Design, synthesis and mechanistic study of new 1,2,4-triazole derivatives as antimicrobial agents

Article abstract of DOI:10.1016/j.bioorg.2021.104841

Novel 5-amino-1,2,4-triazole derivatives and their cyclized 1,2,4-triazolo[1,5-a]pyrimidine analogues were designed, synthesized and evaluated for their antimicrobial activities. They were tested against five bacterial strains (Methicillin Resistant S. aureus (MRSA), E. coli, K. pneumoniae, A. baumannii and P. aeruginosa) using ciprofloxacin as a positive control and against two fungal strains (C. albicans and C. neoformans) using fluconazole and amphotericin B as positive controls. Compounds 9, 13a and 13b showed high to moderate antifungal activities against candida albicans (MIC values = 4–32 μg/ml), with considerable safety profiles; where no cytotoxicity against human embryonic kidney or red blood cells were detected at concentrations up to 32 μg/mL. Furthermore, compound 9 showed significant inhibitory activity against lansterol 14α-demethylase (IC₅₀ = 0.27 μM), compared to the reference drug fluconazole (IC₅₀ = 0.25 μM). Molecular docking of compound 9 into the active site of the cytochrome P450 enzyme revealed comparable binding modes and docking scores to those of fluconazole. Finally, in silico ADME studies prediction and drug-like properties of these compounds revealed favorable oral bioavailability results.

Full text of DOI:10.1016/j.bioorg.2021.104841

Bioorganic Chemistry 111 (2021) 104841
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and mechanistic study of new 1,2,4-triazole derivatives as
antimicrobial agents
,
,
,d
Noha H. Amina *, Mohamed T. El-Saadia b, Ahmed A. Ibrahimc, Hamdy M. Abdel-Rahmanc
a Department of Medicinal Chemistry, Faculty of Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt
b Department of Medicinal Chemistry, Faculty of Pharmacy, Sinai University-Kantra Branch, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni-Suef, Egypt
d Department of Medicinal Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Novel 5-amino-1,2,4-triazole derivatives and their cyclized 1,2,4-triazolo[1,5-a]pyrimidine analogues were
designed, synthesized and evaluated for their antimicrobial activities. They were tested against five bacterial
strains (Methicillin Resistant S. aureus (MRSA), E. coli, K. pneumoniae, A. baumannii and P. aeruginosa) using
ciprofloxacin as a positive control and against two fungal strains (C. albicans and C. neoformans) using flucon-
azole and amphotericin B as positive controls. Compounds 9, 13a and 13b showed high to moderate antifungal
activities against candida albicans (MIC values = 4–32 µg/ml), with considerable safety profiles; where no
cytotoxicity against human embryonic kidney or red blood cells were detected at concentrations up to 32 µg/mL.
1,2,4-Triazoles
1,2,4-Triazolo[1,5-a]pyrimidines
Lansterol 14α-demethylase
Antimicrobial
Antifungal
Furthermore, compound 9 showed significant inhibitory activity against lansterol 14α-demethylase (IC50 = 0.27
µM), compared to the reference drug fluconazole (IC50 = 0.25 µM). Molecular docking of compound 9 into the
active site of the cytochrome P450 enzyme revealed comparable binding modes and docking scores to those of
fluconazole. Finally, in silico ADME studies prediction and drug-like properties of these compounds revealed
favorable oral bioavailability results.
1. Introduction
worldwide [4,5]. Nevertheless, invasive fungal diseases (exemplified by
candidiasis that is caused by microscopic fungi candida albicans) have
Microbial infections have ever since become a burden on all health
care systems and one of the major life threatening problems globally,
owing to its responsibility for numerous hospital acquired infections,
also known as healthcare associated infections (HCAI) [1]. HCAI have
led to extensive health and economic losses, while infections from multi-
drug resistance (MDR) pathogenic bacteria and fungi strains remain
progressively intractable. Consequently, consistent scientific pathways
have been intensified to develop further solutions such as newly syn-
thesized, either chemically or naturally derived, antimicrobial agents in
order to substitute for those that developed resistance or to proceed via
other newly discovered pathways [2,3]. However, the Infectious Disease
Society of America (IDSA) has highlighted six major bacterial species
ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsi-
ella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa and
Enterobacter cloacae) as being the utmost dangerous ones due to their
patterns of antibiotic resistance and responsibility for many infections
formed life-threatening conditions, especially for immune-compromised
hosts (cancer, AIDS or organ transplantations). Moreover, further re-
ported challenges have been encountered by scientists such as the
variability detected in the treatment protocol outcomes due to subjec-
tive factors, resistance emergence, drug toxicity and pharmacodynamic/
kinetic complications [6]. Lansterol 14α-demethylase (CYP51), a
member of the superfamily of heme-containing cytochrome P450 en-
zymes and a principal constitutive and executive component of the
fungal unit, has proved its positive clinical contribution in combating
stubborn fungal infections through its interaction with the heterocyclic
azole moieties of various antifungal agents, thus inhibiting essential
ergosterol biosynthesis [7].
Literature reviews have always highlighted the prominent influence
of the triazole moiety and its fused analogues as effective antimicrobial
agents exemplified by itraconazole, voriconazole, and posaconazole.
Also, fluconazole I (another triazole containing antifungal agent) has
Abbreviations: CO-ADD, The Community for Antimicrobial Drug Discovery; CYP51, cytochrome P450 subtype-51; SEM, standard error of the mean.
* Corresponding author at: Department of Medicinal Chemistry, Faculty of Pharmacy, Beni- Suef University, Beni-Suef 62514, Egypt.
