Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity

Article abstract of DOI:10.1016/j.bmc.2016.09.066

With the appearance of the antifungal resistance, novel antifungal agents need to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of

Full text of DOI:10.1016/j.bmc.2016.09.066

Accepted Manuscript
Synthesis of novel tetrazole derivatives and evaluation of their antifungal ac-
tivity
Edyta Łukowska-Chojnacka, Jolanta Mierzejewska, Małgorzata Milner-
Krawczyk, Małgorzata Bondaryk, Monika Staniszewska
PII:
S0968-0896(16)30827-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.066
BMC 13319
Reference:
Bioorganic & Medicinal Chemistry
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Accepted Date:
6 June 2016
17 September 2016
27 September 2016
Please cite this article as: Łukowska-Chojnacka, E., Mierzejewska, J., Milner-Krawczyk, M., Bondaryk, M.,
Staniszewska, M., Synthesis of novel tetrazole derivatives and evaluation of their antifungal activity, Bioorganic &
Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.09.066
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Synthesis of novel tetrazole derivatives and evaluation of their
antifungal activity
Edyta Łukowska-Chojnacka,a* Jolanta Mierzejewska,a Małgorzata Milner-Krawczyk,a
Małgorzata Bondaryk,b Monika Staniszewskab
aFaculty of Chemistry, Institute of Biotechnology, Warsaw University of Technology,
Noakowskiego St. 3, 00-664 Warsaw, Poland
bIndependent Laboratory of Streptomyces and Fungi Imperfecti, National Institute of Public
Health-National Institute of Hygiene, Chocimska St. 24, 00-791 Warsaw, Poland
KEY WORDS: tetrazole, benzothiazole, benzoxazole, azole, antifungal activity
*Corresponding author: Faculty of Chemistry, Institute of Biotechnology, Warsaw University
of Technology, Noakowskiego St. 3, 00-664 Warsaw, Poland. Tel.: +48 22 234 7677; fax:
+48 22 628 2741. E-mail address: elukowska@ch.pw.edu.pl
ABSTRACT – With the appearance of the antifungal resistance, novel antifungal agents need
to be identified. In this context new 2,5-disubstituted tetrazole derivatives containing
benzothiazole, benzoxazole or phenylsulfonyl moiety were synthesized by N-alkylation of
aryltetrazole
with
2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole
or
2-[(3-
chloropropyl)sulfanyl]-1,3-benzoxazole and Michael-type addition of aryltetrazole to phenyl
vinyl sulfone. The chemical structures of the synthesized compounds were confirmed by
1
means of H NMR, 13C NMR, IR and HRMS spectral data. The compounds were tested
against the moulds: F. sambucinum, F. oxysporum, C. coccodes, A. niger, and the yeast C.
albicans. The results showed that among the moulds only C. coccodes was significantly
sensitive to all the structures examined. All the tetrazole derivatives acted at the same level
against C. albicans and demonstrated a high cell growth inhibition (97-99 %) at the
concentrations ranging from 16 to 0.0313 µg/mL. The mode of action of 2-({3-[5-(4-
chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c) and 2-({3-[5-(2-
chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d) was established by
verifying fungal growth in the presence of osmotic protector-sorbitol. The effect of compound
5c or 5d combined with Fluconazole was determined using the checkerboard method. The
1

calculated fractional inhibitory concentration index (FIC) indicated antagonism (FIC>1).
Additionally, survival experiments with lepidopteran G. mellonella treated with compounds
5c and 5d were performed and demonstrated the lack of toxicity of these compounds.
1.
Introduction
Fungal infections constitute a worldwide health problem and the increase in the number of
human diseases caused by pathogenic fungi is observed. These infections are difficult to treat
and especially dangerous for elderly people and patients with an impaired immune system due
to neutropenia, AIDS, diabetes, cancer and induced immunosuppression after an organ
transplant.1 Another common factor contributing to fungal infections is a long-term treatment
with antibiotics and contraceptives. Among many types of infections the most common are
these caused by Candida spp., Aspergillus spp. and Cryptococcus spp., which lead to
candidiasis, aspergillosis and cryptococcosis, respectively.2-4 The most frequent fungal
pathogens are Candida albicans, Aspergillus fumigatus, Aspergillus niger and Cryptococcus
2-4
neoformans.
The currently used antifungal drugs display various mechanisms of action.
Azoles (e.g. Ketoconazole, Itraconazole, Fluconazole, Voriconazole, Posoconazole,
Ravuconazole),2, 4-6 and allylamines (e.g. Terbinafine) inhibit synthesis of ergosterol, which is
the major component of the fungal cell membrane.2 Polyene antibiotics (e.g. Amphotericin B,
Nystatin) interact with ergosterol and thus disrupt the fungal membrane and increase its
permeability.2, 4, 5 Other compounds inhibit synthesis of the major components of the cell wall
e.g. inhibitors of glucan synthesis (e.g. Echinocandins, Caspofungin, Micafungin) and
inhibitors of chitin synthesis (e.g. Nikkomycin, Polyoxins).2, 4, 5 We also know antifungal
agents that inhibit synthesis of nucleic acids (e.g. Flucytosine) or proteins (e.g. Sordarins) and
many others with a more complex mechanism of action.2, 5 The treatment of fungal infections
is problematic because of an emerging drugs resistance in fungi,5,7-11 therefore studies on the
design and synthesis of new effective antifungal compounds are conducted and focus on
tetrazole derivatives. These azoles significantly inhibit the growth of many pathogenic strains.
For instance, 1-(2,4-dihydroxythiobenzoyl)tetrazoles and tetrazoles containing hydrazone
moiety exhibit a high antifungal activity against Candida spp.12-15 The tetrazole derivatives
bearing both hydrazone and thiazoline moieties display a significant antifungal activity
against: Trichoderma harzianum, Aspergillus ochraceus, Fusarium solani, Fusarium
moniliforme, Fusarium culmorum, and C. albicans.16 It was found that the tetrazole
derivatives containing triazine dendrimeric chalcones exhibit a high fungicidal activity against
2

