The synthesis and activities of novel mononuclear or dinuclear cyclen complexes bearing azole pendants as antibacterial and antifungal agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.075

A series of novel compounds containing 1,4,7,10-tetraazacyclododecane and azoles were synthesized and characterized by ¹H NMR, MS and elemental analysis. Bioactive assay manifested that some target compounds, such as 11a, 11b and 11d, displayed

Full text of DOI:10.1016/j.ejmech.2014.07.075

European Journal of Medicinal Chemistry 84 (2014) 677e686
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis and activities of novel mononuclear or dinuclear cyclen
complexes bearing azole pendants as antibacterial and antifungal
agents
,
,
, *
Shuo Li b *, Jia-Xuan Chen a, Qing-Xiang Xiang a *, Li-Qun Zhang a, Cheng-He Zhou c
,
Jia-Qing Xie b, Lan Yu b, Fang-Zhen Li b
a Department of Chemistry, Leshan Normal University, Leshan, Sichuan 614000, People's Republic of China
b School of Chemical Engineering, Chongqing University of Technology, Chongqing 400054, People's Republic of China
c Laboratory of Bioorganic & Medicinal Chemistry, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715,
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 compounds containing 1,4,7,10-tetraazacyclododecane and azoles were synthesized and
characterized by 1H NMR, MS and elemental analysis. Bioactive assay manifested that some target
compounds, such as 11a, 11b and 11d, displayed good and broad spectrum antimicrobial activities with
relative low MIC values against most of tested strains. These dinuclear complexes gave comparable or
even better antimicrobial efficiencies than the reference drugs Fluconazole and Chloromycin. The result
showed that the metal ions were the key factors to enhance the antimicrobial activities for mononuclear
or dinuclear complexed in varying degrees. The interaction evaluation of compound 11b with bovine
serum albumin (BSA) as an example was tested by fluorescence method. The thermodynamic parameters
indicated that the hydrogen bonds and van der waals forces played the major roles in the strong asso-
ciation between dinuclear compound and BSA. The CCK-8 tests also confirmed the safeties of these
dinuclear compounds in vitro.
Received 7 December 2013
Received in revised form
19 July 2014
Accepted 21 July 2014
Available online 22 July 2014
Keywords:
Azoles
Cyclen
Antibacterial
Antifungal
Fluorescence spectrum
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
benzimidazole drugs like antiparasitic thiabendazole, mebenda-
zole, albendazole, antihistaminic norastemizole and mizolastine, as
The increasing emergence of pathogenic bacterial strains and
concerns about multidrug-resistance, especially the explosion of
New Delhi metallo-b-lactamase 1 (NDM-1) superbugs very
well as antihypertensive telmisartan etc. have been successfully
developed and extensively used in clinic. This has attracted
increasing interest to investigate the possible applications of azole-
based derivatives in medicinal aspects. What's more important is
that numerous azole supermolecules as chemical drugs are under
actively ongoing researches and developments, and have shown
enormous potential [12]. It is undoubted that this research area will
become a new direction for the exploitation of azole complex as
antimicrobial drugs [13], and azole derivatives coordinating with
metal cation to form complexes always showed enhanced antimi-
crobial properties [14]. Besides these azole derivatives, macrocyclic
compounds are already extensively exploited for their medical
applications as MRI agents [15], antibacterial and anticancer agents
recently, had made most of the first-line clinical antibiotics inef-
fective [1]. This situation has stimulated an urgent need to develop
more effective antimicrobial agents with novel chemical structures
which are helpful for overcoming drug-resistance and improving
the antimicrobial potency.
As we know, heterocyclic compounds such as imidazole, benz-
imidazole, pyrazole and triazole have been frequently found to
display a variety of biological activities such as antihelmintic [2],
antihistaminic [3], anticancer [4], antiviral [5], antiinflammatory
[6], antiproliferative [7], antioxidant [8], anticoagulant properties
[9], antitubercular [10], anticonvulsant [11]. Particularly, many
[16]. As
a
well-known macrocyclic polyamine, 1,4,7,10-
tetraazacyclododecane (cyclen) has been widely studied for its
strong coordination ability towards a wide range of cations, and the
safeties of some macrocyclic compounds used in vivo have been
confirmed. Recently, the increasing prevalence strategy to develope
new classes of antimicrobial agents with novel mechanism of
* Corresponding authors.
E-mail addresses: lishuo@cqut.edu.cn, gaoshan210@163.com (S. Li),
qxxiang711@163.com (Q.-X. Xiang).
http://dx.doi.org/10.1016/j.ejmech.2014.07.075
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

678
S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
action was to employ combination of two different active fragments
into one molecule [17]. So that, in view of above observation, it is
reasonable for us with great interest to prepare cyclen complexes
having azoles pendants, and to evaluate their antibacterial and
antifungal behaviours. Herein a series of novel compounds were
prepared for the first time (Schemes 1 and 2). Their antibacterial
and antifungal activities were evaluated, and the structureeactivity
relationships were also investigated.
Serum albumins as the most important and abundant macro-
molecule proteins in the circulatory system have received much
attention for that they could deliver drugs or other bioactive small
molecules to the binding sites [18]. A thorough binding analysis
between drugs or bioactive small molecules and serum albumin
may beneficially provide useful information for the absorption,
transportation, distribution, metabolism and excretion properties
of drugs. It might also be significant for design, modification and
screening of drug molecules. So that there were many important
reasons for us to further investigate the interaction behaviour be-
tween the highly active compound and bovine serum albumin
(BSA) which was used as study model to preliminarily evaluate
their transportation and pharmacokinetic properties by fluores-
cence spectroscopy on molecular level. Further more, the safety or
toxicity of those dinuclear complexes were tested by CCK-8
method.
