The discovery of new potent non-peptide Angiotensin II AT1 receptor blockers: A concise synthesis, molecular docking studies and biological evaluation of N-substituted 5-butylimidazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2012.07.040

A convenient and facile synthesis, in silico docking studies and in vitro biological evaluation of N-substituted 5-butylimidazole derivatives as potent Angiotensin II (ANG II) receptor type 1 (AT1) blockers (ARBs) has been reported in the current study. Our efforts have been directed towards the development of an efficient synthetic route allowing the facile introduction of substituents on the imidazole ring. In particular, a series of imidazole based compounds bearing the biphenyl moiety at the N - 1 position, a halogen atom at the C-4 and polar substituents such as hydroxymethyl, aldo or carboxy group at the C-2 position were designed and synthesized. These compounds were evaluated for binding to human AT1 receptor and for ANG II antagonism in vitro on isolated rat uterus. Among them, 5-butyl-1-[[2′-(2H-tetrazol-5-yl)biphenyl-4-yl] methyl]imidazole-2-carboxylic acid (30) exhibited higher binding affinity compared to the other analogues tested (-log IC₅₀ = 8.46). The latter analogue was also found to be the most active in the rat uterotonic test (pA₂ = 7.83). Importantly, the binding affinity was higher to that of losartan (-log IC₅₀ = 8.25) indicating the importance of carboxy group at the C-2 position. Experimental findings are in good agreement with docking studies, which were undertaken in order to investigate ligand/AT1 receptor interactions.

Full text of DOI:10.1016/j.ejmech.2012.07.040

European Journal of Medicinal Chemistry 55 (2012) 358e374
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The discovery of new potent non-peptide Angiotensin II AT1 receptor blockers:
A concise synthesis, molecular docking studies and biological evaluation
of N-substituted 5-butylimidazole derivativesq
,
g
, ,
,
g
,
1
b
c
d
George Agelis a 1, Amalia Resvani a , Serdar Durdagi , Katerina Spyridaki , Tereza Tumová ,
*
ꢀ
d
e
f
c
ꢁ
Jirina Slaninová , Panagiotis Giannopoulos , Demetrios Vlahakos , George Liapakis ,
Thomas Mavromoustakos e, John Matsoukas a
,g,
*
a Department of Chemistry, University of Patras, Patras 26500, Greece
b Department of Biological Sciences, Institute for Biocomplexity and Informatics, Calgary, Alberta, Canada
c Department of Pharmacology, Faculty of Medicine, University of Crete, Voutes, 71003 Heraklion, Crete, Greece
d Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic
e Department of Chemistry, University of Athens, Athens, Greece
f Department of Internal Medicine, “ATTIKON” University Hospital, Athens, Greece
g Eldrug S.A., Patras, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
A convenient and facile synthesis, in silico docking studies and in vitro biological evaluation of
N-substituted 5-butylimidazole derivatives as potent Angiotensin II (ANG II) receptor type 1 (AT1)
blockers (ARBs) has been reported in the current study. Our efforts have been directed towards the
development of an efficient synthetic route allowing the facile introduction of substituents on the
imidazole ring. In particular, a series of imidazole based compounds bearing the biphenyl moiety at the
N ꢀ 1 position, a halogen atom at the C-4 and polar substituents such as hydroxymethyl, aldo or carboxy
group at the C-2 position were designed and synthesized. These compounds were evaluated for binding
to human AT1 receptor and for ANG II antagonism in vitro on isolated rat uterus. Among them, 5-butyl-1-
[[20-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-2-carboxylic acid (30) exhibited higher binding
affinity compared to the other analogues tested (ꢀlog IC50 ¼ 8.46). The latter analogue was also found to
be the most active in the rat uterotonic test (pA2 ¼ 7.83). Importantly, the binding affinity was higher to
that of losartan (ꢀlog IC50 ¼ 8.25) indicating the importance of carboxy group at the C-2 position.
Experimental findings are in good agreement with docking studies, which were undertaken in order to
investigate ligand/AT1 receptor interactions.
Received 28 May 2012
Received in revised form
19 July 2012
Accepted 20 July 2012
Available online 31 July 2012
Keywords:
Synthesis
Angiotensin II receptor blockers
N-substituted 5-butylimidazole derivatives
Antihypertensive activity
Molecular docking
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
Hypertension is the leading risk factor for human morbidity and
mortality. High blood pressure is associated with cardiovascular
Abbreviations: B(Oi-Pr)3, triisopropyl borate; n-BuLi, n-butyllithium; t-BuOK,
potassium tert-butoxide; Et3N, triethylamine; LTMP, lithium 2,2,6,6-
tetramethylpiperidide; NaBH4, sodium borohydride; NOE, Nuclear Overhauser
Effect; Pd(PPh3)4, tetrakis(triphenylphosphine)palladium; Et3SiH, triethylsilane;
disease (CV) and organ damages. However, despite the large number
of antihypertensive agents, only a few patients achieve to control
their blood pressure. This difficulty is due to the complex patho-
genesis of hypertension and related CV [1]. Renineangiotensin
system (RAS) is among the many factors playing role in the patho-
genesis of hypertension and CV. The RAS is a hormonal cascade
which creates angiotensin peptides and is the main regulator of
blood pressure and fluid and electrolyte balance [2e4]. Inhibition of
RAS has been established as an effective approach for the treatment
of several disorders, including hypertension and congestive heart
failure, as well as for the pathogenesis of diabetes and kidneydisease
TBAF$3H2O,
tetrabutylammonium
fluoride
trihydrate;
TMP,
2,2,6,6-
tetramethylpiperidine; TMSN3, trimethylsilyl azide; Tol, toluene.
q
Docking studies & biological evaluation: Serdar Durdagi; Katerina Spyridaki;
ꢀ
ꢁ
Tereza Tumová; Jirina Slaninová; Panagiotis Giannopoulos; Demetrios Vlahakos;
George Liapakis; Thomas Mavromoustakos.
* Corresponding authors. Department of Chemistry, University of Patras, Patras
26500, Greece.
E-mail addresses: aggelisgeorge@hotmail.com (G. Agelis), eldrug@eldrug.gr
(J. Matsoukas).
1
The first two authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.07.040

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
359
[5]. Research efforts over the last decades have focused on the
development of highly selective angiotensin (ANG II) AT1 receptor
blockers which provided a more specific blockade of the RAS with
virtually no agonist effect and better safety compared to ACE
inhibitors [6,7]. Selective blockade of the AT1 receptor may prevent
all of the known pathologic effects of ANG II associated with its
stimulation, while allowing the positive effects induced by the AT2
receptor.
1,5-disubstituted imidazole ANG II antagonists has demonstrated
that reorientation of the substitution pattern at the C-2 and C-5
positions of the imidazole ring of losartan led to the potent
antagonist V8 [35]. In extending these studies, we have designed
and performed docking calculations in order to synthesize
a variety of synthetic analogues. Indeed, we have developed a new
synthetic strategy for compound 21 and we have synthesized
a series of structurally related compounds 22e24 and 33, which
differ in the orientation of the C-2 and C-5 substituents compared
to losartan [26,35,37] (Fig. 1). These compounds bear the butyl
chain at the C-5 position, the biphenylmethyl group ortho
substituted either with tetrazole or its bioisostere carboxy group at
the N-1 and a halogen atom at the C-4 position since the chloro
substituent in DuPont series interacts with a lipophilic pocket of
the receptor, augmenting affinity [9]. On the other hand, it is well
known that losartan is converted by enzymatic oxidation to the
active metabolite EXP 3174 [38,39] via the aldehyde EXP 3179 and
the former exhibits a 10- to 40-fold higher potency compared to
losartan [40,41]. Taking these findings into account, the oxidized
forms of 21 were synthesized leading to the aldehyde 29 and the
carboxylic acid 30 (Fig. 1).
The discovery of potent and orally active ARBs such as losartan
[8,9] and eprosartan [10] has encouraged the development of
a large number of similar compounds. Among them, candesartan
[11], valsartan [12], irbesartan [13], telmisartan [14], tasosartan
[15], olmesartan [16] have been launched and were established as
ARBs. In 2011, the U.S. Food and Drug Administration (FDA)
approved azilsartan [17,18], which is a newer-generation ARB for
the treatment of high blood pressure in adults. Treatment with an
ARB has been demonstrated to reduce CV events and heart failure
progression as well as to improve renal disease and prevent dia-
betes and this constitutes the importance of their development
[19,20]. The majority of selective ARBs has resulted from the
modification or replacement of several pharmacophore groups of
losartan. The available data from literature and extensive
structureeactivity relationship (SAR) studies have shown that the
important structural features for potential antihypertensive
activity are: (i) a biphenyl moiety at the N-1 position of a hetero-
cyclic ring 11; (ii) an acidic functionality such as a tetrazole group
or an acidic isostere at the ortho position of the biphenyl group
[21e23]; (iii) a short lipophilic alkyl chain substituted on the
heterocyclic ring for efficient binding to the receptor [11,24e26].
Our synthetic approach included efficient and regioselective
reactions in high yield, allowing the facile introduction of the
substituents on the imidazole nucleus. The synthesized analogues
were tested for their ANG II-antagonistic activity on rat uterus (pA2)
and their AT1 receptor affinity (IC50) using binding assays.
2. Results and discussion
The DuPont group recommended
a
lipohilic and electron-
2.1. Chemistry
withdrawing group such as a halogen atom, CF3, ethyl or penta-
fluoroethyl substituents at the C-4 of the imidazole ring and
a small sized group such as CH2OH or CO2H capable of forming
a hydrogen bond at C-5 [9,16].
Our work in recent years focused on the SAR studies of ANG II,
sarilesin, sarmesin and other synthetic linear and cyclic peptides
with antagonistic activity [27e31]. The above data and the
continuous demand for antihypertensive agents prompted us to
search for new and potent ARBs. Thus, we recently synthesized 1,5-
disubstituted imidazole ANG II derivatives found to possess
antagonistic properties [32e36]. The design and synthesis of
Herein, we describe
a concise synthesis of N-substituted
5-butylimidazole derivatives (21e24, 29, 30 and 33, Fig. 1). Specif-
ically, the described synthetic method included efficient reactions
in high yield, rendering it a general approach for the regioselective
substitution on imidazole nucleus in
a sequential manner.
Furthermore, for the synthesis of 5-(40-methyl-2-biphenyl)-2H-
tetrazole (5), a three-step method (Scheme 1) was employed in
order to minimize the use of expensive reagents and hazardous
intermediates via a rapid and mild process [9,42,43]. The interme-
diates (4, 7 and 10) that were used to introduce the biphenylmethyl
Fig. 1. Structures of the synthesized compounds compared to losartan and its metabolites EXP 3179 and EXP 3174.

