Facile and efficient syntheses of a series of N-benzyl and N-biphenylmethyl substituted imidazole derivatives based on (E)-urocanic acid, as angiotensin II AT1 receptor blockers

Article abstract of DOI:10.3390/molecules18077510

In the present work, a facile and efficient route for the synthesis of a series of N-substituted imidazole derivatives is described. Docking studies have revealed that N-substituted imidazole derivatives based on (E)-urocanic acid may be potential antihypertensive leads. Therefore, new AT1 receptor blockers bearing either the benzyl or the biphenylmethyl moiety at the N-1 or N-3 position, either the (E)-acrylate or the propanoate fragment and their related acids at the C-4 position as well as a halogen atom at the C-5 position of the imidazole ring, were synthesized. The newly synthesized analogues were evaluated for binding to human AT1 receptor. The biological results showed that this class of molecules possesses moderate or no activity, thus not always confirming high docking scores. Nonetheless, important conclusions can be derived for their molecular basis of their mode of action and help medicinal chemists to design and synthesize more potent ones. An aliphatic group as in losartan seems to be important for enhancing binding affinity and activity.

Full text of DOI:10.3390/molecules18077510

Molecules 2013, 18, 7510-7532; doi:10.3390/molecules18077510
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Facile and Efficient Syntheses of a Series of N-Benzyl and
N-Biphenylmethyl Substituted Imidazole Derivatives Based on
(
E)-Urocanic acid, as Angiotensin II AT1 Receptor Blockers
1
,2,
1,2
1,2
3
George Agelis *, Konstantinos Kelaidonis , Amalia Resvani , Dimitra Kalavrizioti ,
1
,2
2,3
4
Maria-Eleni Androutsou , Panagiotis Plotas , Demetrios Vlahakos ,
5
1
5
Catherine Koukoulitsa , Theodore Tselios , Thomas Mavromoustakos and
1
,2,
John Matsoukas
*
1
Department of Chemistry, University of Patras, Patras 26500, Greece
Eldrug S.A., Patras Science Park, Patras 26504, Greece
2
3
4
5
Department of Pharmacology, School of Medicine, University of Patras, Patras 26500, Greece
Department of Internal Medicine, ‘ATTIKON’ University Hospital, Athens 12462, Greece
Department of Chemistry, University of Athens, Athens 15771, Greece
*
Authors to whom correspondence should be addressed:
E-Mails: aggelisgeorge@hotmail.com (G.A.); imats@upatras.gr (J.M.)
Received: 24 April 2013; in revised form: 17 June 2013 / Accepted: 20 June 2013 /
Published: 27 June 2013
Abstract: In the present work, a facile and efficient route for the synthesis of a series of
N-substituted imidazole derivatives is described. Docking studies have revealed that
N-substituted imidazole derivatives based on (E)-urocanic acid may be potential
antihypertensive leads. Therefore, new AT1 receptor blockers bearing either the benzyl or
the biphenylmethyl moiety at the N-1 or N-3 position, either the (E)-acrylate or the
propanoate fragment and their related acids at the C-4 position as well as a halogen atom at
the C-5 position of the imidazole ring, were synthesized. The newly synthesized analogues
were evaluated for binding to human AT1 receptor. The biological results showed that this
class of molecules possesses moderate or no activity, thus not always confirming high
docking scores. Nonetheless, important conclusions can be derived for their molecular
basis of their mode of action and help medicinal chemists to design and synthesize more
potent ones. An aliphatic group as in losartan seems to be important for enhancing binding
affinity and activity.

Molecules 2013, 18
7511
Keywords: synthesis; AT1 receptor blockers; (E)-urocanic acid; N-alkylation;
docking studies
1
. Introduction
Angiotensin II (ANG II) is the octapeptide produced by the Renin-Angiotensin System (RAS)
which plays a key role in the pathophysiology of hypertension [1–5]. Inhibitors of the three active sites
of RAS have proven to be effective for the treatment of hypertension and congestive heart failure.
Research efforts over the last decades have focused on the development of highly selective ANG II
AT1 receptor blockers (ARBs) [6]. The DuPont group was the first to develop losartan (DuP 753), an
orally effective angiotensin receptor blocker, which is metabolized in vivo to the more potent full
antagonist EXP 3174 [7,8]. The discovery of losartan has stimulated the design of a large number of
congeners [4,5]. Among them, eprosartan, irbesartan, candesartan, valsartan, olmesartan and azilsartan
have been launched in the market [6,9–11]. Extensive Structure-Activity Relationships (SAR) and
pharmacophore modeling studies [12] of ARBs, as well as the available data from literature, have
illustrated the key elements required for the design of potent AT1 blockers [1]. Lipophilic substituents,
such as the biphenylmethyl fragment substituted with an acidic moiety (tetrazole group, CO H) at the
2
N-1 position of a heterocyclic ring and a linear alkyl group, providing an interaction with a
hydrophobic pocket of the receptor, are required for potent antagonistic activity [6,13–17]. The
DuPont group recommended a lipophilic and electron-withdrawing group such as a halogen atom, CF
ethyl or pentafluoroethyl substituents at the C-4 of the imidazole ring and a small sized group such as
CH OH or CO H at the C-5 capable of forming a hydrogen bond [6,9,18].
3
,
2
2
Our recent work on the synthesis of AT1 receptor antagonists [18,19] indicated that
(5)-butylimidazole-based analogues displayed significant antihypertensive activity. As a continuation
4
of our studies [18–20], we report herein on the preparation of (E)-urocanic acid-based analogues,
focusing our attention on the structural modifications on the imidazole ring which would possibly
enhance potency. Consequently, we have designed using docking studies and synthesized a series of
(
E)-urocanic acid derivatives bearing the benzyl and the biphenylmethyl tetrazole moiety at the N-1 or
N-3 position of the imidazole ring. Furthermore, these analogues bear the (E)-acrylic acid chain of
E)-urocanic acid as well as the corresponding saturated side chain at the C-4, mimicking the carboxy-
(
terminal region of the octapeptide ANG II [21,22] and a bulky lipophilic and electron-withdrawing
group such as a halogen atom at the C-5 of the imidazole ring [6,9]. Additionally, some ARBs
possessing two acidic groups, such as the tetrazole and the carboxyl group, have exhibited low oral
bioavailability (BA) because of their highly polar character [6]. Thus, the rigid acrylic or the saturated
acid side chain was masked by esterification resulting in the methyl ester or the bulky ester group
(
5-methyl-2-oxo-1,3-dioxol-4-yl)methyl or medoxomil [5,9], which is metabolized in vivo to the
carboxyl moiety and may prove to be an effective structural element, emerging to compounds with
improved BA. Finally, the reason for the shortening of the biphenyl group was to evaluate the ability
of the tetrazole group to trigger activity located on a phenyl group instead of a biphenyl moiety and on
the other hand to examine the ability of a single phenyl group to interact appropriately with the

Molecules 2013, 18
7512
receptor [23]. Our synthetic approach included fast, efficient and regioselective reactions in high
yields, allowing the facile introduction of the substituents on the imidazole nucleus. The synthesized
analogues were finally tested for their AT1 receptor affinity using binding assays.
2
. Results and Discussion
2
.1. Chemistry
The intermediates 2 and 4 that were used to introduce the benzyl and the biphenylmethyl moiety to
the imidazole ring were obtained according to reported methods as outlined in Scheme 1 [8,18,24,25].
In this case, protection of the tetrazole ring with the 2-chlorotrityl group by treatment with
2
-chlorotrityl chloride (ClTr-Cl), followed by benzylic bromination provided the requisite alkylating
agents 2 and 4 [18].
Scheme 1. Synthesis of the alkylating agents 2 and 4 [8,18,24,25].
The preparation of N-benzyl imidazole derivatives 10–12 and 14 is depicted in Scheme 2.
(
E)-Urocanic acid (5) was converted to the corresponding methyl ester 6 by esterification in dry
1
methanol (MeOH) [20,26]. The H-NMR spectrum of 6 showed two singlet peaks at δ 7.78 and 7.43
ppm corresponding to the H-2 and H-5 of the imidazole ring, respectively. Additionally, the two
vinylic protons appeared as doublets at δ 6.44 and 7.61 (J = 16.0 Hz), respectively and the methoxy
group at 3.78 ppm. Alkylation of the methyl ester 6 at the N-1 position of the imidazole ring with the
benzyl alkylating agent 2, in the presence of sodium hydride (NaH) in dry N,N-dimethylformamide
1
(
DMF), afforded 7 in 74% yield. The H-NMR spectrum of 7 showed a singlet peak at δ 5.38 assigned
to the methylene protons. Catalytic hydrogenation (Pd/C) of the latter afforded the saturated derivative
1
8
in excellent yield (90%). The H-NMR spectra of 8 showed two triplet peaks at δ 2.76 and 2.56
(J = 7.2 Hz) corresponding to the methylene protons of the saturated side chain. Alkaline hydrolysis [9]
of the methyl esters 7 and 8 under mild conditions using a mixture of KOH in 1:1 H O/dioxane, led to
2
the corresponding acids 13 and 9, respectively. Removal of the ClTr group by treatment with 30%
trifluoroacetic acid (TFA) in dichloromethane (CH Cl ), in the presence of triethylsilane (Et SiH) as
2
2
3
scavenger, provided the final analogues 10–12 and 14.

