Synthesis and mode of action studies of novel {2-(3-R-1H-1,2,4-triazol-5-yl)phenyl}amines to combat pathogenic fungi

Article abstract of DOI:10.1002/ardp.201900092

Due to their high specificity and efficacy, triazoles have become versatile antifungals to treat fungal infections in human healthcare and to control phytopathogenic fungi in agriculture. However, azole resistance is an emerging problem affecting human health as well as food security. Here we describe the synthesis of 10 novel {2-(3-R-1H-1,2,4-triazol-5-yl)phenyl}amines. Their structure was ascertained by liquid chromatography–mass spectrometry, ¹H and ¹³C NMR, and elemental analysis data. Applying an in vitro growth assay, these triazoles show moderate to significant antifungal activity against the opportunistic pathogen Aspergillus niger, 12 fungi (Fusarium oxysporum, Fusarium fujikuroi, Colletotrichum higginsianum, Gaeumannomyces graminis, Colletotrichum coccodes, Claviceps purpurea, Alternaria alternata, Mucor indicus, Fusarium graminearum, Verticillium lecanii, Botrytis cinerea, Penicillium digitatum) and three oomycetes (Phytophtora infestans GL-1, P. infestans 4/91; R+ and 4/91; R?) in the concentration range from 1 to 50 μg/ml (0.003–2.1 μM). Frontier molecular orbital energies were determined to predict their genotoxic potential. Molecular docking calculations taking into account six common fungal enzymes point to 14α-demethylase (CYP51) and N-myristoyltransferase as the most probable fungal targets. With respect to effectiveness, structure–activity calculations revealed the strong enhancing impact of adamantyl residues. The shown nonmutagenicity in the Salmonella reverse-mutagenicity assay and no violations of drug-likeness parameters suggest the good bioavailability and attractive ecotoxicological profile of the studied triazoles.

Full text of DOI:10.1002/ardp.201900092

Received: 21 March 2019
Revised: 10 July 2019
Accepted: 17 July 2019
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DOI: 10.1002/ardp.201900092
F U L L P A P E R
Synthesis and mode of action studies of novel {2‐(3‐R‐1H‐
1,2,4‐triazol‐5‐yl)phenyl}amines to combat pathogenic fungi
Lyudmyla Antypenko1
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Zhanar Sadykova1
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Kostiantyn Shabelnyk2
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Fatuma Meyer1
Karl Steffens1
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Sergiy Kovalenko2 Vera Meyer3
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Leif‐Alexander Garbe1
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1Department of Food and Bioproduct
Technology, Neubrandenburg University,
Neubrandenburg, Germany
Abstract
Due to their high specificity and efficacy, triazoles have become versatile antifungals
to treat fungal infections in human healthcare and to control phytopathogenic
fungi in agriculture. However, azole resistance is an emerging problem affecting
human health as well as food security. Here we describe the synthesis of 10 novel
{2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines. Their structure was ascertained by liquid
2Departments of Pharmaceutical Chemistry,
Organic and Bioorganic Chemistry,
Zaporizhzhya State Medical University,
Zaporizhzhya, Ukraine
3Department of Applied and Molecular
Microbiology, Institute of Biotechnology,
Technische Universität Berlin, Berlin,
Germany
chromatography–mass spectrometry, H and 13C NMR, and elemental analysis data.
1
Applying an in vitro growth assay, these triazoles show moderate to significant
antifungal activity against the opportunistic pathogen Aspergillus niger, 12 fungi
(Fusarium oxysporum, Fusarium fujikuroi, Colletotrichum higginsianum, Gaeumannomyces
graminis, Colletotrichum coccodes, Claviceps purpurea, Alternaria alternata, Mucor indicus,
Fusarium graminearum, Verticillium lecanii, Botrytis cinerea, Penicillium digitatum) and
three oomycetes (Phytophtora infestans GL‐1, P. infestans 4/91; R+ and 4/91; R−) in the
Correspondence
Dr. Lyudmyla Antypenko, Department of Food
and Bioproduct Technology, Neubrandenburg
University, Brodaer Street 2, 17033
Neubrandenburg, Germany.
Email: antypenkol@gmail.com
Funding information
concentration range from
1 to 50 µg/ml (0.003–2.1 μM). Frontier molecular
Bundesministerium für Bildung und
Forschung, Grant/Award Number: FKZ
03FH025IX4
orbital energies were determined to predict their genotoxic potential. Molecular
docking calculations taking into account six common fungal enzymes point to
14α‐demethylase (CYP51) and N‐myristoyltransferase as the most probable fungal
targets. With respect to effectiveness, structure–activity calculations revealed the
strong enhancing impact of adamantyl residues. The shown nonmutagenicity in the
Salmonella reverse‐mutagenicity assay and no violations of drug‐likeness parameters
suggest the good bioavailability and attractive ecotoxicological profile of the studied
triazoles.
K E Y W O R D S
{2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines, Ames test, antifungal activity, drug‐like descriptors,
molecular docking, structure–activity relationship
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1
INTRODUCTION
Abbreviations: 1H NMR, proton nuclear magnetic resonance; CYP51, P‐450‐dependent
14α‐sterol demethylase; DMSO, dimethylsulfoxide; IP, ionization potential; LC–MS, liquid
chromatography–mass spectrometry; LOMO, lowest‐occupied molecular orbital; MFC,
minimum fungicidal concentration; MIC, minimum inhibition concentration; MM+, molecular
mechanics; MNDO, modified neglect of the diatomic overlap; MW, molecular weight; NMT,
N‐myristoyltransferase; PDA, potato dextrose agar; SAR, structure–activity relationship;
Topo II, topoisomerase II; TPSA, molecular topological polar surface areas.
The control of phytopathogenic fungi is of paramount importance to
avoid serious losses in agriculture and to protect food security and
consumer safety for a growing world population. Until the 1940s,
mainly inorganic antifungal substances were used. Since then, new
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chemicals have been developed, which exhibited higher selectivity
and less toxicity towards the environment and food consumers. In
2014, the volume of the global fungicide market was estimated to be
about €10 billion.[1] Given that fungi are eukaryotic systems, it is not
straightforward to find substances with sufficient discriminatory
potential to act against the intended targets with low concomitant
toxicity towards organisms requiring protection. Modern antifungals
mainly act as inhibitors of ergosterol biosynthesis, membrane
disruptors or interfere with biosynthesis of the cell wall, sphingoli-
pids, nucleic acids, proteins, or microtubules. Most of them confer
only fungistatic effects.[2]
single compound had a clear advantage over standard fungicide
flusilazole when considering the spectrum of activity. Applying a
structure–activity relationship (SAR) analysis of aryl or hetaryl
substituents for N‐(4‐(3‐(2,4‐dichlorophenyl)‐3‐oxo‐2‐(1H‐1,2,4‐tria-
zol‐1‐yl)prop‐1‐en‐1‐yl)aryl)benzamides (Figure 1c), Tang et al.[14]
supposed an antifungal activity against Gibberella azea, when a
2‐hydroxy group or 4‐chloro substituent was introduced into the
phenyl ring. Fusarium oxysporum, Cytospora mandshurica, and Pellicu-
laria sasakii were inhibited when the phenyl residue was replaced by a
furan‐2‐yl moiety. The 3‐(adamantan‐1‐yl)‐4‐ethyl‐5‐(R‐methylthio)‐
4H‐1,2,4‐triazoles D1 and D2[15] had a in vitro stronger fungistatic
and fungicidal activity against Candida albicans, than the reference
agent, trimethoprim. Their minimum inhibition concentrations (MIC)
were 31.25 and 7.8 µg/ml, and the minimum fungicidal concentra-
tions (MFC) were 62.5 and 15.6 µg/ml, respectively. Among the
hiourea substituted series, 2‐(2‐((5‐(adamantane‐1‐yl)‐4‐phenyl‐1,2,4‐
triazole‐3‐yl)thio)acetyl)‐N‐ethyl‐hydrazinecarbothioamide (Figure 1e)
appeared the most active in relation to all microbial test‐strains
(Staphylococcus aureus, Escherichia coli, Pseudomоnas aeruginosa, C.
albicans)[16] with an MIC and MFC of 31.25 μg/ml against C. albicans.
The antifungal activities of the myrtenal bearing 1,2,4‐triazoles
F were evaluated against Fusarium wilt on cucumber (F. oxysporum),
apple root spot (Physalospora piricola), tomato early blight (Alternaria
solani), speckle on peanut (Cercospora arachidicola), and wheat scab
(Gibberella zeae) at 50 µg/ml.[17] The i‐propyl F1, O‐nitrobenzyl
F2 and ethyl F3 thioethers exhibited excellent antifungal activity
against P. piricola with inhibition rates of 98.2%, 96.4%, and 90.7%,
respectively, showing better or comparable antifungal activity than
Among ergosterol inhibitors, 1,2,4‐triazoles have attained great
importance in human healthcare[3] and are the most commonly
used type of fungicides in agriculture with a market share of
approximately 20%.[4] This success is based on the extraordinary
activity of this class of antifungals. Spectroscopic analysis on purified
Candida albicans P‐450‐dependent 14α‐sterol demethylase (CYP51)
revealed, that triazoles interact with the sixth coordination position
of the central iron of the P‐450 heme active center.[5] The
three‐dimensional conformation of a full‐length CYP51 14α‐lanos-
terol demethylase from Saccharomyces cerevisiae was elucidated by
crystallization. The enzyme is anchored in the membrane of the
endoplasmic reticulum with an N‐terminal transmembrane α‐helix.
Cocrystallization with sterol substrates confirmed earlier findings of
the involvement of the heme group in the active center and amino
acid residues in the near environment for catalytic function.[6]
The emergence of eucaryotic microorganisms including fungi with
reduced susceptibility or resistance to drugs is a common phenom-
enon also observed with azoles.[7] Disturbingly, in clinical settings,
triazole resistant pathogenic fungus Aspergillus fumigatus carrying
alterations in gene CYP51A could be isolated from infected patients,
who never before were treated with therapeutic azole drugs.
This implies the fungus likely encountered resistance from the
environment.[8,9] Physiological and genetic studies revealed that
overexpression of drug efflux pumps or CYP51, modification of the
ergosterol biosynthetic pathway, or mutations within the CYP51
gene are the main strategies for fungi to become resistant towards
triazoles.[4] For instance, the replacement of a conserved tyrosine
(Y140F/H) in the active site of CYP51 could be identified to confer
resistance against short‐chain azoles to a mutated S. cerevisiae
strain.[10] In this context, the development of new substances is a
necessity to secure the availability of active antifungals for
agriculture and human healthcare in the future.[11]
that of the commercial fungicide azoxystrobin with
a 96.0%
inhibition rate. Molecular docking studies confirmed the experi-
mental results showing that 2‐aryl‐4[{(6‐substituted‐2‐(aryloxy)‐
quinolin‐3‐yl)‐methyl}]‐3H‐1,2,4‐triazol‐3‐ones (Figure 1g) were
active against A. fumigatus and C. albicans (MIC 0.2–1.5 μg/ml).[18]
Analogs containing methoxy and chloro substituents (J1–3)
exhibited the strongest activity. And N‐myristoyltransferase trans-
ferase (NMT) and dihydrofolate reductase were identified as the
most probable target enzymes.
Moreover, it was found that molecular orbital calculations such as
semiempirical methods (MM+ and MNDO) allowed deriving
a
theoretical estimation of the reactivity, and thereby the potential
toxicity of novel substances. Namely, linear dependences are
described between the LUMO energy of the different nitroaromatic
compounds,[19] methylnitro‐ and aminocarbazoles,[20] nitrobenzan-
thrones,[21] and potential gene toxicity as can be detected by
bioassays as the Salmonella mutagenicity test.[22]
In vitro experiments revealed that N‐[2‐hydroxy‐3,3‐dimethyl‐2‐
[(1H‐1,2,4‐triazol‐1‐yl)methyl]butyl]benzamide derivatives (Figure 1a)
bearing a triazole ring with a benzamide moiety displayed high
antifungal activity against Alternaria alternata at 50 μg/ml.[12]
Basarab et al.[13] reported that for most of the 1‐amino‐1,2,4‐
triazole (Figure 1b) fungicides, the basic structural requirement for
activity against Venturia inaequalis, Cercosporidium personatum, Pseu-
docercosporella herpotrichoides, Erysiphe graminis, and Puccinia recondi-
ta was a two‐atom bridge connecting the azole nucleus to a
hydrophobic group, typically an aromatic ring. However, still no
Encouraged by these observations, we explored the antifungal
potential of our {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines with
special focus on phytopathogenic fungi. Here, we present the
synthesis, structural elucidation, and in vitro antifungal activities of
10 novel {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines, bearing cy-
cloalkyl, hetaryl, and halogen residues. Reactivity and thereby the
potential toxicity of the substances is estimated by semiempirical
methods (MM+ and MNDO), namely, frontier molecular orbital