E-mail address: noha.metri@pharm.bsu.edu.eg (N.H. Amin).
https://doi.org/10.1016/j.bioorg.2021.104841
Received 15 December 2020; Received in revised form 20 February 2021; Accepted 17 March 2021
Available online 22 March 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
been widely used in clinical cases owing to its particular molecular
structure, where N4 of 1,2,4-triazole ring has been coordinated to the
heme iron of CYP51. This coordination mode has been considered as an
important key feature for its antifungal activity [8,9].
1,2,4 triazole moiety) exhibited pronounced antimicrobial activities
against several bacterial strains, specifically Staphylococcus aureus and
Escherichia coli (MIC = 3.75 µM/mL), if compared to ampicillin (54 and
46 µM/ml, respectively) [29], Fig. 1.
On the other hand, the 1,2,4-triazolo[1,5-a]pyrimidine nucleus (a
subtype of purine analogs) has displayed a wide range of biological
activities. To name but a few of such activities, triazolopyrimidine de-
rivatives exhibited prominent antimicrobial [10], antitumor [11] and
even anti-inflammatory [12] activities. Although the aforementioned
nucleus under concern constituted two heterocyclic moieties (a pyrim-
idine ring with a triazole one), yet each of them has proven its efficacy
separately where promising scientific results have resulted from such
moieties whether combined or individually [8,10,13,14]. However,
combining them in the same framework has been presumed to result in a
synergistic antimicrobial outcome. A rising example for this fruitful
combination was essramycin II, the first 1,2,4-triazolo[1,5-a]pyrimidine
antibiotic [10,15,16]. Essramycin II, that was isolated from the culture
broth of marine Streptomyces species, has exhibited considerable anti-
microbial activity against Gram-positive and Gram-negative (MIC =
2.0–8.0 µg/ mL) bacterial strains and more than 50% inhibition rate
In view of all the above mentioned scientific literature data and as a
continuation of recent ongoing research programs concerned with
various heterocyclic nuclei [9,10,32], newly designed triazolopyr-
imidine derivatives were synthesized. The design scope of those new
compounds was based on combining substituted amides, thio-
semicarbazide or Schiff’s base moieties with either 5-amino-1,2,4 tri-
azole derivatives or cyclized 1,2,4-triazolo[1,5-a]pyrimidine analogues,
where synergism and toxicity limitation were crucial expected goals,
Fig. 1. The newly synthesized compounds were screened for their anti-
microbial activity against five bacterial strains: Methicillin Resistant
S. aureus (MRSA) (Gram positive), E. coli, K. pneumoniae, A. baumannii
and P. aeruginosa (Gram negative) and two fungal strains: C. albicans and
C. neoformans at CO-ADD (The Community for Antimicrobial Drug
Discovery), followed by cytotoxicity screening against human embry-
onic kidney cell lines and hemolysis of human red blood cells for the
most potent compounds to ensure acceptable safety margin. Further-
against 14 kinds of phytopathogenic fungi at 50
μg/mL for some of the
more, mechanistic studies were performed using lanosterol 14α-deme-
essramycin alkaloid derivatives [10,15–19].
thylase. Moreover, computational molecular docking, in silico ADME
studies prediction and drug-like characters of the compounds were
investigated, Fig. 1.
Similarly, combining thiosemicarbazide and azomethine moieties
with a triazole or a triazolopyrimidine core have preceded successful
pharmacophores as antimicrobial lead compounds [20–25]. 1,2,4-Tria-
zole derivative III (bearing an azomethine moiety) showed a consider-
able antimicrobial activity against both Gram-positive (MRSA) and
Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia, Acineto-
2. Results and discussions:
2.1. Chemistry
bacter baumanni and, Pseudomonas aeruginosa) with MIC values = 0.5 μg/
mL and 0.25–1.0
μ
g/ mL, respectively. On the other hand, compound IV
The synthesis pathways for the target compounds 6a-g, 7, 10a-c, 11,
12a,b, 13a-c and 14a,b were presented in schemes 1-3. The structures
of the newly synthesized compounds were established on their
elemental analyses and spectral data (supplementary CHEMISTRY). In
scheme 1, the starting material 5-amino-1H-[1,2,4] triazole-3-
carboxylic acid 3 was synthesized following a classical condensation
reaction of aminoguanidine bicarbonate and oxalic acid. Esterification
of the acid product 3 gave the corresponding 5-amino-1H- [1,2,4]
triazole-3-carboxylate 4 [33]. Then, the reaction of the ester 4 with
hydrazine hydrate gave the corresponding hydrazide 5. Consequently,
(a thiosemicarbazide moiety linked to a triazolopyrimidine core) was
reported to show a considerable therapeutic activity against Gram-
positive bacteria strains with an interesting DNA displacement activity
(IC50 = 22 μg/mL) [10].
Furthermore, amides have always been considered as important
functional groups for medicinal chemists, where the carboxaimde group
prevailed in more than 25% of known drugs, not to mention its feasible
conjugation with a triazole moiety to result in manifested antimicrobial
activities [26–31]. Compound V (constituted an amide group linked to a
Fig. 1. Synthetic design strategy of the present target compounds 6a-g, 7, 10a-c, 11, 12a,b, 13a-c and 14a,b.
2

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
O
O
C
OH
N
N
NH
NH2
i
HN
H2N
HO
H2CO3
.
