Structure-cytotoxicity relationship of a novel series of miconazole-like compounds

Article abstract of DOI:10.1007/s00044-011-9716-z

In the current study, two novel classes of the carboacyclic nucleosides having miconazole-like scaffolds as imidazole- and pyrimidine-based compounds were examined for their cytotoxic properties. The aim was to establish a relation between cytotoxic activ

Full text of DOI:10.1007/s00044-011-9716-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1921–1928
DOI 10.1007/s00044-011-9716-z
ORIGINAL RESEARCH
Structure–cytotoxicity relationship of a novel series
of miconazole-like compounds
•
Reza Yousefi Ali Khalafi-Nezhad Mohammad Navid Soltani Rad
•
•
•
Somayeh Behrouz Farhad Panahi Mansoore Esmaili
•
•
•
Sayed Mahmoud Ghaffari Ali Niazi Ali Akbar Moosavi-Movahedi
•
Received: 14 December 2010 / Accepted: 16 June 2011 / Published online: 7 July 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In the current study, two novel classes of the
carboacyclic nucleosides having miconazole-like scaffolds
as imidazole- and pyrimidine-based compounds were
examined for their cytotoxic properties. The aim was to
establish a relation between cytotoxic activity and nature of
the synthetic compounds. While Escherichia coli (DH5a)
and human erythromyeloblastoid leukemia cell line (K562)
were the target cells, depending on the type of substitution
made, ranges of antibacterial and antineoplastic activities
were observed. Also the electron-donating and electron-
accepting properties of the ligands were proved to play a
crucial role in their cytotoxic activities. Accordingly, the
substitutions associated with the marked improvement of
cytotoxic activities can be considered as the significant
point in construction of new generation of either antibac-
terial or antineoplastic agents.
Keywords Pyrimidine-based compounds ꢀ
Imidazole-based compounds ꢀ Cytotoxic activities
Introduction
The pharmacokinetic properties and cellular permeability of a
drug can be modulated by derivatization to bio-reversible
forms (Maccari et al., 2002). Hydrazone derivatives are
prominent structural motif in numerous pharmaceutically
active compounds; consequently several well-known drugs
with various chemotherapeutic activities contain a hydrazone
moiety in their structure (Kleeman et al., 1999; Szarvasi et al.,
1973; Pennington et al., 1953; Sah and Peoples, 1953; Szen-
tesi et al., 2001; Murdock et al., 1982). Azoles are a well-
known class of antifungal agents, disrupting biosynthesis of
the major sterol of fungal cell membrane (ergosterol) (Turner
and Rodrigues, 1996; Bodey, 1992). Miconazole and oxi-
conazole are the most established antifungal azoles having
rational versatility in their structures and family (Fig. 1)
(Rossello et al., 2002). Various structurally related miconaz-
ole bioactive hydrazones are known as antimicrobial and
antifungal agents (Mamolo et al., 2004; Dyer et al., 1983).
In general, there are two basic approaches to develop a
new drug for the treatment: (a) synthesis of analogs,
modifications, or derivatives of existing compounds for
shortening and improving treatment, and (b) searching for
novel structures that the organism has never been presented
with before. The current study was carried out to examine
the biological activities of a novel series of carboacyclic
nucleosides having miconazole-like scaffolds as imidazole-
and pyrimidine-based compounds. The general structures
for the parent compounds are shown in Fig. 1a. In these
compounds, the ether and ketoxim-ether linkages were
replaced with hydrazone linkages in miconazole and
R. Yousefi (&)
Protein Chemistry Laboratory (PCL), Department of Biology,
College of Sciences, Shiraz University, Shiraz 71454, Iran
e-mail: ryousefi@shirazu.ac.ir
A. Khalafi-Nezhad ꢀ F. Panahi
Department of Chemistry, College of Sciences,
Shiraz University, Shiraz, Iran
M. N. Soltani Rad ꢀ S. Behrouz
Department of Chemistry, Shiraz University of Technology,
Shiraz, Iran
M. Esmaili ꢀ S. M. Ghaffari ꢀ A. A. Moosavi-Movahedi
Institute of Biochemistry and Biophysics (IBB),
University of Tehran, Tehran, Iran
A. Niazi
Institute of Biotechnology, Shiraz University, Shiraz, Iran
123

1922
Med Chem Res (2012) 21:1921–1928
Fig. 1 Chemical structures and
synthetic routes for preparation
of the hydrazine derivatives.
