Synthesis, antimicrobial evaluation and theoretical prediction of NMR chemical shifts of thiazole and selenazole derivatives with high antifungal activity against Candida spp.

Article abstract of DOI:10.1016/j.molstruc.2015.12.033

Synthesis and investigation of antimicrobial activities of novel thiazoles and selenazoles is presented. Their structures were determined using NMR, FAB(+)-MS, HRMS and elemental analyses. To support the experiment, theoretical calculations of the ¹H NMR shifts were carried out for representative systems within the DFT B3LYP/6-311++G??approximation which additionally confirmed the structure of investigated compounds. Among the derivatives, compounds 4b, 4h, 4j and 4l had very strong activity against reference strains of Candida albicans ATCC and Candida parapsilosis ATCC 22019 with MIC = 0.49-7.81 μg/ml. In the case of compounds 4b, 4c, 4h - 4j and 4l, the activity was very strong against of Candida spp. isolated from clinical materials, i.e. C. albicans, Candida krusei, Candida inconspicua, Candida famata, Candida lusitaniae, Candida sake, C. parapsilosis and Candida dubliniensis with MIC = 0.24-15.62 μg/ml. The activity of several of these was similar to the activity of commonly used antifungal agent fluconazole. Additionally, compounds 4m - 4s were found to be active against Gram-positive bacteria, both pathogenic staphylococci Staphylococcus aureus ATCC with MIC = 31.25-125 μg/ml and opportunistic bacteria, such as Staphylococcus epidermidis ATCC 12228 and Micrococcus luteus ATCC 10240 with MIC = 7.81-31.25 μg/ml.

Full text of DOI:10.1016/j.molstruc.2015.12.033

Journal of Molecular Structure 1108 (2016) 427e437
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Synthesis, antimicrobial evaluation and theoretical prediction of NMR
chemical shifts of thiazole and selenazole derivatives with high
antifungal activity against Candida spp.
,
Krzysztof Z. Ła˛czkowski a *, Katarzyna Motylewska a,
Angelika Baranowska-Ła˛czkowska b, Anna Biernasiuk c, Konrad Misiura a, Anna Malm c,
d
ꢀ
Berta Fernandez
a Department of Chemical Technology and Pharmaceuticals, Faculty of Pharmacy, Collegium Medicum, Nicolaus Copernicus University, Jurasza 2, 85-089
Bydgoszcz, Poland
b Institute of Physics, Kazimierz Wielki University, Plac Weyssenhoffa 11, 85-072 Bydgoszcz, Poland
c
ꢀ
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University, Chodzki 1, 20-093 Lublin, Poland
d Department of Physical Chemistry and Center for Research in Biological Chemistry and Molecular Materials (CIQUS), University of Santiago de
Compostela, 15782 Santiago de Compostela, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthesis and investigation of antimicrobial activities of novel thiazoles and selenazoles is presented.
Their structures were determined using NMR, FAB(þ)-MS, HRMS and elemental analyses. To support the
experiment, theoretical calculations of the 1H NMR shifts were carried out for representative systems
within the DFT B3LYP/6-311þþG** approximation which additionally confirmed the structure of
investigated compounds. Among the derivatives, compounds 4b, 4h, 4j and 4l had very strong activity
against reference strains of Candida albicans ATCC and Candida parapsilosis ATCC 22019 with MIC ¼ 0.49
Received 10 October 2015
Received in revised form
5 December 2015
Accepted 14 December 2015
Available online 18 December 2015
e7.81 mg/ml. In the case of compounds 4b, 4c, 4h e 4j and 4l, the activity was very strong against of
Keywords:
Thiazole
Selenazole
Antimicrobial drugs
DFT calculation
NMR chemical shift
Candida spp. isolated from clinical materials, i.e. C. albicans, Candida krusei, Candida inconspicua, Candida
famata, Candida lusitaniae, Candida sake, C. parapsilosis and Candida dubliniensis with MIC ¼ 0.24
e15.62 mg/ml. The activity of several of these was similar to the activity of commonly used antifungal
agent fluconazole. Additionally, compounds 4m e 4s were found to be active against Gram-positive
bacteria, both pathogenic staphylococci Staphylococcus aureus ATCC with MIC ¼ 31.25e125 g/ml and
opportunistic bacteria, such as Staphylococcus epidermidis ATCC 12228 and Micrococcus luteus ATCC
10240 with MIC ¼ 7.81e31.25 g/ml.
m
m
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
substantial, and the majority of these mycoses-related deaths were
associated with Candida spp., occurring naturally in the human
body, Aspergillus spp., and Cryptococcus spp. The most important
factors that increase the risk of infection are cutaneous barrier
disruption, metabolic dysfunction, defects in cell-mediated im-
munity, and exposures to a variety of endemic fungal pathogens.
Finally widespread and irresponsible use of broad-spectrum anti-
biotics, immunosuppressive agents, anticancer, and anti-AIDS
drugs, leads to the multi-drug resistant microorganisms, which
increased fungal infections significantly [1,4,5]. A possible solution
to the observed drug-resistance of microorganisms is the respon-
sible use of already existing drugs, and the search for innovative
drugs possessing a different mechanism of action. In the present
project we have turned our attention to thiazole and selenazole
Fungal diseases are a growing global public health problem [1].
Although fungal infection can happen to anyone, the most sus-
ceptible are people with immunecompromised and those hospi-
talized with tumours, organ transplantation, HIV/AIDS and with
other serious underlying diseases [2]. Fungal infections in such
people can be very aggressive, and can quickly attack other organs
leading in a short time to death [3].
The morbidity and mortality associated with these infections are
* Corresponding author.
E-mail address: krzysztof.laczkowski@cm.umk.pl (K.Z. Ła˛czkowski).
http://dx.doi.org/10.1016/j.molstruc.2015.12.033
0022-2860/© 2015 Elsevier B.V. All rights reserved.

428
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
derivatives.
corresponding thiazoles 4a-4l and selenazoles 4m-4u with good
The usefulness of thiazole heterocycle as an interesting building
yield (18e94%) and with high chemical purity. The reaction
pathway has been summarized in Scheme 1.
block can be seen in many natural and synthetic compounds. Thi-
azoles are easily metabolized by known biochemical reactions, and
are non-cancerogenic in nature [6]. Their varied biological activ-
ities, such as antibacterial [7e9], antifungal [10,11] and antitumor
[12,13] are widely known. On the other hand, the introduction of
selenium element into organic compounds always influences their
chemical properties and in turn changes their biological activity
[14].
In the recent years, organic selenium derivatives have been re-
ported to have wide and interesting biological properties such as
inhibition of cancer cell proliferation [15e18], protein kinase acti-
vators [19], inactivation of free radicals [20], and antioxidant
[21e24] or antibacterial activity [25].
All obtained thiazoles and selenazoles were purified on silica gel
column chromatography, and in all cases we obtained only pure,
most stable(E)-isomers [30,37]. All compounds were fully charac-
terized spectroscopically using 1H and 13C NMR, EI(þ)-MS, HRMS,
and elemental analyses. The mass spectra of all compounds are
fully consistent with assigned structures. The 1H NMR spectrum of
2 and 3 presents typical three proton signals of NH2 and NH groups
at 7.41, 7.99, 11.03 and 7.90, 8.40, 11.30 ppm, respectively. These
three signals are due to the exchange of H between the terminal
NH2 group and sulphur atom. 1H NMR spectra of azole derivatives
showed singlet at
proton and singlet at
d
(6.28e8.06) due to thiazole-5H/selenazole-5H
(11.17e12.00) indicating the presence of
d
An important example of selenium containing derivatives are
glutathione peroxidase, a key enzyme in antioxidant defence [26],
and selenoproteins occurring widely in nature [27]. The 1,3-
selenazoles show very unique chemical and biological properties
which make them one of the most widely studied selenium de-
rivatives [28]. Toxic effects of selenium derivatives depend strongly
on their chemical form and concentration. For example, the geno-
toxic and cytotoxic effects of organoselenium derivatives in human
leukocyte cells can be associated to the pro-oxidant activity [29].
Given the similarity of the chemical properties between sulfur
and selenium and continuing our previous investigation on the
effect of systematic structural modifications of azole ring on the
antimicrobial activity [30e35], we decided to prepare twelve novel
2,4-disubstituted 1,3-thiazole and eight novel 2,4-disubstituted
1,3-selenazoles. Based on our earlier research study we also inser-
ted fluorine-, chlorine-, bromine-, methoxy-, methyl-, tri-
fluoromethyl-, difluoro-, nitro-, and cyano-groups in the para-
position in the phenyl ring. We also received several derivatives
with methyl-, -COOEt, and eCH2COOEt groups attached directly to
the thiazole ring. These substituents are useful to modulate elec-
tronic effects, and may influence the hydrophilic and hydrophobic
properties of the molecules.
hydrazide NH proton, which confirms the conversion of substrates
into the expected products. The characteristic proton of CH]N
group occurs in the spectra around the 7.30 ppm. The elemental
analyses values are in good agreement with the calculated values
confirming the formation of analytically pure products. All re-
actions were repeated at least two times and are fully reproducible.
