (t, 2H,
J=7.3 Hz, COCH2CH2CH2CH3), 3.42 (d, 1H, J=16.0
Hz, CH2CO2CH3), 3.64 (d, 1H, J=16.0 Hz,
CH2CO2CH3), 3.68 (s, 3H, OCH3), 6.26 (s, 1H, CH),
7.42–7.52 (m, 3H, arom.), 7.74–7.79 (m, 2H, arom.),
9.34 (s, 1H, NH). IR (KBr) cm−1: 3241, 1744, 1716,
1637, 1577. MS m/z (rel. int.%): 356 (M+, 83), 256
(78), 196 (100). Anal. Calc. for C20H24N2O4: C, 67.40;
H, 6.79; N, 7.86. Found: C, 67.40; H, 6.64; N, 7.99%.
Valeryl chloride (181 mg, 1.501 mmol) dissolved in
anhydrous THF (1 ml) was slowly added to a stirred
solution of the 1-aminopyrrole derivatives 2a–e (1
mmol) and anhydrous pyridine (127 mg, 1.605 mmol)
in anhydrous THF (4 ml). After the formation of a
white solid, the reaction mixture was refluxed until
1-aminopyrrole derivatives disappeared (2–5 h, moni-
tored by TLC). After the evaporation of the solvent
under reduced pressure, the residue was solved in ethyl
ether, poured into a separatory funnel and washed
twice with water. The organic layer was dried over
anhydrous sodium sulfate and evaporated under re-
duced pressure to give the residue that afforded the
N-acyl derivatives 3a–e by crystallisation from ethyl
ether–light petroleum (b.p. 40–60°C) or by chromatog-
raphy on a silica gel column by elution with cyclohex-
ane–ethyl acetate mixtures.
1
3e: 93.1% yield as yellow oil. H NMR (CDCl3) l:
0.88 (t, 3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.26–
1.39 (m, 5H, OCH2CH3 and COCH2CH2CH2CH3),
1.54–1.67 (m, 2H, COCH2CH2CH2CH3), 1.83 (s, 3H,
CH3), 1.95 (s, 3H, CH3), 2.08 (s, 3H, CH3), 2.30 (t, 2H,
J=7.1 Hz, COCH2CH2CH2CH3), 4.29 (q, 2H, J=7.1
Hz, OCH2CH3), 9.60 (s, 1H, NH). IR (KBr) cm−1
:
3a: 85.6% yield as colourless plates from ethyl ether–
light petroleum (b.p. 40–60°C), m.p. 73–75°C.
1H NMR l: 0.90 (t, 3H, J=7.2 Hz, COCH2CH2-
CH2CH3), 1.19 (t, 3H, J=7.1 Hz, OCH2CH3), 1.27–
1.38 (m, 2H, COCH2CH2CH2CH3), 1.49–1.61 (m, 2H,
COCH2CH2CH2CH3), 2.22–2.32 (m, 8H, 2CH3 and
COCH2CH2CH2CH3), 3.33 (d, 1H, J=16.8 Hz, CH2-
CO2CH2CH3), 3.58 (d, 1H, J=16.8 Hz, CH2-
CO2CH2CH3), 4.07 (q, 2H, J=7.1 Hz, OCH2CH3),
3243, 1738, 1683, 1645. MS m/z (rel. int.%): 308 (M+,
11), 235 (100). Anal. Calc. for C16H24N2O4: C, 62.32; H,
7.84; N, 9.08. Found: C, 62.42; H, 7.72; N, 9.21%.
6.1.3. Synthesis of the compounds 4a–e
Sodium hydroxide as tritured pellets (80 mg, 2 mmol)
was added to a stirred solution of the N-acyl deriva-
tives 3a–e (1 mmol) and 5-[4%-bromomethyl-1,1%-
biphenyl-2-yl]-1-triphenylmethyl-1H-tetrazole (835 mg,
1.5 mmol) in dichloromethane (4 ml). The reaction
mixture was allowed to stand at r.t. until the N-acyl
derivatives disappeared (7–9 days, monitored by TLC).
