New 1,5 and 2,5-disubstituted tetrazoles-dependent activity towards surface barrier of Candida albicans

Article abstract of DOI:10.1016/j.ejmech.2017.11.089

A series of novel tetrazole derivatives was synthetized using N-alkylation or Michael-type addition reactions, and screened for their fungistatic potential against Candida albicans (the lack of endpoint = 100%). Among them, the selected compounds 2d, 4b, and 6a differing in substituents at the tetrazole ring were non-toxic to Galleria mellonella larvae in vivo and exerted slight toxicity against Caco-2 in vitro (CC₅₀ at 256 μg/mL). An antagonistic effect of tetrazole derivatives 2d, 4b, and 6a respectively in combination with Fluconazole was shown using the checker board and colorimetric methods (fractional inhibitory concentration indexes FICIs >1). The most active 2d and 6a displayed an inverse relation between MICs in the presence of exogenous ergosterol, the effect was opposite to Itraconazole and Amphotericin B. The differences between 6a's and 2d's action mode were noted. Combining both flow cytometry and fluorescence image analyses respectively showed the complexity of planktonic and biofilm cell demise mode under the tetrazole derivatives tested. The following evidences for 6a's interaction with fungal membrane were noted: necrosis-like programmed cell death (97.03 ± 0.88), DNA denaturation (no laddering), mitochondrial damage (XTT assay), reduced adhesion to human epithelium (>50% at 0.0313 μg/mL, p ≤.05), irregular deposit of chitin, and attenuated morphogenesis in mature biofilm. The treatment with 6a reduced pathogenicity of C. albicans during infection in G. mellonella. Contrariwise, 2d enhancing fungal adhesion displayed mechanism targeted to the cell wall (due to the presence of 3-chloropropyl clubbed with aryltetrazole) in the presence of osmotic protector. Under 2d, the accidental cell death (88.60% ± 4.81) was observed. In conclusion, all tetrazole derivatives were obtained in satisfactory yields (60–95%) using efficient, simple and not expensive methods. Fungistatic and slightly anticancer tetrazole derivatives with the novel action mode can circumvent an appearance of antifungal-resistant strains. These results indicate that they are worthy of further studies.

Full text of DOI:10.1016/j.ejmech.2017.11.089

Accepted Manuscript
New 1,5 and 2,5-disubstituted tetrazoles-dependent activity towards surface barrier of
Candida albicans
Monika Staniszewska, Małgorzata Gizińska, Ewa Mikulak, Klaudia Adamus,
Mirosława Koronkiewicz, Edyta Łukowska-Chojnacka
PII:
S0223-5234(17)30992-3
10.1016/j.ejmech.2017.11.089
EJMECH 9964
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 August 2017
Revised Date: 13 November 2017
Accepted Date: 28 November 2017
Please cite this article as: M. Staniszewska, Mał. Gizińska, E. Mikulak, K. Adamus, Mirosł. Koronkiewicz,
E. Łukowska-Chojnacka, New 1,5 and 2,5-disubstituted tetrazoles-dependent activity towards
surface barrier of Candida albicans, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.11.089.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Title:
New 1,5 and 2,5-Disubstituted Tetrazoles-Dependent Activity Towards Surface Barrier
of Candida albicans
Monika Staniszewskaa*, Małgorzata Gizińskaa, Ewa Mikulaka, Klaudia Adamusb, Mirosława
Koronkiewiczc, Edyta Łukowska-Chojnackab
aNational Institute of Public Health-National Institute of Hygiene, Chocimska 24, 00-791
Warsaw, Poland
bFaculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw,
Poland
cNational Medicines Institute, Warsaw, 00-725 Poland
*Correspondence: Monika Staniszewska, Independent Laboratory of Streptomyces and Fungi
Imperfecti, National Institute of Public Health – National Institute of Hygiene, 24 Chocimska,
00-791 Warsaw, Poland
Phone: +48 22 54 21 228. Fax: + 48 22 849 74 84
E-mail: mstaniszewska@pzh.gov.pl

ACCEPTED MANUSCRIPT
Abstract
A series of novel tetrazole derivatives was synthetized using N-alkylation or Michael-
type addition reactions, and screened for their fungistatic potential against Candida albicans
(the lack of endpoint = 100%). Among them, the selected compounds 2d, 4b, and 6a differing
in substituents at the tetrazole ring were non-toxic to Galleria mellonella larvae in vivo and
exerted slight toxicity against Caco-2 in vitro (CC50 at 256 µg/mL). An antagonistic effect of
tetrazole derivatives 2d, 4b, and 6a respectively in combination with Fluconazole was shown
using the checker board and colorimetric methods (fractional inhibitory concentration indexes
FICIs >1). The most active 2d and 6a displayed an inverse relation between MICs in the
presence of exogenous ergosterol, the effect was opposite to Itraconazole and Amphotericin
B. The differences between 6a’s and 2d’s action mode were noted. Combining both flow
cytometry and fluorescence image analyses respectively showed the complexity of planktonic
and biofilm cell demise mode under the tetrazole derivatives tested. The following evidences
for 6a’s interaction with fungal membrane were noted: necrosis-like programmed cell death
(97.03 ± 0.88), DNA denaturation (no laddering), mitochondrial damage (XTT assay),
reduced adhesion to human epithelium (>50% at 0.0313 µg/mL, p≤0.05), irregular deposit of
chitin, and attenuated morphogenesis in mature biofilm. The treatment with 6a reduced
pathogenicity of C. albicans during infection in G. mellonella. Contrariwise, 2d enhancing
fungal adhesion displayed mechanism targeted to the cell wall (due to the presence of 3-
chloropropyl clubbed with aryltetrazole) in the presence of osmotic protector. Under 2d, the
accidental cell death (88.60% ± 4.81) was observed.
In conclusion, all tetrazole derivatives were obtained in satisfactory yields (60-95%)
using efficient, simple and not expensive methods. Fungistatic and slightly anticancer
tetrazole derivatives with the novel action mode can circumvent an appearance of antifungal-
resistant strains. These results indicate that they are worthy of further studies.
Key words: Candida albicans; virulence; antifungal agents; tetrazole derivatives

ACCEPTED MANUSCRIPT
Introduction:
Limited number of the available antifungal agents together with the emergence of
resistant isolates created a need to search for novel antifungal compounds with a new mode of
action and low cytotoxicity [1]. In this context, tetrazole derivatives represent a promising
group of compounds displaying a wide range of biological activities, such as anticancer,
anticonvulsant, anti-inflammatory, analgesic, antiproliferative, antibacterial, antifungal,
antiparasitic and antiviral [2-5]. Tetrazole has poly-nitrogen electron-rich planar structural
features, which allows tetrazole derivatives to easily bind with various enzymes or receptors
via weak interactions, including hydrogen bounds, hydrophobic effect, coordination bonds or
van der Waals force [4, 6]. Moreover, biological and physico-chemical properties of tetrazole
derivatives can be modified by the attachment of several structurally distinct substituents to
the heterocycle ring. This feature makes them desirable templates in many studies aimed at
the development of new bioactive compounds [4]. Recently, new tetrazole-based antifungal
drug candidates, VT-1129, VT-1161 and VT-1598, binding in the sterol 14α-demethylase’s
active site were described [5-9].
Recent evidences suggest that the majority of Candida albicans infections are
associated with biofilm formation [7-9]. Once formed on central venous catheters (CVCs) or
other implanted medical derives, fungal biofilm has the potential to seed disseminated
bloodstream infections [10]. Approximately 8% of bloodstream infections originate in CVCs
infected with C. albicans [11]. Moreover, fungal sessile cells display increased resistance to
antifungal agents and host defence mechanisms, which may carry serious clinical
consequences [7].
Recently there is a particular interest in developing non-mammalian in vivo models to
study fungal virulence [12]. Galleria mellonella (greater wax moth) was introduced as an
alternative host model to study microbial infections and for antimicrobial testing [12-15].
Herein, a series of novel tetrazole derivatives was synthesized and evaluated in vitro for
activity against C. albicans. Afterwards, the three compounds: 2d, 4b and 6a differing in
substituents at the tetrazole ring were further assessed for cytotoxicity. More detailed
mechanisms of anti-C. albicans action mode were elucidated in the case of the most effective
inhibitors 6a and 2d. An effort was made to detect C. albicans cell death under these tetrazole
derivatives. Following the trend in animal studies, we examined whether the novel tetrazole
derivative 6a reduces C. albicans virulence in the G. mellonella model in vivo.
Materials and Methods:
Fungal Strains, Media and Growth Conditions