C. albicans, A. niger, A. fumigatus and Saccharomyces cerevisiae,17 whereas the tetrazoles
with quinolone scaffold are effective against C. albicans and A. niger.18-20 Furthermore, it is
worth mentioning that 2-(1-methyl-1H-1,2,3,4-tetrazol-5-yl)sulfanyl-N-(5-methylisoxazol-3-
yl)acetamide
and
2-(1-phenyl-1H-1,2,3,4-tetrazol-5-yl)sulfanyl-N-(5-methylisoxazol-3-
yl)acetmide inhibit the growth of C. albicans,21 3-aryl-1-(5-phenyl-1H-tetrazol-1-yl)prop-2-
en-1-one exhibits an activity against C. albicans and A. niger,22 pyridyl and pyrimidyl
tetrazole derivatives display antifungal properties against C. albicans, A. fumigatus, and A.
niger,23-25 and selected 5-thiosubstituted tetrazole derivatives show an activity against
Trichophyton mentagrophytes, Penicillium marneffei, Aspergillus flavus and A. fumigatus
comparable to that of Itraconazole.26 Moreover, tertazolyl derivatives of purine,27 thiophene,28
catecholthioethers,29 and organomercurials exhibit an antifungal activity.30 Besides, 1-
aryltetrazole derivatives containing N-substituted piperazine ring with antifungal properties
against A. flavus, A. fumigatus, P. marneffei, and T. mentagrophytes31 are known; the tetrazole
derivatives bearing both N-substituted piperazine and triazole rings are active against C.
neoformans, Candida spp. and Aspergillus spp.,32, 33 as well as many others.34-39
Herein, as a continuation of our previous work,40, 41 we present the synthesis of novel tetrazole
derivatives containing benzothiazole, benzoxazole or phenylsulfonyl scaffold, and evaluation
of their antifungal activity against the yeast C. albicans and selected mould stains: A. niger,
C. coccodes, F. sambucinum, F. oxysporum. Two tetrazole derivatives exhibiting the highest
anti-Candida
activity,
2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-
benzoxazole 5c and 2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-
benzoxazole 5d, were chosen for further studies including the mode of action and the type of
interaction in combination with the synthetic antifungal agent - Fluconazole.
2.
Results and discussion
2.1. Synthetic chemistry
New tetrazole derivatives were obtained in two types of reactions including N-alkylation and
Michael-type addition. The starting 5-aryltetrazoles 1a-d were synthesized from
corresponding nitriles, NaN3 and NH4Cl in DMF according to the described method.42 The 2-
[(3-chloropropyl)sulfanyl]-1,3-benzothiazole
2
and
2-[(3-chloropropyl)sulfanyl]-1,3-
benzoxazole 3 were obtained by S-alkylation of 2-mercapto-1,3-benzothiazole or 2-mercapto-
1,3-benzoxazole. The reactions were carried out in two phase system 10% NaOH/toluene in
3

the presence of tetrabutylammonium bromide (TBAB). Both products were obtained with
satisfactory yields of 76% and 82%, respectively. The phenyl vinyl sulfone 6 was prepared
according to the described method.43
The study on the preparation of new tetrazole derivatives began with the synthesis of 2-{[3-
(5-phenyl-2H-tetrazol-2-yl)propyl]sulfanyl}-1,3-benzothiazole 4a. The first experiment was
performed in acetonitrile with K2CO3 as a base at reflux (Scheme 1). The progress of the
reaction was monitored by thin-layer chromatography. After 47 h the reaction was stopped
and the product was purified on column chromatography with silica gel. The purification was
hampered and repeated three times because of the presence of numerous by-products. The
isolation of the by-products in pure form and their identification was impossible. The yield of
the reaction reached only 38 %, therefore the synthesis was performed again in THF in the
presence of NaOH and TBAB at reflux and in two phase system 10 % NaOH/toluene in the
presence of TBAB at reflux. The product was obtained with lower yield of 32 % and 27 %,
respectively. Other tetrazole derivatives 4b-d and 5a-d were synthesized using K2CO3 and
acetonitrile (Scheme 1).
Scheme 1. Synthesis of tetrazole derivatives 4a-d, 5a-d and 7a-d.
All the reactions were monitored on TLC plates and stopped when the conversion of 2-[(3-
chloropropyl)sulfanyl]-1,3-benzothiazole 2 or 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole 3
reached approximately 100%. In all the cases formation of many by-products was detected,
which significantly complicated the isolation and purification of the main products. In order
to purify the product, column chromatography was repeated several times and in some cases
(compounds: 4b-d, 5a, 5d) additional preparative thin-layer chromatography was done, which
undoubtedly decreased the yield of each reaction (Table 1). All the reactions took place
4

regioselectively and 2,5-disubstituted tetrazoles were formed. However, taking into account
the large number of the by-products, the formation of a trace amounts of 1,5-disubstituded
tetrazoles could not be excluded. As may be seen from the results presented in Table 1,
tetrazole derivatives containing benzoxazole moiety (5a-d) were obtained with higher yields
than their benzothiazole analogs (4a-d).
Significantly better results were obtained in Michael-type addition of 5-aryltetrazoles 1a-d to
phenyl vinyl sulfone 6 (Scheme 1). The reactions were performed in isopropanol in the
presence of Et3N. The first experiment, synthesis of 5-phenyl-2-[2-(phenylsulfonyl)ethyl]-2H-
tetrazole 7a, was performed at room temperature. The reaction was sluggish and even after
120 h of stirring the conversion was below 100 %, and the product was obtained with low
yield of 44%. When the same experiment was performed at reflux, the complete conversion
was already achieved after 2.5 h, and the product 7a was obtained with the yield of 76%.
Consequently, other tetrazole derivatives containing phenylsulfonyl moiety 7b-d were
synthesized at reflux. The time and yields of these reactions were summarized in Table 1.
Table 1. Reaction time and yields of the tetrazole derivatives 4a-d, 5a-d and 7a-d
Compound
Ar
C6H5-
X
S
S
S
S
O
O
O
O
-
Time (h)
47
Yield (%)
38
4a
4b
4c
4d
5a
5b
5c
5d
7a
7b
7c
7d
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
70
120
96
48
70
120
96
2.5
2.5
3
31
33
40
42
39
39
52
76
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
-
-
-
69
59
66
3
Comparing the results presented in Table 1 it may be concluded that N-alkylation reactions
took place with lower yields and significantly longer reaction time than Michael-type
additions. Moreover, the influence of the substituent at the benzene ring of tetrazole
derivatives 1a-d on the rate of N-alkylation reactions was observed. The synthesis time of
benzothiazole derivatives 4a-d and benzoxazole derivatives 5a-d ranged from 47-48 h to 120
h. For comparison the rates of Michael-type addition reactions were independent of the
structure of tetrazole derivatives 1a-d. The reactions took place in a shorter time (2.5-3 h) and
5