The solvent was removed under reduced pressure and
the residue was solved in ethyl acetate, poured into a
separatory funnel and washed with water until neutral-
ity of the aqueous phase. The organic layer was dried
over anhydrous sodium sulfate and evaporated under
reduced pressure. The crude product was purified by
chromatography on a silica gel column by elution with
cyclohexane–ethyl acetate mixtures to obtain the re-
spective products 4a–e.
6.40 (s, 1H, CH), 10.96 (s, 1H, NH). IR (KBr) cm−1
:
3301, 1719, 1690, 1665. MS m/z (rel. int.%): 308 (M+,
47), 235 (41), 208 (100). Anal. Calc. for C16H24N2O4: C,
62.32; H, 7.84; N, 9.08. Found: C, 62.44; H, 7.71; N,
9.21%.
1
3b: 80.4% yield as orange oil. H NMR l: 0.91 (t,
3H, J=7.4 Hz, COCH2CH2CH2CH3), 1.19 (t, 3H,
J=7.3 Hz, OCH2CH3), 1.28–1.40 (m, 2H, COCH2-
CH2CH2CH3), 1.51–1.64 (m, 2H, COCH2CH2CH2-
CH3), 2.28–2.36 (m, 5H, CH3 and COCH2CH2-
CH2CH3), 3.37 (d, 1H, J=17.0 Hz, CH2CO2CH2CH3),
3.63 (d, 1H, J=17.0 Hz, CH2CO2CH2CH3), 4.07 (q,
2H, J=7.3 Hz, OCH2CH3), 6.19 (s, 1H, CH), 7.51–
7.69 (m, 5H, arom.), 11.05 (s, 1H, NH). IR (KBr)
cm−1: 3247, 3190, 1746, 1713, 1684, 1641, 1570. MS
m/z (rel. int.%): 370 (M+, 61), 270 (76), 196 (100).
Anal. Calc. for C21H26N2O4: C, 68.09; H, 7.07; N, 7.56.
Found: C, 68.21; H, 7.17; N, 7.43%.
1
4a: 88.7% yield as white foam. H NMR l: 0.81 (t,
3H, J=7.2 Hz, COCH2CH2CH2CH3), 1.10–1.22 (m,
5H, OCH2CH3 and COCH2CH2CH2CH3), 1.36–1.82
(m, 7H, CH3 and COCH2CH2CH2CH3), 2.28 (s, 3H,
CH3), 3.31 (s, 2H, CH2CO2CH2CH3), 4.04 (q, 2H,
J=7.0 Hz, OCH2CH3), 4.62 (d, 1H, J=14.4 Hz,
CH2–Ar), 4.99 (d, 1H, J=14.4 Hz, CH2–Ar), 6.51 (s,
3c: 94.5% yield as white powder from ethyl ether,
1
m.p. 77–78°C. H NMR l: 0.90 (t, 3H, J=7.0 Hz,
COCH2CH2CH2CH3), 1.23–1.41 (m, 2H, COCH2CH2-
CH2CH3), 1.49–1.69 (m, 2H, COCH2CH2CH2CH3),
2.22–2.32 (m, 8H, 2CH3 and COCH2CH2CH2CH3),
3.55–3.64 (m, 5H, OCH3 and CH2CO2CH3), 6.41 (s,
1H, CH), 10.99 (s, 1H, NH). IR (KBr) cm−1: 3309,
1726, 1689, 1664. MS m/z (rel. int.%): 294 (M+, 43),
194 (100). Anal. Calc. for C15H22N2O4: C, 58.05; H,
7.15; N, 9.03. Found: C, 57.97; H, 7.27; N, 9.17%.
