Primary discovery of 1-aryl-5-substituted-1H-1,2,3-triazole-4-carboxamides as promising antimicrobial agents

Article abstract of DOI:10.1016/j.molstruc.2021.131146

Three series of novel 1H-1,2,3-triazole-4-carboxamides: 1-aryl-5-alkyl/aryl-1H-1,2,3-triazole-4-carboxamides, 1-aryl-5-amino-1H-1,2,3-triazole-4-carboxamides and 1,2,3-triazolo[1,5-a]quinazoline-3-carboxamides were synthesized via base-mediated click azide reactions. Compounds were evaluated for their antimicrobial activities against primary pathogens: Gram-positive and Gram-negative bacterial strains Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, as well as fungal strain Cryptococcus neoformans var. grubii and Candida albicans. Compounds exhibiting moderate to good activities were selected for SAR analysis. Several 5-methyl-1H-1,2,3-triazole-4-carboxamides 4d, 4l, 4r, showed potent antibacterial effect against S. aureus. On the contrary, 5-amino-1H-1,2,3-triazole-4-carboxamide 8b and [1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide 9a were active against pathogenic yeast C. albicans. Thus, compound 4l under 1 μM demonstrated 50% growth inhibition against S. aureus. At the same concentration, the compound 9a killed approx. 40% of C. albicans cells. In general, these compounds demonstrated selective action and no significant impact on the viability of human keratinocytes of HaCaT line.

Full text of DOI:10.1016/j.molstruc.2021.131146

Journal of Molecular Structure 1246 (2021) 131146
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Primary discovery of
1-aryl-5-substituted-1H-1,2,3-triazole-4-carboxamides as promising
antimicrobial agents
Nazariy Pokhodyloa,∗, Nazar Mankoa,b, Nataliya Finiuka,b, Olha Klyuchivskab,
Vasyl Matiychuka, Mykola Obushaka, Rostyslav Stoikaa,b
a Ivan Franko National University of Lviv, Kyryla and Mefodiya Str., 6, 79005 Lviv, Ukraine
b Institute of Cell Biology of National Academy of Sciences of Ukraine, Drahomanov St., 14/16., 79005 Lviv, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Three series of novel 1H-1,2,3-triazole-4-carboxamides: 1-aryl-5-alkyl/aryl-1H-1,2,3-triazole-4-
carboxamides, 1-aryl-5-amino-1H-1,2,3-triazole-4-carboxamides and 1,2,3-triazolo[1,5-a]quinazoline-
3-carboxamides were synthesized via base-mediated click azide reactions. Compounds were evaluated
for their antimicrobial activities against primary pathogens: Gram-positive and Gram-negative bac-
terial strains Escherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa,
Staphylococcus aureus, as well as fungal strain Cryptococcus neoformans var. grubii and Candida al-
bicans. Compounds exhibiting moderate to good activities were selected for SAR analysis. Several
5-methyl-1H-1,2,3-triazole-4-carboxamides 4d, 4l, 4r, showed potent antibacterial effect against S. aureus.
On the contrary, 5-amino-1H-1,2,3-triazole-4-carboxamide 8b and [1,2,3]triazolo[1,5-a]quinazoline-3-
Received 2 June 2021
Revised 4 July 2021
Accepted 18 July 2021
Available online 22 July 2021
carboxamide 9a were active against pathogenic yeast C. albicans. Thus, compound 4l under
1 μM
demonstrated 50% growth inhibition against S. aureus. At the same concentration, the compound 9a
killed approx. 40% of C. albicans cells. In general, these compounds demonstrated selective action and no
significant impact on the viability of human keratinocytes of HaCaT line.
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
genes in Escherichia coli). Thus, inhibition of the SOS mechanism is
a potential way of blocking the formation of antibiotic resistance.
The increasing resistance of pathogenic microorganisms to
available antibiotics is a serious problem and the discovery of new
therapeutic agents and their targets is a big challenge [1, 2]. It
is worth noting that the formation of resistance to antibiotics is
the result of natural selection of the microorganisms. Two main
strategies for elaboration of new antibacterial agents exist: 1) de-
velopment and implementation of antibiotics that differ in struc-
ture and mechanism of action [3, 4]; 2) defining of the mecha-
nisms of formation and realization of the already existing drugs
and their further modification in order to slow down or stop drug
resistance [5]. One of the mechanisms responsible for the forma-
tion of the acquired antibiotic resistance is the bacterial DNA dam-
age response mechanism known as the SOS response induced by
many antibiotics [6, 7]. The SOS system is widespread among mi-
croorganisms and involves the action of many genes (more than 40
Scaffolds
ety, for
containing
1,2,3-triazole-4-carboxamide
moi-
example 5-amino-N-(3,4-dimethylphenyl)-1-(2-((4-
ethoxyphenyl)amino)-2-oxoethyl)-1H-1,2,3-triazole-4-carboxamide
A (GSK1010702A) (Fig. 1), have demonstrated good efficiency in
inhibiting SOS-dependent response [8]. In this regard, studies
of 1H-1,2,3-triazole-4-carboxamides appear to be promising for
screening their antimicrobial activity (Fig. 1). Recently, a series
of novel 1-(2,6-difluorobenzyl)-1H-1,2,3-triazole-4-carboxamides
bearing the piperazine motif (Fig. 1, B) were evaluated for
their antimicrobial activity and found to be efficient against
Gram-positive, Gram-negative bacterial strains (Escherichia coli,
Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus pyogenes,
Klebsiella pneumonia, Streptococcus aureus, Klebsiella terrigena),
as well as fungal strains (Candida albicans, Trichoderma viride,
Aspergillus flavus, and Aspergillus) [9]. Triazole-4-carboxamide C
analogue of N-coumaroyl-tyramine (Fig. 1) was a potent inhibitor
of biofilm formation by Gram-negative strain (Pseudoalteromonas
ulvae TC14) with EC50 close to ampicillin (EC50 = 11 μM) and
without toxic effect on bacterial growth even at high concen-
∗
Corresponding author.
E-mail address: pokhodylo@gmail.com (N. Pokhodylo).
https://doi.org/10.1016/j.molstruc.2021.131146
0022-2860/© 2021 Elsevier B.V. All rights reserved.

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
Fig. 1. Biologically active 1-aryl-1H-1,2,3-triazole-4-carboxamides.
trations (100 μM) [10]. The 1,2,3-triazole-4-carboxyl amide
D
SK-MEL-5 [21, 22]. The 5-(trifluoromethyl)-1H-1,2,3-triazole-4-
carboxamide H and related compounds (Fig. 1) were discovered
as c-Met-targeting and apoptosis-inducing agents for various
tumour cell lines (MCF-7, HepG2, A549, H460, HT-29, MKN-45
and U87MG) with 3–5-fold higher activity than the positive
control drug foretinib [23, 24]. As an example of agents with
other bioactivities, 1,2,3-triazole-4-carboxamides were shown to
be the inhibitors of the Wnt/β-catenin signalling pathway [25],
possessing promising anti-leishmanial activity [26] and exhibiting
virus-inhibitory activities [27].
(Fig. 1) acting as an inhibitor of succinate dehydrogenase enzyme
possessed good fungicidal activity, especially towards Sclerotinia
sclerotiorum. Potentially, it can be used for the development of
novel pesticides [11]. Noteworthy, the compounds containing the
1,2,3-triazole-4-carboxamide moiety are already used as drugs
or are currently under the preclinical studies. Two compounds
are available on the market. For example, the well-known drug
Rufinamide has been used since 2008 for treatment of Lenox-
Gastaut syndrome (a form of epilepsy) [12]. Carboxyamidotriazole
in the form of the orotic acid salt was shown to be an effective
blocker of calcium channels and demonstrated high activity against
chronic myelocytic leukaemia inhibiting the growth of cell lines
LAMA84R and K562R [13]. Other compounds structurally related
to Carboxyamidotriazole were evaluated as calcium-activated potas-
sium channel activators [14]. The 1,2,3-triazole-4-carboxamides
are promising antiproliferative agents for anticancer studies. For
instance, the 1-benzyl-N-(2-(phenylamino)pyridine-3-yl)-1H-1,2,3-
triazole-4-carboxamides have shown cytotoxicity against lung can-
cer A549 cell line by the inhibiting of tubulin polymerization [15].
1,2,3-Triazole-4-carboxamides containing podophyllotoxin were
screened for DNA topoisomerase-IIα inhibitory activity [16]. The
dissymmetric bistriazoles were tested in vitro for their cytotoxic
activity using B16 melanoma cells and showed activity at nanomo-
lar level (< 1 μM) [17, 18]. High activity compounds was found in
1-aryl-5-substituted-1H-1,2,3-triazole-4-carboxamides. For exam-
ple, 1-aryl-5-methyl-1,2,3-triazole-4-carboxamide E (Fig. 1) was an
inhibitor of ER stress-induced CHOP-luciferase [19]. The compound
F (Fig. 1) exhibited inhibition of heat shock protein 90 [20]. Sev-
eral 5-amino-1H-1,2,3-triazole-4-carboxamides structurally close
to 5-methyl-1H-1,2,3-triazole-4-carboxamides were also acting
as potent antiproliferative agents. In particular, the compound G
was active toward CNS SNB-75 and thieno[3,2-e][1,2,3]triazolo[1,5-
a]pyrimidine-3-carboxamide suppressed melanoma cell line
Therefore, the compounds with a 1,2,3-triazole-4-carboxamide
pharmacophore moiety possess considerable potential for the dis-
covery of the biological activity, in particular, the antimicrobial
one.
2. Results and discussion
2.1. Chemistry
In this work, we demonstrated the antimicrobial activity
of the selected 1-aryl-1H-1,2,3-triazole-4-carboxamides possessing
the highest score. Using the developed combinatorial methods for
the synthesis of triazoles [28], a library of 360 compounds was ob-
tained and screened for primary pathogens: Escherichia coli (E. coli,
ATCC 25922), Klebsiella pneumonia (K. pneumoniae, ATCC 700603),
Acinetobacter baumannii (A. baumannii, ATCC 19606), Pseudomonas
aeruginosa (P. aeruginosa, ATCC 27853), Staphylococcus aureus (S. au-
reus, ATCC 43300), Cryptococcus neoformans var. grubii (C. neofor-
mans, H99; ATCC 208821) and Candida albicans (C. albicans, ATCC
90028). These microorganisms are among the pathogens listed
by the World Health Organization with growing multidrug resis-
tance. According to the screening results, the most active com-
pounds were selected to create a structure–activity relationship
(SAR) and further modifications of 1-aryl-5-substituted-1H-1,2,3-
triazole-4-carboxamide scaffolds.
2