ACCEPTED MANUSCRIPT
The quality control C. albicans wild type strains: 90028 and SC5314 were purchased from
American Type Culture Collection (ATCC). Reference strain 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) [16].
Synthesis of Tetrazole Derivatives
All the compounds were synthesized at the Faculty of Chemistry of the Warsaw University of
Technology (Poland). In general, 1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were
recorded on Varian Mercury 500 MHz spectrometer (USA) 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 (thickness 0.2 mm, Merck, Germany)
using UV light as visualizing agent. A column chromatography was performed using
Kieselgel 60 (0.040–0.063 mm, Merck, Germany). Solvents and chemicals were purchased
from POCH (Poland), Sigma-Aldrich (Germany) and Merck (Germany), and were used
without further purification.
N-alkylation of 5-aryltetrazole 1a-d with 1-bromo-3-chloropropane
To the solution of 5-aryltetrazole 1a-d (3.47 mmol) in acetonitrile (20 mL), KOH (15 mmol,
0.84 g) dissolved in MeOH (7.5 mL), and 1-bromo-3-chloropropane (0.12 mol, 12 mL) were
added. The mixture was stirred at reflux and the progress of the reaction was monitored by
TLC using chloroform/methanol (100:1 v/v) as the eluent. After the appropriate time, the
reaction was stopped and cooled to room temperature. Then, the inorganic solid was filtered
off, and the residue evaporated under reduced pressure. Products were separated and purified
on silica-gel column with hexane/ethyl acetate (50:1 v/v) as the eluent.
1-(3-chloropropyl)-5-phenyl-1H-tetrazole 2a. Yield 8%, oil. IR (film, cm-1) 935, 996, 1021,
1037, 1075, 1108, 1120, 1149, 1181, 1405, 1536. 1H NMR (CDCl3)
δ ppm: 2.42-2.47 (m, 2H,
CH2), 3.57-3.59 (m, 2H, CH2Cl), 4.59-4.61 (m, 2H, CH2N), 7.55-7.60 (m, 3H, Ar), 7.68-7.69
13
(m, 2H, Ar). C NMR (CDCl3)
δ ppm: 31.97, 40.96, 45.10, 128.71, 128.85, 129.43, 131.37,
154.62. HRMS [M+H]+ m/z calcd for C10H12N4Cl+ 223.0750 found 223.0643.

ACCEPTED MANUSCRIPT
1-(3-chloropropyl)-5-(4-methylphenyl)-1H-tetrazole 2b. Yield 4%, oil. IR (film, cm-1) 968,
1035, 1067, 1112, 1194, 1344, 1388, 1423, 1452, 1477, 1620. 1H NMR (CDCl3)
δ ppm: 2.41-
2.47 (m, 5H, CH3, CH2), 3.57-3.60 (m, 2H, CH2Cl), 4.59 (t, 2H, CH2N, J= 7.1 Hz), 7.35-7.38
13
(m, 2H, Ar), 7.57-7.60 (m, 2H, Ar). C NMR (CDCl3)
δ ppm: 21.51, 31.96, 41.02, 45.10,
120.70, 126.73, 128.63, 129.58, 130.03, 141.86. HRMS [M+H]+ m/z calcd for C11H15N4Cl+
237.0907 found 237.0760.
5-(4-chlorophenyl)-1-(3-chloropropyl)-1H-tetrazole 2c. Yield 2%, oil. IR (film, cm-1) 971,
1
1012, 1096, 1188, 1385, 1428, 1468, 1531, 1569, 1608. H NMR (CDCl3)
δ ppm: 2.43-2.48
(m, 2H, CH2), 3.57-3.59 (m, 2H, CH2Cl), 4.57-4.60 (m, 2H, CH2N), 7.54-7.56 (m, 2H, Ar),
7.64-7.66 (m, 2H, Ar). 13C NMR (CDCl3)
δ
ppm: 31.85, 40.93, 45.17, 122.11, 129.74, 130.08,
+
137.92, 153.77. HRMS [M+H]+ m/z calcd for C10H11N4Cl2 257.0361 found 257.0812.
5-(2-chlorophenyl)-1-(3-chloropropyl)-1H-tetrazole 2d. Yield 18%, oil. IR (film, cm-1)
1
975, 1016, 1036, 1070, 1112, 1130, 1162, 1410, 1436, 1460, 1534, 1573, 1604. H NMR
(CDCl3)
δ
ppm: 2.36-2.41 (m, 2H, CH2), 3.49-3.52 (m, 2H, CH2Cl), 4.41 (t, 2H, CH2N, J=6.8
13
Hz), 7.46-7.60 (m, 4H, Ar). C NMR (CDCl3)
δ
ppm: 31.60, 40.83, 44.88, 123.59, 127.58,
+
130.35, 132.04, 132.73, 133.85, 152.96. HRMS [M+H]+ m/z calcd for C10H11N4Cl2 257.0361
found 257.0695.
2-(3-chloropropyl)-5-phenyl-2H-tetrazole 3a. Yield 52%, oil. IR (film, cm-1) 924, 971,
1003, 1025, 1045, 1073, 1146, 1191, 1360, 1385, 1448, 1468, 1519, 1531. 1H NMR (CDCl3)
δ
ppm: 2.51-2.56 (m, 2H, CH2), 3.63-3.65 (m, 2H, CH2Cl), 4.84-4.86 (m, 2H, CH2N), 7.45-
13
7.51 (m, 3H, Ar), 8.13-8.17 (m, 2H, Ar). C NMR (CDCl3)
δ ppm: 31.91, 41.00, 50.11,
126.82, 127.26, 128.89, 130.38, 165.29. HRMS [M+H]+ m/z calcd for C10H12N4Cl+ 223.0750
found 223.0533.
2-(3-chloropropyl)-5-(4-methylphenyl)-2H-tetrazole 3b. Yield 58%, oil. IR (film, cm-1)
971, 1044, 1178, 1194, 1366, 1420, 1464, 1512, 1544, 1560, 1624. 1H NMR (CDCl3)
2.41 (s, 3H, CH3), 2.50-2.55 (m, 2H, CH2), 3.62-3.65 (m, 2H, CH2Cl), 4.82-4.85 (m, 2H,
CH2N), 7.30 (d, 2H, Ar, J = 8.3 Hz), 8.02-8.04 (m, 2H, Ar). 13C NMR (CDCl3)
ppm: 21.48,
δ ppm:
δ
31.92, 41.02, 50.04, 124.46, 126.74, 129.58, 140.55, 165.38. HRMS [M+H]+ m/z calcd for
C11H15N4Cl+ 237.0907 found 237.0987.
5-(4-chlorophenyl)-2-(3-chloropropyl)-2H-tetrazole 3c. Yield 71%, colorless crystals mp
48-50°C. IR (nujol, cm-1) 976, 1004, 1019, 1040, 1084, 1105, 1134, 1194, 1420, 1538, 1557,

ACCEPTED MANUSCRIPT
1
1573, 1608. H NMR (CDCl3)
δ ppm: 2.51-2.56 (m, 2H, CH2), 3.62-3.65 (m, 2H, CH2Cl),
4.84-4.87 (m, 2H, CH2N), 7.45-7.48 (m, 2H, Ar), 8.07-8.09 (m, 2H, Ar). 13C NMR (CDCl3)
δ
ppm: 31.85, 40.95, 50.18, 125.75, 128.11, 129.20, 136.44, 164.43. HRMS [M+H]+ m/z calcd
+
for C10H11N4Cl2 257.0361 found 257.0635.
5-(2-chlorophenyl)-2-(3-chloropropyl)-2H-tetrazole 3d. Yield 49%, oil. IR (film, cm-1)
978, 1006, 1036, 1068, 1124, 1146, 1165, 1191, 1360, 1382, 1430, 1444, 1461, 1522, 1576,
1604. 1H NMR (CDCl3)
δ
ppm: 2.53-2.58 (m, 2H, CH2), 3.65-3.67 (m, 2H, CH2Cl), 4.89-4.92
13
(m, 2H, CH2N), 7.37-7.43 (m, 2H, Ar), 7.53-7.55 (m, 1H, Ar). 7.941-7.960 (m, 1H, Ar). C
NMR (CDCl3)
δ
ppm: 31.90, 40.98, 50.23, 126.38, 126.93, 130.84, 131.15, 131.32, 133.07,
+
163.47. HRMS [M+H]+ m/z calcd for C10H11N4Cl2 257.0361 found 257.0458.
Synthesis of tetrazole derivatives containing morpholine moiety 4a-d
To the solution of tetrazole 3a-d (0.42 mmol) and morpholine (10.20 mmol, 0.9 mL) in
acetonitrile (17 mL), K2CO3 (6.2 mmol, 0.857 g) was added. The reaction mixture was stirred
at reflux and the progress of the reaction was monitored by TLC using chloroform/methanol
(30:1 v/v) as the eluent. After 24 h a new portion of morpholine (10.20 mmol, 0.9 mL) was
added. Finally, the reaction was stopped after 168 h, cooled to room temperature, and the
inorganic solid was filtered off. The residue was concentrated under reduced pressure and
purified on silica-gel column using chloroform/methanol (250:1 v/v) as the eluent.
4-[3-(5-phenyl-2H-tetrazol-2-yl)propyl]morpholine 4a. Yield 75%, oil. IR (film, cm-1) 696,
732, 917, 959, 1006, 1028, 1045, 1072, 1116, 1143, 1200, 1360, 1382, 1448, 1461, 1528,
1
1620, 2812, 2858, 2899, 2960. H NMR (CDCl3)
δ ppm: 2.20-2.25 (m, 2H, CH2), 2.42-2.44
(m, 6H, CH2N), 3.66-3,70 (m, 4H, CH2O), 4.74 (t, 2H, CH2tetr., J= 7.1 Hz), 7.45-7.50 (m,
3H, Ar), 8.12-8.15 (m, 2H, Ar). 13C NMR (CDCl3)
ppm: 26.29, 51.31, 53.56, 55.25, 66.93,
δ
126.75, 127.47, 128.87, 130.24. HRMS [M+H]+ m/z calcd for C14H20N5O+ 274.1162 found
274.1162.
4-{3-[5-(4-methylphenyl)-2H-tetrazol-2-yl]propyl}morpholine 4b. Yield 83%, colorless
crystals mp 38–40°C. IR (nujol, cm-1) 685, 756, 916, 955, 1010, 1029, 1044, 1072, 1120,
1143, 1185, 1360, 1375, 1417, 1452, 1464, 1544, 1620, 2817, 2856, 2956. 1H NMR (CDCl3)
δ
ppm: 2.19-2.24 (m, 2H, CH2), 2.41-2.44 (m, 9H, CH3, CH2N), 3.67-3.68 (m, 4H, CH2O),
13
4.73 (t, 2H, CH2tetr., J=6.8 Hz), 7.28-7.30 (m, 2H, Ar), 8.00-8.03 (m, 2H, Ar). C NMR