2.2. Biological Evaluation
2.2.1. Antifungal activity
According to the results presented in Table 1, on the basis of
minimal inhibitory concentration values (MIC) obtained by the
broth microdilution method, it was shown that compounds 4b, 4h,
4j and 4l had very strong activity against the both reference strains
of Candida albicans ATCC and Candida parapsilosis ATCC 22019 with
MIC ¼ 0.49e7.81
m
g/ml and MFC ¼ 3.91 e > 1000
mg/ml. Their
antimicrobial activity was similar to the activity of reference anti-
fungal compound efluconazole. These compounds indicated
fungistatic or fungicidal effect to reference strains of yeasts. Com-
pounds 4c and 4i showed slightly lower activity against these
strains (MIC ¼ 3.91e62.5
m
g/ml, MFC ¼ 31.25 e >1000
mg/ml). In
turn, the other tested compounds, 4d e 4f and 4m e 4t, exhibited
strong, good or moderate activity against reference Candida spp.
Next, these compounds were evaluated for their antimicrobial
activity against a panel of reference strains of 27 microorganisms,
including Gram-positive bacteria, Gram-negative bacteria, and
fungi belonging to yeasts. The microorganisms came from Amer-
ican Type Culture Collection (ATCC), routinely used for the evalu-
ation of antimicrobials. We have also performed Density Functional
Theory (DFT) calculation of NMR chemical shifts of selected thia-
zole derivatives.
strains (MIC ¼ 15.62e250
m
g/ml, MFC ¼ 15.62 e >1000
mg/ml).
Compounds 4c, 4d and 4f showed fungistatic or fungicidal effect to
reference Candida spp., while compounds 4e, 4i and 4m e 4t e
fungicidal activity to these yeasts (MFC/MIC ¼ 1e2).
The tested compounds showed also high activity against some
strains of Candida spp. isolated from clinical materials, i.e. C. albi-
cans, Candida krusei, Candida inconspicua, Candida famata, Candida
lusitaniae, Candida sake, C. parapsilosis and Candida dubliniensis. In
the case of compounds 4b, 4c, 4h e 4j and 4l, the activity against
these isolates was very strong e similar or higher than the activity
of fluconazole. The minimal inhibitory concentration values ranged
2. Results and discussion
2.1. Chemistry
from 0.24 to 15.62
compounds showed fungistatic or fungicidal effect to clinical
strains of Candida spp. (Table 1).
m
g/ml and MFC ¼ 0.98 e > 1000
mg/ml. These
Synthesis of desired azole compounds consists of two steps. In
the first step cyclohexanecarbaldehyde thiosemicarbazone 2 was
synthesized through reaction of cyclohexanecarbaldehyde (1) with
thiosemicarbazide in absolute ethanol in the presence of catalytic
amount of glacial acetic acid and under reflux [36]. The cyclo-
hexanecarbaldehyde selenosemicarbazone 3 was synthesized us-
ing potassium selenocyanate and hydrazine hydrate in the
presence of hydrochloric acid in ethanol and under reflux. The
products 2 and 3 were obtained with 84% and 41% yield, respec-
tively. The selenosemicarbazone 3 is hygroscopic material, the store
and all operations should be carried out under nitrogen atmo-
sphere. In the next step Hantzsch cyclization reaction of thio-
semicarbazone (2), and selenosemicarbazone (3) with appropriate
para-substituted bromoacetophenones, ethyl 4-chloroacetoacetate,
ethyl 2-chloroacetoacetate, and chloroacetone afforded the
Among the remaining newly synthesized compounds, com-
pounds 4s and 4t exhibited slightly lower fungicidal activity against
clinical Candida spp. Minimum concentrations, which inhibited the
growth of yeasts ranged for these compounds from 7.81 to 62.5
ml, and MFC ¼ 15.62e250 g/ml (MFC/MIC ¼ 1e2). Compounds 4d
e 4f indicated similar activity, but with fungistatic or fungicidal
effect against the same isolates (MIC 7.81e125 g/ml,
MFC ¼ 31.25 e >1000 g/ml. In turn, 4m e 4r showed good or
moderate fungicidal activity with MIC 62.5e500 g/ml,
g/ml (MFC/MIC ¼ 1e2) against these yeasts
mg/
m
¼
m
m
¼
m
MFC ¼ 62.5e1000
m
(Table 1).
Moreover, the tested compounds exhibited much lower activity
against two isolates of non-albicans Candida spp., i.e. Candida

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
429
Scheme 1. Synthesis of the target compounds 4a-u.
pulcherrima and Candida lambica with MIC values from 125 to
g/ml (Table 2.). Our results
indicated also that the growth of both the reference and clinical
yeasts strains belonging to Candida spp. was not inhibited by some
of the newly synthesized compounds, i.e. 4a, 4g, 4k and 4u.
attached directly at 4th to the thiazole ring showed very strong
activity. However, replacing the hydrogen atom in compound (4l) at
5th position in the thiazole ring with -COOEt substituent results in
total loss of activity (compound 4k). The replacement of the sulfur
atom in compound (4k) with a selenium atom does not lead to
substantial increase of activity (compound 4u). As can be easily
seen selenazole derivatives (4m-4u) exhibit much lower activity
against tested fungi than thiazole derivatives, but they have a good
antibacterial activity missing in the case of thiazole derivatives. The
study shows that the type of the substituents plays a significant role
in obtaining antimicrobial activity.
ꢀ1000
mg/ml and MFC ¼ 125 e >1000
m
2.2.2. Antibacterial activity
On the basis of minimal inhibitory concentration values ob-
tained by the broth microdilution method, it was shown that
investigated compounds showed some bioactivity. Among them,
the widest spectrum of antibacterial activity possessed compounds
4m e 4s. These compounds were found to be active against Gram-
positive bacteria, both pathogenic staphylococci Staphylococcus
2.3. Calculations
aureus ATCC with MIC ¼ 31.25e125
teria, such as Staphylococcus epidermidis ATCC 12228 and Micro-
coccus luteus ATCC 10240 with MIC ¼ 7.81e31.25 g/ml. The MBC of
these compounds was 31.25e500 g/ml. The compounds showed
mainly good activity with bactericidal effect against these bacteria.
The highest bactericidal or bacteriostatic activity within the
investigated compounds against S. epidermidis ATCC 12228 and
mg/ml and opportunistic bac-
The 1H NMR calculation have been carried out for three thiazole
derivatives (4c, 4f and 4h) using the DFT/B3LYP approximation
(Table 3). The choice of the basis set was made after test calculation
carried out for the set of selected small molecules (methane, ben-
zene, thiazole and 2-aminothiazole). Results of these tests are given
in the Supplementary material. Their analysis reveals that the
B3LYP values are in general close to the experimental results, even
in the case of the very small 6e31þþG** and 6e311þþG** basis
sets. If the two hydrogens of the amino group in the aminothiazole
e i.e. those being the most sensitive to the experimental conditions
m
m
M. luteus ATCC 10240 with MIC ¼ 7.81
mg/ml was shown by com-
pound 4s (Table 2).
Moreover, compounds 4m e 4s showed lower activity against
Bacillus spp., which form spores under adverse environmental
conditions. The minimum concentrations which inhibited the
growth of Bacillus subtilis ATCC 6633 and Bacillus cereus ATCC 10876
e
are excluded from the analysis, both 6e31þþG** and
6e311þþG** basis sets give the same RMSE value, equal to 0.13.
Since the latter set is of a triple-zeta quality and thus should yield
higher quality values, we have chosen the 6-311þþG** basis set for
calculation of chemical shifts in real drugs.
were 250e500
m
g/ml and MBC ¼ 500 e ꢀ 1000
mg/ml (Table 2).
The remaining compounds 4f, 4h, 4l and 4u exhibited some
activity or no activity to reference strains of S. epidermidis, M. luteus
and Bacillus spp. with MIC ¼ 250e1000
m
g/ml and MBC ¼ 500 e ꢀ
The lowest energy optimized structures of the new thiazole
derivatives 4c, 4f and 4h are shown in Fig. 1. The DFT(B3LYP)/6-
311þþG** 1H NMR chemical shifts confirmed the structure of the
studied systems, since the agreement between the theoretical and
the experimental results is good. Chemical shifts obtained in
deuterated chloroform are in better agreement with the gas phase
theoretical values than those measured in the polar solvent DMSO,
which can be explained by the fact that DMSO interacts stronger
with the solute. This observation is consistent with the work [38],
in which the gas phase 1H NMR shifts are closer to the experimental
values recorded in CDCl3 than those measured in DMSO. Particu-
larly large differences between theoretical and experimental values
are observed in the case of hydrogens 35, 34 and 34 for molecules
4c, 4f and 4h, respectively, and can be attributed to external factors
1000 g/ml (Table 2). The results of our study indicated, that the
m
newly synthesized compounds had no inhibitory effect on the
growth of reference strains of Gram-negative bacteria. According to
the data presented in Table 2, the compounds 4a e 4e, 4g, 4i e 4k
and 4t had no influence also on the growth of Gram-positive
bacteria.
In the case of antifungal activity, compounds (4b, 4c, 4h, 4i)
containing eF, eOCH3, eCH3 at 4th position and 2,4-di-F sub-
stituents in the phenyl ring showed the highest activity against
reference strains. It is interesting that such substituents as -Cl and
-Br in (4e, 4f) showed a lower activity compared to the eF sub-
stituent. Derivatives (4g, 4k) with eNO2 and eCF3 groups did not
show activity. Thiazoles (4j, 4l) with eCH2COOEt and eCH3 groups

430
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
Table 1
The antifungal activity data expressed as MIC [mg/ml] and, in parentheses, as MFC [mg/ml] for compounds 4a-4u.
Sp.
