Rational design, efficient syntheses and biological evaluation of N,N'-symmetrically bis-substituted butylimidazole analogs as a new class of potent Angiotensin II receptor blockers

European Journal of Medicinal Chemistry 62 (2013) 352e370

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Original article

Rational design, efﬁcient syntheses and biological evaluation

of N,N⁰-symmetrically bis-substituted butylimidazole analogs as

a new class of potent Angiotensin II receptor blockers

,

l

, ,

,

l

,

1

b

c

George Agelis ^{a 1}, Amalia Resvani ^a, Catherine Koukoulitsa , Tereza Tumová ,

*

ꢀ

c

d

e

f

ꢁ

Jirina Slaninová , Dimitra Kalavrizioti , Katerina Spyridaki , Antreas Afantitis ,

Georgia Melagraki ^f, Athanasia Siafaka ^g, Eleni Gkini ^g, Grigorios Megariotis ^h,

Simona Golic Grdadolnik ⁱ, Manthos G. Papadopoulos ^h, Demetrios Vlahakos ^k,

,

j

Michael Maragoudakis ^d, George Liapakis ^e, Thomas Mavromoustakos ^b,

John Matsoukas ^a

,l,

*

^aDepartment of Chemistry, University of Patras, 26500 Patras, Greece

^bLaboratory of Organic Chemistry, Department of Chemistry, University of Athens, Panepistimiopolis-Zografou 15771, Athens, Greece

^cInstitute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 16610 Prague 6,

Czech Republic

^dDepartment of Pharmacology, School of Medicine, University of Patras, Patras 26500, Greece

^eDepartment of Pharmacology, Faculty of Medicine, University of Crete, Voutes, 71003 Heraklion, Crete, Greece

^fDepartment of Chemoinformatics, NovaMechanics Ltd, John Kennedy, Ave 62-64 Pallouriotissa, Nicosia 1046, Cyprus

^gLaboratory of Biochemistry, Department of Chemistry, University of Athens, Zografou, 15784 Athens, Greece

^hNational Hellenic Research Foundation, Institute of Organic and Pharmaceutical Chemistry, Vas. Constantinou 48, 11635 Athens, Greece

ⁱLaboratory of Biomolecular Structure, National Institute of Chemistry, Hajdrihova 19, SI-1001 Ljubljana, Slovenia

^jEN-FIST Centre of Excellence, Dunajska 156, SI-1000 Ljubljana, Slovenia

^kDepartment of Internal Medicine, ‘ATTIKON’ University Hospital, Athens, Greece

^lEldrug SA, Patras Science Park, 26504 Patras, Greece

a r t i c l e i n f o

a b s t r a c t

Article history:

A series of symmetrically bis-substituted imidazole analogs bearing at the N-1 and N-3 two biphenyl

moieties ortho substituted either with tetrazole or carboxylate functional groups was designed based on

docking studies and utilizing for the ﬁrst time an extra hydrophobic binding cleft of AT1 receptor. The

synthesized analogs were evaluated for their in vitro antagonistic activities (pA₂values) and binding af-

ﬁnities (elogIC₅₀values) to the Angiotensin II AT1 receptor. Among them, the potassium (elogIC₅₀¼ 9.04)

and the sodium (elogIC₅₀¼ 8.54) salts of 4-butyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}

imidazolium bromide (12a and 12b, respectively) as well as its free acid 11 (elogIC₅₀¼ 9.46) and the 4-

butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}imidazolium bromide (14)

(elogIC₅₀¼ 8.37, pA₂¼ 8.58) showed high binding afﬁnity tothe AT1 receptorand high antagonistic activity

(potency). The potency was similar or even superior to that of Losartan (elogIC₅₀¼ 8.25, pA₂¼ 8.25). On the

contrary, 2-butyl-N,N⁰-bis{[2⁰-[2H-tetrazol-5-yl)]biphenyl-4-yl]methyl}imidazolium bromide (27)

(elogIC₅₀¼ 5.77) and 2-butyl-4-chloro-5-hydroxymethyl-N,N⁰-bis{[2⁰-[2H-tetrazol-5-yl)]biphenyl-4-yl]

methyl}imidazolium bromide (30) (elogIC₅₀¼ 6.38) displayed very low binding afﬁnity indicating that the

orientation of the n-butyl group is of primary importance. Docking studies of the representative highly

active 12b clearly showed that this molecule has an extra hydrophobic binding feature compared to pro-

totype drug Losartan and it ﬁts to the extra hydrophobic cavity. These results may contribute to the dis-

covery and development of a new class of biologically active molecules through bis-alkylation of the

imidazole ring by a convenient and cost effective synthetic strategy.

Received 27 September 2012

Received in revised form

25 December 2012

Accepted 26 December 2012

Available online 2 January 2013

Keywords:

AT1 receptor blockers

N,N⁰-Bis-alkylated butylimidazole analogs

Synthesis

Wittig reaction

Hydroxymethylation

Structure elucidation

Docking studies

Molecular dynamics

Biological evaluation

* Corresponding authors. Department of Chemistry, University of Patras, 26500 Patras, Greece. Tel.: þ30 6944514458.

E-mail addresses: aggelisgeorge@hotmail.com (G. Agelis), imats@upatras.gr (J. Matsoukas).

1

The ﬁrst two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.ejmech.2012.12.044

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

353

such as the biphenylmethyl group at the 1-position [12], a linear

alkyl group at the 2-position [13,14] and an acidic group, like tet-

razole, CO₂H, or NHSO₂CF₃on the biphenylmethyl group [4,7,15],

are required for antagonistic activity. Furthermore, the DuPont

group recommended a lipophilic and electron-withdrawing group,

such as iodine or CF₃, as a substituent at the 4-position and a small-

sized group at the 5-position, such as CH₂OH, CH₂OMe, or CO₂H,

which is capable of forming a hydrogen bond [8,9].

We recently described a series of N-substituted 5-butylimidazole

analogs as potent ARBs via efﬁcient synthetic routes, allowing the

facile introduction of substituents on the imidazole ring [16e19].

Based on these ﬁndings, we rationally designed a new class of N,N⁰-

symmetrically bis-substituted butylimidazole analogs as potent

ARBs that occupy an extra hydrophobic binding cleft (Fig.1). Thus, in

the present work, a series of new compounds has been synthesized

bearing (i) an imidazole ring; (ii) the biphenylmethyl moiety

at theN-1 andN-3 of theheterocyclicring;(iii) an acidic functionality

such as a tetrazole group or its bioisostere carboxy group ortho

substituted at the biphenylmethyl group, and (iv) the n-butyl chain

substituted on the heterocyclic ring at the C-2 or C-5 for efﬁcient

binding to the receptor. Furthermore, the hydroxymethyl group was

introduced at the C-2 as well as a halogen atom at the C-4 of the

imidazole ring, since the chloro substituent in the DuPont series

interacts with a lipophilic pocket of the receptor [10].

1. Introduction

The RenineAngiotensin System (RAS) plays a key role in regu-

lating cardiovascular homeostasis and electrolyte/ﬂuid balance in

normotensive and hypertensive subjects. In the RAS the biological

actions of Angiotensin II (ANGII), such as vasoconstriction, release

of aldosterone, stimulation of sympathetic transmission and cel-

lular growth are mediated by the Angiotensin type 1 (AT1) re-

ceptor. Consequently, the RAS has been the prime target for the

therapy of cardiovascular diseases and non-peptide AT1 receptor

antagonists have been developed to speciﬁcally block it [1,2]. In

speciﬁc, the DuPont group pursued a study in this ﬁeld and

developed Losartan, the ﬁrst orally effective non-peptide antago-

nist, which is in vivo metabolized to the more potent EXP 3174 [3]

(Fig. 1). Since then, numerous new antagonists have been reported

by various research groups with nine of them in the clinic i.e.

Candesartan [4], Valsartan [5], Irbesartan [6], Telmisartan [7],

Olmesartan [8] which have been established as strong AT1 Re-

ceptor Blockers (ARBs). On 25 February 2011, the U.S. Food and

Drug Administration (FDA) approved Azilsartan medoxomil [9,10]

which is a newer-generation ARB, for the treatment of high

blood pressure in adults. Despite the plethora of treatment options

for the management of hypertension, 56% of patients do not have

their blood pressure under adequate control [11] suggesting the

need for the development of improved ARBs.

These unusual analogs were in vitro evaluated by means of their

binding afﬁnities for the human AT1 receptor, as well as for their

ability to inhibit the contractility effect of ANGII in isolated rat

uterus, compared to Losartan. The SAR study was performed and

the results suggested that the position of the n-butyl group in this

The majority of selective ARBs have resulted from the strategy of

modifying or replacing several pharmacophore groups of Losartan.

Structure Activity Relationship (SAR) studies of Losartan and other

imidazole blockers have been reported. Lipophilic substituents,

Fig. 1. Selected bis-alkylated butylimidazole synthesized analogs, Losartan, EXP 3174 (in vivo active metabolite) and ARBs marketed for hypertension.

354

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

N,N⁰-bis-alkylated series plays a signiﬁcant role in the interaction

with the AT1 receptor. Finally, structure elucidation and detailed

docking studies have been carried out for the one of the most

potent synthetic analog 12b.

quenching sequence. Wittig reaction of 2c by treatment with NaH

and CH₃CH₂CH₂PPh₃Br in the presence of t-amyl alcohol [26,27] in

THFat 0 ^ꢁC to rt proceeded smoothlyleading tothe anticipated oleﬁn

3c as a mixture of Z and E isomers in a ratio of ca. 3:1 (as indicated by

HPLC) which were isolated and identiﬁed by NMR in 62% and 24%

yield, respectively. Then, the isomers were subjected to catalytic

hydrogenation to afford the saturated 4c in excellent yield (90%).

Finally, hydrolysis of 4c in reﬂuxing 1.5 N HCl [28] afforded the target

compound 2-butylimidazole (5b). The two methods are quite sim-

ilar with respect to yields and ﬁnal products, however, the former

was proven to be more efﬁcient in terms of total steps.

2. Results and discussion

2.1. Chemistry

In the current study, we initially examined various synthetic

protocols for the syntheses of 4(5)- and 2-substituted imidazoles as

key precursors for the preparation of N,N⁰-bis-alkylated analogs as

potentARBs. InScheme1, thesynthesesof4(5)-and2-butylimidazole

via a synthetic sequence involving different N-protected imidazole

groups are depicted. Imidazole (1) was readily transformed through

a four-step synthetic pathway into the corresponding 1-trityl-imi-

dazole-4-carboxaldehyde (2a) [20,21]. Wittig reaction [22] of the

carboxaldehyde 2a by treatment with NaH and the nonstabilized

n-propyl triphenylphosphonium bromide (CH₃CH₂CH₂PPh₃Br) in

THF at rt furnished exclusively the Z-3 isomer 3a in excellent yield

(81%). The Z-conﬁguration was conﬁrmed by ¹H NMR which showed

the presence of two vinylic protons at 6.25 and 5.60 ppm as doubletof

triplets, respectively (J ¼ 11.6 Hz). The latter was subjected to catalytic

hydrogenationunder H₂atmosphere(3bar)inthe presenceofPd/Cat

ambient temperature for 2 h to give 4a in 92% yield. Finally,

demaskingofNHbytreatmentwith20%TFA inCH₂Cl₂inthepresence

of Et₃SiH as scavenger furnished the target 4(5)-butylimidazole (5a).

Two routes were investigated for the conversion of 1 into the

2-butylimidazole (5b). In the ﬁrst approach (Scheme 1, Method A),

the N-1 was protected with the (N-dimethylamino)methyl group

under Mannich reaction conditions [23] resulting in 2b. Then, the

latter compound underwent smooth lithiation, which occurred

regiospeciﬁcally at the 2-position of the imidazole ring, upon

treatment with 1.6 M n-BuLi in the presence of 1,3-dimethyl-3,4,5,6-

tetrahydro-2(1H)-pyrimidinone (DMPU) in THF at ꢀ78 ^ꢁC, followed

by electrophilic quenching with n-BuI [23,24]. Subsequent facile

acidic hydrolysis of the protective group afforded the corresponding

2-butylimidazole (5b) in 85% yield. In the second approach (Scheme

1, Method B), carboxaldehyde 2c was prepared as described in the

published procedure [25] using the N,N-dimethylsulfamoyl group

(DMAS) as NH protective group for the lithiation/electrophilic

In Scheme 2, the syntheses of N,N⁰-bis-alkylated butylimidazole

derivatives bearing the biphenylmethyl moiety ortho substituted

either with the tetrazole or its bioisostere carboxy group as well as

the hydroxymethyl group at the 2-position are depicted. The N,N⁰-

bis-alkylation was generally accomplished under basic conditions

[29] followed by the treatment of the resulting salt with the

appropriate alkylating agent in excess. 4(5)-Butylimidazole (5a)

was converted to the corresponding quaternary salts 7 and 8 under

the general heterogeneous phase alkylation protocol. In particular,

the latter compounds were obtained using excess of the biphe-

nylmethyl bromides 6a and 6b [19] (2.3 equiv.) in the presence of

K₂CO₃in anhydrous DMF at ambient temperature for 8 h in 78e82%

yields. Then, hydroxymethylation was promptly carried out in

a sealed tube by treatment with diisopropylethylamine and 37%

formalin in DMF at 85 ^ꢁC for 1 h [17,19]. This reaction proceeded

quantitatively as indicated by HPLC and led rapidly and exclusively

to the desired hydroxymethylated products 9 and 10. The trityl and

t-Bu groups of 7, 9 and 8, 10, respectively, were promptly removed

upon addition of 20% triﬂuoroacetic acid (TFA) in CH₂Cl₂in the

presence of Et₃SiH as scavenger to obtain the ﬁnal compounds 11,

13, 14, 15 in 72e85% yields. Finally, conversion of 11 into the cor-

responding water soluble bis-potassium and sodium salts 12a and

12b was accomplished by treatment with KOH and NaOH, respec-

tively, in MeOHeH₂O at rt for 5 h in 80e83% yields. The ¹H NMR

spectrum of 12a showed two methylene signals of N-1 and N-3 at

5.33 and 5.32 ppm as singlet peaks, the H-5 of imidazole at

7.41 ppm and H-2 at 8.93 ppm also as singlet peaks. In addition, the

hydroxymethylated analog of 14 showed the absence of the proton

at C-2 and the appearance of a singlet peak at 4.92 ppm, corre-

sponding to the hydroxymethylene protons.

Scheme 1. Reagents and conditions: (a) CH₃CH₂CH₂PPh₃Br, NaH (powdered, 95%), THF, rt, 8 h; (b) H₂(3 bar), 10% Pd/C, MeOH, rt, 2 h; (c) 20% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h; (d) n-BuLi

(1.6 M in hexanes), DMPU, n-BuI, then 2 M HCl; (e) CH₃CH₂CH₂PPh₃Br, NaH (powdered, 95%), t-amyl alcohol, THF, 0 ^ꢁC to rt, 10 h; (f) 1.5 N HCl, THF, reﬂux, 2 h.

