Synthesis and biological activities of novel amine-derived bis-azoles as potential antibacterial and antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2010.06.012

A series of novel amine-derived bis-azole compounds were designed by the systematical structural modification of Fluconazole and synthesized by a convenient and efficient method, and the antimicrobial activities for all prepared compounds were evaluated i

Full text of DOI:10.1016/j.ejmech.2010.06.012

European Journal of Medicinal Chemistry 45 (2010) 4388e4398
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological activities of novel amine-derived bis-azoles as potential
antibacterial and antifungal agents
,
b
Bo Fang a, Cheng-He Zhou a , Xian-Cai Rao
*
a Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China
b Department of Microbiology, College of Medicine, Third Military Medical University, Chongqing 400038, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel amine-derived bis-azole compounds were designed by the systematical structural
modification of Fluconazole and synthesized by a convenient and efficient method, and the antimicrobial
activities for all prepared compounds were evaluated in vitro against six representative bacterial strains
and two fungal strains. Bioactive results indicated that some synthesized compounds exhibited moderate
or even better activities in comparison with the reference drugs. Especially, bis-imidazole 5b and its salts
gave significant antibacterial efficacy against all tested bacteria strains including MRSA, while bis-tri-
azoles 4bec and their corresponding salts exhibited better activities against Candida albicans, Bacillus
proteus than standard drugs Fluconazole and Norfloxacin respectively. Unexpectedly, bis-bromides 3aef
presented excellent activities against all tested microbial strains.
Received 31 March 2010
Received in revised form
4 June 2010
Accepted 5 June 2010
Available online 12 June 2010
Keywords:
Antibacterial
Antifungal
Triazole
Ó 2010 Elsevier Masson SAS. All rights reserved.
Imidazole
Benzimidazole
1. Introduction
Miconazole have been successfully developed and widely used in
clinic. Their inhibitory properties as regard representative fungi
Infectious diseases have been serious and growing threatens to
human health during the past few decades [1,2]. The decrease of
sensibility to antimicrobial agents in current use has also been
increasing for a great variety of pathogens and the resistance to
multiple drugs is more and more prevalent for several microorgan-
isms, especially for Gram-positive bacteria [3] and some intractable
fungi [4]. Furthermore, infections by methicillin-resistant Staphylo-
coccus aureus (MRSA) and Aspergillus presented difficult problems
for medicine [5,6]. Therefore the urgent need for discovery or opti-
mization of antimicrobial agents active against these resistant strains
is of paramount importance [7,8].
Azole derivatives including triazole, imidazole and benzimid-
azole ones etc. are an important class of aromatic heterocyclic
compounds with broad spectrum of biological activities such as
antimicrobial [9e11], anticancer [12], anti-inflammatory [13],
antivirus [14], anticonvulsant [15]. Particularly, the investigations of
azole-based antimicrobial agents have attracted considerable
attention in recent years. Numerous efforts have been directed
toward the development of novel azole antimicrobial agents
[16e19], and some excellent drugs such as Fluconazole and
have been extensively exploited [20e22]. Especially, it is worthy to
note that Fluconazole, the first-line triazole-antifungal drug rec-
ommended by World Health Organization (WHO), has established
an exceptional therapeutic record for Candida infections, and
become the first choice in the treatment of infections by Candida
albicans and Cryptococcus neoformans due to its potent activity,
excellent safety profile, and favorable pharmacokinetic character-
istics [23]. However, Fluconazole is not effective against invasive
aspergillosis and is not fungicidal. In addition, extensive clinical use
of Fluconazole has resulted in the increasing Fluconazole-resistant
C. albicans isolates [24]. Thus, many researches have been devoted
to the further development of Fluconazole in order to broaden its
antimicrobial spectrum and increase its therapeutic indexes. These
works mainly included three aspects: (a) To optimize and modify
the chemical structure of Fluconazole by introducing functional
groups into hydroxyl or triazolyl moiety [25]. Fosfluconazole, an
ester prodrug which was obtained by the esterification of hydroxyl
group of Fluconazole with phosphoric acid, was one of the
successful examples that has been marketed in 2003 [26]; (b) To
expand the new potential applications of Fluconazole in supra-
molecular chemistry and develop the Fluconazole-based supra-
molecular drugs [27,28]; (c) To develop various analogues of
Fluconazole [29,30]. A large number of researches have been
focused on the analogues of Fluconazole in recent years, and some
* Corresponding author. Tel./fax: þ86 23 68254967, þ86 23 68254165.
E-mail address: zhouch@swu.edu.cn (C.-H. Zhou).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.012

B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
4389
outstanding achievements have been obtained in this aspect. Many
azole derivatives, for instance, Voriconazole, Posaconazole and
Ravuconazole were marketed or in the late stages of clinical trials.
Moreover, it was also found that some azoles were effective
inhibitors for enoyl acyl carrier protein reductase (FabI), a novel
antibacterial target [31], while some azole compounds showed
remarkable antibacterial efficacy against MRSA [32].
Prompted by these observations and in continuation of our
researches for bioactive molecules [33,34], we modified the struc-
ture of Fluconazole from the following respects (Fig. 1):
phosphorus tribromide to afford the bromides 3aef in good yields at
20e60 ꢀC. Compounds 3aef reacted with 1H-1,2,4-triazole or 2-
methyl-5-nitro-1H-imidazole in the presence of weak base such as
potassium carbonate in acetonitrile at 40e70 ꢀC to give bis-triazoles
(4aef) and bis-nitroimidazole compound 5g. However, under the
same condition above, it was difficult to produce the corresponding
bis-imidazoles(5aef, 5h, 5i)andbis-benzimidazoles(6aec), whereas
the presence of strong base NaH in anhydrous THF could effectively
result in the formation of target bis-azoles 5aef, 5h, 5i and 6aec in
moderate to high yields at 40e70 ꢀC. The hydrochlorides 7e9 or
nitrates 10e12 were easily prepared by the reaction of compounds
4e6 with hydrochloric acid or nitric acid respectively.
(1) Tertiary amino moiety as bioisoster was selected to replace the
tertiary alcohol of Fluconazole. Compared with tertiary alcohol,
the tertiary amino group could not only be liable to form
hydrogen bonds, but also accept protons or form quaternary
salts which result in the increase of water solubility [35], or
coordinatewith metalions [36], whichprobablylead to enhance
affinity, selectivity and potency in biological properties.
(2) On the basis of above changed structural modification, various
halobenzyl moieties were introduced into target compounds to
substitute 2,4-diflurophenyl group of Fluconazole. Many studies
showed that incorporation of halobenzyl moiety into organic
molecules could greatly improve the pharmacological properties
[37,38], which could enhancetherate of absorption and transport
of drugs in vivo with increased lipid solubility [39]. Moreover,
benzyl moiety with better flexibility compared to phenyl group,
could possibly improve molecular biological properties.
3. Pharmacology
The in vitro antimicrobial screening for all the prepared
compounds were evaluated against three Gram-positive organisms
viz. S. aureus, MRSA, Bacillus subtilis and three Gram-negative
organisms viz. Escherichia coli, Pseudomonas aeruginosa, Bacillus
proteus as well as fungi viz. C. albicans, Aspergillus fumigatus by the
micro-broth dilution method in 96-well microtest plates according
to the methods recommended by the National Committee for
Clinical Laboratory Standards (NCCLS) [43]. The minimum inhibi-
tory concentrations (MIC) were determined and compared to
Chloramphenicol, Norfloxacin and Fluconazole as standard drugs.
All these antimicrobial data are given in Table 4 and 5.
(3) Various azolyl rings like triazolyl and its bioisosters imidazolyl
as well as benzimidazolyl groups were introduced into the
above modified structures to investigate the effect of azolyl
rings on biological activities, since it was found that some bis-
imidazole derivatives [40] and structurally simple alkyl azoles
gave good antimicrobial activities [41].
(4) The methylene bridge between tertiary alcohol group and tri-
azolyl moiety in Fluconazole was replaced by ethylene chain to
increase molecular flexibility, which might be helpful to
improve molecular binding ability to the microbial target [42].
4. Results and discussion
4.1. Chemistry
The structures of the synthesized compounds (Table 1) were
characterized by MS, IR and 1H NMR spectra as well as elemental
analyses. The analytical data were consistent with the assigned
structures and listed in the experimental section. The MS spectra of
all the compounds showed a [M þ H]þ or [M þ H ꢁ 3HY]þ peak, in
agreement with their molecular formula.
In IR spectra, the intermediates 2aef gave broad absorption
bands in 3382e3358 cmꢁ1 region which indicated the presence of
the OH group, while the disappearance of the characteristic
hydroxy group stretching frequencies in bromides 3aef suggested
that the hydroxy group underwent bromination. In addition, the
aryl characteristic CeH bands of compounds 2aef and 3aef
appeared in the region between 3078 and 3027 cmꢁ1. Target bis-
azoles 4e12 can be recognized by the presence of specific absorp-
tion bands of azolyl moiety in 3126e3082 cmꢁ1 region. All the
other absorption bands were also observed at expected regions.
In 1H NMR spectra, intermediates 2aef showed a singlet at
Based on the above considerations, a series of novel halobenzyl
amine-derived bis-azoles and their hydrochlorides and nitrates
including bis-triazoles, bis-imidazoles and bis-benzimidazoles as
the analogues of Fluconazole were designed and synthesized by
a convenient and efficient method. Their antibacterial and anti-
fungal activities were evaluated, and the structureeactivity rela-
tionships were also investigated.
2. Chemistry
d
3.02e3.66 ppm due to the presence of OH protons, protons Hc and
Hd gave signal as triplet at
2.54e2.68 ppm, 3.48e3.59 ppm
The synthetic route of target amine bis-azoles 4e6 and corre-
sponding salts 7e12 was shown in Scheme 1. The intermediates 2aef
couldbeefficientlypreparedwithhighyields(95.8%e98.5%)bytheN-
alkylation of diethanolamine in acetonitrile with halobenzyl halides
1aef, and then diols 2aef were brominated in chloroform using
d
respectively. Compared with the diols 2aef, bromides 3aef dis-
played upfield shifts for Hd (0.15e0.35 ppm), and downfield shifts
for Hc (0.19e0.32 ppm) signal because OH moieties were
substituted by Br groups (some 1H NMR data were listed in Table 2).
When the Br atoms of the compounds (3aef) were replaced by
azolyl groups, the resulting bis-azoles 4e6 showed significant
downfield shifts for Hd and upfield shifts for Ha. The proton
chemical shifts of imiazolyl groups in compounds 5aef were in the
order H-2 > H-4 > H-5, and bis-imidazoles 5aef displayed higher
field chemical shifts for Hd, He and Hf as compared to corresponding
triazoles 4aef. Moreover, the presence of nitro group in compound
5g resulted in large downfield chemical shifts of Hd and He, when
compared with other bis-imidazoles. No large differences were
found in Ha chemical shifts for the protons of phenyl ring in bis-
azoles 4e6.