MIC (MFC) [
m
g/ml] of the tested compound
4c
4a
4b
4d
4e
4f
4g
4h
7.81(62.5)
1.95(3.91)
3.91(62.5)
3.91(31.25)
3.91(31.25)
1.95(31.25)
3.91(31.25)
0.98(7.81)
4i
62.5(250)
7.81(31.25)
15.62(62.5)
1.95(15.62)
15.62(125)
7.81(31.25)
0.98(3.91)
A
B
C
D
E
F
G
H
I
e
e
e
e
e
e
e
e
e
e
e
e
e
7.81(>1000)
1.95(15.62)
7.81(>1000)
0.98(0.98)
1.95(500)
1.95(>1000)
0.98(1.95)
0.49(62.5)
0.24(3.91)
0.98(1.95)
0.98(3.91)
15.62(>1000)
3.91(>1000)
15.62(>1000)
1.95(250)
3.91(500)
3.91(>1000)
1.95(62.5)
1.95(125)
3.91(125)
3.91(62.5)
3.91(62.5)
125(>1000)
125(500)
15.62(31.25)
31.25(125)
15.62(250)
31.25(125)
31.25(>1000)
15.62(250)
7.81(250)
15.62(250)
31.25(250)
62.5(500)
>1000(>1000)
>1000(>1000)
62.5(500)
e
e
e
e
e
e
e
e
e
e
e
e
e
31.25(250)
62.5(1000)
31.25(>1000)
125(>1000)
31.25(250)
15.62(500)
15.62(500)
7.81(250)
15.62(1000)
31.25(1000)
>1000(>1000)
>1000(>1000)
31.25(250)
62.5(250)
31.25(250)
125(500)
62.5(>1000)
31.25(250)
31.25(500)
31.25(125)
62.5(500)
1.95(7.81)
3.91(62.5)
7.81(31.25)
1.95(31.25)
0.98(31.25)
500(>1000)
500(>1000)
J
K
L
0.98(31.25)
1.95(31.25)
>1000(>1000)
>1000(>1000)
62.5(250)
>1000(>1000)
>1000(>1000)
>1000(>1000)
1000(>1000)
>1000(>1000)
>1000(>1000)
M
C. albicans ATCC 2091 (denoted as A), C. albicans ATCC 10231 (B), C. parapsilosis ATCC 22019 (C), C. albicans (D), C. parapsilosis (E), C. sake (F), C. dubliniensis (G), C. famata (H),
C. lusitaniae (I), C. krusei (J), C. inconspicua (K), C. pulcherrima (L), C. lambica (M). The standard antibiotic, fluconazole (FLU) was used as positive controls.
like, for example, the temperature, the concentration of analyte in
the sample, or the type of solvent.
4.1.1. (E)-2-(Cyclohexylmethylene)hydrazinecarbothioamide (2)
Thiosemicarbazide (1.82 g, 0.02 mol) was added to a stirred
solution of cyclohexanecarbaldehyde (1) (2.24 g, 0.02 mol) in ab-
solute ethyl alcohol (30 ml) and then (1 ml) of acetic acid was
added. The reaction mixture was stirred under reflux for 20 h. After
that, the reaction mixture was poured on the crushed ice and kept
aside. The product was filtered off and subsequently washed with
3. Conclusion
In summary, we have developed an efficient and economic
method for the synthesis of 2,4-disubstituted 1,3-thiazoles and 2,4-
disubstituted 1,3-selenazoles. Our results indicate that some newly
synthesized compounds showed high antifungal (4b, 4c, 4h-4j and
4l) and antibacterial (4m-4s) activity. These compounds indicated
fungistatic/fungicidal or bacteriostatic/bactericidal effect, espe-
cially against reference strains and clinical isolates of Candida spp.
(mainly C. albicans, C. krusei, C. inconspicua, C. famata, C. lusitaniae, C.
sake, C. parapsilosis and C. dubliniensis), and Gram-positive bacteria,
both pathogenic staphylococci S. aureus and opportunistic bacteria,
such as S. epidermidis and M. luteus, respectively. The antifungal
activity of obtained compounds was similar or higher than the
activity of commonly used antifungal drugs such as fluconazole.
Thus, these compounds may be regarded as precursor compounds
in the search of new derivatives showing high antifungal activity.
Theoretical calculation of the 1H NMR shifts carried out for systems
4c, 4f and 4h within the DFT(B3LYP)/6-311þþG** approximation
additionally confirmed the structure of investigated compounds.
water to yield 3.10
g
(84%); mp 81e83 ꢁC [36]; eluent-
dichloromethan/methanol (90:10), Rf ¼ 0.67. 1H NMR (DMSO-d6,
700 MHz),
d (ppm): 1.14e1.32 (m, 5H, 3CH2); 1.59e1.65 (m, 1H,
CH2); 1.67e1.73 (m, 2H, CH2); 1.74e1.79 (m, 2H, CH2); 2.13e2.21 (m,
1H, CH); 7.33 (d, 1H, CH, J ¼ 5.0 Hz); 7.41 (s, 1H, NH); 7.99 (s, 1H,
NH); 11.03 (s, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm):
25.46 (2C); 25.97; 30.05 (2C); 40.31; 151.31; 178.18. Anal. Calcd. for
C8H15N3S: C, 51.86; H, 8.16; N, 22.68. Found: C, 51.84; H, 8.15; N,
22.70.
4.1.2. (E)-2-(Cyclohexylmethylene)hydrazinecarboselenoamide (3)
Hydrazine hydrate, 80% (3.3 ml, 0.83 mol), concentrated hy-
drochloric acid (3.5 ml) and a solution of potassium selenocyanate
(2.50 g, 0.017 mol) in water (9 ml) were added to a mixture of ethyl
alcohol (70 ml) and water (9 ml). Subsequently, cyclo-
hexanecarbaldehyde (1) (6.06 ml, 0.054 mol) was added and the
mixture was heated to reflux for 2.5 h under nitrogen atmosphere.
After this time the solution was filtered while hot to remove the
remaining selenium.
4. Experimental
The filtrate was concentrated under vacuum to 20 ml giving a
pale yellow oil which solidified on cooling. The solid was next
isolated by filtration, washed with water and left to dry under
vacuum. The dry solid was then dissolved in chloroform (60 ml),
filtered through celite and the filtrate was evaporated to dryness.
The crude product was recrystallized from benzene to give a pure
product: 1.60 g, 41.0%; mp 82e84 ꢁC. The product is hygroscopic
and all operations must be performed in a nitrogen atmosphere. 1H
4.1. Materials and methods
All experiments were carried out under air atmosphere unless
stated otherwise. Reagents were generally the best quality
commercial-grade products and were used without further purifi-
cation. 1H NMR (700 and 400 MHz) and 13C NMR (100 MHz) spectra
were recorded on a Bruker Avance III multinuclear instrument.
EI(þ)-MS and HRMS analyses were performed by the Team Mass
Spectrometry of the Institute of Organic Chemistry of the Polish
Academy of Sciences in Warsaw. MS spectra were recorded on
AutoSpec Premier (Waters) spectrometer. Elemental analysis was
performed on ELEMENTAR Vario MACRO CHN. Melting points were
determined in open glass capillaries and are uncorrected. Analytical
TLC was performed using Macherey-Nagel Polygram Sil G/UV254
0.2 mm plates. Cyclohexanecarbaldehyde, ethyl 4-chloro-3-
oxobutanoate, ethyl 2-chloro-3-oxobutanoate, 1-chloropropan-2-
one, potassium selenocyanate, hydrazine hydrate and appropriate
bromoketones were commercial materials (Aldrich).
NMR (DMSO-d6, 700 MHz),
d (ppm): 1.10e1.35 (m, 5H, 3CH2);
1.55e1.78 (m, 5H, 3CH2); 2.11e2.18 (m, 1H, CH); 7.41 (d, 1H, CH,
J ¼ 5.0 Hz); 7.90 (s, 1H, NH); 8.40 (s, 1H, NH); 11.30 (s, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz),
40.34; 152.97; 173.53.