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
In Scheme 1, the preparation of the target compounds 4a-
t, 8a-e, 9a-g is described. A convenient synthetic strategy for
the creation of combinatorial libraries of 1-aryl-5-methyl-N-R2-1H-
1,2,3-triazole-4-carboxamides involves cascade reactions of azides
1 with diketene 2 and amines 3 via acylation and cyclocondensa-
tion in one pot (Scheme 1, route A) [28]. By this multicomponent
reaction, 1,2,3-triazole-4-carboxamides 4a-e were prepared in a
short time with high yields and purity. It should be noted that the
reaction of diketene 2 with strongly nucleophilic amines (such as
3-aminomethyl-pyridine) and highly reactive azides underwent at
room temperature, and the reaction yield increased with increasing
the amine basicity. The reaction time correlates with the reactivity
of azides and lasts on average about 30 min. For the synthesis of
other 5-alkyl-1H-1,2,3-triazole-4-carboxamides 4 and 5-methyl-1H-
1,2,3-triazole-4-carboxamides 4k-s from low nucleophilic amines,
the two-step procedure was used (Scheme 1, route B). The or-
ganic azides were condensed with β-ketoesters 5 to afford the
intermediate 1,2,3-triazole-4-carboxylates, which were hydrolyzed
without purification with NaOH to give the corresponding car-
boxylic acids 3 [29, 30, 31, 32]. It is noteworthy that the base sol-
vent system for the Dimroth reaction should be selected depend-
ing on the structure of azides as well as β-ketoesters [29]. Treat-
ment of acids with SOCl2 gave the corresponding acid chlorides,
which were aminolyzed to afford amides 4f-t. It is well known that
derivatives of cyanoacetic acid are highly reactive in the reaction
with azides providing the diversity of 5-amino-1H-1,2,3-triazoles
in a short time at room temperature [33, 34, 35, 36]. We used
the reaction of azides with cyanoacetamides 7 to obtain the target
5-amino-1H-1,2,3-triazole-4-carboxamides 8 (Scheme 1, route С).
The domino-reaction of 2-azidobenzoates 1r-t [37] with cyanoac-
etamides led to [1,2,3]triazolo[1,5-a]quinazoline 9 allowing to “fix”
aryl rotation. Compounds 8 and 9 were formed and sedimented
rapidly after mixing the reagents with yields close to quantitative.
The structures of all title compounds 4, 8, 9 were confirmed by
1H NMR, 13C NMR and LCMS. Amides 4, 8, 9 were formed with
high purity and with no by-products observed. Additionally, the
structural features of 5-cyclopropyl-1,2,3-triazolyl-4-carboxamide,
in which perpendicular or bisected form of cyclopropyl could ex-
ist, has been recently studied by the X-ray crystallographic analysis
[38].
Fig. 3. Distribution diagram of the average percentage inhibition of the compounds
4, 8, 9 toward different pathogens.
note A). In case of amides with fewer than 4 atoms, the activity
was significantly reduced. This is evidenced by the activity data of
structurally related compounds 4a and 4b in which, in contrast to
4d, instead of butyl substituent there is propyl one (Fig. 4, note
A). The second important factor was the increase in the activity
of Candida albicans with a decrease in the substituent at position
5 of the triazole ring. For example, N-butyl-5-propyl-1H-1,2,3-
triazole-4-carboxamide 4j had significantly lower activity than
5-methyl-1H-1,2,3-triazole 4d (GI, % 21.2; 27.4). The 5-isopropyl
triazoles showed lower activity (compounds 4f and 4g) (Fig. 4,
note B). This can be explained by the fact that, the triazole
fragment becomes more like an amide bond and by the fact that
the bulky substituent removes the aryl and triazole ring from the
coplanar position increasing the angle between them [38].
The variation of the substituent at the para-position of aryl (Br
on Et in 4a and 4b, respectively) was not noticeable in the activity
(Fig. 4, note C). A replacement of the aryl moiety in the alkyl sub-
stituent (compound 4e) in the amide moiety or complete replace-
ment with aryl substituent (compounds 4k, 4l, 4m and 4q) leads
to a decrease in the activity, which in some compounds remains
sufficient for further research for its improvement. It is neces-
sary to distinguish compounds that have a substituent in the para-
position of the aryl amide moiety. Thus, the compounds 4l, 4m and
4r were slightly more active than N-phenyl triazole 4k. The com-
pounds 4l and 4m with N-(2,4-dimethoxyphenyl) substituent were
the most active among the aryl structures, however, parallel trials
of the compound 4m were poorly correlated. In the series of Sul-
fanilamide derivatives obtained by acylation of Sulfanilamide with
triazole carboxylic acids 3, only 1-(2-fluorophenyl)-5-methyl-1H-
1,2,3-triazole-4-carboxamide 4r was detected close to partially ac-
tive compounds against C. albicans. In addition, increase in activity
was observed in Sulfanilamide series from phenyl to 2-fluorophenyl
substituent in position 1 of the triazole ring H<4-Me<3-Me<3-
MeO<2-F (GI, % = 1.1; 5.0 (H, 4n); 2.1; 4.2 (4-Me, 4o); 11.8; 8.8
(3-Me; 4p) 21.8, 8.1 (3-MeO, 4q), 40.4, 41.8 (2-F, 4r)).
2.2. Biological activity and structure-activity relationships
2.2.1. Primary antimicrobial screening
According to the results of the preliminary screening of the
newly synthesized 1,2,3-triazolyl-4-carboxamides 4, 8 and 9 (at a
concentration of 32 μg/mL (approx. 100 μM)) on seven pathogens
(S. aureus (ATCC 43300), E. coli (ATCC 25922), K. pneumoniae (ATCC
700603), P. aeruginosa (ATCC 27853), A. baumannii (ATCC 19606),
C. albicans (ATCC 90028) and C. neoformans (H99; ATCC 208821)),
33 compounds were selected. In Table 1, the results of two parallel
trials are presented.
Based on the GI average values of parallel trials, one can see
that most of the tested compounds frequently inhibited the growth
of fungus C. albicans and C. neoformans and Gram-negative bac-
terium A. baumannii (Fig. 3). In addition, there was a compound
highly active toward S. aureus.
The compounds 4 were mainly low active toward the strain
of Cryptococcus neoformans var. grubii. The compound 4p with the
meta-tolyl substituent and the sulfanilamide moiety, as well as
two compounds 4s, t (Fig. 4), containing bulky substituents dis-
rupting the coplanarity of the aryl and triazole rings and obvi-
ously limiting the free rotation of the two rings relative to each
other, were the only ones to show insignificant activity. In partic-
ular, (2,6-dimethylphenyl)-1,2,3-triazole-4-carboxamide 4s and 5-
cyclopropyl-1,2,3-triazole-4-carboxamide 4t inhibited growth of C.
neoformans by 28.6; 37.7 and 17.7; 24.7%, respectively. It is worth
noting that the cyclopropyl substituent in bisector form rotates the
The highest values of the antimicrobial activity were found
toward the strain of diploid fungus Candida albicans. In partic-
ular,
1-(4-bromophenyl)-N-butyl-5-methyl-1H-1,2,3-triazole-4-
carboxamide 4d inhibited strain growth by 63% (GI, % = 63.7 and
67.6) showing good reproducibility (abs Z-Score 12.8; 15.1) (Fig. 4).
This compound was selected as a title structure for the analysis
of the structure–activity relationships, and several determinants
were identified. It should be noted that an important factor is
the length of the carbon framework in the amide moiety (Fig. 4,
3

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
Table 1
Preliminary screening of 1-aryl-N-R2-1H-1,2,3-triazole-4-carboxamides 4, 7
Compnd
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
A. baumannii
C. albicans
C. neoformans
4a
4b
4c
4d
4e
4f
13.3; 14.9a
-10.8; -22.3
-4.0; 5.4
-0.6; 2.7
2.6; 3.0
10.7; 15.9
1.6; 7.3
-1.8; -2.8
0.5; 7.7
-2.4; 2.2
-2.4; 0.3
9.8; 9.9
0.9; 2.7
-1.3; 6.1
-0.9; -0.9
-0.3; -1.6
4.7; 6.5
2.3; 3.2
-0.6; 2.2
-2.5; -4.5
-0.4; 1.9
0.2; 2.2
0.7; 1.7
5.0; 8.9
4.5; 9.2
7.5; 8.2
1.8; 3.4
-1.7; -1.9
0.7; 1.0
-1.4; -5.4
14.1; 7.4
1.9; 7.9
2.5; 4.9
11.9; 9.2
-4.3; -6.7
10.8; 4.9
8.2; 8.7
10.0; 3.0
2.9; 6.9
-0.1; -5.2
11.9; 7.8
-3.6; 11.3
2.0; 6.0
14.1; 16.2
13.2; 18.7
17.9; 31.9b
63.7; 67.6
24.1; 37.0
15.9; 22.9
11.6; 26.1
9.0; 9.3
-19.9; -68.4
2.6; 7.3
-0.0; -6.6
-2.9; -6.8
-3.1; 1.3
-0.2; -2.4
-3.3; -5.9
-9.5; 0.7
-11.7; 1.0
12.5; 9.3
-2.8; 4.3
-1.2; 5.8
-3.1; 1.2
-1.3; -3.8
-12.3; -7.7
-0.5; -1.3
-12.0; -8.0
4.0; 6.8
-2.0; 4.6
-10.4; 0.6
-1.5; -4.3
0.3; 10.2
-0.1; 0.2
-4.2; 1.5
-0.9; -7.5
3.0; 3.4
-29.7; -35.4
-29.4; -34.5
10.1; 4.9
-19.3; -8.7
-11.1; -13.6
7.1; 8.4
-0.5; -7.3
-5.2; 13.1
11.9; 2.2
-3.2; -5.8
-5.8; -8.6
-11.3; -9.0
-1.1; 18.1
-0.9; 4.3
-2.9; -7.6
-0.8; -2.0
-0.1; 8.4
0.2; 1.1
-19.4; -9.8
-20.4; -5.2
-15.1; -65.1
-8.3; -8.7
20.2; 4.4
4g
4h
4i
17.3; 3.2
6.2; 7.8
4.7; 4.7
10.9; 15.8
21.2; 27.4
24.8; 32.8
35.9; 45.9
2.6; 54.4
1.1; 5.0
4j
-16.8; -6.2
-8.2; -8.7
-0.5; 12.0
-2.4; 0.1
4k
4l
-4.0; 6.0
-13.3; 6.2
-12.5; 3.9
5.7; 6.1
-34.3; -36.8
-4.7; -9.5
-4.7; -6.8
-4.4; 3.6
4m
4n
4o
4p
4q
4r
4s
4t
2.4; 6.4
-2.5; -7.2
-3.3; 6.8
1.5; 5.8
2.1; 4.2
1.7; 5.6
-5.8; 1.3
-5.2; 1.1
1.6; 13.2
-0.0; -5.1
1.4; 4.9
3.4; 9.5
11.8; 8.8
21.8; 8.1
40.4; 41.8
5.9; 7.8
24.5; 25.0
20.9; 8.7
-17.8; -9.1
-2.8; -5.2
-14.4; -2.6
1.9; 11.5
12.6; 15.9
-3.3; 3.8
2.8; 4.7
-5.2; -7.6
-1.3; -8.0
-3.6; 2.7
12.2; 22.4
-4.1; -7.3
-5.3; 3.0
19.6; 5.0
28.5; 41.3
23.0; 38.5
15.2; 29.2
-16.4; -7.2
1.2; 9.4
15.1; 17.2
28.6; 37.7
17.7; 24.7
-4.0; -7.2
21.6; 42.7
-0.6; -4.7
-9.4; 17.0
-24.2; -59.0
-78.2; -79.4
-22.4; -3.3
-1.0; -6.2
12.3; 2.2
9.3; 9.6
10.9; 7.4
-1.3; 1.0
-6.5; 1.5
-0.4; -5.5
6.9; 8.5
16.0; 17.2
3.6; 4.7
4u
8a
8b
8c
8d
8e
9a
9b
9c
9d
9e
9f
12.6; 5.5
-3.0; -8.6
-2.1; 1.5
1.2; 6.5
24.9; 32.2
39.1; 41.8
19.5; 25.6
10.5; 19.1
15.6; 21.8
27.3; 32.0
6.9; 8.0
-3.4; -6.4
-5.9; 0.9
7.6; 7.6
3.3; 3.7
-3.7; 7.5
0.5; 9.0
6.8; 7.6
-4.0; 10.1
-1.9; -2.4
-16.5; -9.9
0.1; 1.7
52.6; 63.1
-10.5; -19.4
19.1; 23.5
-23.7; -29.4
21.1; 5.7
15.4; 6.6
-3.5; -6.2
15.5; 9.7
-12.7; -5.8
-1.2; 10.3
-4.2; 7.7
11.6; 15.5
4.5; 7.1
5.8; 6.0
13.4; 9.8
7.9; 8.5
12.3; 4.0
14.3; 3.2
10.5; 16.7
-3.5; -6.7
6.2; 6.7
36.7; 42.7
-0.2; 8.2
4.2; 7.8
-0.8; 1.5
-10.3; -5.8
1.9; 7.4
-2.3; -4.1
1.2; 3.9
9g
-7.6; 7.5
4.4; 6.4
a
Percentage growth inhibition (GI, %).
b
Highest percentage of antibacterial/antifungal growth inhibition is highlighted in bold.
Fig. 4. SAR of 5-alkyl-1H-1,2,3-triazole-4-carboxamides 4 against Candida albicans.
4