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G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
Scheme 1. Synthesis of the alkylating agents 7 and 10a. aReagents and conditions: (a) n-BuLi (1.6 M in hexanes), TMP, B(Oi-Pr)3, THF, ꢀ78 ꢁC to rt, 5 h; (b) 2,2-dimethyl-1,3-
propandiol, Tol, rt, overnight; (c) 4-bromotoluene, Pd(PPh3)4, Tol, EtOH, K2CO3, 100 ꢁC, 4 h; (d) TBAF$3H2O, TMSN3, 120 ꢁC, 36 h; (e) TrCl, Et3N, CH2Cl2, rt, 1 h; (f) NBS, diben-
zoylperoxide, CCl4, reflux, 8 h; (g) 4.5 N HCl, reflux, 12 h; (h) i. oxalyl chloride, CH2Cl2, 0 ꢁC to rt, 3 h, ii. t-BuOK, THF, 10 ꢁC to rt, 1 h.
moiety to the imidazole ring were obtained according to reported
methods [42e45] as outlined in Scheme 1. The biphenyl nitrile 4
was prepared in two steps from the commercially available ortho-
benzonitrile 1 [9,42]. Thus, the synthesis included the conversion
of 1 to the intermediate 2 by in situ trapping of unstable lithio
intermediate, using lithium 2,2,6,6-tetramethylpiperidide (LTMP)
as a base in combination with triisopropylborate (B(Oi-Pr)3) in
THF at ꢀ78 ꢁC. Subsequent treatment with 2,2-dimethyl-1,3-
propanediol afforded the stable arylboronic ester 3 [42]. These
mild conditions led to the corresponding air and chromatography
stable ortho substituted arylboronic ester 3 in good yield (58%) with
excellent purity after recrystallization. The latter was readily con-
verted to the nitrile 4 via Suzuki cross-coupling reaction [42,46,47]
using bromotoluene under catalysis with Pd(PPh3)4 and K2CO3 as
a base at 100 ꢁC for 4 h in 70% yield. The tetrazole derivative 5 was
obtained by [3 þ 2] cycloaddition reaction using trimethylsilylazide
(TMSN3) as azide source in the presence of TBAF$3H2O under
solventless conditions [43] at 120 ꢁC for 36 h in 77% yield.
Protection of the tetrazole ring with the trityl group (Tr) resulted
in the corresponding derivative 6, followed by benzylic bromina-
tion to provide 7 as the sole product in good combined yield (2
steps, 64%). On the other hand, acid hydrolysis of the nitrile 4 led to
the formation of the carboxylic acid 8 in high yield. Protection of the
carboxy group with the tert-butyl group was accomplished
according to an established procedure [9] using oxalyl chloride and
t-BuOK, resulting in the ester 9. Similarly, benzylic bromination of 9
afforded the corresponding derivative 10.
protecting groups was used including the trityl group, the benzyl
group, the N,N-dimethylsulfamoyl group, and the SEM group which
proved an excellent choice.
Most protection conditions have employed NaH in DMF to
deprotonate acidic NHs, followed by addition of SEM-Cl [51]. Using
these standard conditions (SEM-Cl/NaH/DMF) with the imidazole
derivative 11, we found that both regioisomers 12a and 12b were
formed in high combined yield (84%, Scheme 2). These isomers
were separated, characterized by 1H NMR, 13C NMR and their
regiochemistry was unequivocally assigned by 1D NOE experiment.
In this case, substitution at the N-1 position was favored with
a 12a/12b ratio of 2:1 (as indicated by HPLC and 1H NMR). There-
fore, we selected conditions that might improve the regioselectivity
in favor of the isomer 12a. Thus, dicyclohexylmethylamine
(Cyhex2NMe) was used as a hindered non-deprotonating base [52]
and a substantial improvement of regioselectivity was achieved
with a 12a/12b ratio of 3:1 (as indicated by HPLC).
Having the 1-SEM-4-butylimidazole (12a) in hand, two
synthetic routes were investigated for the synthesis of the key
intermediate 16 (Scheme 3). In the first approach (route A), lith-
iation at the C-2 of the imidazole ring 12a by exposure to n-BuLi
at ꢀ78 ꢁC in anhydrous THF and subsequent quenching with
anhydrous DMF from ꢀ78 ꢁC to rt for 16 h, afforded the aldehyde 13
in good yield (65%). The extent of lithiation was monitored by
quenching a reaction sample with D2O, resulting in the absence of
the H-2 signal at 8.82 ppm, thus proved to be quantitatively by 1H
NMR. Furthermore, the proton aldehyde appeared as a singlet at
9.77 ppm, while the carbon signal appeared at 183.47 ppm.
Reduction of 13 in the presence of NaBH4 in MeOH afforded the
alcohol 14 (92%). The 1H NMR spectrum of 14 showed a singlet peak
at 4.69 ppm, which is assigned to the hydroxymethyl protons in
addition to the absence of the aldehyde proton. Alkylation of 14 at
the N-3 of the imidazole ring was carried out selectively upon
addition of the alkylating agent 7 in MeCN under reflux for 12 h to
provide the intermediate salt 16 in poor yield (20%).
The 2-(trimethylsilyl)ethoxymethyl (SEM) group constitutes
a widely used heterocyclic NH protecting group particularly in
indoles, pyrroles [48] and imidazoles [49] but is also an excellent
directing group for lithiaton at the alpha position [50]. In our case,
we have sought an appropriate protecting group at the N-1 for
ortho directed lithiation at the C-2 position with subsequent elec-
trophilic quenching, in combination with regioselective alkylation
at the N-3 of the 4(5)-butylimidazole (11). Therefore, a series of

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
361
Scheme 2. Synthesis and determination of regioisomers 12a and 12b by 1D NOEa. aReagents and conditions: (a) NaH (powdered, 95%), SEM-Cl, DMF, 0 ꢁC to rt, 2 h; (b) Cyhex2NMe,
SEM-Cl, THF, rt, 2 h.
a
Scheme 3. Synthesis of the target compounds 21e24a, Reagents and conditions: (a) SEM-Cl, NaH, DMF, 0 ꢁC to rt, 2 h or Cyhex2NMe, SEM-Cl, THF, rt, 2 h; (b) n-BuLi (1.6 M in
hexanes), THF, DMF, ꢀ78 ꢁC to rt, 18 h; (c) NaBH4, MeOH, 0 ꢁC to rt, 2 h (d) 7, MeCN, reflux, 3 h (e) 37% formalin, diisopropylethylamine, DMF, 85 ꢁC, 1 h (f) 7, MeCN, 70 ꢁC, 12 h; (g)
TBAF (1.0 M in THF), reflux, 8 h; (h) H2, Pd/C, MeOH, 24 h; (i) 20% TFA/CH2Cl2, Et3SiH, rt, 1 h; (j) NXS (X ¼ Cl, Br, I), DMF.

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G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
In order to improve the yield for the formation of 16, we adopted
Having proven that losartan is in vivo metabolized to the main
active metabolite EXP 3174 via the EXP 3179, we then set out to
synthesize 29 and 30, presumably as metabolites of the most
potent hydroxymethyl derivative 21. Scheme 5 depicts the initial
attempt towards the synthesis of 27 as a precursor of the final
aldehyde 29. Thus, treatment of 13 with 3 N HCl in MeOH led to the
cleavage of the SEM group to afford the intermediate 25, which was
alkylated upon addition of 7 in the presence of K2CO3 in DMF. The
expected N-alkylated regioisomers 26 and 27 were separated by
flash column chromatography and their regiochemistry was
confirmed by 2D NOESY experiment. This task was proved rather
simple, however, the desired aldehyde 27 was obtained as the
minor regioisomer (28%) with 26/27 ratio of ca. 2.5:1 (as indicated
by HPLC).
a second approach (route B), which initially included alkylation of
12a. Similarly, the reaction was performed in the presence of the
alkylating reagent 7 in MeCN under reflux for 3 h, resulting in 15 in
78% yield with high purity after recrystallization. At this point, we
were ready to perform the introduction of the hydroxymethyl
group at the C-2 of the imidazole ring of the alkylated compound
15. According to our strategy, the hydroxymethylation was
promptly carried out in a sealed tube by treatment with diisopro-
pylethylamine and 37% formalin in DMF at 85 ꢁC for 1 h. This
reaction proceeded quantitatively as indicated by HPLC, without
the need for purification of the hydroxymethylated product 16. The
1H NMR spectrum of 16 showed the presence of a singlet peak at
4.70 ppm due to the hydroxymethyl protons, while its carbon signal
appeared at 48.92 ppm.
Therefore, our chosen synthetic sequence for the preparation of
the aforementioned 29 and 30 is depicted in Scheme 6 (Method A).
Hence, the alcohol 17 was converted to the aldehyde 27 via Swern
oxidation [53,54] by treatment with dimethyl sulfoxide (DMSO)
and oxalyl chloride in CH2Cl2 at ꢀ78 ꢁC. Subsequently, addition of
Et3N at ꢀ60 ꢁC led to 27 in excellent yield (92%). The proton signal
at 9.77 ppm was attributed to the aldehyde in addition to the
carbon signal at 181.91 ppm. The latter was subjected to Lindgren
oxidation by means of NaClO2 and NaH2PO4 in the presence of
2-methyl-2-butene as chlorine scavenger [55,56] to afford the
carboxylic acid 28, quantitatively. Detritylation of the tetrazole
group of the oxidized intermediates 27 and 28 was accomplished
by treatment with formic acid in Et2O, resulting in the final
compounds 29 and 30, respectively. Alternatively, the carboxylic
acid 30 was also obtained in one step via oxidation of the final
alcohol 21 with KMnO4 (Scheme 6, Method B) in the presence of
18-crown-6 in H2O/acetone in 74% yield. The absence of the
aldehyde proton signal, as well as the carbon signal at 172.10 ppm
of 30, unequivocally confirmed the introduction of the carboxy
group.
A plausible mechanism for this hydroxymethylation involves
the deprotonation by diisopropylethylamine of the more acidic
proton at the C-2 of the imidazolium salt 15 onto a ylide I. The latter
species, to which a carbene form II is an important resonance
contributor, undergoes nucleophilic addition to formaldehyde
(Scheme 4) affording the hydroxymethylated compound 16.
The SEM group of the intermediate salt 16 was then cleaved by
treatment with 1 M TBAF in THF under reflux resulting in the
alcohol 17. Halogenation of 17 at the C-4 position of the imidazole
ring with the appropriate N-halosuccinimide (NXS, X ¼ Cl, Br, I) in
DMF afforded the corresponding halogenated analogues 18e20.
The tetrazole group of 17e20 was promptly deprotected upon
addition of 20% trifluoroacetic acid (TFA) in CH2Cl2 in the presence
of Et3SiH as scavenger to obtain the final compounds 21e24 in
79e85% yield. The 1H NMR spectra of the final halogenated
analogues 22e24 showed the absence of the H-4 signal of the
imidazole ring at 7.36 ppm, appearing at 21. Not surprisingly, the
13C NMR spectra showed an upfield trend in C-4 chemical shifts
produced by bromine and iodine atoms for 23 and 24, at 111.45 and
102.43 ppm, respectively, due to the “heavy atom effect”, while the
C-4 in the chloro derivative 22 was deshielded at 124.43 ppm.
Finally, employing the previously described method, the prep-
aration of the compound 33 where the tetrazole group is replaced
Scheme 4. Proposed mechanism of hydroxymethylation at the C-2 position of the imidazolium salt 15.

a
Scheme 5. Synthesis of the aldehyde 27a. Reagents and conditions: (a) 3 N HCl, EtOH, 60 ꢁC, 3 h; (b) 7, K2CO3, DMF, rt, 18 h.
Scheme 6. Synthesis of the aldehyde 29 and the carboxylic acid 30a. aReagents and conditions: (a) DMSO, oxalyl chloride, CH2Cl2, ꢀ78 ꢁC, 1 h then Et3N, ꢀ60 ꢁC to rt, 1 h; (b)
NaH2PO4, NaClO2, 2-methyl-2-butene, t-BuOH/H2O (1:1), rt, 1 h; (c) formic acid/Et2O (3:2), rt, 2 h; (d) KMnO4, 18-crown-6, acetone/H2O (1:1).