Scheme 1. Synthetic route of target compounds 7aer.

S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
679
Scheme 2. Synthetic route of target compounds 11aef.
2. Results and discussion
then the solvent was dried by anhydrous Na2SO4 and evaporated.
Solution of EtOH (10 mL), the obtained acid free ligand and equal/
double molar weight of perchlorate were mixed and stirred at room
temperature for 24 h. After that, the solid was filtered off, washed
with cold EtOH, the residue was crystallized from H2O/EtOH to
obtained crystals, and the product was dried in vacuo. The synthetic
route of target compounds were outlined in Schemes 1 and 2. The
results of XRD tests shown in Fig. S1 in Supplementary information
released that mono-nuclear complex 7c and di-nuclear complex
11c which was compared with its ligand 13 (shown in Scheme 3)
could form crystal powder but no monocrystal was obtained.
It must be pointed out that the log K values of Cyclen and its
pyridyl derivatives with Cu2þ were 23.4 and 24.0 respectively at the
condition of I ¼ 1 M in KNO3, and stability constant (log K) of Cyclen
with Zn2þ was 16.2 [19]. Meanwhile, the log K values of the other
ligand IDB bonding with Cu2þ and Zn2þ were almost 6.34 and 5.96
at the condition of I ¼ 0.1 M in KNO3 [20], so its coordinative effect
with Cu2þ or Zn2þ was also strong enough to interact with bio-
macromolecules in TriseHCl buffer (pH 7.4) efficiently [21]. Further
more, MS of compounds 7c and 11aef dissolved in DMSO which
was used as the solvent in the antibacterial assays were tested
(shown in Fig. S2eFig. S8 in Supplementary information) and each
molecular ion peak could be found in the spectrogram, so it was
also confirmed the stability of the target material.
2.1. Chemistry
The preparation of target derivatives were synthesized from
3Boc-cyclen. The reactions of protected cyclen with different hal-
ogenides, which were purified by column chromatography with
ethyl acetate and petroleum ether (v/v ¼ 1:2) as eluent. The azoles,
such as 4aed, were reacted with 3aeb on the conditions of NaH/
THF or K2CO3/CHCl3 to get the crude products 5ael, and the yields
after being processed ranged from 22.6% to 95.3%. As for dinuclear
ligands 9aeb were efficiently prepared by substitution reaction
between
compound
3aeb
and
IDB
(N,N-bis(2-
benzimidazolylmethyl)amine) (8) catalysed by K2CO3 in CHCl3,
then removed the residues by filtration and purified with column
chromatography to get target materials, and the yields were 75.6%,
85.6% and 77.3% respectively. It must be pointed out that the sol-
ubility of IDB in CHCl3 was poor, but the target material could
dissolve in CHCl3 very well. The reaction condition between IDB
and compound 3 was mild and the basicity of K2CO3 was weaker
than that of NaH, so that the reaction position in the IDB was the
secondary amine. The residual two benzimidazole molecules could
be used as ligand. Then compounds 5ael and 9aeb were stirred
with saturated HBr/C2H5OH, and their corresponding hydro-
bromates 6ael and 10aeb were filtrated and collected. At last these
compounds were dried in vacuo below 40 ꢀC.
2.2. Pharmacology
All these target complexes were produced followed the routes
described below. The hydrobromide was dissolved in water
(10 mL), and the pH of the solution was adjusted to 12 with aqueous
NaOH. The alkaline solution was extracted with CH2Cl2 (30 mL ꢁ 5),
The in vitro antimicrobial screening for all synthesized com-
pounds were evaluated for three Gram-positive bacteria (Staphy-
lococcus aureus ATCC 6538, Micrococcus luteus ATCC 4698 and

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S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
Scheme 3. The structures of selected ligands 12aeb and 13.
Bacillus subtilis ATCC 21216), three Gram-negative bacteria
(Escherichia coli ATCC 8099, Pseudomonas aeruginosa ATCC 27853
and Bacillus proteus ATCC 13315) and two fungi (Candida albicans
ATCC 76615 and Aspergillus fumigatus ATCC 96918) using two folds
serial dilution technique recommended by National Committee for
Clinical Laboratory Standards (NCCLS) with the positive control of
clinically antimicrobial drugs Fluconazole and Chloromycin [22].
the significant influences of all sorts of aromatic linkers on bio-
logical activities. When the aromatic linkers changed to p-xylyl
group, derivatives still exhibited moderate efficacies against tested
bacteria, which were inferior to 11a and 11b. Complex 11d showed
inhibition potencies against Gram-positive bacteria S. aureus
(MIC ¼ 2
m
g mLꢂ1), M. luteu (MIC ¼ 2
m
g mLꢂ1) and Gram-negative
bacteria P. aeruginosa (MIC ¼ 2
m
g mLꢂ1), meanwhile the MIC values
Minimal inhibitory concentration (MIC,
mg/mL) was defined as the
against these bacteria of Chloromycin was ranging from 2
m
g mLꢂ1
lowest concentration of new compounds that completely inhibited
the growth of pathogenic microorganisms. The values of Clog P (a
partition coefficient) were calculated using ChemDraw Ultra 10.0
software integrated with Cambridge Software (Cambridge Soft
Corporation), and were depicted in Tables 1 and 2 respectively.