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
at a concentration of 50 μM inhibited the HaCaT cell growth by ap-
proximately of 45%.
3. Conclusion
A series of N-substituted 1-aryl-5-substituted-1H-1,2,3-triazole-
4-carboxamides were designed, synthesized and evaluated for
their antimicrobial potential toward the selected pathogens: Es-
cherichia coli, Klebsiella pneumonia, Acinetobacter baumannii, Pseu-
domonas aeruginosa, Staphylococcus aureus, Cryptococcus neofor-
mans var. grubii and Candida albicans. Several 5-methyl-1H-1,2,3-
triazole-4-carboxamides 4d, 4l, 4r, showed potent antibacte-
rial effect against S. aureus. On the contrary, 5-amino-1H-1,2,3-
triazole-4-carboxamide 8b and [1,2,3]triazolo[1,5-a]quinazoline-3-
carboxamide 9a were active against pathogenic yeast C. albicans.
Thus, the compound 4l demonstrated 50% growth inhibition under
1 μM toward S. aureus. At the same concentration, the compound
9a killed approximately 40% of C. albicans cells. In general, these
compounds demonstrated selective action and no impact on the
viability of human keratinocytes of HaCaT line. Taking into account
that the methods for preparing such compounds are highly vari-
able and meet the requirements of click chemistry, and the com-
pounds 4 and 9 themselves are highly active, 1H-1,2,3-triazole-4-
carboxamides scaffolds are valuable scaffolds for the discovery of
selective antimicrobial agents based on their molecules.
Fig. 5. 5-Alkyl-1H-1,2,3-triazole-4-carboxamides most active against C. neoformans.
aryl substituent by almost 90 degrees relative to the triazole plane
[38] (Fig. 5).
A replacement of the alkyl moiety with an amino group did not
enhance the activity leaving the value of antifungal activity against
the strain of Candida albicans at the same level (compounds 8a-b,
e, Fig. 6). The compound 8b was the most active one with reduc-
ing growth by 40%. Interestingly, the compound 7a also showed
activity toward the strain of C. neoformans. On the other hand, two
compounds with substituents in the meta-position inhibited the
growth of Gram-negative bacterium acinetobacteria A. baumannii
(compounds 8e, d Fig. 6). It should be noted that 5-alkyl triazoles
were inactive to A. bedaumannii and only one compound, in par-
ticular, N-(thiazol-2-yl)-1H-1,2,3-triazole-4-carboxamide 4u (GI, %
12.2; 22.4), was low active.
When the rotation of the triazole and aryl rings was “fixed”
via condensation of the amino group to the [1,2,3]triazolo[1,5-
a]quinazoline-3-carboxamide system 9, the activity also changed
dramatically. Representatives of this class were mainly active
against Staphylococcus aureus, and can be considered as a po-
tential scaffold for further antibacterial trials. Most of the
tested compounds showed moderate activity, but it was found
that 5-oxo-N-phenyl-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-
3-carboxamide 9a inhibited more than 50% growth of S. aureus
(Fig. 7). No stable structure–activity correlation was found but the
introduction of the substituent in [1,2,3]triazolo[1,5-a]quinazoline
core led to the loss of activity. Particular emphasis must be placed
on the fact that the compound 9d showed activity against C. neo-
formans (GI, % = 36.7; 42.7).
4. Experiment
4.1. Materials and instrumentation
All chemicals used were of laboratory grade and used without
further purification. Starting azides 1 were prepared from corre-
sponding amines via diazotization by treatment with sodium ni-
trite in hydrochloric and following reaction with sodium azide
via described procedure [37, 39]. The β-ketoesters 5 and 1H-
1,2,3-triazole-4-carboxylic acids were reported in our previous ar-
ticles [29]. Purity of compounds was monitored by TLC on silica
F254 coated aluminum plates (Merck) as adsorbent and U.V. light
and iodine as visualizing agents. 1H and 13C NMR spectra were
recorded on Varian Unity Plus 400 (400 and 101 MHz, respec-
tively) and Bruker 170 Avance 500 (500 and 126 MHz, respectively)
spectrometers in DMSO-d6 solutions using TMS or the deuterated
solvent as internal reference. Melting points of compounds were
determined in open capillary tubes in a silicon oil bath using a
Boetius melting point apparatus and are uncorrected. Mass spectral
analyses were performed using an Agilent 1100 series LC/MSD with
API-ES/APCI mode (200 eV). Elemental analyses were accomplished
using a Carlo Erba 1106 instrument. The absorbance was measured
using a Tecan M1000 Pro monochromator and Biotek Synergy HTX
plate readers.
2.2.2. MIC
The five most active compounds (4d, 4l, 4r, 8b, 9a) were fur-
ther studied at 1 μM concentrations. The compound 4l demon-
strated high antibacterial effect (approximately 50% of growth in-
hibition under 1 μM of compound) against S. aureus, but not so
prominent antibacterial effect against P. aeruginosa in comparison
to other studied compounds (Fig. 8). Furthermore, we observed
that the compound 9a killed nearly 40% of C. albicans cells (Fig. 9).
Other studied compounds showed slight growth inhibition effect.
We assumed that the main effect of those compounds was caused
by the DMSO solvent. Based on the obtained results, we can con-
clude that the proposed chemical structure of compound 9a can be
used for development of potential antifungal agents.
Thus, the compounds 4l, 9a show high enough antimicrobial ef-
fects allowing continuing research on their possible applications in
medicine.
4.2. Synthesis of title compounds 4, 7, 8
4.2.1. General procedure for the synthesis of
1-aryl-5-methyl-1H-1,2,3-triazole-4-carboxyamide 4a-e
2.2.3. Cytotoxicity
An appropriate amine 3 (10.0 mmol), arylazide 1 (10.0 mmol),
and triethylamine 1.4 mL were added to the solution of diketene
(10.0 mmol) in dry acetonitrile (20 mL). The mixture was heated
under reflux during 30 min. Then, it was cooled to room tempera-
ture, and the solid started to sediment. The product was filtered
and washed with methanol to give triazole 3 as a white solid.
When the amide was well soluble in acetonitrile, water was added
dropwise until the solid started to appear. The compounds were
found to be of sufficient purity without further purification.
The cytotoxicity of the five most active compounds (4d, 4l, 4r,
8b, 9a) was evaluated toward human keratinocytes of HaCaT cell
line and the results are summarized in Fig. 10. Two compounds
(4l, 4r) displayed tolerance to keratinocytes even at a high concen-
tration of 50 μM. The compound 4l inhibited the growth of HaCaT
cells by 25.5%, and the compound 4r – by 17.4%. The compound
8a reached the IC50 at 50 μM for HaCaT cells that is 5-fold higher
than the antimicrobial action concentration. Compounds 8b and 4d
5

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
Fig. 6. The most active 5-amino-1H-1,2,3-triazole-4-carboxamides.
Fig. 7. The most active [1,2,3]triazolo[1,5-a]quinazoline-3-carboxamides.
∗∗
∗∗∗
Fig. 8. Antibacterial effect of the tested compounds (1 μM per sample) toward S. aureus and P. aeruginosa.
– p ≤ 0.01,
– p ≤ 0.001 (difference compared with the not
treated control cells).
4.2.1.1. 1-(4-Еthylphenyl)-5-methyl-N-propyl-1H-1,2,3-triazole-4-
carboxamide (4a). White solid; Yield 95%; m.p. 114–115 °C
2H, CH2N), 2.52 (s, 3H, CH3), 1.63 – 1.44 (m, 2H, CH2), 0.87 (t,
J = 7.4 Hz, 3H, CH3); 13C NMR (101 MHz, DMSO-d6): δ 161.07 (СO),
138.89 (СTr-5), 137.08 (СTr-4), 135.14 (СAr-1), 133.15 (2xСHAr-3,5),
127.90 (2xСHAr-2,6), 123.61 (СAr-4), 40.55 (CH2N), 23.00 (CH2),
11.84 (CH3), 9.71 (CH3). MS (m/z): 323, 325 (M++1); anal. calcd.
for C13H15BrN4O: C, 48.31; H, 4.68; N, 17.34; found: C, 48.27; H,
4.75; N, 17.31.
(ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz,
DMSO-d6): δ 8.55 (t, J = 6.3 Hz, 1H, NH), 7.52 (d, J = 7.7 Hz,
2H, HAr-2,6), 7.46 (d, J = 7.9 Hz, 2H, HAr-3,5), 3.31 – 3.15 (m, 2H,
CH2N), 2.72 (q, J = 7.4 Hz, 2H, CH2), 2.50 (s, 3H, CH3), 1.63 – 1.48
(m, 2H, CH2), 1.23 (t, J = 7.4 Hz, 3H, CH3), 0.88 (t, J = 7.1 Hz,
3H, CH3); 13C NMR (126 MHz, DMSO-d6): δ 160.69 (СO), 145.78
(СAr-4), 138.22 (СTr-5), 136.30 (СTr-4), 133.12 (СAr-1), 128.88
(2xСHAr-3,5), 125.23 (2xСHAr-2,6), 40.02 (CH2N), 27.79 (CH2),
22.51 (CH2), 15.36 (CH3), 11.33 (CH3), 9.23 (CH3). MS (m/z): 273
(M++1); anal. calcd. for C15H20N4O: C, 66.15; H, 7.40; N, 20.57;
found: C, 66.23; H, 7.43; N, 20.50.
4.2.1.3. 1-(3-Chloro-2-methylphenyl)-5-methyl-N-propyl-1H-1,2,3-
triazole-4-carboxamide (4c). White solid; Yield 91%; m.p. 114–115
°C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 8.59 (t, J = 5.7 Hz, 1H, NH), 7.81 – 7.67 (m,
1H, HAr), 7.51 – 7.46 (m, 2H, HAr), 3.23 (q, J = 6.7 Hz, 2H, CH2N),
2.33 (s, 3H, CH3), 1.95 (s, 3H, CH3), 1.61 – 1.46 (m, 2H, CH2), 0.88
(t, J = 7.4 Hz, 3H, CH3); 13C NMR (126 MHz, DMSO-d6): δ 161.04
(СO), 138.44 (СTr-5), 137.94 (СTr-4), 135.95 (СAr-2), 135.18 (СAr-1),
134.11 (СAr-3), 131.91 (СHAr-4), 128.59 (СHAr-5), 127.17 (СHAr-6),
40.58 (CH2N), 23.00 (CH2), 15.12 (CH3), 11.87 (CH3), 9.13 (CH3).
4.2.1.2. 1-(4-Bromophenyl)-5-methyl-N-propyl-1H-1,2,3-triazole-4-
carboxamide (4b). White solid; Yield 96%; m.p. 124–125 °C
(ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz,
DMSO-d6): δ 8.56 (t, J = 5.8 Hz, 1H, NH), 7.84 (d, J = 8.6 Hz,
2H, HAr-2,6), 7.60 (d, J = 8.6 Hz, 2H, HAr-3,5), 3.22 (q, J = 6.6 Hz,
6