d (ppm): 25.42 (2C); 25.94; 29.89 (2C);
4.1.3. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
nitrophenyl)thiazole (4a). Typical procedure
Carbothioamide 2 (0.185 g, 1.0 mmol) was added to a stirred
solution of 2-bromo-1-(4-nitrophenyl)ethanone (0.244 g,
1.0 mmol) in absolute ethyl alcohol (30 ml). The reaction mixture

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
431
MIC (MFC) [
4j
mg/ml] of the tested compound
4k
4l
4m
4n
4o
4p
250(500)
4r
4s
62.5(125)
62.5(62.5)
31.25(62.5)
62.5(62.5)
62.5(250)
15.62(31.25)
62.5(62.5)
62.5(62.5)
31.25(62.5)
15.62(15.62)
15.62(31.25)
125(250)
4t
4u
FLU
3.91(15.62)
0.49(7.81)
0.98(3.91)
0.24(0.98)
0.98(7.81)
0.98(7.81)
0.49(1.95)
0.24(1.95)
0.24(3.91)
0.24(0.98)
0.49(15.62)
e
e
e
e
e
e
e
e
e
e
e
e
e
3.91(15.62)
1.95(15.62)
1.95(7.81)
0.98(1.95)
1.95(7.81)
1.95(7.81)
1.95(7.81)
1.95(15.62)
0.98(3.91)
0.24(1.95)
0.49(1.95)
125(>1000)
62.5(500)
125(250)
125(250)
62.5(62.5)
250(250)
250(500)
62.5(125)
250(500)
250(500)
125(250)
62.5(125)
62.5(125)
500(500)
250(500)
125(125)
125(125)
62.5(125)
125(250)
125(250)
62.5(62.5)
250(250)
250(250)
125(125)
62.5(62.5)
62.5(62.5)
500(500)
250(250)
125(250)
125(250)
62.5(125)
250(250)
250(500)
125(125)
250(500)
250(500)
125(125)
62.5(125)
62.5(125)
500(500)
250(500)
125(250)
250(500)
62.5(125)
250(250)
250(1000)
62.5(125)
250(500)
250(500)
125(125)
62.5(125)
62.5(125)
500(1000)
500(500)
31.25(62.5)
15.62(15.62)
15.62(31.25)
31.25(31.25)
15.62(62.5)
15.62(62.5)
31.25(31.25)
31.25(62.5)
31.25(62.5)
7.81(31.25)
15.62(62.5)
500(500)
e
e
e
e
e
e
e
e
e
e
e
e
e
0.24
250(500)
62.5(125)
250(250)
250(1000)
125(250)
250(500)
500(500)
125(250)
125(125)
125(125)
1000(1000)
500(500)
0.98
1.95
1.95
3.91
0.12
1000
15.62
0.24
31.25
62.5
1000(>1000)
1000(>1000)
15.62
7.81
62.5(125)
250(500)
was stirred at room temperature for 20 h. The separated precipitate
was purified on silica gel column chromatography (230e400 mesh)
using (dichloromethane/methanol, 90:10, Rf ¼ 0.81) to afford the
desired product: 0.20 g, 61%; mp 189e192 ꢁC. 1H NMR (DMSO-d6,
J ¼ 9.0 Hz); 11.50 (bs, 1H, NH).13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 25.45 (2C); 26.00; 30.32 (2C); 40.34; 55.58; 101.24; 114.38
(2C); 127.26 (2C); 128.16; 149.79; 150.57; 159.18; 168.99. EI(þ)-MS
(m/z, %): 315.3 [(Mþ), 23.09], 233.2 (15.09), 232.2 (100.00), 206.2
(35.29), 205.2 (12.65),191.2 (10.15),164.2 (35.30),149.2 (18.42), 41.5
(9.95). HRMS (m/z) calculated for C17H21N3OS: 315.1405. Found:
315.1394. Anal. Calcd. for C17H21N3OS: C, 64.73; H, 6.71; N, 13.32.
Found: C, 64.70; H, 6.73; N, 13.35.
700 MHz),
d (ppm): 1.16e1.37 (m, 5H, 3CH2); 1.61e1.68 (m, 1H,
CH2); 1.70e1.76 (m, 2H, CH2); 1.77e1.83 (m, 2H, CH2); 2.20e2.28
(m, 1H, CH); 7.31 (d, 1H, CH, J ¼ 5.0 Hz); 7.64 (s, 1H, CH); 8.10 (d, 2H,
2CH, J ¼ 9.0 Hz); 8.28 (d, 2H, 2CH, J ¼ 9.0 Hz); 11.71 (bs, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz),
d (ppm): 25.44 (2C); 25.99; 30.24 (2C);
40.35; 108.38; 124.55 (2C); 126.75 (2C); 141.27; 146.61; 148.83;
150.51; 169.51. EI(þ)-MS (m/z, %): 330.3 [(Mþ), 10.50], 248.2 (13.01),
247.2 (100.00), 221.2 (34.71), 191.2 (16.21), 175.2 (14.62), 174.2
(13.64), 149.2 (12.70). HRMS (m/z) calculated for C16H18N4O2S:
330.1150. Found: 330.1146. Anal. Calcd. for C16H18N4O2S: C, 58.16; H,
5.49; N, 16.96. Found: C, 58.14; H, 5.50; N, 16.99.
4.1.6. (E)-4-(2-(2-(Cyclohexylmethylene)hydrazinyl)thiazol-4-yl)
benzonitrile (4d)
4-(Bromoacetyl)benzonitrile was reacted with 2. Yield: 0.29 g,
94%, (dichloromethane/methanol, 90:10, Rf
¼
0.76); mp
159e160 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d
(ppm): 1.15e1.33 (m,
5H, 3CH2); 1.58e1.64 (m, 1H, CH2); 1.67e1.73 (m, 2H, CH2);
1.74e1.79 (m, 2H, CH2); 2.18e2.24 (m, 1H, CH); 7.28 (d, 1H, CH,
J ¼ 5.0 Hz); 7.53 (s, 1H, CH); 7.84 (d, 2H, 2CH, J ¼ 8.0 Hz); 7.99 (d, 2H,
2CH, J ¼ 8.0 Hz); 11.64 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
4.1.4. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
fluorophenyl)thiazole (4b)
2-Bromo-1-(4-fluorophenyl)ethanone was reacted with 2.
Yield: 0.25 g, 83%, (dichloromethane/methanol, 90:10, Rf ¼ 0.85);
d
(ppm): 25.43 (2C); 25.98; 30.22 (2C); 40.34; 107.39; 119.43;
mp 135e136 ꢁC. 1H NMR(DMSO-d6, 700 MHz),
d (ppm): 1.16e1.37
126.58 (2C); 128.82; 133.10 (2C); 139.11; 148.69; 150.75; 169.42.
EI(þ)-MS (m/z, %): 310.3 [(Mþ), 12.34], 228.2 (14.24), 227.2 (100.00),
201.2 (44.39), 159.2 (33.35), 41.5 (8.67). HRMS (m/z) calculated for
(m, 5H, 3CH2); 1.60e1.68 (m, 1H, CH2); 1.69e1.76 (m, 2H, CH2);
1.77e1.83 (m, 2H, CH2); 2.19e2.27 (m, 1H, CH); 7.20e7.27 (m, 3H,
3CH); 7.28 (d, 1H, CH, J ¼ 5.0 Hz); 7.84e7.91 (m, 2H, 2CH); 11.57 (bs,
C17H18N4S: 310.1252. Found: 310.1264. Anal. Calcd. for C17H18N4S: C,
1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 25.45 (2C); 26.00;
65.78; H, 5.84; N, 18.05. Found: C, 65.80; H, 5.82; N, 18.04.
30.30 (2C); 40.34; 103.13; 115.72; 115.93; 127.86; 127.94; 131.91;
149.97; 160.78; 163.21; 169.20. EI(þ)-MS (m/z, %): 303.3 [(Mþ),
17.18], 221.2 (13.10), 220.2 (100.00), 194.2 (34.77), 193.2 (9.39),
152.2 (43.79), 122.2 (6.19), 41.5 (9.71). HRMS (m/z) calculated for
4.1.7. (E)-4-(4-Bromophenyl)-2-(2-(cyclohexylmethylene)
hydrazinyl)thiazole (4e)
C
16H18FN3S: 303.1205. Found: 303.1218. Anal. Calcd. for C16H18FN3S:
C, 63.34; H, 5.98; N, 13.85. Found: C, 63.36; H, 5.99; N, 13.87.
2-Bromo-1-(4-bromophenyl)ethanone was reacted with 2.
Yield: 0.16 g, 44%, (n-hexane/ethyl acetate, 80:20, Rf ¼ 0.50); mp
4.1.5. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
methoxyphenyl)thiazole (4c)
145e146 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm): 1.13e1.33 (m,
5H, 3CH2); 1.57e1.64 (m, 1H, CH2); 1.67e1.73 (m, 2H, CH2);
1.74e1.79 (m, 2H, CH2); 2.18e2.24 (m, 1H, CH); 7.26 (d, 1H, CH,
J ¼ 4.0 Hz); 7.30 (s, 1H, CH); 7.57 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.76 (d, 2H,
2CH, J ¼ 9.0 Hz); 11.60 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
2-Bromo-1-(4-methoxyphenyl)ethanone was reacted with 2.
Yield: 0.25 g, 79%, (dichloromethane/methanol, 90:10, Rf ¼ 0.77);
mp 109e112 ꢁC [10]. 1H NMR (DMSO-d6, 700 MHz),
d (ppm):
1.14e1.36 (m, 5H, 3CH2); 1.58e1.64 (m, 1H, CH2); 1.67e1.73 (m, 2H,
CH2); 1.74e1.79 (m, 2H, CH2); 2.16e2.23 (m, 1H, CH); 3.77 (s, 3H,
CH3); 6.94 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.03 (s, 1H, CH); 7.24 (d, 1H, CH,
J ¼ 5.0 Hz); 7.74 (d, 2H, 2CH, J ¼ 9.0 Hz); 11.50 (bs, 1H, NH). 1H NMR
d (ppm): 25.43 (2C); 25.99; 30.25 (2C); 40.36; 104.41; 120.95;
128.02 (2C); 131.96 (2C); 134.18; 149.12; 150.57; 169.27. EI(þ)-MS
(m/z, %): 363.2 [(Mþ), 13.19], 281.1 (13.16), 282.1 (100.00), 281.1
(12.85), 280.1 (97.17), 256.1 (42.31), 254.1 (41.84), 214.1 (12.76),
212.1 (12.71), 175.2 (10.04), 174.2 (65.72), 133.2 (8.93), 89.3 (9.70),
55.5 (10.78), 41.5 (14.92). HRMS (m/z) calculated for C16H18BrN3S:
363.0405. Found: 363.0407. Anal. Calcd. for C16H18BrN3S: C, 52.75;
H, 4.98; N, 11.53. Found: C, 52.73; H, 4.96; N, 11.55.