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FIGURE 1 Reported fungicides containing a triazole ring with types of functional fragments, which were purposefully introduced into novel
synthesized substances (a–j, 1–10), and the antifungal reference hymexazol
calculations. Additionally, triazoles’ affinities are calculated to the six
common fungal enzymatic targets. The findings are discussed with
respect to mutagenicity potential, which is determined with the
reverse Salmonella mutagenicity assay.
in isopropanol media.[26] The latter could be synthesized alternatively
through formation of 4‐chloroquinazolines by treatment of
quinazolin‐4‐ones with PCl5 and POCl3, followed also by
hydrazinolysis.[26] Synthesis of (3H‐quinazolin‐4‐ylidene)hydrazides
of cycloalkyl‐(hetaryl)carboxylic acids f as useful precursors to obtain
the corresponding tricyclic derivatives g was carried out in two ways:
d
b
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2
RESULTS AND DISCUSSION
Synthesis
reaction of substances
d with the appropriate carboxylic acid
hydrazides or acylation of substances e by imidazolides of carboxylic
acids.[27–29] The 2‐cycloalkyl‐(hetaryl)‐[1,2,4]triazolo[1,5‐c]quinazo-
lines g were synthesized by dehydration of substances f in a medium
of glacial acetic acid with usage of a Dean‐Stark receiver.[28–30] As
shown by Kovalenko et al.,[31] derivatives g were electron‐deficient
systems evidenced by the deshielded H‐5 singlet found at
9.85–9.25 ppm in the proton nuclear magnetic resonance (1H NMR)
spectra. The high electrophilic properties of C‐5 promoted the
nucleophilic addition reaction followed by the degradation of the
pyrimidine ring[32–34] to form the appropriate 1–10 in water under
acid catalysis.
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2.1
The antifungal SAR reported by Zhang et al.,[12] Basarab et al.,[13] and
Tang et al.[14] led us to synthesize and analyze novel {2‐(3‐R)‐1H‐
1,2,4‐triazol‐5‐yl]phenyl}amines 1–10 with different cycloalkyl and
hetaryl substituents (Figure 2, Supporting Information).
Quinazolin‐4‐ones b were obtained by heating the corresponding
derivatives of anthranilic acid
reaction).[23,24]
a with formamide (Nimetovsky
Substances b were thionated to c by P2S5 refluxing in xylol.[25]
Then 4‐hydrozinoquinazolines e were obtained by hydrazinolysis of c

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FIGURE 2 The synthetic route of {2‐(3‐R)‐1H‐1,2,4‐triazol‐5‐yl]phenyl}amines (1–10)
According to the spectral data, the products of the above
mentioned reaction were identified as the corresponding {2‐(3‐R‐1H‐
1,2,4‐trizole‐5‐yl)phenyl}amines (1–10). The liquid chromatography–
mass spectrometry (LC–MS) spectra were characterized by intensive
peaks of quasimolecular ions [M+1]+ indicating a high purity of
products. The elemental analysis data confirmed the empirical
formulas of synthesized substances.
compounds 2, 4, 5, and 10 due to their azole–azole tautomerism.
Significant paramagnetic shift of C1 atom (149.0–145.8 ppm) was
caused by the primary amino group and indicated the hydrolytic
cleavage of the pyrimidine cycle. The other carbon atoms of the aryl
fragment and the substituents in position 2 had chemical shifts that
corresponded to the proposed structures.
In 1H NMR spectra, the broadened proton singlet of NH2 group
was registered at 6.60–6.17 ppm and the doubled or broadened
signal of the triazole ring’s proton turned up in the low field
at 14.54–13.23 ppm. The signals of aminophenyl fragments of
substances 1–3 appeared as unresolved doublets at 7.98/7.54 (H‐3)
and 6.45/6.58 (H‐6) ppm, and triplets at 6.68–6.70 (H‐4) and
6.99–6.11 (H‐5) ppm. The doubling or broadening of the signals
was associated with tautomeric transformations of the synthesized
compounds.[32,33] Moreover, 1H NMR spectra of 1–10 were
characterized by signals of all other substituents with the common
multiplicity and chemical shifts.
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2.2
Molecular docking studies
We modeled the potential interaction of the triazoles with sterol
14α‐demethylase (CYP51) by flexible molecular docking[35] to predict
the antifungal mode of action in silico. The cytochrome
P‐450‐type enzyme is involved in the biosynthesis of ergosterol, an
essential plasma membrane constituent of lower eukayotes. Namely,
azoles are known to block the de novo biosynthesis of ergosterol,[3,5]
and thereby not only deplete its source for the buildup of
membranes, but also prevent the formation of physiologically
important intracellular sterols, which are required for cell‐cycle
regulation, multiplication,[36] and cell transformation.[37] The crystal
structure of CYP51 from C. albicans in complex with the tetrazole‐
based antifungal drug candidate VT1161 was downloaded from the
Protein Data Bank (PDB; ID: 5TZ1).[35] As reference, hymexazol
In 13C NMR spectra, it was found that signals caused by C3 and
C5 atoms of triazole cycle were observed as broad singlets at
164.6–157.1 and 163.9–148.7 ppm for 1–10. Doubling of C3 and C5
signals of the aniline fragment was observed in the spectrum for