ACCEPTED MANUSCRIPT
(CDCl3)
δ
ppm: 21.53, 26.30, 51.26, 53.57, 55.27, 66.94, 124.68, 126.66, 129.47, 129.65,
140.37, 165.37. HRMS [M+H]+ m/z calcd for C15H23N5O+ 288.1819 found 288,1919.
4-{3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propyl}morpholine 4c. Yield 77%, colorless
crystals mp 73-75°C. IR (nujol, cm-1) 695, 756, 920, 972, 1016, 1048, 1076, 1092, 1116,
1
1153, 1181, 1200, 1337, 1364, 1420, 1448, 1464, 1522, 1582, 1608, 2816, 2867, 2944. H
NMR (CDCl3)
CH2O), 4.74 (m, 2H, CH2tetr., J= 7.1 Hz), 7.45-7.47 (m, 2H, Ar), 8.06-8.09 (m, 2H, Ar). 13C
NMR (CDCl3) ppm: 26.27, 51.40, 53.56, 55.23, 66.93, 125.98, 128.03, 129.08, 129.26,
δ ppm: 2.19-2.24 (m, 2H, CH2), 2.41-2.44 (m, 6H, CH2N), 3.66-3.69 (m, 4H,
δ
136.27, 164.17. HRMS [M+H]+ m/z calcd for C14H19N5OCl+ 308.1272 found 308.8911.
4-{3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propyl}morpholine 4d. Yield 87%, oil. IR (film,
cm-1) 752, 778, 916, 954, 1012, 1036, 1068, 1120, 1146, 1356, 1385, 1440, 1460, 1520, 1574,
1
1604, 2811, 2856, 2888, 2956. H NMR (CDCl3)
δ ppm: 2.21-2.27 (m, 2H, CH2), 2.43-2.46
(m, 6H, CH2N), 3.66-3.69 (m, 4H, CH2O), 4.77-4.80 (m, 2H, CH2tetr.), 7.36-7.42 (m, 2H,
13
Ar), 7.52-7.54 (m, 1H, Ar), 7.94-7.95 (m, 1H, Ar). C NMR (CDCl3)
δ ppm: 26.28, 51.44,
53.56, 55.24, 66.94, 126.58, 126.91, 130.82, 131.03, 131.29, 133.00, 163.21. HRMS [M+H]+
m/z calcd for C14H19N5OCl+ 308.1272 found 308.8911.
Michael-type addition of ethyl acrylate to 5-aryltetrazole 1a-d
To the solution of 5-arylterazole 1a-d (3.2 mmol) in 2-propanol (10 mL) ethyl acrylate (4.65
mmol, 0.5 mL) and Et3N (2.87 mmol, 0.4 mL) were added. The reaction mixture was stirred
at reflux and the progress of the reaction was controlled by TLC using chloroform/methanol
(100:1 v/v) as the eluent. After the appropriate time, the reaction was stopped. The solvent
and the excess of Et3N were evaporated under reduced pressure, and products were separated
and purified on silica-gel column using chloroform/methanol (1200:1 v/v) as the eluent.
ethyl 3-(5-phenyl-1H-tetrazol-1-yl)propanoate 5a. Yield 9%, oil. IR (film, cm-1) 940, 1029,
1076, 1111, 1200, 1350, 1382, 1404, 1468, 1534, 1608, 1736. 1H NMR (CDCl3)
(t, 3H, CH3, J = 7.3 Hz), 3.07 (t, 2H, CH2CO, J=6.8 Hz), 4.11 (q, 2H, OCH2CH3, J= 7.3 Hz),
4.64-4.67 (m, 2H, CH2N), 7.56-7.59 (m, 3H, Ar), 7.71-7.73 (m, 2H, Ar). 13C NMR (CDCl3)
δ ppm: 1.21
δ
ppm: 14.02, 33.57, 43.53, 61.33, 123.81, 128.87, 129.29, 131.31, 154.61, 169.71. HRMS
+
[M+H]+ m/z calcd for C12H15N4O2 247.1195 found 247.1390.
ethyl 3-[5-(4-methylphenyl)-1H-tetrazol-1-yl]propanoate 5b. Yield 10%, oil. IR (film, cm-
1
1) 946, 1024, 1108, 1196, 1372, 1417, 1452, 1464, 1480, 1620, 1732. H NMR (CDCl3)
δ

ACCEPTED MANUSCRIPT
ppm: 1.21 (t, 3H, CH3, J = 7.1 Hz), 2.45 (s, 3H, CH3Ar), 3.06 (t, 2H, CH2CO, J= 6.8 Hz),
4.11 (q, 2H, OCH2CH3, J = 7.1 Hz), 4.64-4.67 (m, 2H, CH2N), 7.36-7.38 (m, 2H, Ar), 7.60-
7.62 (m, 2H, Ar). 13C NMR (CDCl3)
δ
ppm: 14.03, 21.51, 33.61, 43.49, 61.31, 120.81,
+
128.71, 129.99, 141.77, 154.63, 169.71. HRMS [M+H]+ m/z calcd for C13H17N4O2 261.1352
found 261.1302.
ethyl 3-[5-(2-chlorophenyl)-1H-tetrazol-1-yl]propanoate 5d. Yield 28%, oil. IR (film, cm-
1) 948, 1036, 1075, 1112, 1130, 1196, 1350, 1379, 1408, 1440, 1460, 1532, 1571, 1604, 1736.
1H NMR (CDCl3)
δ
ppm: 1.21 (t, 3H, CH3, J = 7.3 Hz), 3.02 (t, 2H, CH2CO, J= 6.8 Hz), 4.09
13
(q, 2H, OCH2CH3, J = 7.3 Hz), 4.47 (t, 2H, CH2N, J= 6.8 Hz), 7.45-7.58 (m, 4H, Ar). C
NMR (CDCl3)
δ ppm: 13.99, 33.35, 43.35, 61.26, 123.71, 127.53, 130.21, 132.13, 132.68,
133.82, 152.93, 169.61. HRMS [M+H]+ m/z calcd for C12H14N4O2Cl+ 281.0805 found
281.1228.
ethyl 3-(5-phenyl-2H-tetrazol-2-yl)propanoate 6a. Yield 86%, oil. IR (film, cm-1) 924, 946,
1028, 1041, 1073, 1192, 1360, 1372, 1404, 1452, 1464, 1534, 1736. 1H NMR (CDCl3)
1.26 (t, 3H, CH3, J = 7.3 Hz), 3.11-3.13 (m, 2H, CH2CO), 4.19 (q, 2H, OCH2CH3, J=7.3 Hz),
4.93-4.96 (m, 2H, CH2N), 7.46-7.50 (m, 3H, Ar), 8.12-8.14 (m, 2H, Ar). 13C NMR (CDCl3)
δ ppm:
δ
ppm: 14.10, 33.58, 48.53, 61.27, 126.81, 127.30, 128.86, 130.32, 165.19, 169.62. HRMS
[M+H]+ m/z calcd for C12H15N4O2 247.1195 found 247.1148.
+
ethyl 3-[5-(4-methylphenyl)-2H-tetrazol-2-yl]propanoate 6b. Yield 80% solidifying oil. IR
1
(nujol, cm-1) 936, 1024, 1045, 1070, 1115, 1192, 1407, 1449, 1538, 1560, 1617, 1744. H
NMR (CDCl3)
δ ppm: 1.26 (t, 3H, CH3, J = 7.3 Hz), 2.41 (s, 3H, CH3Ar), 3.10-3.13 (m, 2H,
CH2CO), 4.19 (q, 2H, OCH2CH3, J = 7.3 Hz), 4.92-4.95 (m, 2H, CH2N), 7.28-7.29 (m, 2H,
13
Ar), 8.01-8.02 (m, 2H, Ar). C NMR (CDCl3)
δ
ppm: 14.10, 21.47, 33.60, 48.47, 61.26,
+
124.50, 126.73, 129.55, 140.48, 165.27, 169.65. HRMS [M+H]+ m/z calcd for C13H17N4O2
261.1352 found 261.1302.
ethyl 3-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]propanoate 6c. Yield 85%, colorless crystals
1
mp 49-50°C. IR (nujol, cm-1) 1004, 1016, 1041, 1092, 1172, 1404, 1420, 1608, 1736. H
NMR (CDCl3)
2H, OCH2CH3, J= 7.3 Hz), 4.93-4.96 (m, 2H, CH2N), 7.44-7.47 (m, 2H, Ar), 8.05-8.08 (m,
2H, Ar). 13C NMR (CDCl3)
ppm: 14.15, 33.51, 48.60, 61.32, 125.79, 128.10, 129.09,
δ ppm: 1.26 (t, 3H, CH3, J = 7.3 Hz), 3.12 (t, 2H, CH2CO, J= 6.8 Hz), 4.19 (q,
δ
129.25, 136.38, 164.31, 169.57. HRMS [M+H]+ m/z calcd for C12H14N4O2Cl+ 281.0805
found 281.0549.