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

355

Scheme 2. Reagents and conditions: (a) K₂CO₃, DMF rt, 8 h; (b) 37% formalin, diisopropylethylamine, DMF, 80 ^ꢁC, 1 h; (c) 30% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h; (d) KOH, MeOHeH₂O, rt,

5 h for 12a, NaOH, MeOHeH₂O, rt, 5 h for 12b.

In Scheme 3, the syntheses of N,N⁰-bis-alkylated halogenated

butylimidazole derivatives bearing the biphenylmethyl moiety ortho

substituted either with the tetrazole or its bioisostere carboxy group

as well as the hydroxymethyl group at the 2-position are depicted.

The N,N⁰-bis-alkylation was advantageously accomplished under

basic conditionsbysolideliquid phasetransfercatalysis (PTC)[30,31].

4(5)-Butylimidazole (5a) was converted to the corresponding halo-

genated analogs 16cee by treatment with NXS (X ¼Cl, Br, I) in DMF in

71e73% yields. The ¹H NMR spectra of 16cee showed the absence of

the H-4 signal of the imidazole ring at 6.74 ppm. Furthermore, the ¹³C

NMR spectra showedanupﬁeld trend inC-4 chemicalshifts produced

by bromine and iodine atoms for 16d and 16e, at 114.53 and

81.92 ppm, respectively, while the C-4 in the chloro derivative 16c

appeared at 124.24 ppm. Alkylation of 16cee with the alkylating

agent 6a, in the presence of K₂CO₃and catalytic amount 18-crown-6-

ether to assist by chelating potassium in anhydrous THF under reﬂux

for 12 h, furnished the 17cee in 52e57% yields. PTC was also suc-

cessfully adopted for the syntheses of 18cee by treatment with 6b in

the presence of ﬁne powdered KOH and catalytic amount of 18-

crown-6-ether in anhydrous Tol at 80 ^ꢁC for 12 h in 58e64% yields.

Acidolysisofprotective groupsin17ceeand 18ceewasaccomplished

in the same manner and afforded 19cee and 20cee, respectively, in

73e82% yields. On the other hand, hydroxymethylation of 17cee and

18cee by treatment with 37% formalin in the presence of diisopro-

pylethylamine in DMF at 80 ^ꢁC for 1 h afforded 21cee and 22cee in

70e78% yields. The latter were subjected to acid hydrolysis with TFA,

resulting in 23cee and 24cee in 75e81% yields.

Finally, in Scheme 5, the syntheses of bis-alkylated analogs 30

and 31 with the implementation of a facile PTC alkylatione

deprotection reaction sequence in one-pot procedure are depic-

ted. Thus, alkylation under basic conditions of the commercially

available (2-n-butyl-4-chloro-1H-imidazole-4yl)methanol 29,

which is an important precursor for the preparation of Losartan,

followed by acid hydrolysis, afforded the corresponding analogs 30

and 32 in 77% and 74% yield over two steps, respectively.

2.2. Structure elucidation of analog 12b

The chemical structure of 12b (Fig. 2) has been elucidated using

a combination of 2D DQF-COSY and 2D ROESY experiments

(Table 1). The alkyl chain of the molecule was easily identiﬁed from

the 2D DQF-COSY experiment, the multiplicity and integration of

the peaks (Fig. 2). Due to the asymmetry of the molecule the aro-

matic biphenyl protons showed distinct resonances conﬁrmed by

the integration of the peaks (see the alkyl chain extended only to

one side of biphenyltetrazole moiety). The H10 and H10⁰protons

are magnetically un-equivalent and are depicted as separate single

peaks (Fig. 2). 2D DQF-COSY (Supporting information SF1) made

possible to assign all aromatic peaks in combination with 2D ROESY

experiment. H10 showed ROE with the single peak at 6.85 ppm,

which according to the integration corresponds to the resonance of

four protons. These were assigned as the H12, H13, H15, H16 pro-

tons of the aromatic ring A (Supporting information SF2). 2D ROESY

spectrum reveals that this peak is correlated with the protons of the

alkyl chain H6, H7, and H8 (Supporting information SF3). Similarly

H10⁰showed ROE with the doublet at 7.04 ppm attributed to the

protons 13⁰, 15⁰and 18⁰(Supporting information SF2). The 2D DQF-

COSY experiment conﬁrmed the proposed assignment (Supporting

information SF2). The doublet resonating at 6.86 ppm and assigned

Identical methodology was employed for the preparation of 2-

butylimidazole analogs 27 and 28 (Scheme 4) through a two-step

sequence. In particular, alkylation of 5b by treatment with the

alkylating agents 6a and 6b and subsequent acid hydrolysis affor-

ded the expected analogs 27 and 28.

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G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

Scheme 5. Reagents and conditions: (a) 29 / 30: 6a, K₂CO₃, 18-crown-6, THF, reﬂux,

12 h then 30% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h; 29 / 31: 6b, KOH, 18-crown-6, Tol, 80 ^ꢁC,

12 h then 30% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h.

expected to be the most magnetically deshielded ones, because

they are close to the tetrazole ring. Apparently, the protons of the

two B and B⁰rings have identical or close chemical shifts since they

are far away from the butyl chain that differentiates mainly the

environment of aromatic protons of the A and A⁰rings.

The broad singlet at 6.99 ppm has no coupling through bond

correlation. 2D ROESY experiment shows that this peak is corre-

lated through space with H10⁰and with the protons of the alkyl

chain H6, H7, and H8 (Supporting information SF2 and SF3). This

peak is attributed to H5 of imidazole ring. The two different pat-

terns of biphenyl rings are observed in our previous studies using

commercial and synthetic ARBs. In Losartan [32], Valsartan [33] and

Eprosartan [34] the A ring shows clearly the doublet of doublet

pattern common for 1,4 bis-substituted aromatic compounds.

However, in the synthetic analog V8 a singlet peak is observed that

is attributed to the same ring [17,35]. It is obvious that the equiv-

alency of the protons depends on the environment that surrounds

biphenyltetrazole ring. A small peak at 8.47 ppm as a remaining

peak is assigned to H2. It is the most downﬁeld peak, which can be

explained with the position of H2 between two nitrogens.

2.3. Docking studies of analog 12b at the binding site of AT1

Scheme 3. Reagents and conditions: (a) NXS (X ¼ Cl, Br, I), DMF 0 ^ꢁC / rt; (b) 16ce

e / 17cee: 6a, K₂CO₃, 18-crown-6, THF, reﬂux, 12 h and 16cee / 18cee: 6b, KOH, 18-

crown-6, Tol, 80 ^ꢁC, 12 h; (c) 30% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h; (d) 37% formalin, dii-

sopropylethylamine, DMF, 80 ^ꢁC, 1 h.

The synthesis of 12b and its derivatives has been based on

docking studies. The docking studies of 12b at AT1 and its in-

teractions with the amino acids of the active site are of particular

interest. Fig. 3 illustrates the best binding pose of 12b at the binding

to 12⁰and 16⁰protons is correlated through coupling with the

doublet at 7.04 ppm. Such correlation is evident also with Losartan

molecule that contains biphenyltetrazole segment [32]. As the

integration of the doublet shows the existence of three protons, the

other one is assigned to H18⁰. This is because H18⁰shows correla-

tion with the triplet peak resonating at 7.25 ppm and assigned to

H19⁰. Consequently, H19⁰is correlated with multiplet at 7.31 ppm,

assigned to H20⁰, which in turn is correlating with the doublet peak

at 7.55 ppm assigned to H21⁰. The doublet at 7.55 ppm is integrated

to two protons assigned to 21, 21⁰. This is in accordance with results

obtained with Losartan [30]. In addition, these protons are

site of AT1 (

D

G ¼ ꢀ16.21 kcal/mol) with the hydrophilic and hy-

drophobic areas around the molecule. The two biphenyl moieties

point to the widely extended hydrophobic area, whereas the hy-

drophilic area mainly surrounds the imidazole ring and is less

extend around the tetrazole.

Docking calculations using the same program and protocol have

pointed out the best binding pose of Losartan at the active site [17].

In comparison to 12b, Losartan is anchored to the binding site with

a lower binding afﬁnity (

potency.

DG ¼ ꢀ12.30 kcal/mol) reﬂecting its lower

In Fig. 4, the superimposition of the best binding poses of 12b

and Losartan is depicted. Both ligands show similar orientation at

the binding site of AT1 except the biphenyl moiety tetrazole (see A⁰,

B⁰rings) of 12b which has no equivalent groups. Interestingly, this

additional lipophilic part resides in a lipophilic cavity surrounded

by the amino acids Leu112, Ala181, Phe182, Ile288, Ala291. These

hydrophobic interactions are responsible for the enhanced binding

afﬁnity of 12b in comparison to Losartan. It is this ability of 12b to

reside in an extra cavity that triggered our interest for synthesizing

new drugs having a variety of activity. This drug molecule has

resulted after trying manually various derivative compounds which

could ﬁt the extra cavity. 12b has been suggested by the synthetic

group as the drug lead bearing the technognosy of the synthetic

Scheme 4. Reagents and conditions: (a) 5b / 25: 6a, K₂CO₃, DMF, rt, 8 h and

5b / 26: 6b, K₂CO₃, DMF, rt, 8 h; (b) 30% TFA in CH₂Cl₂, Et₃SiH, rt, 1 h.

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

357

1

Fig. 2. H NMR spectrum of analog 12b in D₂O solvent divided into six regions for better representation, recorded on a Varian INOVA 800 MHz spectrometer at a temperature of 25 ^ꢁC.

feasibility of such molecules. The syntheses of this molecule and its

derivatives shows that collaboration of the medicinal chemists and

computational chemists can lead to new ideas and to the rational

design of new classes of molecules with optimized biological ac-

tivity, taking into account pharmacophore orientation and charges.

biological activity [36e38]. For this reason, we have studied not

only the interaction of 12b at the binding site of AT1 receptor but

also its incorporation into plasma membrane.

Before starting the analysis of our results obtained by the MD

simulation, a graphical representation of the simulated system is

provided in Fig. 5(a) where 12b (black color) included in the inte-

rior of the bilayer (dipalmitoyl phospatidylcholine (DPPC): green

color). Fig. 5(b) and (c) depicts the most preferable conformations

of 12b in decreasing order as obtained from a clustering algorithm

available, which is available in the UCSF Chimera software [39]. As

already mentioned, 12b was initially placed into the water phase

and during the course of the simulation penetrated into the interior

of the bilayer. The mass density proﬁles of all components along the

2.4. Molecular dynamics (MD) of 12b at the binding site of AT1

It has been postulated that the molecular basis of AT1 anti-

hypertensive action can be interpreted by a two-step model. In the

ﬁrst step, the ARB is incorporated into the membrane bilayer

through the lipidewater interface and secondly laterally diffuses to

reach the binding site of the AT1 receptor in order to exert its

358

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

Table 1

¹H NMR (800 MHz) chemical shift assignments of 12b in D₂O solution at 25 ^ꢁC.

Hydrogen

Chemical

No. of

Multiplicity

shift (ppm)

hydrogens

9

8

7

6

10

10⁰

0.54

0.97

1.07

2.16

5.06

5.13

6.85

6.86

6.97

6.99

7.04

7.21

7.25

7.31

7.55

8.47

3

2

4

2

1

3

1

2

1

t

m

t

s

bs

d

s

d

t

12, 13, 15, 16

12⁰, 16⁰

18

5

13⁰and 15⁰, 18⁰

19

19⁰

20, 20⁰

21, 21⁰

2

t

m

d

s

(a)

Z-axis are provided in Fig. 6; it is clearly seen that 12b under study

is found inside the bilayer (red curve). The peak of its distribution is

nearly 1.1 nm far from the center of the bilayer which is deﬁned as

the minimum of the DPPC distribution (blue color). Another

Fig. 3. Binding mode of analog 12b at the binding site of AT1 receptor. The hydro-

phobic region of the binding site is shown in yellow, and the hydrophilic site that

ꢀ

surrounds it by a 6 A radius is shown in blue. The Phe 249 (red color) is situated be-

(b)

tween the two biphenyltetrazole moieties of analog 12b. (For interpretation of the

references to colour in this ﬁgure legend, the reader is referred to the web version of

this article.)

12b

(c)

Losartan

Fig. 5. (a) A graphical representation of the simulated system. (b), (c) Most preferable

conformations of analog 12b in the DPPC bilayer.

interesting observation is that 12b is found in a depth that permits

an interaction with some water molecules as seen by the intercept

of the distribution of water (black curve) and drug. A comple-

mentary view is given by Fig. 7, in which the distance between

Fig. 4. Superimposition of Losartan (blue) and analog 12b (brown). (For interpretation

of the references to colour in this ﬁgure legend, the reader is referred to the web

version of this article.)

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

359

Fig. 6. Mass density proﬁles along the Z-axis of all components, i.e. water, DPPC and

analog 12b.

Fig. 8. PMF (along the Z-axis) of analog 12b from water to DPPC phase.

DPPC and drug centers of mass is monitored over the last 40 ns of

the MD simulation. The zero in the aforementioned diagram cor-

responds to the center of bilayer. In order to give a clearer picture of

the simulated system, the average distance of phosphorous atoms

(DPPC molecules) in both leaﬂets is depicted as well, indicating

a stable membrane with a thickness around 3.8 nm. It may also be

concluded that the drug ﬂuctuates around a distance of 1.1 nm from

the bilayer center. However, it does not move signiﬁcantly and

therefore there is no tendency for crossing events, i.e. the move of

a host molecule from one leaﬂet to the other through the middle of

the bilayer.

interaction of this drug with DPPC and water molecules in terms of

hydrogen bonding. To this end, a GROMACS tool was applied which

makes use of standard geometrical criteria such as distance be-

tween acceptor and donor (<0.35 nm) and the angle among

acceptorehydrogenedonor (<30^ꢁ). The average number of hydro-

gen bonds of 12b with DPPC and the penetrated water molecules is

1.366 and 1.519, respectively, showing that indeed there is a sig-

niﬁcant interaction with these components. In particular, DPPC

oxygen atoms participate in a hydrogen-bond network with two

nitrogen atoms of 12b which are indicated by two arrows in

Fig. 5(b). An additional conﬁrmation of the previous observation is

provided in Fig. 9 by the fully overlapping density proﬁles of oxygen

(DPPC) and nitrogen atoms (12b).