OH
H2N
+
ii
N
O
H
2
1
3
O
C
OH
N
O
C
NHNH2
N
N
O
C
OCH3
HN
N
N
HN
H2N
HN
H2N
iv
iii
N
H2N
3
4
5
v
vi
O
C
H
N
H
O
N
N
N
R
HN
HN
N
C
N
H
N
N
H
N
H2N
S
H2N
7
6a-g
6a
; R=H
6e
; R=3,4,5-OCH3
6b
; R=4-F
6f
; R=2,4-Cl
6c
; R=4-Cl
6g
; R=4-N(CH3)2
6d
; R=3-OCH3, 4-OH
Scheme 1. Synthesis of compounds 1–7.
the condensation reaction of compound 5 with different appropriate
aromatic aldehydes (molar ratio 1:1) and in presence of an acidic
catalyst afforded the corresponding hydrazones 6a-g. On the other hand,
the reaction of compound 5 with phenyl isothiocyanate resulted in N-
phenyl thiosemicarbazide 7. IR spectra of compounds 6a-g were char-
acterized by the disappearance of the forked peak of NH2 attributed to
the hydrazide moiety. Also, their 1H NMR spectra showed singlet signals
at δ = 8.34–8.90 ppm and at 11.1–11.8 ppm (exchangeable with D2O),
the forked peak of NH2, attributed to the hydrazide moiety and the
corresponding appearance of a sharp single band, attributed to the
endocyclic NH group. Also, the 1H NMR spectra showed the appear-
–
ance of singlet signals at δ = 2.3–2.34 ppm and at 5.85–5.89 ppm,
attributed to the aliphatic CH3 group and the aromatic proton of the
pyrimidine ring, respectively. Similarly, 1H NMR spectrum of compound
11 showed the appearance of singlet signals at δ = 2.30 ppm and at 5.77
ppm, attributed to the aliphatic CH3 and the aromatic proton of the
pyrimidine ring. Further structure elucidation of the synthesized com-
pounds 10b and 11 using DEPT135 NMR spectra was performed to
distinguish between different carbon atoms; CH3, CH2 and CH. (Sup-
plementary DEPT).
–
–
attributed to N CH and CONH moieties of compounds 6a-g, respec-
–
tively, in addition to the characteristic signals of aromatic protons.
Moreover, DEPT analysis was conducted for compound 6b as a tool for
its structural verification of the different carbon atoms types (Supple-
mentary DEPT), while 1H NMR spectrum of compound 7 exhibited a
singlet signal at δ = 9.88 ppm (exchangeable with D2O), attributed to the
Furthermore in scheme 3, nucleophilic substitution reactions for the
amide compounds 12a,b formation proceeded through the reaction of
the ester derivative 4 with different appropriate aliphatic amines. On the
other hand, the reaction of the ester 4 with aromatic amines, in presence
of ammonium chloride, yielded the corresponding amides 13a-c.
Additionally, cyclization of compounds 13a,b with ethyl acetoacetate
(molar ratio 1:2) yielded the corresponding triazolo[1,5-a]pyrimidines
14a,b. IR spectra of compounds 13a,b revealed the disappearance of the
band corresponding to the ester carbonyl group at 1722 cmꢀ 1 and the
appearance of that corresponding to the amide group at 1640–1690
cmꢀ 1. Also, their 1H NMR spectra revealed a clear upgrade in the
appearance of signals attributed to the aliphatic and aromatic protons of
compounds 12a,b and 13a-c, respectively. Additionally, 1H NMR
–
CONH group, in addition to the characteristic signals of aromatic
proton at δ = 7.13–7.49 ppm.
As for scheme 2, cyclocondensation reaction of compound 4 with
ethyl acetoacetate in glacial acetic acid gave the corresponding 1,2,4-
triazolo[1,5-a]pyrimidine 8, as reported [15]. Hydrazinolysis of the
ester 8 with hydrazine hydrate gave the corresponding hydrazide 9
[15,17]. Furthermore, the condensation reaction of compound 9 with
different appropriate aromatic aldehydes furnished the corresponding
hydrazones 10a-c. On the other hand, the reaction of compound 9 with
phenyl isothiocyanate resulted in N-phenyl thiosemicarbazide 11. IR
spectra of compounds 10a-c were characterized by the disappearance of
3

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
O
O
O
N
N
HN
H2N
O
O
N
N
N
N
9
ii
i
N
N
C
C
C
OCH3
OCH3
NHNH2
N
H
N
H
4
8
iii
iv
O
O
O
C
H
N
H
O
N
N
N
N
N
N
R
C
N
N
N
H
N
S
H
N
H
N
H
10a-c
11
10a; R=H
10b; R=4-F
10c; R=2,4-di-Cl
Scheme 2. Synthesis of compounds 8–11.
O
O
N
N
N
N
HN
H2N
HN
H2N
i
C
C
NH N
N
N
12a; R
=
R
OCH3
12b; R=
O
4
12a,b
ii
O
O
C
N
HN
iii
O
C
HN
N
N
13a, 14a; R1=H
13b, 14b; R1=-F
13c; R1=-NO2
N
HN
R1
H2N
N
R1
N
H
13a-c
14a,b
Scheme 3. Synthesis of compounds 12a,b, 13a-c and 14a-b.
spectra of compounds 14a-c showed the appearance of singlet signals at
δ = 2.38 ppm and at 5.96 ppm, attributed to the aliphatic CH3 and the
aromatic proton of the pyrimidine ring, respectively. Further structure
elucidation of the synthesized compounds 13a, 13b, 14a and 14b using
DEPT135 NMR spectra was performed to distinguish between different
carbon atoms; CH3, CH2 and CH. (Supplementary DEPT).
aldehydes, EtOH, AcOH, reflux, 4 h; iv) C6H5-NCS, EtOH, reflux, 3 h.
Reagents and Conditions: i) Appropriate aliphatic amines, TEA,
reflux, 6 h. ii) Appropriate aromatic amines, NH4Cl, reflux, 7 h; iii) Ethyl
acetoacetate, AcOH, reflux, 7 h.