a The comparison between the
chemical structure of
miconazole, oxiconazole, and
the parent compounds. The
general synthetic routes for the
preparation of b imidazole-
based compounds and
c pyrimidine-based compounds
oxiconazol, respectively. With the aim to establish a rela-
tion between cytotoxic activity and nature of the com-
pounds, the cytotoxic activities of the miconazole-like
compounds were examined and compared using Esche-
richia coli (DH5a) and human erythromyeloblastoid leu-
kemia cell line (K562) as the target cells.
the procedures used for synthesis of ethanone intermediates
considering the differences in chemical behaviors of pyrim-
idine nucleobases in comparison with azoles family. The
ethanone derivatives for ligands 1a–6a were prepared from
the substituted 2-bromoacetophenones and the appropriate
azole derivatives in the presence of triethylamine in refluxing
acetonitrile (Fig. 1b). To synthesize ligands 1b–5b, the cor-
responding intermediates were obtained by the reaction of
substituted 2-bromoacetophenones and pyrimidine nucleo-
bases in refluxing N,N-dimethyl formamide (DMF) (Fig. 1c).
Chemistry
Recently, we reported an efficient procedure for preparation
of new hydrazon derivatives (Soltani Rad et al., 2010). As
shown in Fig. 1b, c, treating the synthesized ethanone
derivatives with phenyl hydrazine in refluxing ethanol,
resulted in the formation of corresponding hydrazone
derivatives. Compounds 1a–6a (Fig. 2) and 1b–5b (Fig. 3)
were synthesized using two different strategies according to
Biochemistry and pharmacology
Azole antifungals are well known for inhibiting a cyto-
chrome P-450-dependent enzyme which is crucial for
ergosterol biosynthesis in fungi, and moreover they involve
123

Med Chem Res (2012) 21:1921–1928
1923
Fig. 2 Chemical structures of
the imidazole-based hydrazon
derivatives
Fig. 3 Chemical structures of
pyrimidine-based hydrazone
derivatives
in hormone synthesis or drug metabolism in mammalian
cells. Furthermore, antifungal azoles have previously been
reported to exhibit antibacterial activity, although their
mechanism of action is not fully understood (Rossello
et al., 2002; Mamolo et al., 2004; Dyer et al., 1983). In this
study, two novel classes of miconazole-like scaffolds as
imidazole- and pyrimidine-based compounds are examined
for their cytotoxic activities. Six imidazole- (1a–6a) and
five pyrimidine-based compounds (1b–5b) were tested for
both antibacterial and antineoplastic activities with the aim
to establish a relation between chemical structure and
cytotoxic activity. The data of antibacterial activities of
these compounds are shown in Tables 1 and 2, while Fig. 4
displays those of their antiproliferation activities.
123

1924
Med Chem Res (2012) 21:1921–1928
Results and discussion
Table 2 Log P and cytotoxicity of the pyrimidine-based compounds
against E. coli (DH5a)
Antibacterial activities of the miconazole-like
compounds
Entry
1b
57.3 3.45 6.2 0.57 4.8 0.71 7.1 0.48 14.6 1.47
1.84 1.72 1.88 2.19 1.95
2b
3b
4b
5b
IC50
(lM)
Log P
Hydrazide–hydrazone has been demonstrated to possess
antibacterial activity (Aussel et al., 1994; Pawelec et al.,
1991). In order to evaluate contribution of the new sub-
stitution on cytotoxic profile of the miconazole-like com-
pounds, a novel series of imidazole- and pyrimidine-based
derivatives were synthesized. In this study, both antimi-
crobial and antineoplastic activities of the synthetic com-
pounds were examined. The antimicrobial agents applied in
this study may be either bacteriostatic or bactericidal. The
cytotoxic activities against E. coli (DH5a) of the imidaz-
ole-based compounds are shown in Table 1. In order to
compare the cytotoxic activity among the different com-
pounds of this class, Ligand 1a with the simplest chemical
structure was considered as the reference compound
(Fig. 2). As shown in Fig. 2 and Table 1, the addition of
electron-donating phenyl moiety to the imidazole (Ligand
2a), markedly decreases the cytotoxic activity. This finding
suggests that the molecule after this modification may gain
certain electronic and/or stereo chemical properties which
abolish its antibacterial activity. On the other hand, the
existence of two carbonyl electron-withdrawing moieties in
hydantoin (Ligand 3a), significantly increases the cytotoxic
effect against E. coli (DH5a). As the imidazole was
replaced with benzimidazole in ligand 4a and 5a, the
cytotoxic activities against E. coli (DH5a) were decreased.