with higher yields (59-76%). In all the reactions the formation of 2,5-disubstituted tetrazole
derivatives was preferred.
2.2. Biological activity
A panel of synthesized tetrazole derivatives was screened against the reference yeast C.
albicans strain ATCC 90028 and the mould strains: A. niger ATCC 16404, C. coccodes MC
1, F. sambucinum MF 1 and F. oxysporum MF 5. Moreover, the cytotoxicity of compounds
5c and 5d was determined against the invertebrate host Galleria mellonella model.
The susceptibility of C. albicans to tetrazole derivatives: 4b, 4d, 5c-d, 7a, 7c-d was evaluated
at the concentrations ranging from 0.0313 to 16 µg/mL using the method M27-A3 (CLSI).44
The obtained results were summarized in Table 2. Although compounds provided a high cell
growth inhibition (from 97 % to 99 %) at all the concentrations tested, they did not show
MICs at this range (visually assessed quality in the method M27-A344). Thus, the MIC values
of compounds exceeded the highest concentration tested of 16 µg/mL. There were no
significant differences in the cell growth for all the compounds tested. Two tetrazole
derivatives, 5c and 5d, were selected for further studies. It can be assumed, that these
compounds damaged the cell wall (Fig. 1), when an osmotic protector (sorbitol at 0.8 M) was
used. Compounds 5c and 5d exhibited MIC at 1 µg/mL (after 48 h) and the effect was
sustained after 7 days of incubation. Compound 5c at MIC exerted the fungicidal effect and
killed 100 % of inoculum of C. albicans (Fig. 1 A, B), whereas 5d killed 97 % of inoculum
after 7 day-incubation (Fig. 1 C, D). Thus, the anti-Candida effect promoted by 5c in the
presence of sorbitol was excellent within the range of concentrations tested, except for 8
µg/mL. We suggested that this is a consequence of an adaptive response of C. albicans to
these tetrazole derivatives, which is more distinct for compound 5d.
6

Table 2. Antifungal activity (% of cell inhibition; means SD) of tetrazole derivatives 4b, 4d, 5c, 5d, 7a, 7c and 7d against the reference C.
albicans strain ATCC 90028 after 48h
C. albicans ATCC 90028
Compd
16 µg/mL
8 µg/mL
4 µg/mL
2 µg/mL
1 µg/mL
0.5 µg/mL 0.25 µg/mL 0.125 µg/mL 0.0625 µg/mL 0.0313 µg/mL
4b
4d
5c
5d
7a
7c
7d
98.82 0.13 99.00 0.08 98.89 0.05 98.58 0.09 98.92 0.05 98.73 0.08 98.56 0.14
98.77 0.04 98.60 0.09 98.70 0.05 98.00 0.14 98.73 0.12 98.64 0.17 98.70 0.09
99.16 0.11 99.44 0.19 98.67 0.22 99.52 0.05 99.32 0.08 99.36 0.01 99.18 0.19
99.07 0.04 99.16 0.03 99.16 0.22 99.14 0.05 99.07 0.15 98.87 0.03 99.40 0.02
99.07 0.05 99.08 0.04 98.93 0.07 99.02 0.03 99.03 0.03 98.94 0.11 98.95 0.18
99.16 0.02 99.48 0.05 99.42 0.08 99.44 0.09 99.37 0.05 99.37 0.12 99.32 0.05
98.49 0.15 97.97 0.24 97.77 0.05 97.86 0.12 98.15 0.07 98.22 0.15 98.06 0.18
98.75 0.06
99.02 0.06
99.12 0.04
99.38 0.04
98.73 0.05
99.26 0.05
97.80 0.09
98.81 0.05
98.91 0.21
99.09 0.05
99.35 0.02
99.04 0.07
99.20 0.09
98.41 0.05
98.80 0.14
98.81 0.30
99.00 0.02
99.36 0.04
99.06 0.06
98.97 0.05
98.58 0.10
*Amphotericin B at the concentration of 0.5 µg/mL was used as a control (inhibition %=100)
7

Figure 1. Antifungal properties of 5c (A, B) and 5d (C, D) in the presence of sorbitol (0.8 M). (A, B) The mode of action of 5c and its combined
fungal growth reduction with sorbitol after 48 h and 7 days. (C, D) The mode of action of 5d combined with sorbitol displayed after 48 h and 7
days
8

Additionally, we tested the effect of Fluconazole against C. albicans (MIC=0.25 μg/mL
assessed visually; MFC=0.5 μg/mL). In a checkerboard microtiter plate format, MICs of all
the tested partner compounds-tetrazole derivatives were decreased when these compounds
were combined with supra-MICs of Fluconazole (1.25 µg/mL). Fluconazole brought down
MIC of 5c and 5d form >16 to 1.25 μg/mL, respectively. The fractional inhibitory
concentration (FIC) of both tetrazole derivatives was considerably low (0.08). Thus the
combination was synergistic when FIC=1.25 μg/mL (combining Fluconazole with partner
agent) and MIC=16 μg/mL of 5c or 5d were considered (Table 3). For both partner
compounds, the endpoint was 100% at 1.25 μg/mL against C. albicans. Contrariwise, the FIC
index increased (antagonism) when FIC of Fluconazole with 5c and 5d respectively were
summed up. Combining Fluconazole with the antagonistic benzoxazole derivatives, which
themselves showed a moderate antifungal activity, resulted in an increased antifungal effect
expressed by the antifungal index.
Table 3. MIC results of checkerboard microtiter plate format testing the combination of
Fluconazole with partner compound (5c or 5d) against C. albicans ATCC 90028
Fluconazole [μg/mL]
Partner compound - benzoxazole
derivatives
80
40
20
98
10
99
5
2.5
100
100
1.25
100*
100*
5c
100
100
100
100
99
5d
100
100
100
* MICs of the listed partner compounds were obtained when tetrazole derivatives were
combined with Fluconazole at the concentration of 1.25 μg/mL. All MICs of the tetrazole
derivatives were decreased, and the corresponding FICs were <1 (FICs=0.08), which
indicates synergism; FIC of Fluconazole in combination with the partner compound at
1.25 µg/mL indicates antagonism (FIC>1). Finally, the FIC index resulting from the sum
of FICs of the listed partner compounds and Fluconazole were all >1 (FIC index=5.08),
which indicates antagonism.
The cytotoxicity of 5c and 5d was determined by applying the G. mellonella model. Knowing,
that both above-mentioned compounds showed comparable antifungal activity at all the tested
concentrations, the highest one (16 µg/mL) was chosen for the cytotoxicity experiment. In
these conditions, compounds 5c and 5d showed the lack of cytotoxicity against G. mellonella
and a 100 % survival within 96 h after inoculation was observed. We also found that DMSO
at the concentration of 0.33 % did not cause the death of G. mellonella. The control consisting
9