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ethyl 3-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]propanoate 6d. Yield 47%, oil. IR (film, cm-
1) 946, 1036, 1064, 1095, 1127, 1192, 1350, 1375, 1398, 1423, 1444, 1461, 1519, 1576, 1601,
1736. 1H NMR (CDCl3)
δ ppm: 1.25 (t, 3H, CH3, J = 7.1 Hz), 3.14 (t, 2H, CH2CO, J=7.1 Hz),
4.19 (q, 2H, OCH2CH3, J = 7.1 Hz), 4.98-5.01 (m, 2H, CH2N), 7.36-7.24 (m, 2H, Ar), 7.52-
13
7.54 (m, 1H, Ar), 7.93-7.95 (m, 1H, Ar). C NMR (CDCl3)
δ ppm: 14.09, 33.54, 48.66,
61.29, 126.40, 126.89, 130.83, 131.09, 131.31, 133.05, 163.37, 169.58. HRMS [M+H]+ m/z
calcd for C12H14N4O2Cl+ 281.0805 found 281.0425.
Antifungal Activity Assay against C. albicans
A susceptibility of planktonic cells of C. albicans reference strain 90028 to novel tetrazole
derivatives was determined using M27-A3 method [17]. The final inoculum of 1.8x103
cells/mL was prepared in the synthetic RPMI 1640 medium at pH 7.0 buffered with
morpholinepropanesulfonic acid (MOPS). Compounds’ concentrations ranging from 0.0313
to 16 µg/mL were prepared with stock solution of compounds (1600 µg/mL) dissolved either
in 40% ethanol (3c, 4c, 6c-d), or 50% (6a-b, 5a-b, 5d, 3a, 2a, 2d, 4a-b, 4d) and 70% DMSO
(3b, 3d). 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 – clear endpoint). Microtiter plates were incubated at 35°C without
agitation for 48 h. After incubation, the growth of cells was measured by microtiter plate
reader Spark Control M10 (Tecan Group Ltd., Austria). Minimal inhibitory concentration
(MIC) was defined as the lowest concentration of the compounds that yielded no visible
growth. The endpoint was calculated as a 100% reduction in OD405 as compared to the growth
in control wells. Percentage of the cell growth inhibition was calculated according to the
following formula: % of inhibition = 100 - (OD405 CTW - OD405 SCW)/(OD405 GCW - OD405
SCW) [18]. CTW were prepared with compound, medium and inoculum; SCW wells
contained compound, RPMI 1640 medium and sterile water replacing inoculum; GCW were
prepared with inoculum, RPMI 1640 medium and the same amount of DMSO/ethanol used in
CTW.
The minimal fungicidal concentration (MFC) was determined as described previously [16, 19-
20]. Briefly, 50 µL aliquots from the selected wells of plates were removed after 48 h of
incubation at 35°C. Next, after 1/10,000 dilutions, 100 µL aliquots were placed onto agent-
free Sabouraud dextrose agar plates and incubated at 35ºC for 48 h. The MFC results for each
tetrazole concentration tested were defined as the reduction viability record (R) and calculated
using the formula: R = lg CFU/mL control cells – lg CFU/mL tetrazole treated cells. The

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lowest tetrazole concentration demonstrating a minimum 103 reduction in viable count (lg R ≥
3) was defined as MFC [16, 19-20].
Cytotoxicity Assay
To test the in vitro cytotoxicity, the lead fungistatic compounds: 2d, 4b, and 6a derived from
different chemical syntheses were screened. In order to discriminate three cytotoxicity
potency values (increasing, optimal, and decreasing) the following concentrations were tested:
256, 16 and 0.0313 µg/mL. Briefly, cytotoxicity against Caco-2 cells (ATCC HTB-37, LGC,
Poland) using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide
(XTT, Roche Diagnostics GmbH, Mannheim, Germany) was evaluated as described in [21].
Each well was treated with 50 µL of XTT reagent for 4-6 h. The concentration at which the
cell viability had dropped by 50% was recorded as the cytotoxic concentration (CC50) [22].
The optical densities at 475 nm (660 nm reference wavelength) were measured using a plate
reader Spark Control M10 (Tecan Group Ltd., Austria).
Additionally, based on the in vitro cytotoxicity data, the leading agents: 2d and 6a, displaying
the best cytotoxic efficiency against Caco-2, were tested in the G. mellonella-based model in
vivo. The larvae were injected with the RPMI-obtained compounds’ concentrations of 256, 16
and 0.0313 µg/mL, with the mixture free of the Candida suspension, as described previously
[23]. A group of 10 larvae were injected with the above compounds’ concentrations in
parallel. Then, the larvae were placed in 9.2 cm Petri dishes, kept at 37°C in darkness up to 96
h, and monitored daily. As larvae’s response to the RPMI injection is comparable with the
PBS one (both non-toxic) [24], in the experiment we included only PBS. Thus, the PBS-
inoculated larvae were used as the controls, to ensure that the death was not due to injection
trauma. Insects were considered dead when they did not respond to physical stimuli [22].
Additionally, the larvae health status was evaluated by assessing larvae mobility, cocoon
formation, melanization and survival. The each attribute contributing to an overall health of
an individual larva was scored and health index was calculated as described previously [14].
No significant difference in the larval survival between the tetrazole-treated larvae and control
group would indicate that the tested agent is not toxic at the examined doses [25].
Assessment of the Fractional Inhibitory Concentration (FIC)
To determine the respective interactions of compounds 2d, 4b, or 6a with Fluconazole (Flc),
the microdilution assays were carried out following the method M27-A3 [17] in the following
endpoint criterion [20]: total inhibition – the compound concentration yielding 100% growth

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inhibition. Fluconazole (Sigma–Aldrich, USA) stock solution (5120 µg/mL) was prepared
with water and subsequently final concentrations were diluted with the RPMI medium.
Fluconazole was tested against C. albicans according to the method M27-A3 [17]. The
fractional inhibitory concentration (FIC) of each compound tested and their total FIC indexes
were determined by using a checkerboard assay [23, 26]. Briefly, the checkboards were
prepared by using serial dilutions of Flc (80-1.25 µg/mL) in the horizontal wells and
compounds: 2d, 4b, or 6a respectively at the final concentrations from 80 to 1.25 µg/mL in
the vertical wells. Yeast suspension was prepared according to the method M27-A3 [17] and
100 µL was inoculated into the each well of a 96-well microtiter plate. 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 Control M10, Tecan Group Ltd., Austria) after 18
and 48 h. Additionally, the combined activity of tetrazoles: 2d, 4b, or 6a respectively with Flc
against C. albicans’ metabolic activity was determined after 48 h using the metabolic
reduction assay (XTT, Roche Diagnostics GmbH, Mannheim, Germany), with reading at 475
nm (660 nm reference wavelength). The antifungal effect was measured by comparing the
reduction in the mean absorbance of the compound-treated well to that of untreated growth
control [27]. In order to characterize the interaction of each combination tested, the fractional
inhibitory concentrations (FICs) of each agent tested and their sums are used to calculate the
FIC index for the cells’ metabolic reduction and endpoint (100% growth reduction). As
defined, the FIC index of <1 is the expression of the agents’ synergism, whereas the FIC
index of >1 represents an antagonism [23, 28].
Adhesion Assay
As the loss of adherence is demonstrated by the necrotic treatment we assessed the cell
adhesion inhibition under 2d, 4b, and 6a to Caco-2 monolayer as described in [22, 29]. For
the experiment, Caco-2 cells were seeded at the density of 1.2x105 cells/mL into 24-well-
plates (Corning, USA) and cultured in the EMEM medium (10 vol. % FCS, 1 mM pyruvic
acid; ATCC, USA) without antibiotics or antifungal agents for 18 h [29].
Yeast cells were grown for 18 h in YEPD at 30°C, and then pre-treated with 2d, 4b, and 6a,
respectively. Two hundred microliters of blastoconidial suspension (at final density of
104/mL) were pre-incubated with 1,800 µL of the RPMI medium (containing selected
concentration of the compound) for 2 h on a shaker at 35°C. Then, cells were washed twice
with phosphate-buffered saline (PBS) and re-suspended in 2,000 µL of fresh RPMI medium.
The adherent endothelial Caco-2 cells were washed twice with PBS before incubation with