(CDCl3, 700 MHz),
d (ppm): 1.10e1.22 (m, 3H, 2CH2); 1.24e1.33 (m,
2H, CH2); 1.62e1.67 (m, 1H, CH2); 1.70e1.80 (m, 4H, 2CH2);
2.16e2.24 (m, 1H, CH); 3.83 (s, 3H, CH3); 6.65 (s, 1H, CH); 6.84e6.88
(m, 1H, CH); 6.91 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.70 (d, 2H, 2CH,

432
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
4.1.8. (E)-4-(4-Chlorophenyl)-2-(2-(cyclohexylmethylene)
hydrazinyl)thiazole (4f)
2-Bromo-1-(4-chlorophenyl)ethanone was reacted with 2.
Yield: 0.16 g, 50%, (dichloromethane/methanol, 90:10, Rf ¼ 0.75);
mp 154e155 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm): 1.13e1.32
(m, 5H, 3CH2); 1.58e1.64 (m, 1H, CH2); 1.66e1.73 (m, 2H, CH2);
1.74e1.79 (m, 2H, CH2); 2.18e2.24 (m, 1H, CH); 7.29 (d, 2H, 2CH,
J ¼ 5.0 Hz); 7.44 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.82 (d, 2H, 2CH,
J ¼ 9.0 Hz); 11.70 (bs, 1H, NH). 1H NMR (CDCl3, 700 MHz),
d (ppm):
1.19e1.38 (m, 5H, 3CH2); 1.67e1.73 (m, 1H, CH2); 1.77e1.82 (m, 2H,
CH2); 1.83e1.88 (m, 2H, CH2); 2.32e2.39 (m, 1H, CH); 6.69 (s, 1H,
CH); 7.46 (d, 2H, 2CH, J ¼ 9.0 Hz); 7.57 (d,1H, CH, J ¼ 6.0 Hz); 7.66 (d,
2H, 2CH, J ¼ 9.0 Hz); 11.50 (bs, 1H, NH). 13C NMR (DMSO-d6,
100 MHz),
d (ppm): 25.43 (2C); 25.99; 30.23 (2C); 40.36; 104.38;
127.74 (2C); 129.06 (2C); 132.45; 133.62; 148.74; 150.87; 169.28.
EI(þ)-MS (m/z, %): 319.3 [(Mþ), 12.94], 238.2 (37.26), 237.2 (13.41),
236.2 (100.00), 212.1 (15.21), 210.2 (40.80), 174.2 (21.29), 170.1
(10.26), 168.1 (28.11), 82.2 (9.39), 80.2 (9.62), 41.5 (10.89). HRMS
(m/z) calculated for C16H18ClN3S: 319.0910. Found: 319.0898. Anal.
Calcd. for C16H18ClN3S: C, 60.08; H, 5.67; N, 13.14. Found: C, 60.07;
H, 4.69; N, 13.11.
4.1.9. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
(trifluoromethyl)phenyl)thiazole (4g)
2-Bromo-1-(4-trifluoromethylphenyl)ethanone was reacted
with 2. Yield: 0.20 g, 57%, (n-hexane/ethyl acetate, 80:20,
Rf ¼ 0.43); mp 138e140 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm):
1.14e1.34 (m, 5H, 3CH2); 1.58e1.65 (m, 1H, CH2); 1.66e1.73 (m, 2H,
CH2); 1.74e1.80 (m, 2H, CH2); 2.18e2.25 (m, 1H, CH); 7.27 (d, 1H,
CH, J ¼ 5.0 Hz); 7.47 (s, 1H, CH); 7.74 (d, 2H, 2CH, J ¼ 8.0 Hz); 8.02 (d,
2H, 2CH, J ¼ 8.0 Hz); 11.62 (bs, 1H, NH). 13C NMR (DMSO-d6,
100 MHz),
d (ppm): 25.44 (2C); 26.00; 30.27 (2C); 40.35; 106.22;
123.48; 125.98 (2C); 126.46 (2C); 127.90; 138.96; 149.30; 150.24;
169.38. EI(þ)-MS (m/z, %): 353.3 [(Mþ), 10.94], 271.2 (14.29), 270.2
(100.00), 244.2 (39.44), 202.1 (29.18), 173.2 (11.46), 41.5 (10.10).
HRMS (m/z) calculated for C17H18F3N3S: 353.1174. Found 353.1166.
Anal. Calcd. for C17H18F3N3S: C, 57.78; H, 5.13; N, 11.89. Found: C,
57.81; H, 5.15; N, 11.90.
4.1.10. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-p-
tolylthiazole (4h)
2-Bromo-1-(4-methylphenyl)ethanone was reacted with 2.
Yield: 0.15 g, 50%, (n-hexane/ethyl acetate, 80:20, Rf ¼ 0.33); mp
157e158 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm): 1.14e1.33 (m,
5H, 3CH2); 1.58e1.64 (m, 1H, CH2); 1.67e1.73 (m, 2H, CH2);
1.74e1.79 (m, 2H, CH2); 2.18e2.25 (m, 1H, CH); 2.30 (s, 3H, CH3);
7.15 (s, 1H, CH); 7.20 (d, 2H, 2CH, J ¼ 8.0 Hz); 7.32 (d, 1H, CH,
J ¼ 5.0 Hz); 7.68 (d, 2H, 2CH, J ¼ 8.0 Hz); 11.50 (bs, 1H, NH). 1H NMR
(CDCl3, 700 MHz), d (ppm): 1.20e1.37 (m, 5H, 3CH2); 1.67e1.73 (m,
1H, CH2); 1.77e1.82 (m, 2H, CH2); 1.83e1.87 (m, 2H, CH2);
2.31e2.37 (m, 1H, CH); 2.39 (s, 3H, CH3); 6.61 (s, 1H, CH); 7.28 (d,
2H, 2CH, J ¼ 8.0 Hz); 7.56 (d, 1H, CH, J ¼ 6.0 Hz); 7.59 (d, 2H, 2CH,
J ¼ 8.0 Hz); 11.60 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 21.29; 25.72 (2C); 25.95; 30.11 (2C); 40.45; 103.30; 126.14;
126.29 (2C); 129.73 (2C); 138.36; 146.18; 154.19; 169.35. EI(þ)-MS
(m/z, %): 299.3 [(Mþ), 15.16], 217.2 (14.11), 216.2 (100.00), 190.2
(34.93), 189.2 (9.30), 148.2 (28.00), 147.2 (14.53), 82.2 (19.11), 80.2
(18.27). HRMS (m/z) calculated for C17H21N3S: 299.1456. Found:
299.1469. Anal. Calcd. for C17H21N3S: C, 68.19; H, 7.07; N, 14.03.
Found: C, 68.19; H, 7.09; N, 14.00.
4.1.11. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(2,4-
difluorophenyl)thiazole (4i)
2-Bromo-1-(204’-difluorophenyl)ethanone was reacted with 4
in acetone. The product was obtained after cooling to ꢂ20 ꢁC. Yield:

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
433
Table 3
DFT(B3LYP)/6-311þþG** chemical shifts (ppm) of compounds 4c, 4f and 4h. The average value for the two most stable conformers is reported, together with the experimental
values in DMSO and CDCl3.
Thiazole (4c)
Atom H
Thiazole (4f)
Atom H
Thiazole (4h)
Average
d
DMSO
CDCl3
Average
d
DMSO
CDCl3
Atom H
Average
d
DMSO
CDCl3
29
32
25
24
28
26
30
33
23
27
31
42
43
41
39
36
34
38
40
35
37
1.19
1.20
1.30
1.44
1.44
1.71
1.71
1.71
1.80
1.80
2.31
3.66
3.66
4.02
6.71
6.76
6.75
7.13
7.85
7.93
8.37
1.14e1.36
1.10e1.33
28
31
24
23
27
29
32
25
22
26
30
33
35
38
37
39
34
36
1.20
1.21
1.30
1.44
1.44
1.71
1.71
1.72
1.81
1.81
2.32
6.79
6.94
7.39
7.45
7.79
7.93
8.34
1.13e1.32
1.19e1.38
28
31
24
23
27
25
32
29
22
26
42
30
41
40
33
35
37
38
39
34
36
1.19
1.20
1.30
1.44
1.44
1.71
1.71
1.72
1.80
1.80
2.00
2.31
2.51
2.54
6.77
6.90
7.31
7.34
7.82
7.94
8.32
1.14e1.33
1.20e1.37
1.58e1.73
1.74e1.79
1.62e1.80
1.58e1.73
1.74e1.79
1.67e1.82
1.83e1.88
1.58e1.73
1.74e1.79
1.67e1.82
1.83e1.87
2.16e2.23
3.77
3.77
3.77
6.94
7.03
7.24
6.94
7.74
2.16e2.24
3.83
3.83
3.83
6.91
6.65
6.84e6.88
6.91
7.70
11.50
7.70
2.18e2.24
7.29
7.29
7.44
7.44
7.82
11.70
7.82
2.32e2.39
7.57
6.69
7.46
7.46
7.66
11.50
7.66
2.30
2.18e2.25
2.30
2,30
7.32
7.15
7.20
7.20
7.68
2.39
2.31e2.37
2.39
2,39
7.56
6.61
7.28
7.28
7.59
11.50
7.74
11.50
7.68
11.60
7.59
0.16 g, 50%, (dichloromethane/methanol, 90:10, Rf ¼ 0.94); mp
134e135 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
(ppm): 1.15e1.37 (m,
5H, 3CH2); 1.60e1.67 (m, 1H, CH2); 1.68e1.75 (m, 2H, CH2);
1.76e1.82 (m, 2H, CH2); 2.20e2.27 (m, 1H, CH); 7.14 (d, 1H, CH,
J ¼ 3.0 Hz); 7.18 (td, 1H, CH, J1 ¼ 3.0 Hz, J2 ¼ 9.0 Hz); 7.32e7.38 (m,
2H, 2CH); 7.99 (m, 1H, CH); 11.70 (bs, 1H, NH). 13C NMR (DMSO-d6,
1.67e1.82 (m, 4H, 2CH2); 2.16e2.29 (m, 1H, CH); 2.44 (s, 3H, CH3);
4.18 (q, 2H, CH2, J ¼ 7.0 Hz); 7.33 (d,1H, CH, J ¼ 5.0 Hz); 11.93 (bs,1H,
d
NH). 13C NMR DMSO-d6, 100 MHz),
d (ppm): 14.75; 17.70; 25.43
(2C); 25.99; 30.05 (2C); 40.34: 60.40; 109.07; 152.88; 159.21;
162.43; 170.02. EI(þ)-MS (m/z, %): 295.3 [(Mþ), 7.65], 213.2 (10.82),
212.2 (100.00), 186.2 (9.40), 184.1 (20.67), 112.2 (11.48), 41.5 (10.05).