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
Fig. 10. Cytotoxicity of triazole-4-carboxamides 4d, 4l, 4r, 8b, 9a towards human
keratinocytes of HaCaT line.∗After a total experimental time (72 h), cell vitality was
∗∗
∗∗∗
detected by the MTT assay. – p ≤ 0.05;
– p ≤ 0.01;
– p ≤ 0.001 (difference
compared with the not treated control cells).
Fig. 9. Antifungal effects of the tested compounds (1 μM per sample) towards C.
albicans ATCC 885653. C0 – background (basic number) Colony Formation Units
ꢀꢀꢀ
(CFU), C4 – CFU number after 4 h of incubation.
– p ≤ 0.001 (difference com-
∗∗∗
pared with not treated control cells of C0),
– p ≤ 0.001 (difference compared
DMSO-d6): δ 8.55 (t, J = 5.6 Hz, 1H, NH), 7.84 (d, J = 8.5 Hz,
2H, HAr-2,6), 7.60 (d, J = 8.5 Hz, 2H, HAr-3,5), 3.31 – 3.20 (m, 2H,
CH2N), 2.52 (s, 3H, CH3), 1.58 – 1.42 (m, 2H, CH2), 1.35 – 1.23
(m, 2H, CH2), 0.89 (t, J = 7.3 Hz, 3H, CH3); 13C NMR (126 MHz,
DMSO-d6): δ 161.03 (СO), 138.89 (СTr-5), 137.07 (СTr-4), 135.13
(СAr-1), 133.15 (2xСHAr-3,5), 127.90 (2xСHAr-2,6), 123.60 (СAr-4),
38.43 (CH2N), 31.85 (CH2), 20.07 (CH2), 14.19 (CH3), 9.71 (CH3). MS
(m/z): 337, 339 (M++1); anal. calcd. for C14 H17 BrN4O: C, 49.77; H,
5.21; N, 16.71; found: C, 49.87; H, 5.20; N, 16.61.
with the not treated control cells of C4).
MS (m/z): 293 (M++1); anal. calcd. for C14 H17 ClN4O: C, 57.44; H,
5.85; N, 19.14; found: C, 57.49; H, 5.79; N, 19.21.
4.2.1.4. 1-(4-Bromophenyl)-N-butyl-5-methyl-1H-1,2,3-triazole-4-
carboxamide (4d). White solid; Yield 97%; m.p. 144–145 °C
(ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz,
Scheme 1. Synthetic routes to the target 1H-1,2,3-triazole-4-carboxamides.
7

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
4.2.1.5. 1-(3,4-Dimethylphenyl)-5-methyl-N-(pyridin-3-ylmethyl)-1H-
1,2,3-triazole-4-carboxamide (4e). White solid; Yield 95%; m.p.
(CH3). MS (m/z): 273 (M++1); anal. calcd. for C15H20N4O: C, 66.15;
H, 7.40; N, 20.57; found: C, 66.23; H, 7.43; N, 20.50.
157–158 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(400 MHz, DMSO-d6): δ 9.29 – 9.23 (m, 1H), 8.56 (d, J = 2.7 Hz,
1H, HPy), 8.44 (t, J = 4.0 Hz, 1H, HPy), 7.77 – 7.71 (m, 1H), 7.48 –
7.17 (m, 4H, HArom.), 4.50 – 4.46 (m, 2H, CH2), 2.48 (s, 3H, CH3),
2.30 (s, 3H, CH3), 2.29 (s, 3H, CH3). 13C NMR (126 MHz, DMSO-d6):
δ 161.48 (СO), 149.41 (СHPy-2), 148.51 (СHPy-6), 138.98 (СTr-5),
138.55 (СAr-3), 138.32 (СTr-4), 137.17 (СAr-4), 135.71 (СAr-1), 135.66
(СHPy-4), 133.54 (СPy-3), 130.81 (СHAr-5), 126.52 (СHAr-2), 123.91
(СHAr-6), 123.01 (СHPy-5), 40.13 (CH2N), 19.72 (CH3), 19.58 (CH3),
9.76 (CH3). MS (m/z): 322 (M++1); anal. calcd. for C18 H19 N5O: C,
67.27; H, 5.96; N, 21.79; found: C, 67.32; H, 5.91; N, 21.71.
4.2.2.4. 1-(4-Chlorophenyl)-N,5-dipropyl-1H-1,2,3-triazole-4-
carboxamide (4i). White solid; Yield 93%; m.p. 88–89 °C (ethanol);
IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (500 MHz, DMSO-d6):
δ 8.58 (t, J = 5.3 Hz, 1H, NH), 7.71 (d, J = 7.5 Hz, 2H), 7.65 (d,
J = 7.5 Hz, 2H), 3.28 – 3.16 (m, 2H, CH2N), 3.00 – 2.87 (m, 2H),
1.60 – 1.48 (m, 2H), 1.45 – 1.33 (m, 2H), 0.87 (t, J = 7.5 Hz,
3H, CH3), 0.71 (s, 3H, CH3); 13C NMR (126 MHz, DMSO-d6): δ
160.40 (СO), 140.35 (СTr-5), 138.28 (СTr-4), 134.85 (СAr-1), 134.37
(СAr-4), 129.81 (2xСHAr-3,5), 127.71 (2xСHAr-2,6), 40.05 (CH2N),
24.15 (CH2), 22.50 (CH2), 21.27 (CH2), 13.42 (CH3), 11.31 (CH3). MS
(m/z): 307 (M++1); anal. calcd. for C15H19ClN4O: C, 58.73; H, 6.24;
N, 18.26; found: C, 58.81; H, 6.29; N, 18.17.
4.2.2. General procedure for the synthesis of
1-aryl-5-substituted-1H-1,2,3-triazole-4-carboxyamide 4f-t
The solution of the appropriate 1,2,3-triazole-4-carboxylic acid
6 (2.0 mmol) in SOCl2 (2 mL) was heated under reflux for 1h.
Then, the mixture was evaporated to dryness to give a residue,
which was washed, and hexane dissolved in anhydrous methylene
dichloride (10 mL). This solution was added in a dropwise man-
ner to a solution of organic amine 3 (2.0 mmol) and TEA (0.5 mL,
3.5 mmol) in anhydrous methylene dichloride (10 mL), and the re-
sulting mixture was heated under reflux for 30 min. The reaction
mixture was cooled to ambient and washed sequentially with 0.5
N HCl (10 mL), 0.5 M Na2CO3 (10 mL) and water (10 mL). The or-
ganic layer was dried over Na2SO4 and evaporated to dryness to
give the desired product as a solid, which found to be of sufficient
purity without further purification.
4.2.2.5. N-Butyl-5-propyl-1-p-tolyl-1H-1,2,3-triazole-4-carboxamide
(4j). White solid; Yield 92%; m.p. 72–73 °C (ethanol); IR (KBr)
νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 8.64
– 8.45 (m, 1H, NH), 7.54 – 7.34 (m, 4H, HAr), 3.30 – 3.22 (m, 2H,
CH2), 2.96 – 2.84 (m, 2H, CH2), 2.41 (d, J = 3.1 Hz, 3H, CH3), 1.56
– 1.46 (m, 2H, CH2), 1.44 – 1.35 (m, 2H, CH2), 1.34 – 1.26 (m,
2H, CH2), 0.94 – 0.85 (m, 3H, CH3), 0.74 – 0.64 (m, 3H, CH3); 13C
NMR (101 MHz, DMSO-d6): δ 161.00 (СO), 140.61 (СAr-4), 140.46
(СTr-5), 138.66 (СTr-4), 133.60 (СAr-1), 130.62 (2xСHAr-3,5), 126.14
(2xСHAr-2,6), 38.44 (CH2N), 31.87 (CH2), 24.70 (CH2), 21.78 (CH2),
21.23 (CH2), 20.09 (CH3), 14.17 (CH3), 13.93 (CH3). MS (m/z): 301
(M++1); anal. calcd. for C17 H24N4O: C, 67.97; H, 8.05; N, 18.65;
found: C, 67.90; H, 8.11; N, 18.74.
4.2.2.1. N,5-Diisopropyl-1-p-tolyl-1H-1,2,3-triazole-4-carboxamide
(4f). White solid; Yield 89%; m.p. 102–103 °C (ethanol); IR (KBr)
4.2.2.6. 1-(2-Fluorophenyl)-5-methyl-N-phenyl-1H-1,2,3-triazole-
4-carboxamide (4k). White solid; Yield 94%; m.p. 114–115 °C
νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 8.27
(ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz,
(d, J = 8.1 Hz, 1H, NH), 7.44 (d, J = 8.0 Hz, 2H, HAr-2,6), 7.39 (d,
J = 8.0 Hz, 2H, HAr-3,5), 4.21 – 4.08 (m, 1H, CHN), 3.17 (hept,
J = 7.0 Hz, 1H, CH), 2.43 (s, 3H, CH3), 1.26 (d, J = 7.0 Hz, 6H, CH3),
1.19 (d, J = 6.5 Hz, 6H, CH3); 13C NMR (126 MHz, DMSO-d6): δ
159.67 (СO), 144.92 (СAr-4), 140.27 (СTr-5), 137.95 (СTr-4), 133.35
(СAr-1), 130.01 (2xСHAr-3,5), 126.42 (2xСHAr-2,6), 40.23 (CH2N),
24.18 (CH), 22.22 (2xCH3), 20.75 (CH3), 19.99 (2xCH3). MS (m/z):
287 (M++1); anal. calcd. for C16 H22N4O: C, 67.11; H, 7.74; N, 19.56;
found: C, 67.05; H, 7.78; N, 19.49.
DMSO-d6): δ 10.54 (s, 1H, NH), 7.86 (d, J = 7.7 Hz, 2H, HArN-2,6),
7.79 – 7.70 (m, 2H, HAr), 7.62 (t, J = 8.9 Hz, 1H, HAr-4), 7.51 (t,
J = 7.6 Hz, 1H, HAr-5), 7.34 (t, J = 7.9 Hz, 2H, HArN-3,5), 7.10
(t, J = 7.4 Hz, 1H, HArN-3,5), 2.48 (s, 3H); 13C NMR (101 MHz,
1
DMSO-d6): δ 159.72 (СO), 156.35 (d, JС-F = 251.7 Hz, СAr-2),
3
139.53 (СTr-5), 139.04 (СTr-4), 138.51 (СPh-1), 133.63 (d, JC-F = 7.9
Hz, СHAr-4), 129.59 (СHPh-4), 129.07 (2xСHPh-3,5), 126.20 (d,
2
3JC-F = 3.8 Hz, СHAr-6), 124.26 (СHAr-5), 123.16 (d, JC-F = 12.1
2
Hz, СAr-1), 120.97 (2xСHPh-2,6), 117.58 (d, JC-F = 19.1 Hz, СAr-3),
11.06 (CH3). MS (m/z): 297 (M++1); anal. calcd. for C16 H13FN4O:
4.2.2.2. 1-(4-Сhlorophenyl)-5-isopropyl-N-propyl-1H-1,2,3-triazole-4-
carboxamide (4g). White solid; Yield 90%; m.p. 87–88 °C (ethanol);
C, 64.86; H, 4.42; N, 18.91; found: C, 64.94; H, 4.47; N, 18.94.
IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6):
4.2.2.7. N-(2,4-Dimethoxyphenyl)-1-(4-ethoxyphenyl)-5-methyl-1H-
1,2,3-triazole-4-carboxamide (4l). White solid; Yield 92%; m.p.
δ 8.58 (t, J = 5.4 Hz, 1H, NH), 7.72 (d, J = 8.5 Hz, 2H, HAr-2,6), 7.60
(d, J = 8.5 Hz, 2H, HAr-3,5), 3.24 (q, J = 6.6 Hz, 2H, CH2N), 3.16
(hept, J = 7.0 Hz, 1H, CH), 1.61 – 1.48 (m, 2H, CH2), 1.27 (d, J = 7.0
Hz, 6H, CH3), 0.88 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (101 MHz,
DMSO-d6): δ 160.89 (СO), 145.56 (СTr-5), 138.60 (СTr-4), 135.67
(СAr-1), 135.14 (СAr-4), 130.22 (2xСHAr-3,5), 129.08 (2xСHAr-2,6),
40.70 (CHN), 24.71 (CH2), 23.00 (CH2), 20.48 (2xCH3), 11.84 (CH3).
MS (m/z): 307 (M++1); anal. calcd. for C15H19ClN4O: C, 58.72; H,
6.24; N, 18.26; found: C, 58.77; H, 6.34; N, 18.21.
174-175 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(400 MHz, DMSO-d6): δ 9.39 (s, 1H, NH), 8.10 (d, J = 7.4 Hz, 1H,
HArN-6), 7.53 (d, J = 8.5 Hz, 2H, HAr-2,6), 7.15 (d, J = 8.3 Hz, 2H,
HAr-3,5), 6.70 (s, 1H, HArN-3), 6.55 (d, J = 8.4 Hz, 1H, HArN-5),
4.19 – 4.08 (m, 2H, CH2O), 3.91 (s, 3H, CH3O), 3.77 (s, 3H, CH3O),
2.52 (s, 3H, CH3), 1.37 (t, J = 5.9 Hz, 3H, CH3); 13C NMR (101
MHz, DMSO-d6): δ 160.01 (СAr-4), 158.81 (СO), 157.03 (СArN-4),
150.57 (СArN-2), 138.22 (СTr-5), 137.82 (СTr-4), 128.35 (СAr-1),
127.42 (2xСHPh-2,6), 121.40 (СHArN-6), 120.66 (СArN-1), 115.63
(2xСHAr-3,5), 104.73 (СHArN-5), 99.37 (СHArN-3), 64.12 (CH2O),
56.57 (CH3O), 55.85 (CH3O), 15.02 (CH3), 9.77 (CH3). MS (m/z):
383 (M++1); anal. calcd. for C20H22N4O4: C, 62.82; H, 5.80; N,
14.65; found: C, 62.79; H, 5.91; N, 14.71.
4.2.2.3. 1-Phenyl-N,5-dipropyl-1H-1,2,3-triazole-4-carboxamide (4h).
White solid; Yield 97%; m.p. 49–50 °C (ethanol); IR (KBr) νmax
1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 8.56 (t,
J = 5.8 Hz, 1H, NH), 7.67 – 7.61 (m, 3H, HPh), 7.60 – 7.55 (m, 2H,
HPh), 3.23 (q, J = 6.6 Hz, 2H, CH2N), 2.99 – 2.86 (m, 2H, CH2), 1.63
– 1.47 (m, 2H, CH2), 1.45 – 1.32 (m, 2H, CH2), 0.87 (t, J = 7.4 Hz,
3H, CH3), 0.70 (t, J = 7.4 Hz, 3H, CH3); 13C NMR (101 MHz, DMSO-
d6): δ 161.01 (СO), 140.66 (СTr-5), 138.72 (СTr-4), 136.06 (СPh-1),
130.68 (СHPh-4), 130.23 (2xСHAr-3,5), 126.36 (2xСHAr-2,6), 40.55
(CH2N), 24.70 (CH2), 23.02 (CH2), 21.76 (CH2), 13.93 (CH3), 11.83
4.2.2.8. 1-(3-Chloro-4-methylphenyl)-N-(2,4-dimethoxyphenyl)-5-
methyl-1H-1,2,3-triazole-4-carboxamide (4m). White solid; Yield
91%; m.p. 193-194 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (500 MHz, DMSO-d6): δ 9.37 (s, 1H, NH), 8.11 (d, J = 8.6
Hz, 1H, HArN-6), 7.77 (s, 1H, HAr-2), 7.63 (d, J = 7.9 Hz, 1H, HAr-6),
8