N
N
Az Az
R1
N
N
N
N
N
N N
N
N
N
Az =
N
N
OH
R3
R2
X1
X2
R4
R5
X1, X2, X3 = H, Cl, or F
F
R1, R2, R3, R4, R5 = H, CH3, C2H5
NO2 or phenyl
X3
Target compounds
F
Fluconazole
Fig. 1. Structures of Fluconazole and amine-derived bis-azoles.

4390
B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
X
N
R5
R4
N
N
OH
OH
Br
X1
X2
X1
X2
X1
N
N
Br
IV
I
II
N
X2
X1
X2
X3
1a–f
X3
2a–f
X3
R5
N
3a–f
R4
III or IV
6a–c
X3
III
R2
N
R1
N
R1
R3
N
N
N
V
N
X1
X2
X1
N
N
N
N
N
R3
R5
R4
X2
5a–i
N
N
R2
X3
X3
4a–f
V
N
N
N
R2
R3
N
V
R1
X1
X2
R5
N
N
R1
N
N
N
N
•3HY
9a or 12a
X1
X2
R4
N
X1
X2
X3
N
N
N
N
R3
•3HY
N
R2
X3
7a–c or 10a–c
•3HY
X3
8a–d or 11a–d
Scheme 1. Synthetic route of intermediates 2, 3 and amine-derived bis-azoles 4e12. Reagents and reaction conditions: (I) NH(CH2CH2OH)2, CH3CN, 50 ꢀC; (II) PBr3, CHCl3, 60 ꢀC;
(III) 1H-1,2,4-triazole or 2-methyl-5-nitro-1H-imidazole, K2CO3, CH3CN, 60 ꢀC; (IV) 1H-imidazole or substituted 1H-imidazole or 1H-benzimidazole or substituted 1H-benzimid-
azole, NaH, THF, 60 ꢀC; (V) Et2O/CHCl3, 4 mol/L HCl or HNO3.
In comparison with free bis-azoles 4e6, their corresponding
hydrochlorides 7e9 and nitrates 10e12 showed large downfield
shifts for some protons (some 1H NMR data were listed in Table 3).
Especially, compounds 7e12 displayed remarkable downfield shifts
for protons Hd (0.68e1.23 ppm) and He, Hf (0.48e2.56 ppm). These
facts demonstrated that the positive charge in nitrogen atoms could
obviously affect the chemical shifts of bis-azoles.
7e12 gave better activities than their corresponding precursors
4e6.
For the tested bis-triazoles 4aef, some compounds showed poor
to moderate antibacterial activities, and dihalobenzyl bis-triazoles
4aec displayed better activities against the tested bacterial strains
than the corresponding monochlorobenzyl compounds 4dee.
Particularly, 3,4-dichlorobenzyl compound 4c exhibited excellent
activity against B. proteus (MIC ¼ 0.5
mg/mL), which was 2-fold and
16-fold more potent than Norfloxacin and Chloramphenicol
respectively. These results suggested that the presence of dichlor-
obenzyl moiety in bis-triazole compounds could improve effec-
tively the antibacterial activity to some extent.
4.2. Antibacterial activity
The obtained results (Table 4) showed that most of dihalobenzyl
amine bis-azoles and all bromides 3aef exhibited moderate to
excellent efficacy against tested bacterial strains, especially all salts
Compared to bis-triazoles 4aef, bis-imidazoles 5aef exhibited
stronger efficacy against S. aureus, MRSA, B. subtilis and E. coli, and
Table 1
Structures of intermediates 1e3 and amine-derived bis-azoles 4e12.
Compd.
X1
X2
X3
X
R1
R2
R3
Compd.
X1
X2
X3
R1
R2
R3
R4
R5
Y
1ae3a
1be3b
1ce3c
1de3d
1ee3e
1fe3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
5g
F
H
H
Cl
H
Cl
H
H
H
Cl
H
Cl
H
H
H
Cl
H
Cl
H
H
H
F
Br
Cl
Cl
Cl
Cl
Cl
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
H
H
H
H
H
H
H
H
5i
6a
6b
6c
7a
7b
7c
8a
8b
8c
8d
9a
10a
10b
10c
11a
11b
11c
11d
12a
Cl
F
Cl
Cl
F
Cl
H
F
Cl
H
Cl
F
H
H
H
H
H
H
Cl
H
H
Cl
H
H
H
H
Cl
H
H
Cl
H
H
Cl
F
Cl
Cl
F
Cl
Cl
F
Cl
Cl
Cl
F
C6H5
e
H
e
e
e
e
e
e
H
H
H
H
H
e
e
e
H
H
H
H
H
H
e
e
e
e
e
e
H
H
H
H
e
e
e
e
H
H
H
H
e
e
H
H
CH3
e
e
e
e
e
e
e
H
e
e
e
e
e
e
e
H
e
H
H
CH3
e
e
e
e
e
e
e
H
e
e
e
e
e
e
e
H
e
Cl
H
Cl
H
H
F
Cl
H
Cl
H
H
F
Cl
H
Cl
H
H
Cl
Cl
Cl
Cl
H
H
Cl
F
Cl
Cl
H
H
Cl
F
Cl
Cl
H
H
Cl
Cl
Cl
e
e
e
e
e
e
e
e
e
e
e
e
e
e
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
NO3
NO3
NO3
NO3
NO3
NO3
NO3
NO3
e
e
e
e
e
e
e
e
H
H
H
C6H5
H
e
e
e
e
e
e
e
e
H
H
H
H
H
H
CH3
C2H5
H
H
H
H
H
H
NO2
CH3
F
F
e
Cl
H
F
Cl
H
Cl
F
Cl
Cl
F
Cl
Cl
Cl
F
e
e
H
H
H
C6H5
H
5h

B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
4391
Table 2
Some 1H NMR data (
d
ppm) of intermediates 2def, 3def and corresponding amine-derived bis-azoles 4e6.
Hc
He
Hg
X2
X1
Ha
R
R
N
Br, Cl, OH,
N
Z
N
N
N
R =
X3
Hb
Hd
Hf
Compd.
2d
2e
2f
3d
3e
3f
4d
4e
4f
5d
5e
5f
5g
6b
6c
Ha
Hb
Hc
Hd
He
Hf
7.15
3.71
2.63
3.52
e
7.20
3.61
2.62
3.56
e
7.31
3.79
2.66
3.58
e
7.19
3.79
2.95
3.30
e
7.14
3.60
2.86
3.25
e
7.17
3.59
2.85
3.24
e
6.89
3.66
2.92
3.96
7.75
7.79
e
6.83
3.56
2.94
4.03
7.82
7.89
e
6.89
3.54
2.93
4.03
7.81
7.88
e
7.09
3.77
2.80
3.82
7.34
7.01
e
6.96
3.58
2.76
3.82
7.34
7.02
e
7.06
3.61
2.78
3.80
7.38
7.01
e
6.95
3.71
2.83
4.20
e
6.70
3.67
2.83
3.90
e
6.72
3.65
2.82
3.87
e
e
e
e
e
e
e
7.86
e
e
e
Hg
e
e
e
e
e
e
7.73
7.45
especially dihalobenzyl compounds 5aec presented remarkable
antibacterial activities against all the tested strains. It seemed that
imidazolyl ring be favorable for antibacterial efficacy in comparison
with triazolyl moiety. Among compounds 5aec, 2,4-dichlorobenzyl
bis-imidazole 5b was the most active one which showed noticeable
and broad spectrum antibacterial activities with MIC values in the
precursor 4c and even 4-fold and 32-fold more potent than stan-
dard drugs Norfloxacin and Chloramphenicol respectively. More-
over, hydrochlorides 8bed and nitrate 11d exhibited stronger
activities against MRSA (MIC ¼ 8
mg/mL) than Chloramphenicol.
The enhanced activities for all these bis-azole hydrochlorides and
nitrates salts 7e12 might be attributed to the improvement of the
water solubility in comparison with their corresponding
precursors.
range of 4e32
mg/mL. Compound 5b exhibited excellent activity
against P. aeruginosa with the MIC value of 4
mg/mL, which was 16-
fold more potent than the reference drug Chloramphenicol. It also
revealed equivalent activities against S. aureus and MRSA with the
MIC values of 8 and 16 mg/mL, respectively. Interestingly, the three
In order to further investigate different groups in titled tertiary
amines on the antimicrobial activities, intermediate diols 2aef and
dibromides 3aef, which may be considered as the bioisosteres of
chlorine in nitrogen mustards, were also evaluated for their anti-
microbial activities, since much works have been found that
nitrogen mustards as clinical anticancer drugs [44] in extensive use
have significant antimicrobial efficacy [45,46]. To our surprise,
amine-derived dibromides 3aef exhibited moderate to excellent
antibacterial activities against all tested strains. Especially,
compound 3b with 2,4-dichlorobenzyl group displayed prominent
activities towards the tested bacterial strains with MIC values
substituted-imidazoles 5ge5i, whose activities were inferior to bis-
imidazole 5b, showed that the introduction of electron-donor or
electron-withdrawing substituent into imidazolyl ring seemed to
be unfavorable for their antibacterial efficacy. However, bis-2-
phenylimidazoles 5i displayed better activities against all tested
strains than compounds 5g and 5h with 2-methyl-4-nitro-
imidazolyl and 2-ethyl-4-methylimidazolyl group respectively, this
might be attributed to the large lipophilic phenyl groups at 2-
position of imidazolyl ring.
between 0.25 mg/mL and 2 mg/mL, which was equivalent or even
Bis-benzimidazoles 6aec showed comparable antibacterial
efficiency with triazole compounds 4aef, but weaker activities
than imidazole derivatives 5aei. Particularly, compound 6b gave
relatively good activities against all tested bacterial strains with the
much better in comparison with the standard drug Norfloxacin.
However, the bis-hydroxy compounds 2aef as the precursors of
bromides 3aef, displayed poor or no obvious activities against the
tested strains.
MIC values ranging from 16 to 128
m
g/mL.
In general, the target compounds 4c, 5aec, hydrochlorides 7c,
8aed, nitrates 10c, 11aed as well as all bromides 3aef showed
moderate or even excellent antibacterial activities in comparison
with the reference drugs (Norfloxacin and Chloramphenicol)
against tested bacteria strains including MRSA. Meanwhile, the
above discussion demonstrated that the antibacterial efficacy
should be related to azolyl ring, halobenzyl group as well as water
solubility of prepared compounds to some extent. For these serial
compounds, the dihalobenzyl and imidazolyl moieties in
compounds 2e6 seemed to be two favorable factors for antibac-
terial activities, and the salts 7e12 gave better inhibitory activities
In addition, some azole compounds with good activities were
chosen to transform into water-soluble hydrochlorides 7e9 and
nitrates 10e12. Such modification resulted in remarkable
improvement in their antibacterial efficacy. Among these salts,
compound 8d showed best antibacterial activities (MIC values
ranging from 2 to 8 mg/mL) against all tested bacteria strains except
B. proteus, which displayed much better efficacy than standard drug
Chloramphenicol. While the strain B. proteus were much sensitive
to hydrochloride 7c and nitrate 10c with the same MIC value of
0.25
mg/mL, which were 2-fold more potent than that of their
Table 3
Some 1H NMR data (
d
ppm) of amine-derived bis-azoles 4e6 and corresponding salts 7e12.