to 32
m
g mLꢂ1. Regretfully, pyridyl-containing compounds 11e and
11f were unfavourable for the antibacterial efficiencies even at high
concentration, this might be explained by the nitrogen atom of
pyridine ring in complex reducing the positive charge density of
each nucleus in 11e and 11f, which made both of them unfav-
ourable for binding target sites. Therefore, suitable linker between
two nuclei is necessary for the excellent antibacterial activities in
drug design. As a comparison, antibacterial activities of some
representative ligands 6f, 12a, 12b and 13 (the structures of com-
pounds 12a, 12b and 13 shown in Scheme 3) were tested, and they
all showed weak antibacterial activities (shown in Table 3). It must
be pointed out that complex 7g which was composed of ligand 12a
and Cu2þ, and the MIC values of 7g against P. aeruginosa and
2.2.1. Antibacterial activity
The antibacterial results as shown in Table 1 revealed that most
of the prepared mononuclear azole derivatives 7aer showed weak
activities against Gram-positive bacteria in vitro, whereas some
mononuclear compounds showed moderate efficacy against Gram-
negative bacteria, especially for B. proteus or P. aeruginosa. Com-
pound, such as 7g, gave quite low inhibitory concentration
B. proteus. were 8
mg/mL and 32
mg/mL respectively which were
(MIC ¼ 8
P. aeruginosa than Chloromycin (MIC ¼ 32
ever its inhibitory potency against B. proteus (MIC ¼ 32
relatively weaker than the corresponding potency of Chloromycin
(MIC ¼ 2 g/mL). Besides the imidazolyl complex 7g, the MIC values
of benzimidazolyl compounds 7e and pyrazolyl compound 7h
against B. proteus were between 16 and 32
g mLꢂ1. These results
m
g/mL), which showed better inhibitory potency against
g/mL) (Table 1). How-
g/mL) was
much lower than that of 12a ranging from 256
m
g/mL to 512 g/mL
m
m
against this two kinds Gram-negative bacteria. Further more, the
enhance effect was more significantly for complex with two metal
ionic nuclei. So it could be concluded that the metal ion was the key
factor for the antibacterial activity.
It could be concluded that the enhanced activities of dinuclear
compound might be attributed to the better adhesion with electro-
negative member outside of bacteria for two cation nuclei, and the
corresponding precursors 7e and 7d with only benzimidazole-
pended and single cation nucleus showed no antibacterial activity.
In a word, compounds 11aec displayed remarkable antibacterial
activities, which indicated that they could be employed as potent
novel board-spectrum antimicrobial agents for further study.
m
m
m
indicated that imidazolyl moiety seemed to be more helpful for the
antibacterial activities than benzimidazolyl or other azole groups.
Unfortunately, compounds like 7a and 7b containing imidazolyl
groups showed no obvious inhibition antibacterial results and
these MIC values were not shown in Table 1. Imidazolyl complexes
having m-xylyl linkers showed better antibacterial activities,
because the introduction of aryl chain with different length had
remarkable effection on antibacterial activities, and it was unfav-
ourable to place the imidazole too near or too far from the nucleus.
As for imidazolyl complex 7m with 2,6-dimethyl pyridyl linker, the
nitrogen atom of pyridinyl ring in the complex might reduce the
positive charge of Cu2þ [23], and could reduce the interaction be-
tween the cell membrane with negative charge and Cu2þ nucleus.
Among these mononuclear salts, different cations in compounds
didn't showed obviously different inhibitory potency.
2.2.2. Antifungal activity
The in vitro antifungal evaluation of mononuclear complexes
shown in Table 1 revealed that the activities of most compounds
were relatively weak. However, the antifungal potencies of mono-
nuclear compounds were more effective than their corresponding
antibacterial properties. As for the compound 7l, the inhibitory
potency of C. albicans (MIC ¼ 2
Fluconazole (MIC ¼ 0.5 g/mL) (Table 1). Meanwhile, complex 7c
also showed fine inhibitory potency against C. albicans, and its MIC
value was 16 g/mL. Especially to A. fumigatus, some mononuclear
complexes showed comparable or even better inhibitory potencies
than Fluconazole (MIC ¼ 256 g/mL), such as 7h (MIC ¼ 128 g/mL)
g/mL) (Table 1). The antifungal potencies of
mg/mL) was comparable with the
To our surprise, the antibacterial results showed that com-
pounds 11aed exhibited excellent efficacies against most of tested
bacterial strains (Table 2). Particularly, compounds 11a, 11b with m-
xylyl linker like 7g displayed the best antibacterial activities,
especially for B. proteus and P. aeruginosa with the MIC values
m
m
m
m
ranging from 0.5 to 1
Chloromycin against the two strains with MIC values of 2
and 32
g mLꢂ1 respectively. Results in Table 2 also demonstrated
m
g mLꢂ1, which was more potent than
g mLꢂ1
and 7j (MIC ¼ 64
m
m
dinuclear compounds 11aef were far more better than their ho-
mologue mononuclear compounds. Compound such as 11a gave
m

S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
681
Table 1
The data of antimicrobial activities of mononuclear cyclen azole complexes and Clog P values.