1H, CH), 6.90–7.77 (m, 23H, arom.). IR (KBr) cm−1
:
1741, 1692, 1669. Anal. Calc. for C49H48N6O4: C, 74.98;
H, 6.16; N, 10.71. Found: C, 74.86; H, 6.28; N, 10.60%.
1
4b: 73.1% yield as white foam. H NMR l: 0.80 (t,
3H, J=7.2 Hz, COCH2CH2CH2CH3), 1.08–1.24 (m,
5H, OCH2CH3 and COCH2CH2CH2CH3), 1.39–1.49
(m, 2H, COCH2CH2CH2CH3), 1.72–1.82 (m, 5H, CH3

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
73
and COCH2CH2CH2CH3), 3.28 (s, 2H, CH2CO2CH2-
CH3), 3.99 (q, 2H, J=7.1 Hz, OCH2CH3), 4.65 (d, 1H,
J=14.4 Hz, CH2–Ar), 4.97 (d, 1H, J=14.4 Hz, CH2–
Ar), 6.25 (s, 1H, CH), 6.86–7.79 (m, 28H, arom.). IR
(KBr) cm−1: 1738, 1689, 1643, 1601. Anal. Calc. for
C54H50N6O4: C, 76.57; H, 5.95; N, 9.92. Found: C,
76.44; H, 6.07; N, 9.78%.
7a: 74.8% yield as white powder from chloroform–
light petroleum (b.p. 40–60°C), m.p. 119–126°C (dec.).
1H NMR l: 1.13 (t, 3H, J=7.1 Hz, OCH2CH3),
1.32–1.50 (m, 15H, cyclic CH2 and OBut), 1.64 (s, 3H,
CH3), 3.30–3.42 (m, 4H, cyclic CH2), 4.00 (q, 2H,
J=7.1 Hz, OCH2CH3), 4.38 (d, 1H, J=14.7 Hz,
CH2–Ar), 4.59 (br s, 2H, NH2), 5.02 (d, 1H, J=14.7
Hz, CH2–Ar), 6.86–7.79 (m, 23H, arom.). IR (KBr)
cm−1: 3420, 3320, 1690, 1642, 1601. Anal. Calc. for
C52H54N8O5: C, 71.70; H, 6.25; N, 12.86. Found: C,
71.56; H, 6.37; N, 12.72%.
7b: 57.0% yield as white powder from chloroform–
light petroleum (b.p. 40–60°C), m.p. 118–125°C (dec.).
1H NMR l: 1.32–1.62 (m, 18H, cyclic CH2, OBut and
CH3), 3.32–3.43 (m, 4H, cyclic CH2), 3.55 (s, 3H,
OCH3), 4.37 (d, 1H, J=14.7 Hz, CH2–Ar), 4.73 (br s,
2H, NH2), 5.06 (d, 1H, J=14.7 Hz, CH2–Ar), 6.88–
7.81 (m, 23H, arom.). IR (KBr) cm−1: 3411, 3314,
1707, 1691, 1604. Anal. Calc. for C51H52N8O5: C, 71.48;
H, 6.12; N, 13.07. Found: C, 71.36; H, 6.26; N, 13.07%.
1
4c: 59.2% yield as white foam. H NMR l: 0.78 (t,
3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.07–1.22 (m,
2H, COCH2CH2CH2CH3), 1.38–1.49 (m, 2H,
COCH2CH2CH2CH3), 1.68–1.83 (m, 5H, CH3 and
COCH2CH2CH2CH3), 2.26 (s, 3H, CH3), 3.30 (s, 2H,
CH2CO2CH3), 3.55 (s, 3H, OCH3), 4.64 (d, 1H, J=
14.3 Hz, CH2–Ar), 4.95 (d, 1H, J=14.3 Hz, CH2–Ar),
6.49 (s, 1H, CH), 6.89–7.79 (m, 23H, arom.). IR (KBr)
cm−1
: 1742, 1687, 1662, 1578. Anal. Calc. for
C48H46N6O4: C, 74.78; H, 6.01; N, 10.90. Found: C,
74.90; H, 6.15; N, 10.76%.