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(3‐hydroxy‐5‐methylisoxazole; Figure 1) was chosen owing to its
structural resemblance and comparable activity.[14] The calculated
affinity score with the minimum energy indicate a high structural
ligand–protein complex matching for all substances (Table 1).
The affinity of all compounds towards CYP 51 exceeded that of
hymexazol. The strongest affinity was calculated for adamantyl
substituted triazoles: 2‐(3‐(adamantan‐1‐yl)‐1H‐1,2,4‐triazol‐5‐yl)‐5‐
fluoroaniline (9), 4‐bromoaniline (7), and 4‐chloroaniline (6). In
addition to hydrophobic interactions of adamantyl residues, sub-
stances 6, 7, and 9 formed several hydrogen bonds with serine 507,
378, and methionine 508, involving triazole NH‐groups and halogen
substituents (Figure 3, Supporting Information). Apart from these
observations, Hargrove et al.[36] emphasized that C. albicans CYP51
was more strongly inhibited by clinical azoles with elongated
side chains.
FIGURE 3 Growth inhibition of Colletotrichum higginsianum by
substances 6, 7, and 9 and antifungal reference hymexazol (hym).
Error bars indicate standard deviation of at least three experiments
TABLE 2 The calculated distribution coefficient (log D) at pH 2–9
in order of lipophilicity decrease
Interestingly, the interaction of triazoles with the P‐450
demethylase heme group, as described by Hitchcock et al.,[5] could
only be modeled for hymexazol and substances without the
adamantyl fragment (1, 3–5, 8, 10), but not for substances 2, 6, 7,
and 9, which show the highest scoring functions (Supporting
Information). However, this is not the only enzyme that may be
affected by the studied compounds. So, it was decided to conduct in
silico molecular docking to further five common antifungal targets.[35]
The calculations revealed that the tested triazoles indeed interact
with all of them and even with a higher probability than the reference
agent, hymexazol (Table 1). The triazoles in this study show a strong
affinity score to NMT only after CYP51, like the previously reported
by Somagond et al.[18] substituted quinolines containing a 1,2,4‐
triazole moiety. Notably, affinity prediction to enzymes taken from
Candida albicans (CYP51, NMT, and secreted aspartic proteinase) has
shown the best scores. The lowest affinity (mean for 1–10: −6.7 kcal/
mol) was determined for L‐glutamine:D‐fructose‐6‐phosphate amino-
transferase (GlcN‐6‐P). Averaged over all calculated targets,
substances 9, 2, 6, and 7 showed the highest level of affinity
(−8.9 to −8.4 kcal/mol).
Log D at pH
Substance
2
4
7.4
4.70
9
7
3.88
3.87
3.66
2.77
3.08
2.90
2.18
2.51
2.50
4.72
4.52
4.11
3.91
3.54
3.53
3.34
3.00
3.11
1.81
0.81
3.63
3.40
3.09
2.91
2.75
2.61
2.54
2.45
2.19
0.78
6
4.49
4.09
3.94
3.53
3.52
3.38
3.00
3.10
1.97
−0.97
9
2
8
5
1
10
4
3
−0.02
Hymexazol
0.83
−1.3
hydrophobic binding site of CYP51. Therefore, log D was calculated
assuming an average cytoplasmic pH at 7.4 for fungal cells (for
instance, Aspergillus niger[38]; Table 2). At this pH level, except for
The polarity of the substances gives an indication of their
potential to penetrate the cell membrane and to enter the relative
TABLE 1 Calculated affinities of tested triazoles and reference hymexazol (hym) to common antifungal enzymatic targets (from Candida
albicans (CYP51, NMT, SAP2), Escherichia coli (MurD, GlcN‐6‐P), and Sacchromyces cerevisiae (Topo II)
Affinity (kcal/mol)
Target enzyme
PDB code hym
9
2
6
7
8
1
3
5
10
4
Mean
14α‐Demethylase (CYP51)
N‐Myristoyltransferase (NMT)
Secreted aspartic proteinase (SAP2)
Topoisomerase II (Topo II)
5TZ1
1IYL
−4.3 −11.1 −10.5 −11.0 −11.0 −9.8 −9.6 −9.1 −9.2 −8.4 −8.5 −9.8
−4.9 −9.5 −9.2 −9.6 −9.7 −8.9 −8.9 −8.4 −8.7 −8.0 −8.1 −8.9
−3.9 −8.6 −8.4 −8.5 −8.7 −8.0 −8.1 −7.5 −8.1 −7.6 −7.7 −8.1
−4.7 −8.2 −7.8 −7.2 −7.0 −7.6 −6.8 −7.5 −7.2 −7.4 −6.6 −7.3
−4.4 −7.9 −7.6 −7.8 −7.7 −7.1 −6.9 −7.7 −6.8 −7.0 −6.6 −7.3
1EAG
1Q1D
1UAG
UDP‐N‐acetyl‐muramoyl‐L‐alanine:D‐glutamate
ligase (MurD)
L‐Glutamine:D‐fructose‐6‐phosphate
aminotransferase (GlcN‐6‐P)
1XFF
−4.8 −7.8 −7.2 −6.3 −6.3 −6.9 −6.7 −6.5 −6.4 −6.5 −6.6 −6.7
Mean
−4.5 −8.9 −8.5 −8.4 −8.4 −8.1 −7.8 −7.8 −7.7 −7.5 −7.4
–
Note: In the order from highest to lowest average data. Bold values designate substances with the three highest affinities to the mentioned enzyme.