Compd.
4a
4b
5a
5b
6a
7a
7b
8a
8b
9a
6.57
3.85
3.17
4.82
10a
10b
11a
11b
12a
Ha
Hb
Hc
Hd
He
Hf
6.91
3.58
2.92
4.02
7.78
7.84
e
6.90
3.61
2.80
3.84
8.10
8.10
e
6.94
3.60
2.71
3.79
7.26
6.94
e
6.85
3.65
2.74
3.80
7.29
6.96
e
6.52
3.32
2.52
3.59
e
7.14
4.31
3.46
4.75
8.47
9.26
e
7.37
4.09
3.29
4.63
8.58
9.45
e
7.00
3.92
3.10
4.51
9.30
7.76
e
7.11
3.92
3.12
4.54
9.36
7.73
e
7.17
4.39
3.42
4.92
8.54
9.37
e
7.14
3.91
3.18
4.52
8.78
9.59
e
7.05
3.95
3.14
4.54
9.31
7.78
e
7.09
3.90
3.09
4.52
9.25
7.71
e
6.93
3.74
3.10
4.66
e
e
e
e
Hg
7.47
10.03
9.56

4392
B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
Table 4
Table 5
Antifungal data as MIC (
Antibacterial data as MIC (
mg/mL) for intermediates 2, 3 and azole derivatives
m
g/mL) for intermediates 2, 3 and azole derivatives 4e12.a
,b
4e12a
Compd.
C. albicans
A. fumigatus
Compd.
S. aureus MRSA
B. subtilis E. coli P.
aeruginosa
B. proteus
2a
2b
256
128
>256
256
2a
2b
2c
2d
2e
2f
3a
256
128
256
>256
>256
>256
1
>256
256
>256
>256
>256
>256
2
256
256
128
>256
>256
>256
128
256
128 >256
>256 >256
>256 >256
256
128
256
128
128
256
>256
256
4
2c
2d
2e
2f
3a
>256
>256
>256
>256
0.5
>256
>256
>256
>256
64
256
1
256
4
3b
3c
0.25
2
64
128
0.5
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
7a
7b
7c
8a
8b
8c
8d
9a
10a
2
0.5
8
16
32
128
64
64
>256
256
>256
32
8
16
128
64
64
256
16
16
64
32
128
64
16
32
16
4
4
2
32
128
32
16
32
16
4
4
32
32
0.25
4
64
128
32
64
>256
32
>256
>256
>256
32
32
16
>256
128
64
32
64
32
64
32
64
32
128
16
8
8
4
4
>256
32
128
16
16
16
2
2
32
64
16
64
64
32
256
>256
256
64
16
16
16
32
32
16
0.25
0.5
2
16
32
8
>256
16
0.5
>256
32
128
64
32
64
>256
64
128
32
16
16
64
32
32
256
4
0.25
32
32
16
4
64
>256
8
0.25
64
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
5d
5e
5f
5g
5h
5i
6a
6b
6c
7a
7b
7c
8a
8b
8c
8d
9a
32
8
16
128
64
128
1
16
64
16
8
32
64
128
16
16
128
4
64
256
32
64
32
8
>256
0.25
>256
>256
>256
>256
>256
128
>256
>256
>256
>256
>256
32
>256
>256
>256
>256
32
0.25
128
128
64
>256
64
>256
64
0.25
256
128
128
>256
64
>256
0.5
>256
>256
>256
>256
>256
>256
>256
>256
64
>256
>256
>256
>256
>256
>256
>256
>256
>256
256
32
>256
256
256
>256
256
>256
32
16
32
256
>256
64
>256
128
16
>256
64
>256
>256
128
32
16
8
8
8
128
>256
256
64
32
16
256
64
128 >256
64
128
16
64
32
32
16
16
4
32
128
8
16
32
64
4
128
256
>256
>256
32
128
32
2
>256
>256
128
>256
>256
>256
64
256
>256
256
2
10a
10b
10c
11a
11b
11c
11d
12a
Fluconazole
>256 >256
128
64
32
32
32
4
16
16
64
64
4
10b
10c
11a
11b
11c
11d
64
32
16
16
4
16
8
16
8
64
4
4
12a
128
0.25
8
256
0.25
16
>256
1
>256 >256
128
1
8
a
C. albicans, Candida albicans ATCC 76615; A. fumigatus, Aspergillus fumigatus
Norfloxacin
Chloramphenicol
0.5
8
2
64
ATCC 96918.
8
a
Minimum inhibitory concentrations were determined by micro-broth dilution
antifungal activities (MIC ¼ 0.25
m
g/mL), which were two-fold more
method.
b
potent than clinical antifungal drug Fluconazole (MIC ¼ 0.5
mg/mL).
S. aureus, Staphylococcus aureus ATCC 29213; MRSA, Staphylococcus aureus N
Bis-imidazole compounds 5h, 8b, 8d and 11d presented better
antifungal activities against C. albicans than other imidazoles and
315; B. subtilis, Bacillus subtilis ATCC 21216; E. coli, Escherichia coli ATCC 25922; P.
aeruginosa, Pseudomonas aeruginosa ATCC 27853; B. Proteus, Bacillus proteus ATCC
13315.
benzimidazoles with the MIC values ranging from 32 to 64
Unfortunately, most compounds showed poor or no obvious
activity (MIC ꢂ 256 g/mL) against A. fumigatus, with the exception
of 3,4-dicholobenzyl compounds 5c, 8c, 11c and 2,4-dicholobenzyl
salts 7b, 8d and 10b (MIC ¼ 32e128 g/mL), which were found to
exhibit better activity than Fluconazole (MIC ¼ 256 g/mL). In
mg/mL.
than their precursors 4e6. Further study is necessary to elucidate
their mechanism and structureeactivity relationships.
m
m
m
4.3. Antifungal activity
addition, dihalobenzyl compounds 4aec and 5aec have stronger
antifungal efficiency than monohalobenzyl ones 4def and 5def
respectively, similar to the antibacterial activities. These results
indicated that triazolyl moiety seemed to be more helpful than
imidazolyl or benzimidazolyl group for enhancing the antifungal
activities against C. albicans, and their corresponding salts were
favorable for improving their antifungal efficacy.
The antifungal activities showed that the intermediates
bromides 3aef exhibited excellent antifungal activities against C.
albicans and A. fumigatus, especially dihalobenzyl compounds 3a
The antifungal evaluation showed that the activities for most bis-
azoles 4e12 were relatively weak compared to their antibacterial
activities (Table 5). However, some dihalobenzyl compounds, their
corresponding hydrochlorides and nitrates as well as bromides 3aef
exhibited significant antifungal activities against tested strains.
Some triazole compounds and their salts exhibited better
activities against C. albicans than corresponding bis-imidazole and
bis-benzimidazole derivatives, especially 2,4-dicholobenzyl bis-
triazole 4b, its hydrochloride 7b and nitrate 10b gave the best

B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
4393
and 3b gave the MIC value of 0.5, 0.25
m
g/mL against C. albicans and
aqueous sodium bicarbonate and extracted with chloroform.
Subsequently, the organic extracts were collected, washed with
brine, dried over anhydrous sodium sulfate and filtered. The filtrate
was evaporated to afford the crude product, which was used in the
next step without further purification.
64, 64 g/mL against A. fumigatus which were comparable or even
m
superior to Fluconazole.
5. Conclusion
In conclusion, a series of novel halobenzyl amine-derived bis-
azoles were designed by the systematical structural modification of
Fluconazole and successfully synthesized by a convenient and
efficient method. All new compounds were characterized by MS, IR,
1H NMR and elemental analyses. The in vitro antimicrobial activities
of these azole derivatives and their intermediates were determined
against six bacterial strains and two fungal strains. The results
showed that some azole derivatives exhibited significant antibac-
terial and antifungal activities, even comparable or superior to
reference drugs (Fluconazole, Norfloxacin and Chloramphenicol).
Imidazoles 5b, 8aed and 11aed bearing dihalobenzyl groups pre-
sented better antibacterial activities than other compounds against
all tested bacteria strains. However, bis-triazoles 4c, 7c and 10c
6.1.1.1. 2,20-(2,4-Difluorobenzylazanediyl)diethanol (2a). Compound
2a was obtained as yellow oil (11.12 g). Yield: 96.3%; IR (KBr, cmꢁ1
)
n
: 3382, 2953, 2884, 1619, 1504, 1456, 1276, 1137, 961, 851; 1H NMR
(300 MHz, CDCl3, ppm): d 7.25e7.15 (m, 1H, Ph 6-H), 6.73e6.61 (m,
2H, Ph 3, 5-H), 3.59 (s, 2H, PhCH2), 3.48 (t, J ¼ 5.1 Hz, 4H, NCH2CH2),
3.21 (s, 2H, OH), 2.54 (t, J ¼ 5.1 Hz, 4H, NCH2); MS m/z: 232
[M þ H]þ.
6.1.1.2. 2,20-(2,4-Dichlorobenzylazanediyl)diethanol (2b). Compound
2b was obtained as yellow oil (12.89 g). Yield: 97.6%; IR (KBr, cmꢁ1
) n:
3374, 2950, 1588, 1561, 1470, 1374, 1244, 1081, 867, 824; 1H NMR
(300 MHz, CDCl3, ppm): 7.46e7.19 (m, 3H, Ar-H), 3.75 (s, 2H,
d
PhCH2), 3.59 (t, J ¼ 5.1 Hz, 4H, NCH2CH2), 3.32 (s, 2H, OH), 2.68 (t,
gave remarkable efficacy (MIC values of 0.25e0.5 mg/mL) against B.
proteus. Moreover, bis-triazoles 4b, 7b and 10b exhibited more
potent antifungal activity than the clinically prevalent antifungal
J ¼ 5.1 Hz, 4H, NCH2); MS m/z: 264 [M þ H]þ.
6.1.1.3. 2,20-(3,4-Dichlorobenzylazanediyl)diethanol (2c). Compound
drug Fluconazole against C. albicans with MIC value of 0.25
mg/mL.
2c was obtained as yellow oil (12.65 g). Yield: 96.2%; IR (KBr, cmꢁ1
) n:
Surprisingly, bromides 3aef also showed excellent antibacterial
and antifungal activities. These antimicrobial results indicated that
structural factors such as the type of azolyl rings, halobenzyl
moieties and the water solubility of target compounds could
significantly affect their antimicrobial activities. In these serial
compounds, it was showed that dihalobenzyl groups are more
helpful for increasing antibacterial and antifungal efficacy in
comparison with the monohalobenzyl ones. For another, imidazolyl
moiety seems to be favorable for antibacterial efficacy against
tested bacteria, while 1,2,4-triazolyl group is conducive to anti-
fungal activities. In addition, the water-soluble salts could further
enhance the inhibitory activities of these compounds.