Reagents and Conditions: i) Water, reflux, 6 h; ii) Aqueous KOH,
reflux, 2 h; iii) MeOH, H2SO4, reflux, 24 h; iv) Hydrazine hydrate, EtOH,
reflux, 7 h; v) Aromatic aldehydes, EtOH, AcOH, reflux, 4 h; vi) C6H5-
NCS, EtOH, reflux, 3 h.
2.2. Biological evaluation
2.2.1. Antibacterial and antifungal activities
Antimicrobial screening for all the newly synthesized compounds
was performed at CO-ADD (The Community for Antimicrobial Drug
Discovery), funded by the Wellcome Trust (UK) and The University of
Queensland, Australia (Supplementary M1). All compounds were
Reagents and Conditions: i) Ethylacetoacetate, AcOH, reflux, 6 h;
ii) Hydrazine hydrate, EtOH, reflux, 4 h.; iii) Appropriate aromatic
4

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
screened for their antibacterial activities at 32 µg/ml against five bac-
terial strains: Methicillin Resistant S. aureus (MRSA) (Gram positive),
E. coli, K. pneumoniae, A. baumannii and P. aeruginosa (Gram negative)
using ciprofloxacin as a positive control and for their antifungal activ-
ities against two fungal strains: C. albicans and C. neoformans using
fluconazole as a positive control. Additionally, compounds displaying
significant growth inhibition percentage (80–90%) were screened for
their minimum inhibitory concentrations (MIC), where promising hits’
minimum inhibitory concentrations (MIC) ranged from 4 to 32 µg/ ml,
table 1. It was observed that all compounds showed weak antibacterial
activity against the tested strains. On the other hand, compound 9
exhibited potent activity against C. albicans (MIC = 4 µg/ml). In addi-
tion, 5-amino-1H-1,2,4-triazole-3-carboxamides 13a and 13b showed
moderate activities against C. albicans, expressed by a common (MIC =
32 µg/ml).
Table 2
Results of the molecular docking study of fluconazole and compounds 9, 13a
and 13b into the active site of lansterol 14 -demethylase. (PDB ID: 1EA1).
α
Compound
Energy
score
(S)
Bond length
between Fe-
Hem and
Nitrogen
atom
Interacting
moiety of
tested
14α-
demethylase
assay IC50
mL)
(μg/
compounds
9
ꢀ 9.33
2.33
2.75
2.34
2.34
N4
N2
N4
N4
0.27
0.46
0.32
0.25
13a
ꢀ 8.991
ꢀ 9.420
ꢀ 9.956
13b
Fluconazole
13a and 13b showed moderate activities with IC50 = 0.46 and 0.32 µg/
mL, respectively.
2.2.2. Cytotoxicity against human embryonic kidney cell line and hemolysis
of human red blood cell effects
2.3. In-silico studies
Screening for Cytotoxicity against human embryonic kidney cell line
and hemolysis of human red blood cells was performed at CO-ADD
institution (Supplementary M1) to ensure the safety margin for the
potent compounds, as shown in table 1. Cytotoxicity of the samples were
classified as toxic when their concentration at 50% cytotoxicity and
HC10 concentration at 10% hemolysis exhibited values higher than 32
µg/mL.(CC50,HC10 ≤ 32 µg/mL). From the obtained results, the inves-
tigated compounds 9, 13a and 13b showed CC50 ˃ 32 µg/mL against the
human embryonic kidney cell line and exhibited HC10 more than 32 µg/
2.3.1. Molecular docking
Molecular docking study was performed in an attempt to explain the
enzymatic activity of the most active compounds (9, 13a and 13b),
based on the results of lansterol 14
α
- demethylase (CYP51) assay,
- deme-
thylase active binding site. Accordingly the three newly synthesized
compounds were docked into the binding site of 14 - demethylase (PDB
through illustrating their positing and fitting into lansterol 14
α
α
ID: 1EA1) [34] to explore their potential binding modes compared to the
co-crystalized ligand fluconazole I. The results were presented in table 2
and Figs. 2 and 3. The figures revealed similar binding modes for the
studied compounds as those of the co-crystallized ligand, showing a
coordination bond between N4 and HEM. Moreover, the cyclized hy-
drazide compound 9 showed an additional coordination bond with HEM
via its hydrazoide moiety, Fig. 3. This positioning supported the prom-
inent in- vivo antifungal activity of the compound against Candida
albicans fungal strains, in contrary to its substituted Schiff’s bases (10a-
c) or amide analogues (14a,b), figure S1 (Supplementary FIGURES).
On the other hand, the schiff’s base derivatives (6a-g) showed different
mL. 2.2.3. Lanosterol 14
Fluconazole is the most commonly used antifungal drug to inhibit
lanosterol 14 -demethylase (CYP51) and ergosterol biosynthesis in the
α-demethylase (CYP51) inhibition activity
α
fungal cell membrane. The assay was performed at confirmatory diag-
nostic unit VACSERA, Egypt (Supplementary M2), using fluconazole as
a reference standard drug with IC50 value = 0.25 µg/mL, as shown in
table 2. The obtained results revealed that compound 9 possessed potent
enzyme inhibitory activity with IC50 = 0.27 µg/mL, compared to 0.25
µg/mL of the reference drug fluconazole. On the other hand, compounds
Table 1
The antimicrobial activities (growth inhibition % at 32 μg/mL concentration, MIC, cytotoxicity and hemolysis) of the examined compounds.
