CrystEngComm
DOI: 10.1039/C7CE02136D
Cytotoxicity assay
In vitro cytotoxicity screening of hydrazones 1aꢀ1c, 2aꢀ2c,
aꢀ3c, 4aꢀ4c and their parent hydrazides 1ꢀ4 was performed
Conflicts of interest
3
There are no conflicts of interest to declare
.
on human acute monocytic leukemia (THPꢀ1, ATCC TIBꢀ202)
and hepatocellular carcinoma (HepG2, ATCC HBꢀ8065) cells
Acknowledgments
5
8
MTS assay was used to ascertain cell viability. HepG2 cells
were grown in DMEM/F12 medium while RPMI 1640
medium was used for THPꢀ1. Media were supplemented with
This work has been fully supported by Croatian Science
Foundation under the project (IPꢀ2016ꢀ06ꢀ4221).
1
0% heat inactivated fetal bovine serum and 1%
4
Notes and references
antibiotic/antimycotic solution. A total of 5·10 cells were
incubated at 37°C in 5% CO atmosphere and 90% relative
a
2
University of Zagreb, Faculty of Science, Department of Chemistry,
humidity overnight in microtiter plates with appropriate cell
medium containing hydrazone or hydrazide, previously
dissolved in DMSO. Hydrazone or hydrazide concentration
range of 0.80–100 ꢁmol/L was used in the case of HepG2 and
Horvatovac, 102a, 10000 Zagreb, Croatia, Fax: ++ 385ꢀ1ꢀ4606341; Tel:
++385ꢀ1ꢀ4606353; Eꢀmail: visnja.vrdoljak@chem.pmf.hr
University of Zagreb, Faculty of Textile Technology, Division of Applied
b
Chemistry, Prilaz baruna Filipovića 28a, 10000 Zagreb, Croatia
c
University of Zagreb, School of Medicine, Department of Chemistry and
–
4
1
0 –100 ꢁmol/L for THPꢀ1. Wells with medium only (blank),
Biochemistry, Šalata 3, 10 000 Zagreb
wells with cells and medium as well as wells with cells,
medium and 1% DMSO served as controls. Wells with cells
†
Electronic Supplementary Information (ESI) available: (1) schemes of
–
6
hydrazones, (2) yields and analytical data, (3) PXRD patterns, (3)
Chemometric Data Analysis, (4) figures for compounds, (5) tables of
selected bond distances and angles and of hydrogen bonds parameters and
and staurosporine applied in concentration range 10 –100
mol/L served as control of MTS test. After incubation, MTS
ꢁ
1
13
reagent (CellTiter® Aqueous One Solution Cell Proliferation
Assay; Promega) was added to each well and incubation was
continued for 1 (HepꢀG2) or 6 hours (THPꢀ1). The absorbance
of the formed purple formazan was measured at 490 nm using
a microplate spectrophotometer Perkin Elmer Victorꢀ2. The
average IC50 value (the concentration required for 50%
decrease in cell viability) was determined from the cell
viability versus concentration curve with GraphPad Prism
software. All experiments were performed in duplicate.
(
7) H and C NMR spectral data. Crystallographic data sets for the
structures 2b·MeOH, 2c·MeOH, 3b, 4a, 4b·EtOH, 4b·H O, 4b·MeOH
and 4c are available through the Cambridge Structural Data base with
deposition numbers CCDC 1588275ꢀ1588282. See DOI:
0.1039/b000000x/
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4
Antibacterial activity assay
Hydrazones 1aꢀ1c, 2aꢀ2c, 3aꢀ3c, 4aꢀ4c and parent hydrazides
1
ꢀ4 were tested for their in vitro antibacterial activity by the
5
broth microdilution method against two Gramꢀpositive
Staphylococcus aureus (ATCC 13709) and Enterococcus
faecalis (ATCC 29212), and two Gramꢀnegative Escherichia
coli (ECM 1556) and Moraxella catarrhalis (ATCC 23246)
bacterial strains according to guidelines of the Clinical and
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Tn10). E. faecalis and M. catarrhalis were grown on
Columbia agar plates with 5% sheep blood while Muellerꢀ
Hinton agar plates were used for S. aureus and E. coli. After
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2 h incubation at 37°C, bacterial inoculum was prepared by
8
direct colony suspension method to a density of 10 CFU/mL,
which corresponds to a 0.5 McFarland standard. All strains
were tested in MuellerꢀHinton II Broth (cationꢀadjusted)
culture medium. Tested compound was dissolved in DMSO
and applied, in the concentration range 0.5–128 ꢁg/mL, to
1
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4
wells inoculated with bacterial suspension (5·10 CFU/well).
1
1
Wells with medium only and wells with medium and bacterial
inoculum were also included for sterility and growth control,
respectively. For antibacterial susceptibility validation
bacteria were also treated with control antibiotic azithromycin
1
1
1
(
0.03–16 ꢁg/mL). Results expressed as minimum inhibitory
concentration (MIC) values were determined by visual
inspection after 22 h incubation at 37°C in ambient air. All
experiments were performed in duplicate.
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