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G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
by the bioisostere carboxy group commenced with alkylation at the
N-3 of 12a by treatment with 10 in MeCN, resulting in the inter-
mediate salt 31 (Scheme 7). The latter was subjected to hydrox-
ymethylation at C-2 of the imidazole ring of 31 in the presence of
diisopropylethylamine and 37% formalin in DMF at 85 ꢁC for 1 h to
afford 32 in 93% yield. Finally, removal of the tert-butyl group by
means of 20% TFA in CH2Cl2 and Et3SiH led to the carboxy
compound 33 in 82% yield.
2.2. Pharmacology
The pharmacological evaluation of the synthesized ARBs was
determined by means of their binding affinities for the human AT1
receptor (using losartan as control), as well as their ability to inhibit
the contractility effect of ANG II in isolated rat uterus. The binding
affinities (ꢀlog IC50) were determined from competition experi-
ments performed under equilibrium conditions in membranes
from HEK 293 cells stably expressing the AT1 receptor and using as
a radioligand the [125IeSar1eIle8] ANG II. As shown in Figs. 2 and 3,
all analogues evaluated herein bound to AT1 receptor in a concen-
tration dose-dependent manner similar to losartan. The binding
affinity of the alcohol 21 (ꢀlog IC50 ¼ 7.52) for the AT1 receptor was
only 5.5-fold lower than that of losartan (ꢀlog IC50 ¼ 8.25, Table 1)
as it was also indicated in our previous work [35]. An interesting
observation is that the introduction of a halogen atom (Cl, Br, I) at
the C-4 of the imidazole ring affording 22e24, exhibited a sharp
drop of affinity (ꢀlog IC50 ¼ 6.20, 6.93, 6.35, respectively, Figs. 2 and
3, Table 1). These results indicate that a lipophilic halogen
substituent at the C-4 position is not beneficial in terms of binding
affinity. On the other hand, the conversion of 21 to the aldehyde 29
did not significantly alter the binding affinity (ꢀlog IC50 ¼ 7.62,
Fig. 3, Table 1). Interestingly, further oxidation of the aldo group of
Fig. 2. Competition binding isotherms of ANG II analogues to human AT1 receptor.
Competition of [125IeSar1eIle8] ANG II specific binding by increasing concentrations of
ANG II analogues, losartan, and compounds 21, 22, 23 and 24 was performed, as
described in Experimental section membranes from HEK 293 cells stably expressing
the human AT1 receptor. The means and S.E. are shown from 2 to 5 different experi-
ments. The data were fit to a one-site competition model by nonlinear regression and
the ꢀlog IC50 values were determined as described in Experimental section.
29 resulted in the carboxylic acid 30, revealing a significant increase
in affinity which was higher than that of losartan (ꢀlog IC50 ¼ 8.46
and 8.25, respectively, Fig. 3, Table 1). In contrast, replacement of
the tetrazole group into 21 with bioisostere carboxy group resulted
in the analogue 33 having 44-fold lower affinity than losartan (ꢀlog
IC50 ¼ 6.61, Fig. 3, Table 1). As expected, these data illustrate the
a
Scheme 7. Synthesis of the carboxy compound 33a. Reagents and conditions: (a) 10, MeCN, reflux, 3 h; (b) 37% formalin, diisopropylethylamine, DMF, 85 ꢁC, 1 h; (c) 20% TFA in
CH2Cl2, Et3SiH, rt, 1 h.

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
365
2.3. Theoretical calculationsemolecular docking studies
Molecular docking results are tabulated at Table 2 together with
experimental activities. Losartan and 30 showed higher (absolute
values) Glide XP docking scores in accordance with experimental
analyses which showed that these analogues have high binding
affinity to the AT1 receptor. Although interaction energy trend for
the halogen derivatives are similar with the in vitro pIC50 values
(23 > 22 > 24), calculated docking for compound 23 is over-
estimated. In Fig. 5 atomistic details of the docking complexes from
top docking poses was represented for compound 30. Corre-
sponding amino acid residues are His256, Gln257, Asn200, Tyr113,
Trp253, Thr260, Phe182, Lys199, Ile197, Val169, Gly196, Val264 and
Phe204 for docking complex of 30 (Fig. 5).
Since compound 23 showed high docking score, this docking
complex was also analyzed (Fig. 6). Close contacts are formed by
Asn200, Phe261, Gly196, Lys199, Tyr184, Pro192, Val264 and
Leu265 for this docking complex. Table 2 also includes Prime
energy components and IFD scores, showing the steric and elec-
tronic contributions of ligand/target complexes. In Fig. 7 are shown
the docking results for 22e24.
Fig. 3. Competition binding isotherms of ANG II analogues to human AT1 receptor.
Competition of [125IeSar1eIle8] ANG II specific binding by increasing concentrations of
ANG II analogues, losartan, and compounds 21, 29, 30 and 33 was performed, as
described in Experimental section, on membranes from HEK 293 cells stably
expressing the human AT1 receptor. The means and S.E. are shown from 3 to 4
different experiments. The data were fit to a one-site competition model by nonlinear
regression and the ꢀlog IC50 values were determined as described in Experimental
section.
3. Conclusions
In this article, a series of N-substituted 5-butylimidazole deriv-
atives has been synthesized, via a convenient and facile synthesis
and evaluated for their in vitro antagonistic activities on ANG II AT1
receptor. In this series, the most potent analogue was found to be
30 bearing the carboxy group at the C-2 position. Previous work has
shown that compound 21 is slightly less potent than losartan and
structurally identical to losartan except that butyl and hydrox-
ymethyl groups are interchanged though is lacking the chlorine
atom [35]. Thus, the polar substituent CH2OH at the C-2 is favorable
for the antagonistic activity. In vitro results showed that generally
substitution at the C-4 of 21 with halogens resulting in 22e24 led to
significant decrease of activity. The latter points out that the lipo-
philic halogen substituents (Cl, Br, I) are unfavorable for the binding
affinity for this class of analogues. Molecular docking calculations
showed that replacement of a hydrogen atom at the C-4 of the
imidazole ring with halogens would have not serious detrimental
effect except for bromine atom whose its interaction energy is
overestimated. To follow a similar strategy with losartan, the
compounds 29 and 30 were synthesized as oxidized forms of 21.
Thus, 29 structurally resembles losartan derivative EXP 3179 and 30
its active metabolite EXP3174. In our case, both the oxidized
compounds 29 and 30 showed similar antagonistic activity in the
uterotonic test as the compound 21 even if the binding affinity of 30
was one order of magnitude higher. This intriguing result showed
clearly that the interchange between butyl and oxidized forms of
hydroxymethyl group can lead to potent analogues. Interestingly,
30 lacks the chlorine atom but still retains equipotency with los-
artan. Moreover, replacement of the tetrazole ring by its bioisostere
CO2H resulting in 33, was detrimental for activity as it was also
shown with losartan series.
importance of tetrazole as pharmacophore group for binding to the
AT1 receptor.
Next the synthesized compounds were tested in rat uterotonic
in vitro test for their ability to inhibit the contractions evoked by
ANG II as described in Experimental section. The results obtained
are summarized in Table 1. As can be seen, the most active
compounds of this series were 29 and 30 (pA2 ¼ 7.76 and 7.83,
respectively). Their activity was only about 2 times lower than that
of losartan (pA2 ¼ 8.25).
In the case of the most active compounds 29 and 30, their
specificity for the AT1 receptor was also tested. Their effect was
found specific, as they did not influence the standard cumulative
dose response curves of bradykinin and oxytocin using even 1000
times higher concentration than that corresponding to pA2 which
completely diminished the response to very high doses of ANG II.
Conclusively, the pharmacological results of this study suggest
that the aldo as well as the carboxy group at the C-2 of the imid-
azole ring was favorable for the interaction with AT1 receptor, with
the carboxy compound 30 to show slightly higher affinity. In Fig. 4,
SAR data of the synthesized compounds 21e24, 29, 30 and 33 are
summarized.
Table 1
In vitro activities of synthesized compounds (competition binding studies were
performed on membrane preparations from HEK 293 cells stably expressing human
AT1 receptor. The ꢀlog IC50 values were obtained by fitting the data from the
competition studies to a one-site competition model by nonlinear regression. The
mean ꢂ S.E. values are from 2 to 5 independent experiments. The IC50 is derived
from the mean value of log IC50).
The Glide XP docking results are in good agreement with experi-
mental findings except for the prediction of interaction energy of
brominated analogue 23. As seen in the Prime energy calculations, 23
hasthe lowest and overestimatedCoulomb energycompared to other
analogues. Since Prime Energy mainly determines the IFD score, they
cannot be discriminated based on their interaction energy profiles.
Nevertheless, considering the limitations of molecular docking algo-
rithms (i.e. simplified approaches of potential solvent effects and
entropy), correct estimation of interaction energies for all docked
structures except one; encourages us to use molecular docking algo-
rithms in binding energy predictions for this class of molecules.
Compound
pA2
IC50 (nM)
ꢀlog IC50
Losartan
8.25 ꢂ 0.13
7.97 ꢂ 0.07
7.83 ꢂ 0.20
7.76 ꢂ 0.47
7.58 ꢂ 0.15
7.35 ꢂ 0.24
6.98 ꢂ 0.19
6.19 ꢂ 0.26
6.0
30.0
3.0
8.25 ꢂ 0.06
7.52 ꢂ 0.16
8.46 ꢂ 0.29
7.62 ꢂ 0.13
6.93 ꢂ 0.16
6.35 ꢂ 0.11
6.20 ꢂ 0.13
6.61 ꢂ 0.15
21
30
29
23
24
22
33
24.0
117.0
446.0
634.0
245.0

366
G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
-logIC50
Compound
R1
Cl
H
H
H
Br
I
R2
R3
R4
pA2
8.25 0.06
Losartan
n-butyl
CH2OH
COOH
CHO
CN4H
CN4H
CN4H
CN4H
CN4H
CN4H
CN4H
COOH
CH2OH
n-butyl
n-butyl
n-butyl
n-butyl
n-butyl
n-butyl
n-butyl
8.25 0.13
7.97 0.07
7.83 0.20
7.76 0.47
7.58 0.15
7.35 0.24
6.98 0.19
6.19 0.26
7.52 0.16
8.46 0.29
7.62 0.13
6.93 0.16
6.35 0.11
6.20 0.13
6.61 0.15
21
30
29
23
24
22
33
CH2OH
CH2OH
CH2OH
CH2OH
Cl
H
Fig. 4. SAR data of the synthesized compounds.
In conclusion, the new series of analogues not only show that
4. Experimental section
4.1. General
like losartan interact with the AT1 receptor in a similar fashion in
the AT1 pocket of the active site but also follow the same trend as
its analogues. However, the only difference is that these analogues
do not demand the presence of lipophilic halogen atom at the C-4
position of the imidazole ring. Finally, the data clearly demonstrate
the importance of having a carboxylic acid at the C-2 as in 30 in
order to enhance the binding affinity to the AT1 receptor, thereby
contributing to potentiation of activity (Fig. 4). The efficient, short-
step, high yield synthesis of 1,5-disubstituted ARBs, the properly
effective reorientation of the pharmacophore groups and the
introduction of a carboxy group at the C-2 position resulting in
strong ANG II blockers, is a contribution to the field and may open
new avenues in antihypertensive drug therapy.
Starting materials were obtained by Aldrich and used as
received. All reactions involving air sensitive reagents were per-
formed under argon atmosphere using syringe-septum cap tech-
niques. All reactions were carried out in anhydrous solvents and
distilled according to literature. Melting points were determined
with a Stuart SMP 10 apparatus and are uncorrected. 1H NMR and
13C NMR spectra were recorded on a Bruker Avance DPX spec-
trometer at 400.13 MHz and 161.76 MHz, respectively. The 1D NOE
spectra were recorded on a Bruker 600 MHz spectrometers.
Chemical shifts are reported in units, parts per million (ppm)
Table 2
Docking results of synthesized compounds compared to losartan.
Compound
Glide XP score
(kcal/mol)
IFD score
(kcal/mol)
Prime Coulomb
energy (kcal/mol)
Prime Covalent
energy (kcal/mol)
Prime VDW
energy (kcal/mol)
Prime solv. SA
Prime solv. GB
Prime
energy (kcal/mol)
Losartan
ꢀ12.114
ꢀ11.464
ꢀ11.103
ꢀ10.691
ꢀ12.894
ꢀ10.410
ꢀ10.912
ꢀ9.148
ꢀ565.712
ꢀ565.848
ꢀ565.583
ꢀ564.090
ꢀ567.479
ꢀ565.233
ꢀ565.325
ꢀ565.085
ꢀ9668.993
ꢀ9576.544
ꢀ9438.741
ꢀ9545.308
ꢀ9706.724
ꢀ9570.051
ꢀ9568.922
ꢀ9538.172
2510.356
2474.498
2489.792
2479.365
2501.439
2472.930
2479.481
2481.973
ꢀ1547.833
ꢀ1526.175
ꢀ1519.70
0.398
2.807
2.169
2.241
ꢀ0.732
2.755
1.527
5.142
ꢀ2365.894
ꢀ2462.276
ꢀ2623.110
ꢀ2460.108
ꢀ2362.244
ꢀ2459.244
ꢀ2448.225
ꢀ2540.213
ꢀ11072.0
ꢀ11087.7
ꢀ11089.6
ꢀ11068.0
ꢀ11091.7
ꢀ11096.5
ꢀ11088.3
ꢀ11118.8
21
30
29
23
24
22
33
ꢀ1544.166
ꢀ1523.437
ꢀ1542.117
ꢀ1552.117
ꢀ1527.480