Code
Gram positive bacteria
MRSA a
Gram negative bacteria
Fungi
Ab d
Cytotoxicity and hemolysis
Ec b
Kp c
Pa e
Ca f
Cn g
CC50
(μ
g/mL)d
HC10
(
μ
g/mL)e
GI%h
GI%
GI%
GI%
GI%
GI%
MIC
GI%
3
18.01
18.66
15.47
7.31
16.54
22.04
12.65
13.47
14.76
10.46
16.94
16.02
12.93
18.07
40.1
27.49
25.79
23.62
14.08
23.75
9.29
8.49
10.74
6.54
8.49
1.89
7.97
1.12
5.9
8.07
6.56
18.46
7.11
3.67
3.33
3.88
3.67
6.91
12.62
5.81
5.67
2.13
84.29
2.49
4.22
7.4
NTi
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
4
7.63
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>32
NT
NT
NT
NT
NT
NT
>32
>32
NT
NT
NT
–
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
>32
NT
NT
NT
NT
NT
NT
>32
>32
NT
NT
NT
–
4
34.17
16.09
25.87
19.59
13.21
22.49
14.76
16.13
36.93
43.04
6.63
6.86
5
8.79
6a
ꢀ 4.29
7.08
6b
16.21
5.6
6c
ꢀ 14.37
ꢀ 5.36
ꢀ 3.21
ꢀ 4.93
1.2
6d
9.57
15.19
20.5
6e
8.32
6f
9.06
17.28
22.91
37.42
6.01
7.65
3.25
37.4
13.95
8.29
8.71
10.95
8.49
2.08
4.12
4.85
4.71
5.45
14.46
6.06
13.18
–
6g
7.21
7
13.59
16.7
4.72
8
ꢀ 0.46
65.56
1.52
ꢀ 7.64
ꢀ 5.94
ꢀ 5.43
ꢀ 7.64
ꢀ 6.74
ꢀ 0.34
ꢀ 3.21
2.01
9
16.09
15.82
13.64
11.62
12.49
7.31
12.72
9.02
17.93
10.32
10.55
6.21
10a
NT
NT
NT
NT
NT
NT
32
10b
3.48
10.14
7.23
10c
12.83
33.45
17.31
16.21
13.72
16.47
10.87
ꢀ 8.4
11
12.74
20.85
21.95
19.96
23.09
23.04
8.52
2.92
3.43
34.55
25.68
83.99
88.92
4.98
7.69
2.78
–
12a
16.34
23.63
8.19
12b
3.67
13a
9.77
4.93
13b
4.97
12.64
8.55
32
ꢀ 2.41
ꢀ 5.79
ꢀ 6.45
ꢀ 3.76
–
13c
15.39
17.26
14.02
–
NT
NT
NT
0.125
–
14a
4.27
14b
ꢀ 5.32
–
0.007
5.4
5.75
Flu-conazole
–
–
Cipro-floxacin
0.25
0.5
1
0.25
–
–
–
–
aMethicillin Resistant S. aureus, bE. coli, cK. pneumonia, dA. baumannii, eP. aeruginosa, fC. albicans, gC. neoforma, hgrowth inhibition %, inot tested.
5

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
Swiss ADME [40–42] were the software used to predict the aforemen-
tioned pharmacokinetics parameters of the most active compounds, as
shown in table 3. The investigated compounds were satisfying to Lip-
inski’s rule of five, where the molecular weight ranged from 203 to 221
(<500), logP values ranged from ꢀ 0.77 to 0.76 (<5); hydrogen bond
donors were 3 (<5) and finally hydrogen bond acceptors ranged from 3
to 5 (<10). Theoretically speaking, such results would result in com-
pounds with anticipated good oral absorption. Also, the compounds
exhibited NROTB range from 2 to 3 (<10) and PSA range from 96.69 to
118.17 (<140A2), indicating good cell permeability through various
biological membranes.
Additionally, further ADME parameters were evaluated such as the
drug solubility S (a key parameter determining dissolution rate to ach-
ieve desired oral bioavailability) and the drug likeness score, which
described compounds with desirable bioavailability during the early
phases of drug discovery (larger score values tended to be for particular
molecules of higher activity). Herein, Molsoft [39] and SwissADME
[40–42] software were applied to predict drug likeness model scores and
solubility of the compounds, as shown in Table 4. Absorption and dis-
tribution characteristics have been known to be changed by aqueous
solubility (solubility improves when solubility values are more than
0.0001 mg/mL). The tested compounds showed values ranging from
1.08 to 1.91 mg/mL, thus promising solubility characteristics have been
expected. Also, the more positive drug likeness score values, the more
likely the compound was expected to be as a promising therapeutic
pharmacophore. A positive model score was attributed for compounds 9
and 13b (0.45 and 0.95, respectively), in contrast to compound 13a,
which showed a negative value (ꢀ 0.07). In addition, all the compounds
were found to be orally safe (less than 1 violation of the Lipinski’s rule of
five) and thus they could be anticipated to possess good oral bioavail-
ability and cell permeability.
Fig. 2. Superimposition of the co-crystallized ligand, fluconazole (Yellow) and
re-docked ligand (Turquoise). (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
disposition and interaction modes, a case that supported their weak in-
vitro antimicrobial activities against the tested strains. Also, the amides
(12a,b and 13a-c) showed weak antibacterial and moderate antifungal
activities against the tested strains, where the fluorinated phenyl car-
boxamide (13b) was bound to HEM more effectively than its unsub-
stituted analogue (13a), figure 3 and S1 (Supplementary FIGURES).
Moreover, the validity of the docking protocol was ensured via re-
docking of the co-crystallized fluconazole into the active site. Docking
results obtained were presented in Fig. 2, which showed a near perfect
alignment with the original ligand as obtained from the X-ray resolved
PDB file. The re-docked ligand showed rmsd of 0.8 between the docked
pose and the co-crystallized ligand (energy score (S) = ꢀ 9.956 kcal/
mol), and by the ability of the docking poses to reproduce the key in-
teractions accomplished by the co-crystallized ligand with HEM B460.