Molecules 2013, 18
7513
a
Scheme 2. Synthesis of the target N-benzyl analogues 10, 11, 12, 14 .
COOH
N
N
N
N
COOCH3
HN
N
N
N
N
N
HN
14
v
N
COOH
12
N
N
N
N
TrCl
N
N
v
13
iv
COOCH3
COOH
COOCH3
N
N
N
N
ii
TrCl
N
i
HN
N
HN
N
N
5
6
7
iii
COOH
COOCH3
N
N
N
N
N
TrCl
N
N
N
N
TrCl
N
iv
N
N
8
9
v
v
COOH
COOCH3
N
N
N
N
N
N
N
N
HN
HN
N
N
1
1
10
a
2 4 2 4
Reagents and conditions: (i) MeOH, anhydrous Na SO , conc. H SO , reflux, 24 h; (ii) 2, NaH
(
powdered 95%), dry DMF, 0 °C to rt, 4 h; (iii) H , 10% Pd-C, MeOH, rt, 3 h; (iv) KOH,
2
H O/dioxane (1:1), rt, 3 h; (v) 30% TFA in CH Cl , Et SiH, rt, 1 h.
2
2
2
3
The synthesis of the N-biphenylmethyl imidazole derivatives 18–20a–c, 22a–b, 24a–b and 26a–f is
demonstrated in Scheme 3. Likewise, direct alkylation of the methyl ester 6 at the N-1 position of the
imidazole ring with the biphenylmethyl alkylating agent 4 afforded the 1,4-disubstituted analogue 15.
1
The H-NMR data of 15 showed a singlet peak at δ 4.95 due to the methylene protons of the alkylating
moiety. Subsequently, hydrogenation of 15 in the presence of 10% Pd-C as catalyst in MeOH, led to
the intermediate 16. Halogenation of 16 at the C-5 position of the imidazole ring with the appropriate
N-halosuccinimide (NXS, X = Cl, Br, I) [18], afforded the halogenated derivatives 17a–c. The
1
H-NMR spectra showed the absence of the H-5 signal of the imidazole ring at 6.47 ppm appearing in
1
6. Saponification of the methyl esters 15–17a–c was mediated by an aqueous solution of KOH in
dioxane for 3 h at rt, to afford the corresponding acids 21a, 23a, 25a, 25c and 25e, respectively. The
1
H-NMR spectra confirmed the absence of the methoxy group at 3.56–3.77 ppm. Treatment of the
latter acids with medoxomil chloride (4-chloromethyl-5-methyl-2-oxo-1,3-dioxole) in the presence of

Molecules 2013, 18
7514
potassium carbonate (K CO ) in dry N,N-dimethylacetamide (DMA), [9] furnished the esters 21b, 23b,
2
3
2
2
5b, 25d and 25f. The presence of the -OCH protons signal at 5.02–4.71 ppm as well as the methyl
protons signal at 2.02–2.16, unequivocally confirmed the introduction of the medoxomil group.
Detritylation of the tetrazole group was accomplished by treatment with TFA in CH Cl , resulting in
2
2
the target compounds 18–20a–c, 22a–b, 24a–b and 26a–f.
Scheme 3. Synthesis of the final N-biphenylmethyl analogues 18, 19, 20a–c, 22a–b,
a
2
4a–b, 26a–f .
a
2
Reagents and conditions: (i) 4, NaH (powdered 95%), dry DMF, 0 °C to rt, 4 h; (ii) H , 10%
Pd-C, MeOH, rt, 3 h; (iii) NXS, X = Br, I in dry DMF or NCS in MeCN, 0 °C to rt, 4 h; (iv) 30%
TFA in CH Cl , Et SiH, rt, 1 h; (v) KOH, H O/dioxane (1:1), rt, 3 h; (vi) K CO , dry DMA,
2
2
3
2
2
3
4
-chloromethyl-5-methyl-2-oxo-1,3-dioxole, rt, 4 h.

Molecules 2013, 18
7515
Finally, the preparation of the N-biphenylmethyl imidazole derivatives 30 and 32 is depicted in
Scheme 4. Firstly, the imidazole ring was protected at the N-1 by the 2-(trimethylsilyl)ethoxymethyl
(
SEM) group using standard conditions [18,27,28]. Thus, treatment of the unsaturated methyl ester 6
with SEM-Cl in the presence of NaH in dry DMF, at ambient temperature for 2 h, led to 27 in 78%
yield. It is worth noting, that using the latter reaction conditions only the desired 1,4-regioisomer was
1
formed, as indicated by HPLC and H-NMR.
a
Scheme 4. Synthesis of the final N-biphenylmethyl substituted analogues 30 and 32 .
a
Reagents and conditions: (i) SEM-Cl, NaH (powdered 95%), dry DMF, 0 °C to rt, 2 h; (ii) H
2
,
1
0% Pd-C, MeOH, rt, 3 h; (iii) 4, CH Cl , reflux, 3 h; (iv) 30% TFA in CH Cl , Et SiH, rt, 1 h;
2
2
2
2
3
(
v) 37% formalin, diisopropylethylamine, DMF, 85 °C, 1 h.
The resulting derivative 27 was subjected to hydrogenation in the presence of catalyst 10% Pd-C in
MeOH to afford 28, in 91% yield. Regioselective alkylation at the N-3 position was performed in the
2 2
presence of the alkylating reagent 4 in CH Cl under reflux for 3 h, resulting in the intermediate salt 29
in high yield (81%). Thus, the SEM group was proven to be an excellent choice for the protection of
the N-1 followed by regioselective alkylation at the N-3 of the imidazole ring. 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 analogue 29. According to our strategy [18], the hydroxymethylation was promptly carried
out in a sealed tube by treatment with diisopropylethylamine and 37% formalin in DMF at 85 °C
for 1 h. The obtained residue was purified by column chromatography to afford the hydroxymethylated

Molecules 2013, 18
7516
1
product 31 in excellent yield (91%) and purity. The H-NMR spectrum of 31 showed the presence of a
singlet peak at 4.72 ppm due to the hydroxymethyl protons. Removal of the ClTr group by means of
3
0% TFA in CH
.2. Pharmacology
The new synthesized analogues were evaluated in radioligand binding assay at a final concentration
2 2 3
Cl and Et SiH led to the 1,5-disubstituted imidazole analogues 30 and 32.
2
−5
of 10 M. Although the used concentration was high enough, there were indications for moderate
activity of the analogues 12 and 18. Competitive binding experiments revealed that the latter analogues
1
25
1
8
caused 40.1% and 59.4% displacement of [ I]-Sar -Ile -ANG II from the AT1 receptor, respectively,
whereas losartan at the same conditions caused 100% displacement.
2
.3. Docking Studies
The synthesized analogues have been rationalized based on their highest docking scores (Table 1).
We notice that some of these analogues show higher scoring than losartan as reported in our previous
paper [18].
a
Table 1. Highest Docking Scores of the synthesized analogues obtained with GLIDE/IFD .
Compounds
Docking Score
−12.114
−9.625
Losartan
1
1
1
1
0
2
8
9
−10.276
−12.089
−11.395
−10.397
−11.099
−11.899
−11.577
−14.401
−12.321
−13.570
−10.577
−13.463
−9.404
2
0a
0b
2
2
0c
2a
2
22b
24a
24b
26a
26b
2
6c
6d
6e
6f
2
−13.047
−13.168
−12.017
−12.281
−11.012
2
2
3
0
2
3
a
Induced Fit Docking/XP module.
However, high scores could not rationalize the pharmacological results which showed that most of
the synthesized analogues were inactive and only few of them showed moderate activity. In order to
comprehend the pharmacological data, we have used as a template for comparison the putative

Molecules 2013, 18
7517
bioactive conformation of losartan in the AT1 receptor presented by the pose of Figure 1a.
Interestingly, the inactive compounds adopted losartan’s orientation with poses of low scoring. The
highest scoring orientations differed from that of losartan (Figure 1). As a result of this, the inactive
compounds, even though docked in the same cavity, exerted different critical interactions that explain
their inability to possess pharmacological activity. This is also applied with analogues 12 and 18 that
1
25
1
8
showed 40.1% and 59.4% displacement of [ I]-Sar -Ile -ANG II from the AT1 receptor (Figure 2).
Figure 1. Ligand interactions of a losartan; b and c analogue 12; d and e analogue 18 with
the aminoacids of the active site of AT1; (b and d represent orientations of analogues 12 and
18 with the highest scoring and c and e. represent orientations with low scoring).
a.
c.
b.

Molecules 2013, 18
7518
Figure 1. Cont.
e.
d.
As it is shown in Figure 1b,d the poses that showed the highest scoring (12, −10.276 kcal/mol and
8, −12.089 kcal/mol), adopted orientations that did not match that of losartan. For example, analogue
8 is lacking the hydrogen bonding with Lys199 and both 12 and 18 are lacking the hydrogen bonding
1
1
with His183, Gln257 and Tyr113. However, the poses that resembled the orientation of losartan
Figure 1c,e) showed low scorings (12, −8.779 kcal/mol and 18, −5.217 kcal/mol). This is attributed to
(
the fact that both 12 and 18 cannot adopt the maximal critical interactions. For example, 18 forms only
two hydrogen bondings (losartan forms four) and 12 only one. It appears that docking experiments
could shed light on the required molecular interactions for drug activity only when pharmacological
data were obtained.
Figure 2. a. Superimposition of losartan (green), with low scoring orientations of
analogues 12 (orange) and 18 (yellow). b. with the highest scoring orientations.