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
7.54 (d, J = 7.6 Hz, 1H, HAr-5), 6.71 (s, 1H, HArN-3), 6.56 (d, J = 8.3
Hz, 1H, HArN-5), 3.92 (s, 3H, CH3O), 3.79 (s, 3H, CH3O), 2.58 (s,
3H, CH3), 2.46 (s, 3H, CH3); 13C NMR (126 MHz, DMSO-d6): δ
158.17 (СO), 156.61 (СArN-4), 150.17 (СArN-2), 137.90 (СTr-5), 137.90
(СAr-1), 137.54 (СTr-4), 134.03 (СAr-4), 133.88 (СAr-3), 132.04
(СHAr-5), 125.66 (СHAr-2), 124.18 (СHAr-6), 121.05 (СHArN-6),
120.07 (СArN-1), 104.26 (СHArN-5), 98.88 (СHArN-3), 56.07 (CH3O),
55.35 (CH3O), 19.38 (CH3), 9.24 (CH3). MS (m/z): 387 (M++1);
anal. calcd. for C19 H19 ClN4O3: C, 58.99; H, 4.95; N, 14.48; found:
C, 58.91; H, 4.93; N, 14.41.
4.2.2.13. 1-(2-Fluorophenyl)-5-methyl-N-(4-sulfamoylphenyl)-1H-
1,2,3-triazole-4-carboxamide (4r). White solid; Yield 88%; m.p.
283-284 °C (ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H
NMR (500 MHz, DMSO-d6): δ 10.91 (s, 1H, NH), 8.06 (d, J = 8.6
Hz, 2H, HAr-3,5), 7.81 (d, J = 8.6 Hz, 2H, HAr-2,6), 7.79 – 7.72
(m, 2H, HAr-4,6), 7.63 (t, J = 9.0 Hz, 1H), 7.52 (t, J = 7.5 Hz, 1H,
HAr-5), 7.29 (s, 2H, NH2), 2.49 (s, 3H, CH3); 13C NMR (126 MHz,
1
DMSO-d6): δ 159.57 (СO), 155.83 (d, JC-F = 251.7 Hz, СAr-2),
141.53 (СArN-1), 139.54 (СArN-4), 138.84 (СTr-5), 137.68 (СTr-4),
3
133.19 (d, JC-F = 7.6 Hz), 129.07 (СHAr-5), 126.44 (2xСHArN-3,5),
3
2
125.71 (d, JC-F = 3.6 Hz, СHAr-4), 122.56 (d, JC-F = 12.5 Hz,
2
С
Ar-1), 120.03 (2xСHArN-2,6), 117.09 (d, JC-F = 18.9 Hz, СHAr-3),
4.2.2.9. 5-Methyl-1-phenyl-N-(4-sulfamoylphenyl)-1H-1,2,3-triazole-
4-carboxamide (4n). White solid; Yield 97%; m.p. 308-309 °C
8.82 (CH3). MS (m/z): 376 (M++1); anal. calcd. for C16 H14 FN5O3S:
(ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
C, 51.19; H, 3.76; N, 18.66; found: C, 51.05; H, 3.71; N, 18.75.
MHz, DMSO-d6): δ 10.80 (s, 1H, NH), 8.05 (d, J = 8.7 Hz, 2H,
HAr-3,5), 7.80 (d, J = 8.6 Hz, 2H, HAr-2,6), 7.71 – 7.58 (m, 5H,
HPh), 7.24 (s, 2H, NH2), 2.58 (s, 3H, CH3); 13C NMR (101 MHz,
DMSO-d6): δ 160.35 (СO), 142.11 (СArN-1), 139.27 (СArN-4), 138.66
(СTr-5), 138.43 (СTr-4), 135.69 (СPh-1), 130.64 (СHPh-4), 130.24
(2xСHPh-3,5), 126.94 (2xСHArN-3,5), 125.97 (2xСHPh-2,6), 120.48
(2xСHArN-2,6), 10.00 (CH3). MS (m/z): 358 (M++1); anal. calcd. for
C16 H15N5O3S: C, 53.77; H, 4.23; N, 19.60; found: C, 53.84; H, 4.23;
N, 19.60.
4.2.2.14. N-(2,5-Dimethoxyphenyl)-1-(2,6-dimethylphenyl)-5-methyl-
1H-1,2,3-triazole-4-carboxamide (4s). White solid; Yield 93%; m.p.
144-145 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(500 MHz, DMSO-d6): δ 9.62 (s, 1H, NH), 8.03 (s, 1H, HArN-2), 7.47
(t, J = 7.2 Hz, 1H, HAr-4), 7.35 (d, J = 7.2 Hz, 2H, HAr-3,5), 7.05 (d,
J = 8.6 Hz, 1H, HArN-4), 6.68 (d, J = 8.6 Hz, 1H, HArN-5), 3.90 (s,
3H, CH3O), 3.74 (s, 3H, CH3O), 2.35 (s, 3H, CH3), 1.91 (s, 6H, CH3).
13C NMR (126 MHz, DMSO-d6): δ 158.40 (СO), 153.13 (СArN-3),
142.48 (СArN-6), 138.23 (СTr-5), 137.62 (СTr-4), 135.48 (2xСAr-2,6),
133.14 (СAr-1), 130.75 (СHAr-4), 128.66 (2xСHAr-3,5), 127.48 (СArN
-
4.2.2.10. 5-Methyl-N-(4-sulfamoylphenyl)-1-p-tolyl-1H-1,2,3-triazole-
4-carboxamide (4o). White solid; Yield 95%; m.p. 294-295 °C
1), 111.59 (СHArN-5), 107.86 (СHArN-4), 106.14 (СHArN-2), 56.42
(CH3O), 55.37 (CH3O), 16.67 (2xCH3), 8.30 (CH3). MS (m/z): 367
(M++1); anal. calcd. for C20H22N4O3: C, 65.56; H, 6.05; N, 15.29;
found: C, 65.63; H, 6.00; N, 15.35.
(ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 10.84 (s, 1H, NH), 8.06 (d, J = 8.4 Hz, 2H,
HAr-3,5), 7.80 (d, J = 8.4 Hz, 2H, HAr-2,6), 7.54 (d, J = 7.7 Hz,
2H, HTol-2,6), 7.46 (d, J = 7.6 Hz, 2H, HTol-3,5), 7.28 (s, 2H, NH2),
2.57 (s, 3H, CH3), 2.43 (s, 3H, CH3); 13C NMR (126 MHz, DMSO-
d6): δ 159.86 (СO), 141.61 (СArN-1), 139.95 (СArN-4), 138.74 (СTr-5),
138.08 (СTr-4), 137.84 (СTol-4), 132.73 (СTol-1), 130.10 (2xСHTol-3,5),
126.42 (2xСHArN-3,5), 125.25 (2xСHTol-2,6), 119.95 (2xСHArN-2,6),
20.74 (CH3), 9.46 (CH3). MS (m/z): 372 (M++1); anal. calcd. for
C17 H17 N5O3S: C, 54.97; H, 4.61; N, 18.86; found: C, 54.90; H, 4.73;
N, 18.93.
4.2.2.15. 5-Cyclopropyl-N-(4-fluorophenyl)-1-(4-methoxyphenyl)-1H-
1,2,3-triazole-4-carboxamide (4t). White solid; Yield 96%; m.p.
156-157 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(400 MHz, DMSO-d6): δ 10.51 (s, 1H, NH), 7.87 (dd, J = 7.9, 3.7 Hz,
2H, HArN-2,6), 7.59 (d, J = 7.9 Hz, 2H, HAr-2,6), 7.24 – 7.13 (m, 4H,
HArom.), 3.86 (s, 3H, CH3O), 2.11 – 1.99 (m, 1H, CH), 0.98 – 0.77
(m, J = 9.5 Hz, 4H, CH2). 13C NMR (101 MHz, DMSO-d6): δ 160.64
1
(СO), 159.51 (СAr-4), 158.76 (d, JC-F = 240.1 Hz, СArN-4), 142.13
(СTr-5), 138.99 (СTr-4), 135.63 (СArN-1), 129.15 (СAr-1), 127.82
3
4.2.2.11. 5-Methyl-N-(4-sulfamoylphenyl)-1-m-tolyl-1H-1,2,3-triazole-
4-carboxamide (4p). White solid; Yield 91%; m.p. 253-254 °C
(2xСHAr-2,6), 122.58 (d, JC-F = 7.7 Hz, 2xСHArN-2,6), 115.61 (d,
2JC-F = 22.2 Hz, 2xСHArN-3,5), 115.03 (2xСHAr-3,5), 56.09 (CH3O),
8.10 (2xCH2), 5.77 (CH). MS (m/z): 353 (M++1); anal. calcd. for
C19 H17 FN4O2: C, 64.76; H, 4.86; N, 15.90; found: C, 64.71; H, 4.97;
N, 15.97.
(ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 10.86 (s, 1H, NH), 8.06 (d, J = 8.6 Hz, 2H,
HAr-3,5), 7.80 (d, J = 8.5 Hz, 2H, HAr-2,6), 7.53 (t, J = 7.5 Hz, 1H,
HTol-3), 7.50 – 7.42 (m, 3H, HTol), 7.29 (s, 2H, NH2), 2.58 (s, 3H,
CH3), 2.43 (s, 3H, CH3); 13C NMR (101 MHz, DMSO-d6): δ 160.35
(СO), 142.12 (СArN-1), 140.13 (СHTol-2), 139.26 (СArN-4), 138.57
(СTr-5), 138.37 (СTr-4), 135.62 (СTol-3), 131.22 (СTol-1), 129.98
(СHTol-5), 126.94 (2xСHArN-3,5), 126.31 (СHTol-4), 122.98 (СHTol-6),
120.47 (2xСHArN-2,6), 21.25 (CH3), 10.00 (CH3). MS (m/z): 372
(M++1); anal. calcd. for C17 H17 N5O3S: C, 54.97; H, 4.61; N, 18.86;
found: C, 54.95; H, 4.55; N, 18.81.
4.2.2.16. 1-(4-Chlorophenyl)-5-isopropyl-N-(thiazol-2-yl)-1H-1,2,3-
triazole-4-carboxamide (4u). White solid; Yield 83%; m.p. 226-227
°C (ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(400 MHz, DMSO-d6): δ 12.36 (s, 1H, NH), 7.74 (d, J = 8.7 Hz, 2H,
HAr-2,6), 7.66 (d, J = 8.6 Hz, 2H, HAr-3,5), 7.57 (d, J = 3.5 Hz, 1H,
HThiazole-4), 7.31 (d, J = 3.5 Hz, 1H, HThiazole-5), 3.21 (hept., J = 7.0
Hz, 1H), 1.31 (d, J = 7.0 Hz, 6H); 13C NMR (101 MHz, DMSO-d6): δ
159.52 (СO), 157.96 (СThiazol-1), 147.72 (СHThiazol-4), 138.35 (СTr-5),
136.85 (СTr-4), 135.95 (СAr-1), 134.81 (СAr-4), 130.33 (2xСHAr-3,5),
129.11 (2xСHAr-2,6), 114.47 (СHThiazol-5), 24.88 (CH), 20.36 (CH3)..
MS (m/z): 348 (M++1); anal. calcd. for C15H14 ClN5OS: C, 51.80; H,
4.06; N, 20.13; found: C, 51.74; H, 4.14; N, 20.21.
4.2.2.12. 1-(3-Methoxyphenyl)-5-methyl-N-(4-sulfamoylphenyl)-1H-
1,2,3-triazole-4-carboxamide (4q). White solid; Yield 87%; m.p.
234-235 °C (ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H
NMR (400 MHz, DMSO-d6): δ 10.85 (s, 1H, NH), 8.05 (d, J = 8.8
Hz, 2H, HArN-3,5), 7.80 (d, J = 8.7 Hz, 2H, HArN-2,6), 7.56 (t, J = 8.1
Hz, 1H, HAr-5), 7.28 (s, 2H, NH2), 7.26 – 7.18 (m, 3H, HAr-5), 3.84
(s, 3H, CH3O), 2.59 (s, 3H, CH3); 13C NMR (101 MHz, DMSO-d6):
δ 160.43 (СAr-3), 160.34 (СO), 142.11 (СArN-1), 139.27 (СArN-4),
138.74 (СTr-5), 138.36 (СTr-4), 136.68 (СAr-1), 131.09 (СHAr-3),
126.94 (2xСHArN-3,5), 120.48 (2xСHArN-2,6), 118.07 (СHAr-6),
116.40 (СHAr-4), 111.74 (СHAr-2), 56.19 (CH3O), 10.01 (CH3). MS
(m/z): 388 (M++1); anal. calcd. for C17 H17 N5O4S: C, 52.70; H,
4.42; N, 18.08; found: C, 52.77; H, 4.48; N, 18.25.
4.2.3. General procedure for the synthesis of
1H-1,2,3-triazol-5-amines 8 and
[1,2,3]triazolo[1,5-a]quinazolin-5(4H)-ones 9
To the solution of sodium methoxide (108 mg, 2.0 mmol) in
dry methanol (5 mL), an appropriate substituted cyanoacetamide
7 (2.0 mmol) was added. To this solution, substituted methyl 2-
azidobenzoate 1r-t (10.0 mmol) in dry methanol (2 mL) was added
dropwise, and the solid started to precipitate. The mixture was
9