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blastoconidial suspension (pre-treated with compound) for 2 h at 37°C in a humidified
atmosphere with 5% CO2 (vol/vol). Afterwards, the non-adherent cells were removed by
standard rinsing. The Caco-2 cell line was lysed by adding sterile water resulting with C.
albicans cells’ recovery. After 18-h growth on the Sabouraud dextrose agar plates at 30°C, the
number of adherent cells was determined by colony counting and compared with the controls.
The adhesion of the untreated cells was assessed as described above (but without antifungal
compound). Adherence was expressed as a percentage of the total number of cells added
(control cells) [22, 29].
Test with Sorbitol
Since both 2d and 6a were the leading tetrazole derivatives with the anticancer
efficiency and reduced toxicity, their MICs in the presence of an osmotic protector were
determined using the method M27-A3 [17] and previously described protocols [16, 30].
Sabouraud dextrose broth (SDB) medium was added to the each well of the microplate and
the serial dilutions of tetrazole derivatives (1.04–400 µg/mL) were carried. Afterwards, 100
µL of C. albicans inoculum (1.7x103 CFU/mL) prepared in Sabouraud dextrose broth medium
supplemented with sorbitol at final concentration of 0.8 M (Sigma-Aldrich, USA) was
transferred to wells as described previously [16, 30]. Plates were incubated at 35°C and then
read visually and spectrophotometrically at 405 nm (Spark Control M10, Tecan Group Ltd.,
Austria) after 2 and 7 days.
C. albicans Biofilm Cell-Death Assessment with Nuclear Staining Techniques under
Confocal Laser Scanning Microscope
Since 6a reduced significantly (at p≤0.05) the adherence of C. albicans (initial step of
biofilm formation) to Caco-2, the compound was then used to assess the mature biofilm cell-
death. TR146 cells were cultured in D-MEM with 10% of fetal bovine serum (FBS) and 0.1%
of gentamicin solution (50 mg/mL) at 37°C and 5% of CO2. For the experiment, TR146 cells
were seeded at the density of 1.2x105 cells/mL into 12-well-plates with polycarbonate filter
inserts (Corning, USA). To assess the influence of 6a on the C. albicans biofilm formation,
the TR146 cell line after 21-day post seeding was inoculated with 1.0-1.2x105/mL of the log-
phase yeast cells of 90028 ATCC. Afterwards, the biofilms were pre-treated with 6a at 256,
16, or 0.0313 µg/mL separately for 18-48 h (37°C in 5 vol % CO2). After double washing
with PBS, we separately used three staining methods that provided the illustration of cells’
abnormality and their content in fluorescence microscopy. Accordingly, propidium iodide (PI,
5 µg/mL, Roche Diagnostics GmbH, Mannheim, Germany) was used as a marker for
degenerating cells (without fixation). Acridine orange (Sigma, USA) staining was elaborated

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to show DNA denaturation in situ in the apoptotic cells under the tested tetrazole derivatives.
Additionally, the cells treated with tetrazole derivatives were stained with Calcofluor white
(10 mg/mL, Sigma Aldrich, USA) to depict chitin content according to the manufacturer’s
instruction. The influence of 6a on the C. albicans biofilm generated on TR146 was assessed
using the Olympus FLUOROVIEW FV1000 confocal laser scanning microscope CLSM
(Olympus, USA). Images were assembled using the Photoshop software (Adobe Photoshop
CS3 Extended, France).
Additionally, a Comet assay was used to detect DNA damage in the 6a-pretreated cells and
suspended in 1% melted agarose gel, cast on microscope slides and immersed in alkaline
detergent (2.5 M NaCL, 100 mM Na2EDTA, Tris-HCL, 1% Na-sarcosinate, pH 10) following
gelling in order to lyse cells. The experiment was prepared according to the protocol
described by Olive and Banáth [31] subsequently including: low-gelling-temperature agarose,
slide precoating, 6a-treated and untreated samples, and alkaline lysis followed by
electrophoresis (5 V/cm for 5 min) under alkaline conditions, consecutively by DNA staining
with fluorescent ethidium bromide generating red emission signals under the fluorescence
microscope CLSM (Olympus, USA). This method was confirmed by the detection of
apoptosis in the 6a-treated cells with the technique that involves the extraction of nuclear
DNA and assessment of the oligonucleosomal ladder with gel electrophoresis [32-33].
Briefly, the extracted DNA from the 6a-treated and untreated cells was loaded on 1.5%
agarose gel containing 1.4 µg/mL ethidium bromide. Additionally, DNA denaturated at 100°C
for 10 min was included as a negative control. The gel was examined and photographed by
the ultraviolet gel documentation system (iNTAS, Goettingen, Germany).
Flow Cytometry Analysis
Additionally, flow cytometry technique was adapted for the aborted apoptosis measurement.
Generation and labelling of Candida protoplasts with the Annexin-V-FLUOS method was
performed as described in [34]. In compliance with the statement of the cell wall
impermeability for Annexin-V and our prediction that tetrazoles 2d and 6a interact with the
plasma membrane, we generated protoplasts followed by their sensitivity to 5% SDS lysis as
described previously [34]. After the pre-treatment of the protoplasts with 6a or 2d, they were
stained with Fluorescein-labelled (FITC) annexin-V and propidium iodide (PI) using the
Annexin-V-FLUOS Staining Kit (Roche Diagnostics GmbH, Mannheim, Germany). The
annexin-V and (PI) status of a minimum of 5,000 protoplasts was recorded for each treatment
and the measurement was repeated in three independent experiments. Flow cytometry

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analyses were run on a FACSCanto II flow cytometer (BD Biosciences, San Jose CA, USA),
and analyzed using the BD FACSDiva software.
Efficacy of 6a in G. mellonella Infected with C. albicans
Since solely 6a was active against the initial and mature biofilm formation, appearing in the
mucosal candidiasis in vivo [24], this agent was used in further studies, as described below.
The compound was tested in the lowest most effective anti-biofilm concentration, namely 16
µg/mL. The later concentration was obtained by dilution of the stock conc. of 1600 µg/mL in
the RPMI medium. Larvae were accordingly selected by weight (195±17 mg) and disinfected
using ethanol 70% (vol/vol). The larvae killing assays were performed as described in [22].
Briefly, the larvae were injected with 10 µL of C. albicans suspension into the last right
proleg using a Hamilton syringe. Afterwards, larvae were injected with 10 µL of single
treatment dose of 6a (at 16 µg/mL RPMI) into last left proleg after 30 min or 1 h,
respectively. To assess the ability of 6a to protect larvae against subsequent C. albicans
infection, single treatment doses of the compound were given 1 h or 30 min prior inoculation.
The larvae injected with C. albicans or PBS instead of 6a were used as the positive or
negative control respectively. The larvae survival was monitored every 24 h for 2 days. The
health index was calculated as described previously [14]. At least three independent
experiments were performed; with experimental values expressed as means±standard error of
the mean (SEM) [14].
Test with Ergosterol
6a’s and 2d’s interaction with exogenous ergosterol (Sigma-Aldrich, St. Louis, MO, USA)
was determined using the method M27-A3 [17] with a few modifications. The following four
compounds were tested: 6a, 2d, Itraconazole (ITR), and Amphotericin B (AmB), respectively
at the range of concentrations from 512 to 1 µg/mL (tetrazole and ITR) and from 125 to 0.24
µg/mL (AmB). Briefly, the amount of 200 µL of the tested compound at the concentration of
1.024 µg/mL or 250 µg/mL was put into the first column of the microtiter plate.
Simultaneously, RPMI (100 µL) was added to the tested wells except for the wells of the first
column. Then, the compound was serially diluted and subsequently it was removed (100 µL)
from the tenth well. The final inoculum of yeast suspension recommended by M27-A3 (103
CFU/mL) was prepared in RPMI supplemented with ergosterol and distributed to the wells in
the volume of 100 µL, giving ergosterol’s final concentration of 400 µg/mL in the tested
wells. The ergosterol had been prepared directly before the experiment. It was dissolved in
10% DMSO (v/v), and 1% Tween 80 (v/v), heated to augment the solubility, and then diluted