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substance 3, all triazoles appeared to be more lipophilic than the
reference compound hymexazol.
compound hymexazol 50 µg/ml (0.5 μM). As shown in Table 3,
a significant antifungal activity could be observed for all tested
substances in a concentration of 50 µg/ml (0.13–0.21 μM) by mycelial
growth rate assay[14] against other opportunistic pathogen A. niger, 12
fungal (F. oxysporum, Fusarium fujikuroi, Colletotrichum higginsianum,
Gaeumannomyces graminis, C. coccodes, Claviceps purpurea, A. alternata,
Mucor indicus, Fusarium graminearum, Verticillium lecanii, Botrytis cinerea,
Penicillium digitatum) and three oomycete (Phytophtora infestans GL‐1, P.
infestans 4/91; R+ and 4/91; R−) strains.
Danby et al.[39] investigated the influence of pH on the in vitro
antifungal activity level of drugs and demonstrated that under
conditions of lowered pH (4–5), Candida glabrata isolates remained
susceptible to caspofungin and flucytosine. At the same time, there
was a dramatic increase of sensitivity to amphotericin B, fluconazole,
voriconazole, posaconazole, itraconazole, ketoconazole, and other
triazole agents. Therefore, log D was also calculated for the newly
synthesized substances at various pH (Table 2).
The effects were very different depending on the type of triazole
and tested organism. The synthesized substances inhibited majority
of the fungi, among which 10, 6, 9, 2, and 8 had the best results. F.
fujikuroi was the most sensitive strain (89.7% of inhibition), whereas
at the other end, A. niger showed the highest level of resistance
(13.9% of inhibition). Interestingly, with respect to therapeutic
triazoles, acquired resistance of A. fumigatus is an often described
phenomenon, which may be based on similar mode(s) of action.[41] In
addition, generally, the tested Phytophtora strains (24.2–34.2% of
inhibition) were difficult to inhibit by triazoles (including reference
substance hymexazol), which may reflect their distinct physiology as
belonging to the oomycete group. Interestingly, hymexazol and
substance 3 were completely ineffective towards M. indicus. And, at
the same time substances 5 and 10 at least brought about 70% of
inhibition.
Basically, log D remained unchanged at pH 7.4 and 4.0. Only at
pH 2 and 9 was the ionization state of substances affected with a
concomitant reduced lipophilicity by protonation and deprotonation,
respectively. In this context, it may also be considered that after the
addition of antifungal agents, the intracellular pH of fungi could be
changed. Ullah et al.[40] monitored the intracellular pH of C. glabrata
under the influence of antifungals caspofungin or amphotericin B
with the pH‐sensitive probe GFP ratiometric pHluorin: in media at
pH 4, the drugs caused an acidification of the cytoplasm, whereas in
media at pH 7.4, a slight alkalization was observed. These broad
physiological reactions may interfere with normal cell metabolism
and fungal growth as with the susceptibility towards antifungal drugs
such as triazole inhibitors of CYP51 demethylase.
The growth of strains G. graminis, C. higginsianum, C. coccodes, C.
purpurea, and A. alternata was inhibited practically at 60% by all
substances. But, when calculating the average activity of the
strongest substances (10, 6, 9, 2, and 8), the above mentioned fungi
were sensitive at almost 70–80%. Moreover, in comparison to N‐(4‐
(3‐(2,4‐dichlorophenyl)‐3‐oxo‐2‐(1H‐1,2,4‐triazol‐1‐yl)prop‐1‐en‐1‐yl)
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2.3
Fungal proliferation and antifungal activity
The in silico modeling approach and log D data strongly suggest that the
newly synthesized triazoles should penetrate the cell membrane and
interact with the CYP51 active site, thereby provoking an antifungal
effect at a similar or higher level as compared to the reference
TABLE 3 Growth inhibition rate of tested triazoles 1–10 and hymexazol at 50 μg/ml
Growth inhibition rate (%)
Substance
μM FF
GG
CH
CC
CP
AA FO
MI
FG
VL
BC
PD PI GL‐1 PI p‐3 PI p‐4 AN Mean
hym.
0.50 85.7 28.0 32.9 56.1 50.9 95.8 41.6 0.0 34.4 100.0 95.6 0.0 51.5
0.19 95.2 98.8 70.2 70.3 100.0 87.8 61.4 69.4 59.8 100.0 47.8 18.9 49.5
0.15 81.0 100.0 100.0 72.5 57.1 63.4 59.5 52.5 59.1 33.5 37.7 41.8 56.8
0.16 90.1 100.0 100.0 70.1 46.7 63.4 34.5 53.9 61.2 31.2 57.2 41.1 29.0
0.17 93.8 52.9 48.8 72.5 49.2 57.1 62.6 72.1 46.0 36.4 64.6 37.7 45.5
0.19 88.6 52.0 47.2 57.4 94.2 69.0 63.3 59.3 49.5 43.9 35.7 33.0 41.6
0.19 64.5 34.9 60.7 57.4 100.0 57.1 58.8 71.4 51.5 12.6 29.0 31.6 44.2
0.20 85.7 35.5 49.6 52.3 56.1 58.1 34.5 42.4 45.4 33.5 18.9 27.6 41.6
0.13 100.0 86.3 68.7 65.8 26.4 41.8 28.8 18.9 21.3 23.7 9.8 18.9 23.8
0.21 100.0 16.1 34.5 50.5 49.2 32.8 39.0 25.6 20.6 47.4 27.0 17.5 7.9
33.2
32.3
16.7
38.1
35.3
36.7
22.9
27.0
15.9
13.9
14.1
25.3
31.8
30.4
18.7
35.3
13.8
18.7
34.6
28.9
18.6
11.3
33.3
28.4
24.2
24.2
17.1 47.1
16.3 62.3
41.2 56.8
−2.4 51.7
19.4 50.8
−4.8 50.1
42.0 48.0
−7.1 38.7
−0.1 35.1
−2.4 32.1
−8.6 21.3
10
6
9
2
8
5
4
7
1
3
0.21 100.0 28.7 18.7 21.9
3.3 25.7 25.6 0.0 6.9 39.9 25.6 8.1 2.0
89.9 60.5 59.8 59.1 58.2 55.6 46.8 46.5 42.1 40.2 35.3 27.6 34.2
89.7 80.7 73.2 68.6 69.4 68.1 56.3 61.4 55.1 49.0 48.6 34.5 44.5
Mean (1–10)
Mean (2, 6, 8–10)
–
–
9.35
13.9
–
–
Note: Bold values indicate more than 60.0% of inhibition.
Abbreviations: AA, Alternaria alternata; AN, Aspergillus niger; BC, Botrytis cinerea; CC, Colletotrichum coccodes; CH, Colletotrichum higginsianum; CP, Claviceps
purpurea; FF, Fusarium fujikuroi; FG, Fusarium graminearum; FO, Fusarium oxysporum; GG, Gaeumannomyces graminis; hym., hymexazol; MI, Mucor indicus; PD,
Penicillium digitatum; PI GL‐1, Phytophtora infestans; PI‐3, P. infestans p‐3; PI‐4, P. infestans p‐4; VL, Verticillium lecanii.

ANTYPENKO ET AL.
7 of 14
|
aryl)benzamides (Figure 1c) studied by Tang et al.[14] at 50 µg/ml, half
of the {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}amines presented here
were more active against F. oxysporum and practically all are stronger
against P. infestans.
TABLE 4 List of calculated bonds between substance 10 and
N‐myristoyltransferase (1IYL) or 14α‐demethylase (5TZ1) active sites
Name
Å
Category
Type
(a) N‐Myristoyltransferase (1IYL)
A moderate growth level (27–47%) was shown by F. oxysporum,
M. indicus, F. graminearum, V. lecanii, B. cinerea, and P. digitatum.
A residual concentration of 1% DMSO (dimethylsulfoxide) in
potato dextrose agar (PDA) test plates was necessary to ensure an
even distribution of the lipophilic triazoles. Notably, this caused a
stress phenotype on A. niger, which became evident by a black
colorization of the mycel instead of yellow. Similar effects were not
observed with the other strains.
:UNL1:H ‐ B:TYR335:OH
:UNL1:H ‐ B:LEU450:O
2.05 Hydrogen bond Conventional
2.58 Hydrogen bond Conventional
:UNL1:H ‐ B:LEU451:OXT 2.33 Hydrogen bond Conventional
:UNL1:C ‐ B:LEU451:O
:UNL1:C ‐ B:TYR119:OH
B:LEU451:OXT ‐ :UNL1
:UNL1 ‐ B:PHE117
3.57 Hydrogen bond Carbon
3.32 Hydrogen bond Carbon
3.32 Electrostatic
4.37 Hydrophobic
4.82 Hydrophobic
5.25 Hydrophobic
4.13 Hydrophobic
5.49 Hydrophobic
4.74 Hydrophobic
5.29 Hydrophobic
Pi‐anion
Pi‐pi stacked
Pi‐pi T‐shaped
Alkyl
B:TYR354 ‐ :UNL1
In accordance with docking studies, 9, 8, 6, and 2 were among the
five most active substances. And, in summary the growth inhibition
data reflect the result from in silico docking analysis and calculated
lipophilicity. However, a notable exception is compound 10 combin-
ing the second lowest affinity with the best antifungal activity.
Generally, the data reveal that chemicals may have quite different
antifungal potentials. Nevertheless, in this context, docking studies
are valuable tools to select most promising structures to develop
highly active antifungals.
:UNL1:CL ‐ B:LEU394
B:TYR225 ‐ :UNL1:CL
B:TYR354 ‐ :UNL1:CL
:UNL1 ‐ B:LEU394
Pi‐alkyl
Pi‐alkyl
Pi‐alkyl
:UNL1 ‐ B:LEU337
(b) 14α‐Demethylase (5TZ1)
:UNL1:H ‐ A:TYR132:OH
Pi‐alkyl
2.71 Hydrogen bond Conventional
A:TYR132:OH ‐ :UNL1
A:HEM601 ‐ :UNL1
:UNL1 ‐ A:TYR118
:UNL1 ‐ A:TYR118
:UNL1 ‐ A:TYR132
3.59 Hydrogen bond Pi‐donor
So, due to the revealed strong activity, it was decided to test
substances 6, 7, and 9 along with the reference compound against
one of the fungi, namely C. higginsianum, at lower concentrations to
demonstrate the good antifungal potential of substances for further
studies. In the concentration range of 0.0027–0.2 μM, all three
compounds (6, 7, and 9) still had a higher antifungal activity than
hymexazol at 0.01–0.2 μM (Figure 3).
3.97 Hydrophobic
4.03 Hydrophobic
4.95 Hydrophobic
5.36 Hydrophobic
3.43 Hydrophobic
Pi‐pi stacked
Pi‐pi stacked
Pi‐pi T‐shaped
Pi‐pi T‐shaped
Alkyl
A:HEM601:CMD ‐
:UNL1:CL
:UNL1:CL ‐ A:ILE131
A:HEM601 ‐ :UNL1:CL
3.39 Hydrophobic
4.90 Hydrophobic
Alkyl
Compound 9 showed decrease of antifungal activity, when the
concentration was increased from 5 to 10 µg/ml (0.016–0.032 μM).
This counter‐intuitive characteristic, which to a lesser extent was
also observed for substance 6 at 15 µg/ml (0.046 μM), may reflect
a hormesis effect (toxicological concept characterized by low‐dose
stimulation and high‐dose inhibition). Such phenomena have to be
considered to adjust the dosage of antifungal agents to secure the
most effective protection at the lowest necessary input. Substance
Pi‐alkyl
Pi‐alkyl
Pi‐alkyl
Pi‐alkyl
:UNL1 ‐ A:HEM601:CMD 4.31 Hydrophobic
:UNL1 ‐ A:LEU121
:UNL1 ‐ A:LEU376
5.00 Hydrophobic
5.28 Hydrophobic
|
2.3.2
Structure–activity relationship
7
increased the level of inhibition in proportion to applied
It was found that change of the electron‐withdrawing substituent
(pyridine‐3‐yl) to electron‐donating ones (cyclohexyl and adamantyl)
(3 < 1 < 2), expansion of the cyclobutyl ring to adamantyl, introduc-
tion of the furan‐3‐yl substituent into the third position of the
triazole ring (4 < 5 < 6 < 10), and fluorine introduction to the 2‐phenyl
fragment (1 < 8) led to an increase of the antifungal activity rate as
well as a widened range of susceptible fungi (Figure 5).
concentrations.
|
2.3.1
Molecular docking visualization
Moreover, for the most active substance 10 (Table 4), a visual
representation of the binding in the active sites of N‐myristoyl-
transferase (1IYL) and 14‐α‐demethylase (5TZ1) is shown in Figure
4a,b, and these are calculated as targets of its strongest affinity
(Table 1).
It is notable that for fluorine containing substances 8 and 9, there
was practically no difference of antifungal activity, when the
cyclohexyl residue was replaced by adamantyl, aiming at a higher
fluorine impact on the activity rate. But, for 9 and 6 there was a
difference for fluorosubstitution and chlorosubstitution, when bear-
ing the adamantyl residue. Besides this, chloroderivative 6 had better
activity than bromosubstituted 7, and practically the same inhibition
rate was observed for substance 2 bearing no halogen, showing the
supremacy of adamantyl ring in possessing antifungal properties.
As listed in Table 4, substance 10 formed 13 bonds with
N‐myristoyltransferase, among which three are conventional
hydrogen bonds due to connection to oxygen (with TYR 335 and
LEU 450 and 451). In contrast, for 14α‐demethylase, only one
conventional hydrogen bond was shown with TYR 123, among 12
possible ones.