Further reduction of the cytotoxic activity was observed in
the case of compound 5a which was bearing an electron-
donating methoxy in its structure. Surprisingly, the addi-
tion of an electron-withdrawing element such as nitrogen
(position 2) in compound 6a markedly enhances its cyto-
toxic activity. Overall, these findings show the importance
of electronic and chemical properties in the cytotoxicity of
the synthetic compounds.
markedly increases their cytotoxic activities as compared
with that of the reference compound (Ligand 1b). Also, the
methoxy bearing compound (Ligand 2b) exhibits slightly
higher cytotoxic activity than the compound with methyl
moiety (Ligand 4b). As methyl group was substituted with
a halogen (chloride) in ligand 3b, a significant enhance-
ment in the cytotoxicity was observed. Similar to what was
observed for the imidazole-based compound (Ligand 6a),
addition of the nitrogen element to pryimidine ring (posi-
tion 6) in ligand 5b noticeably enhanced the cytotoxic
activity of this compound, displaying the vital role which
might be played by the existence of additional hydrogen-
accepting nitrogen element in the cytotoxic compounds.
Overall, the electron-donating and electron-accepting
properties of the synthetic miconazole-like compounds are
identified to be very important for their biological
properties.
Antineoplastic activities of the synthetic miconazole-
like derivatives
As shown previously, miconazole-like compounds may
slowdown proliferation of the activated human T-cells and
human acute myelogenous leukemia (AML) cells (Bruse-
¨
rud, 2001; Kramer et al., 2009; Thomae et al., 2007;
Mossman, 1983). In this study, all of the synthetic mico-
nazole-like derivatives (20, 40, and 80 lM) were further
examined for their toxicity against K562 cell line. After
24-h exposure of K562 leukemic cells to the different
synthetic compounds, viability was assessed on the basis of
cellular conversion of 3-(4,5-dimethtl-2-thiazolyl)-2,5-di-
phenyl-2H-tetrazoliun bromide (MTT) into a formazan
product, using MTT-based cell proliferation assay (Gurkok
In this study, the different pyrimidine-based compounds
were also examined for their cytotoxic activities against
E. coli (DH5a), and the results are listed as Table 2. In
order to compare the cytotoxic activities between different
members of this class, ligand 1b which possesses the
simplest chemical structure is considered as the reference
compound (Fig. 3). In contrast to the imidazole-based
compounds, addition of the electron-donating moieties
such as methoxy in compound 2b and methyl in Ligand 4b
´
et al., 2009; Rufian-Henaresa and Morales, 2008). As the
leukemic cells (2 9 104 cells/ml) were cultured in 96-well
plates for 24 h the cytotoxic activities were assessed, and a
range of antitumor activities was observed for the synthetic
compounds (Fig. 4). Among the imidazole-based deriva-
tives, ligand 6a proved to be the most active compound.
Table 1 Log P and cytotoxicity of the imidazole-based compounds against Escherichia coli (DH5a)
Entry
1a
2a
3a
4a
5a
6a
IC50 (lM)
7.6 0.81
2.13
16.8 1.71
4.18
3.1 0.61
1.26
12.3 0.23
3.60
13.7 1.42
3.48
3.4 0.20
4.01
Log P
123

Med Chem Res (2012) 21:1921–1928
1925
Fig. 4 Antiproliferation
140
120
100
80
activities of the synthetic
ligands against K562 cells
(2 9 104 cells/ml) were
cultured with varying
concentrations of the ligands
(20, 40, and 80 lM) for 24 h,
and cell proliferation was
assessed by MTT assay as
described in ‘‘Materials’’
Section. Results (Cytotoxicity)
are presented relative to cell
growth under control conditions
(the absence of the ligands) and
expressed as percentage of
control (as 100%). The vertical
bars represent SD of triplicate
determinations and asterisks
indicate *P \ 0.05, **P \ 0.01,
and ***P \ 0.001 compared to
control (the absence of the
ligands).