The potential of mean force (PMF) of 12b from water to lipid

phase is depicted in Fig. 8, in which the zero at the abscissa cor-

responds to the bilayer center while remote there is pure water. The

position of minimum is about 1 nm far from the bilayer center and

it is compatible to the position found by the MD simulation with

12b initially placed in the aqueous phase. Another interesting

observation is the high energy barrier (41 kJ mol^ꢀ1) that 12b has to

overcome so as to move from one leaﬂet to the other through the

middle of the bilayer. Besides, the free energy for partitioning into

the membrane was estimated by the difference of PMF at the water

phase (large distances) and at the location of the minimum. This

2.5. HOMO and LUMO quantum chemical calculations

Table 2 summarizes the results of HOMO and LUMO levels of the

compounds studies. Based on these results, binding afﬁnities and

the in vitro ANGII antagonist properties, several remarks can be

made. First of all, inclusion of halogens (Br, Cl, and I) in R¹position

reduced HOMO values as expected. As already mentioned HOMO

energy values decrease with the presence of electron withdrawing

groups (EWGs) such as halogens. A decrease in in vitro binding

afﬁnity as determined in radioligand binding studies for com-

pounds 19c and 19d was also reported. The inclusion of a small

value is approximately equal to ꢀ81 kJ mol^ꢀ1

.

Having deﬁned the preferred position of 12b in the membrane

from the mass density proﬁles and the PMF, we analyzed the

Fig. 7. Distance between analog 12b and bilayer centers of mass. This plot is over the

Fig. 9. Density proﬁles along Z-axis of DPPC oxygens and analog 12b nitrogens. The

last 40 ns of the simulation. No crossing events were observed.

points of black curve have been multiplied by 10 for visualization purposes.

360

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

Table 2

variable (HOMO & LUMO). The accuracy of the Decision Tree is 92%.

The graphical representation of the Decision Tree for the data set is

presented in Fig.11. According to the DecisionTree, the design of new

molecules as ARBs can been accomplished by high HOMO and LUMO

energy values. More speciﬁcally, in the case of HOMO values are

greater than ꢀ0.31916 eV and LUMO greater than ꢀ0.14252, the

compounds exert high binding afﬁnity to the receptor. HOMO and

LUMO ﬁelds are graphically depicted in Fig. 11 for 11.

HOMO and LUMO energies, binding afﬁnity expressed as elogIC₅₀values and clas-

siﬁcation of synthesized analogs according to the afﬁnity.

Compd

HOMO (eV)

LUMO (eV)

Binding afﬁnity

elogIC₅₀

Classiﬁcation

11

ꢀ0.3185

ꢀ0.1449

9.46 ꢃ 0.44

9.04 ꢃ 0.38

8.54 ꢃ 0.18

8.37 ꢃ 0.15

6.08 ꢃ 0.14

6.15 ꢃ 0.22

5.19 ꢃ 0.42

7.37 ꢃ 0.29

5.77 ꢃ 0.46

6.38 ꢃ 0.02

6.48 ꢃ 0.10

6.30 ꢃ 0.08

High afﬁnity

Low afﬁnity

High afﬁnity

Low afﬁnity

12a

12b

14

19c

19d

23c

23d

27

ꢀ0.26218

ꢀ0.28058

ꢀ0.31844

ꢀ0.30837

ꢀ0.30956

ꢀ0.31631

ꢀ0.31508

ꢀ0.32167

ꢀ0.31916

ꢀ0.33018

ꢀ0.33136

ꢀ0.13175

ꢀ0.1382

ꢀ0.14207

ꢀ0.14742

ꢀ0.14558

ꢀ0.14252

ꢀ0.13845

ꢀ0.13413

ꢀ0.14134

ꢀ0.13951

ꢀ0.15484

2.6. Pharmacological characterization and SAR

In order to pharmacologically characterize the synthesized

analogs, we determined their binding afﬁnities (elogIC₅₀) for the

human AT1 receptor in radioligand competition binding assay

(Table 3, Fig. 12(a) and (b)), as well as their ability to antagonize the

activity of ANGII in the rat uterus in vitro test (pA₂) of the high

afﬁnity compounds (elogIC₅₀ꢂ 6.50) using Losartan as a reference

compound (Table 3). Interestingly, the introduction of an addi-

tional biphenyltetrazole moiety on the 4(5)-butylimidazole led

to the N,N⁰-bis-substituted imidazolium salt 11, which is bound to

AT1 receptor with higher afﬁnity (elogIC₅₀¼ 9.46) than Losartan

(elogIC₅₀¼ 8.25) (Table 3, Fig. 12). Furthermore, the water soluble

potassium (elogIC₅₀¼ 9.04) and sodium (elogIC₅₀¼ 8.54) salts of

compound 11 (12a and 12b, respectively) showed slightly

higher binding afﬁnities compared to Losartan (elogIC₅₀¼ 8.25)

(Table 3, Fig. 12). Similar to binding afﬁnities, the potency of 12b

(pA₂¼ 8.45) to antagonize the activity of ANGII was higher com-

pared to Losartan (pA₂¼ 8.25). Acquisition of high binding afﬁnity

and potency has also been achieved by the introduction of the

hydroxymethyl group at the C-2, resulting in 14 (elogIC₅₀¼ 8.37,

pA₂¼ 8.58) (Table 3, Fig. 12). Replacement of the tetrazole with

the bioisostere carboxy group, thus resulting in 13 and 15 sig-

niﬁcantly decreased binding afﬁnity (Fig. 12) in line with previous

studies in which tetrazole is a superior non-classical isostere of

carboxylate [10,19]. More importantly, the halogenated analogs

19c, 19d, 23c, 23d (Table 3, Fig. 12) showed remarkable drop in

binding afﬁnity in contrast to the halogenated series of

Losartan which retained afﬁnity [10] indicating the ambiguous

function of substituents at C-4 of imidazole [19]. Interestingly,

the bis-alkylation of 2-butylimidazole and 2-butyl-4-chloro-5-

hydroxymethylimidazole, the key precursor in the synthesis

of Losartan, resulted in compounds 27 (elogIC₅₀¼ 5.77) and 30 (e

logIC₅₀¼ 6.38) (Table 3, Fig. 12) displaying dramatic loss of binding

afﬁnity compared to 11, in which the n-butyl group at the C-5 is

exposed, thus enhancing binding to the receptor. These results

suggested that the position of the n-butyl group plays a signiﬁcant

role in their interaction with the AT1 receptor.

30

13

15

sized group such as eCH₂OH (14 and 15) did not signiﬁcantly

change HOMO and LUMO values but resulted also in a reduced

binding afﬁnity. The afﬁnity was further reduced when halogens

were also included in the R¹position (23c, 23d) as expressed in

in vitro activity (pA₂). Inclusion of K and Na (12a and 12b) sig-

niﬁcantly reduced HOMO values and a decrease in binding afﬁnity

(elogIC₅₀) was also reported. A switch of the substituents in R²and

R³position for 11 and 27, 19c and 30 also resulted in a decreased

activity while HOMO increased and LUMO decreased.

We have further explored the HOMO LUMO energies by devel-

oping a simple Decision Tree with the primary purpose of discrim-

inating compounds intotwo categories, lowafﬁnityand high afﬁnity,

(threshold elogIC₅₀< 6.48). HOMO and LUMO values were used as

inputvariablesforthedevelopmentof theDecisionTree(Fig.10)[40].

The visual inspection of the Decision Tree helps in the analysis and

interpretation of the structural data, the existence of clusters and

outliers, the relationship between samples and the inﬂuence of each

Fig. 10. Graphical representation of the Decision Tree.

Fig. 11. HOMO and LUMO orbitals of analog 11.

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

361

Table 3

Biological activity of synthesized analogs.

Compd

R¹R²

R³

R

Binding afﬁnity Uterotonic activity

elogIC₅₀

in vitro pA₂

11

H

Bu

H

K

9.46 ꢃ 0.44

9.04 ꢃ 0.38

12a

12b

14

19c

19d

23c

23d

27

Na 8.54 ꢃ 0.18

8.45 ꢃ 0.39

8.58 ꢃ 0.39

CH₂OH

H

CH₂OH

Bu

H

8.37 ꢃ 0.15

6.08 ꢃ 0.14

6.15 ꢃ 0.22

5.19 ꢃ 0.42

7.37 ꢃ 0.29

5.77 ꢃ 0.46

6.38 ꢃ 0.02

8.25 ꢃ 0.06

Cl Bu

Br Bu

Cl Bu

Br Bu

7.13 ꢃ 0.13

8.25 ꢃ 0.13

H

30

Losartan

Cl CH₂OH Bu

13

15

H

Bu

H

6.48 ꢃ 0.10

6.30 ꢃ 0.08

CH₂OH

3. Conclusions

The present study refers to the design and syntheses of bis-

alkylated 4(5)-butylimidazole analogs as potent ARBs. In partic-

ular, a rational design was performed to synthesize a series of N,N⁰-

symmetrically bis-substituted imidazole analogs bearing at the N-1

and N-3 two biphenylmethyl moieties ortho substituted either with

tetrazole or carboxylate functional groups. This work describes for

the ﬁrst time potent N,N⁰-bis-alkylated analogs in contrast to the

marketed ARBs, which their heterocyclic core is monoalkylated.

Furthermore, we elaborated general synthetic methodology for the

assembly of the n-butyl group onto the imidazole ring via the

Wittig reaction as well as the bis-alkylation of the N-1 and N-3 of

4(5)-butylimidazole and 2-butylimidazole with the biphe-

nylmethyl moiety. A remarkably efﬁcient approach was also

adopted for the introduction of the hydroxymethyl group at the C-2

position, as we have previously shown [19]. The SAR studies

demonstrated the importance of the nature and the position of

pharmacophores. In particular, the presence of two anionic tetra-

zole groups along with the lipophilic n-butyl group at the C-5 led to

the potent analogs 11 and its water soluble salts 12a and 12b. The

introduction of the electronegative hydroxymethyl group at the C-2

(analog 14) led to acquisition of afﬁnity and potency, while the

presence of a halogen atom at the C-4 of the imidazole ring (analogs

19cee and 23cee) led to an essential decrease in afﬁnity. Therefore,

Fig. 12. Competition binding isotherms of ANGII analogs to human AT1 receptor.

Competition of [¹²⁵I-Sar¹-Ile⁸] ANGII speciﬁc binding by increasing concentrations of

ANGII analogs was performed as described under “materials and methods” using

membranes from HEK 293 cells stably expressing the human AT1 receptor. The means

and S.E. are shown from 2 to 4 different experiments. The data were ﬁt to a one-site

competition model by nonlinear regression and the elogIC₅₀values were deter-

mined as described under “materials and methods”. (a) Losartan and analogs 11, 12a,

12b, 13, 14, 15, 27 and 30 (b) Losartan and analogs 19c, 19d, 23c and 23d.

the presence of two tetrazole rings seems to be optimal for high

afﬁnity in contrast to the carboxy group (analogs 13, 15) which

represents poor bioisostere for the tetrazole group, since these

analogs showed remarkably low afﬁnity. The position of the n-butyl

group plays a signiﬁcant role, since reorientation from the 4- (an-

alogs 11, 12a, 12b) to the 2-position (27, 30) of the imidazole ring

resulted in a sharp drop of binding afﬁnity. This dramatic loss of

activity may be explained by the concealment of the n-butyl group

between the two biphenyltetrazole moieties.

362

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

Of paramount importance were the biological results of the

2.22 mmol) in anhydrous THF (8 mL), was stirred at rt for 8 h under

Ar atmosphere. Then, the reaction mixture was quenched with

saturated aqueous NaHCO₃(5 mL) solution and extracted with CH₂Cl₂

(3 ꢄ 15 mL). The organic layer was washed with H₂O (1 ꢄ 10 mL),

dried over Na₂SO₄and concentrated in vacuo. The residue was pu-

riﬁed by ﬂash column chromatography (silica gel, EtOAc/Hex 1:1) to

furnish the Z-3 isomer (0.44 g) which is the sole isolated product.

Yield: 81%; R_f0.63 (EtOAc/Hex 4:1); t_R12.42 min (30% MeCN / 100%

MeCN in 30 min); ESI-MS (m/z): 123.18 [M þ H], 243.26 [Tr]; ¹H NMR

synthetic analog 12b. Its higher afﬁnity and potency to antagonize

ANGII compared to Losartan can be explained by docking studies

which demonstrated an additional hydrophobic cavity. MD calcu-

lations on lipid bilayers showed that this molecule is incorporated

at the interface and exerts a network of interactions with polar

region. Thus, this molecule does not only interact with the same

binding site as other sartans but also acts through lipid bilayer at

the same topographical location [41e44]. Particularly, it anchors at

the interface as other sartans where exerts its amphiphilic in-

teractions both with the polar region and upper hydrophobic seg-

ments of the phospholipid bilayers. Biophysical studies are in

progress to provide more information on this issue. Another

important ﬁnding from these studies is the critical role of the ori-

entation of the lipophilic n-butyl group. The highly hydrophobic

residues of receptor that surround this group and interact through

van der Waals interactions can explain its orientation signiﬁcance.

(400 MHz, CDCl₃):

d 7.64 (br s, 1H), 7.36e7.35 (m, 9H), 7.16e7.13 (m,

6H), 6.75 (br s, 1H), 6.25 (dt, 1H, J ¼ 1.5, 11.6 Hz), 5.60 (dt, 1H, J ¼ 7.5,

11.6 Hz), 2.28 (quint d, 2H, J ¼ 1.7, 7.5 Hz), 1.00 (t, 3H, J ¼ 7.5 Hz); ¹³C

NMR (160 MHz, CDCl₃):

d 141.70, 137.64, 137.15, 134.53, 129.66,

128.29, 128.18, 120.20, 119.00, 76.03, 22.55, 13.79.

4.2.3. 1-Trityl-4-butylimidazole (4a)

A solution of 3a (0.30 g, 0.82 mmol) in anhydrous MeOH (10 mL)

was transferred in a hydrogenation ﬂask and Pd/C (10% wt/wt,

0.04 g, 0.04 mmol) was added. The reaction mixture was allowed to

proceed at rt, under a H₂atmosphere at a pressure of 3 bar (Parr

hydrogenator apparatus) for 2 h. Then, the catalyst was ﬁltered

through a pad of Celite^Òand washed with MeOH. The combined

ﬁltrates were concentrated in vacuo to furnish 4a as a white foam

(0.28 g), which was directly used without further puriﬁcation.

Yield: 92%; R_f0.53 (EtOAc/Hex 4:1); t_R13.15 min (30%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 367.50 [M þ H],

4. Experimental section

4.1. Chemistry

4.1.1. General

Starting materials were obtained by Aldrich and used as

received. All reactions involving air sensitive reagents were per-

formed under Ar atmosphere using syringe-septum cap techniques.