of untouched larvae or those which had received 10 µL of RPMI or PBS showed also a 100 %
survival. DMSO at this concentration was safe and can be used for compounds delivery in G.
mellonella. Melanisation for both the larvae inoculated or not after 96 h was not observed.
Concluding, both benzoxazole derivatives 5c and 5d were well tolerated in vivo by the larvae.
The model provided rapid evaluation of cytotoxicity of the tetrazole derivatives 5c and 5d in
vivo.
The tetrazole derivatives: 4a-d, 5a-d and 7a, 7c-d were also assessed for antifungal activity
against the mould strains including the human pathogen A. niger, and the plant pathogens: C.
coccodes, F. sambucinum and F. oxysporum. Unfortunately, all the compounds tested at the
concentration of 64 µg ml-1 showed either a weak or lack of an antifungal activity (Table 4).
The highest antifungal properties were observed for the compounds 5a-c, 7a, and 7d, which
reduced the growth of C. coccodes by more than 40%.
Table 4. Antifungal activity (% of growth inhibition) of the tetrazole derivatives 4a-d, 5a-d
and 7a-d at the concentration of 64 μg ml-1
A. niger ATCC
C. coccodes
F. sambucinum
F. oxysporum
Compound
16404
MC1
MF 1
MF 5
4a
4b
4c
4d
5a
5b
5c
5d
7a
4.4
3
0
23.4
33.4
36.7
16.7
43.4
43.4
46.7
33.4
41.1
18.4
43.4
86.7
100
13
0
5
5
27
0
0
0
0
6
0
4
0
17.7
1.7
31.2
77
17.6
0
20.6
11.8
11.8
6
18.5
11.2
17.14
100
52.9
16
14.5
14.5
14.5
1.8
35.6
86
7c
7d
control*
control **
67.7
*Amphotericin B - standard antifungal agent at the concentration of 16 μg ml-1;
**Fluconazole - standard antifungal agent at the concentration of 32 μg ml-1.
3.
Conclusion
New tetrazole derivatives containing benzothiazole, benzoxazole or phenylsulfonyl moiety
were synthesized and their antifungal activity was evaluated. The chemical step included N-
10

alkylation and Michael-type addition reactions. In all the cases only 2,5-disubstituted tetrazole
derivatives were isolated. Depending on the substrate, the products were obtained with yields
ranging from 31 % to 76 %. The lowest yields were achieved for N-alkylation reactions,
which undoubtedly was caused by repeated purification of the products. Biological tests
showed, that the synthesized compounds exhibit moderate to high antifungal activity against
C. coccodes MC 1 and C. albicans ATCC 90028, respectively. Herein, it was also reported
that the benzoxazole derivative 5c can modify the cell wall architecture. We noticed that
chlorine substituents (4-Cl) at benzene ring of the benzoxazole derivative 5c can play a role in
this mechanism. Our preliminary data showed that the antifungal activity of benzoxazoles
against C. albicans can be potentiated by a combination with supra-MICs concentrations of
Fluconazole. The benzoxazole derivatives 5c and 5d exhibited activity against C. albicans at
concentrations that are non-toxic to the invertebrate host G. mellonella, which should give
insight into the clinical relevance of this phenomenon.
4.
Experimental section
4.1 General
13
1H NMR (500MHz) and C NMR (100MHz) spectra were recorded on Varian Mercury 400
MHz spectrometer (Palo Alto, CA) in CDCl3 solution; chemical shifts (δ) were reported in
ppm. IR spectra were taken on a Carl Zeiss Specord M80 instrument (Germany). HRMS
spectra were recorded on a Micro-mass ESI Q-ToF Premier instrument (Micromass UK
Limited, Manchester UK). Melting points were determined in open capillaries and are
uncorrected. The reactions were monitored by thin-layer chromatography (TLC) aluminum
plates with silica gel Kieselgel 60 F254 (0.2 mm thickness film, Merck, Germany) using UV
light as visualizing agent. Column chromatography was performed using Kieselgel 60 (0.040-
0.063 mm, Merck, Germany). Preparative-layer chromatography (PLC) was performed using
glass plates (20 cm × 20 cm) with silica gel 60 F254 (2 mm thickness film, Merck, Germany).
Solvents and chemicals were purchased from POCH (Poland), Merck (Germany) and Sigma-
Aldrich (Germany) and were used without further purification.
4.2. Fungal strains, media and growth conditions
The reference strains: Candida albicans ATCC 90028 and Aspergillus niger ATCC 16404
were purchased from American Type Culture Collection (ATCC). Colletotrichum coccodes
11