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
367
Fig. 5. Top docking pose of 30 at the active site of the receptor.
downfield from tetramethylsilane (TMS) and coupling constants (J)
4.2. Chemistry
are given in Hertz (Hz). HPLC analysis was performed on an Alliance
Waters 2695 equipped with a Waters 2996 Photodiode Array De-
4.2.1. 2-(5,5-dimethyl-[1,3,2]dioxaborinan-2-yl)-benzonitrile (3) [42]
To a solution of TMP (3.28 mL, 19.4 mmol) in anhydrous THF
(20 mL), n-BuLi (1.6 M in hexanes, 11.7 mL, 18.8 mmol) was added
at ꢀ10 ꢁC and the resulting mixture was stirred for 10 min. Then,
B(Oi-Pr)3 (5.50 mL, 25 mmol) was added dropwise at ꢀ78 ꢁC and
stirred for additional 5 min before benzonitrile 1 (1.28 mL,
12.5 mmol) was added via a syringe in a single portion and the
reaction mixture was stirred at ꢀ78 ꢁC for 2 h. The solution was left
to warm to rt while being stirred for 3 h and then quenched with
saturated aqueous NH4Cl (60 mL). The resulting mixture was
extracted with EtOAc (3 ꢃ 70 mL), the combined organic extracts
were dried (Na2SO4) and concentrated in vacuo. The intermediate
tector UV vis, using the XBridge C18 column (4.6 ꢃ 150 mm, 3.5
mm)
as stationary phase and a gradient of H2O/MeCN bothcontaining
0.08% TFA as mobile phase. Electrospray-ionization mass spectra
(ESI-MS) were obtained on a UPLC (ultra performance liquid
chromatography) equipped with SQ detector AcquityÔ by Waters.
Analytical TLC was performed on silica gel 60 F254 plates (Merck,
Germany) and visualized by UV irradiation, iodine and Brady’s
reagent. Purification of compounds 12a and 12b was performed by
Waters preparative HPLC equipped with Waters Prep LC Controller
and Photodiode Array Detector 2996 using SunFire Prep C18
column (50 ꢃ 100 mm) with 5
mm packing material. Separation was
achieved using H2O/MeCN as a mobile phase with a stepped linear
gradient from 40% MeCN to 100% MeCN in 50 min with a flow rate
of 9 mL/min.
product 2 was dissolved in anhydrous Tol (50 mL) and
2,2-dimethyl-1,3-propandiol (1.57 g, 15 mmol) was added and then
stirred overnight at rt. The organic phase was washed with H2O

368
G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
Fig. 6. Top docking pose of 23 at the active site of the receptor.
(3 ꢃ 30 mL) and the aqueous extracts were washed with CH2Cl2
(3 ꢃ 30 mL). The CH2Cl2-phase was washed with H2O (1 ꢃ 30 mL),
combined with Tol extract, dried (Na2SO4) and concentrated in
vacuo. Recrystallization of the crude product from heptane afforded
pure 3 (58%, 2 steps) as a white crystalline solid: M.p. 109e111 ꢁC;
ESI-MS (m/z): 216.64 [MH]; 1Η ΝΜR (400 MHz, CDCl3):
7.88 (d, 1Η,
d
J ¼ 7.5 Hz), 7.68 (d, 1Η, J ¼ 7.5 Hz), 7.54 (td, 1Η, J ¼ 7.5 Hz, 1.0 Hz),
7.48 (td, 1Η, J ¼ 7.5 Hz, 1.0 Hz), 3.83 (s, 4H), 1.05 (s, 6H) ppm; 13C
Fig. 7. Superimposition of top docking poses of 22, 23 and 24 at the active site of the receptor. The arrows show the positions of the halogens (highlighted region). Clearly, chlorine
and iodine are positioned in a spatial vicinity while bromine is localized in a different position.