3358, 2927,1593,1562,1471,1288,1204,1130,1031, 880, 822, 770; 1H
NMR (300 MHz, CDCl3, ppm):
d
7.42 (s,1H, Ph 2-H), 7.35 (d, J ¼ 8.2 Hz,
1H, Ph 5-H), 7.16 (d, J ¼ 8.2 Hz, 1H, Ph 6-H), 3.62 (s, 2H, PhCH2), 3.59
(t, J ¼ 5.0 Hz, 4H, NCH2CH2), 3.40 (s, 2H, OH), 2.65 (t, J ¼ 5.0 Hz, 4H,
NCH2); MS m/z: 264 [M þ H]þ.
6.1.1.4. 2,20-(2-chlorobenzylazanediyl)diethanol (2d). Compound 2d
was obtained as yellow oil (11.11 g). Yield: 97.0%; IR (KBr, cmꢁ1
) n:
3382, 3066, 2927, 1594, 1572, 1472, 1275, 1203, 1144, 1035, 876, 753;
1H NMR (300 MHz, CDCl3, ppm):
d
7.37e7.34 (m, 1H, Ph 3-H),
7.28e7.25 (m, 1H, Ph 5-H), 7.20e7.09 (m, 2H, Ph 4,6-H), 3.71 (s, 2H,
PhCH2), 3.52 (t, J ¼ 5.2 Hz, 4H, NCH2CH2), 3.02 (s, 2H, OH), 2.63 (t,
J ¼ 5.2 Hz, 4H, NCH2); MS m/z: 230 [M þ H]þ.
6. Experimental protocol
6.1.1.5. 2,20-(3-Chlorobenzylazanediyl)diethanol (2e). Compound 2e
6.1. General methods
was obtained as yellow oil (11.28 g). Yield: 98.5%; IR (KBr, cmꢁ1
) n:
3368, 3072, 2927, 1596, 1568, 1285, 1203, 1135, 1032, 878, 762; 1H
NMR (300 MHz, CDCl3, ppm):
Melting points were determined on X-6 melting point apparatus
and are uncorrected. Infrared (IR) spectra were recorded on a Bio-
Rad FTS-185 spectrometer by using KBr disks. 1H NMR spectra were
recorded using TMS as an internal reference standard either on
a Bruker AV 300 or Varian-Mercury 400 spectrometer. Chemical
d
7.48 (d, J ¼ 7.9 Hz, 1H, Ph 4-H),
7.38e7.32 (m, 1H, Ph 5-H), 7.23e7.16 (m, 2H, Ph 2, 6-H), 3.61 (s, 2H,
PhCH2), 3.56 (t, J ¼ 5.2 Hz, 4H, NCH2CH2), 3.18 (s, 2H, OH), 2.62 (t,
J ¼ 5.2 Hz, 4H, NCH2); MS m/z: 230 [M þ H]þ.
shifts are reported in parts per million (
d
). Coupling constants (J) are
6.1.1.6. 2,20-(4-Chlorobenzylazanediyl)diethanol (2f). Compound 2f
reported in hertz (Hz). The peak shapes are denoted as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; br, broad. The following
abbreviations were used to designate azolyl groups: Tri, triazolyl;
Im, imidazolyl; Benim, benzoimidazolyl. The mass spectra were
recorded on FINNIGAN TRACE GCeMS 2000 mass spectrometer.
Elemental analyses were carried out on a Carlo Erba model EA 1106
elemental analyzer. TLC analyses were done using pre-coated silica
gel plates. Column chromatographies were performed on silica gel
(Merck; 70e230 mesh) column. Developed plates were visualized
by UV light. All solvents were reagent grade and, if necessary, were
purified and dried by standard methods.
was obtained as yellow oil (10.97 g). Yield: 95.8%; IR (KBr, cmꢁ1
) n:
3359, 2949, 2882, 2827, 1615, 1597, 1490, 1275, 1143, 1087, 901, 876,
841, 808; 1H NMR (300 MHz, CDCl3, ppm):
d
7.36e7.26 (m, 4H, Ph
2,3,5, 6-H), 3.79 (s, 2H, PhCH2), 3.66 (s, 2H, OH), 3.58 (t, J ¼ 4.9 Hz,
4H, NCH2CH2), 2.66 (t, J ¼ 4.9 Hz, 4H, NCH2); MS m/z: 230 [M þ H]þ.
6.1.2. General procedure for the preparation of compounds 3aef
To a stirred solution of compound 2 (2aef) (0.04 mol) in chlo-
roform was added tribromophosphine (13.54 g, 0.05 mol) drop-
wise. The resulting mixture was stirred at room temperature for
2 h, and then heated at 60 ꢀC. After the reaction was completed
(monitored by TLC, methanol/chloroform (1/20e1/8, V/V)), the
reaction was cooled to room temperature and treated with ice
water. After basified with saturated aqueous sodium bicarbonate,
the mixture was extracted with chloroform, and the combined
organic extracts were washed with brine, dried over sodium sulfate
and filtered. The filtrate was concentrated to give crude product,
and the crude product was purified by column chromatography on
silica gel eluting with dichloromethane/light petroleum (1/20e1/6,
6.1.1. General procedure for the preparation of compounds 2aef
A mixture of diethanolamine (6.31 g, 0.06 mol) and compound 1
(1aef) (0.05 mol) in acetonitrile was stirred and heated at 50 ꢀC.
After the reaction was completed (monitored by TLC, dichloro-
methane/light petroleum (1/100e1/20, V/V)), the mixture was
cooled to room temperature and the solvent was removed under
reduced pressure, and then the residue was basified with saturated

4394
B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
V/V) to afford the pure target 3aef. Characterization data of 3aef
are given below.
sulfate and filtered. After the filtrate was concentrated, the crude
product was purified by column chromatography on silica gel
eluting with acetone/ethylacetate (1/8e1/1, V/V). Characterization
data of 4aef and 5g are given below.
6.1.2.1. 2-Bromo-N-(2-bromoethyl)-N-(2,4-difluorobenzyl)ethan-
amine (3a). Compound 3a was obtained as yellow oil (12.25 g).
Yield: 85.8%; IR (KBr, cmꢁ1
)
n
: 3078, 2965, 2846, 1618, 1502, 1457,
6.1.3.1. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine (4a). Compound 4a was obtained
1278, 1136,1094, 970, 851; 1H NMR (300 MHz, CDCl3, ppm):
d
7.32
(d, J ¼ 7.1 Hz, 1H, Ph 6-H), 6.76e6.71 (m, 2H, Ph 3, 5-H), 3.64 (s, 2H,
PhCH2), 3.25 (t, J ¼ 7.2 Hz, 4H, NCH2CH2), 2.85 (t, J ¼ 7.2 Hz, 4H,
NCH2); MS m/z: 358 [M þ H]þ.
as a yellow solid (0.84 g). Yield: 83.8%, mp: 90e93 ꢀC; IR (KBr, cmꢁ1
)
1
n: 3118, 2955, 2841, 1619, 1506, 1431, 1275, 1140, 962, 855; H NMR
(400 MHz, CDCl3, ppm):
d
7.84 (d, J ¼ 3.6 Hz, 2H, Tri 3-H), 7.78 (d,
J ¼ 4.0 Hz, 2H, Tri 5-H), 6.93e6.88 (m, 1H, Ph 6-H), 6.77e6.72 (m, 2H,
Ph 3, 5-H), 4.03 (t, J ¼ 6.4 Hz, 4H, Tri-CH2), 3.58 (s, 2H, PhCH2), 2.93 (t,
J ¼ 6.4 Hz, 4H, Tri-CH2CH2); MS m/z: 334 [M þ H]þ; Anal. Calcd. for
C15H17F2N7: C, 54.05; H, 5.14; N, 29.41. Found: C, 54.17; H, 5.12; N,
29.68.
6.1.2.2. 2-Bromo-N-(2-bromoethyl)-N-(2,4-dichlorobenzyl)ethan-
amine (3b). Compound 3b was obtained as yellow oil (14.88 g).
Yield: 95.6%; IR (KBr, cmꢁ1
)
n
: 2960, 2923, 2850, 1588, 1561, 1469,
1379, 1282, 1206, 1096, 1048, 865, 822; 1H NMR (300 MHz, CDCl3,
ppm): 7.26e7.54 (m, 3H, Ar-H), 3.82 (s, 2H, PhCH2), 3.44 (t,
d
J ¼ 7.1 Hz, 4H, NCH2CH2), 3.00 (t, J ¼ 7.1 Hz, 4H, NCH2); MS m/z: 390
6.1.3.2. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
[M þ H]þ.
(1H-1,2,4-triazol-1-yl)ethanamine (4b). Compound 4b was obtained
as colorless oil (0.90 g). Yield: 82.0%; IR (KBr, cmꢁ1
)
n
: 3123, 3106,
3067, 3056, 2968, 2829,1508,1473,1450,1383,1201,1141,1046,1007,
876, 816; 1H NMR (300 MHz, CDCl3, ppm):
8.10 (m, 4H, Tri 3-H, 5-
6.1.2.3. 2-Bromo-N-(2-bromoethyl)-N-(3,4-dichlorobenzyl)ethan-
amine (3c). Compound 3c was obtained as yellow oil (14.36 g).
d
Yield: 92.3%; IR (KBr, cmꢁ1
)
n
: 3058, 3012, 2963, 2926, 2830, 1593,
1562, 1469, 1282, 1205, 1093, 1030, 819; 1H NMR (300 MHz, CDCl3,
ppm):
H), 7.10e6.69 (m, 3H, Ar-H), 3.84 (t, J ¼ 5.6 Hz, 4H, Tri-CH2), 3.61 (s,
2H, PhCH2), 2.80 (t, J ¼ 5.6 Hz, 4H, Tri-CH2CH2); MS m/z: 366
[M þ H]þ; Anal. Calcd. for C15H17Cl2N7: C, 49.19; H, 4.68; N, 26.77.
Found: C, 49.32; H, 4.63; N, 26.59.
d
7.36 (s, 1H, Ph 2-H), 7.28 (d, J ¼ 8.2 Hz, 1H, Ph 5-H), 7.10 (d,
J ¼ 8.2 Hz, 1H, Ph 6-H), 3.57 (s, 2H, PhCH2), 3.24 (t, J ¼ 7.1 Hz, 4H,
NCH2CH2), 2.84 (t, J ¼ 7.1 Hz, 4H, NCH2); MS m/z: 390 [M þ H]þ.
6.1.3.3. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
6.1.2.4. 2-Bromo-N-(2-bromoethyl)-N-(2-chlorobenzyl)ethanamine
(1H-1,2,4-triazol-1-yl)ethanamine (4c). Compound 4c was obtained
(3d). Compound 3d was obtained as yellow oil (11.97 g). Yield:
as colorless oil (0.83 g). Yield: 75.6%; IR (KBr, cmꢁ1
)
n
: 3118, 2953,
2832, 1508, 1470, 1275, 1139, 960, 875; 1H NMR (400 MHz, CDCl3,
ppm):
84.3%; IR (KBr, cmꢁ1
)
n
: 3064, 3015, 2966, 2837, 1593, 1572, 1471,
1441, 1282, 1206,1092, 1037, 754; 1H NMR (300 MHz, CDCl3, ppm):
d
7.86 (s, 2H, Tri 3-H), 7.80 (s, 2H, Tri 5-H), 7.23 (t, J ¼ 4.8 Hz,
d
7.49 (d, J ¼ 7.0 Hz, 1H, Ph 3-H), 7.42e7.34 (m, 1H, Ph 5-H),
1H, Ph 5-H), 7.00 (d, J ¼ 1.6 Hz, 1H, Ph 2-H), 6.72e6.70 (m, 1H, Ph 6-
H), 4.03 (t, J ¼ 6.0 Hz, 4H, Tri-CH2), 3.48 (s, 2H, PhCH2), 2.90 (t,
J ¼ 6.0 Hz, 4H, Tri-CH2CH2); MS m/z: 366 [M þ H]þ; Anal. Calcd. for
C15H17Cl2N7: C, 49.19; H, 4.68; N, 26.77. Found: C, 49.28; H, 4.60; N,
26.82.