100 MHz),
d
(ppm): 25.41 (2C); 25.98; 30.18 (2C); 40.30; 104.96;
HRMS (m/z) calculated for C14H21N3O2S: 295.1354. Found:
107.69; 112.27; 119.00; 130.96; 142.39; 151.29; 158.69; 161.20;
168.59. EI(þ)-MS (m/z, %): 321.3 [(Mþ), 12.18], 239.2 (13.11), 238.2
(100.00), 212.2 (42.16), 211.2 (10.43), 170.2 (54.56), 41.5 (10.91).
HRMS (m/z) calculated for C16H17F2N3S: 321.1111. Found: 321.1125.
Anal. Calcd. for C16H17F2N3S: C, 59.79; H, 5.33; N, 13.07. Found: C,
59.82; H, 5.32; N, 13.05.
295.1367. Anal. Calcd. for C14H21N3O2S: C, 56.92; H, 7.17; N, 14.22.
4.1.12. (E)-Ethyl 2-(2-(2-(cyclohexylmethylene)hydrazinyl)thiazol-
4-yl)acetate (4j)
Ethyl 4-chloro-3-oxobutanoate was reacted with 2. Yield: 0.20 g,
68%, (dichloromethane/methanol, 90:10, Rf
¼
0.70); mp
131e132 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d
(ppm): 1.18 (t, 3H, CH3,
J ¼ 7.0 Hz); 1.20e1.35 (m, 5H, 3CH2); 1.57e1.65 (m, 1H, CH2);
1.66e1.79 (m, 4H, 2CH2); 2.13e2.24 (m, 1H, CH); 3.53 (s, 2H, CH2);
4.06 (q, 2H, CH2, J ¼ 7.0 Hz); 6.53 (s, 1H, CH); 7.20 (d, 1H, CH,
J ¼ 5.0 Hz); 11.34 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 14.56; 25.43 (2C); 25.99; 30.31 (2C); 37.54; 40.29; 60.65;
105.25; 145.35; 149.64; 168.94; 170.51. EI(þ)-MS (m/z, %): 295.3
[(Mþ), 9.20], 222.2 (9.97), 213.2 (10.47), 212.2 (100.00), 186.2
(11.59), 184.1 (11.95), 140.2 (11.59), 114.2 (15.64), 113.2 (30.26), 112.2
(36.33), 95.3 (10.06), 83.4 (24.62), 81.4 (14.49), 71.3 (28.60), 69.4
(18.78), 67.4 (33.51), 56.4 (12.86), 55.5 (50.64), 45.4 (16.28), 43.5
(20.87), 42.5 (10.77), 41.5 (61.10), 39.5 (22.44). HRMS (m/z) calcu-
lated for C14H21N3O2S: 295.1354. Found: 295.1368. Anal. Calcd. for
C
14H21N3O2S: C, 56.92; H, 7.17; N, 14.22. Found: C, 56.90; H, 7.15; N,
14.24.
4.1.13. (E)-Ethyl 2-(2-(cyclohexylmethylene)hydrazinyl)-4-
methylthiazole-5-carboxylate (4k)
Ethyl 2-chloro-3-oxobutanoate was reacted with 2. Yield: 0.20 g,
68%, (dichloromethane/methanol, 90:10, Rf
164e167 ꢁC. 1H NMR (DMSO-d6, 400 MHz),
(ppm): 1.14e1.35 (m,
5H, 3CH2); 1.25 (t, 3H, CH3, J ¼ 7.0 Hz); 1.57e1.66 (m, 1H, CH2);
¼
0.74); mp
d
Fig. 1. Optimized structures of investigated thiazoles 4c, 4f and 4h calculated within
the B3LYP/6-311þþG** approximation.

434
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
Found: C, 56.89; H, 7.14; N, 14.23.
358.3 [(Mþ), 12.09], 277.2 (19.05), 276.2 (14.01), 275.2 (100.00),
273.2 (49.61), 272.2 (17.83), 271.2 (18.95), 249.1 (26.42), 247.1
(14.58), 207.1 (41.71), 205.1 (20.68), 127.2 (21.24), 55.5 (14.12), 41.5
(18.03). HRMS (m/z) calculated for C17H18N4Se: 358.0697. Found:
358.0679. Anal. Calcd. for C17H18N4Se: C, 57.14; H, 5.08; N, 15.68.
Found: C, 57.17; H, 5.10; N, 15.67.
4.1.14. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-
methylthiazole (4l)
1-Chloropropan-2-one was reacted with 2. Yield: 0.07 g, 31%,
(dichloromethane/methanol, 90:10, Rf ¼ 0.78); mp 136-138 ꢁC. 1H
NMR (DMSO-d6, 700 MHz),
d (ppm): 1.13e1.35 (m, 5H, 3CH2);
1.57e1.66 (m, 1H, CH2); 1.67e1.78 (m, 4H, 2CH2); 2.12 (s, 3H, CH3);
2.13e2.23 (m, 1H, CH); 6.28 (s, 1H, CH); 7.20 (d, 1H, CH, J ¼ 5.0 Hz);
4.1.18. (E)-4-(4-Bromophenyl)-2-(2-(cyclohexylmethylene)
hydrazinyl)-1,3-selenazole (4p)
11.17 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 17.66;
2-Bromo-1-(4-bromophenyl)ethanone was reacted under ni-
trogen atmosphere with 3. Yield: 0.14 g, 52%, (n-hexane/ethyl ac-
etate, 80:20, Rf ¼ 0.62); mp 159-160 ꢁC. 1H NMR (DMSO-d6,
25.45 (2C); 26.00; 30.35 (2C); 40.92; 102.21; 148.05; 149.53;
168.72. EI(þ)-MS (m/z, %): 223.2 [(Mþ), 11.77], 140.2 (100.00), 114.2
(29.50), 69.4 (10.85), 55.5 (10.49), 41.5 (12.26). HRMS (m/z) calcu-
lated for C11H17N3S: 223.1143. Found: 223.1138. Anal. Calcd. for
700 MHz),
d (ppm): 1.13e1.33 (m, 5H, 3CH2); 1.58e1.65 (m, 1H,
CH2); 1.66e1.73 (m, 2H, CH2); 1.74e1.81 (m, 2H, CH2); 2.18e2.25 (m,
1H, CH); 7.37 (d, 1H, CH, J ¼ 5.0 Hz); 7.57 (d, 2H, 2CH, J ¼ 9.0 Hz);
C
11H17N3S: C, 59.16; H, 7.67; N, 18.81. Found: C, 59.16; H, 7.66; N,
18.80.
7.70 (s, 1H, CH); 7.74 (d, 2H, 2CH, J ¼ 9.0 Hz); 11.60 (bs, 1H, NH). 13
C
NMR (DMSO-d6, 100 MHz),
d (ppm): 25.40 (2C); 25.99; 30.07 (2C);
4.1.15. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
nitrophenyl)-1,3-selenazole (4m)
40.37; 108.47; 121.11; 128.43 (2C); 131.97 (2C); 134.14; 148.09;
152.89; 172.73. EI(þ)-MS (m/z, %): 410.2 [(Mþ), 1.73], 330.1 (50.35),
328.1 (63.95), 327.1 (10.24), 326.1 (29.73), 304.1 (12.42), 302.1
(17.16), 262.0 (12.01), 260.0 (15.95), 222.1 (34.07), 220.1 (17.03),
182.1 (15.45), 180.1 (12.13), 102.3 (12.53), 101.3 (16.21), 89.3 (11.24),
82.2 (97.39), 81.2 (39.68), 80.2 (100.00), 79.2 (39.49), 55.5 (63.73).
41,5 (99.40), 39.5 (40.06). HRMS (m/z) calculated for C16H18BrN3Se:
410.9849. Found: 410.9848. Anal. Calcd. for C16H18BrN3Se: C, 46.73;
H, 4.41; N, 10.22. Found: C, 46.71; H, 4.40; N, 10.25.