Melting points were determined with a Stuart SMP 10 apparatus

and are uncorrected. Hydrogenation reaction was carried out in

a Parr Hydrogenation Apparatus equipped with a 4 L Hydrogen

tank. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker

Avance DPX spectrometer at 400.13 MHz and 161.76 MHz, respec-

tively. Chemical shifts are reported in units, parts per million (ppm)

downﬁeld from tetramethylsilane (TMS) and coupling constants (J)

are given in Hertz (Hz). The 2D DQF-COSY and ROESY NMR spectra

were recorded on a Varian DirectDrive 800 MHz spectrometer at

25 ^ꢁC. 2 mg of 12b were dissolved in 0.7 mL of D₂O. Experiments

were recorded in the phase-sensitive mode using the pulse se-

quences in the Varian library of pulse programs. Spectra were ac-

quired with a spectral width of 8012 Hz, 4096 data points in t₂, 4e

32 scans, 512 complex points in t₁, and a relaxation delay of 1.5 s.

The mixing time in 2D ROESY was 75 ms. HPLC analysis was per-

formed on an Alliance Waters 2695 equipped with a Waters 2996

Photodiode Array Detector UVevis, using the XBridge C18 column

243.38 [Tr]; ¹H NMR (400 MHz, CDCl₃):

d 7.64 (s, 1H), 7.33e7.31 (m,

10H), 7.15e7.13 (m, 6H), 6.50 (s, 1H), 2.53 (t, 2H, J ¼ 7.5 Hz), 1.59 (q,

2H, J ¼ 7.5 Hz), 1.33 (sext, 2H, J ¼ 7.5 Hz), 0.91 (t, 3H, J ¼ 7.5 Hz); ¹³C

NMR (160 MHz, CDCl₃):

d 148.81, 142.63, 142.12, 138.16, 129.79,

127.93, 127.89, 127.85, 127.15, 117.60, 74.99, 31.48, 28.17, 22.41, 13.91.

4.2.4. 4(5)-Butylimidazole (5a)

Compound 4a (0.20 g, 0.55 mmol) was dissolved in a solution of

20% TFA in CH₂Cl₂(2 mL) and then Et₃SiH (0.09 mL, 0.55 mmol) was

added as scavenger. The resulting solution was stirred at rt for 1 h

and concentrated in vacuo. Precipitation from Et₂O furnished the

TFA salt of 5a as an amorphous powder. The TFA salt was neutralized

by treatment with saturated aqueous NaHCO₃solution for 30 min at

rt and extracted with CH₂Cl₂(3 ꢄ 10 mL). The organic phase was

dried over Na₂SO₄, ﬁltered and concentrated in vacuo to afford 5a

(0.06 g) as a yellow solid. Yield: 87%; m.p. 45e47 ^ꢁC; R_f0.60 (CHCl₃/

MeOH 9:1); t_R6.82 min (5% MeCN / 100% MeCN in 30 min); ESI-

MS (m/z): 125.19 [M þ H]; ¹H NMR (400 MHz, CD₃OD):

d 7.54 (s,

(4.6 ꢄ 150 mm, 3.5

mm) as stationary phase and a gradient of H₂O/

1H), 6.74 (s, 1H), 2.59 (t, 2H, J ¼ 7.2 Hz), 1.62 (quint, 2H, J ¼ 7.2 Hz),

MeCN both containing 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. All reactions were carried out in anhydrous

solvents. Analytical TLC was performed on silica gel 60 F₂₅₄plates

(Merck, Germany) and visualized by UV irradiation and iodine.

1.38 (sext, 2H, J ¼ 7.2 Hz), 0.97 (t, 3H, J ¼ 7.2 Hz); ¹³C NMR (160 MHz,

CD₃OD):

d 136.83, 134.47, 117.28, 31.76, 26.04, 22.28, 13.17.

4.3. Synthesis of 2-butylimidazole (5b), method A

4.3.1. 1-[(N,N-Dimethylamino)methyl]imidazole (2b)

4.2. Synthesis of 4(5)-butylimidazole (5a)

Prepared from imidazole (1) according to the literature method

[23]. Data were consistent with literature. R_f0.44 (CHCl₃/MeOH

9:1); t_R2.41 min (5% MeCN / 100% MeCN in 30 min); ESI-MS (m/

4.2.1. 1-Trityl-imidazole-4-carboxaldehyde (2a)

Prepared from imidazole (1) as starting material according to

the literature method [20,21]. Data are consistent with literature. R_f

0.53 (EtOAc/Hex 4:1); t_R11.59 min (30% MeCN / 100% MeCN in

z): 126.58 [M þ H]; ¹H NMR (400 MHz, CDCl₃):

d 7.44 (s, 1H), 7.01 (s,

1H), 6.90 (s, 1H), 4.61 (s, 2H), 2.23 (s, 6H); ¹³C NMR (160 MHz,

CDCl₃):

d 137.63, 129.08, 119.74, 69.25, 41.49.

30 min); ¹H NMR (400 MHz, CDCl₃):

d

9.88 (s, 1H), 7.61 (d, 1H,

J ¼ 1.3 Hz), 7.53 (s, 1H, J ¼ 1.3 Hz), 7.38e7.36 (m, 9H), 7.12e7.10 (m,

6H); ¹³C NMR (160 MHz, CDCl₃):

186.51, 141.47, 140.79, 140.56,

129.60, 128.49, 128.34, 127.88, 126.72.

4.3.2. 2-Butylimidazole (5b)

d

To a solution of 2b (0.25 g, 2 mmol) in anhydrous THF (10 mL)

under Ar atmosphere was added n-BuLi (1.6 M in hexanes, 1.37 mL,

2.20 mmol) dropwise via a syringe at ꢀ78 ^ꢁC. After 30 min, DMPU

(0.48 mL, 4.0 mmol) was added at ꢀ70 ^ꢁC and stirred for additional

30 min before n-BuI (0.27 mL, 2.40 mmol) was added at ꢀ78 ^ꢁC. The

reaction mixture was allowed to warm to rt over a period of 16 h.

4.2.2. (Z)-1-Trityl-4-(but-1-enyl)imidazole (3a)

A suspension of 2a (0.50 g, 1.48 mmol), propyl triphenylphos-

phonium bromide (0.63 g,1.63 mmol) and NaH (powered 95%, 0.05 g,

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

363

Then, aqueous HCl (2 M, 8 mL) was added, the organic solvent was

removed and the resulting mixture was neutralized with saturated

aqueous NaHCO₃solution and extracted with CH₂Cl₂(3 ꢄ15 mL) The

organic phase was dried over Na₂SO₄and concentrated in vacuo to

furnish 5b as a pale yellow oil (0.21 g). Data were consistent with

literature. Yield: 85%; R_f0.54 (CHCl₃/MeOH 9:1) t_R5.52 min (5%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 125.17 [M þ H]; ¹H

CH₂Cl₂(3 ꢄ 10 mL). The combined organic extracts were washed

with brine, dried over Na₂SO₄and concentrated in vacuo to afford 7

as a pale yellow oil (0.09 g, 86%).

4.5. General procedure 1: bis-alkylation of 4(5)-butylimidazole (5a)

4.5.1. 4-Butyl-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]biphenyl-4-yl]

methyl}imidazolium bromide (7)

NMR (400 MHz, CDCl₃):

(quint, 2H, J ¼ 7.5 Hz),1.34 (sext, 2H, J ¼ 7.5 Hz), 0.93 (t, 3H, J ¼ 7.5 Hz);

¹³C NMR(160MHz, CDCl₃):

148.97,120.84, 30.81, 27.64, 22.20,12.95.

d

6.87 (s, 2H), 2.68 (t, 2H, J ¼ 7.5 Hz), 1.67

To a solution of 4(5)-butylimidazole (5a) (1.00 g, 8.05 mmol) in

anhydrous DMF (30 mL) were sequentially added K₂CO₃(2.22 g,

16.10 mmol) and the alkylating agent 6a (10.31 g, 18.52 mmol)

under Ar atmosphere and stirred at rt for 8 h. Then, the mixture

was diluted with CH₂Cl₂(90 mL), washed with H₂O (3 ꢄ 50 mL),

the organic phase was dried over Na₂SO₄, ﬁltered and concentrated

in vacuo. The resulting residue was precipitated from cold Et₂O to

afford 7 as a white solid. Yield 78%; R_f0.45 (CHCl₃/MeOH, 9:1); t_R

15.93 min (70% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):

d

4.4. Synthesis of 2-butylimidazole (5b), method B

4.4.1. 1-(N,N-Dimethylsulfamoyl)imidazole-2-carboxaldehyde (2c)

Prepared from imidazole (1) according to the literature method

[25]. Data are consistent with literature. R_f0.20 (EtOAc/Hex 7:3); t_R

5.00 min (5% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):

1078.56 [M ꢀ Br], 243.17 [Tr]; ¹H NMR (400 MHz, CDCl₃):

d 11.03 (s,

204.29 [M þ H]^þ; ¹H NMR (400 MHz, CDCl₃):

d 9.93 (s, 1H), 7.58 (d,

1H), 7.90e7.84 (m, 2H), 7.43e7.30 (m, 5H), 7.30e7.09 (m, 25H),

6.88e6.83 (m, 14H), 6.46 (s, 1H), 5.31 (s, 4H), 2.20 (t, 2H, J ¼ 7.5 Hz),

1.26e1.09 (m, 4H), 0.72 (t, 3H, J ¼ 7.5 Hz).

1H, J ¼ 1.5 Hz), 7.30 (d, 1H, J ¼ 1.5 Hz), 3.00 (s, 6H); ¹³C NMR

(160 MHz, CDCl₃):

d 179.09, 143.45, 130.16, 125.87, 38.56.

4.4.2. 1-(N,N-Dimethylsulfamoyl)-2-(but-1-enyl)imidazole (3c)

To a stirred suspension of propyl triphenylphosphonium bro-

mide (1.43 g, 3.72 mmol) and anhydrous t-amyl alcohol (0.45 mL,

4.09 mmol) in anhydrous THF (14 mL) was added NaH (powdered

95%, 0.10 g, 4.09 mmol) at 0 ^ꢁC under Ar atmosphere and stirred at

rt for 2 h. To the resulting yellow phosphorous ylide was added

a solution of 2c (0.38 g, 1.86 mmol) in anhydrous THF (2 mL)

dropwise at 0 ^ꢁC. The mixture was stirred at rt over a period of 8 h

before being quenched with saturated aqueous NaHCO₃solution

and extracted with CH₂Cl₂(3 ꢄ 20 mL). The organic layer was

washed with H₂O (2 ꢄ 10 mL), dried over Na₂SO₄and concentrated

in vacuo. The residue was puriﬁed by ﬂash column chromatography

(silica gel, EtOAc/Hex 3:2) to afford the pure Z- and E-9 isomers as

yellow oils (0.26 g and 0.10 g, respectively). Z-Isomer: yield: 62%; R_f

0.46 (EtOAc/Hex 7:3); t_R10.08 min (5% MeCN / 100% MeCN in

30 min); ESI-MS (m/z): 230.33 [M þ H]^þ; ¹H NMR (400 MHz,

4.5.2. 4-Butyl-N,N⁰-bis{[2⁰-(tert-butoxycarbonyl)biphenyl-4-yl]

methyl}imidazolium bromide (8)

The same procedure was employed for the alkylation of 5a by

treatment with the alkylating agent 6b. Yield 82%; R_f0.32 (CHCl₃/

MeOH 9:1); t_R9.59 min (60% MeCN / 100% MeCN in 30 min); ESI-

MS (m/z): 657.63 [M ꢀ Br]; ¹H NMR (400 MHz, CDCl₃):

d 11.02 (s,

1H), 7.80 (dd, 2H, J ¼ 7.5 Hz, J ¼ 1.2 Hz), 7.47 (t, 2H, J ¼ 7.6 Hz), 7.40

(t, 2H, J ¼ 7.6 Hz), 7.35e7.22 (m, 8H), 7.11 (d, 2H, J ¼ 7.5 Hz), 6.79 (s,

1H), 5.56 (s, 2H), 5.50 (s, 2H), 2.43 (t, 2H, J ¼ 7.4 Hz), 1.44 (quint, 2H,

J ¼ 7.4 Hz), 1.30 (sext, 2H, J ¼ 7.4 Hz), 1.21 (s, 18H), 0.82 (t, 3H,

J ¼ 7.4 Hz).

4.6. General procedure 2: hydroxymethylation at C-2 position of 7

4.6.1. 4-Butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]

biphenyl-4-yl]methyl}imidazolium bromide (9)

CDCl₃):

d

7.27 (d, 1H, J ¼ 1.3 Hz), 7.07 (d, 1H, J ¼ 1.3 Hz), 6.66 (dt, 1H,

In

a sealed tube were sequentially added 7 (0.50 g,

J ¼ 1.3, 11.7 Hz), 5.98 (dt, 1H, J ¼ 7.4, 11.7 Hz), 2.85 (s, 6H), 2.70 (quint

0.43 mmol), DMF (1 mL), 37% formalin (0.11 mL, 1.48 mmol) and

diisopropylethylamine (0.51 mL, 3.01 mmol). The resulting mix-

ture was stirred at 80 ^ꢁC until HPLC showed no starting material

left (ca. 1 h). Then, the mixture was diluted with CH₂Cl₂(30 mL),

washed with 5% aqueous citric acid (2 ꢄ 10 mL), brine (ꢄ10 mL),

the organic phase was dried over Na₂SO₄, ﬁltered and con-

centrated in vacuo. The resulting residue was precipitated from

cold Et₂O to afford 9 as a white powder. Yield 84%; R_f0.36 (CHCl₃/

MeOH 9:1); t_R15.32 min (70% MeCN / 100% MeCN in 30 min);

ESI-MS (m/z): 1108.06 [M ꢀ Br]; ¹H NMR (400 MHz, CDCl₃):

d, 2H, J ¼ 1.9, 7.4 Hz), 1.08 (t, 3H, J ¼ 7.4 Hz); ¹³C NMR (160 MHz,

CDCl₃):

d 145.14, 142.46, 128.00, 119.69, 114.76, 38.37, 22.86, 13.73.