Compds
Clog P
MIC50 (m )
g mLꢂ1
C. albicans
A. fumigatus
M. luteus
S. aureus
B. subtilis
P. aeruginosa
B. proteus
E. coli
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7q
A
2.7389
4.0630
3.8629
2.4689
2.2689
2.7389
2.5389
4.0630
1.7391
1.5391
0.9720
1.2420
0.2421
2.5659
ꢂ1.09
16
>512
512
128
256
256
128
>512
512
64
>512
512
256
512
512
e
>512
>512
128
>512
>512
>512
>512
>512
>512
512
>512
>512
>512
>512
>512
512
>512
>512
256
>512
512
>512
>512
>512
8
>512
>512
>512
>512
512
>512
>512
>512
>512
>512
>512
>512
512
512
>512
>512
512
8
128
512
512
>512
>512
256
256
128
512
32
>512
64
32
512
32
16
>512
>512
>512
128
512
512
64
>512
>512
>512
>512
512
>512
>512
>512
>512
>512
>512
>512
512
256
>512
512
256
64
256
128
64
2
512
512
64
>512
e
>512
>512
>512
256
512
4
256
2
512
4
2
B
ꢂ0.44
0.5
256
e
e
e
e
A: Chloromycin; B: Fluconazole.
quite low inhibitory concentration (MIC ¼ 8
m
g/mL) against
which induced fungus to death. Similarly, Chloromycin
(Clog P ¼ ꢂ1.09) also needed to enter into cytoplasm and binded
50S ribosomal subunit to interference the synthesis of protein in
bacterial, and finally resulted bacterial to death. However, As seen
from Table 2, all dinuclear compounds showed relative large Clog P
values ranging from 4.9190 to 6.8159, it meaned that they could
blend with lipid membrane conveniently and destruct the member
of fungus straightly. And there was another key point needed to be
mentioned, the two cationic nuclei could regulate the water-
solubility appropriately at the same time, even the relative high
positive charge density would also enhance the antifungal effect by
reinforcing electrostatic interaction between the surface of micro-
organism and complex. So the proposed mechanism of antimicro-
bial activity of compounds 11aed might be quite different from
Fluconazole or Chloromycin, and it proned to destroy the cell
member like the quaternary ammonium salt antimicrobial agents
[24]. Unlike the dinuclear compounds, Clog P values of the mono-
nuclear compounds were ranging from 0.0421 to 4.0630 (Table 1),
none of them were similar to either lipophilicity of the dinuclear
compound or hydrophilicity of the standard drug, so that their
antimicrobial activities were relatively poor. The Clog P values of
cyclen azole complexes represented their antimicrobial efficiencies
and were in accordance with the antimicrobial results discussed
above.
A. fumigatus, which was 32-fold more effective than the first-line
antifungal drug Fluconazole (MIC ¼ 256 g/mL), and the inhibi-
tory potency of C. albicans (MIC ¼ 0.5 g/mL) was same as the
g/mL) (Table 2). To our surprise, all most
m
m
Fluconazole (MIC ¼ 0.5
m
newly synthesized dinuclear compounds, except 11e and 11f, dis-
played better or comparable antifungal activity with standard drug,
and their MIC values were between 0.5
mg/mL and 128
mg/mL to
against C. albicans and ranging from 8
mg/mL to 256
mg/mL for
A. fumigatus. Target materials 11e and 11f were unfavourable for the
antifungal efficiency, for that the nitrogen atom in pyridine might
decrease the positive charge density on nucleus, and also impeded
the approach between nucleus and fungus by electrostatic in-
teractions. Like their antibacterial activities, the data in Tables 1 and
2 revealed that the inhibitory potencies of dinuclear compounds
against fungi were much stronger than mononuclear analogue. At
the same time, it could be found that complex coordinated Cu2þ
showed better antifungal activity, and it could be explained by the
oxygen radicals which enhanced the destructiveness originated
from Cu2þ. To further explain the key effect of metal ion, we also
test the antifungal activities of some representative ligands, such as
compounds 6f, 12a, 12b and 13, and they showed weak antifungal
activities (shown in Table 3). So that, it could be concluded that the
metal ion was also the key factor for the antifungal activity.
Therefore, a significant structureeactivity relationship could be
concluded from these data to design the antifungal cyclen azole
complex in future. Compounds with two nuclei conjugated azoles
by m-xylyl groups and coordinated Cu2þ showed relative stronger
antifungal potencies, and the length between two metal nuclei
influenced the antimicrobial property greatly.
2.3. Binding discussion
2.3.1. Fluorescence quenching mechanism
In this experiment, the concentration of BSA solution was
1.0 ꢁ 10ꢂ5 M, and the concentrations of compound 11b solution
increased progressively from 0 to 5.5 ꢁ 10ꢂ5 M at an increment of
0.5 ꢁ 10ꢂ5 M. The effect of compound 11b on fluorescence of BSA
intensity at 298 K was shown in Fig. 1A. It was observed that a
gradual decrease of the fluorescence intensity was caused by the
increment of compound 11b, accompanied with the red shift of
wavelength (from 345 to 352 nm) in the albumin spectrum. In
Fig. 1A, the blue line showed the only emission spectrum of com-
pound 11b, which indicated that compound 11b did not posses
significant fluorescence feature, thus at the excitation wavelength
(295 nm), the effect of compound 11b on fluorescence of BSA could
be negligible. The decreased fluorescence intensity is usually
described by the SterneVolmer [25] equation:
2.2.3. Effect of Clog P values on antimicrobial activity
These antimicrobial results could also be analysed by Clog P
values. lipophilicity/hydrophilicity plays
a significant role in
determining where drugs are distributed and how rapidly they are
metabolized and excreted in the body, for example, hydrophobic
drugs are preferentially distributed to hydrophobic compartments
such as lipid bilayers of cells while hydrophilic drugs are prefer-
entially found in hydrophilic compartments like blood serum. For
example, Fluconazole (Clog P ¼ ꢂ0.49) with good water-solubility,
had good hydrophilicity and could go into cytoplasm easily, and
prohibited the P450 enzyme to synthesize ergosterol in fungus

682
S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
Table 2
The data of antimicrobial activities of dinuclear cyclen complexes and Clog P values.