2-Bromo-1-(4-nitrophenyl)ethanone was reacted under nitro-
gen atmosphere with 3. Yield: 0.16 g, 42%, (n-hexane/ethyl acetate,
80:20, Rf ¼ 0.47); mp 163-164 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d
(ppm): 1.14e1.34 (m, 5H, 3CH2); 1.58e1.65 (m, 1H, CH2);
1.67e1.74 (m, 2H, CH2); 1.75e1.81 (m, 2H, CH2); 2.18e2.24 (m, 1H,
CH); 7.32 (d, 1H, CH, J ¼ 5.0 Hz); 8.07 (m, 3H, 3CH); 8.24 (d, 2H, 2CH,
J ¼ 9.0 Hz); 11.88 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d
(ppm): 25.42 (2C); 25.99; 30.09 (2C); 40.30; 112.87; 124.49 (2C);
127.14 (2C); 141.44; 146.49; 148.56; 152.13; 172.68. EI(þ)-MS (m/z,
%): 378.3 [(Mþ), 11.52], 297.2 (20.76), 296.2 (13.81), 259.2 (100.00),
293.2 (53.67), 292.2 (19.19), 291.2 (20.53), 269.1 (23.78), 267.1
(12.57), 227.1 (10.37), 222.1 (12.63), 55.5 (12.03), 41.5 (13.31). HRMS
(m/z) calculated for C16H18N4O2Se: 378.0595. Found: 378.0609.
Anal. Calcd. for C16H18N4O2Se: C, 50.93; H, 4.81; N, 14.85. Found: C,
50.96; H, 4.78; N, 18.83.
4.1.19. (E)-4-(4-Chlorophenyl)-2-(2-(cyclohexylmethylene)
hydrazinyl)-1,3-selenazole (4r)
2-Bromo-1-(4-chlorophenyl)ethanone was reacted under ni-
trogen atmosphere with 3. Yield: 0.18 g, 49%, (dichloromethane/
methanol, 95:5, Rf ¼ 0.88); mp 156e157 ꢁC. 1H NMR (DMSO-d6,
400 MHz),
d (ppm): 1.13e1.38 (m, 5H, 3CH2); 1.55e1.84 (m, 5H);
2.16e2.30 (m, 1H, CH); 7.36e7.41 (m, 1H, CH); 7.44 (d, 2H, 2CH,
J ¼ 9.0 Hz); 7.69 (s, 1H, CH); 7.82 (d, 2H, 2CH, J ¼ 9.0 Hz); 11.70 (bs,
4.1.16. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-(4-
fluorophenyl)-1,3-selenazole (4n)
1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 25.40 (2C); 25.98;
2-Bromo-1-(4-fluorophenyl)ethanone was reacted under ni-
trogen atmosphere with 3. Yield: 0.15 g, 41%, (n-hexane/ethyl ac-
etate, 80:20, Rf ¼ 0.29); mp 152e154 ꢁC. 1H NMR (DMSO-d6,
30.06 (2C); 40.43; 108.40; 128.15 (2C); 129.06 (2C); 132.56; 133.72;
147.85; 153.05; 172.77. EI(þ)-MS (m/z, %): 367.2 [(Mþ), 11.93], 286.1
(44.11), 285.1 (12.64), 284.1 (100.00), 283.1 (10.49), 282.1 (48.47),
281.1 (15.47), 280.1 (17.43), 260.1 (11.13), 258.1 (25.63), 256.1
(13.83), 222.1 (15.94), 218.1 (19.07), 216.1 (36.64), 136.2 (21.24),
55.5 (13.99), 41.5 (18.09). HRMS (m/z) calculated for C16H18ClN3Se:
367.0354. Found: 367.0357. Anal. Calcd. for C16H18ClN3Se: C, 52.40;
H, 4.95; N, 11.46. Found: C, 52.43; H, 4.99; N, 11.49.
700 MHz),
d (ppm): 1.13e1.34 (m, 5H, 3CH2); 1.58e1.65 (m, 1H,
CH2); 1.67e1.73 (m, 2H, CH2); 1.74e1.81 (m, 2H, CH2); 2.19e2.26 (m,
1H, CH); 7.19e7.25 (m, 2H, 2CH); 7.40 (d, 1H, CH, J ¼ 5.0 Hz); 7.59 (s,
1H, CH); 7.79e7.84 (m, 2H, 2CH); 11.60 (bs, 1H, NH). 13C NMR
(DMSO-d6, 100 MHz), d (ppm): 25.38 (2C); 25.97; 29.99 (2C); 40.32;
107.48; 115.95 (2C); 128.63 (2C); 130.88; 154.03; 161.00; 163.44;
173.04. EI(þ)-MS (m/z, %): 351.3 [(Mþ), 13.55], 270.1 (19.12), 269.2
(12.48), 268.1 (100.00), 266.1 (49.50), 265.1 (17.62), 264.1 (18.74),
242.1 (24.14), 241.1 (11.81), 240.1 (13.48), 200.1 (49.60), 198.1
(24.47), 120.2 (31.40), 55.5 (12.92), 41.5 (16.85). HRMS (m/z)
calculated for C16H18FN3Se: 351.0650. Found: 351.0656. Anal. Calcd.
for C16H18FN3Se: C, 54.86; H, 5.18; N,12.00. Found: C, 54.90; H, 5.20;
N, 12.03.
4.1.20. (E)-2-(2-(Cyclohexylmethylene)hydrazinyl)-4-p-tolyl-1,3-
selenazole (4s)
2-Bromo-1-(4-methylphenyl)ethanone was reacted under ni-
trogen atmosphere with 3. Yield: 0.18 g, 52%, (n-hexane/ethyl ac-
etate, 80:20, Rf ¼ 0.44); mp 158e160 ꢁC.1H NMR (DMSO-d6,
700 MHz),
d (ppm): 1.13e1.37 (m, 5H, 3CH2); 1.58e1.67 (m, 1H,
CH2); 1.68e1.82 (m, 4H, 2CH2); 2.17e2.28 (m, 1H, CH); 2.31 (s, 3H,
CH3); 7.19 (d, 2H, 2CH, J ¼ 8.0 Hz); 7.35 (d, 1H, CH, J ¼ 5.0 Hz); 7.53
(s, 1H, CH); 7.69 (d, 2H, 2CH, J ¼ 8.0 Hz); 11.70 (bs, 1H, NH). 13C NMR
4.1.17. (E)-4-(2-(2-(Cyclohexylmethylene)hydrazinyl)-1,3-
selenazol-4-yl)benzonitrile (4o)
4-(Bromoacetyl)benzonitrile was reacted under nitrogen at-
mosphere with 3. Yield: 0.15 g, 42%, (n-hexane/ethyl acetate, 80:20,
(DMSO-d6, 100 MHz), d (ppm): 21.28; 25.37 (2C); 25.97; 29.95 (2C);
40.42; 106.75; 126.55 (2C); 129.70 (2C); 131.06; 138.04; 146.83;
154.82; 173.21. EI(þ)-MS (m/z, %): 347.3 [(Mþ), 20.25], 345.3 (10.81),
266.2 (20.07), 264.2 (100.00), 262.2 (49.68), 261.2 (17.92), 260.2
(18.51), 238.2 (30.12), 196.1 (35.23), 194.1 (18.21), 116.3 (17.44),
115.3 (32.44), 82.2 (26.43), 80.2 (27.70), 55.5 (16.49), 41.5 (23.01).
HRMS (m/z) calculated for C17H21N3Se: 347.0901. Found: 347.0914.
Anal. Calcd. for C17H21N3Se: C, 58.96; H, 6.11; N, 12.13. Found: C,
58.93; H, 6.12; N, 12.10.
Rf ¼ 0.37); mp 166-167 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm):
1.13e1.37 (m, 5H, 3CH2); 1.56e1.66 (m, 1H, CH2); 1.67e1.83 (m, 4H,
2CH2); 2.16e2.27 (m, 1H, CH); 7.35 (d, 1H, CH, J ¼ 5.0 Hz); 7.83 (d,
2H, 2CH, J ¼ 8.0 Hz); 7.97 (s, 1H, CH); 7.99 (d, 2H, 2CH, J ¼ 8.0 Hz);
11.70 (bs, 1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 25.39
(2C); 25.97; 30.05 (2C); 40.29; 111.76; 119.39; 127.01 (2C); 129.44;
133.09 (2C); 139.12; 147.99; 152.87; 172.81. EI(þ)-MS (m/z, %):

K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
435
4.1.21. (E)-Ethyl 2-(2-(2-(cyclohexylmethylene)hydrazinyl)-1,3-
selenazol-4-yl)acetate (4t)
(for bacteria) and RPMI 1640 with MOPS (for fungi) were inoculated
with the suspensions of bacterial or fungal species. Microbial sus-
pensions were prepared in sterile 0.85% NaCl with an optical den-
sity of McFarland standard scale 0.5 e approximately 1.5 ꢃ 108 CFU
(Colony Forming Units)/ml for bacteria and 0.5 McFarland standard
scale e approximately 5 ꢃ 105 CFU/ml for fungi.