Molecules 2013, 18
7519
3. Experimental
3.1. General
Starting materials were purchased by Aldrich (Patras, Greece) and used as received. Hydrogenation
reaction was carried out in a Parr hydrogenation apparatus equipped with a 4 L hydrogen tank. The
1
13
H-NMR and C-NMR spectra were recorded on a Bruker Avance DPX spectrometer at 400.13 MHz
and 161.76 MHz, respectively. Chemical shifts are given in δ values (ppm) using tetramethylsilane as
the internal standard and coupling constants (J) are given in Hertz (Hz). HPLC analysis was performed
on an Alliance Waters 2695 equipped with a Waters 2996 Photodiode Array Detector UV-Vis, using
the XBridge Waters C18 column (4.6 × 150 mm, 3.5 μm) as stationary phase and a gradient of
H O/MeCN both containing 0.08% TFA as mobile phase. Electrospray-ionization mass spectra
2
(
ESI-MS) were obtained on a UPLC (ultra performance liquid chromatography) equipped with SQ
TM
detector Acquity by Waters. All reactions were carried out in anhydrous solvents. Analytical TLC
was performed on silica gel 60 F254 plates (Merck, Germany) and visualized by UV irradiation and
iodine. Silica gel 60N (particle size 0.04–0.063 mm) was used for column chomatography.
3.2. Synthesis
3
.2.1. General Procedure 1: Alkylation of the (E)-urocanic Methyl Esters at the Ν-1 Position
To a solution of 6 (2.0 g, 13.16 mmol) in dry DMF (25 mL), dry ΝaH (powdered 95%, 0.35 g,
1
4.47 mmol) was added and the resulting suspension was stirred for 30 min at 0 °C under nitrogen
Then, 4 (8.56 g, 14.48 mmol) was added in two portions and the mixture was stirred for 4 h at RT. The
mixture was diluted in H O, extracted with CH Cl and the organic phase was washed successively
with 5% w/v citric acid, brine, dried over Na SO and concentrated. The residue was purified by flash
column chromatography (7:3 EtOAc:hexanes) to afford 15.
2
2
2
2
4
3
.2.2. General Procedure 2: Catalytic Hydrogenation of the Ν-1 Alkylated (E)-Urocanic methyl Esters
A mixture of 15 (5.0 g, 7.54 mmol), 10% w/w Pd-C (0.50 g) in MeOH (20.0 mL) was stirred under
a hydrogen atmosphere (3 bar) at ambient temperature for 3 h. The catalyst was filtered off by a Celite
pad and the filtrate was concentrated in vacuo. The residue was purified by flash column
chromatography (EtOAc) to provide 16.
3
.2.3. General Procedure 3: Halogenation of the Ν-1 Alkylated Imidazole Derivatives
To a solution of 16 (1.0 g, 1.5 mmol) in dry DMF (5.0 mL) at 0 °C under nitrogen, NBS
(
0.29 g, 1.65 mmol) was added in three portions and the mixture was allowed to cool at room
temperature. After 4 h, the solvent was removed and the residue was purified by flash column
chromatography (8:2, EtOAc:hexanes) to provide 17b.

Molecules 2013, 18
.2.4. General Procedure 4: Alkaline Hydrolysis of the Ν-Substituted Imidazole Methyl Esters
To a solution of 16 (2.50 g, 3.75 mmol) in H O/dioxane (10.0 mL, 1:1) was added fine powdered
7520
3
2
ΚΟΗ (2.10 g, 37.50 mmol) and the resulting mixture was stirred at ambient temperature for 3 h. The
dioxane was removed by distillation in vacuo and to the residual solution was added 1 N HCl to give a
white precipitate 23a which was collected by vacuum filtration.
3
.2.5. General Procedure 5: Esterification of the Ν-Substituted Imidazole Carboxylic Acids with the
(
5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl or Medoxomil Group
2 3
To a solution of 23a (2.0 g, 3.07 mmol) in dry DMA (5.0 mL) under nitrogen was added K CO
(0.88 g, 6.41 mmol) and the mixture was stirred at ambient temperature for 30 min. A solution
of 4-chloromethyl-5-methyl-2-oxo-1,3-dioxole (0.67 g, 4.56 mmol) in dry DMA was added dropwise
and the resulting mixture was stirred for 4 h. Then, the mixture was diluted with EtOAc and the
organic phase was washed with H O, brine, dried over Na SO and concentrated in vacuo. The crude
2
2
4
product was purified by flash column chromatography (8:2 EtOAc:hexanes) to afford 23b.
.2.6. General Procedure 6: Removal of the 2-Chlorotrityl Protecting Group
To a solution of 16 (0.50 g, 0.75 mmol) in 30% TFA in CH Cl (2.0 mL), TES (0.10 mL, 0.63 mmol)
3
2
2
was added dropwise and the resulting solution was stirred for 1 h at ambient temperature. The reaction
mixture was concentrated and recrystallized from diethyl ether to afford 19.
3
.2.7. Synthesis of 1,4-Disubstituted Benzyl Analogues 10, 11, 12, 14
N-(2-Chlorotrityl)-5-(2-bromobenzyl)tetrazole (2). Prepared from 1 according to the literature method
+
[
(
8,18,24,25]. Yield 78%; R
f
= 0.46 (2:8 EtOAc:hexanes); ESI-MS (m/z): 238.27 (M+H -ClTr), 277.78
1
ClTr); H-NMR (CDCl ): δ 8.22–8.18 (m, 1H), 7.51–7.32 (m, 12H), 7.25–7.21 (m, 4H), 6.86 (d, 1H,
3
1
3
J = 7.6 Hz), 4.92 (s, 2H); C-NMR (CDCl
3
): δ 162.90, 141.28, 136.87, 131.64, 130.46, 129.96,
28.94, 128.42, 127.96, 127.84, 127.29, 126.43, 83.45, 32.42. Anal. Calcd for C27 ClBr (%): C:
2.87; H: 3.91; N: 10.86. Found (%): C: 62.99; H: 4.00; N: 10.54.
1
6
20 4
H N
Methyl 3-(1H-imidazol-4-yl)acrylate (6). Prepared from 5 according to the literature method [20,26].
+
1
Yield 95%; M.p. 92–94 °C; R = 0.42 (9:1 CHCl :MeOH); ESI-MS (m/z): 153.22 (M+H ); Η-ΝΜR
f
3
(
3
CD OD): δ 7.78 (s, 1H), 7.61 (d, 1Η, J = 16.0 Hz), 7.43 (s, 1H), 6.44 (d, 1H, J = 16.0 Hz), 3.78 (s,
1
3
3
H); C-NMR (CDCl
(%): C: 55.26; H: 5.30; N: 18.41. Found (%): C: 55.21; H: 5.39; N: 18.37. All data were
consistent with literature [26].
3
): δ 168.14, 137.43, 135.25, 134.35, 124.0, 114.35, 50.61. Anal. Calcd for
7 8 2 2
C H N O
(
E)-Methyl 1-[[1-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]phenyl-2-yl]methyl]imidazole-4-acrylate (7).
General procedure 1 was employed for the preparation of 7 using 2 as alkylating agent. Yield 74%;
+
+
R
f
= 0.38 (8:2 EtOAc:hexanes); ESI-MS (m/z): 587.27 (M+H ), 309.37 (M+H -ClTr), 277.88 (ClTr);
): δ 8.28 (dd, 1H, J = 2.0, 7.6 Hz), 7.53–7.10 (m, 19H), 6.76 (s, 1H), 6.48 (d, 1H,
1
Η-ΝΜR (CDCl
3