MC 1, Fusarium oxysporum MF 5 and Fusarium sambucinum MF 1 were received from the
IHAR-PIB collection of Plant Breeding and Acclimatization Institute, Młochów. Candida
albicans ATCC 90028 was stored on ceramic beads in a Microbank tube (Prolab Diagnostics,
Canada) at -70°C. Prior to the respective examinations, routine cultures were conducted at
30°C for 18 h in yeast extract-peptone-dextrose medium (YEPD).45 Mould strains were stored
on Potato dextrose agar plates (PDA, Merck) at 4°C and subcultured every 4 months.
4.3 Synthesis of 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole
chloropropyl)sulfanyl]-1,3-benzoxazole 3
2
and 2-[(3-
To the solution of 2-mercapto-1,3-benzothiazole (4 g, 0.024 mol) or 2-mercapto-1,3-
benzoxazole (4 g, 0.0265 mol) in toluene (10 mL), 10 % NaOH (10 mL), TBAB (0.1 g) and
1-bromo-3-chloropropane (0.192 mol, 19 mL in the case of 2-mercapto-1,3-benzothiazole or
0.212 mol, 21 mL in the case of 2-mercapto-1,3-benzoxazole) were added. The mixture was
stirred at room temperature and the progress of the reaction was monitored by TLC using
hexane:ethyl acetate (6:1 v/v) as the eluent. After 1 h the reaction was stopped by adding H2O
and the product was extracted with diethyl ether (5 x 30 mL). The organic phase was washed
with water (4 x 40 mL) and dried under anhydrous MgSO4. Product was purified on a silica-
gel column with hexane: ethyl acetate gradient (100:1 v/v, 100:2 v/v and 100:5 v/v) as the
eluent.
2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole (2). Yield 76%, colorless crystals m.p. 45-
1
47oC. H NMR (CDCl3) δ ppm: 2.31-2.34 (m, 2H, CH2CH2CH2); 3.51 (t, 2H, CH2S, J=6.8
Hz); 3.72 (t, 2H, CH2Cl, J=6.2 Hz); 7.28-7.32 (m, 1H, Ar); 7.40-7.44 (m, 1H, Ar); 7.75-7.77
(m, 1H, Ar); 7.86-7.88 (m, 1H, Ar).46 HRMS [M+H]+ m/z calcd for C10H10ClNS+ 244.0021,
found 244.3024.
2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole (3). Yield 82%, oil. 1H NMR (CDCl3) δ ppm:
2.31-2.36 (m, 2H, CH2CH2CH2); 3.45 (t, 2H, CH2S, J=7.0 Hz); 3.72 (t, 2H, CH2Cl, J=6.2
Hz); 7.22-7.30 (m, 2H, Ar); 7.42-7.44 (m, 1H, Ar); 7.58-7.60 (m, 1H, Ar).46 HRMS [M+H]+
m/z calcd for C10H10ClNO+ 228.0250, found 228.0580.
12

4.4 Synthesis of tetrazole derivatives containing benzothiazole moiety 4a-d
To the solution of 2-[(3-chloropropyl)sulfanyl]-1,3-benzothiazole 2 (0.5 g, 2.05 mmol) in
acetonitrile (60 mL), K2CO3 (1.13 g, 8.21 mmol) and appropriate aryltetrazole 1a-d (4.10
mmol) were added. The reaction mixture was stirred at reflux and the progress of the reaction
was monitored by TLC using hexane: ethyl acetate (6:1 v/v) as the eluent. After the time
indicated in Table 1, the reaction was cooled to the room temperature, chloroform (20 mL)
was added and an inorganic solid filtered off. The residue was concentrated under reduced
pressure and purified. Product 4a was purified three times on a silica-gel column with hexane:
ethyl acetate (6:1 v/v) as the eluent. Product 4b was purified in two steps. The first was a
column chromatography on silica gel, and the second a preparative-layer chromatography
with a hexane:ethyl acetate (6:1v/v and 10:1v/v). The extraction of product 4b from a silica
gel was performed with methylene chloride (5 x 30 mL). Products 4c and 4d were purified in
three steps. The first and the second was column chromatography on a silica-gel with hexane:
ethyl acetate 25:1 v/v and 35:1 v/v as the eluent, respectively. The third step was preparative-
layer chromatography with hexane: ethyl acetate 10:1 (v/v) as the eluent. Products were
extracted from silica gel with methylene chloride (5 x 30 mL).
2-{[3-(5-phenyl-2H-tetrazol-2-yl)propyl]sulfanyl}-1,3-benzothiazole (4a). Yield 38%,
colorless crystals m.p. 64-66oC. IR (nujol, cm-1) 1678; 1525; 1452; 1428; 1404; 1185; 1140;
1076; 1048; 1019; 992; 965; 924. 1H NMR (CDCl3) δ ppm: 2.63-2.66 (m, 2H, CH2CH2CH2);
3.44-3,48 (m, 2H, CH2S); 4.85-4.88 (m, 2H, CH2N); 7.27-7.30 (m, 1H, Ar); 7.40-7.41 (m, 1H,
Ar); 7.48-7.50 (m, 3H, Ar); 7.73-7.76 (m, 1H, Ar); 7.82-7.84 (m, 1H, Ar); 8.14-8.16 (m, 2H,
13
Ar). C NMR (CDCl3) δ ppm: 29.14; 29.88; 51.54; 120.98; 121.56; 124.37; 126.06; 126.81;
127.29; 128.86; 130.31; 135.22; 152.98; 165.20; 165.51. HRMS [M+H]+ m/z calcd for
+
C17H16N5S2 354.0847, found 354.0883.
2-({3-[5-(4-methylphenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzothiazole (4b). Yield
31%, colorless crystal m.p. 65-67oC. IR (nujol, cm-1) 1680; 1614; 1592; 1458; 1428; 1366;
1
1178; 1124; 1080; 1045; 1022; 996. H NMR (CDCl3) δ ppm: 2.42 (s, 3H, CH3); 2.62-2.65
(m, 2H, CH2CH2CH2); 3.46-3.48 (m, 2H, CH2S); 4.84-4.86 (m, 2H, CH2N); 7.28-7.32 (m,
3H, Ar); 7.39-7.42 (m, 1H, Ar); 7.73-7.75 (m, 1H, Ar); 7.84-7.86 (m, 1H, Ar); 8.02-8.04 (m,
2H, Ar). 13C NMR (CDCl3) δ ppm: 21.51; 29.12; 29.87; 51.48; 110.49; 120.98; 121.55;
13