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
369
NMR (160 MHz, CDCl3):
d
135.07, 133.64, 131.44, 130.47, 119.63,
4.2.7. Tert-butyl-4΄-methylbiphenyl-2-carboxylate (9)
116.56, 72.49, 31.84, 21.83 ppm.
Prepared according to reported procedure [9]. Rf 0.41 (hex-
anes:EtOAc, 4:1); tR 18.25 min (30% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 254.24 [MH ꢀ CH3], 212.25 [MH ꢀ C(CH3)3];
4.2.2. 40-methyl-2-biphenylcarbonitrile (4) [9,42]
To a solution of the boronic ester 3 (0.80 g, 3.72 mmol) in Tol
(30 mL) and EtOH (3 mL) under argon atmosphere, 4-bromotoluene
(0.50 mL, 4.10 mmol), 2 M K2CO3 (3 mL) and Pd(PPh3)4 (3 mol%,
0.19 g) were successively added and the resulting reaction mixture
was stirred at 100 ꢁC for 4 h. After cooling the mixture to rt, satu-
rated aqueous NH4Cl (70 mL) was added, followed by extraction
with EtOAc (3 ꢃ 70 mL), drying (Na2SO4) and evaporation in vacuo.
Flash column chromatography (hexanes:EtOAc, 3:1) afforded
nitrile 4 (70%) as a white solid: M.p. 50e52 ꢁC; Rf 0.52 (hex-
anes:EtOAc, 1:4); tR 15.15 min (30% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 194.06 [MH]; 1Η ΝΜR (400 MHz, CDCl3):
1Η ΝΜR (400 MHz, CD3OD):
d
7.69 (d, 1H, J ¼ 7.6 Hz), 7.53 (t, 1H,
J ¼ 7.4 Hz), 7.41 (t, 1Η, J ¼ 7.4 Hz), 7.34 (d, 1H, J ¼ 7.6 Hz), 7.23 (d, 2H,
J ¼ 8.0 Hz), 7.19 (d, 2H, J ¼ 8.0 Hz), 2.41 (s, 3H), 1.29 (s, 9H) ppm; 13C
NMR (160 MHz, CD3OD):
d 167.77, 140.67, 137.61, 135.64, 135.49,
131.83, 129.65, 129.51, 129.04, 129.01, 128.94, 127.83, 125.43, 80.19,
28.20, 17.17 ppm.
4.2.8. Tert-butyl-4΄-(bromomethyl)biphenyl-2-carboxylate (10)
Prepared according to reported procedure [9]. Rf 0.39 (hex-
anes:EtOAc, 4:1); tR 18.37 min (30% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 347.27 [MH] (79Br), 349.29 [MH þ 2] (81Br);
d
7.55e7.51 (m, 1Η), 7.50e7.48 (m, 1H), 7.42e7.34 (m, 4H), 7.22 (d,
1Η ΝΜR (400 MHz, CDCl3):
d
7.74 (d, 1H, J ¼ 7.6 Hz), 7.44e7.41
2H, J ¼ 7.6 Hz), 2.37 (s, 3H) ppm; 13C NMR (160 MHz, CDCl3):
(m, 1H), 7.37e7.34 (m, 3Η), 7.25e7.19 (m, 3H), 4.49 (s, 2H), 1.19 (s,
d
145.53, 138.69, 135.27, 133.71, 132.76, 129.97, 129.45, 128.61,
9H) ppm; 13C NMR (160 MHz, CDCl3):
d
167.82, 142.16, 141.37,
127.27, 118.88, 111.18, 21.25 ppm.
136.61, 132.87, 130.73, 130.36, 129.61, 129.77, 129.01, 128.69, 127.34,
127.72, 81.44, 33.39, 27.57 ppm.
4.2.3. 5-(40-methyl-2-biphenyl)2H-tetrazole (5) [9,42,43]
To a sealed tube were successively added TBAF 3H2O (0.35 g,
1.10 mmol), 4 (0.42 g, 2.20 mmol) and TMSN3 (0.38 g, 3.30 mmol)
and the resulting mixture was heated at 120 ꢁC for 36 h. The
resulting reaction mixture was extracted with EtOAc (30 mL) and
TBAF was removed by washing the organic phase with 1 M HCl
aqueous solution (3 ꢃ 10 mL). The organic extract was dried
(Na2SO4), filtered and concentrated in vacuo. Recrystallization from
diisopropyl ether furnished pure 5 (77%) as a white solid: M.p.
145e147 ꢁC; Rf 0.57 (CHCl3:MeOH, 9:1); tR 11.54 min (30%
MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 237.0 [MH]; 1Η
4.2.9. 4-butyl-1-[(2-(trimethylsilyl)ethoxy)methyl]imidazole (12a)
and its regioisomer 12b
To a solution of 4(5)-butylimidazole (11) (0.50 g, 4.03 mmol) in
anhydrous DMF (15 mL) under argon atmosphere at 0 ꢁC was
added powdered dry NaH (95%, 0.12 g, 4.84 mmol) and the
suspension was left at the same temperature for 15 min. Then,
SEM-Cl (0.68 mL, 4.84 mmol) was added in three portions and the
reaction mixture was allowed to warm to rt for 2 h. The reaction
was quenched with 0.5 N NaOH (15 mL) and extracted with CH2Cl2
(3 ꢃ 20 mL). The combined organic phases were washed with
brine (ꢃ2), dried (Na2SO4) and concentrated in vacuo. Purification
by preparative RP-HPLC afforded 12a (56%) as a yellow oil (ca. 2:1
12a/12b regioisomers by RP-HPLC and 1H NMR spectroscopic
analysis).
ΝΜR (400 MHz, CD3OD):
d 7.69e7.64 (m, 2Η), 7.56e7.52 (m, 2Η),
7.13 (d, 2H, J ¼ 7.8 Hz), 7.00 (d, 2H, J ¼ 7.8 Hz), 2.33 (s, 3H) ppm; 13C
NMR (160 MHz, CD3OD):
d 155.42, 142.17, 137.43, 136.24, 131.05,
130.39, 130.17, 128.76, 127.27, 122.83, 19.72 ppm.
Alternatively, to a solution of 4(5)-butylimidazole (11) (1.50 g,
12.10 mmol) in anhydrous THF (30 mL) were added sequentially
Cyhex2NMe (2.90 mL, 14.40 mmol) and SEM-Cl (1.72 mL,
9.68 mmol). After stirring the solution for 2 h, the reaction was
quenched with 0.5 N NaOH (30 mL) and extracted with EtOAc
(3 ꢃ 40 mL). The combined organic extracts were washed with
brine, dried (Na2SO4), filtered and concentrated in vacuo. The
crude oily residue was purified by preparative RP-HPLC (40%
MeCN / 100% MeCN in 50 min) to afford 12a (69%) as a yellow oil
(ca. 3:1 12a/12b regioisomers by RP-HPLC). 12a: Rf 0.53
(CHCl3:MeOH, 9.5:0.5); tR 16.24 min (5% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 255.08 [ΜH]; 1Η ΝΜR (400 MHz, CDCl3):
4.2.4. N-(trityl)-5-[4΄-(methyl)biphenyl-2-yl]tetrazole (6)
Prepared according to reported procedure [9]. M.p. 161e163 ꢁC;
Rf 0.48 (hexanes:EtOAc, 4:1); tR 13.68 min (70% MeCN / 100%
MeCN in 30 min); ESI-MS (m/z): 236.67 [MH-Tr], 243.09 [Tr]; 1Η
ΝΜR (400 MHz, CDCl3):
7.39e728 (m, 10H), 7.03e6.94 (m, 10H), 2.32 (s, 3H) ppm; 13C NMR
(160 MHz, CDCl3): 164.18, 142.27, 141.25, 138.17, 136.34, 130.65,
d
7.88 (d, 1H, J ¼ 7.6 Hz), 7.50e7.43 (m, 2H),
d
129.21, 129.08, 128.58, 128.16, 127.92, 127.57, 127.24, 126.43, 82.84,
21.16 ppm.
4.2.5. N-(trityl)-5-[4΄-(bromomethyl)biphenyl-2-yl]tetrazole (7)
Prepared according to reported procedure [9]. M.p. 136e138 ꢁC;
Rf 0.40 (hexanes:EtOAc, 4:1); tR 13.07 min (70% MeCN / 100%
MeCN in 30 min); ESI-MS (m/z): 315.41 [MH ꢀ Tr] (79Br), 317.17
d
8.82 (s, 1H), 6.92 (s, 1H), 5.40 (s, 2H), 3.54 (t, 2H, J ¼ 8.0 Hz), 2.71
(t, 2H, J ¼ 7.4 Hz), 1.65 (quint, 2H, J ¼ 7.4 Hz), 1.36 (sext, 2H,
J ¼ 7.4 Hz), 0.94e0.89 (m, 5H), ꢀ0.02 (s, 9H) ppm; 13C NMR
[MH ꢀ Tr þ 2] (81Br), 242.95 [Tr]; 1Η ΝΜR (400 MHz, CDCl3):
d
8.01
(160 MHz, CDCl3): d 138.86, 136.17, 117.12, 79.51, 68.51, 31.69, 26.17,
(d, 1H, J ¼ 7.6 Hz), 7.66e7.58 (m, 2H), 7.53e7.33 (m, 10H), 7.28
(d, 2H, J ¼ 8.0 Hz), 7.20 (d, 2H, J ¼ 8.0 Hz), 7.04e7.02 (m, 6H), 4.53
23.50, 19.23, 15.06, 0.0 ppm. 12b: Yellow oil. Yield 24%; Rf 0.53
(CHCl3:MeOH, 9.5:0.5); tR 16.58 min (5% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 255.14 [ΜH]; 1Η ΝΜR (400 MHz, CDCl3):
(s 2H) ppm; 13C NMR (160 MHz, CDCl3):
d 163.82, 141.11, 138.17,
130.62, 130.32, 130.21, 129.96, 129.61, 129.36, 128.48, 128.22, 127.91,
127.72, 82.96, 33.21 ppm.
d
8.98 (s, 1H), 7.10 (s, 1H), 5.47 (s, 2H), 3.57 (t, 2H, J ¼ 8.0 Hz), 2.69
(t, 2H, J ¼ 7.5 Hz), 1.66 (quint, 2H, J ¼ 7.5 Hz), 1.44 (sext, 2H,
J ¼ 7.5 Hz), 0.99e0.91 (m, 5H), 0.00 (s, 9H) ppm; 13C NMR
4.2.6. 4΄-methylbiphenyl-2-carboxylic acid (8)
(160 MHz, CDCl3): d 137.28, 132.20, 126.70, 73.78, 65.65, 30.28,
Prepared according to reported procedure [9]. M.p. 139e140 ꢁC;
Rf 0.59 (CHCl3:MeOH, 9.5:0.5); tR 6.48 min (30% MeCN / 100%
MeCN in 30 min); ESI-MS (m/z): 312.02 [MH]; 1Η ΝΜR (400 MHz,
23.30, 22.29, 17.55, 13.74, ꢀ1.55 ppm.
4.2.10. 4-butyl-1-[(2-(trimethylsilyl)ethoxy)methyl]imidazole-2-
carboxaldehyde (13)
To a solution of 12a (0.50 g,1.96 mmol) in anhydrous THF (10 mL)
at ꢀ78 ꢁC under argon was added n-BuLi (1.6 M in hexanes, 1.34 mL,
2.16 mL) dropwise via a syringe. The resulting yellow solution was
CDCl3):
d
7.77 (d, 1H, J ¼ 7.6 Hz), 7.66e7.62 (m, 1H), 7.53e7.41 (m,
4H), 7.31 (d, 2H, J ¼ 8.0 Hz), 2.43 (s, 3H) ppm; 13C NMR (160 MHz,
CDCl3):
d 171.49, 139.86, 137.79, 137.23, 134.25, 130.50, 130.45,
129.42, 129.08, 128.66, 127.38, 19.72 ppm.