Ethyl 4-chloro-3-oxobutanoate was reacted with 3. Yield:
0.061 g, 18%, (dichloromethane/methanol, 90:10, Rf ¼ 0.75); mp
92e94 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d (ppm): 1.14e1.35 (m, 5H,
3CH2); 1.18 (t, 3H, CH3, J ¼ 7.0 Hz); 1.56e1.80 (m, 5H, 3CH2);
2.13e2.25 (m, 1H, CH); 3.47 (s, 2H, CH2); 4.06 (q, 2H, CH2,
J ¼ 7.0 Hz); 6.80 (bs, 1H, CH); 7.25 (d, 1H, CH, J ¼ 5.0 Hz); 11.60 (bs,
Samples containing 50 mg of examined compounds were first
dissolved in 1 ml dimethylsulfoxide (DMSO) and then appropriately
diluted. Furthermore, bacterial and fungal suspensions were put
1H, NH). 13C NMR (DMSO-d6, 100 MHz),
d (ppm): 14.57; 25.42 (2C);
26.02; 30.25 (2C); 38.25; 40.32; 60.62; 107.62; 145.38; 151.24;
170.41; 171.80. Anal. Calcd. for C14H21N3O2Se: C, 49.12; H, 6.18; N,
12.28. Found: C, 49.16; H, 6.15; N, 12.28.
onto Petri dishes with solid media containing 2000 mg/ml of the
tested compounds 4a-4u followed incubation under similar con-
ditions as previously. The inhibition of microbial growth was
judged by comparison with a control culture prepared without any
sample tested. Ciprofloxacin (for bacteria) or fluconazole (for fungi)
were used as a reference antimicrobial compounds (Sigma).
Subsequently MIC (Minimal Inhibitory Concentration), defined
as the lowest concentration of the samples showing complete
bacterial or fungal growth inhibition, was examined by the
microdilution broth method using their two-fold dilutions from
4.1.22. (E)-Ethyl 2-(2-(cyclohexylmethylene)hydrazinyl)-4-methyl-
1,3-selenazole-5-carboxylate (4u)
Ethyl 2-chloro-3-oxobutanoate was reacted with 3. Yield: 0.11 g,
32%, (dichloromethane/methanol, 90:10, Rf
¼
0.58); mp
140e143 ꢁC. 1H NMR (DMSO-d6, 700 MHz),
d
(ppm): 1.08e1.36 (m,
5H, 3CH2); 1.22 (t, 3H, CH3, J ¼ 7.0 Hz); 1.56e1.82 (m, 5H, 3CH2);
2.18e2.28 (m, 1H, 1CH); 2.38 (s, 3H, CH3); 4.14 (q, 2H, CH2,
J ¼ 7.0 Hz); 7.45 (s, 1H, CH); 12.00 (bs, 1H, NH). Anal. Calcd. for
1000 to 0.488
RPMI 1640 broth with MOPS (for fungi) prepared in microtiter
plates. Next 2 l of each bacterial or fungal suspension with an
optical density of 0.5 McFarland standard, was added per each well
containing 200 l broth with various concentrations of the exam-
mg/ml, in Mueller-Hinton broth (for bacteria) and
C
14H21N3O2Se: C, 49.12; H, 6.18; N,12.28. Found: C, 49.15; H, 6.20; N,
m
12.31.
m
4.2. Biological activity
ined compounds. The microplates were incubated at suitable con-
ditions (37 ꢁC or 30 ꢁC, 24h for bacteria or fungi, respectively) and
then MIC was assessed spectrophotometrically. Appropriate DMSO,
growth and sterile controls were carried out. The medium with no
tested substances was used as control.
The MBC (Minimal Bactericidal Concentration) or MFC (Minimal
Fungicidal Concentration) are described as the lowest concentra-
tion of the compounds that is required to kill a particular bacterial
The examined compounds: 4a-4u were screened in vitro for
antibacterial and antifungal activities using the broth microdilution
method according to European Committee on antimicrobial sus-
ceptibility testing (EUCAST) [39] and clinical and Laboratory stan-
dards Institute guidelines (CLSI) [40]. In this study were used
reference strains of microorganisms from American Type Culture
Collection (ATCC), including Gram-positive bacteria (Staphylococcus
aureus ATCC 6538, S. aureus ATCC 43300, S. aureus ATCC 25923,
Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC 6633,
Bacillus cereus ATCC 10876, Micrococcus luteus ATCC 10240), Gram-
negative bacteria (Escherichia coli ATCC 3521, E. coli ATCC 25922,
Klebsiella pneumoniae ATCC 13883, Proteus mirabilis ATCC 12453,
Bordetella bronchiseptica ATCC 4617, Salmonella typhimurium ATCC
14028, Pseudomonas aeruginosa ATCC 9027) and fungi belonging to
yeasts (Candida albicans ATCC 2091, C. albicans ATCC 10231, Candida
parapsilosis ATCC 22019)
In the study of antifungal activity of the compounds 4a-4u were
also used 10 clinical strains of yeasts from Candida species, e.g. C.
albicans, C. dubliniensis C. famata, C. inconspicua, C. krusei, C. lambica,
C. lusitaniae, C. parapsilosis, C. pulcherrima and C. sake, These fungi
were isolated by the authors (from Department of Pharmaceutical
Microbiology of Medical University in Lublin, Poland) from
different clinical materials, e.g. from upper respiratory tract of
hospitalized patients including cancer persons (i.e. with non-small
cell lung cancer or hematological malignancies). Some patients
were after pre- or post-operative chemotherapy (treated with
Vepesid or cis-platin given in doses according to the standard
procedures), patients with chronic hepatitis C (undergoing pegin-
terferon and ribavirin therapy or without antiviral therapy), pa-
tients with diabetes, elderly people, aged of 65 years old or older,
staying in close population, such as a care centre and people staying
outside the home care. The Ethical Committee of the Medical
University of Lublin approved the study protocol (No. KE-0254/75/
2011). The isolates were identified by standard diagnostic methods
e biochemical microtest, e.g. API 20 C AUX, ID 32 C, API Candida
or fungal species. MBC/MFC was determined by removing 20 ml of
the culture using for MIC determinations from each well and
spotting onto appropriate agar medium. After incubation (37 ꢁC or
30 ꢁC, 24h for bacteria or fungi, respectively), the lowest com-
pounds concentrations with no visible growth observed was
assessed as a bactericidal/fungicidal concentration. All the experi-
ments were performed three times and representative data are
presented [41].
In this study, no bioactivity was defined as a MIC >1000
mild bioactivity as a MIC in the range 501e1000 g/ml, moderate
bioactivity with MIC from 126 to 500 g/ml, good bioactivity as a
MIC in the range 26e125 g/ml, strong bioactivity with MIC be-
tween 10 and 25 g/ml and very strong bioactivity as a MIC <10 g/
mg/ml,
m
m
m
m
m
ml. The MBC/MIC or MFC/MIC ratios were calculated in order to
determine bactericidal/fungicidal (MBC/MIC ꢄ 4, MFC/MIC ꢄ 4) or
bacteriostatic/fungistatic (MBC/MIC > 4, MFC/MIC > 4) effect of the
tested compounds [42].
5. Computational details
We evaluated the 1H nuclear shieldings within the Dalton 2011
program [43], while geometry parameter optimizations were car-
ried out using the Gaussian 09 package [44]. In the present work we
are interested in the ab initio shielding tensors, that can be evalu-
ated as mixed energy derivatives calculated with respect to the
magnetic field and the nuclear magnetic moment. The chemical
shift, d, is defined in terms of the chemical shielding, as the dif-
ference between that of a reference (here tetramethylsilane, TMS)
and that of the sample.
ꢀ
(bioMerieux) on the basis of assimilation of various substrates.
Considering the size of the molecules in the present investiga-
tion, we resorted to Density Functional Theory (DFT), and started by
carrying out test calculations of chemical shifts for a set of selected
small molecules: methane, benzene, thiazole, and aminothiazole.
The microbial cultures were subcultured on nutrient agar or
Sabouraud agar at 35 ꢁC for 18e24 h or 30 ꢁC for 24e48 h for
bacteria and fungi, respectively. The surface of Mueller-Hinton agar

436
K.Z. Ła˛czkowski et al. / Journal of Molecular Structure 1108 (2016) 427e437
P. Chimenti, A. Granese, S. Carradori, D. Rivanera, D. Lilli, A. Zicari, S. Distinto,
Synthesis and in vitro activity of 2-thiazolylhydrazone derivatives compared
with the activity of clotrimazole against clinical isolates of Candida spp. Bio-
org. Med. Chem. Lett. 17 (2007) 4635e4640.
We used the B3LYP functional and a wide range of basis sets, among
them Dunning's aug-cc-pVXZ (abbreviated as aVXZ), and Pople's 6-
31G**, 6-31þþG**, 6-311G** and 6-311þþG** basis sets. Next, on
the basis of these results the DFT(B3LYP)/6-311þþG** approxima-
tion was chosen to carry out the evaluation of the NMR shifts for the
2,4-disubstituted 1,3-thiazoles (4c, 4f and 4h). For benzene we took
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parameters for methane, thiazole, aminothiazole, and the new
thiazole derivatives were optimized within the B3LYP/6-311þþG**
approximation. For molecules 4c, 4f and 4h, four starting confor-
mations were used per system in the geometrical parameter opti-
mizations, and the chemical shifts were computed for the two
lowest energy conformers of each potential drug. The nuclear
shieldings were calculated using the GIAO method. In our
approximation molecular rotation and vibration of the molecules
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The authors confirm that this article content has no conflicts of
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This study was supported by the Nicolaus Copernicus University
(project No. 786/2014) and the Foundation for Polish Science within
the PARENT/BRIDGE programme (Pomost/2013-7/1), cofinanced
from European Regional Development Fund within Innovative
Economy Operational Programme.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.molstruc.2015.12.033.
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