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
6H, CH3). 13C NMR (101 MHz, DMSO-d6): δ 161.01 (СO), 155.73
(СArN-4), 145.37 (СTr-5), 139.71 (2xСAr-3,5), 135.04 (СTr-4), 132.46
stirred for 1 h. The resulting suspension was filtered and the
solid product was washed with water and methanol to give the
corresponding 1H-1,2,3-triazol-5-amines 8 and [1,2,3]triazolo[1,5-
a]quinazolin-5(4H)-ones 9.
(СHAr-4), 130.90 (СAr-1), 122.14 (2xСHAr-2,6), 122.09 (2xСHArN
-
2,6), 122.02 (СArN-1), 114.14 (2xСHArN-3,5), 55.63 (CH3), 21.26
(2xCH3). MS (m/z): 338 (M++1); anal. calcd. for C18 H19 N5O2: C,
64.08; H, 5.68; N, 20.76; found: C, 64.14; H, 5.78; N, 20.71.
4.2.3.1. N-(4-Acetamidophenyl)-5-amino-1-(3-bromophenyl)-1H-
1,2,3-triazole-4-carboxamide (8a). White solid; Yield 98%; m.p.
235-236 °C (ethanol-DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H
4.2.3.6. 5-Oxo-N-phenyl-4,5-dihydro-[1,2,3]triazolo[1,5-a]quinazoline-
3-carboxamide (9a). White solid; Yield 97%; m.p. >250 °C (DMF);
NMR (400 MHz, DMSO-d6): δ 10.15 (s, 1H, NH), 9.88 (s, 1H, NH),
7.94 – 7.42 (m, 8H, HArom.), 6.68 (s, 2H, NH2), 2.03 (s, 3H, CH3). 13C
IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6):
NMR (126 MHz, DMSO-d6): δ 167.93 (СO), 160.43(СO), 145.29 (СTr
-
δ 11.78 (br.s, 1H, NH), 10.43 (s, 1H, NH), 8.38 (d, J = 8.1 Hz, 1H,
Hquin-6), 8.29 (d, J = 7.6 Hz, 1H, Hquin-9), 8.01 (t, J = 7.6 Hz, 1H,
Hquin-8), 7.88 (d, J = 8.4 Hz, 2H, HPh-2,6), 7.71 (t, J = 7.6 Hz, 1H,
Hquin-7), 7.31 (t, J = 7.7 Hz, 2H, HPh-3,5), 7.06 (t, J = 7.1 Hz, 1H,
HPh-4). 13C NMR (126 MHz, DMSO-d6) δ 160.24 (CONH), 159.48
(CONH), 149.72 (Cquin-3a), 139.27 (Cquin-9a), 138.15 (СPh-1), 134.46
(CHquin-8), 129.36 (2xCHPh-3,5), 129.17 (СHPh-4), 126.26 (CHquin-7),
125.80 (CHquin-6), 123.81 (Cquin-5a), 119.17 (2xCHPh-2,6), 117.32
(Cquin-3), 115.17 (CHquin-9).MS (m/z): 306 (M++1); anal. calcd. for
C16 H11 N5O2: C, 62.95; H, 3.63; N, 22.94; found: C, 63.01; H, 3.74;
N, 22.91.
5), 135.97 (СTr-4), 134.85 (СArN-4), 134.03 (СArN-1), 132.04 (СAr-1),
131.59 (СHAr-5), 127.02 (СHAr-4), 123.50 (СHAr-6), 122.12 (СHAr-2),
121.48(СAr-3), 120.50 (2xСHArN-2,6), 119.17 (2xСHArN-3,5), 23.88
(CH3). MS (m/z): 415, 417 (M++1); anal. calcd. for C17 H15BrN6O2:
C, 49.17; H, 3.64; N, 20.24; found: C, 49.09; H, 3.71; N, 20.21.
4.2.3.2. 5-Amino-N-(2,3-dimethylphenyl)-1-(4-fluorophenyl)-1H-1,2,3-
triazole-4-carboxamide (8b). White solid; Yield 97%; m.p. 209-210
°C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 9.69 (s, 1H, NH), 7.67 (dd, J = 8.9, 4.9 Hz, 2H,
HAr-2,6), 7.47 (t, J = 8.8 Hz, 2H, HAr-3,5), 7.28 (d, J = 7.5 Hz, 1H,
HArN-6), 7.08 (t, J = 7.6 Hz, 1H, HArN-5), 7.03 (d, J = 7.1 Hz, 1H,
HArN-4), 6.48 (s, 2H, NH2), 2.27 (s, 3H, CH3), 2.14 (s, 3H, CH3).
4.2.3.7. N-(2-Fluorophenyl)-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-
1
13C NMR (101 MHz, DMSO-d6): δ 162.57 (d, JC-F = 246.1 Hz),
a]quinazoline-3-carboxamide (9b). White solid; Yield 91%; m.p.
161.27 (СO), 145.72 (СTr-5), 137.27 (СArN-1), 136.25 (СTr-4), 131.99
>250°C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
4
3
(СArN-3), 131.59 (d, JC-F = 2.5 Hz, СAr-1), 127.58 (d, JC-F = 9.1 Hz,
MHz, DMSO-d6): δ 12.01 (br.s, 1H, NH), 9.79 (s, 1H, NH), 8.38
(d, J = 8.2 Hz, 1H, Hquin-6), 8.27 (d, J = 8.0 Hz, 1H, Hquin-9),
8.01 (t, J = 7.9 Hz, 1H, Hquin-8), 7.98 – 7.92 (m, 1H, HAr-3), 7.71
(t, J = 7.7 Hz, 1H, Hquin-7), 7.26 – 7.16 (m, 3H, HAr). 13C NMR
(126 MHz, DMSO-d6) δ 159.02 (CONH), 158.87 (CONH), 155.70
2xСHAr-2,6), 127.43 (СHArN-4), 125.63 (СHArN-5), 124.26 (СHArN-6),
2
121.93 (СArN-2), 117.15 (d, JC-F = 23.1 Hz, 2xСHAr-3,5), 20.67 (CH3),
14.52 (CH3). MS (m/z): 326 (M++1); anal. calcd. for C17 H16 FN5O:
C, 62.76; H, 4.96; N, 21.53; found: C, 62.77; H, 4.90; N, 21.57.
1
(d, JC-F = 246.4 Hz, CAr-2), 136.91 (Cquin-3a), 135.96 (CHquin-8),
4.2.3.3. 5-Amino-N-(2,3-dimethylphenyl)-1-m-tolyl-1H-1,2,3-triazole-
4-carboxamide (8c). White solid; Yield 96%; m.p. 134-135 °C
134.79 (Cquin-9a), 128.91 (CHquin-7), 128.79 (CHAr-5), 127.05 (d,
2
3JC-F = 7.7 Hz, CHAr-4), 126.50 (CHquin-6), 125.57 (d, JC-F = 11.4
(ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz,
3
Hz, CAr-1), 124.81 (d, JC-F = 3.0 Hz, CHAr-6), 123.89 (Cquin-5a),
DMSO-d6): δ 9.68 (s, 1H, NH), 7.51 (t, J = 7.6 Hz, 1H, HAr-5),
7.46 – 7.35 (m, 3H, HAr), 7.30 (d, J = 7.4 Hz, 1H, HArN-6), 7.09 (t,
J = 7.5 Hz, 1H, HArN-5), 7.04 (d, J = 7.1 Hz, 1H, HArN-4), 6.45 (s,
2H, NH2), 2.43 (s, 3H, CH3), 2.28 (s, 3H, CH3), 2.15 (s, 3H, CH3).
13C NMR (126 MHz, DMSO-d6): δ 160.83 (СO), 144.88 (СTr-5),
139.50 (СArN-1), 136.75 (СTr-4), 135.74 (СTol-3), 134.67, 131.46
(СArN-3), 129.73 (СTol-1), 129.55 (СHTol-5), 126.91(СHArN-4), 125.12
(СHArN-5), 124.55 (СHArN-6), 123.73 (СHTol-4), 121.52 (СArN-2),
121.20 (СHTol-6), 20.85 (CH3), 20.17 (CH3), 14.02 (CH3). MS (m/z):
322 (M++1); anal. calcd. for C18 H19N5O: C, 67.27; H, 5.96; N,
21.79; found: C, 67.33; H, 5.99; N, 21.72.
2
117.59 (Cquin-3), 116.17 (d, JC-F = 19.6 Hz, CHAr-3), 115.70 (CHquin
-
9). MS (m/z): 324 (M++1); anal. calcd. for C16 H10FN5O2: C, 59.44;
H, 3.12; N, 21.66; found: C, 59.41; H, 3.19; N, 21.74.
4.2.3.8. N-(3,4-dichlorophenyl)-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-
a]quinazoline-3-carboxamide (9c). White solid; Yield 97%; m.p.
>250°C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 11.81 (br.s, 1H, NH), 10.79 (s, 1H, NH), 8.38 (d,
J = 7.2 Hz, 1H, Hquin-6), 8.29 (d, J = 7.3 Hz, 1H, Hquin-9), 8.26 (s,
1H, HAr-2), 8.01 (t, J = 7.9 Hz, 1H, Hquin-8), 7.85 (d, J = 8.9 Hz,
1H, HAr-6), 7.71 (t, J = 7.5 Hz, 1H, Hquin-7), 7.45 (d, J = 8.7 Hz, 1H,
HAr-5). 13C NMR (126 MHz, DMSO-d6) δ 159.34 (CONH), 158.99
(CONH), 137.25 (Cquin-3a), 136.01 (CHquin-8), 134.78 (Cquin-9a),
133.78 (СAr-1), 131.29 (СAr-3), 130.99 (СHAr-5), 128.96 (CHquin-7),
4.2.3.4. 5-Amino-N,1-di-m-tolyl-1H-1,2,3-triazole-4-carboxamide
(8d). White solid; Yield 96%; m.p. 153-155 °C (ethanol); IR (KBr)
νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 9.81 (s,
1H, NH), 7.68 (s, 1H, HArN-2), 7.59 (d, J = 8.2 Hz, 1H, HArN-6), 7.47
(t, J = 7.7 Hz, 1H, HAr-5), 7.44 – 7.39 (m, 2H, HAr), 7.32 (d, J = 7.5
Hz, 1H, HAr-4), 7.14 (t, J = 7.8 Hz, 1H, HArN-5), 6.83 (d, J = 7.5 Hz,
1H, HArN-4), 6.44 (s, 2H, NH2), 2.47 (s, 3H, CH3), 2.34 (s, 3H, CH3).
13C NMR (126 MHz, DMSO-d6): δ 160.58, 144.85, 140.28, 138.12,
136.47, 134.27, 130.43, 129.80, 128.59, 128.35, 127.27, 123.88,
122.51, 120.61, 120.54, 21.21, 20.26. MS (m/z): 308 (M++1); anal.
calcd. for C17 H17 N5O: C, 66.43; H, 5.58; N, 22.79; found: C, 66.55;
H, 5.70; N, 22.71.
128.86 (CHquin-6), 125.57 (СAr-4), 124.02 (Cquin-5a), 121.84 (СHAr
-
2), 120.69 (СHAr-6), 117.63 (Cquin-3), 115.72 (CHquin-9). MS (m/z):
374 (M++1); anal. calcd. for C16 H9Cl2N5O2: C, 51.36; H, 2.42; N,
18.72; found: C, 51.44; H, 2.52; N, 18.71.
4.2.3.9. N-(2,4-dichlorophenyl)-5-oxo-4,5-dihydro-[1,2,3]triazolo[1,5-
a]quinazoline-3-carboxamide (9d). White solid; Yield 95%; m.p.
>250°C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400
MHz, DMSO-d6): δ 12.19 (br.s, 1H, NH), 9.72 (s, 1H, NH), 8.40 (d,
J = 8.2 Hz, 1H, Hquin-6), 8.29 (d, J = 8.0 Hz, 1H, Hquin-9), 8.25 (d,
J = 8.9 Hz, 1H, HAr-2), 8.02 (t, J = 7.5 Hz, 1H, Hquin-8), 7.73 (t,
J = 7.4 Hz, 1H, Hquin-7), 7.57 (d, J = 2.2 Hz, 1H, HAr-3), 7.40 (dd,
J = 8.8, 2.1 Hz, 1H, HAr-5). MS (m/z): 374 (M++1); anal. calcd. for
C16 H9Cl2N5O2: C, 51.36; H, 2.42; N, 18.72; found: C, 51.40; H, 2.35;
N, 18.75.
4.2.3.5. 5-Amino-1-(3,5-dimethylphenyl)-N-(4-methoxyphenyl)-1H-
1,2,3-triazole-4-carboxamide (8e). White solid; Yield 96%; m.p.
163-164 °C (ethanol); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR
(400 MHz, DMSO-d6): δ 10.09 (s, 1H, NH), 7.74 (d, J = 8.9 Hz, 2H,
HArN-2,6), 7.21 (s, 2H, HAr-2,6), 7.17 (s, 1H, HAr-4), 6.89 (d, J = 8.9
Hz, 2H, HArN-3,5), 6.49 (s, 2H, NH2), 3.73 (s, 3H, CH3O), 2.37 (s,
10