7.22e7.17 (m, 2H, Ph 4,6-H), 3.79 (s, 2H, PhCH2), 3.30 (t, J ¼ 7.2 Hz,
4H, NCH2CH2), 2.95 (t, J ¼ 7.2 Hz, 4H, NCH2); MS m/z: 356 [M þ H]þ.
6.1.2.5. 2-Bromo-N-(2-bromoethyl)-N-(3-chlorobenzyl)ethanamine
(3e). Compound 3e was obtained as yellow oil (10.31 g). Yield:
72.6%; IR (KBr, cmꢁ1
)
n
: 3060, 3014, 2964, 2830, 1597, 1575, 1474,
1430, 1282, 1204,1099, 1077, 971, 889, 780; 1H NMR (300 MHz,
CDCl3, ppm): 7.17e7.11 (m, 4H, Ph 2,4,5, 6-H), 3.60 (s, 2H, PhCH2),
6.1.3.4. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2-chlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine (4d). Compound 4d was obtained
d
as a white solid (0.66 g). Yield: 66.3%, mp: 91e93 ꢀC; IR (KBr, cmꢁ1
) n:
3.25 (t, J ¼ 7.2 Hz, 4H, NCH2CH2), 2.86 (t, J ¼ 7.2 Hz, 4H, NCH2); MS
3121, 3110, 3058, 2951, 2881, 2826, 1594, 1507, 1465, 1136, 1045, 857,
m/z: 356 [M þ H]þ.
779; 1H NMR (400 MHz, CDCl3, ppm):
d
7.79 (s, 2H, Tri 3-H), 7.75 (s,
2H, Tri 5-H), 7.26e7.23 (m, 1H, Ph 3-H), 7.13e7.04 (m, 2H, Ph 4, 5-H),
6.91e6.87 (m,1H, Ph 6-H), 3.96 (t, J ¼ 6.0 Hz, 4H, Tri-CH2), 3.66 (s, 2H,
PhCH2), 2.92 (t, J ¼ 6.0 Hz, 4H, Tri-CH2CH2); MS m/z: 332 [M þ H]þ;
Anal. Calcd. for C15H18ClN7: C, 54.30; H, 5.47; N, 29.55. Found: C,
54.18; H, 5.41; N, 29.76.
6.1.2.6. 2-Bromo-N-(2-bromoethyl)-N-(4-chlorobenzyl)ethanamine
(3f). Compound 3f was obtained as yellow oil (12.31 g). Yield:
86.7%; IR (KBr, cmꢁ1
)
n
: 3027, 2925, 2852, 1596, 1541, 1490, 1281,
1225,1091, 878, 833, 806; 1H NMR (300 MHz, CDCl3, ppm):
7.21e7.13 (m, 4H, Ph 2,3,5, 6-H), 3.59 (s, 2H, PhCH2), 3.24 (t,
d
J ¼ 7.2 Hz, 4H, NCH2CH2), 2.85 (t, J ¼ 7.2 Hz, 4H, NCH2); MS m/z: 356
6.1.3.5. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(3-chlorobenzyl)-2-
[M þ H]þ.
(1H-1,2,4-triazol-1-yl)ethanamine (4e). Compound 4e was obtained
as a white solid (0.50 g). Yield: 50.2%, mp: 93e94 ꢀC; IR (KBr, cmꢁ1
)
6.1.3. General procedure for the preparation of compounds 4aef
and 5g
n
: 3117, 3059, 2957, 2882, 2816, 1598, 1575, 1509, 1466, 1382, 1207,
1141, 880, 768; 1H NMR (400 MHz, CDCl3, ppm):
d
7.89 (s, 2H, Tri 3-
To
a
stirred suspension of potassium carbonate (1.04 g,
H), 7.82 (s, 2H, Tri 5-H), 7.20e7.13 (m, 2H, Ph 4, 5-H), 6.98 (d,
J ¼ 1.6 Hz, 1H, Ph 2-H), 6.84e6.82 (m, 1H, Ph 6-H), 4.03 (t, J ¼ 6.0 Hz,
4H, Tri-CH2), 3.56 (s, 2H, PhCH2), 2.94 (t, J ¼ 6.0 Hz, 4H, Tri-CH2CH2);
MS m/z: 332 [M þ H]þ; Anal. Calcd. for C15H18ClN7: C, 54.30; H, 5.47;
N, 29.55. Found: C, 54.15; H, 5.58; N, 29.84.
7.5 mmol) in 10 mL of acetonitrile was added 1H-1,2,4-triazole
(0.48 g, 6.9 mmol) or 2-methyl-5-nitro-1H-imidazole (0.88 g,
6.9 mmol). The mixture was stirred at 60 ꢀC for 1 h. After cooled to
room temperature, the compound 3 (3aef) (3.0 mmol) was added
and the mixture was stirred at 60 ꢀC. The absence of compound 3
monitored by TLC (dichloromethane/light petroleum) (1/20e1/2,
V/V) indicated that the reaction was completed. Subsequently, the
mixture was cooled to room temperature, the solvent was removed
under reduced pressure, and the residue was partitioned between
chloroform and saturated aqueous sodium bicarbonate. The organic
extracts were collected, washed with brine, dried over sodium
6.1.3.6. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(4-chlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine (4f). Compound 4f was obtained
as colorless oil (0.57 g). Yield: 57.2%; IR (KBr, cmꢁ1
)
n
: 3114, 2953,
2894, 2831, 1670, 1597, 1577, 1507, 1275, 1207, 1141, 846, 752; 1H
NMR (400 MHz, CDCl3, ppm): 7.88 (s, 2H, Tri 3-H), 7.81 (s, 2H, Tri
5-H), 7.18 (d, J ¼ 6.4 Hz, 2H, Ph 3, 5-H), 6.89 (d, J ¼ 8.4 Hz, 2H, Ph 2,
d

B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
4395
6-H), 4.03 (t, J ¼ 6.0 Hz, 4H, Tri-CH2), 3.54 (s, 2H, PhCH2), 2.93 (t,
J ¼ 6.0 Hz, 4H, Tri-CH2CH2); MS m/z: 332 [M þ H]þ; Anal. Calcd. for
C15H18ClN7: C, 54.30; H, 5.47; N, 29.55. Found: C, 54.52; H, 5.38; N,
29.79.
Im-CH2CH2); MS m/z: 364 [M þ H]þ; Anal. Calcd. for C17H19Cl2N5: C,
56.05; H, 5.26; N, 19.23. Found: C, 56.32; H, 5.19; N, 19.04.
6.1.4.4. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2-chlorobenzyl)-2-(1H-
imidazol-1-yl)ethanamine (5d). Compound 5d was obtained as
6.1.3.7. N-(2,4-Dichlorobenzyl)-2-(2-methyl-4-nitro-1H-imidazol-1-
yl)-N-(2-(2-methyl-4-nitro-1H-imidazol-1-yl)ethyl)ethanamine
(5g). Compound 5g was obtained as a white solid (1.22 g). Yield:
84.4%, mp: 189e191 ꢀC; IR(KBr, cmꢁ1): 3126, 2815,1587, 1536, 1496,
colorless oil (0.62 g). Yield: 62.6%; IR (KBr, cmꢁ1
)
n
: 3112, 2986,
2951, 2899, 2825, 1594, 1571, 1550, 1506, 1377, 1229, 1079, 1035,
888, 820, 755; 1H NMR (400 MHz, CDCl3, ppm):
7.35e7.33 (m, 3H,
d
Im 2-H, Ph 3-H), 7.34 (s, 2H, Im 2-H), 7.20e7.17 (m, 2H, Ph 4, 5-H),
7.10e7.07 (m, 1H, Ph 6-H), 7.01 (t, J ¼ 1.2 Hz, 2H, Im 4-H), 6.76 (t,
J ¼ 1.2 Hz, 2H, Im 5-H), 3.82 (t, J ¼ 6.0 Hz, 4H, Im-CH2), 3.77 (s, 2H,
PhCH2), 2.80 (t, J ¼ 6.0 Hz, 4H, Im-CH2CH2); MS m/z: 330 [M þ H]þ;
Anal. Calcd. for C17H20ClN5: C, 61.91; H, 6.11; N, 21.23. Found: C,
61.78; H, 6.20; N, 21.08.
1386, 1183, 1112, 826, 750; 1H NMR (300 MHz, CDCl3, ppm):
d 7.86
(s, 2H, Im 4-H), 7.62e6.95 (m, 3H, Ph 3, 5, 6-H), 4.20 (t, J ¼ 5.8 Hz,
4H, Im-CH2), 3.71 (s, 2H, PhCH2), 2.83 (t, J ¼ 5.8 Hz, 4H, Im-CH2CH2),
2.31 (s, 6H, Im-CH3); MS m/z: 482 [M þ H]þ; Anal. Calcd. for
C19H21Cl2N7O4: C, 47.31; H, 4.39; N, 20.33. Found: C, 47.09; H, 4.46;
N, 20.12.
6.1.4.5. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(3-chlorobenzyl)-2-(1H-
6.1.4. General procedure for the preparation of compounds 5e6
To a stirred suspension of sodium hydride (0.18 g, 7.5 mmol) in
10 mL of tetrahydrofuran was added 1H-imidazole (0.47 g,
6.9 mmol) or 4-ethyl-2-methyl-1H-imidazole (0.76 g, 6.9 mmol) or
2-phenyl-1H-imidazole (0.99 g, 6.9 mmol) or 1H-benzoimidazole
(0.82 g, 6.9 mmol) or 5,6-dimethyl-1H-benzoimidazole (1.00 g,
6.9 mmol), the mixture was heated at 60 ꢀC for 1 h. Then the
mixture was cooled to room temperature, the compound 3 (3aef)
(3.0 mmol) was added and the mixture was stirred at 60 ꢀC. The
reaction was monitored by TLC (dichloromethane/light petroleum
(1/20e1/2, V/V)) when the absence of compound 3 indicated that
the reaction was completed. Then the reaction was cooled to room
temperature and quenched with ice water, the mixture was parti-
tioned between chloroform and saturated aqueous sodium bicar-
bonate, the combined organic extracts were washed with brine,
dried over sodium sulfate and filtered, the filtrate was concen-
trated. The crude product was purified by column chromatography
on silica gel eluting with acetone/ethylacetate (1/8e1/2, V/V).