Molecules 2013, 18
7521
J = 15.6 Hz), 5.38 (s, 2H), 3.77 (s, 3H). Anal. Calcd for C H N O Cl (%): C: 74.02; H: 4.30; N:
3
4
27
6
2
1
3.28. Found (%): C: 73.96; H: 4.22; N: 13.32.
Methyl 1-[[1-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]phenyl-2-yl]methyl]imidazole-4-propanoate (8).
General procedure 2 was employed for the preparation of 8. Yield 90%; R = 0.45 (9:1 CHCl :MeOH);
f
3
+
+
1
ESI-MS (m/z): 589.17 (M+H ), 311.45 (M+H -ClTr), 277.38 (ClTr); Η-ΝΜR (CDCl
3
): δ 8.18–8.15
(
m, 1H), 7.42–7.26 (m, 9H), 7.07–6.99 (m, 9H), 6.37 (s, 1H), 5.27 (s, 2H), 3.57 (s, 3H), 2.76 (t, 2H,
J = 7.2 Hz), 2.56 (t, 2H, J = 7.2 Hz). Anal. Calcd for C H N O Cl (%): C: 69.32; H: 4.96; N: 14.27.
3
4
29
6
2
Found (%): C: 69.45; H: 4.88; N: 14.32.
1-[[1-[[N-(2-Chlorotrityl)]-1H-tetrazol-5-yl]phenyl-2-yl]methyl]imidazole-4-propanoic acid (9).
General procedure 4 was employed for the preparation of 9. Yield 93%; R = 0.31 (8.5:1.5
f
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 576.19 (M+H ), 299.63 (M+H -ClTr), 277.77 (ClTr); Η-ΝΜR
(
CDCl ): δ 8.31 (d, 1Η, J = 7.2 Hz), 7.54–7.11 (m, 17H), 6.85 (d, 1H, J = 7.6 Hz), 6.42 (s, 1H), 5.40
3
(
s, 2H), 2.73 (bs, 2H), 2.65 (bs, 2H). Anal. Calcd for C H N O (%): C: 68.92; H: 4.73; N: 14.61.
33 27 6 2
Found (%): C: 68.92; H: 4.73; N: 14.61.
Methyl 1-[[1-(1H-tetrazol-5-yl)phenyl-2-yl]methyl]imidazole-4-propanoate (10). General procedure 6
was employed for the preparation of 10. Yield 91%; R = 0.48 (8:2 CHCl :MeOH); ESI-MS (m/z):
OD): δ 8.80 (s, 1H), 7.96-7.93 (m, 1H), 7.64–7.60 (m, 3H), 7.33 (s,
Η), 5.75 (s, 2H), 3.65 (s, 3H), 2.94 (t, 2H, J = 7.2 Hz), 2.69 (t, 2H, J = 7.2 Hz). Anal. Calcd for
·CF COOH (%): C: 47.89; H: 4.02; N: 19.71. Found (%): C: 47.78; H: 3.95; N: 19.87.
f
3
+
1
3
13.34 (M+H ); Η-ΝΜR (CD
3
1
C
15
H
16
N
6
O
2
3
1
-[[1-(1H-Tetrazol-5-yl)phenyl-2-yl]methyl]imidazole-4-propanoic acid (11). General procedure 6
was employed for the preparation of 11. Yield 88%; R = 0.37 (4:1:1 n-butanol:acetic acid:H O);
OD): δ 8.83 (s, 1H), 7.97–7.93 (m, 1H), 7.65–7.59 (m,
H), 7.36 (s, 1H), 5.76 (s, 2H), 2.93 (t, 2H, J = 7.2 Hz), 2.66 (t, 2H, J = 7.2 Hz). Anal. Calcd for
·CF COOH (%): C: 46.11; H: 3.67; N: 20.38. Found (%): C: 46.02; H: 3.57; N: 20.48.
f
2
+
1
ESI-MS (m/z): 297.24 (M+H ); Η-ΝΜR (CD
3
3
C
14
H
14
N
6
O
2
3
(
E)-Methyl 1-[[1-(1H-tetrazol-5-yl)phenyl-2-yl]methyl]imidazole-4-acrylate (12). General procedure 6
was employed for the preparation of 12. Yield 95%; R = 0.48 (8:2 CHCl :MeOH); ESI-MS (m/z):
11.24 (M+H ); Η-ΝΜR (DMSO-d ): δ 8.46 (s, 1H), 7.90 (s, 1H), 7.79 (s, 1H), 7.62 (bs, 2H), 7.58
f
3
+
1
3
6
(
m, 4Η), 7.50 (d, 1H, J = 15.6 Hz), 7.34 (s, 1H), 6.46 (d, 1H, J = 15.6 Hz), 5.68 (s, 2H), 3.78 (s, 3H).
Anal. Calcd for C15 ·CF COOH (%): C: 48.12; H: 3.56; N: 19.81. Found (%): C: 48.22; H:
.47; N: 19.71.
H N
14 6
O
2
3
3
(
E)-1-[[1-[[N-(2-Chlorotrityl)]-1H-tetrazol-5-yl]phenyl-2-yl]methyl]imidazole-4-acrylic acid (13).
General procedure 4 was employed for the preparation of 13. Yield 95%; R
f
= 0.30 (9:1
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 574.15 (M+H ), 297.54 (M+H -ClTr), 277.78 (ClTr); Η-ΝΜR
(CDCl ): δ 8.28 (d, 2Η, J = 5.6 Hz), 7.52–7.23 (m, 12H), 7.10 (d, 6H, J = 5.6 Hz), 6.74 (bs, 1H), 6.44
3
25 6 2
(d, 1H, J = 15.6 Hz), 5.36 (s, 2H). Anal. Calcd for C33H N O Cl (%): C: 69.17; H: 4.40; N: 14.67.
Found (%): C: 69.11; H: 4.29; N: 14.72.

Molecules 2013, 18
7522
(
E)-1-[[1-(1H-Tetrazol-5-yl)phenyl-2-yl]methyl]imidazole-4-acrylic acid (14). General procedure 6
was employed for the preparation of 14. Yield 95%; R = 0.50 (4:1:1 n-butanol:acetic acid:H O);
f
2
+
1
ESI-MS (m/z): 297.30 (M+H ); Η-ΝΜR (CD
.72–7.64 (m, 3H), 7.48 (d, 1H, J = 16.0 Hz), 6.49 (d, 1H, J = 16.0 Hz), 5.80 (s, 2H). Anal. Calcd for
C H N O ·CF COOH (%): C: 48.12; H: 3.56; N: 19.81. Found (%): C: 48.22; H: 3.47; N: 19.71.
3
OD): δ 8.85 (s, 1H), 7.98–7.95 (m, 1H), 7.84 (s, 1H),
7
1
4
12
6
2
3
3
.2.8. Synthesis of 1,4-Disubstituted biphenylmethyl analogues 18, 19, 20a–c, 22a–b, 24a–b, 26a–f
Ν-(2-Chlorotrityl)-5-[4′-(bromomethyl)biphenyl-2-yl]tetrazole (4). Prepared from 3 according to the
literature method [8,19,25,26]. Yield 80%; M.p. 155–157 °C; R = 0.33 (1.5:8.5 EtOAc:hexanes);
f
+
1
3
ESI-MS (m/z): 315.48 (M+H -ClTr), 277.22 (ClTr); Η-ΝΜR (CDCl ): δ 8.02–7.89 (m, 1H),
1
3
7
1
1
.60–6.87 (m, 20H), 6.79 (dd, 1H, J = 1.5, 8.0 Hz), 4.43 (s, 2H); C-NMR (CDCl ): δ 163.95, 141.63,
3
41.47, 140.64, 139.49, 136.34, 132.13, 131.75, 130.79, 130.53, 130.15, 129.69, 128.87, 128.66,
24 4
27.97, 127.91, 126.68, 81.96, 33.45. Anal. Calcd for C33H Ν ClBr (%): C: 66.96; H: 4.09; N: 9.47.
Found (%): C: 66.88; H: 4.15; N: 9.42.
(E)-Methyl 1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-acrylate (15).
General procedure 1 was employed for the preparation of 15 using 4 as alkylating agent. Yield 73%;
+
+
R
f
= 0.50 (EtOAc); ESI-MS (m/z): 664.19 (M+H ), 387.29 (M+H -ClTr), 277.38 (ClTr);
): δ 8.28 (dd, 1H, J = 1.8, 7.6 Hz), 7.53–7.12 (m, 22H), 6.84 (m, 2H), 6.54 (d, 1H,
1
Η-ΝΜR (CDCl
3
1
3
J = 15.6 Hz), 4.95 (s, 2H), 3.77 (s, 3H); C-NMR (CDCl
3
): δ 168.20, 137.43, 162.87, 140.48, 139.14,
1
1
1
38.89, 138.22, 136.33, 134.05, 132.22, 131.84, 130.99, 130.54, 129.29, 128.86, 128.30, 126.97,
26.31, 122.18, 115.53, 82.66, 51.63, 49.53. Anal. Calcd for C40
2.67. Found (%): C: 72.38; H: 4.77; N: 12.62.
31 6 2
H N O Cl (%): C: 72.44; H: 4.71; N:
Methyl 1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-propanoate (16).
General procedure 2 was employed for the preparation of 16. Yield 88%; R
f
= 0.55 (9:1
+
+
1
CHCl :MeOH); ESI-MS (m/z): 666.19 (M+H ), 388.39 (M+H -ClTr), 277.77 (ClTr); Η-ΝΜR
3
(
CDCl
J = 1.2, 8.0 Hz), 6.47 (s, 1H), 4.87 (s, 2H), 3.65 (s, 3H), 2.84 (t, 2H, J = 7.6 Hz), 2.64 (t, 2H,
J = 7.6 Hz). Anal. Calcd for C40 Cl (%): C: 72.23; H: 5.00; N: 12.63. Found (%): C: 72.18; H:
3
): δ 7.98 (dd, 1H, J = 2.0, 7.6 Hz), 7.53–7.12 (m, 14H), 6.89–6.83 (m, 7H), 6.72 (dd, 1H,
33 6 2
H N O
5
.09; N: 12.57.
Methyl
5-chloro-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-
propanoate (17a). General procedure 3 was employed for the preparation of 17a using NCS in MeCN.
+
+
Yield 85%; R
f
= 0.49 (EtOAc); ESI-MS (m/z): 700.59 (M+H ), 422.86 (M+H -ClTr), 277.77 (ClTr);
): δ 7.97 (dd, 1Η, J = 1.2, 7.2 Hz), 7.52–7.14 (m, 15H), 6.90–6.84 (m, 6H), 6.72 (d,
H, J = 8.0 Hz), 4.89 (s, 2H), 3.68 (s, 3H), 2.87 (t, 2H, J = 7.6 Hz), 2.70 (t, 2H, J = 7.6 Hz).
C H Cl N O . Anal. Calcd for C H N O Cl (%): C: 68.67; H: 4.61; N: 12.01. Found (%): C:
1
Η-ΝΜR (CDCl
3
1
4
0
32
2
6
2
40 32
6
2
2
6
7.92; H: 4.78; N: 11.84.
Methyl
5-bromo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-
propanoate (17b). General procedure 3 was employed for the preparation of 17b using NBS in DMF.