8 of 14
ANTYPENKO ET AL.
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FIGURE 4 Visual representation (2D and 3D) of the substance 10 showing bond formation and position in the active site of (a)
N‐myristoyltransferase (1IYL) and (b) 14‐α‐demethylase (5TZ1). Light blue: carbon hydrogen bond, green: classical conventional hydrogen bond,
purple: hydrophobic π–π stacked bond, pink: hydrophobic alkyl and π‐alkyl bonds
And, also substances with no halogen (1) or with pyridin‐3‐yl (3)
substituents exhibited the lowest growth inhibition effects. It is
worth mentioning that 3 has the lowest lipophilicity according to
calculated log D (Table 2).
And according to the calculated descriptors the investigated
substances are not highly reactive, hard electrophiles with
µ = −4.86 0.18 eV, η = 4.32 0.14 eV, and ω = 2.73 0.16 eV; the
values are roughly at the level as those for reference hymexazol
(µ = −5.12 eV, η = 5.00 eV, and ω = 2.62 eV; Supporting Information).
Due to the presence of additional heterocyclic rings in the molecular
structure (pyrimidine or furan, respectively), substances 3 and 10
showed the lowest hardness with η = 4.09 and 4.04 eV, correspond-
ingly pointing to an elevated biochemical reactivity. Still, the
And the findings on furan‐3‐yl 10, which has strong activity,
corresponds to the SAR analysis on furan‐2‐yl substituted com-
pounds of Tang et al.[14]
|
2.4
Safety evaluation of triazoles
electrophilicity index of substance
3 was lower than that of
|
2.4.1
Frontier molecular orbitals calculations
substance 10 with 2.52 against 2.72 eV, making the latter the most
reactive among all synthesized substances besides 8 and 9 with an
ω = 2.93 and 2.91 eV, respectively. Additionally, the substances were
screened for pan‐assay interference compounds[35] by means of a
Badapple (bioactivity data associative promiscuity pattern learning
engine) algorithm for identifying likely promiscuous compounds via
associated scaffolds. This analysis reveals that only substances 3 and
10 have p scores higher than 300.
Each novel substance needs a thorough safety evaluation, at least
with respect to human health and environmental friendliness, before
application in industrial processes, agriculture or medical care.
Generally, it was found that lipophilic compounds with low‐energy
LUMOs were highly likely to be mutagenic, hence possibly
carcinogenic. Hence, these parameters should be evaluated in the
course of the development of drugs.[19–21] We investigated these
characteristics for the novel triazoles by calculating important
physical descriptors using density functional theory with the help
of the calculated HOMO–LUMO (highest occupied molecular
orbital–lowest unoccupied molecular orbital) energies[35] (Supporting
Information) and testing the mutagenic potential with Salmonella
reverse‐mutagenicity in vitro assay (Ames test).
Interestingly, the calculated log D of substances 3 and 10
indicates the lowest lipophilicity index, thus should have the lowest
permeability into cells. There was no correlation between the log D
and LUMO energies (R2 = 0.0107) or log D and electrophilicity
(R2 = 0.1575) of all substances. Thus, for the investigated series of
substances, the log D–LUMO relationship is not an indicator of

ANTYPENKO ET AL.
9 of 14
|
FIGURE 5 The structure–activity relationship of investigated triazoles 1–10 in order of decreasing activities (averages from Table 2)
mutagenicity. Besides this, an elevated level of potential carcino-
genicity of substances may be expected from the calculated high
values of electrophilicity. However, as discussed by Tiwary et al.[42]
flavonoids interacting with DNA confer prooxidative stress, which, in
turn, results in positive mutagenic activity in the reverse Salmonella
mutagenicity test. Nevertheless, with respect to transformation to
cancer cells, flavonoids act protectively on mammalian organisms.
Also, Gopalakrishnan et al.[43] calculated electrophilicity of silymarin
as 3.24 eV, which is above that of the substances presented here.
However, it was also demonstrated that silibinin, a major active
component of silymarin, can form complexes with DNA, and at the
same time confer radioprotective and anticancer activity. Thus, the
electrophilic profile of molecules may give a strong indication
towards reactivity with DNA; with consideration to carcinogenicity
the interrelation may be less straightforward and correspondingly
interpretation of data should not be overstressed.
cytochrome P‐450,[45] followed by an enzymatic acetylation or
sulfonation, which further leads to the formation of a nitrenium
ion. This highly reactive electrophilic species could bind covalently
to
biomacromolecules‐generating
aminoaryl
derivatives.[46]
N‐Hydroxylamines also could be metabolically converted to nitroso
species leading to the formation of reactive oxygen species. The level
of mutagenicity is also related to the degree of oxidizability of the
amino group and the stability of the generated nitrenium ion.[45] The
electron‐withdrawing groups destabilize the nitrenium ion, reducing
the mutagenic potential of the parent amine. So, substance 3 with the
pyridine‐3‐yl substituent and 10 with furan‐3‐yl should have the
lowest mutagenic potential. On the contrary, it was found that
electron‐donating (ED) groups as para‐alkyl substituents increase
mutagenic activity by stabilization of the nitrenium ion. In addition,
the elongation of alkyl substituents up to adamantyl should increase
their mutagenic activity. But for larger substituents, especially in the
ortho‐position, such as the bulky 1,2,4‐triazole ring, the mutagenicity
of anilines primarily depends on steric effects[47] leading to less toxic
compounds. Moreover, aromatic amines with more than one ring
forming a conjugated system, either fused or nonfused, showed
greater mutagenic activity by charge delocalization, but not
stabilization of nitrenium species.[48]
Recently, Gadaleta et al.[44] reported on a knowledge‐based
classification algorithm, which implemented a series of defined rules
to predict the mutagenic potency of aromatic amines. Applying
these rules, the 1,2,4‐triazole compounds presented here should be
nonmutagens. Namely,
a possible metabolization consists of
N‐oxidation of the amine group to N‐hydroxylamine mediated by