E-Isomer: yield: 24%; R_f0.25 (EtOAc/Hex 7:3); t_R10.26 min (5%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 230.29 [M þ H]^þ;

¹H NMR (400 MHz, CDCl₃):

d

7.25 (d, 1H, J ¼ 1.5 Hz), 6.99 (s, 1H,

J ¼ 1.5 Hz), 6.90 (dt, 1H, J ¼ 7.4, 15.6 Hz), 6.78 (d, 1H, J ¼ 15.6 Hz),

2.85 (s, 6H), 2.82 (quint d, 2H, J ¼ 1.4, 7.4 Hz), 1.10 (t, 3H, J ¼ 7.4 Hz);

¹³C NMR (160 MHz, CDCl₃):

38.33, 26.00, 12.91.

d 146.38, 141.41, 128.06, 119.89, 115.86,

4.4.3. 1-(N,N-Dimethylsulfamoyl)-2-butylimidazole (4c)

d 7.96e7.93 (m, 2H), 7.50e7.43 (m, 5H), 7.37e7.14 (m, 20H), 7.07

Compound 4c was prepared in an analogous manner to that

described for 4a. A mixture of the isolated Z- and E- was subjected

to catalytic hydrogenation. Yield: 90%; R_f0.44 (CHCl₃/MeOH 95:5);

t_R10.45 min (5% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):

(d, 2H, J ¼ 8.0 Hz), 6.94e6.91 (m, 15H), 6.85 (d, 2H, J ¼ 8.0 Hz),

6.46 (s, 1H), 5.53 (s, 2H), 5.47 (s, 2H), 4.64 (s, 2H), 2.34 (t, 2H,

J ¼ 7.5 Hz), 1.40 (quint, 2H, J ¼ 7.5 Hz), 1.26 (sext, 2H, J ¼ 7.5 Hz),

0.80 (t, 3H, J ¼ 7.5 Hz).

231.97 [M þ H]^þ; ¹H NMR (400 MHz, CD₃OD):

d 7.38 (d, 1H,

J ¼ 1.4 Hz), 6.64 (d, 1H, J ¼ 1.4 Hz), 3.30 (s, 6H), 2.95 (t, 2H,

4.6.2. 4-Butyl-2-hydroxymethyl-N,N⁰-bis{[[2⁰-(tert-butoxycarbonyl)]

biphenyl-4-yl]methyl}imidazolium bromide (10)

J ¼ 7.6 Hz),1.73 (quint, 2H, J ¼ 7.6 Hz),1.40 (sext, 2H, J ¼ 7.6 Hz), 0.95

(t, 3H, J ¼ 7.6 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

144.16, 126.16,

The same procedure was employed for the hydroxymethylation

of 8. Yield: 81%; R_f0.31 (CHCl₃/MeOH 9:1); t_R8.91 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 687.56 [M ꢀ Br];

119.66, 37.16, 30.08, 27.68, 21.97, 12.66.

4.4.4. 2-Butylimidazole (5b)

¹H NMR (400 MHz, CDCl₃):

d

7.72 (t, 2H, J ¼ 7.0 Hz), 7.47e7.40 (m,

A solution of 4c (0.20 g, 0.86 mmol) in THF (2 mL) was reﬂuxed

with 1.5 N HCl (3 mL) for 2 h and then cooled. After neutralization

by addition of saturated aqueous NaHCO₃solution, the solvent was

concentrated in vacuo to give a residue, which was extracted with

2H), 7.36e7.17 (m, 10H), 7.04 (d, 2H, J ¼ 8.0 Hz), 6.85 (s, 1H), 5.66

(s, 4H), 4.82 (s, 2H), 2.48 (t, 2H, J ¼ 7.5 Hz), 1.49 (quint, 2H,

J ¼ 7.5 Hz), 1.31 (sext, 2H, J ¼ 7.5 Hz), 1.21 (s, 9H), 1.20 (s, 9H), 0.83

(t, 3H, J ¼ 7.5 Hz).

364

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

4.7. General procedure 3: removal of protective groups

4.9. 4-Butyl-N,N⁰-bis{[2-carboxybiphenyl-4-yl]methyl}imidazolium

bromide (13)

4.7.1. 4-Butyl-N,N⁰-bis{[[2⁰-(2H-tetrazol-5-yl)]biphenyl-4-yl]

methyl}imidazolium bromide (11)

Compound 7 (0.40 g, 0.34 mmol) was dissolved in a solution of

30% TFA (1.2 mL) in CH₂Cl₂(2.8 mL) and then Et₃SiH (54

General procedure 3 was employed for the preparation of 13.

Yield 79%; R_f0.24 (CHCl₃/MeOH/gl. AcOH, 8:2:0.2); t_R16.38 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 545.45

mL,

0.34 mmol) was added as a scavenger. The resulting solution was

stirred at rt for 1 h before being concentrated in vacuo. Precipi-

tation from Et₂O furnished an amorphous solid, then, dissolved in

CH₂Cl₂(20 mL) and the resulting mixture was washed with satu-

rated aqueous NaHCO₃solution (2 ꢄ 5 mL), dried over Na₂SO₄,

ﬁltered and concentrated in vacuo to furnish 11 as a white solid:

yield 72%; m.p. 201e203 ^ꢁC; R_f0.35 (CHCl₃/MeOH/gl. AcOH,

9:1:0.1); t_R12.35 min (20% MeCN / 100% MeCN in 30 min); ESI-

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d 9.09 (s, 1H), 7.87 (d, 2H,

J ¼ 7.6 Hz), 7.57 (t, 2H, J ¼ 7.6 Hz), 7.53 (s, 1H), 7.49e7.34 (m, 12H),

5.46 (s, 2H), 5.44 (s, 2H), 2.65 (t, 2H, J ¼ 7.3 Hz), 1.59 (quint, 2H,

J ¼ 7.3 Hz), 1.40 (sext, 2H, J ¼ 7.3 Hz), 0.93 (t, 3H, J ¼ 7.3 Hz); ¹³C

NMR (160 MHz, CD₃OD):

d 173.38, 144.83, 143.73, 139.69, 138.18,

134.16, 134.58, 133.57, 133.35, 132.72, 131.86, 131.50, 131.45, 130.21,

129.58, 121.68, 54.78, 52.25, 31.19, 25.23, 24.05, 14.81.

MS (m/z): 593.57 [M ꢀ Br]; ¹H NMR (400 MHz, DMSO-d₆):

d

9.35

4.9.1. 4-Butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)-

biphenyl-4-yl]methyl}imidazolium bromide (14)

(d, 1H, J ¼ 1.2 Hz), 7.71e7.67 (m, 4H), 7.62 (d, 2H, J ¼ 7.0 Hz), 7.59

(br s, 1H), 7.55 (d, 2H, J ¼ 7.0 Hz), 7.37 (d, 2H, J ¼ 8.1 Hz), 7.25 (d,

2H, J ¼ 8.1 Hz), 7.19e7.16 (m, 4H), 5.45 (s, 2H), 5.41 (s, 2H), 1.44

(quint, 2H, J ¼ 7.5 Hz), 1.28 (sext, 2H, J ¼ 7.5 Hz), 0.94 (t, 3H,

General procedure 3 was employed for the preparation of 14.

Yield: 83%; R_f0.29 (CHCl₃/MeOH/gl. AcOH, 9:1:0.1); t_R11.59 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 623.37

J ¼ 7.5 Hz); ¹H NMR (400 MHz, CD₃OD):

d

9.02 (br s, 1H), 7.71e7.67

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d 7.56e7.54 (m, 2H), 7.50e7.47

(m, 4H), 7.61e7.56 (m, 4H), 7.46 (br s, 1H), 7.36 (d, 2H, J ¼ 8.0 Hz),

7.26e7.20 (m, 6H), 5.40 (s, 2H), 5.38 (s, 2H), 2.57 (t, 2H, J ¼ 7.5 Hz),

1.56 (quint, 2H, J ¼ 7.5 Hz), 1.38 (sext, 2H, J ¼ 7.5 Hz), 0.92 (t, 3H,

(m, 2H), 7.44e7.41 (m, 4H), 7.37 (s, 1H), 7.20 (d, 2H, J ¼ 8.1 Hz), 7.16e

7.13 (m, 4H), 7.00 (d, 2H, J ¼ 8.1 Hz), 5.46 (s, 2H), 5.42 (s, 2H), 2.51 (t,

2H, J ¼ 7.5 Hz), 1.49 (quint, 2H, J ¼ 7.5 Hz), 1.32 (sext, 2H, J ¼ 7.5 Hz),

J ¼ 7.5 Hz); ¹³C NMR (160 MHz, DMSO-d₆):

d

160.54, 160.19, 143.46,

0.86 (t, 3H, J ¼ 7.5 Hz); ¹H NMR (400 MHz, DMSO-d₆):

d 7.69e7.53 (m,

142.99, 142.92, 138.99, 138.29, 136.00, 135.48, 133.30, 133.26,

133.10, 133.02, 132.30, 132.26, 132.22, 132.03, 130.57, 130.47, 130.29,

130.22, 130.12, 129.99, 122.21, 54.46, 51.98, 31.30, 25.27, 24.24,

16.25.

9H), 7.35 (d, 2H, J ¼ 8.0 Hz), 7.17e7.12 (m, 6H), 6.26 (s, 1H), 5.53 (s,

2H), 5.48 (s, 2H), 4.92 (s, 2H), 2.40 (t, 2H, J ¼ 7.4 Hz), 1.36 (quint, 2H,

J ¼ 7.4 Hz),1.22 (sext, 2H, J ¼ 7.4 Hz), 0.79 (t, 3H, J ¼ 7.4 Hz); ¹³C NMR

(160 MHz, CD₃OD):

d 161.22, 160.00, 145.06, 142.04, 141.62, 140.90,

135.96, 132.11, 132.02, 129.74, 129.71, 129.66, 125.68, 125.63, 119.28,

119.06, 51.38, 51.17, 48.09, 28.82, 23.05, 21.68, 12.54.

4.8. General procedure 4: conversion to the potassium salt 12a

4.8.1. Bis-potassium salt of 4-butyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)]

biphenyl-4-yl]methyl}imidazolium bromide (12a)

4.9.2. 4-Butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-carboxybiphenyl-4-yl]

methyl}imidazolium bromide (15)

To a suspension of 11 (0.20 g, 0.30 mmol) in MeOHeH₂O, 1:1

(4 mL) was added KOH (33 mg, 0.60 mmol) and the reaction

mixture was stirred at rt for 5 h. Activated charcoal was added to

the mixture, stirred for 10 min before being ﬁltered through a pad

of Celite^Ò. The ﬁltrate was concentrated in vacuo to remove ca. half

the solvent volume. The residue was precipitated with acetone to

afford a white solid. Lyophilization of the resulting solid furnished

the potassium salt 12a as a white foam. Yield 80%; R_f0.30 (CHCl₃/

MeOH/gl. AcOH, 9:1:0.1); t_R12.19 min (20% MeCN / 100% MeCN

in 30 min); ESI-MS (m/z): 631.23 [(M ꢀ K) þ H ꢀ Br]; ¹H NMR

General procedure 3 was employed for the preparation of 15.

Yield: 85%; R_f0.22 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R16.01 min (10%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 575.50 [M ꢀ Br]; ¹H

NMR (400 MHz, CD₃OD):

d

7.86 (t, 2H, J ¼ 7.0 Hz), 7.60e7.56 (m, 2H),

7.51 (s, 1H), 7.49e7.35 (m, 10H), 7.24 (d, 2H, J ¼ 8.0 Hz), 5.62 (s, 2H),

5.59 (s, 2H), 5.00 (s, 2H), 2.60 (t, 2H, J ¼ 7.4 Hz), 1.55 (quint, 2H,

J ¼ 7.4 Hz),1.37 (sext, 2H, J ¼ 7.4 Hz), 0.89 (t, 3H, J ¼ 7.4 Hz); ¹³C NMR

(160 MHz, CD₃OD):

d 171.46, 145.12, 142.36, 141.95, 140.76, 140.72,

136.11, 132.99, 132.71, 132.67, 130.44, 130.19, 129.08, 127.47, 127.14,

127.05, 125.97, 119.30, 51.37, 51.24, 28.82, 23.13, 21.73, 12.51.

(400 MHz, CD₃OD): d 8.93 (s, 1H), 7.60e7.58 (m, 2H), 7.53e7.50 (m,

2H), 7.47e7.45 (m, 4H), 7.41 (s, 1H), 7.27 (d, 2H, J ¼ 8.0 Hz), 7.21e

7.15 (m, 6H), 5.33 (s, 2H), 5.32 (s, 2H), 2.59 (t, 2H, J ¼ 7.5 Hz), 1.56

(quint, 2H, J ¼ 7.5 Hz), 1.39 (sext, 2H, J ¼ 7.5 Hz), 0.93 (t, 3H,

4.10. General procedure 5: halogenation of 4(5)-butylimidazole

4.10.1. 5-Butyl-4-chloro-1H-imidazole (16c)

J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

161.22, 161.21, 144.27,

To a solution of 5a (1.00 g, 8.05 mmol) in anhydrous DMF (5 mL)

was added NCS (1.18 g, 8.86 mmol) in three portions at 0 ^ꢁC. The

reaction mixture was allowed to warm to rt over a period of 2 h

before being quenched with H₂O (5 mL) and extracted with CH₂Cl₂

(3 ꢄ 20 mL). The combined organic phases were washed with brine,

dried over Na₂SO₄, ﬁltered and concentrated in vacuo. The oily

residue was puriﬁed by ﬂash column chromatography (silica gel,

CHCl₃/MeOH 95:5) to afford the title compound 16c as a white solid.

Yield: 73%; m.p. 103e105 ^ꢁC; R_f0.21 (CHCl₃/MeOH, 95:5); t_R

8.82 min (5% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 159.16

[M þ H] (³⁵Cl),161.11 [M þ H þ 2] (³⁷Cl); ¹H NMR (400 MHz, CD₃OD):

142.15, 143.16, 140.86, 140.82, 136.61, 131.93, 130.52, 130.50,

129.95, 129.88, 129.81, 129.73, 129.69, 128.75, 128.55, 127.62,

127.27, 127.19, 126.94, 119.25, 52.40, 49.89, 28.86, 22.86, 21.74,

12.58.

4.8.2. Bis-sodium salt of 4-butyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)]

biphenyl-4-yl]methyl}imidazolium bromide (12b)

The same procedure was employed for the preparation of so-

dium salt 12b. Yield 83%; t_R12.21 min (20% MeCN / 100% MeCN in

30 min); ESI-MS (m/z): 615.12 [(M ꢀ Na) þ H ꢀ Br]; ¹H NMR

(400 MHz, CD₃OD):

d

8.56 (s, 1H), 7.60e7.58 (m, 2H), 7.55e7.50 (m,

d

7.44 (s, 1H), 2.68 (t, 2H, J ¼ 7.5 Hz), 1.66 (quint, 2H, J ¼ 7.5 Hz), 1.46

2H), 7.47e7.45 (m, 4H), 7.42 (s,1H), 7.26 (d, 2H, J ¼ 8.2 Hz), 7.19e7.16

(m, 6H), 5.32 (s, 2H), 5.31 (s, 2H), 2.58 (t, 2H, J ¼ 7.4 Hz), 1.57 (quint,

2H, J ¼ 7.4 Hz),1.38 (sext, 2H, J ¼ 7.4 Hz), 0.93 (t, 3H, J ¼ 7.4 Hz) ppm;

(sext, 2H, J ¼ 7.5 Hz), 0.93 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz,

CD₃OD):

d 133.04, 126.14, 124.24, 31.05, 23.23, 22.0, 12.98.