Molecules 2013, 18
7523
+
+
Yield 68%; R = 0.47 (EtOAc); ESI-MS (m/z): 745.54 (M+H ), 467.80 (M+H -ClTr), 277.80 (ClTr);
f
1
Η-ΝΜR (CDCl
H, J = 1.6, 8.0 Hz), 4.88 (s, 2H), 3.56 (s, 3H), 2.73 (t, 2H, J = 7.6 Hz), 2.58 (t, 2H, J = 7.6 Hz). Anal.
Calcd for C H N O ClBr (%): C: 64.57; H: 4.33; N: 11.29. Found (%): C: 64.68; H: 4.40; N: 11.23.
3
): δ 7.84 (dd, 1H, J = 1.2, 7.2 Hz ), 7.46-7.24 (m, 8H), 7.18-6.79 (m, 13H), 6.68 (dd,
1
4
0
32
6
2
Methyl
5-iodo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-
propanoate (17c). General procedure 3 was employed for the preparation of 17c using NIS in DMF.
+
+
Yield 70%; R = 0.46 (EtOAc); ESI-MS (m/z): 792.48 (M+H ), 514.44 (M+H -ClTr), 277.76 (ClTr);
f
1
3
Η-ΝΜR (CD OD): δ 7.58 (d, 1H, J = 7.6 Hz), 7.49–7.23 (m, 13H), 7.16–7.00 (m, 8H), 6.83 (d, 1H,
J = 8.0 Hz), 5.15 (s, 2H), 3.57 (s, 3H), 2.78 (t, 2H, J = 7.6 Hz), 2.57 (t, 2H, J = 7.6 Hz). Anal. Calcd
for C H N O ClI (%): C: 60.73; H: 4.08; N: 10.62. Found (%): C: 60.66; H: 4.14; N: 10.56.
4
0
32
6
2
(
E)-Methyl 1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-acrylate (18). General procedure 6
was employed for the preparation of 18. Yield 94%; R = 0.51 (8.5:1.5 CHCl :MeOH); ESI-MS (m/z):
87.15 (M+H ); Η ΝΜR (400 MHz, DMSO-d ): δ 8.49 (s, 1H), 7.87 (s, 1H), 7.68–7.54 (m, 4H), 7.53
f
3
+
1
3
6
(
(
(
d, 1H, J = 15.6 Hz), 7.27 (d, 2H, J = 7.6 Hz), 7.13 (d, 2H, J = 8.0 Hz), 6.48 (d, 1H, J = 15.6 Hz), 5.32
s, 2H), 3.71 (s, 3H). Anal. Calcd for C21
%): C: 55.11; H: 3.72; N: 16.84.
18 6 2 3
H N O ·CF COOH (%): C: 55.20; H: 3.83; N: 16.79. Found
Methyl 1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoate (19). General procedure
was employed for the preparation of 19. Yield 89%; R = 0.49 (8.5:1.5 CHCl :MeOH); ESI-MS
OD): δ 7.91 (d, 1H, J = 7.5 Hz), 7.62-6.76 (m, 9H), 6.62 (s, 1H),
6
f
3
+
1
(
m/z): 389.13 (M+H ); Η-ΝΜR (CD
3
1
3
5
1
1
3
.04 (s, 2H), 3.64 (s, 3H), 2.76 (t, 2H, J = 7.2 Hz), 2.56 (t, 2H, J = 7.2 Hz); C-NMR (CD OD): δ
67.99, 163.90, 141.79, 141.32, 140.68, 139.38, 138.96, 137.53, 136.06, 135.08, 131.47, 130.72,
30.30, 129.66, 128.36, 127.80, 127.31, 126.52, 114.82, 50.85, 50.37, 35.75, 29.83. Anal. Calcd for
21 20 6 2 3
C H N O ·CF COOH (%): C: 54.98; H: 4.21; N: 16.73. Found (%): C: 54.86; H: 4.33; N: 16.61.
Methyl 5-chloro-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoate (20a). General
procedure 6 was employed for the preparation of 20a. Yield 95%; R = 0.53 (8.5:1.5 CHCl :MeOH);
OD): δ 8.30 (s, 1H), 7.71-7.60 (m, 2H), 7.23–7.18 (m,
H), 5.32 (s, 2H), 3.66 (s, 3H), 2.92 (t, 2H, J = 7.2 Hz), 2.70 (t, 2H, J = 7.2 Hz). Anal. Calcd for
C H N O Cl·CF COOH (%): C: 51.45; H: 3.75; N: 15.65. Found (%): C: 51.37; H: 3.83; N: 16.51.
f
3
+
1
ESI-MS (m/z): 423.92 (M+H ); Η-ΝΜR (CD
3
4
2
1
19
6
2
3
Methyl 5-bromo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoate (20b). General
procedure 6 was employed for the preparation of 20b. Yield 93%; R = 0.52 (8.5:1.5 CHCl :MeOH);
OD): δ 8.80 (s, 1H), 7.70–7.56 (m, 4H), 7.26–7.18 (m,
H), 5.40 (s, 2H), 3.66 (s, 3H), 2.97 (t, 2H, J = 7.2 Hz), 2.73 (t, 2H, J = 7.2 Hz). Anal. Calcd for
Br·CF COOH (%): C: 47.52; H: 3.47; N: 14.46. Found (%): C: 47.63; H: 3.36; N: 14.55.
f
3
+
1
ESI-MS (m/z): 468.39 (M+H ); Η-ΝΜR (CD
3
4
C
21
H
19
N
6
O
2
3
Methyl 5-iodo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoate (20c). General
procedure 6 was employed for the preparation of 20c. Yield 95%; R = 0.53 (8.5:1.5 CHCl :MeOH);
OD): δ 8.37 (s, 1H), 7.62–7.51 (m, 4H), 7.09 (s, 4H),
f
3
+
1
ESI-MS (m/z): 515.38 (M+H ); Η-ΝΜR (CD
3

Molecules 2013, 18
7524
5
.23 (s, 2H), 3.58 (s, 3H), 2.84 (t, 2H, J = 7.2 Hz), 2.60 (t, 2H, J = 7.2 Hz). Anal. Calcd for
C H N O I·CF COOH (%): C: 43.96; H: 3.21; N: 13.37. Found (%): C: 43.87; H: 3.34; N: 13.44.
2
1
19
6
2
3
(E)-1-[[2′-[[N-(2-Chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-acrylic acid (21a).
General procedure 4 was employed for the preparation of 21a. Yield 90%; R = 0.38 (9:1
f
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 650.17 (M+H ), 372.80 (M+H -ClTr), 277.76 (ClTr); Η-ΝΜR
DMSO-d ): δ 7.81 (d, 1H, J = 7.6 Hz), 7.62–7.49 (m, 4H), 7.45–7.30 (m, 9H), 7.29 (d, 1H, J = 15.6 Hz),
.11–7.06 (m, 4H), 6.78 (d, 4H, J = 7.6 Hz), 6.68 (d, 1Η, J = 8.0 Hz), 6.44 (d, 1H, J = 15.6 Hz), 5.12
(
6
7
(
s, 2H). Anal. Calcd for C H N O Cl (%): C: 72.16; H: 4.50; N: 12.95. Found (%): C: 72.09; H:
39 29 6 2
4
.58; N: 12.89.
(
E)-(5-Methyl-2-oxo-1,3-dioxol)methyl-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]-
methyl]imidazole-4-acrylate (21b). General procedure 5 was employed for the preparation of 21b.
+
+
Yield 84%; R = 0.52 (EtOAc); ESI-MS (m/z): 762.165 (M+H ), 484.44 (M+H -ClTr), 277.76 (ClTr);
f
1
Η-ΝΜR (DMSO-d
.67 (s, 1H), 6.28 (d, 1H, J = 16.0 Hz), 5.12 (s, 2H), 5.02 (s, 2H), 2.16 (s, 3H). Anal. Calcd for
Cl (%): C: 69.42; H: 4.37; N: 11.04. Found (%): C: 69.39; H: 4.32; N: 10.98.
6
): δ 7.80 (s, 2H), 7.60–7.42 (m, 7H), 7.34–7.27 (m, 7H), 7.09 (s, 4H), 6.76 (s, 4H),
6
44 33 6 5
C H N O
(
E)-1-[[2′-(1H-Tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-acrylic acid (22a). General procedure
was employed for the preparation of 22a. Yield 95%; R = 0.38 (4:1:1 n-butanol:acetic acid:H O);
): δ 8.56 (s, 1H), 7.84 (s, 1H), 7.66 (s, 2H), 7.58
s, 1H), 7.52 (s, 1H), 7.42 (d, 1H, J = 16.0 Hz), 7.26 (s, 2H), 7.11 (s, 2H), 6.40 (d, 1H, J = 16.0 Hz),
6
f
2
+
1
ESI-MS (m/z): 373.44 (M+H ); Η-ΝΜR (DMSO-d
6
(
5
5
.31 (s, 2H). Anal. Calcd for C20
4.45; H: 3.41; N: 17.39.
16 6 2 3
H N O ·CF COOH (%): C: 54.32; H: 3.52; N: 17.28. Found (%): C:
(E)-(5-Methyl-2-oxo-1,3-dioxol)methyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-acrylate
(
22b). General procedure 6 was employed for the preparation of 22b. Yield 94%; R = 0.52 (8.5:1.5
f
+
1
CHCl :MeOH); ESI-MS (m/z): 485.52 (M+H ); Η-ΝΜR (DMSO-d ): δ 8.10 (s, 1H), 7.76 (s, 1H),
3
6
7
.65 (s, 2H), 7.57–7.51 (m, 3H), 7.24–7.20 (m, 2H), 7.11–7.07 (m, 2H), 6.38 (d, 1H, J = 16.0 Hz), 5.24
(
s, 2H), 5.04 (s, 2H), 2.16 (s, 3H). Anal. Calcd for C25 ·CF COOH (%): C: 54.18; H: 3.54; N:
H
20
N
6
O
5
3
1
4.04. Found (%): C: 54.08; H: 3.41; N: 14.17.
1-[[2′-[[N-(2-Chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-propanoic acid (23a).
General procedure 4 was employed for the preparation of 23a. Yield 93%; R = 0.33 (9:1
f
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 652.19 (M+H ), 374.77 (M+H -ClTr), 277.77 (ClTr); Η-ΝΜR
DMSO-d ): δ 7.82–7.69 (m, 1H), 7.64–7.44 (m, 6H), 7.40–7.27 (m, 7H), 7.10–7.02 (m, 4H),
.09–6.66 (m, 6H), 5.00 (s, 2H), 2.60 (t, 2H, J = 7.2 Hz), 2.38 (t, 2H, J = 7.2 Hz). Anal. Calcd for
Cl (%): C: 71.94; H: 4.80; N: 12.91. Found (%): C: 71.87; H: 4.86; N: 12.87.
(
6
7
39 31 6 2
C H N O
(5-Μethyl-2-oxo-1,3-dioxol)methyl-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]-methyl]
imidazole-4-propanoate (23b). General procedure 5 was employed for the preparation of 23b. Yield
+
+
7
3%; R
f
1
= 0.45 (9:1 CHCl
3
:MeOH); ESI-MS (m/z): 764.21 (M+H ), 486.56 (M+H -ClTr), 277.76
(
ClTr); Η-ΝΜR (CDCl ): δ 7.96 (d, 1H, J = 7.2 Hz), 7.49–7.31 (m, 9H), 7.22–7.18 (m, 5H), 7.13 (d,
3