1
4d: 68.9% yield as white foam. H NMR (CDCl3) l:
0.88 (t, 3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.22–
1.33 (m, 2H, COCH2CH2CH2CH3), 1.58–1.65 (m, 2H,
COCH2CH2CH2CH3), 1.83–1.95 (m, 2H, COCH2CH2-
CH2CH3), 2.10 (s, 3H, CH3), 3.04 (s, 2H, CH2CO2-
CH3), 3.59 (s, 3H, OCH3), 4.65 (d, 1H, J=14.0 Hz,
CH2–Ar), 4.78 (d, 1H, J=14.0 Hz, CH2–Ar), 6.33 (s,
6.1.5. Synthesis of the N-acyl deri6ati6es (8a–b)
Valeryl chloride (181 mg, 1.501 mmol) dissolved in
anhydrous THF (1 ml) was slowly added to a stirred
solution of the 1-aminopyrrole derivatives 7a–b (1
mmol) and anhydrous pyridine (127 mg, 1.605 mmol)
in anhydrous THF (4 ml). After the formation of a
white solid, the reaction mixture was stirred at r.t. until
compounds 7a–b disappeared (17–24 h, monitored by
TLC). After the evaporation of the solvent under re-
duced pressure, the residue was solved in dichlo-
romethane, poured into a separatory funnel and
washed twice with water. The organic layer was dried
over anhydrous sodium sulfate and evaporated under
reduced pressure to give a residue that directly provided
the N-acyl derivatives 8a–b by crystallisation from
ethyl ether.
8a: 63.8% yield as whitish powder from ethyl ether,
m.p. 105–110°C (dec.). 1H NMR l: 0.90 (t, 3H, J=7.1
Hz, COCH2CH2CH2CH3), 1.16 (t, 3H, J=7.1 Hz,
OCH2CH3), 1.32–1.59 (m, 22H, OBut, cyclic CH2, CH3
and COCH2CH2CH2CH3), 2.24–2.30 (m, 2H,
COCH2CH2CH2CH3), 3.18–3.23 (m, 2H, cyclic CH2),
3.68–3.74 (m, 2H, cyclic CH2), 4.04 (q, 2H, J=7.1 Hz,
OCH2CH3), 4.25–4.36 (m, 1H, CH2–Ar), 4.92–5.23
(m, 1H, CH2–Ar), 6.90–7.81 (m, 23H, arom.), 9.56 (br
s, 1H, NH). IR (KBr) cm−1: 3165, 1710, 1680, 1599.
Anal. Calc. for C57H62N8O6: C, 71.68; H, 6.54; N,
11.73. Found: C, 71.81; H, 6.42; N, 11.73%.
1H, CH), 6.90–7.78 (m, 28H, arom.). IR (KBr) cm−1
:
1742, 1687, 1644, 1585. Anal. Calc. for C53H48N6O4: C,
76.42; H, 5.81; N, 10.09. Found: C, 76.27; H, 5.81; N,
10.22%.
1
4e: 71.0% yield as white foam. H NMR l: 0.75 (t,
3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.10–1.22 (m,
5H, OCH2CH3 and COCH2CH2CH2CH3), 1.40–1.54
(m, 2H, COCH2CH2CH2CH3), 1.61–1.77 (m, 8H,
2CH3 and COCH2CH2CH2CH3), 1.95 (s, 3H, CH3),
4.22 (q, 2H, J=7.1 Hz, OCH2CH3), 4.70 (d, 1H,
J=14.3 Hz, CH2–Ar), 4.90 (d, 1H, J=14.3 Hz, CH2–
Ar), 6.78–7.76 (m, 23H, arom.). IR (KBr) cm−1: 1737,
1685, 1646, 1600. Anal. Calc. for C49H48N6O4: C, 74.98;
H, 6.16; N, 10.71. Found: C, 74.85; H, 6.28; N, 10.56%.