¹³C NMR (160 MHz, CD₃OD):

d

163.52,144.56,143.12,134.25,133.78,

4.10.2. 4-Bromo-5-butyl-1H-imidazole (16d)

132.73, 132.17, 132.10, 132.03, 131.98, 130.96, 130.02, 129.50, 129.35,

121.43, 54.68, 52.18, 31.13, 25.15, 24.04, 14.83 ppm.

The same procedure was employed for the preparation of 16d.

Yield: 71%; m.p. 109e111 ^ꢁC; R_f0.21 (CHCl₃/MeOH/95:5); t_R

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

365

8.42 min (5% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 203.15

[M þ H] (⁷⁹Br), 205.11 [M þ H þ 2] (⁸¹Br); ¹H NMR (400 MHz,

CH₂Cl₂(3 ꢄ 20 mL). The combined organic extracts were dried over

Na₂SO₄, ﬁltered and concentrated in vacuo. Flash column chro-

matography (silica gel, CHCl₃/MeOH 97:3) afforded 18c as a yellow

foam. Yield: 61%; R_f0.35 (CHCl₃/MeOH 9:1); t_R11.52 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 691.53 [M ꢀ Br]

CD₃OD):

d

2.48 (t, 2H, J ¼ 7.5 Hz), 1.50 (quint, 2H, J ¼ 7.5 Hz), 1.27

(sext, 2H, J ¼ 7.5 Hz), 0.88 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz,

CD₃OD):

d 137.91, 132.64, 114.53, 34.66, 27.43, 25.56, 16.58.

(

³⁵Cl), 693.54 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz, CDCl₃):

d 11.46

4.10.3. 5-Butyl-4-iodo-1H-imidazole (16e)

(s, 1H), 7.73 (d, 2H, J ¼ 7.6 Hz), 7.50 (d, 2H, J ¼ 8.0 Hz), 7.43e7.28 (m,

8H), 7.21e7.16 (m, 4H), 5.60 (s, 2H), 5.57 (s, 2H), 2.52 (t, 2H,

J ¼ 7.5 Hz), 1.35e1.24 (m, 4H), 1.20 (s, 9H), 1.17 (s, 9H), 0.82 (t, 3H,

J ¼ 7.5 Hz).

The same procedure was employed for the preparation of 16e.

Yield: 72%; m.p. 93e95 ^ꢁC; R_f0.21 (CHCl₃/MeOH/95:5); t_R8.94 min

(5% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 250.85 [M þ H],

500.80 [2M þ H]; ¹H NMR (400 MHz, CD₃OD):

d 7.73 (s, 1H), 2.59 (t,

2H, J ¼ 7.5 Hz), 1.60 (quint, 2H, J ¼ 7.5 Hz), 1.35 (sext, 2H, J ¼ 7.5 Hz),

4.12.2. 4-Bromo-5-butyl-N,N⁰-bis{[2⁰-(tert-butoxycarbonyl)

biphenyl-4-yl]methyl}imidazolium bromide (18d)

0.96 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 140.56,137.92,

81.92, 34.98, 28.90, 25.62, 16.72.

The same procedure as above was employed for the preparation

of 18d. Yield: 64%; R_f0.36 (CHCl₃/MeOH 9:1); t_R11.34 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 736.45 [M ꢀ Br]

4.11. General procedure 6: bis-alkylation of 16cee with the

alkylating agent 6a

(

d

⁷⁹Br), 738.51 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz, CDCl₃):

11.46 (s, 1H), 7.72 (d, 2H, J ¼ 7.5 Hz), 7.48 (d, 2H, J ¼ 8.0 Hz), 7.41e

4.11.1. 5-Butyl-4-chloro-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]

biphenyl-4-yl]methyl}imidazolium bromide (17c)

7.27 (m, 8H), 7.21e7.16 (m, 4H), 5.59 (s, 2H), 5.56 (s, 2H), 2.54 (t, 2H,

J ¼ 7.5 Hz), 1.37e1.25 (m, 4H), 1.22 (s, 9H), 1.18 (s, 9H), 0.84 (t, 3H,

J ¼ 7.5 Hz).

To a solution of 16c (0.20 g, 1.26 mmol) in anhydrous THF (5 mL)

under Ar atmosphere were sequentially added anhydrous K₂CO₃

(0.34 g, 2.50 mmol), a catalytic amount of 18-crown-6-ether as

a phase transfer catalyst and the alkylating agent 6a (1.61 g,

2.89 mol). The reaction mixture was stirred under reﬂux over

a period of 12 h before being quenched with H₂O (10 mL) and

extracted with CH₂Cl₂(3 ꢄ 20 mL). The combined organic extracts

were dried over Na₂SO₄, ﬁltered and concentrated in vacuo. Flash

column chromatography (silica gel, CHCl₃/MeOH 98:2) afforded 17c

as a yellow foam. Yield: 57%; R_f0.49 (CHCl₃/MeOH/9:1); t_R

16.81 min (70% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):

4.12.3. 5-Butyl-4-iodo-N,N⁰-bis{[2⁰-(tert-butoxycarbonyl)biphenyl-

4-yl]methyl}imidazolium bromide (18e)

The same procedure as above was employed for the preparation

of 18e. Yield: 58%; R_f0.36 (CHCl₃/MeOH 9:1); t_R11.76 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 783.43 [M ꢀ Br]; ¹H

NMR (400 MHz, CDCl₃):

d

11.53 (s, 1H), 7.72 (d, 2H, J ¼ 7.5 Hz), 7.51

(d, 2H, J ¼ 8.0 Hz), 7.43e7.28 (m, 8H), 7.21e7.16 (m, 4H), 5.59 (s, 4H),

2.51 (t, 2H, J ¼ 7.4 Hz), 1.35e1.24 (m, 4H), 1.20 (s, 9H), 1.17 (s, 9H),

0.83 (t, 3H, J ¼ 7.4 Hz).

1111.82 [M ꢀ Br]; ¹H NMR (400 MHz, CDCl₃):

d 11.46 (s, 1H), 7.95e

7.93 (m, H), 7.33e7.14 (m, 32H), 6.93e6.90 (m, 12H), 5.44 (s, 2H),

5.42 (s, 2H), 2.37 (t, 2H, J ¼ 7.5 Hz), 1.28e1.19 (m, 4H), 0.82 (t, 3H,

J ¼ 7.5 Hz).

4.13. 5-Butyl-4-chloro-N,N⁰-bis{[2⁰-[2H-tetrazol-5-yl]biphenyl-4-

yl]methyl}imidazolium bromide (19c)

General procedure 3 was employed for the preparation of 19c.

Yield: 82%; R_f0.40 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R13.29 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 628.55

[M ꢀ Br] (³⁵Cl), 630.50 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz,

4.11.2. 4-Bromo-5-butyl-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]

biphenyl-4-yl]methyl}imidazolium bromide (17d)

The same procedure as above was employed for the preparation

of 17d. Yield: 55%; R_f0.50 (CHCl₃/MeOH 9:1); t_R16.90 min (70%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 1157.11 [M ꢀ Br]; ¹H

CD₃OD): d 9.12 (s,1H), 7.69e7.66 (m, 4H), 7.60e7.54 (m, 4H), 7.34 (d,

2H, J ¼ 8.0 Hz), 7.29 (d, 2H, J ¼ 8.0 Hz), 7.23e7.20 (m, 4H), 5.45 (s,

NMR (400 MHz, CDCl₃): d 11.28 (s,1H), 7.89e7.64 (m, 2H), 7.42e7.09

2H), 5.44 (s, 2H), 2.68 (t, 2H, J ¼ 7.4 Hz), 1.39e1.30 (m, 4H), 0.87 (t,

(m, 32H), 6.86e6.84 (m, 12H), 5.38 (s, 4H), 2.33 (t, 2H, J ¼ 7.5 Hz),

3H, J ¼ 7.4 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 156.35, 145.39,

1.49e1.17 (m, 4H), 0.94 (t, 3H, J ¼ 7.5 Hz).

141.54, 141.08, 140.96, 133.99, 132.72, 132.45, 131.52, 131.49, 130.80,

130.60, 130.22, 130.12, 128.55, 128.38, 128.20, 126.69, 123.57, 119.89,

51.55, 51.10, 29.44, 23.21, 22.12, 13.28.

4.11.3. 5-Butyl-4-iodo-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]

biphenyl-4-yl]methyl}imidazolium bromide (17e)

The same procedure as above was employed for the preparation

of 17e. Yield: 52%; R_f0.50 (CHCl₃/MeOH 9:1); t_R16.31 min (70%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 1203.60 [M ꢀ Br];

4.13.1. 4-Bromo-5-butyl-N,N⁰-bis{[2⁰-[2H-tetrazol-5-yl]biphenyl-4-yl]

methyl}imidazolium bromide (19d)

General procedure 3 was employed for the preparation of 19d.

Yield: 80%; R_f0.43 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R13.17 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 671.37

[M ꢀ Br] (⁷⁹Br), 673.32 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz,

¹H NMR (400 MHz, CDCl₃):

d 10.88 (s, 1H), 7.94e7.89 (m, 2H), 7.48e

7.15 (m, 32H), 6.92e6.89 (m, 12H), 5.38 (s, 2H), 5.36 (s, 2H), 2.60 (t,

2H, J ¼ 7.5 Hz), 1.56e1.19 (m, 4H), 0.83 (t, 3H, J ¼ 7.5 Hz).

CD₃OD): d 9.12 (s, 1H), 7.66e7.63 (m, 4H), 7.57e7.52 (m, 4H), 7.32 (d,

4.12. General procedure 7: bis-alkylation of 16cee with the

alkylating agent 6b

2H, J ¼ 8.0 Hz), 7.26e7.19 (m, 4H), 7.07 (d, 2H, J ¼ 8.0 Hz), 5.44 (s,

2H), 5.41 (s, 2H), 2.67 (t, 2H, J ¼ 7.4 Hz), 1.40e1.30 (m, 4H), 0.88 (t,

3H, J ¼ 7.4 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 156.42, 156.35,

4.12.1. 5-Butyl-4-chloro-N,N⁰-bis{[2⁰-(tert-butoxycarbonyl)

biphenyl-4-yl]methyl}imidazolium bromide (18c)

145.33, 141.10, 135.09, 133.58, 132.17, 131.93, 130.62, 130.30, 130.21,

129.85, 128.68, 127.76, 124.18, 106.82, 51.65, 51.04, 29.23, 22.78,

21.77, 13.74.

To a solution of 16c (0.20 g, 1.26 mmol) in anhydrous Tol (4 mL)

under Ar atmosphere were sequentially added powdered KOH

(0.14 g, 2.52 mmol), a catalytic amount of 18-crown-6-ether as

a phase transfer catalyst and the alkylating agent 6b (1.00 g,

2.90 mol). The reaction mixture was stirred at 80 ^ꢁC over a period of

12 h before being quenched with H₂O (10 mL) and extracted with

4.13.2. 5-Butyl-4-iodo-N,N⁰-bis{[2⁰-[2H-tetrazol-5-yl]biphenyl-4-yl]

methyl}imidazolium bromide (19e)

General procedure 3 was employed for the preparation of 19e.

Yield: 79%; R_f0.43 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R13.0 min (20%

366

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 719.45 [M ꢀ Br]; ¹H

7.25e7.08 (m, 27H), 6.86e6.84 (m, 12H), 5.51 (s, 2H), 4.44 (s, 2H),

2.46 (t, 2H, J ¼ 7.5 Hz), 1.21e1.19 (m, 4H), 0.79 (t, 3H, J ¼ 7.5 Hz).

NMR (400 MHz, CD₃OD):

d 9.24 (s, 1H), 7.67e7.62 (m, 4H), 7.59e

7.53 (m, 4H), 7.30 (d, 2H, J ¼ 8.0 Hz), 7.29e7.21 (m, 4H), 7.09 (d,

2H, J ¼ 8.0 Hz), 5.50 (s, 2H), 5.43 (s, 2H), 2.70 (t, 2H, J ¼ 7.5 Hz),1.37e

1.30 (m, 4H), 0.97 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

4.14.2. 5-Butyl-2-hydroxymethyl-4-iodo-N,N⁰-bis{[2⁰-[2-(trityl)

tetrazol-5-yl]biphenyl-4-yl]methyl}imidazolium bromide (21e)

General procedure 2 was employed for the preparation of 21e.

Yield: 70%; R_f0.46 (CHCl₃/MeOH 9:1); t_R15.31 min (70%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 1234.37 [M ꢀ Br]^þ;

d

157.52, 146.43, 140.59, 135.75, 133.49, 133.07, 131.23, 130.72,

130.55, 130.15, 129.93, 129.41, 128.01 127.56, 126.08, 89.85, 51.80,

50.04, 29.23, 22.78, 21.77, 13.74.

¹H NMR (400 MHz, CDCl₃)

d 7.92e7.85 (m, 2H), 7.43e7.40 (m, 5H),

4.13.3. 5-Butyl-4-chloro-N,N⁰-bis{[2⁰-carboxybiphenyl-4-yl]methyl}

imidazolium bromide (20c)

7.33e7.06 (m, 27H), 6.89e6.80 (m, 12H), 5.69 (s, 2H), 5.61 (s, 2H),

4.48 (s, 2H), 2.54 (t, 2H, J ¼ 7.5 Hz), 1.25e1.19 (m, 4H), 0.86 (t, 3H,

J ¼ 7.5 Hz).

General procedure 3 was employed for the preparation of 20c.

Yield: 79%; R_f0.26 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R17.35 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 579.34

[M ꢀ Br] (³⁵Cl), 581.36 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz,

4.14.3. 5-Butyl-4-chloro-2-hydroxymethyl-N,N⁰-bis{[2⁰-(tert-

butoxycarbonyl)biphenyl-4-yl]methyl}imidazolium bromide (22c)

The same procedure as above was employed for the preparation

of 22c. Yield: 78%; R_f0.32 (CHCl₃/MeOH 9:1); t_R10.59 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 721.45 [M ꢀ Br]

CD₃OD):

d

9.14 (s, 1H), 7.82e7.79 (m, 2H), 7.51 (td, 2H, J ¼ 7.6 Hz,

1.4 Hz), 7.42e7.28 (m, 12H), 5.44 (s, 2H), 5.43 (s, 2H), 2.68 (t, 2H,

J ¼ 7.5 Hz), 1.40e1.22 (m, 4H), 0.83 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR

(160 MHz, CD₃OD):

d

173.58, 148.32, 145.51, 143.69, 134.57, 132.87,

(

³⁵Cl), 723.46 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz, CDCl₃):

d 7.78

132.50, 132.35, 131.59, 130.45, 130.17, 129.82, 129.52, 123.91, 54.78,

52.25, 31.19, 25.23, 24.05, 14.81.