10 of 14
ANTYPENKO ET AL.
|
TABLE 5 Mutagenicity index (Mi) calculated for reference mutagens
and investigated substances relative to negative control plates with
100 µl of DMSO (dimethylsulfoxide)[18]
50 µg/plate with metabolic activation, which was at 2.52 and 2.79
for strain TA98 and TA100, respectively. Increasing the dose to
500 µg per plate resulted in a Mi of 10.33 for TA98 with metabolic
activation. These data reveal the mutagenic potential of substance
10. Nevertheless, compared to the reference substance 2‐amino-
fluorene (Mi = 47.35 [TA98] and 4.58 [TA100]; 10 µg/plate;
metabolic activation), the mutagenicity is quite weak. Only
substance 10 was significantly activated by S9‐mix cytochrome
P‐450. The elevated mutagenicity of substance 10 may be caused
by the enzymatic oxidation of the amino group, but also by the
presence of the furan‐3‐yl ring. This structure is prone to
reversibly intercalate into the DNA and thereby introduce
base pair mutations in the course of DNA‐replication. According
to the highest negative charges calculated by HyperChem 8.0
(Supporting Information), the most probable centers for
electrophilic attacks are nitrogens in NH2‐groups of substances 3
(−0.376) and 10 (−0.369). Considering the in vitro nonmutageni-
city of substance 3, it is obvious that not only the heterocyclic
substituent but also steric effects contribute to the
elevated mutagenicity potential of substance 10.[47] Generally, it
has to be considered that the prokaryotic in vitro Ames assay
reflects in vivo mutagenicity and carcinogenicity in eukaryotic
Dose
(µg/
TA 98
TA 100
+S9‐mix
Substances
plate)
+S9‐mix
2‐Nitrofluorene
10
1
58.40
Methyl
4.20
methansulfonate
2‐Aminofluorene
10
50
50
50
50
50
50
50
50
50
50
50
1.56 47.35
1.01 4.58
1.03 0.90
0.98 0.87
1.00 1.07
0.91 0.94
1.14 1.08
0.89 0.92
1.02 0.81
0.87 1.23
0.97 0.81
1.06 0.75
1.28 2.79
Hymexazol
0.71
0.76
1.04
0.81
1.19
1.21
1.17
1.07
0.90
1.04
1.48
0.85
0.88
0.87
0.63
0.72
0.77
0.79
0.95
0.51
0.91
2.52
1
2
3
4
5
6
7
8
9
10
organisms only to
a certain degree: DNA repair capacities,
distribution and apparent concentration levels of substances and
metabolic conversions are unknowns, which can only be studied by
animal experiments.
|
2.4.2
Ames gene‐toxicity test
So, to examine the above‐predicted characteristics, substances were
tested for mutagenicity by a standard plate incorporation assay with
Salmonella typhimurium strains TA98 and TA100, as described by
Maron and Ames (Table 5).[22,35]
|
2.4.3
Drug‐likeness calculations
The assay was carried out with and without addition of rat liver
S9 extract for metabolic activation. According to the number of
formed revertants, the studied substances were found to be
nonmutagenic (mutagenicity index [Mi], <2), except substance
10. Only this triazole showed an elevated Mi when tested at
Bioavailability of drug candidates is an important parameter to
evaluate the safety of chemicals. Thus, drug‐likeness parameters of
novel triazoles 1–10 and hymexazol were calculated with the
Molinspiration online engine.[35] All substances complied to drug‐
likeness relevant descriptors such as molecular weight (MW),
TABLE 6 Calculated parameters of drug‐likeness optimization of triazoles 1–10 and hymexazol (hym)
Compound no. SMILES MW miLogP TPSA HBA HBD nrotb
99.1 0.55
hym
1
CC1═CC(═NO1)O
46.3
67.6
67.6
80.5
67.6
67.6
67.6
67.6
67.6
67.6
80.7
3
4
4
5
4
4
4
4
4
4
5
1
3
3
3
3
3
3
3
3
3
3
0
2
NC1═CC═CC═C1C2═NC(═N[NH]2)C3CCCCC3
242.33 2.94
294.40 3.96
237.27 1.99
248.72 2.35
262.74 3.08
328.85 4.61
373.30 4.74
260.32 3.08
2
NC1═CC═C═C1C2═NC(═N[NH]2)[C]34CC5[CH2][CH](C[CH]([CH2]5)C3)C4
NC1═CC═CC═C1C2═NC(═N[NH]2)C3═CC═CN═C3
2
3
2
4
NC1═CC═C(Cl)C═C1C2═NC(═N[NH]2)C3CCC3
2
5
NC1═CC═C(Cl)C═C1C2═NC(═N[NH]2)C3CCCC3
2
6
NC1═CC═C(Cl)C═C1C2═NC(═N[NH]2)[C]34CC5[CH2][CH](C[CH]([CH2]5)C3)C4
NC1═CC═C(Br)C═C1C2═NC(═N[NH]2)[C]34CC5[CH2][CH](C[CH]([CH2]5)C3)C4
NC1═CC(═CC═C1C2═NC(═N[NH]2)C3CCCCC3)F
2
7
2
8
2
9
NC1═CC(═CC═C1C2═NC(═N[NH]2)[C]34CC5[CH2][CH](C[CH]([CH2]5)C3)C4)F 312.39 4.09
NC1═CC═C(Cl)C═C1C2═NC(═N[NH]2)C3═COC═C3 260.68 2.66
≤500 ≤5
2
10
2
Drug lead‐like criteria
≤140
≤10 ≤5
≤10
Abbreviations: HBA, hydrogen bond acceptors; HBD, hydrogen bond donors; MW, molecular weight; miLogP, octanol/water partition coefficient; nrotb,
number of rotatable bonds; TPSA, molecular topological polar surface area.

ANTYPENKO ET AL.
11 of 14
|
TABLE 7 Correlation coefficients between average antifungal activity of triazoles 1–10 and their calculated miLogP, Log D, and affinity to the
enzymatic targets
Log D at pH
2
4
7.5
9
MW
TPSA
miLogP
R2
0.0549
0.0132
0.1349
0.3965
0.2048
0.1835
0.3066
Towards affinity to enzymes
CYP51
0.0199
NMT
SAP2
Topo II
0.1368
MurD
0
GlcN‐6‐P
R2
0.0099
0.0572
0.0476
Abbreviations: CYP51, affinity to 14α‐demethylase; GlcN‐6‐P, L‐glutamine:D‐fructose‐6‐phosphate aminotransferase; Log D, distribution coefficient;
miLogP, octanol/water partition coefficient; MurD, UDP‐N‐acetyl‐muramoyl‐L‐alanine:D‐glutamate ligase; MW, molecular weight; NMT, N‐myristoyl-
transferase; SAP2, secreted aspartic proteinase; Topo II, topoisomerase II; TPSA, molecular topological polar surface area.
octanol/water partition coefficient, hydrogen bond acceptors and
donors as well as number of rotatable bonds (Table 6).
potential and ecotoxicology profile to be developed into useful
agrochemicals.
Molecular topological polar surface areas (TPSA) were calculated
to be not more than 140 Å2, but lower than 75 Å2 for all substances
except the 3rd, so they could possibly penetrate the blood–brain
barrier. Also, substances 2, 6, and 9 had good lipophilicity and low
TPSA. Still their molecular surfaces were larger than that of
hymexazol, which is already widely used as a pesticide with no
detected mammalian toxicology.[49]
|
4
EXPERIMENTAL
Chemistry
|
4.1
|
4.1.1
General
Melting points were determined in open capillary tubes and were
uncorrected. The elemental analyses (C, H, N, and S) were
performed using a Vario EL Cube analyzer (Elementar Americas,
NJ). Analyses were indicated by the symbols of the elements or
functions within 0.3% of the theoretical values. The 1H NMR
spectra (400 MHz) and 13C NMR spectra (125 MHz) were
recorded on a Varian‐Mercury 400 (Varian Inc., Palo Alto, CA)
spectrometer with TMS as an internal standard in DMSO‐d6
solution. LC–MS were recorded using the chromatography/mass
spectrometric system consisting of an “Agilent 1100 Series” high‐
performance liquid chromatograph (Agilent, Palo Alto, CA)
equipped with an “Agilent LC/MSD SL” diode matrix.
|
2.4.4
Correlations between calculated descriptors
To analyze what descriptor had
a higher possibility to affect
penetration of triazoles into the fungal cell and bind to the enzymatic
target, we calculated the correlation coefficients between average
antifungal activity of each tested substance and its several found
parameters (Table 7).
The strongest correlation (0.3965) was found between antifungal
activity and Log D at pH 2. Therefore, acidification of the fungal
growth media, when treated with substances could increase their
level of activity and avoid one of the fungal resistance mechanisms.
However, deprotonation at pH 9 also had a positive impact on
correlation with R2 = 0.3066. Interestingly, correlation with MW was
better than with TPSA, still with very low values. Among the affected
target enzymes, topoisomerase II (Topo II, R2 = 0.1368) was found
with a higher calculated probability to be involved in inhibition of all
tested fungal strains.
Synthesis of starting substances (b–g, Figure 2) was done using
the prior reported methods.[23–31] LC–MS and 1H NMR spectra of
1–10 are presented in Supporting Information.
The InChI codes of the investigated compounds together with
some biological activity data are also provided as Supporting
Information.
|
3
CONCLUSION
2‐(3‐Cyclohexyl‐1H‐1,2,4‐triazol‐5‐yl)aniline (1)
Yield, 93.2%. M.p. 152–154°C; 1H NMR: δ, ppm. (J, Hz): 13.53/13.37
(b.s, 1H, NH), 7.92/7.62 (unres.d, 1H, H‐3), 6.99 (unres.t, 1H, H‐5),
6.68 (unres.t, 1H, H‐4), 6.48–6.58 (m, 1H, H‐6), 6.17 (b.s, 2H, NH2),
2.83–2.65 (m, 1H, H‐1 cyclohexane), 2.17–1.15 (m, 10 H, H‐2,2, 3,3,
4,4, 5,5, 6,6 cyclohexane); 13C NMR: δ, ppm: 160.7 (triazole C‐3),
155.0 (triazole C‐5), 147.1 (Ar C‐1), 130.0 (Ar C‐3), 128.0 (Ar C‐5),
116.2 (Ar C‐4), 115.6 (Ar C‐2,6), 36.2 (Cy C‐1), 31.6 (Cy C‐2, 6), 26.0
(Cy C‐4), and 25.8 (Cy C‐3,5); LC–MS, m/z = 285 [M+1]; Anal. calcd.
for C14H18N4: C, 69.39; H, 7.49; N, 23.12; Found: C, 69.36; H, 7.50;
N, 23.13.
We identified several antifungal agents of phytopathogenic signifi-
cance among the novel series of 1,2,4‐triazole derivatives, bearing
bulky alkyl, heterocyclic, and halogen substituents. With respect to
toxicology, our data so far show no restrictive risk. Calculated drug‐
likeness parameters suggest a good bioavailability of the substances
with low toxicity. 14‐α‐Demethylase and N‐myristoyltransferase are
suggested to be the most probable enzymes for binding. Considering
the shown nonmutagenicity of {2‐(3‐R‐1H‐1,2,4‐triazol‐5‐yl)phenyl}‐
amines, they warrant further investigations of their antifungal