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G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
stirred at ꢀ78 ꢁC for another 30 min and then anhydrous DMF
(0.17 mL, 2.16 mmol) was added dropwise at the same temperature.
The mixture was allowed to reach rt while being stirred for another
12 h. Then, the solution was quenched with saturated aqueous
solution NH4Cl (50 mL), the organic phase was extracted with EtOAc
(3 ꢃ 50 mL), dried (Na2SO4), filtered and concentrated in vacuo. The
residue was purified by flash column chromatography (hex-
anes:EtOAc, 1:4) to give the title compound (65%) as a yellow oil: Rf
0.58 (hexanes:EtOAc,1:9); tR 15.61 min (5% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 301.28 [ΜH þ H2O], 283.37 [ΜH]; 1Η ΝΜR
(70% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 761.65
[Μ ꢀ Βr]; 1Η ΝΜR (400 MHz, CDCl3):
d 7.96e7.93 (m, 1H), 7.51e7.44
(m, 2H), 7.37e7.13 (m, 13H), 6.95e6.92 (m, 6H), 6.83 (d, 2H,
J ¼ 8.0 Hz), 5.90 (s, 2H), 5.42 (s, 2H), 4.70 (s, 2H), 3.70 (t, 2H,
J ¼ 8.0 Hz), 2.44 (t, 2H, J ¼ 7.5 Hz), 1.55 (quint, 2H, J ¼ 7.5 Hz), 1.31
(sext, 2H, J ¼ 7.5 Hz), 0.96 (t, 2H, J ¼ 8.0 Hz), 0.87 (t, 3H, J ¼ 7.5 Hz),
0.01 (s, 9H) ppm; 13C NMR (160 MHz, CDCl3):
d 163.81, 146.47,
135.45, 131.79, 130.56, 130.25, 130.00, 128.36, 127.95, 127.88, 127.66,
126.18, 125.58, 117.26, 82.92, 78.24, 68.36, 51.97, 48.92, 28.76, 23.68,
22.06, 17.88, 13.58, 1.39 ppm.
(400 MHz, CDCl3): d 9.77 (s, 1H), 7.09 (s, 1H), 5.72 (s, 2H), 3.55 (t, 2H,
J ¼ 8.0 Hz), 2.84 (t, 2H, J ¼ 7.5 Hz), 1.66 (quint, 2H, J ¼ 7.5 Hz), 1.38
4.2.14. 5-butyl-2-hydroxymethyl-1-[[(20-(2-trityl)-tetrazol-5-yl)
biphenyl-4-yl]methyl]imidazole (17)
(sext, 2H, J ¼ 7.5 Hz), 0.96e0.89 (m, 5H), ꢀ0.02 (s, 9H) ppm; 13C NMR
(160 MHz, CDCl3):
d
183.47, 147.88, 144.11, 123.79, 76.96, 68.26,
Compound 16 (1.10 g, 1.31 mmol) was dissolved in a solution of
1 M TBAF in THF (6 mL) and the resulting mixture was heated to
reflux under argon atmosphere for 8 h. Then, the solvent was
concentrated and the residue was partitioned between CH2Cl2
(40 mL) and H2O (30 mL). The organic phase was washed with
brine, dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by flash column chromatography (CHCl3:MeOH, 9.7:0.3)
afforded the alcohol 17 (90%) as a white foam: Rf 0.24 (CHCl3:MeOH,
9.7:0.3); tR 14.66 min (40% MeCN / 100% MeCN in 30 min); ESI-MS
32.83, 29.45, 23.84, 19.28, 15.32, 0.00 ppm.
4.2.11. 4-butyl-2-hydroxymethyl-1-[(2-(trimethylsilyl)ethoxy)
methyl]imidazole (14)
To a solution of 13 (0.40 g, 1.42 mmol) in MeOH (10 mL) at 0 ꢁC
was added NaBH4 (70 mg, 1.85 mmol) portionwise over 20 min and
stirred at 0 ꢁC for an additional 1 h. The reaction mixture was then
quenched with saturated aqueous solution NH4Cl (40 mL), extrac-
ted with EtOAc (3 ꢃ 40 mL) and the organic extracts were washed
with brine, dried (Na2SO4), filtered and concentrated in vacuo. Pure
alcohol 14 (92%) was obtained as a yellow oil: Rf 0.57 (CHCl3:MeOH,
9.5:0.5); tR 15.90 min (5% MeCN / 100% MeCN in 30 min); ESI-MS
(m/z): 631.49 [ΜH], 243.33 [Τr]; 1Η ΝΜR (400 MHz, CDCl3):
d 7.92
(dd, 1H, J ¼ 7.6, 1.4 Hz), 7.48e7.44 (m, 2H), 7.35e7.23 (m, 10H), 7.08
(d, 2H, J ¼ 7.6 Hz), 6.93e6.71 (m, 9H), 5.07 (s, 2H), 4.46 (s, 2H), 2.29
(t, 2H, J ¼ 7.5 Hz), 1.49 (quint, 2H, J ¼ 7.5 Hz), 1.25 (sext, 2H,
J ¼ 7.5 Hz), 0.83 (t, 3H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
(m/z): 285.39 [ΜH]; 1Η ΝΜR (400 MHz, CDCl3):
d 6.69 (s, 1H), 5.31
(s, 2H), 4.69 (s, 2H), 3.53 (t, 2H, J ¼ 8.0 Hz), 2.51 (t, 2H, J ¼ 7.5 Hz),
d 163.94, 147.39, 135.11, 133.47, 130.73, 130.24, 129.75, 128.28,
1.58 (quint, 2H, J ¼ 7.5 Hz), 1.35 (sext, 2H, J ¼ 7.5 Hz), 0.93e0.90 (m,
127.64, 126.31, 125.27, 124.19, 82.89, 56.79, 46.35, 30.05, 23.98,
22.94, 13.79 ppm.
5H), 0.02 (s, 9H) ppm; 13C NMR (160 MHz, CDCl3):
d 162.85, 147.06,
140.72, 127.90, 74.98, 66.47, 55.99, 31.20, 27.30, 22.32, 17.73, 13.87,
1.00 ppm.
4.2.15. 5-butyl-2-hydroxymethyl-1-[[20-(2H-tetrazol-5-yl)
biphenyl-4-yl]methyl]imidazole (21)
4.2.12. 5-butyl-1-[[(20-(2-trityl)-tetrazol-5-yl)biphenyl-4-yl]
Compound 17 (0.15 g, 0.24 mmol) was dissolved in a solution of
20% TFA in CH2Cl2 (2 mL) and then Et3SiH (0.11 mL, 0.72 mmol)
was added as a scavenger. The resulting solution was stirred at rt
for 1 h before it was concentrated in vacuo. Precipitation from
Et2O furnished the TFA salt as an amorphous powder. The TFA salt
was partitioned between H2O (5 mL) and EtOAc (5 mL), followed
by addition of NaHCO3 (0.21 g, 2.5 mmol). Then, the mixture was
adjusted to pH 3.0 with 1 N HCl, the organic phase was separated,
dried (Na2SO4) and concentrated in vacuo to give 21 (85%) as
a white solid: M.p. 109e111 ꢁC; Rf 0.33 (CHCl3:MeOH:AcOH,
8.5:1.5:0.5); tR 8.52 min (20% MeCN / 100% MeCN in 30 min);
ESI-MS (m/z): 389.13 [ΜH]; 1Η ΝΜR (400 MHz, CD3OD):
methyl]-3-[(2-(trimethylsilyl)ethoxy)methyl]imidazole (15)
To a stirred solution of 12a (0.55 g, 2.16 mmol) in anhydrous
MeCN (10 mL) under argon was added the alkylating agent 7
(1.32 g, 2.38 mmol) in one portion and the resulting mixture was
heated at 70 ꢁC for 3 h. Upon completion (disappearance of
starting material confirmed by RP-HPLC), the solvent was
concentrated, followed by recrystallization with Et2O to afford 15
(78%) as a white powder: Rf 0.29 (CHCl3:MeOH, 9.6:0.4); tR
9.52 min (70% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):
731.72 [Μ ꢀ Br]; 1Η ΝΜR (400 MHz, CDCl3):
d 10.63 (s, 1H), 7.87 (d,
1H, J ¼ 7.6 Hz), 7.55e7.14 (m, 17H), 6.92 (m, 6H), 5.53 (s, 2H), 5.42
(s, 2H), 3.65 (t, 2H, J ¼ 8.0 Hz), 2.61 (t, 2H, J ¼ 7.5 Hz), 1.59 (quint,
2H, J ¼ 7.5 Ηz), 1.38 (sext, 2H, J ¼ 7.5 Hz), 0.98e0.89 (m, 5H), 0.03
(s, 9H) ppm.
d
7.73e7.68 (m, 2H), 7.62e7.56 (m, 2H), 7.36 (s, 1H), 7.19 (d, 2H,
J ¼ 8.3 Hz), 7.13 (d, 2H, J ¼ 8.3 Hz), 5.45 (s, 2H), 4.83 (s, 2H), 2.58 (t,
2H, J ¼ 7.5 Hz), 1.58 (quint, 2H, J ¼ 7.5 Hz), 1.39 (sext, 2H,
J ¼ 7.5 Hz), 0.93 (t, 3H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz,
4.2.13. 5-butyl-2-hydroxymethyl-1-[[(20-(2-trityl)-tetrazol-5-yl)
biphenyl-4-yl]methyl]-3-[(2-(trimethylsilyl)ethoxy)methyl]
imidazole (16)
CD3OD): d 162.35, 149.26, 143.40, 143.27, 137.96, 135.50, 132.62,
132.04, 131.88, 131.41, 130.68, 129.41, 128.31, 128.03, 119.68, 56.97,
31.60, 25.36, 24.05, 14.88 ppm.
In a sealed tube were sequentially added 15 (1.20 g, 1.48 mmol),
DMF (0.5 mL), 37% formalin (0.29 mL, 10.36 mmol) and diisopro-
pylethylamine (0.65 mL, 3.70 mmol). The resulting mixture was
stirred at 85 ꢁC until HPLC showed no starting material left (ca. 1 h).
Then, the mixture was quenched with 5% aqueous citric acid
(10 mL), extracted with CH2Cl2 (3 ꢃ 20 mL) and the combined
organic phases were washed with brine, dried (Na2SO4), filtered
and concentrated in vacuo. Recrystallization from Et2O afforded 16
(89%) as a white powder.
Alternatively, compound 16 was prepared by direct alkylation of
the alcohol 14 (1 eq) with 7 (1.1 eq) in MeCN at 70 ꢁC for 12 h.
Purification by flash column chromatography furnished the title
compound: Yield 20%; Rf 0.25 (CHCl3:MeOH, 9.6:0.4); tR 8.31 min
4.2.16. 5-butyl-4-chloro-2-hydroxymethyl-1-[[(20-(2-trityl)-
tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole (18)
To a solution of 17 (0.30 g, 0.47 mmol) in anhydrous DMF (4 mL)
was added NCS (70 mg, 0.52 mmol) in one portion. After stirring at
70 ꢁC for 8 h, the reaction mixture was quenched with H2O (2 mL)
and extracted with CH2Cl2 (3 ꢃ 30 mL). The combined organic
phases were washed with brine, dried (Na2SO4), filtered and
concentrated in vacuo. The oily residue was purified by flash
column chromatography (CHCl3) to afford the title compound 18
(86%) as a white foam: Rf 0.65 (CHCl3:MeOH, 9.6:0.4); tR 19.11 min
(40% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 665.46 [ΜΗ]
(
35Cl), 667.48 [ΜΗ þ 2] (37Cl); 1Η ΝΜR (400 MHz, CDCl3):
d 7.94 (d,