124.35; 126.04; 126.71; 129.56; 135.22; 140.47; 152.98; 165.28; 165.54. HRMS [M+H]+ m/z
+
calcd for C18H18N5S2 368.1004, found 368.0471.
2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzothiazole (4c). Yield
33%, colorless crystals m.p. 65-67oC. IR (nujol, cm-1) 1681; 1576; 1560; 1541; 1519; 1456;
1428; 1185; 1140; 1092; 1076; 1045; 1019; 992; 968. 1H NMR (CDCl3) δ ppm: 2.63-2.66 (m,
2H, CH2CH2CH2); 3.47-49 (m, 2H, CH2S); 4.85-4.88 (m, 2H, CH2N); 7.31-7.33 (m, 1H, Ar);
7.40-7.47 (m, 3H, Ar); 7.74-7.76 (m, 1H, Ar); 7.84-7.86 (m, 1H, Ar); 8.07-8.09 (m, 2H, Ar).
13C NMR (CDCl3) δ ppm: 29.06; 29.78; 51.59; 120.99; 121.52; 124.39; 125.75; 126.06;
128.07; 129.15; 135.20; 136.33; 152.94; 164.31; 165.43. HRMS [M+H]+ m/z calcd for
+
C17H15ClN5S2 388.0457, found 388.0356.
2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzothiazole (4d). Yield
40%, oil. IR (film, cm-1) 1676; 1604; 1566; 1522; 1458; 1428; 1379; 1356; 1194; 1162; 1146;
1128; 1068; 1036; 1019; 996; 962. 1H NMR (CDCl3) δ ppm: 2.64-2.68 (m, 2H, CH2CH2CH2);
3.46-3.50 (m, 2H, CH2S); 4.90-4.93 (m, 2H, CH2N); 7.25-7.32 (m, 1H, Ar); 7.37-7.44 (m, 3H,
Ar); 7.53-7.55 (m, 1H, Ar); 7.74-7.76 (m, 1H, Ar); 7.83-7.85 (m, 1H, Ar); 7.94-7.97 (m, 1H,
13
Ar). C NMR (CDCl3) δ ppm: 29.20; 29.93; 51.67; 120.94; 121.51; 124.31; 126.00; 126.34;
126.84; 130.75; 131.03; 131.25; 132.98; 135.17; 152.93; 163.28; 165.40. HRMS [M+H]+ m/z
+
calcd for C17H15ClN5S2 388.0457, found 388.0234.
4.5 Synthesis of tetrazole derivatives containing benzoxazole moiety 5a-d
To the solution of 2-[(3-chloropropyl)sulfanyl]-1,3-benzoxazole 3 (0.5g, 2.19 mmol) in
acetonitrile (60 mL), K2CO3 (1.21g, 8.79 mmol) and appropriate aryltetrazole 1a-d (4.4
mmol) were added. The reaction mixture was stirred at reflux and the progress of the reaction
was monitored by TLC using toluene: ethyl acetate (5:1 v/v) as the eluent. After the time
indicated in Table 2, the reaction was cooled to the room temperature, chloroform (20 mL)
was added and an inorganic solid filtered off. The residue was concentrated under reduce
pressure and purified. Products 5a and 5d were purified in two steps. The first was twice
chromatography on a silica-gel column with toluene: ethyl acetate (30:1 v/v) and hexane:
ethyl acetate (25:1 v/v) as the eluent. The second was preparative-layer chromatography with
hexane: ethyl acetate 10:1 (v/v) as the eluent. Products were extracted from silica gel with
methylene chloride (5 x 30 mL). Products 5b and 5c were purified twice on a silica-gel
14

column with toluene: ethyl acetate (20:1 v/v/) and hexane: ethyl acetate (15:1v/v and 35:1
v/v), respectively.
2-{[3-(5-phenyl-2H-tetrazol-2-yl)propyl]sulfanyl}-1,3-benzoxazole (5a). Yield 42%,
colorless crystals m.p. 97-99oC. IR (nujol, cm-1) 1601; 1528; 1493; 1181; 1132; 1092; 1073;
1
1048; 1010; 975; 927. H NMR (CDCl3) δ ppm: 2.66-2.68 (m, 2H, CH2CH2CH2); 3.39-3.42
(m, 2H, CH2S); 4.86-4.89 (m, 2H, CH2N); 7.24-7.29 (m, 2H, Ar); 7.42-7.44 (m, 1H, Ar);
13
7.48-7.50 (m, 3H, Ar); 7.57-7.59 (m, 1H, Ar); 8.14-8.16 (m, 2H, Ar). C NMR (CDCl3) δ
ppm: 28.82; 29.00; 51.36; 109.93; 118.42; 124.02; 124.35; 126.80; 127.22; 128.87; 130.35;
141.68; 151.85; 163.89; 165.24. HRMS [M+H]+ m/z calcd for C17H16N5OS+ 338.1076, found
338.0663.
2-({3-[5-(4-methylphenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5b). Yield
39%, colorless crystals m.p. 96-98oC. IR (nujol, cm-1) 1624; 1598; 1496; 1347; 1185; 1132;
1
1092; 1051; 1006; 975; 924. H NMR (CDCl3) δ ppm: 2.42 (s, 3H,CH3); 2.62-2.69 (m, 2H,
CH2CH2CH2); 3.37-3.40 (m, 2H, CH2S); 4.84-4.87 (m, 2H, CH2N); 7.22-7.30 (m, 4H, Ar);
13
7.42-7.44 (m, 1H, Ar); 7.57-7.59 (m, 1H, Ar); 8.02-8.04 (m, 2H, Ar). C NMR (CDCl3) δ
ppm: 21.52; 28.83; 28.99; 51.30; 109.93; 118.43; 124.00; 124.34; 124.41; 126.71; 129.57;
140.52; 141.70; 151.85; 163.91; 165.33. HRMS [M+H]+ m/z calcd for C18H18N5OS+
352.1232, found 352.1602.
2-({3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5c). Yield
39%, colorless crystals m.p. 93-95oC. IR (nujol, cm-1) 1598; 1544; 1504; 1452; 1420; 1140;
1092; 1041; 1016; 975; 930. 1H NMR (CDCl3) δ ppm: 2.64-2.68 (m, 2H, CH2CH2CH2); 3.37-
3.41 (m, 2H, CH2S); 4.85-4.88 (m, 2H, CH2N); 7.23-7.31 (m, 2H, Ar); 7.42-7.48 (m, 3H, Ar);
7.56-7.58 (m, 1H, Ar); 8.07-8.10 (m, 2H, Ar). 13C NMR (CDCl3) δ ppm: 28.81; 28.95; 51.44;
109.94; 118.42; 124.05; 124.38; 125.73; 128.09; 129.18; 136.38; 141.66; 151.85; 163.84;
164.37. HRMS [M+H]+ m/z calcd for C17H15ClN5OS+ 372.0686, found 372.0849.
2-({3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}sulfanyl)-1,3-benzoxazole (5d). Yield
52%, oil. IR (filml, cm-1) 1617; 1600; 1573; 1541; 1496; 1452; 1356; 1096; 1068; 1036;
1
1006; 927. H NMR (CDCl3) δ ppm: 2.67-2.70 (m, 2H, CH2CH2CH2); 3.40-3.43 (m, 2H,
CH2S); 4.91-4.94 (m, 2H, CH2N); 7.23-7.31 (m, 2H, Ar); 7.37-7.45 (m, 3H, Ar); 7.53-7.60
13
(m, 2H, Ar); 7.95-7.97 (m, 1H, Ar). C NMR (CDCl3) δ ppm: 28.83; 29.00; 51.48; 109.93;
15