Molecules 2013, 18
7525
2
2
6
H, J = 8.0 Hz), 6.92-6.84 (m, 5H), 6.82 (s, 1H), 6.72 (d, 1H, J = 8.0 Hz), 4.88 (s, 2H), 4.81 (s, 2H),
.83 (t, 2H, J = 7.2 Hz), 2.68 (t, 2H, J = 7.2 Hz), 2.16 (s, 3H). Anal. Calcd for C H N O Cl (%): C:
4
4
35
6
5
9.24; H: 4.62; N: 11.01. Found (%): C: 69.19; H: 4.67; N: 11.08.
1-[[2′-(1H-Tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoic acid (24a). General procedure 6
was employed for the preparation of 24a. Yield 92%; R = 0.33 (4:1:1 n-butanol:acetic acid:H O);
f
2
+
1
ESI-MS (m/z): 375.41 (M+H ); Η-ΝΜR (CD
m, 2H), 7.39 (s, 1H), 7.35 (d, 2H, J = 7.2 Hz), 7.20 (d, 2H, J = 7.2 Hz), 5.50 (s, 2H), 2.98 (t, 2H
J = 7.2 Hz), 2.71 (t, 2H, J = 7.2 Hz). Anal. Calcd for C H N O ·CF COOH (%): C: 54.10; H: 3.92;
3
OD): δ 8.89 (s, 1H), 7.71–7.67 (m, 2Η), 7.60–7.56
(
2
0
18
6
2
3
N: 17.21. Found (%): C: 54.17; H: 3.81; N: 17.32.
(
5-Methyl-2-oxo-1,3-dioxol)methyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoate
24b). General procedure 6 was employed for the preparation of 24b. Yield 87%; R = 0.49 (8.5:1.5
CHCl :MeOH); ESI-MS (m/z): 487.56 (M+H ); Η-ΝΜR (DMSO-d ): δ 9.06 (s, 1H),
(
f
+
1
3
6
7.67–7.12 (m, 9H), 5.35 (s, 2H), 4.94 (s, 2H), 2.87 (t, 2H, J = 7.2 Hz), 2.74 (t, 2H, J = 7.2 Hz), 2.12 (s,
3
H). Anal. Calcd for C H N O ·CF COOH (%): C: 54.00; H: 3.86; N: 13.99. Found (%): C: 53.91;
2
5
22
6
5
3
H: 3.91; N: 14.11.
5-Chloro-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-propanoic
acid (25a). General procedure 4 was employed for the preparation of 25a. Yield 94%; R
f
= 0.39 (9:1
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 686.66 (M+H ), 408.92 (M+H -ClTr), 277.81 (ClTr); Η-ΝΜR
3
(CDCl ): δ 7.98–7.94 (m, 1H), 7.48-7.08 (m, 15H), 6.85 (d, 6H, J = 7.6 Hz), 6.71 (d, 1H, J = 8.0 Hz),
4
1
.84 (s, 2H), 2.79 (bs, 2H), 2.58 (bs, 2H). Anal. Calcd for C39
2.26. Found (%): C: 68.08; H: 4.53; N: 12.57.
30 6 2 2
H N O Cl (%): C: 68.31; H: 4.41; N:
(5-Methyl-2-oxo-1,3-dioxol)methyl-5-chloro-1-[[2΄-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-
yl]methyl]imidazole-4-propanoate (25b). General procedure 1 was employed for the preparation of 7.
+
+
Yield 82%; R
f
= 0.50 (EtOAc); ESI-MS (m/z): 798.72 (M+H ), 521.03 (M+H -ClTr), 277.78 (ClTr);
): δ 7.95 (d, 1Η, J = 7.2 Hz), 7.48–7.34 (m, 6H), 7.34–7.32 (m, 5H), 7.23 (m, 5H),
.11 (d, 2H, J = 8.0 Hz), 6.91–6.86 (m, 8H), 4.93 (s, 2H), 4.83 (s, 2H), 2.87 (t, 2H, J = 7.2 Hz), 2.74 (t,
H, J = 7.2 Hz), 2.15 (s, 3H). Anal. Calcd for C44 Cl (%): C: 66.25; H: 4.30; N: 10.54. Found
1
Η-ΝΜR (CDCl
3
7
2
H
34
N
6
O
5
2
(%): C: 66.24; H: 4.18; N: 10.33.
5-Bromo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-propanoic
acid (25c). General procedure 4 was employed for the preparation of 25c. Yield 91%; R
f
= 0.37 (9:1,
+
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 731.11 (M+H ), 453.36 (M+H -ClTr), 277.76 (ClTr); Η-ΝΜR
(CDCl
3
): δ 8.04 (d, 1H, J = 7.6 Hz), 7.67–7.28 (m, 15H), 7.09 (d, 2H, J = 8.0 Hz), 6.97 (d, 4H, J = 7.6
Hz), 6.87 (d, 1H, J = 8.0 Hz), 5.12 (s, 1H), 2.96–2.92 (m, 2H), 2.81–2.78 (m, 2H). Anal. Calcd for
C H N O ClBr (%): C: 64.16; H: 4.14; N: 11.51. Found (%): C: 64.11; H: 4.21; N: 11.44.
3
9
30
6
2
(5-Methyl-2-oxo-1,3-dioxol)methyl-5-bromo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-
yl]methyl]imidazole-4-propanoate (25d). General procedure 5 was employed for the preparation of
+
+
2
5d. Yield 72%; R = 0.48 (EtOAc); ESI-MS (m/z): 843.18 (M+H ), 565.43 (M+H -ClTr), 277.78
f