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
4.2.3.10. 7-Fluoro-N-(furan-2-ylmethyl)-5-oxo-4,5-dihydro-
compared to the negative control (Bacteria/fungi only, set to 0% in-
hibition). The growth rates for all bacteria and fungi have a varia-
tion of -/+ 10%, which is within the reported normal distribution of
bacterial/fungal growth. Any significant variation (or outliers/hits)
is identified by the modified Z-Score, and actives are selected by a
combination of inhibition value and Z-Score.
[1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide (9e). White solid;
Yield 89%; m.p. >250 °C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 11.83 (br.s, 1H, NH), 8.92 (t,
J = 5.9 Hz, 1H, NH), 8.41 (dd, J = 9.0, 4.2 Hz, 1H, Hquin-6), 7.91
(dd, J = 8.3, 2.7 Hz, 1H, Hquin-9), 7.81 (td, J = 8.8, 3.0 Hz, 1H,
Hquin-8), 7.43 (d, J = 0.8 Hz, 1H, HFur-5), 6.32 (dd, J = 2.9, 1.9 Hz,
1H, HFur-4), 6.25 (d, J = 2.7 Hz, 1H, HFur-3), 4.50 (d, J = 5.9 Hz, 2H,
1
CH2N). 13C NMR (126 MHz, DMSO-d6) δ 161.25 (d, JC-F = 247.0 Hz,
CFquin-7), 160.21 (CONH), 158.23 (CONH), 152.88 (CFur-2), 142.36
(CHFur-5), 136.07 (Cquin-3a), 131.68 (Cquin-9a), 124.16 (Cquin-3),
4.3.2. Antimicrobial studies
2
3
Antibacterial activity was determined by using MTT test. Experi-
ments were conducted at pH 7.2. Subsequent bacterial culture in
the logarithmic phase of growth in Sabouraud medium, pH 7.2,
was centrifuged for 10 min at 500 g, and sediment of bacteria was
washed with sterile saline and resuspended in a small volume of
sterile saline. A defined volume of this suspension was introduced
into Sabouraud medium with pH 7.2 for obtaining OD 0.4-0.6 at
590 nm (optical path 1.0 cm). Then, 100 μL of each suspension
were introduced into series of 1.5 mL Eppendorf tubes, and there-
after inoculation with 10, 5, and 2 μL of the tested sample solution
was conducted. Each experiment was repeated in triplicate. Tubes
were incubated for 4 h at 37 °C. Thereafter, 10 μL of MTT solution
(5 mg/mL) was introduced, and incubation was continued for 1 h.
Cells were harvested by centrifugation for 5 min at 1,500 g, the su-
pernatant was discarded and small sediment was suspended in 1
mL of DMSO. After the incubation for 1 h at 37°C, the OD of liq-
uid was measured at 580 nm on the spectrophotometer ULAB 102
UV (Ukraine). The effect of tested compound upon bacteria viabil-
ity was compared with that in a control.
123.63 (d, JC-F = 24.7 Hz, CHquin-8), 119.77 (d, JC-F = 7.9 Hz,
3
2
Cquin-5a), 118.59 (d, JC-F = 8.6 Hz, CHquin-9), 114.55 (d, JC-F = 24.8
Hz, CHquin-6), 110.92 (CHFur-4), 107.28 (CHFur-3), 35.65 (СH2N).
MS (m/z): 328 (M++1); anal. calcd. for C15H10FN5O2: C, 55.05; H,
3.08; N, 21.40; found: C, 55.15; H, 3.17; N, 21.37.
4.2.3.11. N-(2-Chlorophenyl)-7-fluoro-5-oxo-4,5-dihydro-
[1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide (9f). White solid;
Yield 94%; m.p. >250 °C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 12.39 (br.s, 1H, NH), 9.63 (s, 1H,
NH), 8.45 (dd, J = 8.9, 4.4 Hz, 1H, Hquin-6), 8.31 (dd, J = 8.1, 1.3
Hz, 1H, HAr-3), 7.94 (dd, J = 8.4, 2.9 Hz, 1H, Hquin-9), 7.84 (td,
J = 8.5, 2.8 Hz, 1H, Hquin-8), 7.49 (dd, J = 8.0, 1.3 Hz, 1H, HAr-6),
7.36 (dt, J = 7.6, 1.3 Hz, 1H, HAr-5), 7.16 (dt, J = 7.7, 1.4 Hz, 1H,
HAr-4). 13C NMR (126 MHz, DMSO-d6) δ 161.38 (d, JC-F = 248.4
1
Hz, CFquin-7), 158.56 (2xCONH), 134.74 (Cquin-3a), 131.68 (Cquin-9a),
129.97 (CAr-1+CHAr-3), 128.21 (CAr-2+CHAr-5), 126.78 (CHAr-4),
125.16 (CHAr-6), 123.92 (Cquin-3), 123.70 (d, J = 24.5 Hz, CHquin-8),
3
3
119.89 (d, JC-F = 7.3 Hz, Cquin-5a), 118.66 (d, JC-F = 8.3 Hz,
Antifungal activity was studied on C. albicans strains in the
Sabouraud medium by using the Colony Forming Units (CFU)
method. The quality of peptone as the component of the
Sabouraud medium and control of pH are of great importance for
the reproducibility of results. In the experiments, the enzymatic
peptone (Kyiv, Ukraine) was used that provided a rapid growth of
C. albicans and the appearance of distinct colonies after 24 h of
incubation at 37 °C.
2
CHquin-9), 114.57 (d, JC-F = 24.7 Hz, CHquin-6). MS (m/z): 358
(M++1); anal. calcd. for C16 H9ClFN5O2: C, 53.72; H, 2.54; N, 19.58;
found: C, 53.70; H, 2.59; N, 19.51.
4.2.3.12. 7-Chloro-N-(2-chlorophenyl)-5-oxo-4,5-dihydro-
[1,2,3]triazolo[1,5-a]quinazoline-3-carboxamide (9g). White solid;
Yield 96%; m.p. >250°C (DMF); IR (KBr) νmax 1670 (s, C=O) cm–1
;
1H NMR (400 MHz, DMSO-d6): δ 12.46 (br.s, 1H, NH), 9.66 (s,
1H, NH), 8.41 (d, J = 8.6 Hz, 1H, HAr-3), 8.27 (d, J = 8.3 Hz, 1H,
Hquin-8), 8.19 (s, 1H, Hquin-6), 8.02 (d, J = 9.0 Hz, 1H, Hquin-9),
7.50 (d, J = 8.1 Hz, 1H, H6Ar), 7.36 (t, J = 7.6 Hz, 1H, H5Ar), 7.17
(t, J = 7.6 Hz, 1H, H4Ar). MS (m/z): 374 (M++1); anal. calcd. for
C16 H9Cl2N5O2: C, 51.36; H, 2.42; N, 18.72; found: C, 51.45; H, 2.36;
N, 18.81.
A suspension of Candida sp containing 107 cells/mL was pre-
pared by suspending cells taken from the colonies grown on the
Sabouraud agar, pH 5.8. Cells’ number was counted in the hemo-
cytometric chamber, since a size of Candida cells (2.5-4 μm) per-
mitted doing that accurately. The tested compound solution in 10,
5 and 2,5 μL volume was introduced into 3 round bottom Eppen-
dorf tubes, and thereafter, 100 μL of Candida cells suspension was
added. Two control tubes were prepared: at the start (time 0) and
at the end (4 h) of incubation. The tubes were incubated for 4 h
at 37 °C (except control 0 kept at 4 °C). Then, 10 μL aliquote was
withdrawn at the end of incubation from each tube after thorough
mixing, diluted 10,000 fold with water and 0.2 mL of this dilu-
tion was distributed on the surface of Sabouraud agar medium,
pH 5.8, in the Petri dish. They were incubated at 37 °C and af-
ter distinct formation of colonies (usually after 24 h) the image
was scanned and colonies were counted using the Photoshop pro-
gram. The number of colonies in control tube at 0 h time must
be 200 50 per dish, in control after 4 h of incubation colonies
number must be 1.5-2.5 fold higher. The experiment was abol-
ished when the increase in a number of colonies in control after
the incubation was less than 1.5 fold. The effect of tested com-
pound upon the viability of Candida cells was expressed as a ratio
of colony number.
4.3. Biological screening
4.3.1. Primary screening data collection via CO-ADD
[http://www.co-add.org/]
An inhibition of bacterial growth was determined via measur-
ing the absorbance at 600 nm. The percentage of growth inhibi-
tion was calculated for each well, using the negative control (me-
dia only) and positive control (bacteria without inhibitors) on the
same plate as references. Growth inhibition of C. albicans was de-
termined by measuring absorbance at 530 nm, while the growth
inhibition of C. neoformans was determined by measuring the dif-
ference in absorbance between 600 and 570 nm, after the addition
of resazurin (0.001% final concentration) and incubation at 35°C for
additional 2 h. The percentage of growth inhibition was calculated
for each well, using the negative control (media only) and posi-
tive control (bacteria without inhibitors) on the same plate as ref-
erences. Degree of growth inhibition of an individual sample was
calculated based on negative controls (media only) and positive
controls (bacterial/fungal media without inhibitors). Negative inhi-
bition values indicate that the growth rate (or OD600) is higher
The index lower than control (0-hour time) was considered as
candidacidal effect, while higher than control (0-hour time) but
less than in control after 4 h of incubation was classified as growth
inhibition, and the index higher than in control after 4 h of incu-
bation indicated a stimulation of growth [40].
11

