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PHOSPHORUS, SULFUR, AND SILICON AND THE RELATED ELEMENTS
3
Figure 1. Relative ergosterol level (REL) of C. albicans (ATCC 24433) after treatment with compound 5b, fluconazole and ketoconazole.
aromatic protons (C4–H and C5–H) of imidazole were and hence inhibition of ergosterol biosynthesis causes the end
observed at 7.72 and 6.90–7.15 ppm as a doublet, respect- of life functions. Hence, in order to understand the effect of
ively. Aromatic protons of 1,4-disubstitutedphenyl appeared the most active compound 5b on ergosterol biosynthesis, an in
at 7.15–7.72 as two different doublets. The other peaks
belonging to aromatic and aliphatic protons of variable side
chains were recorded at estimated areas. Elemental analysis
vitro assay, based on quantification of ergosterol level in fungi
was performed.
An LCMSMS based method was used for quantitative deter-
for C, H, N, and S elements were satisfactory within calcu- mination of ergosterol level of C. albicans. Before the analytical
lated values of the compounds. No more than 0.4% devi- measurements, total intracellular sterols were extracted from
ation between calculated and found results were determined, fungi as reported by Breivik and Owades.[24] Ergosterol stand-
which indicated target compounds were synthesized purely.
ard (Product No: 45480, Sigma-Aldrich, Germany) was used for
quantification of ergostrerol in both inhibitor-free (negative
control) and inhibitor including samples. The most active com-
pound 5b and reference drugs were used at 6.25, 12.5, and
25mg/mL concentrations. Ergosterol quantity in negative con-
trol samples was regarded as 100%. All concentrations were
analyzed in quadruplicate, and the results were expressed as
mean standard deviation (SD) (Figure 1).
Ergosterol quantification studies revealed that compound
5b and reference agents significantly decreased the level of
ergosterol at all tested concentrations. A concentration
dependant decrease in the ergosterol level was seen for all
tested agents. Thus, it can be clearly suggested that compounds
5e have a role in the ergosterol biosynthesis pathway.
Antifungal activity
Antifungal activity was investigated by finding minimum
inhibitory concentration (MIC) of the synthesized compounds
(5a–5i) against C. glabrata (ATCC 90030), C. krusei (ATCC
6258), C. parapsilosis (ATCC 22019) and C. albicans (ATCC
24433) comparing with fluconazole and ketoconazole as stand-
ard drugs. The MIC value of the compounds and control drugs
The MIC values were generally within the range of
12.5–25 lg/mL. Most of the compounds showed significant
antifungal activity against C. albicans (ATCC 24433) and C.
krusei (ATCC 6258). In consideration of synthesized com-
pounds’ MIC values with standard drug, only the compound
5b indicated similar antifungal activity to ketoconazole and
fluconazole against C. parapsilosis (ATCC 22019) and eval-
uated as the most active derivative in the series. Observation of
very similar antifungal activity displayed by the compounds
(5a–5i) indicates that there is no important difference between
contributions of various dithiocarbamoyl moieties to antifun-
gal activity and makes consideration of the SARs very difficult.
Hence, it can be assumed that antifungal activity of the com-
pounds (5a–5i) is related to their general structural character-
istics provided by both dithiocarbamoyl moieties and N-[4-
((1H-imidazol-1-yl)methyl)phenyl]acetamide substructure.
ADME prediction
The absorption, distribution, metabolism, and excretion
(ADME) studies are critical in modern drug discovery. Early
characterization of these properties is essential for successful
drug discovery studies to eliminate compounds with
unacceptable pharmacokinetic properties. In silico ADME
and toxicological screening systems can provide an oppor-
tunity to predict performance in an in-vivo setting. Thus,
predictions of ADME properties of the all synthesized com-
pounds (5a–5i) were performed by QikProp 4.8 software.[25]
This software evaluates the ADME profiles of drug candi-
dates according to the Lipinski’s rule of five[26] and Jorgensen’s
rule of three.[27] The data of the software such as molecular
weight, log P, polar surface area (PSA), number of hydrogen
Inhibition of ergosterol biosynthesis
Most antifungal therapies target the ergosterol biosynthesis donors, number of hydrogen acceptors, number of rotatable
pathway. It is the main sterol of fungi cell membranes and is bonds and volume, are presented in Table S2 (Supplemental
necessary for fluidity, permeability and protein function[23] Materials) along with the violations of rule of three and five.