118.43; 124.02; 124.35; 126.92; 130.82; 131.13; 131.30; 133.02; 141.69; 151.85; 163.42;
163.89. HRMS [M+H]+ m/z calcd for C17H15ClN5OS+ 372.0686, fund 372.0992.
4.6 Synthesis of tetrazole derivatives containing phenylsulfonyl moiety 7a-d
The solution of appropriate 5-aryltetrazole 1a-d (2.05 mmol), phenyl vinyl sulfone 6 (0.345 g,
2.05 mmol) and Et3N (0.3 ml, 2.15 mmol) in isopropanol (6 mL) was stirred at reflux. The
progress of the reaction was monitored by TLC with hexane:ethyl acetate 3:1 (v/v) as the
eluent. After the appropriate time, indicated in Table 1, the reaction was stopped and cooled
to 0oC. Then products 7a, 7b and 7c were filtered off, and the residues containing also
products were concentrated under reduce pressure and purified on a silica-gel column using
hexane:ethyl acetate 10:1 v/v (compound 7a) or 5:1 v/v (compounds 7b and 7c) as the eluent.
Product 7d (did not crystallize from the reaction mixture) was purified on column
chromatography with hexane:ethyl acetate 5:1 (v/v).
5-phenyl-2-[2-(phenylsulfonyl)ethyl]-2H-tetrazole (7a). Yield 76%, colorless crystals m.p.
91-92oC. IR (nujol, cm-1) 1584; 1542; 1529; 1448; 1408; 1360; 1328; 1306; 1200; 1156;
1
1088; 1072; 1044; 1024; 992; 928. H NMR (CDCl3) δ ppm: 3.92 (m, 2H, CH2N); 5.06 (m,
2H, CH2S); 7.46-7.51 (m, 5H, Ar); 7.52-7.58 (m, 1H, Ar); 7.84-7.87 (m, 2H, Ar); 7.99-8.02
(m, 2H, Ar). 13C NMR (CDCl3) δ ppm: 46.83; 54.28; 126.72; 126.75; 127.94; 128.84; 129.44;
130.52; 134.28; 137.98; 165.30. HRMS [M+H]+ m/z calcd for C15H15N4O2S+ 315.0916, fund
315.0982.
5-(4-methylphenyl)-2-[2-(phenylsulfonyl)ethyl]-2H-tetrazole (7b). Yield 69%, colorless
crystals m.p. 106-108oC. IR (nujol, cm-1) 1584; 1540; 1414; 1300; 1245; 1148; 1130; 1111;
1
1088; 1037; 1021; 992. H NMR (CDCl3) δ ppm: 2.41 (s, 3H, CH3); 3.92 (m, 2H, CH2N);
5.04 (m, 2H, CH2S); 7.25-7.28 (m, 3H, Ar); 7.47-7.51 (m, 1H, Ar); 7.54-7.56 (m, 1H, Ar);
7.84-7.90 (m, 4H, Ar). 13C NMR (CDCl3) δ ppm: 21,51; 46.78; 54.31; 123.93; 126.68;
127.95; 129.44; 129.55; 134.28; 138.00; 140.75; 165.41. HRMS [M+H]+ m/z calcd for
C16H18N4O2S+ 329.1072, fund 329.1347.
5-(4-chlorophenyl)-2-[2-(phenylsulfonyl)ethyl]-2H-tetrazole (7c). Yield 59%, colorless
crystals m.p. 138-140oC. IR (nujol, cm-1) 1414; 1304; 1200; 1152; 1136; 1114; 1088; 1034;
16

1008; 954. 1H NMR (CDCl3) δ ppm: 3.91 (m, 2H, CH2N); 5.06 (m, 2H, CH2S); 7.43-7.51 (m,
4H, Ar); 7.55-7.58 (m, 1H, Ar); 7.84-7.87 (m, 2H, Ar); 7.94-7.96 (m, 2H, Ar). 13C NMR
(CDCl3) δ ppm: 46.89; 54.23; 125.21; 127.30; 127.95; 128.03; 129.17; 129.46; 134.30;
136.61; 137.96. HRMS [M+H]+ m/z calcd for C15H14ClN4O2S+ 349.0526, fund 349.1031.
5-(2-chlorophenyl)-2-[2-(phenylsulfonyl)ethyl]-2H-tetrazole (7d). Yield 66%, colorless
crystals m.p. 84-85oC. IR (nujol, cm-1) 1600; 1580; 1564; 1513; 1449; 1414; 1320; 1200;
1
1148; 1111; 1080; 1059; 1031; 1000; 957; 925. H NMR (CDCl3) δ ppm: 3.94 (m, 2H,
CH2N); 5.10 (m, 2H, CH2S); 7.35-7.43 (m, 2H, Ar); 7.50-7.54 (m, 3H, Ar); 7.58-7.62 (m, 1H,
13
Ar); 7.83-7.89 (m, 3H, Ar). C NMR (CDCl3) δ ppm: 46.99; 54.24; 125.77; 126.90; 128.01;
13
129.51; 130.87; 131.20; 131.29; 132.87; 134.35; 137.95; 163.51. C NMR (CDCl3) δ ppm:
HRMS [M+H]+ m/z calcd for C15H14ClN4O2S+ 349.0526, fund 349.1169.
4.7 Antifungal activity assay against C. albicans ATCC 90028
Activity of the tetrazole derivatives 4b, 4d, 5c, 5d, 7a, 7c, 7d was assayed against C. albicans
ATCC 90028 using the broth microdilution method M27-A3 (CLSI, 2008).44, 47 In brief, the
final inoculum ranging from 0.5x102 to 2.5x103 CFU/mL was prepared in RPMI 1640
medium at pH 7.0 buffered with morpholinepropanesulfonic acid (MOPS). The stock solution
of the compounds (1600 µg/ml) was dissolved in water 33% DMSO. Then, yeast suspension
and the compound (final dilution 1:100) were dispensed at a final volume of 250 µL/well into
96-well plates. The microplates were read with the Infinite M200 PRO NANOQuant (Tecan
Group Ltd., Austria). Percentage of the cells growth inhibition was read
spectrophotometrically (optical density OD405) and the endpoint was calculated as a 100%
reduction in OD405 as compared to the growth in the control well. The growth reduction for
each compound concentration was calculated as follows: % of inhibition=100 - (OD405 CTW -
OD405 SCW)/ (OD405 GCW - OD405 SCW),48 where (1) CTW were prepared with compound,
medium and inoculum; (2) SCW contained compound and medium; (3) GCW means medium,
inoculum and the same amount of DMSO used in CTW. Amphotericin B (Sigma-Aldrich,
USA) was diluted in DMSO (1600 µg/ml) to be subsequently used in the assay as a reference
antifungal at the concentration of 0.5 µg/mL (100% cell inhibition).48 Fluconazole (Sigma-
Aldrich, USA) stock solution (5120 μg/mL) was prepared with water and subsequently final
concentrations were diluted with RPMI medium v/v.44 We used two endpoint criteria for MIC
determination: (i) total inhibition (MICTI), the lowest concentration of the compounds that
17