Compds
Clog P
MIC50 (m )
g mLꢂ1
C. albicans
A. fumigatus
M. luteus
S. aureus
B. subtilis
P. aeruginosa
B. proteus
E. coli
11a
11b
11c
11d
11e
11f
6.8159
6.4160
6.8159
6.4160
5.3190
4.9190
0.5
1
128
16
64
256
8
16
8
8
16
8
2
>512
>512
16
64
256
2
512
>512
32
1
0.5
1
4
>512
64
256
256
64
64
32
512
>512
128
128
64
256
512
>512
>512
2
128
512
8
256
>512
A
B
ꢂ1.09
ꢂ0.44
e
e
2
8
e
4
e
32
e
2
e
4
e
0.5
256
A: Chloromycin; B: Fluconazole.
Table 3
The data of antimicrobial activities of ligands and their Clog P values.
Compds
Clog P
MIC50 (m )
g mLꢂ1
C. albicans
A. fumigatus
M. luteus
S. aureus
B. subtilis
P. aeruginosa
B. proteus
E. coli
6f
ꢂ0.726
0.004
0.101
1.22
256
256
128
64
512
256
256
128
256
256
512
512
>512
512
>512
128
>512
512
>512
512
>512
256
512
256
>512
512
512
256
256
126
512
512
12a
12b
13
A
B
ꢂ1.09
ꢂ0.44
e
e
8
8
e
8
e
4
e
8
e
2
e
4
256
A: Chloromycin; B: Fluconazole.
the calculated results were depicted in Table 5. Fig. 2 showed the
Modified SterneVolmer and Scatchard plots for the compound 11b-
BSA system at different temperatures. The decreasing trend of Ka
with increasing temperatures was in accordance with KSV's
dependence on temperatures. Moreover, the interaction between
BSA and compound 11b accelerated with the temperature
increasing, hence the high affinity binding sites of the interaction
were slightly strengthened, which were accordance with the re-
sults shown in Table 5. The results also showed that the binding
constant was moderate and the effect of temperature was not
significant, thus compound 11b might be stored and carried by this
protein [28].
ꢀ
ꢁ
F0
F
¼ 1 þ KSV
Q
(1)
In this equation the F0 and F represent steady-state fluorescence
intensities in the absence and presence of compound 11b, respec-
tively. KSV is the SterneVolmer quenching constant, and [Q] is the
concentration of compound 11b. Hence, KSV was calculated by
linear regression of plot of F0/F versus [Q] (Fig. 1BeD). Quenching
mechanisms are often classified as dynamic quenching, static
quenching etc. depending on temperature and viscosity. Because
higher temperatures result in larger diffusion coefficients, the
quenching constants are expected to increase with a gradually
increasing temperature in dynamic quenching. However, the
increasing of temperature is likely to result in a smaller static
quenching constant due to the dissociation of weakly bound
complexes [26]. Therefore, the KSV was calculated from
SterneVolmer plots at each temperature. The results (Table 4)
demonstrated that the quenching constant KSV was inversely
correlated with temperature, which indicated that the probable
quenching mechanism of compound 11b-BSA binding reaction was
initiated by ground-state complex formation (static quenching)
[27].
2.3.3. Binding mode and thermodynamic parameters
The interaction forces between bioactive small molecules or
drugs and biomolecules include electrostatic interactions, multiple
hydrogen bonds, van der Waals interactions, hydrophobic and
steric contacts within the antibody-binding site and so on. If the
enthalpy change (DH) does not vary significantly over the studied
temperature range, then its value and that of entropy change (
can be evaluated from the van't Hoff equation:
DS)
DH DS
LnK ¼ ꢂ
þ
(3)
RT
R
2.3.2. Binding constant
For a static quenching procedure, the data was analysed ac-
cording to the Modified SterneVolmer [28] equation:
K is analogous to the associative binding constants at the cor-
responding temperature and R is the gas constant. In order to
explain the interaction between compound 11b and BSA, the
thermodynamic parameters were calculated from the van't Hoff
F0
1
1
1
¼
þ
(2)
DF faKa ½Qꢃ fa
plots. The enthalpy change (DH) was estimated from the slope of
the van't Hoff relationship (Fig. 3). The free energy change (
then calculated from the following equation:
DG) was
DF is the difference of fluorescence in the absence and presence
of compound 11b at concentration [Q], fa is the fraction of accessible
fluorescence, and Ka is the effective quenching constant for the
DG ¼ DH ꢂ TDS
(4)
accessible fluorophores. The dependence of F0/DF on the reciprocal
value of concentration [Q]ꢂ1 is linear with the slope equalling to the
value of (faKa)ꢂ1. The Modified SterneVolmer plots were Fig. 2 and
Table 6 summarized the values of
DH, DG and DS. The negative
values of free energy G suggested the binding process was
D

S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
683
Fig. 1. (A) Emission spectra of BSA in the presence of various concentrations of compound 11b. c(BSA) 1.0 ꢁ 10ꢂ5 M; c(compound 11b)/(10ꢂ5 M), ael: from 0.0 to 5.5 at increments of
0.50; blue line shows the emission spectrum of compound 11b only; T ¼ 298 K, lex ¼ 295 nm. (BeD) SterneVolmer plot of 11b-BSA at three different temperatures. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
spontaneous, the negative entropy
D
S and enthalpy
D
H values of
activities of these azole cyclen derivatives were evaluated against
six bacterial strains and two fungal strains. The biological results
revealed that the most mono-nucleus complexes exhibited poor or
the interaction of compound 11b with BSA indicated that the
binding was mainly enthalpy-driven and the entropy was unfav-
ourable for it, moreover, hydrogen bonds and van der Waals forces
played a major role in the reaction [29].
