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Synthesis, Antituberculotic And Cytotoxic Properties Of New Hydrazone
Derivatives Bearing Pyrimidine-Alkylsulfanyl Moiety
Leyla Yurttaş1,*, Zafer Asım Kaplancıklı1, Zerrin Cantürk2, Hülya Karaca Gencer2
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, 26470,
Eskisehir, Turkey
b
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Anadolu University, 26470
Eskisehir, Turkey
Email: lyurttas@anadolu.edu.tr
Supplemental Materials
Biological Activity Evaluation
The title compounds (3a-3m) were screened for their antituberculosis activity by MABA
method and MICs of the compounds were determined by Broth Microdilution method. Rifampicin
(RIF) was used as standard drug and results are presented in Table S 1. The MICs were found to
be 100 and 400 µg/mL. Among tested compounds, 3a, 3b, 3c, 3e, 3f, 3g exhibited moderate
activity with a MIC value of 100 µg/mL whereas Rifampicin have a MIC value of 25 µg/mL. The
physico-chemical properties was reported one of the factor influencing the antimycobacterial
activity.39 When MIC and the lipophilicity (calculated log P) were compared, there is no clear
dependence; however, chlorine including molecules 3e, 3f and 3g possess the lowest MIC values
with the highest log P values among the other compounds.
Mycobacterium tuberculosis is hard
to treat organism being equipped with lipid and waxed cell wall rendering most of the antibiotics
unable to penetrate.38 In this study, N’-(arylidene)-4-[(pyrimidine-2-yl)thio]butanohydrazide
derivatives (3a-3m) also did not show a significant antituberculotic effect.
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In vitro cytotoxicity of the title compounds was assessed by MTT bioassay in one cancer
cell line (human alveolar adenocarcinoma cells-A549) and one non-malignant cell line (murine
fibroblasts cells-NIH/3T3), at 24h of drug administration. Cisplatin was used as a positive control.
After treatment of A549 cell line for 24 h with compounds 3a-3m in the range of concentration
3.9-500 µg/mL. The 50 % inhibitory concentrations (IC50) of the compounds were calculated and
listed in Table S 2. Compounds 3c and 3l failed to inhibite A549 cell line at the highest tested
concentration, therefore IC50 could not calculated for these compounds. Compound 3m including
p-nitro substitution on phenyl moiety exhibited the highest cytotoxicity against A549 cells with a
IC50 value of 53.25 µg/mL, whereas cisplatin possess the IC50 value of 40.3 µg/mL. Compound
3m also effected NIH/3T3 healty cells with a value of 158.34 µg/mL which is greater than 53.23
µg/mL. Compound 3m can be considered to show selective toxicity and this compound can be
evaluated for further studies. In this regard, compounds 3b and 3f can be evaluate to exhibite
selectivity that they showed higher cytotoxicity against tumor cells than normal cells. The other
compounds 3a, 3d, 3e, 3f, 3g, 3h, 3j and 3k exhibited moderate activity against both of the cell
lines. In general, all of the compounds showed weak cytotoxicity (IC50 > 158 µg/mL) against
NIH/3T3 cell line in high concentrations, consequently it may thought compounds 3a, 3b, 3c, 3e,
3f and 3g possess antitubercular activity with lower cytotoxicity.
Antituberculosis Activity
MABA (Microplate Alamar Blue Assay)
M. tuberculosis H37RV (ATCC 27294), was provided from ATCC cell bank. The cells
were grown in ATCC® Medium 1395: Middlebrook 7H9 broth with ADC enrichment at a
temperature of 37oC for 10 day. The turbidity of the cultures was adjusted to McFarland standard
no. 1. The following concentrations of chemicals were used: 0.97-500 µg/mL concentrations for
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Rifampicin (Sigma, R3501). All black, clear-bottomed, 96-well plates (Corning) were incubated
at 37°C in 5% CO2 for 7 days. On day 7 of the incubation, freshly prepared 1:1 mixture of Alamar
Blue reagent (1:10 dilution, Invitrogen, 156703SA)) and 10% Tween 80 was added to one well
among the positive controls. The plates were further incubated at 37 °C for 24 h. If the contents of
the well turned pink, the reagent mixture was added to all the wells of the microplate. The results
were expressed as MIC (at which all bacteria were inhibited).40
Cytotoxicity
All the final compounds synthesized were further examined for cytotoxicity against
NIH/3T3 mouse embryonic fibroblasts cell line (NIH/3T3) and adenocarcinomic human alveolar
basal epithelial cell line (A549) by cell proliferation analysis using standard 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay previously described in the
literature with small modifications [40-42]. After 24 h of preincubation, the tested compounds (3ah) were added to give final concentration in the range of 3.9-500 µg/mL and the cells were
incubated for 24 h. At the end of this period, MTT was added to a final concentration of 0.5 mg/mL
and the cells were incubated for 4 h at 37 oC. Purple formazan was occurred at the end of the
process which is the reduction product of MTT agent by the mitochondrial dehydrogenase enzyme
of intact cells. Formazan crystals were dissolved in 100 µL DMSO and the absorbance was read
by ELISA reader (OD570nm). The percentage of viable cells was calculated based on the medium
control. Every concentration was repeated in three wells and IC50 values (µM) were defined as the
compound concentrations that reduced absorbance to 50 % of control values.41-43
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Table S 1: Antitubercular activities against M. tuberculosis H37RV (ATCC 27294) and log Pa
measurements of compounds 3a-3m
a
Compounds
MIC (µg/mL)
Log P
3a
100
2.89
3b
100
2.89
3c
100
2.92
3d
400
2.94
3e
100
3.52
3f
100
3.54
3g
100
3.56
3h
400
3.0
3i
400
3.02
3j
400
3.05
3k
400
1.97
3l
400
1.99
3m
400
2.01
Rifampicin
25
2.62
Calculated by http://www.molinspiration.com/.
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Table S 2. IC50 valuesa (µg/mL) for compounds 3a-3m for 24 h against NIH/3T3 normal cells and
A549 cancer cells
a
Compounds
NIH/3T3
A549
3a
220.60
437.6
3b
283.06
273.75
3c
246.46
>500
3d
253.07
328.5
3e
278.93
397.05
3f
347.37
299.75
3g
229.0
234.25
3h
262.98
336.89
3i
237.09
>500
3j
248.33
335.25
3k
199.75
464.8
3l
355.63
399.39
3m
158.34
53.25
Cisplatin
238.18
40.3
Cytotoxicity of the compounds. Incubation for 24 hours. IC50 is the drug concentration required to inhibit 50 % of the
cell growth.
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Figure S 1: 1H NMR of 3a
Figure S 2: 13C NMR of 3a
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Figure S 3: 1H NMR of 3m
Figure S 4: 13C NMR of 3m