6.1.4. Synthesis of the compounds 7a–b
Sodium hydroxide as tritured pellets (80 mg, 2 mmol)
was added to a stirred solution of the 1-NH-Boc pro-
tected 1,2-diaminopyrroles 6a–b (1 mmol) and 5-[4%-
bromomethyl-1,1%-biphenyl-2-yl]-1-triphenylmethyl-1H-
tetrazole (835 mg, 1.5 mmol) in dichloromethane (4
ml). The reaction mixture was allowed to stand at r.t.
until the pyrrole derivatives disappeared (5–7 days,
monitored by TLC). The reaction mixture was poured
into a separatory funnel and washed with brine until
neutrality of the aqueous phase. The organic layer was
dried over anhydrous sodium sulfate and the extraction
solvent was removed under reduced pressure. The
residue was chromatographed on a silica gel column by
elution with cyclohexane–ethyl acetate mixtures to af-
ford the pertinent pure products 7a–b.
8b: 64.0% yield as light yellow powder from chloro-
form–ethyl ether–light petroleum (b.p. 40–60°C), m.p.
1
157–158°C (dec.). H NMR l: 0.89 (t, 3H, J=7.1 Hz,
COCH2CH2CH2CH3), 1.30–1.56 (m, 22H, OBut, cyclic
CH2, CH3 and COCH2CH2CH2CH3), 2.25 (t, 2H, J=
6.6 Hz, COCH2CH2CH2CH3), 3.17–3.22 (m, 2H, cyclic
CH2), 3.47–3.51 (m, 2H, cyclic CH2), 3.58 (s, 3H,

74
O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
OCH3), 4.23–4.34 (m, 1H, CH2–Ar), 4.91–5.19 (m,
1H, CH2–Ar), 6.87–7.79 (m, 23H, arom.), 9.48 (br s,
1H, NH). IR (KBr) cm−1: 3165, 1710, 1670, 1600.
Anal. Calc. for C56H60N8O6: C, 71.47; H, 6.43; N,
11.91. Found: C, 71.61; H, 6.31; N, 12.04%.
2H, COCH2CH2CH2CH3), 1.43–1.50 (m, 2H, COCH2-
CH2CH2CH3), 1.79–1.88 (m, 2H, COCH2CH2CH2-
CH3), 1.92 (s, 3H, CH3), 3.34–3.62 (m, 5H, CH2CO2-
CH3 and OCH3), 4.76 (d, 1H, J=14.3 Hz, CH2–Ar),
4.97 (d, 1H, J=14.3 Hz, CH2–Ar), 6.28 (s, 1H, CH),
7.04–7.16 (m, 4H, arom.), 7.47–7.73 (m, 9H, arom.),
16.25 (br s, 1H, NH). IR (KBr) cm−1: 3151, 1743,
1690, 1643, 1572. Anal. Calc. for C34H34N6O4: C, 69.14;
H, 5.80; N, 14.23. Found: C, 69.28; H, 5.68; N, 14.38%.
6.1.6. Synthesis of the biphenyltetrazole deri6ati6es
(5a–e) and (9a–b)
1
The compounds 4a–e and 8a–b were heated in
EtOH or MeOH under reflux until complete deprotec-
tion (1.5–3 h, monitored by TLC). The solvent was
eliminated under reduced pressure and the crude
product was chromatographed on a silica gel column
by elution with cyclohexane–ethyl acetate mixtures to
yield the biphenyltetrazole derivatives 5a–e and 9a–b.