no biological activities, some compounds such as 7g (MIC ¼ 8
mg/
mL against P. aeruginosa) and 7l (MIC ¼ 2 g/mL against C. albicans)
m
showed moderate antibacterial and antifungal activities respec-
tively. Meanwhile, most of newly synthesized di-nuclei compounds
11 displayed moderate to excellent antibacterial and antifungal
activities against all the tested strains compared to the reference
drugs Chloromycin and Fluconazole. Compounds 11aed with aro-
matic chains gave better antimicrobial efficiency than compounds
11eef incorporated pyridyl groups, especially for m-xylyl linked
compounds 11a and 11b which exhibited the best bacterial growth
inhibitory activities. The MIC value of 11a against P. aeruginosa was
2.4. Cytotoxicity
The cytotoxic activities of these complexes were tested by CCK8
method. The in vitro cytotoxicity was evaluated in HL-7702 cells. As
shown in Fig. 4, none of them displayed serious cytotoxicity in this
cell-line and the relative cell viability of 11a,11c,11e were more than
70% when its concentration was over 100 mg/mL. To our surprise,
three Cu2þ complexes showed relative high cytotoxicity in the cell-
line. Data obtained from in vitro and cell culture studies were largely
supportive of copper's capacity to initiate oxidative damage and
interfere with important cellular events [30]. But the results also
reflected that all of these complexes 11aef showed relative low
toxicity to normal human hepatic cells at high concentration. And
especially at the dose of MIC to several kinds of pathogenic micro-
organisms, dinuclear compounds were still safe for use.
1
0.5
m
g/mL, and the MIC values of 11a and 11b against B. proteus were
g/mL and 1 g/mL respectively, which were 4e12 fold to that of
Chloromycin. As for antifungal activity, the MIC values of 11a and
11b against A. fumigatus were 8 g/mL and 16 g/mL, and it indi-
m
m
m
m
cated 16e32 fold to the MIC values of Fluconazole. And it also could
be concluded that the metal ion was the key factor for the anti-
microbial activity, especially for the complexes with two nuclei.
The specific interaction of compound 11b with BSA was studied
by fluorescence spectroscopy. The experimental results displayed
that the quenching mechanism of fluorescence of BSA by com-
pound 11b was a static quenching procedure, binding constant and
binding sites were obtained according to the classical equations.
3. Conclusion
In conclusion, all the new compounds were characterized by 1H
NMR, MS, and elemental analysis spectra. The in vitro antimicrobial

684
S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
Table 4
The thermodynamic parameters indicated that the binding process
was spontaneous, the negative entropy S and enthalpy H values
SterneVolmer quenching constants for the interaction of compound 11b with BSA at
various temperatures.
D
D
of the interaction of compound 11b and BSA suggested that the
binding should be mainly enthalpy-driven and the entropy was
unfavourable for it, moreover, hydrogen bonds and van der Waals
forces played major roles in the interaction. The CCK-8 tests certi-
fied that the cytotoxicities of these effective compounds 11aed
were low. These results indicate that the new type of antimicrobial
complexes could be a series of promising reagents for further
exploitation.
pH
T (K)
10ꢂ4 Ksv/(L/moL)
Ra
Standard error
7.4
7.4
7.4
300
303
306
0.198
0.196
0.185
0.989
0.979
0.991
0.00485
0.00762
0.0043
a
Is the correlation coefficient.
6.0
T = 300K
T = 303K
T = 306K
4. Experimental protocols
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
4.1. General methods
Melting points were uncorrected and recorded on X-6 melting
point apparatus without any corrections. TLC analysis was done
using pre-coated silica gel plates. All of reagents and solvents used
were commercially available without further purification. MS
spectra data were recorded on Finnigan MAT-4510 and VG Auto
3000 mass spectrometer and Agilent 6200 LCMS-IT-TOF mass
spectrometer, respectively. 1H NMR spectra were recorded on
Brucker AV-300 and AV-400. MHz and chemical shifts are reported
relative to internal Me4Si. Elemental analyses were performed by
using a Carlo-Elba 1106 elemental analytical instrument. XRD data
were tested by Shimazduo DIFFRACTOMETER-6000. CHCl3 and THF
were purified according to the standard method. N, N-Diisopro-
pylethylamine (DIPEA) and Halides 2aec were purchased from
Sigma or prepared follow the reference (Supplementary
information). All aqueous solutions were prepared using deion-
ized and redistilled water. All other chemicals and solvents were
commercially available, and were used without further purification.
All fluorescence spectra were recorded on Horiba Jobin Yvon
Fluoromax-4 spectrofluorometer; corrected for the system
response equipped with 1.0 cm quartz cells, the widths of both the
excitation and emission slit were set as 2.5 nm, and the excitation
wavelength was 295 nm. Fluorescence spectra were recorded at
298, 300, 303, 306 K in the range of 300e450 nm. The temperature
were controlled with PolyScience temperature controller. The BSA
was obtained from SigmaeAldrich (St. Louis, MO, USA). Tris, NaCl,
HCl, ethanol were analytical purity. BSA was dissolved in TriseHCl
buffer solution (0.05 M Tris, 0.15 M NaCl, pH ¼ 7.4), compound 11b
was dissolved in redistilled water [31].