*
**
**
**
**
***
***
***
***
***
***
***
***
***
** ***
***
60
***
***
40
***
***
***
***
***
20
***
0
C
1a
2a
3a
4a
5a
6a
1b
2b
3b
4b
5b
Ligand
When pyrimidine derivatives were examined for their
cytotoxic activity against K562 leukemia cell line, ligand
3b and 5b were the most potent of all the compounds tested
for their antiproliferation activities. Two ligands 6a and 5b
possessed additional nitrogen element in the imidazole and
pyrimidine rings, respectively (Figs. 2, 3). The obtained
results confirm that the addition of further nitrogen element
with the ability for hydrogen accepting in the synthetic
compounds could significantly enhance the cytotoxic
properties of the miconazole-based compounds. As shown
in Fig. 3, the substitution of hydrogen (in compound 1b)
with chloride yielded compound 3b with significantly
improved activity against the tumor cells. Thus, electron-
donating and electron-accepting natures of the synthetic
ligands are proven to be of importance in their antineo-
plastic activities. Furthermore, the lipophilicity of the
synthesized compounds, which varied significantly when
compared with the parent compound, might be considered
as an important parameter in their cytotoxic activities
Conclusion
This study describes the cytotoxic properties of several
imidazole- and pyrimidine-based derivatives against both
E. coli (DH5a) and K562 leukemia cell line. Overall, the
addition of the electron-withdrawing elements (N and
carbonyl) with the capability for hydrogen being accepted
into the imidazole scaffolds displayed a critical role in the
enhancement of the antibacterial activities of the studied
compounds. Conversely, the addition of the electron-
donating moieties such as benzyl, methyl, and methoxy to
these ligands significantly suppresses their antibacterial
activities. The similar pattern of antibacterial properties
was not observed in the case of the pyrimidine-based
ligands since the methoxy- and methyl-bearing pyrimidine
compounds exhibited greater antibacterial activities than
that of their corresponding reference ligand. The additional
nitrogen element existing in the structure of both imidazole
and pyrimidine moieties of the synthetic ligands signifi-
cantly enhanced their antibacterial activities. As the leu-
kemic cells were used as the target; the synthetic ligands
6a, 3b, and 5b exhibited the highest antiproliferation
properties. These ligands also showed higher antibacterial
activities than their corresponding reference compounds
against E. coli (DH5a). Therefore, results of the current
study suggest that addition of either halogen or nitrogen
elements to the scaffold of the synthetic ligands signifi-
cantly enhances their cytotoxic activities against both
E. coli (DH5a) and K562 leukemic cells. In conclusion, the
obtained results suggest that the electron-donating and
electron-accepting properties and/or stereo chemical fea-
tures of the ligands may play critical roles in their cytotoxic
activities. As several synthetic compounds were screened
´
(Sanchez et al., 2008; Berney et al., 2006). The lipophil-
icity may render them more capable of penetrating various
biomembranes, consequently improving their permeation
´
properties through the cell membranes (Sanchez et al.,
2008; Berney et al., 2006). Thus, LogP of the synthetic
ligands was calculated using CS Chem Draw Ultra 8/CS
Chem 3D Ultra 8 (Cambridge Soft, 2004) and expressed in
Tables 1 and 2. The variation of Log P as shown in these
tables is more significant in the case of imidazole-based
compounds compared to that of pyrimidine-based ligands.
However, the results of this study did not show regular
pattern of correlation between Log P and antiprolifereation
activities of the synthetic compounds except in a few cases
(Tables 1, 2; Fig. 4).
123

1926
Med Chem Res (2012) 21:1921–1928
for their cytotoxic properties; the compounds with signifi-
cant activity can also represent a starting point for a new
generation of cytotoxic agents. However, additional studies
are required to determine whether these synthetic com-
pounds could induce apoptosis in the cancer cells.
(1.38 g, 0.01 mol) and catalytic amounts of TBAB (0.1 g)
was dissolved in dry DMF (30 ml). Then, the mixture was
heated at reflux for 2–3 h (TLC control). The solvent was
evaporated at reduced pressure, the residue was dissolved
in CHCl3 (200 ml), and washed with H2O (2 9 100 ml).