Characterization data of 5e6 are given below.
imidazol-1-yl)ethanamine (5e). Compound 5e was obtained as
colorless oil (0.58 g). Yield: 58.2%; IR (KBr, cmꢁ1
)
n
: 3109, 2946,
2829, 1634, 1596 1509, 1473, 1374, 1231, 747; 1H NMR (400 MHz,
CDCl3, ppm):
d
7.34 (s, 2H, Im 2-H), 7.20 (d, J ¼ 2.4 Hz, 1H, Ph 4-H),
7.18 (d, J ¼ 8.0 Hz, 1H, Ph 5-H), 7.11 (s, 1H, Ph 2-H), 7.02 (s, 2H, Im 4-
H), 6.97e6.94 (m, 1H, Ph 6-H), 6.74 (t, J ¼ 1.2 Hz, 2H, Im 5-H), 3.82 (t,
J ¼ 6.0 Hz, 4H, Im-CH2), 3.58 (s, 2H, PhCH2), 2.76 (t, J ¼ 6.0 Hz, 4H,
Im-CH2CH2); MS m/z: 330 [M þ H]þ; Anal. Calcd. for C17H20ClN5: C,
61.91; H, 6.11; N, 21.23. Found: C, 61.69; H, 6.06; N, 21.41.
6.1.4.6. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(4-chlorobenzyl)-2-(1H-
imidazol-1-yl)ethanamine (5f). Compound 5f was obtained as
colorless oil (0.62 g). Yield: 62.2%; IR (KBr, cmꢁ1
)
n
: 3113, 2937, 1597,
1508, 1493, 1285, 1233, 1108, 1081, 915, 808, 748; 1H NMR
(400 MHz, CDCl3, ppm):
d
7.38 (s, 2H, Im 2-H), 7.25 (d, J ¼ 2.4 Hz, 2H,
Ph 3, 5-H), 7.06 (d, J ¼ 2.4 Hz, 2H, Ph 2, 6-H), 7.01 (s, 2H, Im 4-H),
6.81 (t, J ¼ 1.2 Hz, 2H, Im 5-H), 3.80 (t, J ¼ 6.0 Hz, 4H, Im-CH2), 3.61
(s, 2H, PhCH2), 2.78 (t, J ¼ 6.0 Hz, 4H, Im-CH2CH2); MS m/z: 330
[M þ H]þ; Anal. Calcd. for C17H20ClN5: C, 61.91; H, 6.11; N, 21.23.
Found: C, 61.75; H, 6.17; N, 21.09.
6.1.4.1. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-(1H-
imidazol-1-yl)ethanamine (5a). Compound 5a was obtained as
6.1.4.7. N-(2,4-Dichlorobenzyl)-2-(2-ethyl-4-methyl-1H-imidazol-1-
yl)-N-(2-(2-ethyl-4-methyl-1H-imidazol-1-yl)ethyl)ethanamine
(5h). Compound 5h was obtained as a white solid (1.14 g). Yield:
84.8%, mp: 142e144 ꢀC; IR (KBr, cmꢁ1): 3132, 2975, 2510, 1644,
1588, 1503, 1382, 1101, 1047, 863, 823, 750; 1H NMR (300 MHz,
colorless oil (0.86 g). Yield: 86.3%; IR (KBr, cmꢁ1
)
n
: 3112, 2947,
2834, 1618, 1505, 1452, 1280, 1137, 961, 850; 1H NMR (400 MHz,
CDCl3, ppm):
d
7.26 (s, 2H, Im 2-H), 6.94 (d, J ¼ 3.0 Hz, 3H, Im 4-H,
Ph 6-H), 6.76e6.71 (m, 4H, Im 5-H, Ph 3, 5-H), 3.79 (t, J ¼ 4.5 Hz, 4H,
Im-CH2), 3.60 (s, 2H, PhCH2), 2.71 (t, J ¼ 4.5 Hz, 4H, Im-CH2CH2); MS
m/z: 332 [M þ H]þ; Anal. Calcd. for C17H19F2N5: C, 61.62; H, 5.78; N,
21.13. Found: C, 61.81; H, 5.72; N, 21.34.
CDCl3, ppm): d 7.86e7.32 (m, 3H, Ph 3, 5, 6-H), 6.97 (s, 2H, Im 5-H),
3.83 (t, J ¼ 6.0 Hz, 4H, Im-CH2), 3.62 (s, 2H, PhCH2), 2.84 (t,
J ¼ 6.0 Hz, 4H, Im-CH2CH2), 2.59 (q, J ¼ 5.4 Hz, 4H, Im-CH2CH3), 2.27
(s, 6H, Im-CH3), 1.53 (t, J ¼ 5.4 Hz, 6H, Im-CH2CH3); MS m/z: 448
[M þ H]þ; Anal. Calcd. for C23H31Cl2N5: C, 61.60; H, 6.97; N, 15.62.
Found: C, 61.81; H, 6.88; N, 15.46.
6.1.4.2. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine (5b). Compound 5b was obtained as
colorless oil (0.93 g). Yield: 85.2%; IR (KBr, cmꢁ1
)
n
: 2924, 2853,
1644, 1590, 1562, 1505, 1453, 1287, 1079, 821, 736; 1H NMR
(300 MHz, CDCl3, ppm): 7.29 (s, 2H, Im 2-H), 7.23 (s, 1H, Ph 3-H),
6.1.4.8. N-(2,4-Dichlorobenzyl)-2-(2-phenyl-1H-imidazol-1-yl)-N-
(2-(2-phenyl-1H-imidazol-1-yl)ethyl)ethanamine (5i). Compound
5i was obtained as a white solid (1.35 g). Yield: 87.2%, mp:
146e145 ꢀC; IR (KBr, cmꢁ1): 3106, 2958, 2804, 1587, 1363, 1136,
d
7.05 (d, J ¼ 8.3 Hz, 1H, Ph 5-H), 6.96 (bs, 2H, Im 4-H), 6.85 (d,
J ¼ 8.3 Hz, 1H, Ph 6-H), 6.69 (bs, 2H, Im 5-H), 3.80 (t, J ¼ 6.0 Hz, 4H,
Im-CH2), 3.65 (s, 2H, PhCH2), 2.74 (t, J ¼ 6.0 Hz, 4H, Im-CH2CH2); MS
m/z: 364 [M þ H]þ; Anal. Calcd. for C17H19Cl2N5: C, 56.05; H, 5.26; N,
19.23. Found: C, 56.23; H, 5.35; N, 19.37.
1115, 836, 776; 1H NMR (300 MHz, CDCl3, ppm):
d 7.37 (m, 10H, Ar-
H), 7.22e6.64 (m, 7H, Ar-H), 3.83 (t, J ¼ 5.6 Hz, 4H, Im-CH2), 3.36 (s,
2H, PhCH2), 2.52 (t, J ¼ 5.6 Hz, 4H, Im-CH2CH2); MS m/z: 516
[M þ H]þ; Anal. Calcd. for C29H27Cl2N5: C, 67.44; H, 5.27; N, 13.56.
Found: C, 67.66; H, 5.35; N, 13.42.
6.1.4.3. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine (5c). Compound 5c was obtainꢁed1 as
a white solid (0.79 g). Yield: 72.3%, mp: 91e93 ꢀC; IR (KBr, cm
)
n
:
6.1.4.9. N-(2-(1H-Benzoimidazol-1-yl)ethyl)-2-(1H-benzoimidazol-
1-yl)-N-(2,4-difluorobenzyl)ethanemine (6a). Compound 6a was
obtained as a white solid (1.02 g). Yield: 78.9%, mp: 114e115 ꢀC; IR
3093, 3050, 2965, 2930, 2859, 1594, 1505, 1486, 1265, 1212, 1139,
847, 752,720; 1H NMR (400 MHz, CDCl3, ppm):
d
7.25 (s, 2H, Im 2-
H), 7.24 (d, J ¼ 2.4 Hz, 1H, Ph 5-H), 7.10 (s, 1H, Ph 2-H), 6.96 (bs, 2H,
Im 4-H), 6.80e6.78 (m, 1H, Ph 6-H), 6.68 (bs, 2H, Im 5-H), 3.80 (t,
J ¼ 6.0 Hz, 4H, Im-CH2), 3.49 (s, 2H, PhCH2), 2.70 (t, J ¼ 6.0 Hz, 4H,
(KBr, cmꢁ1
)
n
: 3100, 2941, 2854,1619,1501,1459,1285,1139, 957, 846,
742; 1H NMR (400 MHz, CDCl3, ppm):
7.47 (d, J ¼ 8.0 Hz, 2H, Benim
2-H), 7.26 (bs, 2H, Benim 4-H) 6.96e6.88 (m, 4H, Benim 6,7-H),
d

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B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
6.78e6.76 (m, 2H, Benim 5-H, Ph 3, 5-H), 6.54e6.49 (m,1H, Ph 6-H),
6.40e6.34 (m, 1H, Ph 5-H), 6.26e6.22 (m, 1H, Ph 3-H), 3.59 (t,
J ¼ 6.4 Hz, 4H, Benim-CH2), 3.32 (s, 2H, PhCH2), 2.52 (t, J ¼ 6.0 Hz, 4H,
Benim-CH2CH2); MS m/z: 432 [M þ H]þ; Anal. Calcd. for C25H23F2N5:
C, 69.59; H, 5.37; N, 16.23. Found: C, 69.82; H, 5.45; N, 16.41.
2-H), 7.51 (s, 1H, Ph 6-H), 4.74 (s, 4H, Tri-CH2), 4.33 (s, 2H, PhCH2),
3.47 (s, 4H, Tri-CH2CH2); MS m/z: 366 [M þ Hꢁ3HCl]þ; Anal. Calcd.
for C15H20Cl5N7: C, 37.88; H, 4.24; N, 20.61. Found: C, 37.72; H, 4.20;
N, 20.69.
6.1.5.4. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine hydrochloride (8a). Compound 8a
6.1.4.10. N-(2-(1H-Benzoimidazol-1-yl)ethyl)-2-(1H-benzoimidazol-
1-yl)-N-(2,4-dichlorobenzyl)ethanemine (6b). Compound 6b was
obtained as a white solid (1.25 g). Yield: 89.8%, mp: 144e146 ꢀC; IR
(KBr, cmꢁ1): 3082, 2939 2823, 1587, 1496, 1455, 1365, 1286, 1202,
was obtained as
107e109 ꢀC; IR (KBr, cmꢁ1
1276, 1141, 960, 856; 1H NMR (400 MHz, DMSO, ppm):
a
white solid (0.15 g). Yield: 68.0%, mp:
: 3089, 2954, 2857, 1620, 1501, 1457,
9.30 (s, 2H,
)
n
d
864, 742; 1H NMR (300 MHz, CDCl3, ppm):
d
7.73 (s, 2H, Benim 2-H),
Im 2-H), 7.76 (s, 2H, Im 4-H), 7.63 (s, 2H, Im 5-H), 7.22e7.18 (m, 2H,
Ph 2, 5-H), 7.00 (t, J ¼ 7.6 Hz, 1H, Ph 6-H), 4.51 (s, 4H, Im-CH2), 3.92
(s, 2H, PhCH2), 3.10 (s, 4H, Im-CH2CH2); MS m/z: 332
[M þ H ꢁ 3HCl]þ; Anal. Calcd. for C17H22Cl3F2N5: C, 46.33; H, 5.03;
N, 15.89. Found: C, 46.49; H, 4.97; N, 15.70.