yielded no visible growth (a clear well); and (ii) partial inhibition (MICPI), the lowest
concentration that produced a prominent decrease in turbidity compared to that of the drug-
free control (visual assessment).49
4.8 Test with sorbitol
The MIC of tetrazole derivatives 5c and 5d against the C. albicans ATCC 90028 in the
presence of sorbitol (an osmotic protector) was assessed using the method M27-A3 (CLSI,
2008).44 Briefly, 50 μL of Sabouraud Dextrose Broth (SDB; Sigma-Aldrich, USA) was added
to each well of the microtiter plate. Subsequently, serial dilutions of tetrazole derivatives 5c
and 5d (533 to 0.3 µg/mL) were carried. Then, 50 μL of C. albicans inoculum (103 CFU/mL)
prepared in SDB supplemented with sorbitol at the final concentration of 0.8 M (Sigma-
Aldrich, USA) was transferred to wells as described.50, 51 Simultaneously, the fungal growth
control was assessed by placing 50 μL of SDB and 50 μL of the fungal inoculum with sorbitol
at 0.8 M. The sterility testing control was performed as follows: 50 μL of SDB with sorbitol at
0.8 M and 50 μL of tetrazole derivative solutions (5c or 5d) were mixed without fungal
suspension. The plates were incubated at 35°C and results were read visually and
spectrophotometrically at 405 nm (Spark Tecan, Swiss) after 48 h and again after 7 days.51
4.9 Synergism test
The combined effect of compounds 5c or 5d with Fluconazole was determined to derive the
fractional inhibitory concentration (FIC) of each compound tested and their sum, called the
FIC index, by using a checkerboard assay with the method M27-A3 (CLSI, 2008).44, 52 The
checkeboards were prepared by using serial dilutions of Fluconazole (80-1.25 µg/mL) in the
horizontal wells and compound (5c or 5d) at the final concentrations (160-2.5 µg/mL) in the
vertical wells. C. albicans suspension was prepared according to the method M27-A3 (CLSI,
2008) and 100 µL was inoculated into each well of a 96-well microtiter plate. The assay was
performed in triplicate, and the microplates were incubated at 35°C for 48 h. The growth rate
of C. albicans ATCC 90028 was assessed spectrophotometrically at 405 nm (Spark Tecan,
Swiss) after 18 and 48 h. Based on the results obtained the FIC index was calculated. FIC of
the tetrazole derivative is the concentration that kills when used in combination with
Fluconazole divided by the concentration that has the same effect when used individually.
18

FIC values of approximately 1 shows additive interaction while FIC values <1 and >1 have
synergistic and antagonistic interactions respectively.53
4.10 G. mellonella cytotoxicity test
G. mellonella larvae were randomly selected during the last instar of the larval development
and inoculated with 5c or 5d by injecting 10 µl of each agent at the concentration of 16
µg/mL into the hemocoel through the last pro-leg using a Hamilton syringe. Before injection,
the area was disinfected with an alcohol swab (70%). Larvae were stored in dark and
incubated in Petri dishes at 35ºC. The experiment was performed with control groups: (I)
regarding the physical trauma into hemocoel of larvae, inoculated with 10 µl of RPMI or
PBS; (II) regarding DMSO cytotoxicity at the concentration of 0.33%; (III) received no
injection. Mortality was assessed after 96 hours based on the black color of cuticle
(melanisation) and the absence of movement in response to stimulation. All the experiments
were performed at least in triplicate. A group of 10 larvae were used per treatment.
4.11 Antifungal activity assay against mould stains A.niger ATCC 16404, C. coccodes
MC 1, F. sambucinum MF 1 and F. oxysporum MF 5
The antifungal activity of compounds 4a-d, 5a-d and 7a,c,d against the mould strains A. niger
ATCC 16404, C.coccodes MC 1, F. sambucinum MF 1, and F. oxysporum MF 5 was
determined as was described previously.40 The compound dissolved in dimethyl sulfoxide
(DMSO, Sigma Aldrich) was added to a potato dextrose agar (PDA, Merck) medium at the
final concentration of 64 µg ml-1 (w/v). The final DMSO concentration, less than 0.2 % (v/v),
was identical in both control (without tested compound) and treated culture. The radial fungal
growth on the PDA plate was measured after 5-7 days of incubation at 25 °C. The inhibitory
activity of each compound was evaluated as a percentage of the control growth using a
following equation: Inhibition = [(control – treated)/control] × 100%. As a control to the
antifungal activity assay, the PDA plates supplemented with either Amphotericin B (BioShop)
or Fluconazole (Sigma Aldrich) at the final concentration of 16 µg ml-1 or 32 µg ml-1 (w/v),
respectively were used.
19

ACKNOWLEDGMENTS
This work was financially supported by Warsaw University of Technology. We thank
undergraduate student Klaudia Adamus and graduate student Monika Cieślak for assistance in
chemical and biological experiments. MB and MS were supported by the National Science
Centre in Poland [grant number: UMO-2014/15/N/NZ6/03710].
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Graphical abstract
Highlights
• New tetrazole derivatives were synthesized.
• Synthesized compounds exhibited high activity against C. albicans.
• Examined compounds showed moderate activity against C. coccodes.
• Compounds 5c and 5d combined with Fluconazole demonstrated antagonism.
23