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
371
1H, J ¼ 7.6 Hz), 7.51e7.44 (m, 3H), 7.36e7.23 (m, 8H), 7.10 (d, 2H,
4.2.21. 5-butyl-2-hydroxymethyl-4-iodo-1-[[20-(2H-tetrazol-5-yl)
biphenyl-4-yl]methyl]imidazole (24)
Compound 24 was prepared in an analogous manner to that
described for 21. Yield 80%; M.p. 101e102 ꢁC; Rf 0.47 (CHCl3:MeO-
H:AcOH, 90:15:5); tR 9.94 min (20% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 515.28 [ΜH]; 1Η ΝΜR (400 MHz, CD3OD):
J ¼ 8.0 Hz), 6.93e6.91 (m, 7H), 6.72 (d, 2H, J ¼ 8.0 Hz), 5.07 (s, 2H),
4.24 (s, 2H), 2.36 (t, 2H, J ¼ 7.5 Hz), 1.37e1.19 (m, 4H), 0.82 (t, 3H,
J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
d 163.94, 146.28,
134.77, 131.10, 130.58, 130.35, 130.24, 128.69, 128.02, 126.58, 125.86,
125.62, 83.24, 57.21, 47.37, 31.22, 22.53, 22.94, 14.09 ppm.
d
7.69e7.65 (m, 2H), 7.55 (m, 2H), 7.14 (d, 2H, J ¼ 8.1 Hz), 7.07 (d, 2H,
4.2.17. 4-bromo-5-butyl-2-hydroxymethyl-1-[[(20-(2-trityl)-
tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole (19)
J ¼ 8.1 Hz), 5.44 (s, 2H), 4.60 (s, 2H), 2.37 (t, 2H, J ¼ 7.5 Hz),
1.32e1.25 (m, 4H), 0.84 (t, 3H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz,
To a solution of 17 (0.25 g, 0.40 mmol) in anhydrous DMF (4 mL)
was added NBS (78 mg, 0.44 mmol) in one portion. After stirring at
rt for 30 min the reaction mixture was quenched with H2O (2 mL)
and extracted with CH2Cl2 (3 ꢃ 30 mL). The combined organic
phases were washed with brine, dried (Na2SO4), filtered and
concentrated in vacuo. The oily residue was purified by flash
column chromatography (CHCl3) to afford the title compound 19
(92%) as a white foam: Rf 0.64 (CHCl3:MeOH, 9.6:0.4); tR 18.54 min
(40% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 709.50 [ΜΗ]
CD3OD): d 154.73, 149.37, 141.49, 139.07, 136.17, 130.27, 130.22,
129.27, 127.57, 126.07, 123.25, 102.43, 55.61, 30.66, 24.03, 21.87,
12.65 ppm.
4.2.22. 4-butyl-1H-imidazole-2-carboxaldehyde (25)
To a solution of the aldehyde 13 (120 mg, 0.42 mmol) in EtOH
(2 mL) was added 3 N HCl (0.5 mL) and the resulting mixture was
heated at 60 ꢁC. After being stirred for 3 h, the reaction mixture was
cooled to rt, quenched with saturated aqueous solution of K2CO3
(20 mL) and extracted with EtOAc (3 ꢃ 20 mL). The combined
organic extracts were dried (Na2SO4), filtered and concentrated in
vacuo. Purification by flash column chromatography (CHCl3:MeOH,
9.6:0.4) afforded 25 (63%) as a white solid: M.p. 97e99 ꢁC; Rf 0.46
(CHCl3:MeOH, 9.5:0.5); tR 6.45 min (5% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 153.66 [ΜH]; 1Η ΝΜR (400 MHz, DMSO-d6):
(
79Br), 711.51 [MΗ þ 2] (81Br); 1Η ΝΜR (400 MHz, CDCl3):
d 7.94 (d,
1H, J ¼ 7.6 Hz), 7.49e7.44 (m, 3H), 7.36e7.24 (m, 8H), 7.10 (d, 2H,
J ¼ 7.6 Hz), 6.94e6.92 (m, 7H), 6.74 (d, 2H, J ¼ 8.0 Hz), 5.09 (s, 2H),
4.43 (s, 2H), 2.36 (t, 2H, J ¼ 7.5 Hz), 1.37e1.21 (m, 4H), 0.83 (t, 3H,
J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
d 167.52, 147.28,
135.27, 132.10, 130.69, 130.25, 130.13, 129.48, 128.52, 126.58,
125.72, 125.26, 111.39, 83.55, 56.11, 47.15, 30.16, 22,56, 22.47,
15.52 ppm.
d
9.52 (s, 1H), 7.13 (s, 1H), 2.57 (t, 2H, J ¼ 7.4 Hz), 1.58 (quint, 2H,
J ¼ 7.4 Hz),1.29 (sext, 2H, J ¼ 7.4 Hz), 0.88 (t, 2H, J ¼ 7.4 Hz) ppm; 13C
NMR (160 MHz, DMSO-d6):
d 184.94, 149.57, 145.46, 130.52, 35.14,
4.2.18. 5-butyl-2-hydroxymethyl-4-iodo-1-[[(20-(2-trityl)-tetrazol-
5-yl)biphenyl-4-yl]methyl]imidazole (20)
29.69, 26.08, 18.06 ppm.
Compound 17 was prepared in an analogous manner to that
described for 18 using NIS (1.5 eq). Yield 90%. Rf 0.64 (CHCl3:MeOH,
9.6:0.4); tR 16.50 min (40% MeCN / 100% MeCN in 30 min); ESI-MS
4.2.23. 5-butyl-1-[[(20-(2-trityl)-tetrazol-5-yl)biphenyl-4-yl]
methyl]imidazole-2-carboxaldehyde (27)
To a solution of the aldehyde 25 (0.10 g, 0.66 mmol) in anhy-
drous DMF (10 mL) under argon atmosphere were added sequen-
(m/z): 757.71 [ΜΗ], 631.49 [MH ꢀ I]; 1Η ΝΜR (400 MHz, CDCl3):
d 7.94
(dd,1H, J ¼ 7.3 Hz,1.7 Hz), 7.51e7.44 (m, 3H), 7.36e7.24 (m, 8H), 7.11
(d, 2H, J ¼ 8.0 Hz), 6.93e6.91 (m, 7H), 6.74 (d, 2H, J ¼ 8.0 Hz), 5.15 (s,
2H), 4.45 (s, 2H), 2.37 (t, 2H, J ¼ 7.5 Hz),1.36e1.21 (m, 4H), 0.84 (t, 3H,
tially anhydrous K2CO3 (0.18 g, 1.32 mmol) and 7 (0.41 g,
0.73 mmol). The reaction mixture was stirred at rt for 18 h before
being quenched with H2O (20 mL) and extracted with CH2Cl2
(3 ꢃ 20 mL). The combined organic extracts were washed with H2O
(3 ꢃ 40 mL), dried (Na2SO4), filtered and concentrated in vacuo.
Purification by flash column chromatography (hexanes:EtOAc, 6:4)
furnished the title compound 27 (28%) as white foam (ca. 2.5:1
26/27 regioisomers by RP-HPLC analysis).
J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
d 164.25, 149.41, 141.61,
134.70, 130.57, 130.27, 128.70, 128.29, 128.02, 127.62, 125.64, 83.25,
81.70, 57.30, 47.86, 31.65, 24.97, 22.68, 14.13 ppm.
4.2.19. 5-butyl-4-chloro-2-hydroxymethyl-1-[[20-(2H-tetrazol-5-
yl)biphenyl-4-yl]methyl]imidazole (22)
Alternatively, aldehyde 27 was prepared by Swern oxidation. To
Compound 22 was prepared in an analogous manner to that
described for 21. Yield 82%; M.p. 105e107 ꢁC; Rf 0.51 (CHCl3:MeO-
H:AcOH, 90:15:5); tR 10.73 min (20% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 423.41 [ΜH] (35Cl), 425.87 [ΜΗ þ 2] (37Cl);
a solution of anhydrous dimethyl sulfoxide (85 mL, 1.20 mmol) in
anhydrous CH2Cl2 (3 mL) at ꢀ78 ꢁC was added oxalyl chloride
(48
mL, 0.56 mmol) such that the temperature did not
exceed ꢀ65 ꢁC. After 15 min, a solution of the alcohol 17 (0.20 g,
0.31 mmol) in CH2Cl2 (1 mL) was added and the reaction mixture
was stirred at the same temperature for 50 min. Then, anhydrous
1Η ΝΜR (400 MHz, CD3OD):
d 7.71e7.67 (m, 2H), 7.57 (m, 2H), 7.14
(d, 2H, J ¼ 8.0 Hz), 7.06 (d, 2H, J ¼ 8.0 Hz), 5.35 (s, 2H, Η-11), 4.55 (s,
2H), 2.49 (t, 2H, J ¼ 7.5 Hz), 1.35e1.27 (m, 4H), 0.86 (t, 3H, J ¼ 7.5 Hz)
Et3N (160 mL, 1.24 mmol) was added and the reaction mixture was
ppm; 13C NMR (160 MHz, CD3OD):
d
157.81, 145.28, 141.46, 139.38,
allowed to warm to rt for 1 h. The mixture was diluted with CH2Cl2
(20 mL) and the organic phase was washed once with H2O (20 mL),
brine, dried (Na2SO4), filtered and concentrated. Purification by
flash column chromatography (hexanes:EtOAc, 6:4) furnished the
title compound 27 (92%) as a white foam. 26: Rf 0.43 (hex-
anes:EtOAc, 3:7); tR 14.24 min (40% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 629.48 [ΜH], 243.21 [Tr]; 1Η ΝΜR (400 MHz,
135.77, 130.27, 130.18, 129.50, 129.27, 129.23, 128.08, 125.91, 124.43,
55.70, 30.27, 22.19, 21.73, 12.60 ppm.
4.2.20. 4-bromo-5-butyl-2-hydroxymethyl-1-[[20-(2H-tetrazol-5-
yl)biphenyl-4-yl]methyl]imidazole (23)
Compound 23 was prepared in an analogous manner to that
described for 21. Yield 79%; M.p. 103e105 ꢁC; Rf 0.46
(CHCl3:MeOH:AcOH, 9:1.5:0.5); tR 10.55 min (20% MeCN / 100%
MeCN in 30 min); ESI-MS (m/z): 467.20 [ΜΗ] (79Βr), 469.16
CDCl3):
d
9.77 (s, 1H), 7.92 (d, 1H, J ¼ 7.6 Hz), 7.51e7.44 (m, 3H),
7.36e7.25 (m, 8H), 7.15 (s, 1H), 7.09 (d, 2H, J ¼ 7.9 Hz), 6.94e6.92
(m, 7H), 6.80 (d, 2H, J ¼ 7.9 Hz), 5.60 (s, 2H), 2.41 (t, 2H,
J ¼ 7.5 Hz),1.54 (quint, 2H, J ¼ 7.5 Hz),1.29 (sext, 2H, J ¼ 7.5 Hz), 0.87
[ΜΗ þ 2] (81Βr); 1Η ΝΜR (400 MHz, CD3OD):
d 7.74e7.67 (m, 2H),
7.58 (m, 2H), 7.16 (d, 2H, J ¼ 8.0 Hz), 7.08 (d, 2H, J ¼ 8.0 Hz), 5.43 (s,
2H), 4.65 (s, 2H), 2.53 (t, 2H, J ¼ 7.5 Hz), 1.32e1.25 (m, 4H), 0.87 (t,
(t, 2H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
d 181.57, 163.91,
146.87, 143.56, 141.44, 141.22, 140.75, 140.18, 134.52, 130.30, 130.74,
130.23, 129.94, 129.74, 128.25, 127.64, 126.27, 125.89, 82.89, 47.43,
27.70, 23.62, 22.31, 13.73 ppm. 27: Rf 0.69 (hexanes:EtOAc, 3:7); tR
13.75 min (40% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):
3H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CD3OD):
d 155.76, 146.79,
141.50, 139.08, 136.02, 130.93, 130.23, 129.24, 127.58, 127.54, 126.03,
123.50, 111.45, 55.72, 30.12, 24.05, 21.87, 12.65 ppm.