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with the RPMI medium. ITR and AmB were tested as positive controls, and yeast growth and
sterility were also controlled. The plates were incubated at 35°C and then read after 72 h. This
assay was carried out in triplicate and then repeated on three different days. The geometric
mean values were calculated for each experiment. MIC was determined as the lowest
concentration of the compound which inhibited the visible fungal growth.
Statistical Analysis
Each experiment was performed at least three times on three different occasions; with
the experimental values expressed as means±SD. Statistical differences between the control
and test values were determined by means of the Wilcoxon signed-rank matched-pair test.
The p values ≤ 0.05 were considered to be statistically significant.
Results
Chemistry
A chemical synthesis involved preparation of tetrazole derivatives including 1-(3-
chloropropyl)-5-aryltetrazoles 2a-d and 3a-d, tetrazole derivatives containing morpholine
moiety 4a-d and propionate esters substituted by various 5-arylterazole 5a-d and 6a-d. 5-
Aryltetrazole 1a-d required for these syntheses were prepared according to the described
method [35]. Compounds 2a-d and 3a-d were obtained by N-alkylation of appropriate 5-
aryletrazole 1a-d with 1-bromo-3-chloropropane (Fig. 1). The reaction was carried out in the
presence of KOH in solution of acetonitrile and MeOH at reflux. The progress of the reaction
was monitored by thin-layer chromatography.
Cl
N
N
N
N
Y
N
N
NH
N
N
KOH, MeOH
CH3CN, reflux
N
N
N
+
+
Cl
Br
X
X
Y
X
Y
1a-d
X
2a-d
3a-d
Cl
Y
a - H
b - CH3
c - Cl
H
H
H
d - H Cl
Fig. 1. N-alkylation of 5-aryltetrazole 1a-d
In all cases the formation of two isomers, 1,5- and 2,5-disubstituted tetrazoles, was observed.
The reaction was highly regioselective and preferential alkylation at the 2-position of tetrazole
ring took place (Table 1). The weight ratio of isomers 2a-d to 3a-d depended on the aryl
substituent at 5-position of tetrazole ring. The highest regioselectivity (isomers ratio 50:1) was
observed for N-alkylation of 5-(4-chlorophenyl)-1H-tetrazole 1c, whereas the lowest for N-

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alkylation of 5-(2-chlorophenyl)-1H-tetrazole 1d (isomers ratio 3:1). Furthermore, depending
on the aryl substituent at tetrazole ring the reaction took place in various time ranging from
3.5 to 5.5 h indicating the dependency of the reaction rate from steric congestion (Table 1).
Table 1 Reaction time, total yields and isomers ratios of tetrazole derivatives 2a-d to 3a-d
Compound X
Y
H
H
H
Time (h) Yield (%) Isomers ratio (2:3)
2a + 3a
2b + 3b
2c + 3c
2d + 3d
H
3.5
3.5
4.5
60
62
67
73
7:1
CH3
Cl
H
15:1
50:1
3:1
Cl 5.5
Isomers 2a-d and 3a-d were separated and purified on a column chromatography on silica gel
and obtained with satisfactory total yields of 60-73%. It is worth pointing out that the
regioselective alkylation of tetrazoles was previously mentioned in the work concerning
synthesis of compound 3a (authors did not state the isomers ratio) [36], and in other works
[37-38].
In order to obtain more complex molecules and study the structure-activity relationships
chlorine atom in tetrazole derivative 3a-d was replaced by morpholine moiety. Thus
disubstituted tetrazole derivatives 3a-d were used as alkylating agents in N-alkylation of
morpholine (Fig. 2). The reaction was performed in the presence of K2CO3, in acetonitrile at
reflux. The progress of the reaction was monitored on TLC plates using chloroform/methanol
as the eluent.
O
Cl
N
N
N
N
Y
N
Y
O
N
N
CH3CN, K2CO3
reflux
+
N
N
N
H
X
X
3a-d
4a-d
Fig. 2. Synthesis of tetrazole derivatives bearing morpholine moiety 4a-d
Regardless of the substrate the 100% conversion was reached after 168 h. All products were
purified on a column chromatography on silica gel and obtained with high yields ranging
from 75 to 87% (Table 2).

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Table 2 Reaction time and yields of synthesis of tetrazole derivatives containing morpholine
moiety 4a-d
Compound X
Y
H
H
H
Cl
Time (h) Yield (%)
H
75
4a
4b
6c
4d
CH3
Cl
H
83
168
77
87
The third group of synthesized tetrazole derivatives were propionate esters 5a-d and 6a-d.
These compounds were obtained by Michael-type addition of 5-aryltetrazole 1a-d to ethyl
acrylate (Fig. 3). The reaction was carried out in 2-propanol, in the presence at Et3N at reflux.
The progress of the reaction was controlled using TLC plates.
N
N
N
N
X
Y
O
5a-d
O
N
N
Y
NH
2-propanol
O
N
+
+
Et3N, reflux
O
X
O
N
N
1a-d
N
O
N
X
Y
6a-d
Fig. 3. Michael-type addition of 5-aryltetrazole 1a-d to ethyl acrylate
As in the case of N-alkylation of 5-arylterazole 1a-d with 1-bromo-3-chloropropan, these
reactions also took place regioselectively and formation of 2,5-disubstituted tetrazole 6a-d
was preferred, but not exclusive. The weight isomers ratios were summarized in Table 3.

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Table 3 Reaction time, total yields and isomers ratio of tetrazole derivatives 5a-d to 6a-d
Compounds X
Y
H
H
H
Time (h) Yield (%) Isomers ratio (5:6)
5a + 6a
5b + 6b
6c
H
168
95
90
85
75
9.2:1
8.6.1
1:0
CH3
Cl
H
168
162.5
5d + 6d
Cl 162.5
2.6:1
1,5-Disubtiuted tetrazoles, were separated and purified in three cases (5a, 5b, 5d). Isomer 5c
was formed in trace amounts, which was sufficient only for HRMS analysis, therefore the
isomers ratio was calculated as 1:0.
Biological Evaluation
Terazole Derivatives as Inhibitors of C. albicans Growth without Fungicidal Properties
The antifungal screening revealed that all the synthesized tetrazole derivatives were active
against C. albicans, demonstrating the moderate-to-good cell growth inhibition at 0.0313-16
µg/mL (Table 4). Additionally, four compounds (3c, 4c, and 6c-d) fully inhibited the growth
of C. albicans (100% cell reduction) at 16 µg/mL after 18 h. This showed small structure-
activity relationships, the four compounds are 2,5-disubstituted tetrazoles and all contain
chlorine substituent at the benzene ring. The substituent types at N-1 or N-2 of the tetrazole
ring exhibited little antifungal effect of the synthesized derivatives. Neither clear endpoint nor
fungicidal activity (R≥3 lg) (Table 5) were observed after 48-h incubation of the reference C.
albicans cells with the tested compounds in the microdilution method M27-A3. No visual
MIC was observed either after 18 or 48 h of the yeast cells incubation with each tetrazole
derivative.

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Table 4 Antifungal Activity (Cells Inhibition %; Means±SD) of Tetrazole Derivatives Against the Reference C. albicans Strain ATCC 90028
Concentration (µg/mL)a
Incubation
Compound
time (h)
18
16
8
4
2
1
0.5
0.25
0.125
0.0625
0.0313
98.95±0.49 98.57±0.29 98.66±0.49 95.82±0.45 96.69±0.22 97.53±0.22 94.68±0.35 98.12±0.42 99.45±0.24 99.65±0.27
99.05±0.29 98.81±0.11 98.90±0.23 97.23±0.14 98.80±0.07 98.82±0.11 98.19±0.11 99.16±0.18 99.12±0.17 99.02±0.27
2a
2d
3a
3b
3c
3d
4a
4b
4c
4d
5a
48
18
99.62±0.30 99.44±0.21 99.62±0.24 97.40±0.39 93.49±0.06 98.40±0.37 99.96±0.01
100±0.02
99.90±0.07 99.51±0.40
48
99.39±0.44 99.06±0.15 99.41±0.46 99.14±0.44 98.22±0.33 99.22±0.47 99.62±0.24 99.43±0.32 99.34±0.06 99.38±0.45
99.36±0.22 99.54±0.31 99.53±0.19 99.25±0.20 62.02±0.27 99.10±0.25 98.55±0.36 99.65±0.09 99.99±0.00 99.51±0.40
99.22±0.21 99.63±0.45 99.29±0.33 99.57±0.44 97.60±0.22 99.16±0.49 99.16±0.55 99.19±0.29 99.76±0.18 99.38±0.27
98.46±0.08 99.06±0.31 99.63±0.19 98.39±0.20 99.60±0.33 97.59±0.46 99.45±0.45 98.66±0.14 99.06±0.46 98.95±0.39
98.26±0.05 99.08±0.18 98.84±0.10 98.87±0.18 98.65±0.18 98.95±0.08 98.79±0.09 98.92±0.05 99.12±0.14 98.94±0.04
18
48
18
48
18
100±0.02
98.47±0.04 99.02±0.05 99.22±0.02 91.33±0.02 96.41±0.08 99.99±0.002 99.94±0.05 99.94±0.04 96.23±0.13
48
98.74±0.14 98.75±0.19 98.93±0.15 99.24±0.38 99.10±0.20 98.74±0.12 99.04±0.16 98.90±0.09 98.83±0.09 99.00±0.04
94.16±0.30 98.90±0.40 99.32±0.43 95.00±0.41 98.99±0.11 98.90±0.41 99.95±0.02 99.62±0.23 98.61±0.21 99.97±0.02
98.34±0.13 99.05±0.19 98.50±0.06 98.60±0.11 98.69±0.06 98.89±0.08 98.96±0.11 99.13±0.08 99.03±0.08 99.17±0.05
97.02±0.11 99.94±0.03 99.64±0.03 85.92±0.02 80.42±0.18 99.52±0.04 99.96±0.06 99.81±0.07 99.93±0.07 94.63±0.02
99.31±0.20 98.82±0.04 99.15±0.20 98.84±0.12 98.87±0.08 99.59±0.08 99.13±0.20 97.95±0.03 99.55±0.03 98.98±0.03
99.87±0.11 99.97±0.02 99.75±0.16 95.72±0.11 57.60±0.30 98.99±0.46 98.25±0.35 99.57±0.21 99.50±0.34 99.87±0.09
99.44±0.78 99.39±0.38 99.45±0.46 98.30±0.07 96.58±0.05 99.18±0.48 98.68±0.18 98.96±0.44 99.03±0.34 99.56±0.61
18
48
18
48
18
48
18
100±0.02
99.35±0.21 98.81±0.04 98.97±0.11 99.61±0.17 99.36±0.22 99.32±0.33 99.24±0.29 98.92±0.11 99.28±0.30 99.12±0.29
99.64±0.16 98.97±0.53 98.62±0.23 92.76±0.30 98.01±0.47 98.11±0.37 90.79±0.47 98.25±0.24 98.70±0.14 100±0.03
96.30±0.09 98.10±0.15 98.31±0.02 98.79±0.01 92.34±0.18 99.91±0.07 99.95±0.05
100±0.01
97.92±0.05
48
18
48
99.20±0.23 98.97±0.31 98.91±0.05 97.16±0.12 99.02±0.34 98.83±0.09 98.01±0.17 98.94±0.18 99.00±0.15 99.12±0.08
99.13±0.37 99.40±0.18 98.44±0.43 90.79±0.18 95.93±0.44 98.87±0.50 95.76±0.43 99.99±0.01 99.37±0.33 99.69±0.31
98.96±0.10 99.02±0.13 98.91±0.17 97.06±0.06 98.87±0.08 98.96±0.18 98.01±0.20 99.01±0.05 99.26±0.28 99.23±0.24
18
48