Furthermore, a pre-ADMET software was applied to estimate the
various ADMET factors that might affect the pharmacokinetics of the
synthesized compounds, exemplified by the blood brain barrier partition
coefficient BBB (a key parameter for brain therapeutically active tar-
geted drugs), the Caco2 (human colon adenocarcinoma) permeability
coefficient (a human colon epithelial cancer cell line that differentiates
to form tight junctions between cells to serve as a model of paracellular
movement of compounds across the monolayer), the human intestinal
absorption HIA (one of the key processes determining factors of the drug
bioavailability upon oral administration as there is a correlation be-
tween the structures of organic compounds and their human small in-
testinal absorption and the human plasma protein binding PPB (an
essential criteria to estimate the fraction of drug bound to plasma pro-
teins and thus may predict the efficiency of the free drug that can cross
various biological membranes and thus can bind to the intended mo-
lecular target). Last but not least, inhibition of cytochrome P4502D6
(CYP4502DC) and Madin-Darby canine kidney cells MDCK permeability
coefficient provided helpful in vitro tools to assess a drug’s permeability
and transporter interactions during drug discovery and development.
Herein, ADME data of the most active compounds was calculated using
preADMET software [35,36], as shown in table 5. All the investigated
compounds showed a medium CNS absorption range from 0.132 to
0199, comparable to the normal medium range ≤ 0.1–2. Also the tested
compounds showed medium permeability in Caco2 model range from
4.3 to 18.9 nm/s (normal range 4–70 nm/s), in addition to high
permeability in MDCK model, ranging from 2.35 to 220.6 nm/s (normal
range 4–70 nm/s). Moreover, the compounds showed no inhibition of
CYP2D6 enzyme and thus no interaction with CYP2D6 inducers or in-
hibitors. Last but not least, the tested compounds showed moderate
absorption, ranging from 60.3 to 71.2% (normal range 30–70 %) and
low plasma protein binding, ranging from 9.4 to 23.9 (normal range ≤
90).
2.3.2. Computational analysis: In-silico prediction of physicochemical
properties, pharmacokinetics and drug-likeness profile
In-silico ADMET prediction studies have played an increasingly
important part in drug research and development through providing an
effective way to assess multiple pharmacokinetic properties in hit-to-
lead and lead-optimization campaigns. The importance of such a strat-
egy alongside the evolution of chemoinformatics has grown since the
1960 s, not to mention the widely applied drug-likeness concepts of the
1990 s. Accordingly, predicted absorption, distribution, metabolism and
excretion (ADME) studies were performed for the most active com-
pounds (9, 13a and 13b) and results were presented in tables 3-5.
Herein, pre-ADME [35,36], molinspiration [37,38], Molsoft [39] and
Swiss ADME [40–42] were the software used to predict the aforemen-
tioned pharmacokinetic parameters of the most active compounds. The
SMILE molecular structures of the compounds were obtained from
PubChem (https://pubchem.ncbi.nlm.nih.gov). The performed in-silico
studies were used as a predictive tool to investigate the expected phar-
macokinetic properties of the target compounds to improve their in- vivo
outcomes through
a pharmacokinetic-pharmacodynamic balance
[35–42].
Literature reports have proved that compounds were most likely to
have poor absorption for various reasons, such as having a molecular
weight (MW) ˃ 500, a calculated octanol/water partition coefficient logP
˃ 5, number of H-bond donors ˃ 5 or the number of H-bond acceptors ˃10.
Also, assessing other parameters has been helpful as virtual screening
tools, such as calculating the partition coefficient (a descriptive
parameter for the lipophilicity of a drug) and topological polar surface
area PSA (a criteria that allows the prediction of transport properties of
polar atoms through membranes). Moreover, the number of rotatable
bonds (NROTB) has been an important descriptor, associated with any
single non-ring bond, bounded to nonterminal heavy (i.e., non-
hydrogen) atom, where a large number of rotatable bonds (≥10) indi-
cated poor oral bioavailability. Herein, molinspiration [37,38] and
6

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 3. 2D and 3D interaction diagrams of fluconazole (a) and (b), compound 9 (c) and (d), compound 13a (e) and (f) and compound 13b (g) and (h), respectively,
with 14- demethylase (PDB: 1EA1).
α
7

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
screening in order to generate efficient antifungal agents with consid-
erable safety profiles.
Table 3
Estimated physicochemical properties of compounds 9, 13a and 13b using
SwissADME & Molinspiration software.
4. Experimental
Physicochemical properties
log P
Code
TPSAc
H-bond
donor
H-bond
NORTBb
MWa
(A2)
acceptor
4.1. Chemistry
118.17
96.7
3
3
3
5
3
4
2
3
3
208.18
203.2
ꢀ 0.77
0.43
9
Chemical reagents and solvents were all obtained from Sigma
Aldrich, Germany. Reactions were monitored by TLC: Pre-coated plastic
sheets, 0.2 mm silica gel with fluorescent indicator (E. Merck). Melting
points were determined on Stuart electrothermal melting point appa-
ratus and were uncorrected. IR spectra were recorded as KBr disks on a
Bruker spectrophotometer (Karlsruhe, Germany) at Nahda University,
Beni-Suef, Egypt. 1H NMR spectra were carried out on Bruker apparatus
400 MHz (Karlsruhe, Germany) at Sohag University, Sohag, Egypt, using
TMS as internal reference, chemical shifts (values are given in parts per
million (ppm) using DMSO‑d6 or CDCl3 as solvents. 13C NMR and DEPT
135 were recorded using Bruker 100 MHz NMR, using TMS as internal
standard and chemical shifts were recorded in ppm on δ scale (Karls-
ruhe, Germany) at Sohag University,Sohag, Egypt. Elemental analyses
(C, H and N) and mass spectrometry were performed on FLASH 2000
CHNS/O analyser, VARIO-Elementer apparatus at The Regional Center
for Mycology and Biotechonology, Al-Azhar University, Cairo, Egypt.