7.50e6.70 (m, 11H, Ar-H), 3.90 (t, J ¼ 7.2 Hz, 4H, Benim-CH2), 3.67 (s,
2H, PhCH2), 2.83 (t, J ¼ 7.2 Hz, 4H, Benim-CH2CH2); MS m/z: 464
[M þ H]þ; Anal. Calcd. for C25H23Cl2N5: C, 64.66; H, 4.99; N, 15.08.
Found: C, 64.91; H, 5.06; N, 15.29.
6.1.4.11. N-(2,4-Dichlorobenzyl)-2-(5,6-dimethyl-1H-benzoimidazol-
1-yl)-N-(2-(5,6-dimethyl-1H-benzoimidazol-1-yl)ethyl)ethanamine
(6c). Compound 6c was obtained as a white solid (1.36 g). Yield:
87.2%, mp: 190e192 ꢀC; IR (KBr, cmꢁ1): 3085, 2936, 2830, 1585,
1499, 1466, 1356, 1221, 1139, 845; 1H NMR (300 MHz, CDCl3, ppm):
6.1.5.5. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine hydrochloride (8b). Compound 8b
was obtained as
127e128 ꢀC; IR (KBr, cmꢁ1
1504, 1455, 1268, 1200, 1138, 958, 853; 1H NMR (400 MHz, DMSO,
ppm): 9.36 (s, 2H, Im 2-H), 7.73 (s, 2H, Im 4-H), 7.68 (s, 2H, Im 5-
a
white solid (0.16 g). Yield: 67.5%, mp:
)
n
: 3121, 3038, 2961, 2931, 2846, 1601,
d
7.45 (s, 2H, benzimi 2-H), 7.19e6.72 (m, 7H, Ar-H), 3.87 (t,
d
J ¼ 6.8 Hz, 4H, Benim-CH2), 3.65 (s, 2H, PhCH2), 2.82 (t, J ¼ 6.8 Hz,
4H, Benim-CH2CH2), 2.28 (s, 12H, Ar-CH3); MS m/z: 520 [M þ H]þ;
Anal. Calcd. for C29H31Cl2N5: C, 66.92; H, 6.00; N, 13.46. Found: C,
66.64; H, 6.08; N, 13.57.
H), 7.55 (s, 1H, Ph 3-H), 7.30 (s, 1H, Ph 5-H), 7.11 (s, 1H, Ph 6-H), 4.54
(s, 4H, Im-CH2), 3.92 (s, 2H, PhCH2), 3.12 (s, 4H, Im-CH2CH2); MS m/
z: 364 [M þ H ꢁ 3HCl]þ; Anal. Calcd. for C17H22Cl5N5: C, 43.11; H,
4.68; N, 14.79. Found: C, 43.32; H, 4.62; N, 14.92.
6.1.5. General procedure for the preparation of hydrochlorides 7e9
Hydrochloric acid (4 mol/L) was added dropwise into a solution
of compound 4a (167 mg, 0.5 mmol), or 4b (183 mg, 0.5 mmol), or
4c (183 mg, 0.5 mmol), or 5a (166 mg, 0.5 mmol), or 5b (182 mg,
0.5 mmol), or 5c (182 mg, 0.5 mmol), or 5i (258 mg, 0.5 mmol), or
6a (216 mg, 0.5 mmol) in ethyl ether/chloroform (4/1, V/V), stopped
the addition when no more precipitate formed. After the addition,
the formed precipitate was filtered, and washed with chloroform
and then with light petroleum ether and dried by freeze drying
oven to afford hydrochlorides 7e9.
6.1.5.6. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine hydrochloride (8c). Compound 8c
was obtained as a white solid (0.18 g). Yield: 75.9%, mp: 129e131 ꢀC;
IR (KBr, cmꢁ1
)
n
: 3109, 3046, 2958, 2928, 2856, 1602, 1509, 1452,
1261, 1201, 1140, 949, 850, 750; 1H NMR (400 MHz, DMSO, ppm):
9.34 (s, 2H, Im 2-H), 7.79 (s, 2H, Im 4-H), 7.66 (s, 2H, Im 5-H), 7.52 (d,
d
J ¼ 8.0 Hz,1H, Ph 2-H), 7.26 (s,1H, Ph 5-H), 7.18 (s,1H, Ph 6-H), 4.50 (s,
4H, Im-CH2), 3.84 (s, 2H, PhCH2), 3.08 (s, 4H, Im-CH2CH2); MS m/z:
364 [M þ H ꢁ 3HCl]þ; Anal. Calcd. for C17H22Cl5N5: C, 43.11; H, 4.68;
N, 14.79. Found: C, 43.33; H, 4.73; N, 14.61.
6.1.5.1. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine hydrochloride (7a). Compound
7a was obtained as a white solid (0.16 g). Yield: 72.2%, mp:
6.1.5.7. N-(2,4-Dichlorobenzyl)-2-(2-phenyl-1H-imidazol-1-yl)-N-
(2-(2-phenyl-1H-imidazol-1-yl)ethyl)ethanamine hydrochloride (8d).
Compound 8d was obtained as a white solid (0.23 g). Yield: 73.5%,
mp: 137e139 ꢀC; IR (KBr, cmꢁ1): 3101, 3038, 2952, 2921, 2843,1598,
1505, 1458, 1402, 1271, 1109, 831, 772; 1H NMR (400 MHz, DMSO,
126e128 ꢀC; IR (KBr, cmꢁ1
)
n
: 3068, 3021, 2941, 1637, 1572, 1520,
1377, 1111, 886, 825, 721; 1H NMR (400 MHz, DMSO, ppm):
d
9.26 (s,
2H, Tri 3-H), 8.47 (s, 2H, Tri 5-H), 7.67 (d, J ¼ 6.0 Hz,1H, Ph 3-H), 7.34
(t, J ¼ 8.4 Hz, 1H, Ph 5-H), 7.14 (t, J ¼ 8.4 Hz, 1H, Ph 6-H), 4.75 (s, 4H,
Tri-CH2), 4.31 (s, 2H, PhCH2), 3.46 (s, 4H, Tri-CH2CH2); MS m/z: 334
[M þ H ꢁ 3HCl]þ; Anal. Calcd. for C15H20Cl3F2N7: C, 40.69; H, 4.55;
N, 22.15. Found: C, 40.83; H, 4.61; N, 22.32.
ppm): d 7.72 (s, 2H, Im 4-H), 7.63 (s, 2H, Im 5-H), 7.43e7.35 (m, 4H,
Ar-H), 6.95e6.82 (m, 9H, Ar-H), 4.52 (s, 4H, Im-CH2), 3.91 (s, 2H,
PhCH2), 3.13 (s, 4H, Im-CH2CH2); MS m/z: 516 [M þ H ꢁ 3HCl]þ;
Anal. Calcd. for C29H30Cl5N5: C, 55.65; H, 4.83; N, 11.19. Found: C,
55.43; H, 4.77; N, 11.32.
6.1.5.2. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine hydrochloride (7b). Compound
7b was obtained as a white solid (0.18 g). Yield: 75.6%, mp:
6.1.5.8. N-(2-(1H-Benzoimidazol-1-yl)ethyl)-2-(1H-benzoimidazol-
1-yl)-N-(2,4-difluorobenzyl)ethanamine hydrochloride (9a).
Compound 9a was obtained as a white solid (0.20 g). Yield:
110e112 ꢀC;IR(KBr, cmꢁ1
)
n
:3093, 3046,3021, 2938,1630,1582,1528,
73.9%, mp: 119e121 ꢀC; IR (KBr, cmꢁ1
)
n
: 3117, 3025, 2921, 2841,
1619, 1552, 1503, 1381, 1280, 1101, 820, 732; 1H NMR (400 MHz,
DMSO, ppm):
1380, 1101, 904, 850, 823; 1H NMR (400 MHz, DMSO, ppm):
d
9.45 (s,
2H, Tri3-H),8.58(s, 2H, Tri5-H), 7.61(s,1H, Ph3-H),7.37(s, 2H, Ph5,6-
H), 4.63 (s, 4H, Tri-CH2), 4.09 (s, 2H, PhCH2), 3.29 (s, 4H, Tri-CH2CH2);
MS m/z: 366 [M þ Hꢁ3HCl]þ; Anal. Calcd. for C15H20Cl5N7: C, 37.88; H,
4.24; N, 20.61. Found: C, 37.69; H, 4.19; N, 20.72.
d
10.03 (s, 2H, Benim 2-H), 7.98 (d, J ¼ 8.0 Hz, 2H,
Benim 4-H), 7.84 (d, J ¼ 8.0 Hz, 2H, Benim 7-H), 7.60e7.52 (m,
4H, Benim 5, 6-H), 7.02 (s, 1H, Ph 3-H), 6.80 (s, 1H, Ph 5-H), 6.57
(s, 1H, Ph 6-H), 4.82 (s, 4H, Benim-CH2), 3.85 (s, 2H, PhCH2), 3.17
(s, 4H, Benim-CH2CH2); MS m/z: 432 [M þ Hꢁ3HCl]þ; Anal.
Calcd. for C25H26Cl3F2N5: C, 55.52; H, 4.85; N, 12.95. Found: C,
55.39; H, 4.90; N, 12.86.
6.1.5.3. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine hydrochloride (7c). Compound 7c
was obtained as
124e126 ꢀC; IR (KBr, cmꢁ1
1140, 958, 876; 1H NMR (400 MHz, DMSO, ppm):
H), 8.42 (s, 2H, Tri 5-H), 7.80 (s,1H, Ph 5-H), 7.67 (d, J ¼ 8.4 Hz,1H, Ph
a
white solid (0.16 g). Yield: 67.2%, mp:
: 3116, 2952, 2838, 1506, 1476, 1278,
9.19 (s, 2H, Tri 3-
)
n
6.1.6. General procedure for the preparation of nitrates 10e12
The nitric acid (4 mol/L) was added dropwise into a solution of
compound 4a (0.17 g, 0.5 mmol), or 4b (0.18 g, 0.5 mmol), or 4c
d

B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
4397
(0.18 g, 0.5 mmol), or 5a (0.17 g, 0.5 mmol), or 5b (0.18 g, 0.5 mmol),
6.1.12. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
or 5c (0.18 g, 0.5 mmol), or 5i (0.26 g, 0.5 mmol), or 6a (0.22 g,
0.5 mmol) in ethyl ether/chloroform (4/1, V/V), stopped the addi-
tion when no more precipitate formed. After the addition, the
formed precipitate was filtered, and the precipitate was washed
with chloroform and light petroleum ether by turns, and then dried
by freeze drying oven to afford compounds 10e12.