5e: 86.2% yield as light yellow oil. H NMR l: 0.79
(t, 3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.09–1.29
(m, 5H, OCH2CH3 and COCH2CH2CH2CH3), 1.40–
1.61 (m, 2H, COCH2CH2CH2CH3), 1.67–1.78 (m, 5H,
CH3 and COCH2CH2CH2CH3), 1.87 (s, 3H, CH3), 2.02
(s, 3H, CH3), 4.30 (q, 2H, J=7.1 Hz, OCH2CH3), 4.76
(d, 1H, J=14.3 Hz, CH2–Ar), 4.90 (d, 1H, J=14.3
Hz, CH2–Ar), 7.03–7.16 (m, 4H, arom.), 7.50–7.72 (m,
1
5a: 89.4% yield as yellowish foam. H NMR l: 0.79
(t, 3H, J=7.2 Hz, COCH2CH2CH2CH3), 1.10–1.23
(m, 5H, OCH2CH3 and COCH2CH2CH2CH3), 1.37–
1.49 (m, 2H, COCH2CH2CH2CH3), 1.72–1.91 (m, 5H,
CH3 and COCH2CH2CH2CH3), 2.32 (s, 3H, CH3), 3.21
(d, 1H, J=17.1 Hz, CH2CO2CH2CH3), 3.34 (d, 1H,
J=17.1 Hz, CH2CO2CH2CH3), 4.04 (q, 2H, J=7.1
Hz, OCH2CH3), 4.65 (d, 1H, J=14.3 Hz, CH2–Ar),
4.98 (d, 1H, J=14.3 Hz, CH2–Ar), 6.51 (s, 1H, CH),
7.01–7.12 (m, 4H, arom.), 7.49–7.69 (m, 4H, arom.),
16.21 (br s, 1H, NH). IR (KBr) cm−1: 3471, 1738,
1692, 1666, 1579. Anal. Calc. for C30H34N6O4: C, 66.40;
H, 6.32; N, 15.49. Found: C, 66.54; H, 6.20; N, 15.37%.
4H, arom.), 16.20 (br s, 1H, NH). IR (KBr) cm−1
:
3060, 1737, 1686, 1644, 1597. Anal. Calc. for
C30H34N6O4: C, 66.40; H, 6.32; N, 15.49. Found: C,
66.26; H, 6.32; N, 15.60%.
9a: 79.9% yield as white powder from ethyl ether,
m.p. 195–197°C (dec.). 1H NMR l: 0.88 (t, 3H, J=7.1
Hz, COCH2CH2CH2CH3), 1.14–1.65 (m, 25H,
OCH2CH3, COCH2CH2CH2CH3, OBut, CH3 and cyclic
CH2), 2.19–2.25 (m, 2H, COCH2CH2CH2CH3), 3.08–
3.71 (m, 4H, cyclic CH2), 4.09 (q, 2H, J=7.1 Hz,
OCH2CH3), 4.25–4.37 (m, 1H, CH2–Ar), 4.94–5.24
(m, 1H, CH2–Ar), 7.05–7.18 (m, 4H, arom.), 7.50–7.89
(m, 4H, arom.), 9.28 and 9.63 (2 br s, 1H, NH), 16.31
(br s, 1H, NH). IR (KBr) cm−1: 3360, 1712, 1695,
1585. Anal. Calc. for C38H48N8O6: C, 64.03; H, 6.79; N,
15.72. Found: C, 64.17; H, 6.66; N, 15.77%.
9b: 95.1% yield as white powder from ethyl ether,
m.p. 179–182°C (dec.). 1H NMR l: 0.88 (t, 3H, J=7.1
Hz, COCH2CH2CH2CH3), 1.17–1.64 (m, 22H, OBut,
cyclic CH2, CH3, and COCH2CH2CH2CH3), 2.18–2.24
(m, 2H, COCH2CH2CH2CH3), 3.12–3.60 (m, 4H, cyclic
CH2), 3.64 (s, 3H, OCH3), 4.24–4.36 (m, 1H, CH2–Ar),
4.95–5.23 (m, 1H, CH2–Ar), 7.09–7.18 (m, 4H, arom.),
7.50–7.69 (m, 4H, arom.), 9.31 and 9.61 (2 br s, 1H,
NH), 16.31 (br s, 1H, NH). IR (KBr) cm−1: 3339,
1718, 1709, 1602, 1575. Anal. Calc. for C37H46N8O6: C,
63.59; H, 6.63; N, 16.03. Found: C, 63.48; H, 6.69; N,
16.17%.