The organic layer was dried (10 g of Na2SO4) and con-
centrated to afford the crude product which was purified by
column chromatography on silica gel eluting with proper
solvents.
Experimental
Synthesis of ligands
General procedure for the synthesis of compounds 1a–6a
and 1b–5b
The ligands as shown in Figs. 1 and 2 were prepared
according to the methods reported in the literatures (Soltani
Rad et al., 2010).
In a double-neck round-bottomed flask (100 ml) equipped
with a condenser, a mixture of appropriate ketone
(0.01 mol), phenylhydrazine (1.62 g, 0.015 mol), and
acetic acid (3 drops) was dissolved in ethanol (15 ml), and
the mixture was heated at reflux for 15 h (TLC control).
Afterward, the reaction mixture was kept at refrigerator
overnight. Then, it was filtered and washed with cool
ethanol (2 9 5 ml) and recrystallized from MeOH/H2O to
afford pure phenylhydrazone derivatives.
General
All the chemicals were purchased from Fluka or Merck
chemical companies. The progress of reaction was fol-
lowed with thin layer chromatography (TLC) using silica
gel SILG/UV 254 plates. Silica gel 60, 0.063–0.200 mm
(70–230 mesh ASTM) was used for column chromatogra-
phy. Infrared (IR) spectra were run on a Shimadzu FTIR-
8300 spectrophotometer. The 1H NMR (250 MHz) and 13
C
Biology
NMR (62.5 MHz) were run on a Bru¨ker Avanced DPX-
250, FT-NMR spectrometer; d in ppm, J in Hz. Mass
spectra were recorded on a Shimadzu GC MS-QP 1000 EX
apparatus. Microanalyses were performed on a Perkin–
Elmer 240-B microanalyzer. Melting points (mp) were
recorded on a Bu¨chi 510 apparatus in open capillary tubes
and are uncorrected.
Materials
Fetal calf serum (FCS) was obtained from veterinary fac-
ulty at the University of Tehran. RPMI 1640 medium,
Penicillin, Streptomycin, and all other reagents were pur-
chased from the Sigma (Deisenhofen, Germany) and were
at least of analytic grade. All solutions were made in
double-distilled water.
General procedure for the synthesis of ketones 1aa–6aa
In a double-neck round-bottomed flask (100 ml) equipped
with a condenser, a mixture of appropriate N-heterocyle
(0.01 mol), 2-bromo acetophenones (0.012 mol), anhy-
drous tetra ethyl amine (TEA,1.01 g, 0.01 mol), and cata-
lytic amounts of tetra butyl ammonium bromide
(TBAB,0.1 g) was dissolved in dry acetonitrile (40 ml).
Then, the mixture was heated at reflux for 10 h (TLC
control). The solvent was evaporated at reduced pressure,
the residue was dissolved in CHCl3 (200 ml), and washed
with H2O (2 9 100 ml). The organic layer was dried (10 g
of Na2SO4) and concentrated to afford the crude product
which was purified by column chromatography on silica
gel eluting with proper solvents.
Determination of antimicrobial activity
The synthetic ligands were dissolved and diluted in ethanol
to a given concentration, and from this stock solution serial
twofold dilutions were made in the well of microtitre plate.
Final concentration ranged from 5 to 80 lM. DH5a, an
antibiotic sensitive strain of E. coli was grown in LB agar
media containing 1% Trypton, 0.5% NaCl, and 0.5% yeast
extract. The antibacterial activity of the ligands was
assessed on Microtiter plate-based assay according to the
method reported previously (El-Ziney et al., 1998). In each
well, 200 ll of overnight cultured E. coli (DH5a) and 10 ll
of each diluted miconazole-like compound were added. As
the control experiment, 200 ll of the bacteria and 10 ll of
ethanol/ddH2O (1:1) were inoculated into the wells. The
mixture was shaken at 37°C for 16 h, and the optical
density at 600 nm was measured every 30 min until the
end of the experiment using an Elisa Reader Expert 96
(ASYS Hitech, Eugendorf, Austria). The specific growth
General procedure for the synthesis of ketones 1bb–5bb
In a double-neck round-bottomed flask (100 ml) equipped
with a condenser, a mixture of appropriate nucleobase
(0.01 mol), 2-bromo acetophenones (0.012 mol), K2CO3
123

Med Chem Res (2012) 21:1921–1928
1927
Acknowledgments The authors would like to thank the financial
support of Iran National Science Foundation (INSF)—Grant nos.