N. Pokhodylo, N. Manko, N. Finiuk et al.
Journal of Molecular Structure 1246 (2021) 131146
4.3.3. Cell culture and cytotoxicity assay (MTT and Trypan Blue dye
assays)
against cancer and pathogenic microorganisms”), the Ministry of
Education and Science of Ukraine (Grant No 0121U107777) and the
Research Grant of the Program “Genomic, molecular and cellular
bases of development of innovative biotechnologies” awarded by
the National Academy of Sciences of Ukraine (No 0120U103077,
2020–2024). The authors are grateful to CO-ADD (The Community
for Antimicrobial Drug Discovery) funded by the Welcome Trust
(UK) and The University of Queensland (Australia) for the support
with antimicrobial screening.
Human keratinocytes of HaCaT line were obtained from a Col-
lection at the Institute of Molecular Biology and Genetics, National
Academy of Sciences of Ukraine (Kyiv, Ukraine). Cells were grown
in the DMEM (Biowest, Nuaille, France) culture medium supple-
mented with 10% fetal bovine serum (Biowest, Nuaille, France) un-
der standard conditions. In vitro evaluation of cytotoxic activity of
the synthesized compounds compared with the doxorubicin used
as a reference control, toward human keratinocytes of HaCaT line
was measured by the MTT test [41]. Briefly, cells were seeded
for 24 h in 96-well microtiter plates at a concentration of 5,000
cells/well (100 μL/well); after that, cells were incubated for 72 h
with various additions of the synthesized compounds (0–50 μM).
MTT, which is converted to dark blue, water-insoluble formazan
by the mitochondrial dehydrogenases, was used to determine vi-
able cells according to the Sigma-Aldrich protocol. Formazan was
dissolved in the DMSO, and the results of the reaction were de-
termined by an Absorbance Reader BioTek ELx800 (BioTek Instru-
ments, Inc., Winooski, VT, USA). The IC50 of the tested compounds
was calculated as a concentration of drug killing 50% of cells com-
pared with the untreated culture.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131146.
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