(d, 2H, J ¼ 7.6 Hz), 7.48 (t, 2H, J ¼ 7.5 Hz), 7.40 (t, 2H, J ¼ 7.5 Hz),

7.35e7.22 (m, 8H), 7.11 (d, 2H, J ¼ 7.6 Hz), 5.86 (s, 2H), 5.79 (s, 2H),

4.83 (s, 2H), 2.68 (t, 2H, J ¼ 7.5 Hz), 1.41e1.26 (m, 4H), 1.25 (s, 9H),

1.23 (s, 9H), 0.90 (t, 3H, J ¼ 7.5 Hz).

4.13.4. 4-Bromo-5-butyl-N,N⁰-bis{[2⁰-carboxybiphenyl-4-yl]

methyl}imidazolium bromide (20d)

General procedure 3 was employed for the preparation of 20d.

Yield: 76%; R_f0.26 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R17.27 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 623.30

[M ꢀ Br] (⁷⁹Br), 625.32 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz,

4.14.4. 4-Bromo-5-butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-(tert-

butoxycarbonyl)biphenyl-4-yl]methyl}imidazolium bromide (22d)

The same procedure as above was employed for the preparation

of 22d. Yield: 75%; R_f0.33 (CHCl₃/MeOH 9:1); t_R10.42 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 765.56 [M ꢀ Br]

CD₃OD):

d

9.24 (s, 1H), 7.81e7.78 (m, 2H), 7.49 (td, 2H, J ¼ 7.5 Hz,

1.3 Hz), 7.42e7.27 (m, 12H), 5.46 (s, 2H), 5.39 (s, 2H), 2.67 (t, 2H,

J ¼ 7.4 Hz), 1.33e1.29 (m, 4H), 0.84 (t, 3H, J ¼ 7.4 Hz); ¹³C NMR

(

⁷⁹Br), 767.57 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz, CDCl₃):

d 7.79

(d, 2H, J ¼ 7.4 Hz), 7.49 (t, 2H, J ¼ 7.3 Hz), 7.41 (t, 2H, J ¼ 7.3 Hz),

7.35e7.23 (m, 8H), 7.12 (d, 2H, J ¼ 7.6 Hz), 5.92 (s, 2H), 5.84 (s, 2H),

4.82 (s, 2H), 2.70 (t, 2H, J ¼ 7.5 Hz), 1.58e1.36 (m, 4H), 1.27 (s, 9H),

1.25 (s, 9H), 0.93 (t, 3H, J ¼ 7.1 Hz).

(160 MHz, CD₃OD):

d 173.30, 148.38, 145.00, 144.89, 143.18, 137.39,

135.14, 134.32, 133.75, 132.91, 132.55, 131.59, 131.47, 130.12, 129.81,

129.56, 129.52, 109.14, 54.67, 53.52, 31.56, 25.18, 24.16, 14.77.

4.13.5. 5-Butyl-4-iodo-N,N⁰-bis{[2⁰-carboxybiphenyl-4-yl]methyl}

imidazolium bromide (20e)

General procedure 3 was employed for the preparation of 20e.

Yield: 73%; R_f0.26 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R17.42 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 671.31

4.14.5. 5-Butyl-2-hydroxymethyl-4-iodo-N,N⁰-bis{[2⁰-(tert-

butoxycarbonyl)biphenyl-4-yl]methyl}imidazolium bromide (22e)

The same procedure as above was employed for the preparation

of 22e. Yield: 74%; R_f0.35 (CHCl₃/MeOH 9:1); t_R10.42 min (60%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 813.48 [M ꢀ Br]; ¹H

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d

9.20 (s, 1H), 7.81e7.79 (m,

NMR (400 MHz, CDCl₃):

d

7.79 (d, 2H, J ¼ 7.6 Hz), 7.49 (t, 2H,

2H), 7.50 (td, 2H, J ¼ 1.2, 7.5 Hz), 7.42e7.28 (m, 12H), 5.45 (s, 2H),

5.42 (s, 2H), 2.67 (t, 2H, J ¼ 7.3 Hz), 1.38e1.23 (m, 4H), 0.83 (t, 3H,

J ¼ 7.4 Hz), 7.41 (t, 2H, J ¼ 7.4 Hz), 7.35e7.19 (m, 8H), 7.10 (d, 2H,

J ¼ 7.6 Hz), 5.90 (s, 2H), 5.83 (s, 2H), 4.79 (s, 2H), 2.73 (t, 2H,

J ¼ 7.5 Hz), 1.52e1.35 (m, 4H), 1.27 (s, 9H), 1.26 (s, 9H), 0.94 (t, 3H,

J ¼ 7.5 Hz).

J ¼ 7.3 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 173.41, 147.88, 144.86,

144.68, 143.40, 141.99, 134.32, 134.11, 133.08, 132.58, 132.55, 131.53,

131.37, 129.98, 129.64, 129.50, 80.86, 56.12, 53.28, 31.93, 26.47,

24.20, 14.75.

4.15. 5-Butyl-4-chloro-2-hydroxymethyl-N,N⁰-bis{[2⁰-[2H-tetrazol-

5-yl]biphenyl-4-yl]methyl}imidazolium bromide (23c)

4.14. 5-Butyl-4-chloro-2-hydroxymethyl-N,N⁰-bis{[2⁰-[2-(trityl)

tetrazol-5-yl]biphenyl-4-yl]methyl}imidazolium bromide (21c)

General procedure 3 was employed for the preparation of 23c.

Yield: 77%; R_f0.35 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R12.78 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 657.20

[M ꢀ Br] (³⁵Cl), 659.23 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz,

General procedure 2 was employed for the preparation of 2c.

Yield: 76%; R_f0.45 (CHCl₃/MeOH 9:1); t_R16.68 min (70%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 1141.47 [M ꢀ Br],

901.43 [(M ꢀ Tr) þ H ꢀ Br], 242.98 [Tr]; ¹H NMR (400 MHz, CDCl₃):

CD₃OD): d 7.68e7.66 (m, 4H), 7.60e7.56 (m, 4H), 7.24e7.07 (m, 8H),

5.64 (s, 2H), 5.62 (s, 2H), 4.95 (s, 2H), 2.66 (t, 2H, J ¼ 7.5 Hz), 1.36e

d

7.92e7.86 (m, 2H), 7.47e7.45 (m, 5H), 7.32e7.13 (m, 27H), 6.98e

1.29 (m, 4H), 0.83 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

6.71 (m, 12H), 5.76 (s, 2H), 5.67 (s, 2H), 4.73 (s, 2H), 2.51 (t, 2H,

d 155.32,146.23,143.96,141.18,140.06,133.14,132.62,131.98,131.12,

J ¼ 7.5 Hz), 1.29e1.19 (m, 4H), 0.84 (t, 3H, J ¼ 7.5 Hz).

130.39, 130.17, 129.68, 129.53, 129.03, 127.89, 127.82, 126.96, 126.44,

125.86, 122.98, 54.39, 49.09, 29.14, 22.26, 21.68, 12.40.

4.14.1. 4-Bromo-5-butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-[2-(trityl)

tetrazol-5-yl]biphenyl-4-yl]methyl}imidazolium bromide (21d)

General procedure 2 was employed for the preparation of 21d.

Yield: 72%; R_f0.44 (CHCl₃/MeOH 9:1); t_R15.90 min (70%

MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 1187.21 [M ꢀ Br];

4.15.1. 4-Bromo-5-butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-[2H-

tetrazol-5-yl]biphenyl-4-yl]methyl}imidazolium bromide (23d)

General procedure 3 was employed for the preparation of 23d.

Yield: 77%; R_f0.34 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R12.78 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 701.40

¹H NMR (400 MHz, CDCl₃)

d 7.91e7.88 (m, 2H), 7.42e7.40 (m, 5H),

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

367

[M ꢀ Br] (⁷⁹Br), 703.51 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz,

1078.89 [M ꢀ Br], 835.61 [(M ꢀ Tr) þ H ꢀ Br], 243.20 [Tr]; ¹H NMR

CD₃OD):

d

7.65e7.63 (m, 4H), 7.55e7.51 (m, 4H), 7.18e7.03 (m, 8H),

(400 MHz, CDCl₃): d 7.96e7.94 (m, 2H), 7.50e7.47 (m, 6H), 7.34e

5.60 (s, 2H), 5.59 (s, 2H), 4.90 (s, 2H), 2.61 (t, 2H, J ¼ 7.5 Hz), 1.33e

7.07 (m, 28H), 6.93e6.91 (m, 12H), 5.31 (s, 4H), 2.86 (t, 2H,

J ¼ 7.3 Hz), 1.30e1.22 (m, 4H), 0.79 (t, 3H, J ¼ 7.3 Hz).

1.23 (m, 4H), 0.83 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

156.74,156.67, 146.69,143.99,141.12,141.10,140.53,134.81,133.58,

132.91, 132.45, 130.26, 129.76, 129.56, 127.77, 126.65, 126.27, 124.61,

107.35, 50.29, 49.22, 29.35, 23.06, 21.74, 12.46.

4.16.1. 2-Butyl-N,N⁰-bis{[2⁰-(tert-butoxycarbonyl)]biphenyl-4-yl]

methyl}imidazolium bromide (26)

General procedure 1 was employed for the preparation of 26.

Yield: 84%; m.p. 148e151 ^ꢁC; R_f0.51 (CHCl₃/MeOH, 9:1); t_R9.84 min

(60% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 657.63

4.15.2. 5-Butyl-2-hydroxymethyl-4-iodo-N,N⁰-bis{[2⁰-[2H-tetrazol-

5-yl]biphenyl-4-yl]methyl}imidazolium bromide (23e)

General procedure 3 was employed for the preparation of 23e.

Yield: 75%; R_f0.36 (CHCl₃/MeOH/gl. AcOH 9:1:0.1); t_R12.89 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 749.45

[M ꢀ Br]; ¹H NMR (400 MHz, CDCl₃)

d

7.80 (dd, 2H, J ¼ 1.2, 7.6 Hz),

7.52 (s, 2H), 7.48 (dd, 2H, J ¼ 1.2, 7.6 Hz), 7.42e7.36 (m, 10H), 7.27e

7.25 (m, 2H), 5.58 (s, 4H), 3.26 (t, 2H, J ¼ 7.2 Hz), 1.42e1.23 (m, 22H),

0.88 (t, 3H, J ¼ 7.2 Hz).

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d 7.65e7.58 (m, 4H), 7.49e

7.42 (m, 4H), 7.14e7.09 (m, 8H), 5.59 (s, 4H), 4.91 (s, 2H), 2.61 (t,

2H, J ¼ 7.5 Hz), 1.24e1.23 (m, 4H), 0.79 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR

4.17. 2-Butyl-N,N⁰-bis{[2⁰-(2H-tetrazol-5-yl)biphenyl-4-yl]methyl}

imidazolium bromide (27)

(160 MHz, CD₃OD):

d 158.32, 146.58, 141.49, 137.75, 134.59, 134.17,

131.53, 130.82, 130.25, 130.01, 129.51, 128.51, 127.77, 125.58, 89.58,

52.50, 51.54, 29.75, 22.48, 21.52, 12.55.

General procedure 3 was employed for the preparation of 27.

Yield: 84%; R_f0.30 (CHCl₃/MeOH/gl. AcOH, 9:1:0.1); t_R11.66 min

(20% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 593.39

4.15.3. 5-Butyl-4-chloro-2-hydroxymethyl-N,N⁰-bis{[2⁰-

carboxybiphenyl-4-yl]methyl}imidazolium bromide (24c)

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d 7.67e7.54 (m, 10H), 7.26e

General procedure 3 was employed for the preparation of 24c.

Yield: 81%; R_f0.23 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R16.99 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 609.39

[M ꢀ Br] (³⁵Cl), 611.34 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR (400 MHz,

7.19 (m, 8H), 5.43 (s, 4H), 3.03 (t, 2H, J ¼ 7.2 Hz), 1.36e1.28 (m, 4H),

0.84 (t, 3H, J ¼ 7.2 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 161.99, 147.76,

143.85, 140.88, 134.24, 132.98, 130.49, 130.27, 130.21, 129.77, 129.40,

127.74, 127.43, 124.22, 122.01, 51.07, 30.70, 21.98, 20.31, 12.35.

CD₃OD):

d

7.80 (d, 2H, J ¼ 7.4 Hz), 7.49 (t, 2H, J ¼ 7.4 Hz), 7.40e7.27

(m, 12H), 5.64 (s, 4H), 4.97 (s, 2H, H-6), 2.66 (t, 2H, J ¼ 7.5 Hz), 1.30e

4.17.1. 2-Butyl-N,N⁰-bis{[2⁰-carboxybiphenyl-4-yl]methyl}

imidazolium bromide (28)

General procedure 3 was employed for the preparation of 28.

Yield: 80%; m.p. 129e132 ^ꢁC; R_f0.22 (CHCl₃/MeOH/gl. AcOH,

8:2:0.2); t_R16.13 min (10% MeCN / 100% MeCN in 30 min); ESI-MS

1.23 (m, 4H), 0.78 (t, 3H, J ¼ 7.5 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

171.25. 147.78, 144.60, 142.62, 140.76, 134.59, 134.33, 134.08,

132.40, 132.32, 131.50, 131.38, 129.43, 128.82, 128.40, 121.96, 51.62,

51.48, 31.53, 24.68, 24.07, 14.70.

(m/z): 545.46 [M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d 7.87 (d, 2H,

4.15.4. 4-Bromo-5-butyl-2-hydroxymethyl-N,N⁰-bis{[2⁰-

carboxybiphenyl-4-yl]methyl}imidazolium bromide (24d)

J ¼ 7.5 Hz), 7.65 (s, 2H), 7.59 (t, 2H, J ¼ 7.5 Hz), 7.47e7.45 (m, 12H),

5.51 (s, 4H), 3.13 (t, 2H, J ¼ 7.4 Hz), 1.39e1.30 (m, 4H), 0.87 (t, 3H,

J ¼ 7.4 Hz); ¹³C NMR (160 MHz, CD₃OD):

d 170.29, 147.84, 142.40,

141.35, 132.70, 131.29, 131.05, 131.40, 129.58, 129.18, 127.29, 122.03,

51.25, 28.23, 23.00, 22.07, 12.34.

General procedure 3 was employed for the preparation of 24d.