Molecules 2013, 18
7526
1
(
ClTr); Η-ΝΜR (CDCl ): δ 8.03 (d, 1H, J = 7.2 Hz), 7.65–7.33 (m, 13H), 7.24 (d, 2H, J = 7.6 Hz),
3
6
.99 (m, 4H), 6.87 (d, 1H, J = 7.6 Hz), 5.07 (s, 2H), 4.93 (s, 2H), 2.94 (t, 2H, J = 6.4 Hz), 2.82 (t, 2H,
34 6 5
J = 6.4 Hz), 2.24 (s, 3H). Anal. Calcd for C44H N O ClBr (%): C: 62.75; H: 4.07; N: 9.98. Found
(%): C: 62.68; H: 4.15; N: 9.91.
5-Iodo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]imidazole-4-propanoic acid
(
25e). General procedure 4 was employed for the preparation of 25e. Yield 95%; R = 0.37 (9:1
f
+
+
1
CHCl :MeOH); ESI-MS (m/z): 778.13 (M+H ), 500.35 (M+H -ClTr), 277.76 (ClTr); Η-ΝΜR
3
(CDCl
3
): δ 7.97 (d, 1H, J = 7.6 Hz), 7.72 (s, 1H), 7.49–7.17 (m, 15H), 6.93 (d, 2H, J = 7.6 Hz ), 6.88
(d, 4H, J = 7.6 Hz), 6.73 (d, 1H, J = 7.2 Hz), 4.98 (s, 2H), 2.88 (t, 2H, J = 7.2 Hz), 2.73 (t, 2H,
J = 7.2 Hz). Anal. Calcd for C H N O ClI (%): C: 60.28; H: 3.89; N: 10.82. Found (%): C: 60.21; H:
3
9
30
6
2
3
.96; N: 10.73.
(5-Methyl-2-oxo-1,3-dioxol)methyl-5-iodo-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-
yl]methyl]imidazole-4-propanoate (25f). General procedure 5 was employed for the preparation of
+
+
2
5f. Yield 74%; R = 0.51 (EtOAc); ESI-MS (m/z): 890.21 (M+H ), 612.45 (M+H -ClTr), 276.88
f
1
(
ClTr); Η-ΝΜR (CDCl
3
): δ 7.77 (d, 1H, J = 7.6 Hz), 7.45–7.22 (m, 8Η), 7.15–6.80 (m, 14H), 4.86 (s,
2
H), 4.71 (s, 2H), 2.71 (t, 2H, J = 7.2 Hz), 2.59 (t, 2H, J = 7.2 Hz), 2.02 (s, 3H); Anal. Calcd for
ClI (%): C: 59.44; H: 3.85; N: 9.45. Found (%): C: 59.36; H: 3.94; N: 4.37.
44 34 6 5
C H N O
5
-Chloro-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoic acid (26a). General
procedure 6 was employed for the preparation of 26a. Yield 90%; R = 0.32 (4:1:1 n-butanol:acetic
OD): δ 8.62 (s, 1H), 7.70 (d, 2H, J = 7.2
Hz), 7.62–7.56 (m, 2H), 7.27 (d, 2H, J = 7.6 Hz), 7.19 (d, 2H, J = 7.6 Hz), 5.38 (s, 2H), 2.94 (t, 2H,
f
+
1
2 3
acid:H O); ESI-MS (m/z): 409.31 (M+H ); Η-ΝΜR (CD
17 6 2 3
J = 7.2 Hz), 2.69 (t, 2H, J = 7.2 Hz). Anal. Calcd for C20H N O Cl·CF COOH (%): C: 50.54; H: 3.47;
N: 16.07. Found (%): C: 50.43; H: 3.58; N: 16.17.
(5-Methyl-2-oxo-1,3-dioxol)methyl-5-chloro-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-
4
-propanoate (26b). General procedure 6 was employed for the preparation of 26b. Yield 89%;
+
1
R
f
3 6
= 0.56 (8.5:1.5 CHCl :MeOH); ESI-MS (m/z): 522.00 (M+H ); Η-ΝΜR (DMSO-d ): δ 8.24 (s,
1
2
1
H), 7.71–7.54 (m, 4H), 7.17–7.08 (m, 4H), 5.24 (s, 2H), 4.92 (s, 2H), 2.76 (t, 2H, J = 7.6 Hz), 2.69 (t,
H, J = 7.6 Hz), 2.13 (s, 3H). Anal. Calcd for C H N O Cl·CF COOH (%): C: 51.07; H: 3.49; N:
2
5
21
6
5
3
3.24. Found (%): C: 51.13; H: 3.41; N: 13.35.
5
-Bromo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoic acid (26c). General
= 0.31 (4:1:1 n-butanol:acetic
OD): δ 7.67–7.55 (m, 5H), 7.12 (s, 4H),
.21 (s, 2H), 2.83 (t, 2H, J = 7.2 Hz), 2.60 (t, 2H, J = 7.2 Hz). Anal. Calcd for
C H N O Br·CF COOH (%): C: 46.58; H: 3.20; N: 14.81. Found (%): C: 46.63; H: 3.32; N: 14.69.
procedure 6 was employed for the preparation of 26c. Yield 93%; R
f
+
1
2 3
acid:H O); ESI-MS (m/z): 454.37 (M+H ); Η-ΝΜR (CD
5
2
0
17
6
2
3
(5-Methyl-2-oxo-1,3-dioxol)methyl-5-bromo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-
4
-propanoate (26d). General procedure 6 was employed for the preparation of 26d. Yield 91%;
+
1
R = 0.55 (8.5:1.5 CHCl :MeOH); ESI-MS (m/z): 566.45 (M+H ); Η-ΝΜR (DMSO-d ): δ 7.91 (s,
f
3
6

Molecules 2013, 18
7527
1
4
H), 7.67–7.64 (m, 2H), 7.58–7.53 (m, 2H), 7.09 (s, 4H), 5.17 (s, 2H), 4.92 (s, 2H), 2.72–2.63 (m,
H), 2.13 (s, 3H). Anal. Calcd for C H N O Br·CF COOH (%): C: 47.73; H: 3.26; N: 12.37. Found
2
5
21
6
5
3
(%): C: 47.83; H: 3.34; N: 13.27.
5-Iodo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-propanoic acid (26e). General
procedure 6 was employed for the preparation of 26e. Yield 91%, R = 0.29 (4:1:1 n-butanol:acetic
f
+
1
acid:H
.56–7.31 (m, 4H), 7.19 (d, 2H, J = 8.0 Hz), 5.35 (s, 2H), 2.95 (t, 2H, J = 7.2 Hz), 2.68 (t, 2H,
J = 7.2 Hz). Anal. Calcd for C H N O I·CF COOH (%): C: 43.01; H: 2.95; N: 13.68. Found (%): C:
2 3
O); ESI-MS (m/z): 501.38 (M+H ); Η-ΝΜR (CD OD): δ 8.79 (s, 1H), 7.68–7.64 (m, 2H),
7
2
0
17
6
2
3
4
3.13; H: 2.87; N: 13.79.
(
5-Methyl-2-oxo-1,3-dioxol)methyl-5-iodo-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]imidazole-4-
propanoate (26f). General procedure 6 was employed for the preparation of 26f. Yield 90%; R = 0.56
8.5:1.5 CHCl :MeOH); ESI-MS (m/z): 613.44 (M+H ); Η-ΝΜR (CD OD): δ 8.00 (s, 1H), 7.66–7.56
f
+
1
(
(
3
3
m, 4H), 7.15 (bs, 4Η), 5.22 (s, 2H), 4.88 (s, 2H), 2.86 (t, 2H, J = 7.2 Hz), 2.70 (t, 2H,
J = 7.2 Hz), 2.16 (s, 3H). Anal. Calcd for C H N O I·CF COOH (%): C: 44.64; H: 3.05; N: 11.57.
2
5
21
6
5
3
Found (%): C: 44.72; H: 3.14; N: 11.65.
3
.2.9. Synthesis of 1,5-Disubstituted Benzyl Analogues 30, 32
(E)-Methyl 3-[1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-imidazole-4-yl]acrylate (27). To a solution of
(
E)-urocanic methyl ester (6) (0.61 g, 4.03 mmol) in dry DMF (15.0 mL) under argon atmosphere at
0
°C was added dry NaH (powdered 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 4 h. The reaction was quenched with 0.5 N NaOH
(
2 2
15 mL) and extracted with CH Cl (3 × 20 mL). The combined organic phases were washed with
brine (× 2), dried (Na SO ) and concentrated in vacuo. Purification by flash column chromatography
2
4
(
(
7:3 EtOAc:hexanes) afforded 27. Yield 78%; R = 0.35 (8:2 EtOAc:hexanes); ESI-MS (m/z): 283.29
f
+
1
M+H ); Η-ΝΜR (CDCl
3
): δ 8.58 (s, 1H), 7.52 (d, 1H, J = 16.0 Hz), 7.35 (s, 1H), 6.68 (d, 1H,
J = 16.0 Hz), 5.42 (s, 2H), 3.82 (s, 3H), 3.58 (t, 2H, J = 8.0 Hz), 0.96 (t, 2H, J = 8.0 Hz), 0.01 (s, 9H);
1
3
C-NMR (CDCl
8.22, 1.44. Anal. Calcd for C13
H:7.72; N:9.87.
3
): δ 166.64, 137.98, 134.48, 130.47, 121.21, 120.59, 50.85, 76.79, 68.14, 51.95,
1
22 2 3
H N O
Si (%): C: 55.29; H: 7.85; N:9.92. Found (%): C:55.36;
Methyl 3-[1-[(2-(trimethylsilyl)ethoxy)methyl]-1H-imidazole-4-yl]propanoate (28). General procedure
2
2
was employed for the preparation of 28. Yield 91%; R
f
= 0.44 (9:1 CHCl
3
:MeOH); ESI-MS (m/z):
+
1
84.99 (M+H ); Η-ΝΜR (CDCl
3
): δ 7.91 (s, 1H), 6.88 (s, 1H), 5.25 (s, 2H), 3.67 (s, 3H), 3.50 (t, 2H,
J = 8.0 Hz), 3.00 (t, 2H, J = 7.2 Hz), 2.72 (t, 2H, J = 7.2 Hz), 0.91 (t, 2H, J = 8.0 Hz), 0.01 (s, 9H);
1
3
C-NMR (CDCl
3
): δ 173.13, 136.14, 116.19, 76.88, 67.08, 120.59, 51.65, 33.27, 22.27, 17.89, 1.44.
Si (%): C:54.90; H:8.51; N:9.85. Found (%): C:54.81; H:8.61; N:9.74.
Anal. Calcd for C13
24 2 3
H N O
Methyl
1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]-3-[(2-(trimethylsilyl)-
ethoxy)methyl]imidazolium-5-propanoate bromide (29). To a stirred solution of 28 (2.0 g, 7.03 mmol)