12 of 14
ANTYPENKO ET AL.
|
2‐(3‐(Adamantan‐1‐yl)‐1H‐1,2,4‐triazol‐5‐yl)aniline (2)
C‐5), 145.9 (Ar C‐1), 129.3 (Ar C‐3), 127.1 (Ar C‐5), 118.8 (Ar C‐4),
117.7 (Ar C‐2,6), 41.1 (Ad C‐1,2,8,9), 36.5 (Ad C‐4,6,10), and 28.1 (Ad
C‐3,5,7); LC–MS, m/z = 329 [M+1]; Anal. calcd. for C18H21ClN4: C,
65.74; H, 6.44; Cl, 10.78; N, 17.04; Found: C, 65.72; H, 6.45; Cl,
10.79; N, 17.03.
Yield: 89.3%. M.p. 150–152°C; 1H NMR: δ, ppm. (J, Hz): 13.58/13.23
(b.s, 1H, NH), 7.98/7.54 (unres.d, 1H, H‐3), 7.00 (unres.t, 1H, H‐5), 6.70
(unres.t, 1H, H‐4), 6.58–6.45 (m, 1H, H‐6), 6.19 (b.s, 2H, NH2), 2.12–1.98
(m, 9H, Ad), 1.85–1.74 (m, 6H, Ad); 13C NMR: δ, ppm: 163.7 (triazole
C‐3), 161.3 (triazole C‐5), 146.9 (Ar C‐1), 130.8/129.6 (Ar C‐3), 128.4/
127.1 (Ar C‐5), 116.0 (Ar C‐4), 115.6 (Ar C‐2), 113.6 (Ar C‐6), 41.7 (Ad
C‐1), 41.1 (Ad C‐2,8,9), 36.8/36.5 (Ad C‐4,6,10), and 28.3/28.1 (Ad
C‐3,5,7); LC–MS, m/z = 295 [M+1]; Anal. calcd. for C18H22N4: C, 73.44;
H, 7.53; N, 19.03; Found: C, 73.42; H, 7.54; N, 19.04.
2‐(3‐(Adamantan‐1‐yl)‐1H‐1,2,4‐triazol‐5‐yl)‐4‐bromoaniline (7)
Yield: 88.9%. M.p. 249–251°C; 1H NMR: δ, ppm. (J, Hz): 13.55/13.35
(b.s, 1H, NH), 8.01 (s, 1H, H‐3), 7.07 (d, J = 8.6 Hz, 1H, H‐5), 6.68 (d,
J = 8.7 Hz, 1H, H‐6), 6.36 (b.s, 2H, NH2), 2.13–1.98 (m, 9H, Ad), 1.80
(s, 6H, Ad); LC–MS, m/z = 374 [M+1]; Anal. calcd. for C18H21BrN4: C,
57.92; H, 5.67; Br, 21.40; N, 15.01; Found: C, 57.90; H, 5.68; Br,
21.41; N, 15.01.
2‐(3‐(Pyridin‐3‐yl)‐1H‐1,2,4‐triazol‐5‐yl)aniline (3)
Yield: 95.9%. M.p. 247–249°C; 1H NMR, δ, ppm. (J, Hz): 14.54/14.10
(b.s, 1H, NH), 9.26 (s, 1H, H‐2 Pyr), 8.58 (d, J = 8.0 Hz, 1H, H‐6 Pyr),
8.39 (d, J = 7.9 Hz, 1H, H‐4 Pyr), 7.72 (d, J = 7.9 Hz, 1H, H‐3), 7.41 (t,
J = 7.6 Hz, 1H, H‐5 Pyr), 7.11 (t, J = 7.7 Hz, 1H, H‐5), 6.88–6.51 (m,
2H, H‐4, H‐6), 6.26 (b.s, 2 H, NH2); 13C NMR: δ, ppm: 157.1 (triazole
C‐3), 150.6 (triazole C‐5), 147.6 (Pyr C‐4), 147.5 (Ar C‐1, Pyr C‐2),
133.8 (Pyr C‐6), 131.3 (Ar C‐3), 127.6 (Ar C‐5), 124.4 (Pyr
C‐1,5), 116.7 (Ar C‐4), and 115.7 (Ar C‐2,6); LC–MS, m/z = 238
[M+1]; Anal. calcd. for C13H11N5: C, 65.81; H, 4.67; N, 29.52; Found:
C, 65.80; H, 4.68; N, 29.51.
2‐(3‐Cyclohexyl‐1H‐1,2,4‐triazol‐5‐yl)‐5‐fluoroaniline (8)
Yield: 97.9%. M.p. 151–153°C; 1H NMR: δ, ppm. (J, Hz): 13.55/13.28
(b.s, 1H, NH), 7.96/7.59 (unres.d, 1H, H‐3), 6.66–6.33 (m, 3H, H‐6,
NH2), 6.26 (d, J = 8.9 Hz, 1H, H‐4), 2.85–2.63 (m, 1H, H‐1 cyclohex-
ane), 2.08–1.75 (m, 4H, H‐2,2, 6,6 cyclohexane), 1.74–1.23 (m, 6H, H‐
3,3, 4,4, 5,5 cyclohexane); 13C NMR: δ, ppm: 163.5 (d, J = 242.3 Hz, Ar
C‐5), 160.8 (triazole C‐3), 160.4 (triazole C‐5), 148.9 (d, J = 10.4 Hz,
Ar C‐1), 130.2 (d, J = 10.6 Hz, Ar C‐3), 110.2 (Ar C‐2), 102.5 (d,
J = 23.7 Hz, Ar C‐4), 101.3 (d, J = 24.0 Hz, Ar‐C‐6), 35.7 (Cy C‐1), 31.4
(Cy C‐2,6), 25.8 (Cyc C‐4), and 25.7 (Cyc C‐3,5); LC–MS,
m/z = 261 [M+1]; Anal. calcd. for C14H17FN4: C, 64.60; H, 6.58;
F, 7.30; N, 21.52; Found: C, 64.59; H, 6.58; F, 7.31; N, 21.50.
4‐Chloro‐2‐(3‐cyclobutyl‐1H‐1,2,4‐triazol‐5‐yl)aniline (4)
Yield: 88.7%. M.p. 176–178°C; 1H NMR: δ, ppm. (J, Hz): 13.70/13.43
(b.s, 1H, NH), 7.97/7.68 (unres.d, 1H, H‐3), 6.96 (d, J = 8.7 Hz, 1H, H‐
5), 6.72 (d, J = 8.6 Hz, 1H, H‐6), 6.33 (b.s, 2H, NH2), 3.78–3.52 (m, 1H,
H‐1 cyclobutane), 2.47–2.30 (m, J = 18.0 Hz, 4H, H‐2,2, 4,4 cyclobu-
tane), 2.05 (dt, J = 19.6, 10.4 Hz, 2H, H‐3,3 cyclobutane); 13C NMR: δ,
ppm: 160.6 (triazole C‐3), 159.7 (triazole C‐5), 145.8 (Ar
C‐1), 130.7/129.3 (Ar C‐3), 127.2/126.2 (Ar C‐5), 118.8 (Ar C‐4),
117.7 (Ar C‐2), 114.5 (Ar C‐6), 31.6 (cyclobutane C‐1), 28.0
(cyclobutane C‐2,4), and 18.7 (cyclobutane C‐3); LC–MS, m/z = 249
[M+1]; Anal. calcd. for C12H13ClN4: C, 57.95; H, 5.27; Cl, 14.25; N,
22.53; Found: C, 57.93; H, 5.29; Cl, 14.27; N, 22.52.
2‐(3‐(Adamantan‐1‐yl)‐1H‐1,2,4‐triazol‐5‐yl)‐5‐fluoroaniline (9)
Yield: 95.2%. M.p. 224–226°C; 1H NMR: δ, ppm. (J, Hz): 13.33 (s, 1H,
NH), 7.98/7.60 (unres.d, 1H, H‐3), 6.60–6.35 (m, 3H, H‐6, NH2), 6.23
(d, J = 8.7 Hz, 1H, H‐4), 2.13–1.96 (m, H, Ad), 1.79 (s, 6H, Ad); 13C
NMR: δ, ppm: 164.6 (triazole C‐3), 163.9 (triazole C‐5), 161.6 (d,
J = 256.0 Hz, Ar C‐5), 149.0 (d, J = 14.4 Hz, Ar C‐1), 130.3 (Ar C‐6),
110.4 (Ar C‐1), 102.5 (d, J = 25.6 Hz, Ar C‐6), 101.4 (d, J = 24.1 Hz, Ar
C‐4), 41.1 (Ad C‐1,2,8,9), 36.5 (Ad C‐4,6,10), and 28.1 (Ad C‐3,5,7);
LC–MS, m/z = 313 [M+1]; Anal. calcd. for C18H21FN4: C, 69.21; H,
6.78; F, 6.08; N, 17.94; Found: C, 69.20; H, 6.79; F, 6.06; N, 17.96.
4‐Chloro‐2‐(3‐cyclopentyl‐1H‐1,2,4‐triazol‐5‐yl)aniline (5)
Yield: 85.5%. M.p. 161–163°C; 1H NMR: δ, ppm. (J, Hz): 13.51
(s, 1H, NH), 7.88 (s, 1H, H‐3), 6.96 (d, J = 8.6 Hz, 1H, H‐5), 6.72 (d,
J = 8.8 Hz, 1H, H‐6), 6.33 (b.s, 2H, NH2), 3.21 (t, J = 8.4 Hz, 1H, H‐1
cyclopropane), 2.19–1.51 (m, 8H, H‐2,2 3,3, 4,4, 5,5 cyclopropane);
13C NMR: δ, ppm: 160.5 (triazole C‐3, C‐5), 145.8 (Ar C‐1), 130.6/
129.2 (Ar C‐3), 127.2/126.2 (Ar C‐5), 118.8 (Ar C‐4), 117.7 (Ar C‐2),
114.6 (Ar C‐6), 36.8 (cyclopentan C‐1), 32.2 (cyclopentan C‐2, 5),
25.4 (cyclopentan C‐3,4); LC–MS, m/z = 263 [M+1]; Anal. calcd. for
C13H15ClN4: C, 59.43; H, 5.75; Cl, 13.49; N, 21.32; Found: C, 59.42;
H, 5.76; Cl, 13.50; N, 21.31.
4‐Chloro‐2‐(3‐(furan‐3‐yl)‐1H‐1,2,4‐triazol‐5‐yl)aniline (10)
Yield 97.9%. M.p. 248–250°C; 1H NMR: δ, ppm. (J, Hz): 14.20/13.98 (b.s,
1H, NH), 8.31–7.52 (m, 3H, H‐3, furan H‐2, furan H‐5), 7.10–6.68 (m,
3H, H‐4, H‐5, furan H‐4), 6.38 (b.s, 2H, NH2); 13C NMR: δ, ppm:
(126 MHz, DMSO‐d6) δ 161.3 (triazole C‐3), 148.7 (triazole C‐5), 147.0/
146.0 (Ar C‐1), 145.3/144.6 (furan C‐2), 143.1/142.1 (furan C‐5), 131.0/
129.6 (Ar C‐3), 127.3/126.4 (Ar C‐5), 118.9 (Ar C‐4), 118.4 (Ar C‐2),
117.8 (Ar C‐6), 109.0 (furan C‐3,4); LC–MS, m/z = 261 [M+1]; Anal.
calcd. for C12H9ClN4O: C, 55.29; H, 3.48; Cl, 13.60; N, 21.49; O, 6.14;
Found: C, 55.27; H, 3.49; Cl, 13.61; N, 21.47; O, 6.15.
2‐(3‐(Adamantan‐1‐yl)‐1H‐1,2,4‐triazol‐5‐yl)‐4‐chloroaniline (6)
Yield: 83.1%. M.p. 256–258°C; 1H NMR: δ, ppm. (J, Hz): 13.65/13.40
(b.s, 1H, NH), 7.90 (s, 1H, H‐3), 6.95 (d, J = 8.6 Hz, 1H, H‐5), 6.71 (d,
J = 8.9 Hz, 1H, H‐6), 6.32 (b.s, 2H, NH2), 2.14–1.98 (m, 9H, Ad), 1.79
(s, 6H, Ad); 13C NMR: δ, ppm: 164.1 (triazole C‐3), 160.1 (triazole
|
4.2
Antifungal studies
The mycelial growth rate assay was used for antifungal studies.[14]
Strains of filamentous fungi were obtained from the following