Yield: 77%; R_f0.24 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R16.91 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 653.34

[M ꢀ Br] (⁷⁹Br), 655.31 [M þ 2 ꢀ Br] (⁸¹Br); ¹H NMR (400 MHz,

CD₃OD):

d

7.78 (d, 2H, J ¼ 7.3 Hz), 7.50 (t, 2H, J ¼ 7.5 Hz), 7.41e7.23

4.18. 2-Butyl-4-chloro-5-hydroxymethyl-N,N⁰-bis{[2⁰-(2H-tetrazol-

5-yl)biphenyl-4-yl]methyl}imidazolium bromide (30)

(m, 12H), 5.65 (s, 4H), 4.98 (s, 2H), 2.66 (t, 2H, J ¼ 7.3 Hz), 1.30e1.25

(m, 4H), 0.79 (t, 3H, J ¼ 7.3 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

172.13,147.14,142.65,142.50,140.94,136.11,135.26,132.84,132.39,

General procedures 6 and 3 were employed for the preparation

of 30. Yield: 77%; m.p. 159e162 ^ꢁC; R_f0.34 (CHCl₃/MeOH/gl. AcOH,

9:1:0.1); t_R10.93 min (20% MeCN / 100% MeCN in 30 min); ESI-MS

(m/z): 657.44 [M ꢀ Br] (³⁵Cl), 659.46 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR

130.70, 130.64, 130.50, 129.59, 129.45, 127.55, 127.51, 126.81, 126.51,

107.77, 52.62, 50.79, 49.79, 29.78, 23.57, 22.23, 12.83.

4.15.5. 5-Butyl-2-hydroxymethyl-4-iodo-N,N⁰-bis{[2⁰-

(400 MHz, CD₃OD):

d 7.68e7.64 (m, 4H), 7.59e7.30 (m, 5H), 7.27e

carboxybiphenyl-4-yl]methyl}imidazolium bromide (24e)

General procedure 3 was employed for the preparation of 24e.

Yield: 75%; R_f0.25 (CHCl₃/MeOH/gl. AcOH 8:2:0.2); t_R16.80 min

(10% MeCN / 100% MeCN in 30 min); ESI-MS (m/z): 701.35

7.20 (m, 7H), 5.66 (s, 2H), 5.58 (s, 2H), 4.65 (s, 2H), 2.71 (t, 2H,

J ¼ 7.5 Hz), 1.32e1.24 (m, 2H), 0.73 (t, 3H, J ¼ 7.5 Hz).

[M ꢀ Br]; ¹H NMR (400 MHz, CD₃OD):

d

7.78 (d, 2H, J ¼ 7.4 Hz), 7.50

4.19. 2-Butyl-4-chloro-5-hydroxymethyl-N,N⁰-bis{[2⁰-

carboxybiphenyl-4-yl]methyl}imidazolium bromide (31)

(td, 2H, J ¼ 0.9, 7.4 Hz), 7.41e7.20 (m, 12H), 5.64 (s, 4H), 4.95 (br s,

2H), 2.67 (t, 2H, J ¼ 7.4 Hz), 1.30e1.19 (m, 4H, H-8), 0.80 (t, 3H,

J ¼ 7.4 Hz); ¹³C NMR (160 MHz, CD₃OD):

d

171.53, 150.39, 144.49,

General procedures 7 and 3 were employed for the preparation

of 31. Yield: 74%; m.p. 128e131 ^ꢁC; R_f0.20 (CHCl₃/MeOH/gl. AcOH,

8:2:0.2); t_R16.11 min (10% MeCN / 100% MeCN in 30 min); ESI-MS

(m/z): 609.26 [M ꢀ Br] (³⁵Cl), 611.21 [M þ 2 ꢀ Br] (³⁷Cl); ¹H NMR

144.30, 142.76, 141.80, 134.95, 134.55, 132.38, 131.48, 131.33, 130.12,

129.43, 128.58, 128.37, 120.49, 120.16, 82.07, 54.73, 51.70, 32.12,

26.94, 24.23, 14.78.

(400 MHz, CD₃OD):

d

7.73 (d, 2H, J ¼ 7.5 Hz), 7.46 (t, 2H, J ¼ 7.5 Hz),

4.16. 2-Butyl-N,N⁰-bis{[2⁰-[2-(trityl)tetrazol-5-yl]biphenyl-4-yl]

methyl}imidazolium bromide (25)

7.38e7.36 (m, 6H), 7.27e7.25 (m, 4H), 7.20 (d, 2H, J ¼ 8.0 Hz), 5.62

(s, 2H), 5.54 (s, 2H), 4.63 (s, 2H), 3.01 (t, 2H, J ¼ 7.2 Hz), 1.21 (quint,

2H, J ¼ 7.2 Hz) 1.10 (sext, 2H, J ¼ 7.2 Hz), 0.70 (t, 3H, J ¼ 7.2 Hz); ¹³C

General procedure 1 was employed for the preparation of 25.

Yield: 85%; m.p. 152e154 ^ꢁC; R_f0.50 (CHCl₃/MeOH, 96:4); t_R

15.61 min (70% MeCN / 100% MeCN in 30 min); ESI-MS (m/z):

NMR (160 MHz, CD₃OD): d 174.11, 151.61, 144.60, 142.78, 135.77,

134.76, 134.30, 132.56, 132.39, 131.50, 131.31, 129.47, 128.56, 128.30,

122.46, 53.73, 51.76, 51.07, 30.59, 26.33, 24.30, 14.52.

368

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

4.20. Docking studies

4.22. Umbrella sampling simulations

The 3D model of the AT1 receptor used in ourdocking studies was

kindly provided by Tuccinardi et al. [45]. The construction of this

model is based on X-ray bovine rhodopsin structure, molecular

procedure and available site-directed mutagenesis data [46]. Mo-

lecular Docking studies were performed using Glide extra precision

(XP) implemented Induced Fit Docking (IFD) protocol (v5.0) [47e49]

docking programs under the Linux operating system.

The PMF of the 12b from aqueous to lipid phase was computed

by the Weighted Histogram Analysis Method (WHAM) [60,61] that

is available in GROMACS 4.5.4. In particular, thirty one biasing MD

simulations were conducted along a reaction coordinate

which is deﬁned as the distance of the 12b center of mass from the

bilayer center (

z (Z-axis),

z

¼ 0). In addition, a harmonic potential was applied

(k ¼ 500 kJ/mol/nm²) between bilayer and 12b centers of mass. It

has also to be reported that the simulation system contains 3021

water molecules so as to avoid the interaction of the drug with the

periodic image of the opposite leaﬂet of the bilayer. The duration of

the biasing MD simulations was 24 ns of which the ﬁrst 10 ns was

regarded as an equilibration and therefore they were not taken into

account for the computation of PMF. The rest of the MD parameters

were the same with those given in the previous subsection.

ꢀ

The binding site was deﬁned by 20 A inner cubic grid box,

centered on the point that is the center of mass of residues Lys199

and 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 PolakeRibiere conjugate gradient (PRCG)

4.23. Quantum chemical calculations

ꢀ^ꢀ1

minimization (0.0001 kJA mol^ꢀ1convergence criteria). Proton-

Geometry optimization and HOMO and LUMO calculations were

performed for the studied compounds. The geometries were fully

optimized using Density Functional Theory (DFT) hybrid method

with the Becke’s three-parameter exchange functional and

gradient-corrected functional of Lee, Yang and Parr (B3LYP) [62]

using the following standard basis set: 3e21 g. All geometry opti-

mization were followed by calculations of frequencies in order to

identify obtained structures as energy minima (no imaginary fre-

quencies). All minima for all the compounds were veriﬁed by

establishing that the matrix of energy second derivatives (Hessian)

has only positive eigenvalues (all vibrational frequencies are real).

All calculations were carried out with the Gaussian 09W program.

HOMO and LUMO frontier orbitals of the molecule were also

computed at the same level of theory. Molecular orbital (MO) sur-

faces visually represent the various stable electron distributions of

a molecule. According to Frontier orbital theory [63] the shapes and

symmetries of the highest-occupied and lowest-unoccupied mo-

lecular orbitals (HOMO and LUMO) are crucial in predicting the

reactivity of a species and the stereochemical and regiochemical

outcome of a chemical reaction. Molecules with high HOMO

(highest occupied molecular orbital energy) values are able to

donate electron density more easily than molecules with low

HOMO energy values. The HOMO energy value can be increased

with the presence of electron-donating groups (EDG) such as e

NMe₂, eNH₂, eNHEt, and eOMe and decreased with the pres-

ence of electron-withdrawing groups (EWG) such as halogens, eCN

and eNO₂groups [64].

ation 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 [48,49]

to account for ﬂexibility of the receptor. Schrodinger’s IFD proto-

col 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 A. (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 A of ligand

poses were minimized in order to form suitable conformations of

poses at the binding site of the receptor. (iv) Glide re-docking of

each proteineligand complex.

4.21. Molecular dynamics simulations

A lipid bilayer of 72 DPPC molecules was simulated under the

CHARMM 36 force ﬁeld [50,51] with all hydrogen atoms explicit,

whereas the aqueous phase (2162 water molecules) was described

by the TIP3P [52] (transferable intermolecular potential 3P) model.

The initial structure for the bilayer was downloaded as a PDB ﬁle by

the following web page: http://terpconnect.umd.edu/wjbklauda/

research/download.html. As far as the 12b molecule, its topology

ﬁle was created by the SwissParam server which is available online:

http://swissparam.ch/ [53]. The abovementioned topology ﬁles are

compatible with the CHARMM force ﬁeld.

4.24. Pharmacological evaluation

All simulations were performed with the molecular dynamics

package GROMACS 4.5.4 [54e57] in the context of NP_zAT ensemble

with a constant area per lipid, A, equal to 0.64 nm²/lipid. The

equations of motion were integrated with a time step equal to 2 fs

while temperature was kept constant at 317 K using the Berendsen

thermostat [58] with a coupling time constant equal to 0.1 ps.

Regarding to the pressure along the normal to two DPPC leaﬂets (Z-

axis), it was regulated by the Berendsen barostat [58] at 1 bar, with

a coupling time constant to be 1 ps. LennardeJones and Coulomb

interactions were calculated using a 1.0 nm cut-off radius while the

long range electrostatics were treated with the Particle Mesh Ewald

(PME) method [59]. The system was energy-minimized employing

the steepest descent method [57] and after that the MD simulation

was commenced with the 12b molecule initially placed in the

aqueous phase. The whole duration of the simulation is equal to

120 ns whereas the last 40 ns were employed for the calculation of

the properties.

4.24.1. AT1 receptor binding assay of synthesized ANGII analogs

4.24.1.1. 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

mg of plasmid DNA encoding the human AT1 receptor, using

9

mL 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) con-

taining 3.15 g/L glucose,10% bovine calf serum and 700 mg/mL of the

antibiotic, Geneticin. The antibiotic used ensured the selection of

a stably transfected pool of cells.

4.24.1.2. Harvesting cells and membrane preparation. HEK 293 cells

(grown in 100 mm dishes) stably expressing the human AT1 re-

ceptor were washed with phosphate-buffered saline (PBS; 4.3 mM

G. Agelis et al. / European Journal of Medicinal Chemistry 62 (2013) 352e370

369

Na₂HPO₄$7H₂O,1.4 mM KH₂PO₄,137 mM NaCl, 2.7 mM KCl, pH 7.2e

Author contribution

7.3 at room temperature), brieﬂy treated with PBS containing 2 mM

EDTA (PBS/EDTA), and then dissociated in PBS/EDTA. Cell

suspension was centrifuged at 1000ꢄ g for 5 min at room

temperature, 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

Organic Synthesis: George Agelis, Amalia Resvani, John Matsou-

kas, Structure Elucidation, Docking Studies, Molecular Dynamics &

ꢀ

ꢁ

Biological Evaluation: Catherine Koukoulitsa, Tereza Tumová, Jirina

Slaninová, Dimitra Kalavrizioti, Katerina Spyridaki, Antreas Afan-

titis, Georgia Melagraki, Athanasia Siafaka, Eleni Gkini, Grigorios

Megariotis, Simona Golic Grdadolnik, Manthos G. Papadopoulos,

Demetrios Vlahakos, George Liapakis, Michael Maragoudakis,

Thomas Mavromoustakos.

T25 homogenizer (at setting w20, 10e15 s,

4

^ꢁC). The

homogenate was centrifuged at 250ꢄ g for 5 min at room

temperature. The pellet was discarded and the supernatant was

centrifuged (16,000ꢄ g, 10 min, 4 ^ꢁC). The membrane pellet was

resuspended (0.6e0.7 mL/100 mm dish) in buffer B (50 mM Trise

HCl containing 1 mM EDTA, 10 mM MgCl₂, 0.2% BSA, 0.2 mg/mL

Acknowledgments

bacitracin, and 0.93 m

g/mL aprotinin, pH 7.4 at 4 ^ꢁC) and used for

This project was supported by the pharmaceutical companies

ELDRUG SA and VIANEX SA. We also acknowledge Demetrios

Vahliotis (Instrumental Analysis Laboratory, University of Patras)

for recording the NMR spectra. Prof. T. Mavromoustakos acknowl-

edges EENC program for covering the travel and residence expenses

to perform NMR experiments in the Slovenian. The NMR studies

were supported by EN-FIST Center of Excellence (Dunajska 156,

SI-1000 Ljubljana, Slovenia) and the Slovenian Research Agency.

K.K. acknowledges Greek national funds through the Operational

Program “Education and Lifelong Learning” of the National Strate-

gic Reference Framework(NSRF)eResearch Funding Program:

Heracleitus II, investing in knowledge society through the Euro-

pean Social Fund.

radioligand binding studies.

4.24.1.3. [¹²⁵I-Sar¹-Ile⁸] ANGII binding. The [¹²⁵I-Sar¹-Ile⁸] ANGII

competition binding was performed as follows. Aliquots of diluted

membrane suspension (50 mL) were added into tubes, containing

buffer B and 100,000e120,000 cpm [¹²⁵I-Sar¹-Ile⁸] ANGII with or

without increasing concentrations of ANGII analogs in a ﬁnal vol-

ume 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% polyethyleneimine at 4 ^ꢁC. The

ﬁlters were washed 10 times with 1e2 mL of ice-cold 50 mM Trise

HCl 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 concentration of the radioligand added. Speciﬁc [¹²⁵I-Sar¹-

Ile⁸] ANGII binding was deﬁned as the total binding minus non-

speciﬁc binding in the presence of 1000 nM [Sar¹-Ile⁸] ANGII. Data

analysis for competition binding was performed by nonlinear

regression analysis, using GraphPad Prism 4.0 (GraphPad Software,

San Diego, CA). elogIC₅₀values were obtained by ﬁtting the data

from competition studies to a one-site competition model and

presented as mean plus/minus standard error (SE).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2012.12.044.

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Rational design, efficient syntheses and biological evaluation of N,N'-symmetrically bis-substituted butylimidazole analogs as a new class of potent Angiotensin II receptor blockers

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