Molecules 2013, 18
7528
in dry CH Cl (20.0 mL) under argon was added the alkylating agent 4 (4.31 g, 7.73 mmol) in one
2
2
portion and the resulting mixture was heated under reflux for 3 h. Upon completion (disappearance of
starting material confirmed by RP-HPLC), the solvent was concentrated, followed by chromatographic
purification (97:3 CHCl :MeOH) to afford 29 as a white powder. Yield 81%; R = 0.40 (9:1
3
f
+
1
CHCl
3
:MeOH); ESI-MS (m/z): 796.19 (M -Br); Η-ΝΜR (CDCl
3
): δ 7.93 (dd, 1H, J = 1.6, 7.6 Hz),
7
.52–7.46 (m, 3H), 7.40–7.13 (m, 13H), 7.11 (s, 1H), 6.94 (d, 6H, J = 7.6 Hz), 5.67 (s, 2H), 5.46 (s,
H), 3.75–3.64 (m, 5H), 2.75 (t, 2H, J = 7.2 Hz), 2.47 (t, 2H, J = 7.2 Hz), 0.94 (t, 2H, J = 8.0 Ηz),
.002 (s, 9H). Anal. Calcd for C H N O SiBr (%): C:63.04; H:5.52; N:9.59. Found (%): C:62.93;
2
0
4
6
48
6
3
H:5.57; N:9.51.
Methyl 1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3-[(2-(trimethylsilyl)ethoxy)methyl]imidazole-
-propanoate (30). General procedure 6 was employed for the preparation of 30. Yield 90%; R = 0.53
8.5:1.5 CHCl :MeOH); ESI-MS (m/z): 389.51 (M+H ); Η-ΝΜR (DMSO-d ): δ 9.13 (s, 1H), 7.68 (d,
5
f
+
1
(
3
6
1
H, J = 8.0 Hz), 7.59 (d, 1H, J = 7.6 Hz), 7.54 (d, 1H, J = 7.6 Hz), 7.50 (s, 1H), 7.21 (d, 2H, J = 8.0 Hz),
.14 (d, 1H, J = 8.0 Hz), 5.48 (s, 2H), 3.60 (s, 3H), 2.77 (t, 2H, J = 7.2 Hz), 2.64 (t, 2H, J = 7.2 Hz). Anal.
7
Calcd for C H N O ·CF COOH (%): C:54.98; H:4.21; N:16.73. Found (%): C:54.91; H:4.13; N:16.61.
21
20
6
2
3
Methyl
2-hydroxymethyl-1-[[2′-[[N-(2-chlorotrityl)]-1H-tetrazol-5-yl]biphenyl-4-yl]methyl]-3-[(2-
(
trimethylsilyl)ethoxy)methyl]imidazolium-5-propanoate bromide (31). In a sealed tube were
sequentially added 29 (2.0 g, 2.38 mmol), DMF (1.0 mL), 37% formalin (2.65 mL, 35.63 mmol) and
diisopropylethylamine (2.02 mL, 11.90 mmol). The resulting mixure 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
(
(
2 2
10 mL), extracted with CH Cl and the combined organic phases were washed with brine, dried
Na
2
SO
4
), filtered and concentrated in vacuo. Purification by flash column chromatography (96:4
+
CHCl
1
3
:MeOH) afforded 31. Yield 91%; R
f
= 0.28 (9:1 CHCl
3
:MeOH); ESI-MS (m/z): 826.32 (M -Br);
3
Η-ΝΜR (CDCl ): δ 7.95 (d, 1H, J = 6.8 Hz), 7.52–7.42 (m, 3H), 7.40–7.26 (m, 9H, ), 7.19 (s, 1H),
7
5
.15 (d, 2H, J = 8.0 Hz), 6.94–6.89 (m, 8H), 5.76 (s, 2H), 5.52 (s, 2H), 4.72 (s, 2H), 3.70–3.62 (m,
H), 2.76 (t, 2H, J = 6.8 Hz), 2.54 (t, 2H, J = 6.8 Hz), 0.95 (t, 2H, J = 8.0 Ηz), 0.001 (s, 9H). Anal.
50 6 4
Calcd for C47H N O SiBr (%): C:62.28; H:5.56; N:9.27. Found (%): C:62.37; H:5.43; N:9.19.
Methyl 2-hydroxymethyl-1-[[2′-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-3-[(2-(trimethylsilyl)ethoxy)-
methyl]imidazole-5-propanoate (32). General procedure 6 was employed for the preparation of 32.
+
1
Yield 88%; R
f
= 0.32 (8:2 CHCl
.67 (dd, 2H, J = 2.0, 7.6 Hz), 7.59 (d, 1H, J = 7.6 Hz), 7.52 (d, 1H, J = 7.6 Hz), 7.44 (s, 1H), 7.12 (s,
H), 6.26 (bs, 1H), 5.45 (s, 2H), 4.74 (s, 2H), 3.58 (s, 3H), 2.71 (t, 2H, J = 6.8 Hz), 2.61 (t, 2H, J = 6.8
3 6
:MeOH); ESI-MS (m/z): 419.56 (M+H ); Η-ΝΜR (DMSO-d ): δ
7
4
Hz). Anal. Calcd for C22
H:4.25; N:15.86.
22 6 3 3
H N O ·CF COOH (%): C:54.14; H:4.35; N:15.78. Found (%): C:54.23;
3
.3. Pharmacological Evaluation
.3.1. Radioligand Binding Assay
Radioligand binding assay was performed as previously described [20].
3

Molecules 2013, 18
.4. Docking Studies
The 3D model of the AT1 receptor used in our docking studies was kindly provided by
7529
3
Tuccinardi et al. [29]. The construction of this model is based on X-ray bovine rhodopsin structure,
molecular procedure and available site-directed mutagenesis data [30]. Molecular Docking studies
were performed using Glide extra precision (XP) implemented Induced Fit Docking (IFD) protocol
(
v 5.0) [31–33] docking programs under the Linux operating system. The active site was defined by
0 Å inner cubic grid box, centered on the point that is the center of mass of residues Lys199 and
2
His256. The IFD protocol under the Schrodinger molecular modeling package was used in order to
eliminate clashes between receptor and ligand atoms and for the receptor to gain partial flexibility to
the receptor. Before the docking simulations, the complexes were submitted to the protein preparation
module of Schrodinger. Ligands were constructed using the Schrodinger’s Maestro module and then
geometry optimization was performed for these ligands using Polak-Ribiere conjugate gradient
−1
−1
(
PRCG) minimization (0.0001 kJÅ mol convergence criteria). Protonation states of residues were
created using LigPrep and Protein Preparation modules under the Schrodinger package at neutral pH.
IFD uses the Glide docking program to account the ligand flexibility and the refinement module and
the Prime (v.1.6) program [32,33] to account for flexibility of the receptor. Schrodinger’s IFD protocol
model uses the following steps (the description below is taken from the IFD user manual):
(
i) Constrained minimization of the receptor with an RMSD cutoff of 0.18 Å. (ii) Initial Glide docking
of each ligand using soft potentials (0.5 van der Waals radii scaling of non-polar atoms of ligands and
receptor using partial charge cutoff of 0.15). (iii) Derived docking poses were refined using the Prime
Induced Fit module of Schrodinger. Residues within 5.0 Å of ligand poses were minimized in order to
form suitable conformations of poses at the active site of the receptor. (iv) Glide re-docking of each
protein–ligand complex.
4
. Conclusions
In the present study, we have demonstrated an efficient and convenient strategy for the syntheses of
a series of N-benzyl and N-biphenylmethyl imidazole derivatives substituted either at the N-1 or N-3
positions of (E)-urocanic acid. A facile and clean methodology with a few-step synthetic protocol in
high yields has been developed. Biological evaluation of the synthesized analogues concerning their
binding affinity for the AT1 receptor revealed that certain analogues (compounds 12 and 18) are
moderate inhibitors. In particular, the methyl acrylate analogue 18 which bears the biphenylmethyl
moiety, showed relevant higher activity compared to the others. In addition, the lack of a lipophilic
alkyl chain may also explain the lower activity of 18 which seems to be critical for binding affinity,
compared to losartan. Docking results showed that flexibility of these molecules is an important factor
that governs their drug activity. The synthesized analogues adopt different orientations in the active
site as indicated by the docking studies. It is reasonable to assume that the studied molecules first
adopt the most comfortable conformation and orientation and show the highest scoring when these are
approaching the receptor. Such a hypothesis can explain the experimental data which have indicated
the poor activity of the studied molecules. The propensity of some molecules to adopt the appropriate
orientation and thus exert all favored but not maximal interactions can explain their moderate activity.

Molecules 2013, 18
7530
Acknowledgments
This research project was supported by the research programmes PRAXE B-EPAN &
NEW_B_2012, General Secretariat of Research and Technology of Greece, Ministry of Development
4
and the pharmaceutical companies ELDRUG SA and VIANEX SA. We also acknowledge Demetrios
Vahliotis (Instrumental Analysis Laboratory, University of Patras) for recording the NMR spectra. C.
Koukoulitsa acknowledges Greek national funds through the Operational Program “Education and
Lifelong Learning” of the National Strategic Reference Framework (NSRF)-Research Funding
Program: Heracleitus II, investing in knowledge society through the European Social Fund.
Conflict of Interest
The authors have no financial/commercial conflicts of interest to declare.
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