ACCEPTED MANUSCRIPT
18
48
18
48
18
48
18
48
18
48
18
48
98.97±0.25 98.51±0.39 99.94±0.03 89.93±0.16 98.46±0.26 98.88±0.30 95.87±0.44 99.61±0.09 99.59±0.29 99.74±0.29
98.82±0.21 98.86±0.10 99.01±0.22 97.05±0.10 99.04±0.10 99.08±0.07 98.72±0.37 98.94±0.01 99.11±0.22 99.34±0.08
99.55±0.02 99.99±0.00 99.90±0.01 92.75±0.03 87.68±0.17 97.11±0.12 99.78±0.15 99.93±0.06 99.98±0.01 99.33±0.00
99.28±0.26 99.17±0.23 99.25±0.08 99.07±0.04 99.17±0.23 99.06±0.14 99.02±0.16 99.24±0.05 99.19±0.23 99.20±0.26
99.95±0.04 99.01±0.42 99.96±0.02 99.35±0.12 85.06±0.29 98.97±0.24 98.40±0.33 99.13±0.46 99.57±0.20 99.34±0.06
99.65±0.30 98.86±0.25 99.68±0.48 99.70±0.14 97.51±0.04 99.31±0.43 98.70±0.08 99.18±0.49 99.12±0.15 99.12±0.11
99.98±0.005 99.61±0.15 98.99±0.34 98.19±0.30 45.15±0.50 99.07±0.43 98.38±0.38 99.76±0.05 99.51±0.19 99.31±0.43
99.37±0.43 99.14±0.57 98.95±0.33 98.95±0.55 96.34±0.36 99.16±0.53 98.95±0.30 98.72±0.17 98.79±0.22 98.81±0.23
5b
5d
6a
6b
6c
6d
100±0.03
98.78±0.09 98.86±0.11 99.07±0.10 99.12±0.16 98.78±0.08 99.00±0.07 98.87±0.07 98.85±0.13 98.80±0.17 99.14±0.12
100±0.03 99.23±0.04 100±0.00 99.98±0.01 99.38±0.01 96.86±0.05 99.86±0.16 99.97±0.03 99.86±0.05 97.51±0.10
98.83±0.14 98.84±0.17 99.05±0.16 99.41±0.31 98.86±0.12 98.84±0.14 99.09±0.14 98.82±0.09 98.78±0.06 99.27±0.29
97.97±0.07 99.85±0.02 99.74±0.02 84.36±0.03 98.32±0.04 99.95±0.04 99.92±0.09 99.88±0.14 99.32±0.02
Legend: aAmphotericin B at the concentration of 0.5 µg/mL was used as a control (inhibition % = 100).

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Table 5. Fungicidal Activity of Tetrazole Derivatives against C. albicans 90028
C. albicans (CFU x 105) after
Untreated starting
Logarithm
reduction (R) of C.
albicans cells
treatment with tetrazole derivative at
inoculum of C. albicans
90028 ATCC (CFU x 105)
Compound
the concentration of 16 µg/mL
(Growth control cells)
(Tetrazole treated cells)
176
83
115
115
115
565
439
565
115
115
439
115
115
115
115
115
115
439
439
-0.18
0.18
-0.23
0.65
0.26
0.51
-0.34
0.56
0.34
-0.08
0.16
-0.54
-0.01
0.33
-0.23
0.29
0.19
2a
2d
3a
3b
3c
3d
4a
4b
4c
4d
5a
5b
5d
6a
6b
6c
6d
196
126
241
177
261
35
203
152
87
432
119
59
213
222
282
Legend: Tetrazole derivative reducing the C. albicans cells at least for lg R≥3 was defined as fungicidal.
Reduction was expressed as decimal log reduction separately using the formula: lg R = lg CFU/mL control cells
– lg CFU/mL tetrazole treated cells. The fungicidal activity was determined using the microdilution method
M27-A3. Accordingly, 50-µL aliquots from the wells of microplates containing tetrazole treated cells (PTW:
tetrazole at the concentration of 16 µg/mL and yeast suspension) and the growth control cells (GCW: solvent
DMSO or ethanol and yeast suspension) after 48-h incubation at 35°C were placed in volume of 100 µL onto
Sabouraud dextrose agar plates after 104 dilution in PBS and incubated at 35°C for the next 48 h.
In Vitro and In Vivo Cytotoxicity of Tetrazole Derivatives
Due to the similar antifungal activity of all synthesised tetrazole derivatives the
cytotoxicity of the following representative structures 2d, 4b, and 6a derived from three
different chemical syntheses was tested against the Caco-2 cell line in vitro after 18 h
incubation. The data are summarized in Table 6 and show a relationship between the structure
of the compound and the cytotoxicity. 2d at 256 µg/mL (CC50) was highly cytotoxic and
reduced the Caco-2 viability to 63%. 2d at 16 µg/mL displayed a 25%-reduction in viability
in vitro. 6a exhibited CC50 at 16 µg/mL against Caco-2 after 18-h treatment. Interestingly,
toxicity of 6a did not increase at the concentration of 256 µg/mL. Both 2d and 6a were non-

ACCEPTED MANUSCRIPT
toxic at the concentration of 0.0313 µg/mL. Morpholine derivative 4b did not reach CC50 at
the tested concentrations. The close structurally related molecules with similar values (3.06-
3.16) of log P (logarithm of the partition coefficient of inhibitor between n-octanol and water
according to ChemBioDraw Ultra 13.0 software) exhibit remarkable differences in
cytotoxicity. The replacement of the aliphatic chain with the morphine moiety lowered
cytotoxicity.
Accordingly, tetrazole derivatives 2d and 6a were chosen for the in vivo toxicity assay.
2d and 6a had no effect on the larval survival vs the PBS-treated control (p≤0.05), and were
therefore considered to be non-toxic at all the doses tested (Table 6).
Table 6 In vitro and in vivo Cytotoxicity of Tetrazole Derivatives 2d, 4b, and 6a
Cytotoxicity assay
In vitro
In vivo
Concentration
(µg/mL)
Compound
Toxicity of
Viability of
Caco-2 cells
(%)
Larvae
Larvae
Health
Score
9±0.40
9±0.60
10±0.00
nd
Tetrazole to Caco-2
Survival (%)
after 96 h
100±0.00
100±0.00
100±0.00
nd
cells (%)
256
16
63.27±0.22a
25.49±0.53
0.00±0.52
36.73±0.22
74.51±0.53
100±0.52
86.96±0.04
72.27±0.24
81.24±0.29
54.51±0.17
49.54±0.10
100±0.01
nd
2d
4b
6a
0.0313
256
13.04±0.04
27.73±0.24
18.76±0.29
45.49±0.17
50.46±0.10a
0.00±0.01
16
nd
nd
0.0313
256
nd
nd
100±0.00
100±0.00
100±0.00
100±0.00
9±0.60
10±0.20
10±0.00
10±0.00
16
0.0313
nd
PBS-treated control
a
Legend: nd stands for not determined; stands for cytotoxic effect (CC50) of tetrazole against Caco-2. The
analysis of Caco-2 cells’ viability was assayed after 18-h treatment with compounds 2d, 4b, and 6a respectively.
Toxicity of 2d and 6a to G. mellonella larvae was assessed after 96-h incubation. Data are expressed as the mean
± SD of three independent experiments. All samples were tested in duplicate.
Antifungal Activity of Tetrazole Derivatives in the Presence of Sorbitol
To provide osmotic protection during the treatment with tetrazole derivatives, the cells
were suspended in the isotonic RPMI medium containing sorbitol in the final concentration of
0.8 M. This allowed us to test whether exposure to sorbitol protects C. albicans against the
tetrazole-induced inhibition. The tetrazole 6a-induced growth inhibition was enhanced at 100
µg/mL notably after 7 days (Fig. 4 A, B). This result demonstrated the appearance of a surface
barrier defect that increases corresponding with 6a’s higher concentration. Compared with the
untreated control cells, the cells exposed to sorbitol displayed substantial protection against

ACCEPTED MANUSCRIPT
the 2d tetrazole-induced inhibition (Fig. 4 C, D). Notably, the MIC values of both tetrazole
derivatives tested, displayed no evident changes in the antifungal activity using sorbitol.
Contrariwise, the antifungal effect of 2d (especially at lower concentrations) was reversed in
the medium containing sorbitol. Thus it is possible to consider the fungal cell wall as a target
for the tested propionate esters.