372
G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
629.54 [ΜH], 243.18 [Tr]; 1Η ΝΜR (400 MHz, CDCl3):
d
9.73 (s, 1H),
(CHCl3/MeOH, 9.6:0.4); tR 8.79 min (60% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 521.58 [M ꢀ Br]; 1Η ΝΜR (400 MHz, CDCl3):
7.90 (dd, 1H, J ¼ 7.6 Hz, 1.2 Hz), 7.44e7.38 (m, 2H), 7.30e7.26
(m, 4H), 7.19e7.16 (m, 6H), 7.04 (d, 2H, J ¼ 8.0 Hz), 6.88 (d, 2H,
J ¼ 8.0 Hz), 6.84e6.83 (m, 6H), 6.64 (s, 1H), 5.38 (s, 2H), 2.46 (t, 2H,
J ¼ 7.5 Hz),1.51 (quint, 2H, J ¼ 7.5 Hz),1.29 (sext, 2H, J ¼ 7.5 Hz), 0.84
d
10.95 (s, 1H), 7.77 (d, 1H, J ¼ 7.6 Hz), 7.47e7.23 (m, 7H), 7.10 (s, 1H),
5.72 (s, 2H), 5.59 (s, 2H), 3.70 (t, 2H, J ¼ 8.0 Hz), 2.54 (t, 2H,
J ¼ 7.5 Hz),1.56 (quint, 2H, J ¼ 7.5 Hz),1.35e1.25 (m,11H), 0.97e0.89
(m, 5H), 0.0 (s, 9H) ppm.
(t, 2H, J ¼ 7.5 Hz) ppm; 13C NMR (160 MHz, CDCl3):
d 181.91, 163.87,
146.30, 142.29, 141.39, 141.21, 134.51, 130.68, 130.21, 129.97, 129.79,
128.27, 127.62, 127.62, 127.19, 126.24, 123.15, 82.89, 50.24, 31.40,
28.05, 22.46, 13.87 ppm.
4.2.27. 5-butyl-1-[[20-(tert-butoxycarbonyl)biphenyl-4-yl]methyl]-
2-hydroxymethyl-3-[(2-(trimethylsilyl)ethoxy)methyl]imidazole
(32)
4.2.24. 5-butyl-1-[[20-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]
imidazole-2-carboxaldehyde (29)
Compound 32 was prepared in an analogous manner to that
described for 16 by direct hydroxymethylation. Yield 93%; Rf 0.26
(CHCl3/MeOH, 9.6:0.4); tR 7.25 min (60% MeCN / 100% MeCN in
30 min); ESI-MS (m/z): 551.80 [M ꢀ Βr]; 1Η ΝΜR (400 MHz, CDCl3):
To a solution of the aldehyde 27 (0.16 g, 0.25 mmol) in Et2O
(2 mL) was added formic acid (3 mL) and the resulting mixture was
stirred at rt for 2 h. Then, the solvent was concentrated in vacuo,
followed by recrystallization from diisopropyl ether to afford the
title compound 29 (83%) as a white amorphous solid: Rf 0.45
(CHCl3/MeOH/AcOH, 8.5:1.5:0.5); tR 8.25 min (20% MeCN / 100%
MeCN in 30 min); ESI-MS (m/z): 405.25 [MH þ Η2Ο], 387.23 [ΜH];
d
7.78 (d,1H, J ¼ 7.6 Hz), 7.49 (t,1H, J ¼ 7.4 Hz), 7.41 (t,1H, J ¼ 7.4 Hz),
7.32 (d, 2H, J ¼ 8.0 Hz), 7.24 (s, 1H), 7.18 (m, 1H), 7.08 (d, 2H,
J ¼ 8.0 Hz), 5.78 (s, 2H), 5.67 (s, 2H), 4.87 (s, 2H), 3.68 (t, 2H,
J ¼ 8.0 Hz), 2.56 (t, 2H, J ¼ 7.5 Hz), 1.62 (quint, 2H, J ¼ 7.5 Hz),
1.56e1.25 (m, 11H), 0.96e0.88 (m, 5H), 0.0 (s, 9H) ppm.
1Η ΝΜR (400 MHz, CD3OD):
d 9.68 (s, 1H), 7.57e7.51 (m, 2H),
7.47e7.45 (m, 2H), 7.17 (s, 1H), 7.09 (d, 2H, J ¼ 8.0 Hz), 6.92 (d, 2H,
J ¼ 8.0 Hz), 5.68 (s, 2H), 2.57 (t, 2H, J ¼ 7.5 Hz), 1.57 (quint, 2H,
J ¼ 7.5 Hz),1.37 (sext, 2H, J ¼ 7.5 Hz), 0.91 (t, 3H, J ¼ 7.5 Hz) ppm; 13C
4.2.28. 5-butyl-1-[(20-carboxybiphenyl-4-yl)methyl]-2-
hydroxymethylimidazole (33)
Compound 33 was prepared in an analogous manner to that
described for 21. Yield 82%; Rf 0.29 (CHCl3/MeOH/AcOH,
8.5:1.5:0.5); tR 12.43 min (10% MeCN / 100% MeCN in 30 min);
ΝΜR (160 MHz, CD3OD):
d 180.45, 157.68, 147.58, 141.24, 139.12,
135.23, 130.72, 130.30, 129.72, 129.67, 129.55, 128.49, 128.23, 127.57,
126.53, 126.06, 124.25, 55.72, 29.41, 23.11, 21.88, 12.62 ppm.
ESI-MS (m/z): 365.17 [MH]; 1Η ΝΜR (400 MHz, CDCl3):
d 7.89 (d, 1H,
J ¼ 7.6 Hz), 7.50 (t, 1H, J ¼ 7.4 Hz), 7.40 (t, 1Η, J ¼ 7.4 Hz), 7.32 (d, 2H,
J ¼ 7.6 Hz), 7.29 (s, 1H), 7.05-6.97 (m, 3H), 5.26 (s, 2H), 4.61 (s, 2H),
2.48 (t, 2H, J ¼ 7.5 Hz), 1.53 (quint, 2H, J ¼ 7.5 Hz), 1.34 (sext, 2H,
J ¼ 7.5 Hz), 0.87 (t, 3H, J ¼ 7.5 Hz) ppm; 13C ΝΜR (160 MHz, CDCl3):
4.2.25. 5-butyl-1-[[20-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl]
imidazole-2-carboxylic acid (30)
To a solution of the aldehyde 27 (0.10 g, 0.16 mmol) in t-BuOH
(1 mL) were sequentially added 2-methyl-2-butene (0.8 mL),
NaH2PO4 (0.13 g, 1.10 mmol) and a solution of 80% NaClO2 (0.19 g,
1.44 mmol) in H2O (1 mL). The reaction mixture was stirred at rt
d
172.82, 147.37, 142.55, 141.69, 135.49, 132.43, 131.55, 130.77,
130.55, 129.84, 127.97, 126.53, 125.35, 54.02, 48.07, 29.06, 23.88,
22.44, 13.94 ppm.
for
1 h. The resulting solution was extracted with CH2Cl2
(3 ꢃ 20 mL) and the combined organic extracts were washed with
brine, dried (Na2SO4), filtered and concentrated to afford the
carboxylic acid 28 (95%) as a white foam, which was used directly
without further purification. Then, detritylation of 28 was
accomplished by an analogous manner to that described for 29 to
furnish 30 (86%).
4.3. Pharmacological evaluation
4.3.1. AT1 receptor binding assay of synthesized ANG II analogues
cell culture and transfection
Human embryonic kidney (HEK 293) cells were grown in
DMEM/F12 (1:1) containing 3.15 g/L glucose and 10% bovine calf
serum at 37 ꢁC and 5% CO2. 60 mm dishes of HEK 293 cells at
80e90% confluence were transfected with 3
encoding the human AT1 receptor, using 9 L of Lipofectamine and
2 mL of OPTIMEM. To generate stably transfected pools of cells
expressing the AT1 receptor 5e12 h after transfection, the medium
was replaced by DMEM/F12 (1:1) containing 3.15 g/L glucose, 10%
Alternatively, the title compound 30 was prepared by oxidation
with KMnO4. To a solution of the alcohol 21 (80 mg, 0.21 mmol) in
a mixture of H2O/acetone 1:1 (2 mL) at 0 ꢁC were sequentially
added KMnO4 (44 mg, 0.28 mmol) and a catalytic amount of 18-
crown-6 (5 mg). The resulting mixture was allowed to warm to rt
over a period of 1 h before it was quenched with MeOH (1 mL),
followed by filtration through a pad of CeliteÒ. The filtrate was
adjusted to pH 4 with 0.1 M HCl, extracted with EtOAc (3 ꢃ 20 mL)
and the combined organic extracts were washed with brine, dried
(Na2SO4), filtered and concentrated in vacuo. Recrystallization from
diisopropyl ether afforded the title compound 30 (74%) as an
amorphous solid. 30: Rf 0.21 (CHCl3/MeOH/AcOH, 8.5:1.5:0.5); tR
8.48 min (20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):
805.64 [2M þ Η], 403.30 [ΜH], 359.26 [ΜH-COOH]; 1Η ΝΜR
mg of plasmid DNA
m
bovine calf serum and 700 mg/mL of the antibiotic, Geneticin. The
antibiotic use ensured the selection of a stably transfected pool of
cells.
4.3.2. Harvesting cells and membrane preparation
HEK 293 cells (grown in 100 mm dishes) stably expressing the
human AT1 receptor were washed with phosphate-buffered saline
(PBS; 4.3 mM Na2HPO4$7H2O, 1.4 mM KH2PO4, 137 mM NaCl,
2.7 mM KCl, pH 7.2e7.3 at rt), briefly treated with PBS containing
2 mM EDTA (PBS/EDTA), and then dissociated in PBS/EDTA. Cell
suspension was centrifuged at 1000ꢃ g for 5 min at rt, and the
pellet was homogenized in 1 mL of buffer O (50 mM TriseHCl
containing 0.5 mM EDTA, 10% sucrose, 10 mM MgCl2, pH 7.4 at
4 ꢁC) using a Janke & Kunkel IKA Ultra Turrax T25 homogenizer (at
setting w20, 10e15 s, 4 ꢁC). The homogenate was centrifuged at
250ꢃ g for 5 min at rt. The pellet was discarded and the supernatant
was centrifuged (16,000ꢃ g, 10 min, 4 ꢁC). The membrane pellet
was resuspended (0.6e0.7 mL/100 mm dish) in buffer B (50 mM
TriseHCl containing 1 mM EDTA, 10 mM MgCl2, 0.2% BSA,
(400 MHz, CD3OD): d 7.68e7.67 (m, 2H), 7.58e7.54 (m, 2H), 7.29 (s,
1H), 7.15 (br s, 4H), 6.02 (s, 2H), 2.54 (t, 2H, J ¼ 7.5 Hz), 1.55 (quint,
2H, J ¼ 7.5 Hz),1.38 (sext, 2H, J ¼ 7.5 Hz), 0.91 (t, 3H, J ¼ 7.5 Hz) ppm;
13C ΝΜR (160 MHz, CD3OD):
d 172.10, 160.27, 146.60, 141.28, 139.42,
134.65, 130.75, 130.26, 129.32, 128.50, 128.22, 127.62, 126.58,
123.86, 51.52, 25.06, 24.05, 21.88, 14.85 ppm.
4.2.26. 5-butyl-1-[[20-(tert-butoxycarbonyl)biphenyl-4-yl]methyl]-
3-[(2-(trimethylsilyl)ethoxy)methyl]imidazole (31)
Compound 31 was prepared in an analogous manner to that
described for 15, using 10 as an alkylating agent. Yield 87%; Rf 0.31

G. Agelis et al. / European Journal of Medicinal Chemistry 55 (2012) 358e374
373
0.2 mg/mL bacitracin, and 0.93
used for radioligand binding studies.
m
g/mL aprotinin, pH 7.4 at 4 ꢁC) and
receptor was mapped with grid-based calculations. In grid-based
calculation procedure the target protein is embedded in a three-
dimensional grid. Then, a probe atom is sequentially located at
each grid point, the interaction energy between the probe atom and
the target atoms is computed, and the value is stored in the grid.
The energy of interaction of this single atom with the protein is
assigned to the grid point. The active site was defined by 20 and
46 Å inner and outer cubic grid boxes, centered on the point that is
the center of mass of residues K199 and H256. The Glide XP (extra
precision) (v5.0) [66] combined with Induced Fit Docking (IFD)
have been used for the docking calculations. IFD uses the Glide
ligand docking program to account the ligand flexibility, and the
Refinement module and the Prime algorithm to account for flexi-
bility of the receptor. Schrodinger’s IFD protocol model uses the
following steps (the description below is from the IFD user
manual): (i) constrained minimization of the receptor with an
RMSD cut-off of 0.18 Å; (ii) initial Glide docking of each ligand using
a soft potentials (0.5 van der Waals radii scaling of non-polar atoms
of ligands and receptor using partial charge cut-off of 0.15); (iii)
derived docking poses were refined using the Prime module of the
Schrodinger. Residues within the 5.0 Å of ligand poses were mini-
mized in order to form suitable conformations of poses at the active
site of the receptor; (iv) Glide re-docking of each proteineligand
complex.
4.3.3. [125IeSar1eIle8] ANG II binding
The [125IeSar1eIle8] ANG II competition binding was performed
as follows. Aliquots of diluted membrane suspension (50
added into tubes, containing buffer B and 100,000e120,000 cpm
125IeSar1eIle8] ANG II with or without increasing concentrations
mL) were
[
of ANG II analogues in a final volume of 0.15 mL. The mixtures were
incubated (1 h, 24 ꢁC) and then, filtered using a Brandel cell
harvester through Whatman GF/C glass fiber filters, presoaked for
1 h in 0.5% polyethylenimine at 4 ꢁC. The filters were washed 10
times with 1e2 mL of ice-cold 50 mM TriseHCl containing NaCl
120 mM, pH 7.4 at 4 ꢁC. Filters were assessed for radioactivity in
a gamma counter (LKB Wallac 1275 minigamma, 80% efficiency).
The amount of membrane used was adjusted to ensure that specific
binding was always equal to or less than 10% of the total concen-
tration of the radioligand added. Nonspecific binding of
[
125IeSar1eIle8] ANG II binding was estimated in the presence of
1000 nM [Sar1eIle8] ANG II. Specific binding was defined as total
binding minus nonspecific binding [57]. Data analysis for compe-
tition binding was performed by nonlinear regression analysis,
using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). ꢀlog
IC50 values were obtained by fitting the data from competition
studies to a one-site competition model and presented as mean
plus/minus standard error (SE).
Acknowledgments
4.3.4. Rat uterotonic test in vitro
This research project was supported by the research program
PRAXE B-EPAN, General Secretariat of Research and Technology of
Greece, Ministry of Development and the pharmaceutical compa-
nies ELDRUG SA and VIANEX SA. We also acknowledge Dr Pan-
agiotis Zoumpoulakis (National Hellenic Research Foundation) and
Demetrios Vahliotis (Instrumental Analysis Laboratory, University
of Patras) for recording the NMR spectra.
The test was performed in the same way as described for
oxytocin and vasopressin [58e63] or bradykinin [64] analogues.
The excised and longitudinally cut strips of rat uterus were placed
into a bathing chamber into media without magnesium ions and
hooked up to recorder of contractions. The height of the single
isometric contraction of a uterine strip was measured. The cumu-
lative doseeresponse curves of standard ANG II were constructed,
i.e. doses of standard (in the presence or absence of analogues)
were added successively to the uterus in the organ bath in doubling
concentrations and at 1 min intervals without the fluid being
changed until the maximal response (the highest contraction) was
obtained. The shift of the curves in the presence of the compounds
was determined. The concentration of the compounds leading to
the shift corresponding to 0.3 in logarithmic scale (it means that
twice higher concentration of the standard was necessary to reach
the half maximal effect) was determined. Negative logarithm on
the base of 10 of that concentration is pA2. All samples were dis-
solved in DMSO to make stock solution of 1e4 mg/mL. Further
dilutions were made in physiol. solution. Standard ANG II, oxytocin
or bradykinin were dissolved in physiol. solution. Each analogue
was tested using uteri from 3 to 5 different animals; the values in
the Table 1 are averages ꢂ SEM. Specificity of the effect was tested
using construction of bradykinin and oxytocin doseeresponse
curves in the presence of the analogues.
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