Compounds 3, 4 [33], 5, 13a [26] and 8, 9 [15,17] were prepared ac-
cording to their reported procedures.
13a
13b
96.69
221.19
0.76
aMW, molecular weight; bNROTB, number of rotatable bonds cTPSA, topological
polar surface area.
Table 4
Predicted drug likeness using Molsoft & SwissADME software.
Code
Solubility
(mg/L)
Drug likeness
model score
Lipinski’s rule
Bioavailability
score
violations
9
1.08
1.91
1.09
0.45
0
0
0
0.55
0.55
0.55
13a
13b
ꢀ 0.07
0.94
Table 5
ADME data of most active compounds calculated using preADMET software.
Code
Pharmacokinetics
BBBa
Caco-2b
HIAc
(%)
MDCKd (nm/
PPBe
CYP
4.1.1. General procedure for the synthesis of compounds 6a-g
(nm/s)
s)
2D6f
A mixture of compound 5 (0.0017 mol), appropriate aldehyde
(0.0017 mol) and catalytic glacial acetic acid (1 mL) in absolute ethanol
(20 mL) was heated under reflux for 4 h. The reaction was monitored by
TLC; then the obtained precipitate from the hot ethanolic solution was
filtered and re-crystallized from ethanol to offer products 6a-g.
9
0.132
0.177
0.199
4.3
60.3
71.2
71.1
2.35
220.6
81.0
9.4
No
No
No
13a
13b
7.02
18.9
23.9
23.4
aBBB:blood brain barrier penetration; bCACO-2: permeability through cells
derived from human colon adenocarcinoma; cHIA: percentage human intestinal
absorption; dMDCK: permeability through Madin-Darby canine kidney cells;
ePPB: plasma protein binding; fCYP2D6: cytochrome P450 2D6.
4.1.1.1. 5-
Amino-N′-benzylidene-1H-1,2,4-triazole-3-carbohydrazide
6a. White powder, yield: 55.6%, m.p.: 265–267 ◦C; FT-IR (KBr (ʋmax
/
cmꢀ 1): 3220 (forked NH ), 3307 (NH), 1692 (C O amide), 3047 (CH
–
–
2
3. Conclusion
aromatic); 1H NMR (DMSO‑d6, 400 MHz) δ = 6.06 (s, 2H, NH2), δ =
–
–
7.45–7.70 (m, 5H, Ar-H), 8.51 (s, 1H,
N CH), 11.41 (s, 1H,
–
A novel series of 5-amino-1,2,4 triazole derivatives and their cyclized
1,2,4-triazolo[1,5-a]pyrimidine analogues, were designed, synthesized
and their structures were characterized by melting points, IR, 1H NMR,
13C NMR, DEPT 135, elemental analyses and mass spectroscopy. The
antimicrobial activities of all the compounds were evaluated against five
bacterial strains (Methicillin Resistant S. aureus (MRSA), E. coli, K.
pneumoniae, A. baumannii and P. aeruginosa) using ciprofloxacin as a
positive control and against two fungal strains (C. albicans and
C. neoformans) using fluconazole and amphotericin B as positive controls
at CO-ADD, Australia. Compounds 9, 13a and 13b showed promising
anti-candida albicans activities (MIC values = 4–32 µg/ml), associated
with significant safety profiles against certain cytotoxic parameters.
They were not cytotoxic against neither human embryonic kidney cell
line nor 10% red blood cell hemolysis, at concentrations up to 32 µg/
mL. Further mechanistic studies highlighted the interesting potency of
the hit hydrazide derivative 9 due to its considerable ability of lanosterol
–
–
– –
CONH , D2O exchangeable), 12.49 (s, 1H, cyclic NH , D2O
exchangeable); EIMS m/z (%): 230.98 [M+] (31.26), 208.17 (100);
Elemental analysis calculated for C10H10N6O (230.23): C, 52.17; H,
4.38; N, 36.50; Found C, 52.31; H, 4.52; N, 36.73.
4.1.1.2. 5- amino -N′-(4-fluorobenzylidene)-1H-1,2,4-triazole-3-carbohy-
drazide 6b. White powder, yield: 60.4%, m.p.: 241–243 ◦C; FT-IR (KBr
1
–
(ʋmax/ cm- ): 3273 (forked NH ), 3440 (NH), 1687 (C O amide), 3035
–
2
(CH aromatic); 1H NMR (DMSO‑d6, 400 MHz) δ = 6.08 (s, 2H, NH2),
7.50, 7.52 (d, J = 8.00 Hz, 2H, Ar-H), 7.70, 7.72 (d, J = 8.00 Hz, 2H, Ar-
–
–
– –
N CH), 11.48 (s, 1H, CONH , D2O exch.) 12.50 (s,
–
H), 8.50 (s, 1H,
1H, cyclic NH , D2O exchangeable); 13C NMR (DMSO‑d6):(100 MHz)
δ = 116.19, 116.41, 129.62, 129.70, 131.53, 147.34, 152.35, 164.80;
DEPT C135 (CDCl3, 100 MHz, δ ppm); 116.27 (phenyl CH-3 + CH-5),
– –
–
–
116.49 (phenyl CH-2 + CH-6), 147.19 (
N CH);); EIMS m/z (%):
–
248.79 [M+] (27.51), 206.82 (100); Elemental analysis calculated for
C10H9FN6O (248.22): C, 48.39; H, 3.65; N, 33.86; Found C,48.59;
H,3.75;N,33.68.