88001578 and 88001894). Also the financial support of the research
council of Shiraz University is gratefully acknowledged.
rate (l) was calculated from the plot of OD600 versus time
as follows:
Ln OD600
l ¼
Dt
where t is the time of incubation (Ku¨c¸u¨kgu¨zel et al., 2003;
Bruserud, 2001). The lethal concentration 50 (LC50) was
determined by probit analysis using the Pharm. PCS sta-
tistical package (Springer-Verlage, New York).
References
Aussel C, Breittmayer JP, Ticchioni M, Pelassy C, Bernard A (1994)
Regulation of T cell activation by cytochrome P450 inhibitors.
Cell Immunol 155:436–445
Berney M, Weilenmann HU, Ihssen J, Bassin C, Egli T (2006)
Specific growth rate determines the sensitivity of Escherichia
coli to thermal, UVA, and solar disinfection. Appl Environ
Microbiol 72:2586–2593
Bodey GP (1992) Azole antifungal agents. Clin Infect Dis 14:161–
169
Bruserud O (2001) Effects of azoles on human acute myelogenous
leukemia blasts and T lymphocytes derived from acute leukemia
patients with chemotherapy-induced cytopenia. Int Immuno-
pharmacol 1:2183–2195
Dyer RL, Ellames GJ, Hamill BJ, Manley PW, Pope AM (1983)
Synthesis of (E)-1-(5-chlorothien-2-yl)-2-(1H-imidazol-1-yl)eth-
Cancer cell culture
K562 Chronic myelogenous leukemia cells were obtained
from the cell bank of Pasteur Institute of Iran. K562 cells
were grown in RPMI with 10% heat-inactivated fetal calf
serum (FCS), supplemented with 4 mM L-glutamine,
100 U penicillin, and 100 lg/ml streptomycin at pH 7.4, in
a humidified, 5% CO2 incubator and at the temperature
of 37°C.
anone 2,6-dichlorophenylhydrazone hydrochloride,
orally active antifungal agent. J Med Chem 26:442–445
a novel,
K562 proliferation assay
El-Ziney MG, Arneborg N, Uyttendaele M, Debevere J, Jakobsen M
(1998) Characterization of growth and metabolite production of
Lactobacillus reuteri during glucose/glycerolcofermentation in
batch and continuous cultures. Biotechnology Lett 20:913–916
Gurkok G, Altanlar N, Suzen S (2009) Investigation of antimicrobial
activities of indole-3-aldehyde hydrazide/hydrazone derivatives.
Chemotherapy 55:15–19
Kleeman A, Engel J, Kutscher B, Reichert D (1999) Pharmaceutical
substances, 3rd edn. Thieme, Stuttgart
¨
Kramer SD, Lombardi D, Primorac A, Thomae AV, Wunderli-
Allenspach H (2009) Lipid-bilayer permeation of drug-like
compounds. Chem Biodivers 6:1900–1916
To evaluate the cytotoxicity effect of the ligands on
K562, the harvested cells were seeded into 96-well plates
(2 9 104 cells/ml) and incubated for 24 h in the presence
of 20, 40, and 80 lM of the synthetic compounds. The
antiproliferation curve activity was determined based on
´
MTT colorimertic assay (Yousefi et al., 2005; Rufian-
Henaresa and Morales, 2008; Gurkok et al., 2009). Four
hours before the end of incubations, the cells were
washed with PBS three times, changed with fresh med-
ium, and each well loaded with 25 ll MTT solution
(5 mg/ml in PBS) followed by 4-h incubation. The
insoluble formazan produced by living cells were dis-
solved in solution containing 10% SDS and 50% DMF
(Left for 2 h at 37°C in dark conditions), and optical
density (OD) was read against blank with multi-well
scanning spectrophotometer (ELISA reader, Model, Mul-
tiskan MS) at a wavelength of 540 (OD540). The per-
centage of cell viability was calculated using equation:
[mean optical density (OD) of treated cells/mean OD of
control cells] 9 100. The final concentration of ethanol
never exceeds 2.5%, which has no deleterious effect on
the target cells.