ACCEPTED MANUSCRIPT
Fig. 4. Impact of Sugar Alcohol – Sorbitol (0.8 M) on the Antifungal Activity of Tetrazole Derivatives 2d and 6a Against C. albicans. Legend:
PTW +0.8 M Osm stands for tetrazole + yeast suspension + 0.8 M sorbitol; GCW stands for yeast suspension + solvent; PTW stands for tetrazole + yeast suspension. MIC of
2d and 6a assessed with and without Osm protector was unaffected. The tests were performed with the microdilution method M27-A3 in triplicate, and the plates were
incubated at 35°C for 7 days. Reading was performed after 2 and 7 days respectively.

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Tetrazole Derivatives Display Antagonism in Combination with Fluconazole
In the case of the tetrazole derivatives tested on their own, as described above, due to
the absence of a clear endpoint in the range of the concentrations tested using M27-A3
method, in order to calculate the FIC index the assumption was made of the endpoint as >16
µg/mL. As shown in Table 7, Flc alone had good antifungal effect towards the reference strain
displaying MIC and endpoint at 0.25 µg/mL (determined visually and spectrophotometrically
using the microdilution method M27-A3). Next, the effects of tetrazole derivatives 2d, 4b or
6a combined separately with Flc against C. albicans were investigated and the percentage of
growth inhibition was determined (Table 8). The endpoint value of Flc increased 5-fold when
combined with the partner tetrazole 2d. Surprisingly, the combination of 4b and 6a with Flc
respectively produced unidentified MICs. Nevertheless, taking the endpoint values as >80
µg/mL for Flc, 4b, and 6a, the FIC indexes of all the tested combinations were >1, thus
resulting in antagonism (data not shown). Subsequently, these results were confirmed by
testing the metabolic activity of C. albicans cells treated with tetrazoles combined with Flc
with the use of the XTT method (Table 9). When these agents were tested in their
combinations, Flc reduced the cells viability at >90% at 1.25 µg/mL. The active
concentrations of tetrazoles combined with Flc were respectively 20 µg/mL for 4b and 80
µg/mL for both 2d and 6a (50%-reduction in viability). Based on the XTT results, the
recalculated FIC indexes of all the combinations tested fit the definition of antagonism (Table
10).

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Table 7 Antifungal Activity of Fluconazole Against the Reference C. albicans Strain ATCC
90028
Time
(h)
Fluconazole (µg/mL)
64
32
16
8
4
2
1
0.5
100
100
0.25
0.125 0.0625 0.0313
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100 a
99.74
98.89
18
48
100 a 99.96 99.18
Legend: a indicates total growth inhibition named endpoint = 100% calculated based on the growth rate assessed
spectrophotometrically (OD405) and visually MICT; data are expressed as means ± SD.
Table 8 Growth Inhibition in the Checkerboard Microtiter Plate Testing the Combination of
Fluconazole with Partner Tetrazole Derivatives 2d, 4b, or 6a Against C. albicans ATCC
90028 After 48 h
Partner tetrazole
Fluconazole (µg/mL)
Concentration
Name
80
40
20
10
5
2.5
1.25
(µg/mL)
99.78
99.54
99.61
99.54
99.59
99.58
99.33
99.26
99.25
99.43
99.21
99.49
99.34
99.47
99.61
99.49
99.19
99.54
99.49
99.54
99.51
99.79
99.21
99.34
99.43
99.45
99.45
99.34
98.99
99.29
99.29
98.78
99.34
99.25
99.15
99.51
99.32
98.91
99.46
99.16
99.36
99.46
99.74
99.71
99.59
99.53
99.55
99.54
99.69
99.10
99.50
99.61
98.99
99.46
99.67
99.16
99.58
99.53
98.82
99.52
99.58
99.46
99.65
99.78
99.80
99.84
99.59
99.69
99.53
99.73
99.32
99.52
99.67
98.99
99.41
99.66
98.80
99.57
99.75
99.09
99.49
99.66
99.59
99.65
99.27
97.80
99.18
98.95
98.83
98.93
99.02
98.27
98.72
98.90
98.12
98.97
98.81
98.69
99.12
98.94
98.33
99.08
98.74
99.86
98.99
99.67
99.54
99.77
99.14
99.77
99.74
99.25
99.31
99.63
99.41
98.46
99.62
99.14
98.90
99.77
99.42
98.48
99.77
99.64
99.23
99.20
100a
98.77
99.97
99.93
99.57
99.56
100
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
80
40
20
10
5
98.75
99.79
99.86
97.81
99.72
99.79
98.77
99.71
100
98.95
99.79
99.71
99.02
99.81
2.5
1.25
a
Legend: indicates total growth inhibition 100% – endpoint. In case of 4b and 6a combination with Flc no
endpoint was identified. Therefore, endpoint was taken as the highest concentration of 80 µg/mL to calculate the
FIC index.

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Table 9 Metabolic Activity of C. albicans ATCC 90028 After 48 h Treatment with the
Combination of Fluconazole with Partner Tetrazole Derivatives 2d, 4b, or 6a
Metabolic activity (%) of C. albicans cells treated with agents’ combination
Partner tetrazole
Fluconazole (µg/mL)
Concentration
Name
80
40
20
10
5
2.5
1.25
(µg/mL)
53.22
73.44
40.00
63.62
66.33
55.99
81.77
69.42
94.76
80.34
89.30
77.26
77.27
88.19
71.92
80.42
84.01
72.63
62.42
100
89.73
100
70.42
50.83
73.11
74.10
54.14
54.82
67.85
61.69
57.34
87.40
93.01
70.81
73.79
93.31
67.66
68.53
86.25
67.39
60.65
100
73.98
11.87
52.64
70.86
28.26
56.45
68.01
46.94 a
59.93
75.42
99.02
76.18
66.60
81.48
69.35
64.55
85.87
71.01
62.08
100
100
100
100
100
100
100
99.55
100
100
100
100
100
100
100
100
100
100
95.38
100
100
100
59.43
78.22
64.31
75.66
26.03
61.61
81.50
71.88
80.61
79.69
100
2.60 a
100
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
2d
4b
6a
80
97.37
100
41.98 a
13.59
40.06
66.60
0.00
95.31
100
40
20
10
5
82.92
93.50
99.65
100
76.07
90.68
73.65
100
100
94.14
86.34
100
71.14
68.03
100
90.58
79.99
100
94.92
97.62
100
80.73
75.77
100
88.02
0.00
100
2.5
92.82
81.57
100
82.62
71.55
100
90.20
86.44
74.26
87.00
1.25
66.82
86.85
62.20
74.44
75.73
a
Legend: indicates inhibition of metabolic activity <50%; Because of the residual turbidity observed at supra –
endpoint of compounds’ combination (tetrazole 2d combined with Flc) indicating an incomplete metabolic
activity inhibition, accordingly, the lowest concentration could therefore not be used as an endpoint in this
experimental setting. We selected the highest concentration reducing metabolic activity of <50%.

ACCEPTED MANUSCRIPT
Table 10 Effects of Tetrazole Derivatives 2d, 4b, and 6a in Combination with Fluconazole
Against C. albicans Assessed using XTT and Growth Inhibition (Microdilution Method M27-
A3)
Combination
Total
FIC
MICa
No. of
MICb
(µg/mL)
1.25
80
Compound
FIC
combination
Effect
(µg/mL)
index
Fluconazole
2d
0.25
>16
0.25
>16
0.25
>16
5.0
>5.0
5.0
1
2
3
>10
>6.25
>10
Fluconazole
4b
10
antagonism
20
>1.25
5
Fluconazole
1.25
80
>5
6a
Legend: a indicates total growth inhibition (clear end point, MICTI); bstands for metabolic reduction; Total index
means sum up of FIC of Flc and appropriate tetrazole.
Impact of Tetrazole Derivatives 2d, 4b and 6a on Adhesive Properties of C. albicans
As shown in Table 11, the cells pre-treated with tetrazole derivatives 2d, 4b and 6a at
256 µg/mL showed significant changes in the adhesion properties to Caco-2, when compared
with the non-treated control (p≤0.05). Of these, 6a at 256, 16 and 0.0313 µg/mL inhibited
cells’ adhesion respectively as follows: 2.5-, 2.9- and 2.7-fold (p≤0.05, compared to non-
treated control). The remaining compounds 2d and 4b at 256 µg/mL significantly increased
the attachment of fungal cells to Caco-2 vs untreated counterparts (p≤0.05).