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sources: A. niger DSM 246, G. graminis DSM 12044, C. purpurea DSM
715, C. coccodes DSM 2492, A. alternata DSM 1102, F. graminearum
DSM 1095, F. fujikuroi DSM 893, V. lecanii DSM 63098, M. indicus
DSM 2185, and P. digitatum DSM 2731 from DSMZ (Braunschweig,
Germany); F. oxysporum 39/1201 St. 9336, B. cinerea from the
Technische Universität Berlin (Germany); Phytophtora infestans
(GL‐1 01/14 wild strain), p‐3 (4/91; R+) and p‐4 (4/91;
R−) strains were kindly donated by Julius Kühn‐Institut (Quedlinburg,
Germany); C. higginsianum (MAFF 305635), originally isolated by the
Ministry of Agriculture, Forest and Fisheries collection, Japan, via the
Department of Biology, Friedrich Alexander Universität (Erlangen,
Germany). PDA was purchased from C. Roth (Karlsruhe, Germany).
Hymexazol (98%) was obtained from Prosperity World Store (Hebei,
China). The procedure was used as described earlier.[35]
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4.3
Other methods
Molecular docking studies, quantum data, molecular descriptors
calculations and the Salmonella reverse‐mutagenicity test were done
as described earlier.[35]
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ACKNOWLEDGMENTS
Authors gratefully acknowledge Enamine Ltd. (Kiev, Ukraine) and the
German Federal Ministry of Education and Research (Grant: FKZ
03FH025IX4) for financial support for this study; the Federal
Research Centre for Cultivated Plants (Quedlinburg, Germany) for
providing the strain P. infestans; the Department of Biology, Friedrich
Alexander University, Erlangen, Nürnberg, Germany for the strain
Colletotrichum higginsianum, and Dr. Oleksii Antypenko (Zaporizhzhya
State Medical University, Ukraine) for conducting molecular docking.
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CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.
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ORCID
Lyudmyla Antypenko
Zhanar Sadykova
http://orcid.org/0000-0003-0057-1551
http://orcid.org/0000-0002-4807-9008
http://orcid.org/0000-0003-2008-8380
http://orcid.org/0000-0001-8017-9108
Kostiantyn Shabelnyk
Sergiy Kovalenko
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Additional supporting information may be found online in the
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