0.2
0.3
0.4
0.5
0.6
0.7
10-5[Q]-1(L/mol)
Fig. 2. Modified SterneVolmer plots of compound 11b-BSA system at different
temperatures.
Table 5
Modified SterneVolmer quenching constants for the interaction of compound 11b
with BSA at various temperatures.
pH
T (K)
10ꢂ4 Ka/(L/moL)
Ra
S.D.b
7.4
7.4
7.4
300
303
306
0.765
0.431
0.324
0.998
0.999
0.998
0.07879
0.03979
0.07545
a
Is the correlation coefficient.
Is standard deviation.
b
4.1.1. Synthesis of compounds 1e6, 9e10, 12 and 13
Compounds 1e6, 9e10, 12 and 13 were prepared according to
the literature procedures [32] (Supplementary information).
9.0
LnKa = -34.7434 + 13.09229X
= 0.985
4.1.2. Synthesis of compounds 7 and 11
General procedure of the syntheses of 7 and 11. The obtained
acid free ligand and equal or double molar weight of
Zn(ClO4)2$6H2O or Cu(ClO4)2$6H2O were stirred at room temper-
ature for 24 h. After that, the solids were filtered off, washed with
cooled EtOH, the residue was crystallized from H2O/EtOH to ob-
tained colourless crystals. The product was dried in vacuo. Data of
compounds 7aer were shown in Supplementary information.
R
8.8
8.6
8.4
8.2
8.0
Table 6
Thermodynamic parameters of compound 11b-BSA interaction at different
temperatures.
T (K)
△H (kJ/mol)
ꢂ108.78
△G (kJ/mol)
△S (J/moL K)
ꢂ288.72
3.26
3.27
3.28
3.29
3.30
3.31
3.32
3.33
3.34
T(10-3 K-1)
300
303
306
ꢂ22.16
ꢂ21.30
ꢂ20.43
Fig. 3. Van't Hoff plots of compound 11b-BSA system.

S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
685
yl)methanamine Cu(II) complex (11d). A solution of EtOH (10 mL),
the obtained acid free ligand (0.16 g, 0.29 mmoL) and
Cu(ClO4)2$6H2O (0.22 g, 0.60 mmoL) was stirred at room temper-
ature for 24 h. After that, the process followed the procedure
described above. Blue solid (yield 37.3%). m.p. 267ꢂ271 ꢀC. MS(ESI)
m/z: 674 [(M
ꢂ
3Hþ
ꢂ
4ClOꢂ4 )þ, 100]. Anal. Calcd. for
C32H41Cl4N9O16Cu2$2H2O: C 34.54, H 4.08, N 11.42; found: C 34.96,
H 4.05, N 11.02.
4.1.2.5. 1-(6-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)pyridin-2-
yl)-N,N-bis((1H-benzo[d]imidazol-2-yl)methyl)methanamine Zn(II)
complex (11e). A solution of EtOH (10 mL), the obtained acid free
ligand (0.17 g, 0.32 mmoL) and Zn(ClO4)2$6H2O (0.24 g, 0.65 mmoL)
was stirred at room temperature for 24 h. After that, the process
followed the procedure described above. White solid (yield 31.3%).
m.p. 273ꢂ275 ꢀC. MS(ESI) m/z: 715.2184 [(MþKþ ꢂ 4Hþ ꢂ OHꢂ
ꢂ 3ClO4ꢂ)þ], 817.1693 [(MþKþ ꢂ 3Hþ ꢂ OHꢂ ꢂ 2ClOꢂ4 )þ]. Anal. Calcd.
for C31H42Cl3N10O13Zn2$H2O: C 36.58, H 4.36, N 13.76; found: C
Fig. 4. Relative cell viabilities of compounds 11aef in HL-7702 cells.
36.87, H 4.64, N 13.33. 13C NMR (d6-DMSO, 100 MHz)
d: 46.55,
53.70, 76.03, 85.36, 112.66, 115.12, 119.18, 122.27, 137.54, 153.34.
4.1.2.1. N-(3-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-N-
((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-yl)
methanamine Zn(II) complex (11a). A solution of EtOH (10 mL), the
obtained acid free ligand (0.17 g, 0.32 mmoL) and Zn(ClO4)2$6H2O
(0.24 g, 0.65 mmo1) was stirred at room temperature for 24 h. After
that, the process followed the procedure described above. White
solid (yield 29.3%). m.p. 265ꢂ267 ꢀC. MS(ESI) m/z: 714.2311
[(MþKþ ꢂ 4Hþ ꢂ 4ClO4ꢂ)], 752.1704 [(Mþ2Kþ ꢂ 5Hþ ꢂ 4ClOꢂ4 )].
Anal. Calcd. for C32H41Cl4N9O16Zn2$5H2O: C 32.84, H 4.39, N 10.77;
4.1.2.6. 1-(6-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)pyridin-2-
yl)-N,N-bis((1H-benzo[d]imidazol-2-yl)methyl)methanamine Cu(II)
complex (11f). A solution of EtOH (10 mL), the obtained acid free
ligand (0.17 g, 0.32 mmoL) and Cu(ClO4)2$6H2O (0.24 g, 0.65 mmoL)
was stirred at room temperature for 24 h. After that, the process
followed the procedure described above. Blue solid (yield 58.5%).
m.p.