Ku¨c¸u¨kgu¨zel SG, Mazi A, Sahin F, Oztu¨rk S, Stables J (2003)
Synthesis and biological activities of diflunisal hydrazide-
hydrazones. Eur J Med Chem 38:1005–1013
`
Maccari R, Ottana R, Monforte F, Vigorita MG (2002) In vitro
antimycobacterial activities of 2’-monosubstituted isonicotinohyd-
razides and their cyanoborane adducts. Antimicrob Agent Chemo-
ther 46(2):294–299
Mamolo MG, Zampieri D, Falagiani V, Vio L, Fermeglia M, Ferreno M,
PriclS,BanfiE,ScialinoG(2004)Antifungalandantimycobacterial
activity of new N1-[1-aryl-2-(1H-imidazol-1-yl and 1H-1,2,4-
triazol-1-yl)-ethylidene]-pyridine-2-carboxamidrazone derivatives:
a combined experimental and computational approach. ARKIVOC
V: 231–250
Mossman T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Method 65:55–63
Murdock KC, Child RG, Lin Y, Warren JD, Fabio PF, Lee VJ, Izzo
PT, Lang SA, Angier RB, Citarella RV, Wallace RE, Durr FE
(1982) Antitumor agents. 2. Bisguanylhydrazones of anthracene-
9,10-dicarboxaldehydes. J Med Chem 25:505–518
Pawelec G, Ehninger G, Rehbein A, Schaudt K, Jaschonek K (1991)
Comparison of the immunosuppressive activities of the antimy-
cotic agents itraconazole, fluconazole, ketoconazole and mico-
nazole on human T-cells. Int J Immunopharmacol 13:299–304
Pennington FC, Guercio PA, Solomons IA (1953) Streptohydrazid.
J Am Chem Soc 75:2261
Statistical analysis
The experiments were repeated three times for each sam-
ple, and the statistical differences were determined by
analysis of variance (ANOVA) followed by Turkey–Kra-
mer multiple comparison tests on the instant package.
Differences were regarded as significant at P \ 0.05.
Rossello A, Bertini S, Lapucci A, Macchia M, Martinelli A, Rapposelli
S, Herreros E, Macchia B (2002) Synthesis, antifungal activity,
123

1928
Med Chem Res (2012) 21:1921–1928
and molecular modeling studies of new inverted oxime ethers of
oxiconazole. J Med Chem 45:4903–4912
Szentesi P, Collet C, Sarkozi S, Szegedi C, Jona I, Jacquemond V,
Kovacs L, Csernoch L (2001) Effects of dantrolene on steps of
excitation-contraction coupling in mammalian skeletal muscle
fibers. J Gen Physiol 118:355–376
´
Rufian-Henaresa JA, Morales FJ (2008) Microtiter plate-based assay
for screening antimicrobial activity of melanoidins against
E. coli and S. aureus. Food Chem 11:1069–1074
¨
Thomae AV, Koch T, Panse C, Wunderli-Allenspach H, Kramer SD
Sah PPT, Peoples SA (1953) The antitubercular activity of
D-galacturonic acid isonicotinyl hydrazone. J Am Chem Soc
75:2512–2513
(2007) Comparing the lipid membrane affinity and permeation of
drug-like acids: the intriguing effects of cholesterol and charged
lipids. Pharm Res 24:1457–1472
´
Sanchez C, Neves AR, Cavalheiro J, dos Santos MM, Garcıa-
´
Turner WW, Rodrigues MJ (1996) Recent advances in the medicinal
chemistry of antifungal agents. Curr Pharm Design 2:209–224
Yousefi R, Ardestani SK, Saboury AA, Kariminia A, Zeinali M,
Amani M (2005) Investigation on the surface hydrophobicity and
aggregation kinetics of human calprotectin in the presence of
calcium. J Biochem Mol Biol 38:407–413
´
´
Quintans N, Lopez P, Santos H (2008) Contribution of citrate
metabolism to the growth of Lactococcus lactis CRL264 at low
pH. Appl Environ Microbiol 74:1136–1144
Soltani Rad MN, Khalafi-Nezhad A, Behrouz S (2010) Synthesis of
some novel hydrazono acyclic nucleoside analogues. Beilstein J
Org Chem 6:1–8
Szarvasi E, Fontaine L, Betbeder-Matibet A (1973) Antimicrobials:
new nitrofuran derivatives. J Med Chem 16:281–287
123