1
5b: 93.4% yield as yellow foam. H NMR l: 0.79 (t,
3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.09–1.22 (m,
5H, OCH2CH3 and COCH2CH2CH2CH3), 1.39–1.51
(m, 2H, COCH2CH2CH2CH3), 1.76–1.91 (m, 5H, CH3
and COCH2CH2CH2CH3), 3.22 (d, 1H, J=17.3 Hz,
CH2CO2CH2CH3), 3.34 (d, 1H, J=17.3 Hz,
CH2CO2CH2CH3), 4.01 (q, 2H, J=7.1 Hz, OCH2CH3),
4.68 (d, 1H, J=14.3 Hz, CH2–Ar), 4.98 (d, 1H, J=
14.3 Hz, CH2–Ar), 6.27 (s, 1H, CH), 7.03–7.66 (m,
13H, arom.), 16.23 (br s, 1H, NH). IR (KBr) cm−1
:
3431, 1733, 1685, 1638, 1597, 1569. Anal. Calc. for
C35H36N6O4: C, 69.52; H, 6.00; N, 13.90. Found: C,
69.40; H, 6.12; N, 14.05%.
1
5c: 86.2% yield as white foam. H NMR l: 0.78 (t,
3H, J=7.2 Hz, COCH2CH2CH2CH3), 1.09–1.22 (m,
2H, COCH2CH2CH2CH3), 1.38–1.52 (m, 2H, COCH2-
CH2CH2CH3), 1.69–1.79 (m, 2H, COCH2CH2CH2-
CH3), 1.89 (s, 3H, CH3), 2.31 (s, 3H, CH3), 3.18–3.30
(m, 2H, CH2CO2CH3), 3.58 (s, 3H, OCH3), 4.66 (d, 1H,
J=14.3 Hz, CH2–Ar), 4.95 (d, 1H, J=14.3 Hz, CH2–
Ar), 6.50 (s, 1H, CH), 7.01–7.07 (m, 4H, arom.),
7.48–7.68 (m, 4H, arom.), 16.43 (br s, 1H, NH). IR
(KBr) cm−1: 3470, 1742, 1686, 1577. Anal. Calc. for
C29H32N6O4: C, 65.89; H, 6.10; N, 15.90. Found: C,
66.02; H, 6.10; N, 15.78%.
6.2. Pharmacology
6.2.1. Pharmacology [3H]AII binding assay
Rat adrenal cortex membranes were prepared ac-
cording to Chang et al. [30]. AII (1 mM) was used for
the determination of non specific binding. Losartan
(10−11–10−4 M) was tested as a reference standard.
The test compounds were dissolved in 100% DMSO at
the concentration of 10−2 M and then diluted with
1
5d: 92.7% yield as yellow foam. H NMR l: 0.81 (t,
3H, J=7.1 Hz, COCH2CH2CH2CH3), 1.14–1.25 (m,

O.A. Attanasi et al. / Il Farmaco 54 (1999) 64–76
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assay buffer [31] and used in the assay in range of
concentrations from 10−11 to 10−4 M. For the assay,
50 ml of test compound were added into test tubes
containing 100 ml of membrane suspension (0.05 mg of
protein), 50 ml of [3H]AII (Amersham, UK, 1.2 nM,
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Brandel cell harvester and washed rapidly three times
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Acknowledgements
This work was supported by financial assistance from
the Ministero dell’Universita` e della Ricerca Scientifica
e
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