>
247
ꢀC
(dec).
MS(ESI)
m/z:
714.2190
[(MþKþ ꢂ 4Hþ ꢂ 4ClOꢂ4 )þ], 816.1904 [(MþKþ ꢂ 3Hþ ꢂ 3ClOꢂ4 )þ],
Anal. Calcd. for C31H40Cl4N10O16Cu2$C2H5OH: 35.27, H 4.13, N 12.47;
found: C 35.72, H 4.49, N 12.58.
found: C 32.78, H 4.45, N 10.10. 1H NMR (d6-DMSO, 300 MHz)
d:
2.51e3.06 (br, 16H, NCH2CH2N), 3.44e3.49 (br, 2H, CyclenCH2Py),
4.07 (s, 2H, NCH2Py), 4.31 (br, 4H, CH2BIm), 7.13 (br, 2H, BIm-H),
7.30e7.87 (m, 12H, Py-H, BIm-H).
4.2. Antibacterial and antifungal assays
The in vitro minimal inhibitory concentrations (MICs) of the
target compounds were determined using the two-fold serial
dilution technique in 96-well microtest plates according to the
National Committee for Clinical Laboratory Standards. The tested
microorganism strains were provided by the School of Pharma-
ceutical Sciences, Southwest University and the College of Phar-
macy, Third Military Medical University. Chloromycin and
Fluconazole were used as standard drugs.
4.1.2.2. N-(3-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-
N-((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-
yl)methanamine Cu(II) complex (11b). A solution of EtOH (10 mL),
the obtained acid free ligand (0.17 g, 0.32 mmoL) and
Cu(ClO4)2$6H2O (0.24 g, 0.65 mmo1) was stirred at room temper-
ature for 24 h. After that, the process followed the procedure
described above. Blue solid (yield 58.7%). m.p. 254ꢂ256 ꢀC. MS(ESI)
m/z: 697 [(MþNaþ
ꢂ
4Hþ
ꢂ
4ClOꢂ4 ), 61]. Anal. Calcd. for
C
32H41Cl4N9O16Cu2$C2H5OH: C 36.37, H 4.22, N 11.32; found: 36.48,
4.2.1. Antibacterial assays
H 4.49, N 10.99.
The prepared compounds 7aer and 11aef were evaluated for
their antibacterial activities against S. aureus ATCC 6538, M. luteus
ATCC 4698 and B. subtilis ATCC 21216 as Gram-positive, E. coli ATCC
8099, P. aeruginosa ATCC 27853 and B. proteus ATCC 13315 as Gram-
negative bacteria. The bacterial suspension was adjusted with
sterile saline to a concentration of 1 ꢁ 10ꢂ5 CFU. The tested com-
pounds were dissolved in 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 512, 256, 128, 64, 32, 16, 8, 4,
4.1.2.3. N-(4-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-
N-((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-
yl)methanamine Zn(II) complex (11c). A solution of EtOH (10 mL),
the obtained acid free ligand (0.16 g, 0.29 mmoL) and
Zn(ClO4)2$6H2O (0.22 g, 0.60 mmo1) was stirred at room temper-
ature for 24 h. After that, the process followed the procedure
described above. White solid (yield 45.6%). m.p. 255ꢂ257 ꢀC.
MS(ESI) m/z: 752.1786 [(Mþ2Kþ ꢂ 5Hþ ꢂ 4ClO4ꢂ)þ], 818.1784
[(MþKþ
ꢂ
3Hþ
ꢂ
3ClOꢂ4 )þ].
Anal.
Calcd.
for
C
32H41Cl4N9O16Zn2$3H2O: C 33.88, H 4.18, N 11.11; found: C 34.11, H
2, 1, 0.5 mg/mL. These dilutions were inoculated and incubated at
4.47, N 10.77. 1H NMR (d6-DMSO, 300 MHz)
d: 2.69e3.10 (br, 16H,
37 ꢀC for 24 h. To ensure that the solvent had no effect on bacterial
growth, a control test was performed with test medium supple-
mented with DMSO at the same dilutions as used in the
experiment.
NCH2CH2N), 3.62 (s, 2H, CyclenCH2Py), 4.08 (s, 2H, NCH2Py), 4.33
(br, 4H, CH2BIm), 6.95e6.97 (br, 2H, BIm-H), 7.30e7.77 (m, 12H, Py-
H, BIm-H). 13C NMR (d6-DMSO, 100 MHz)
d: 42.20, 43.77, 44.68,
53.39, 60.84, 63.33, 100.92, 103.40, 114.53, 118.01, 122.66, 123.85,
124.17, 131.61, 135.05.
4.2.2. Antifungal assays
The newly synthesized compounds were evaluated for their
antifungal activity against C. albicans ATCC 76615 and A. fumigatus
ATCC 96918. A spore suspension in sterile distilled water was
4.1.2.4. N-(4-((1,4,7,10-Tetraazacyclododecan-1-yl)methyl)benzyl)-
N-((1H-benzo[d]imidazol-2-yl)methyl)-1-(1H-benzo[d]imidazol-2-

686
S. Li et al. / European Journal of Medicinal Chemistry 84 (2014) 677e686
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ꢀ
ꢀ
ꢀ
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This work was financially supported by Natural Science Foun-
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Commission (Grant No. KJ130820), the New Teacher Foundation of
Chongqing University of Technology (2012ZD24), and National
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Appendix A. Supplementary data
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