(1H-imidazol-1-yl)ethanamine nitrate (11c)
Compound 11c was obtained asꢁa1 white solid (0.17 g). Yield:
61.5%, mp: 142e144 ꢀC; IR (KBr, cm
) n: 3118, 3038, 2964, 2921,
2853,1609,1504,1456,1251,1138, 857, 746, 720; 1H NMR (400 MHz,
DMSO, ppm): 9.32 (s, 2H, Im 2-H), 7.78 (s, 2H, Im 4-H), 7.63 (s, 2H,
d
Im 5-H), 7.62 (d, J ¼ 8.0 Hz, 1H, Ph 2-H), 7.23 (s, 1H, Ph 5-H), 7.18 (s,
1H, Ph 6-H), 4.48 (s, 4H, Im-CH2), 3.81 (s, 2H, PhCH2), 3.05 (s, 4H,
Im-CH2CH2); MS m/z: 364 [M þ H ꢁ 3HNO3]þ; Anal. Calcd. for
C17H22Cl2N8O9: C, 36.90; H, 4.01; N, 20.25. Found: C, 36.72; H, 3.96;
N, 20.37.
6.1.7. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine nitrate (10a)
Compound 10a was obtained asꢁa1 white solid (0.17 g). Yield:
65.1%, mp: 147e149 ꢀC; IR (KBr, cm
) n: 3123, 3056, 3034, 2957,
6.1.13. N-(2,4-Dichlorobenzyl)-2-(2-phenyl-1H-imidazol-1-yl)-N-
(2-(2-phenyl-1H-imidazol-1-yl)ethyl)ethanamine nitrate (11d)
Compound 11d was obtained as a white solid (0.23 g). Yield:
65.2%, mp: 143e145 ꢀC; IR (KBr, cmꢁ1): 3112, 3054, 2961, 2928,
2852, 1599, 1503, 1456, 1259, 1106, 829, 770; 1H NMR (400 MHz,
1621, 1563, 1509, 1341, 1123, 837, 821, 720; 1H NMR (400 MHz,
DMSO, ppm): 9.37 (s, 2H, Tri 3-H), 8.54 (s, 2H, Tri 5-H), 7.74 (d,
d
J ¼ 6.0 Hz, 1H, Ph 3-H), 7.38 (t, J ¼ 8.0 Hz, 1H, Ph 5-H), 7.17 (t,
J ¼ 8.0 Hz,1H, Ph 6-H), 4.92 (s, 4H, Tri-CH2), 4.39 (s, 2H, PhCH2), 3.42
(s, 4H, Tri-CH2CH2); MS m/z: 334 [M þ H ꢁ 3HNO3]þ; Anal. Calcd.
for C15H20F2N10O9: C, 34.49; H, 3.86; N, 26.81. Found: C, 34.66; H,
3.81; N, 26.74.
DMSO, ppm):
d 7.83 (s, 2H, Im 4-H), 7.74 (s, 2H, Im 5-H), 7.54e7.46
(m, 4H, Ar-H), 6.93e6.76 (m, 9H, Ar-H), 4.54 (s, 4H, Im-CH2), 3.93 (s,
2H, PhCH2), 3.12 (s, 4H, Im-CH2CH2); MS m/z: 516
[M þ H ꢁ 3HNO3]þ; Anal. Calcd. for C29H30Cl2N8O9: C, 49.37; H,
4.29; N, 15.88. Found: C, 49.56; H, 4.34; N, 15.72.
6.1.8. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine nitrate (10b)
Compound 10b was obtained asꢁa1 white solid (0.16 g). Yield:
1632, 1588, 1560, 1384, 1108, 851, 827, 724; 1H NMR (400 MHz,
6.1.14. N-(2-(1H-Benzoimidazol-1-yl)ethyl)-2-(1H-benzoimidazol-
1-yl)-N-(2,4-difluorobenzyl)ethanamine nitrate (12a)
57.7%, mp: 133e135 ꢀC; IR (KBr, cm
) n: 3098, 3050, 3019, 2946,
Compound 12a was obtained as ꢁa1 white solid (0.17 g). Yield:
DMSO, ppm): 9.59 (s, 2H, Tri 3-H), 8.78 (s, 2H, Tri 5-H), 7.53 (d,
d
54.8%, mp: 124e126 ꢀC; IR (KBr, cm
) n: 3114, 3016, 2907, 2850,
J ¼ 2.0 Hz, 1H, Ph 3-H), 7.32e7.29 (m, 1H, Ph 5-H), 7.14 (d, J ¼ 8.0 Hz,
1H, Ph 6-H), 4.52 (t, J ¼ 6.0 Hz, 4H, Tri-CH2), 3.91 (s, 2H, PhCH2), 3.18
(t, J ¼ 6.0 Hz, 4H, Tri-CH2CH2); MS m/z: 366 [M þ H ꢁ 3HNO3]þ;
Anal. Calcd. for C15H20Cl2N10O9: C, 32.44; H, 3.63; N, 25.22. Found:
C, 32.69; H, 3.56; N, 25.41.
1620, 1548, 1510, 1384, 1279, 1106, 824, 730; 1H NMR (400 MHz,
DMSO, ppm): 9.56 (s, 2H, Benim 2-H), 7.93 (m, 2H, Benim 4-H),
d
7.82 (m, 2H, Benim 7-H), 7.62e7.53 (m, 4H, Benim 5, 6-H),
6.96e6.90 (m, 1H, Ph 6-H), 6.78e6.73 (m, 1H, Ph 5-H), 6.57e6.53
(m, 1H, Ph 3-H), 4.66 (t, J ¼ 6.0 Hz, 4H, Benim-CH2), 3.74 (s, 2H,
PhCH2), 3.10 (t, J ¼ 6.0 Hz, 4H, Benim-CH2CH2); MS m/z: 432
[M þ H ꢁ 3HNO3]þ; Anal. Calcd. for C25H26F2N8O9: C, 48.39; H, 4.22;
N, 18.06. Found: C, 48.04; H, 4.36; N, 18.19.
6.1.9. N-(2-(1H-1,2,4-Triazol-1-yl)ethyl)-N-(3,4-dichlorobenzyl)-2-
(1H-1,2,4-triazol-1-yl)ethanamine nitrate (10c)
Compound 10c was obtained asꢁa1 white solid (0.17 g). Yield:
61.3%, mp: 129e131 ꢀC; IR (KBr, cm
) n: 3121, 2946, 2828, 1502,
6.2. Biological assays
1470, 1288, 1142, 960, 871; 1H NMR (400 MHz, DMSO, ppm):
d 9.21
(s, 2H, Tri 3-H), 8.47 (s, 2H, Tri 5-H), 7.86 (s, 1H, Ph 5-H), 7.72 (d,
J ¼ 8.4 Hz,1H, Ph 2-H), 7.54 (s,1H, Ph 6-H), 4.78 (s, 4H, Tri-CH2), 4.35
(s, 2H, PhCH2), 3.50 (s, 4H, Tri-CH2CH2); MS m/z: 366
[M þ H ꢁ 3HNO3]þ; Anal. Calcd. for C15H20Cl2N10O9: C, 32.44; H,
3.63; N, 25.22. Found: C, 32.62; H, 3.68; N, 25.05.
The in vitro minimal inhibitory concentrations (MICs) of the
target compounds and their precursors were determined by the
micro-broth dilution method in 96-well microtest plates according
to the methods recommended by the National Committee for
Clinical Laboratory Standards (NCCLS). The tested strains were
provided by Department of Microbiology, College of Medicine,
Third Military Medical University, China. Fluconazole and Chlor-
amphenicol obtained from their respective manufacturers served
as the controls.
6.1.10. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-difluorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine nitrate (11a)
Compound 11a was obtained asꢁa1 white solid (0.14 g). Yield:
53.8%, mp: 126e128 ꢀC; IR (KBr, cm
) n: 3063, 2949, 2853, 1616,
1503, 1452, 1266, 1137, 957, 854; 1H NMR (400 MHz, DMSO, ppm):
6.2.1. Antibacterial assay
d
9.31 (s, 2H, Im 2-H), 7.78 (s, 2H, Im 4-H), 7.65 (s, 2H, Im 5-H),
The prepared compounds 2e12 were evaluated for their anti-
bacterial activities against S. aureus ATCC 29213, S. aureus N 315
(MRSA), B. subtilis ATCC 21216 as Gram-positive, E. coli ATCC 25922,
P. aeruginosa ATCC 27853, B. proteus ATCC 13315 as Gram-negative
bacteria. The bacterial suspension was adjusted with sterile saline
to a concentration of 1 ꢃ 104e1 ꢃ 105 CFU. The tested compounds
were dissolved in dimethyl sulfoxide (DMSO) to prepare the stock
solutions. The tested compounds and reference drugs were
prepared in Mueller-Hinton Broth (Guangdong huaikai microbial
sci.& tech co., Ltd, Guangzhou, Guangdong, China) by two-fold
serial dilution to obtain the required concentrations of 256, 128, 64,
7.23e7.20 (m, 2H, Ph 2, 5-H), 7.05 (t, J ¼ 7.2 Hz, 1H, Ph 6-H), 4.54 (s,
4H, Im-CH2), 3.95 (s, 2H, PhCH2), 3.14 (s, 4H, Im-CH2CH2); MS m/z:
332 [M þ H ꢁ 3HNO3]þ; Anal. Calcd. for C17H22F2N8O9: C, 39.24; H,
4.26; N, 21.53. Found: C, 39.45; H, 4.31; N, 21.70.
6.1.11. N-(2-(1H-Imidazol-1-yl)ethyl)-N-(2,4-dichlorobenzyl)-2-
(1H-imidazol-1-yl)ethanamine nitrate (11b)
Compound 11b was obtained asꢁa1 white solid (0.15 g). Yield:
54.2%, mp: 138e140 ꢀC; IR (KBr, cm
) n: 3116, 3054, 2956, 2928,
2856, 1608, 1501, 1457, 1209, 1141, 961, 850; 1H NMR (400 MHz,
DMSO, ppm): 9.25 (s, 2H, Im 2-H), 7.71 (s, 2H, Im 4-H), 7.58 (s, 2H,
d
32, 16, 8, 4, 2, 1, 0.5, and 0.25 mg/mL. These dilutions were inocu-
lated and incubated at 37 ꢀC for 24 h. To ensure that the solvent had
no effect on bacterial growth, a control test was performed with
test medium supplemented with DMSO at the same dilutions as
used in the experiment. The growth was monitored visually and
Im 5-H), 7.51 (s, 1H, Ph 3-H), 7.28 (s, 1H, Ph 5-H), 7.09 (s, 1H, Ph 6-H),
4.52 (s, 4H, Im-CH2), 3.90 (s, 2H, PhCH2), 3.09 (s, 4H, Im-CH2CH2);
MS m/z: 364 [M þ H ꢁ 3HNO3]þ; Anal. Calcd. for C17H22Cl2N8O9: C,
36.90; H, 4.01; N, 20.25. Found: C, 36.68; H, 4.06; N, 20.14.

4398
B. Fang et al. / European Journal of Medicinal Chemistry 45 (2010) 4388e4398
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Acknowledgments
This work was partially supported by the Natural Science
Foundation of Chongqing (CSCT: 2007BB5369, 2009BB5296) and
Talents Introduction Foundation of Southwest University
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