14α-demethylase (CYP51) inhibition (IC50 value = 0.27 μg/mL),
compared to fluconazole (IC50 = 0.25 μg/mL). Additionally, in-silico
docking studies showed similarity in the binding patterns of X-ray
structures of both the synthesized compounds (9, 13a and 13b) and
4.1.1.3. 5- Amino-N′-(4-chlorobenzylidene)-1H-1,2,4-triazole-3-carbohy-
fluconazole, targeting the same pocket of lanosterol 14-α- demethylase
drazide 6c. White powder, yield: 55.9%, m.p.: 236–238 ◦C; FT-IR (KBr
(CYP51) active site (1EA1). Finally, in silico prediction of physico-
chemical properties, pharmacokinetics and drug-likeness profile studies
of the most active compounds showed favorable oral bioavailability, cell
permeability and minimal toxicity. Collectively, compounds 9, 13a and
13b showed coinciding and promising in- vivo, in- vitro and in-silico re-
sults. Accordingly, the aforementioned compounds, topped by com-
pound 9, could be considered as reliable pharmacophores for further
–
(ʋmax/ cm-1): 3267 (forked NH2), 3325 (NH), 1692 (C O amide),
–
3050 (CH aromatic); 1H NMR (DMSO‑d6, 400 MHz) δ = 6.04 (s, 2H,
NH2), 7.22, 7.24 (d, J = 8.00 Hz, 2H, Ar-H), 7.54, 7.56 (d, J = 8.00 Hz,
–
–
N
– –
CONH , D2O
2H, Ar-H), 8.44 (s, 1H,
CH), 11.36 (s, 1H,
–
– –
exchangeable), 12.42 (s, 1H, cyclic NH , D2O exchangeable); EIMS
m/z (%): 263.08 [M+ꢀ H] (79.20), 266.10 [M+2] (27.50), 266.76
8

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
– –
– –
NH , D2O
[M+2] (45.45), 188.07 (100); Elemental analysis calculated for
NH
,
D2O exchangeable), 12.49 (s, 1H, cyclic
C
10H9ClN6O (264.67) C, 45.38; H, 13.40; N, 31.75; Found C, 45.49; H,
exchangeable); EIMS m/z (%): 277.98 [M+] (31.31), 82.23 (100);
Elemental analysis calculated for C10H11N7OS (277.31) C, 43.31; H,
4.00; N, 35.36; Found C,43.41;H,4.3;N,35.48.
3.51; N, 31.91.
4.1.1.4. 5-Amino-N′-(4-hydroxy-3-methoxybenzylidene)-1H-1,2,4-tri-
azole-3-carbohydrazide 6d. White powder, yield: 29.6%, m.p.:
275–277 ◦C; FT-IR (KBr (ʋmax/ cmꢀ 1): 3445 (forked NH2), 3319 (NH),
4.1.3. General procedure for the synthesis of compounds 10a-c
A mixture of compound 9 (0.00072 mol), appropriate aldehyde
(0.00076 mol) and catalytic glacial acetic acid (1 mL) in absolute
ethanol (20 mL) was heated under reflux for 4 h. The reaction was
monitored by TLC; then the obtained precipitate from the hot ethanolic
solution was filtered and re-crystallized from ethanol to offer products
10a-c.
1
–
1674 (C O amide), 3070 (CH aromatic); H NMR (DMSO‑d , 400 MHz)
–
6
δ = 3.84 (s, 3H, OCH3), 6.06 (s, 2H, NH2), 6.83, 6.85 (d, J = 8.00 Hz, 1H,
Ar-H), 7.05, 7.07 (d, J = 8.00 Hz, 1H, Ar-H), 7.30 (s, 1H, Ar-H), 8.37 (s,
–
–
1H,
N CH), 9.36 (s, 1H, phenolic OH, D O exchangeable), 11.18 (s,
–
2
–
–
– –
1H, CONH , D2O exchangeable), 12.46 (s, 1H, cyclic NH , D2O
exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 56.19, 109.88,
116.00, 122.52, 123.33, 126.63, 148.56, 148.99, 149.49, 157.54,
163.22; EIMS m/z (%): 276.60 [M+] (21.83), 107.36 (100); Elemental
analysis calculated for C11H12N6O3 (276.25) C, 47.83; H, 4.38; N, 30.42;
Found C,47.59; H,4.52;N,30.25.
4.1.3.1. Benzylidene-5-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]
pyrimidine-2-carbohydrazide 10a. White powder, yield 62.5%, m.p.:
184–186 ◦C FT-IR (KBr (ʋmax/ cmꢀ 1): 3273–3193 (NH pyrimidine, NH
hydrazone), 1667 (C O amide), 3020 (CH aromatic); 1H NMR
–
–
– –
(DMSO‑d6, 400 MHz) δ = 2.33 (s, 3H, CH3 ), 5.88 (s, 1H, Ar-H),
–
4.1.1.5. 5-Amino-N′-(3,4,5-trimethoxybenzylidene)-1H-1,2,4-triazole-3-
–
–
N CH ), 10.84 (s, 1H, cyclic
–
7.18–7.95 (m, 5H, Ar-H), 8.49 (s, 1H,
carbohydrazide 6e. White powder, yield: 88.6%, m.p.: 232–234 ◦C; FT-
– –
– –
NH , D2O exchangeable), 11.90 (s, 1H, NH , D2O exchangeable);
EIMS m/z (%): 296.10 [M+] (42.43), 203.86 (100); Elemental analysis
calculated for C14H12N6O2 (296.28) C, 56.75; H, 4.08; N, 28.36; Found
C,56.73; H,4.06; N,28.34.
IR (KBr (ʋmax/ cmꢀ 1): 3439.73 (forked NH ), 3320 (NH), 1662 (C
O
–
–
amide), 3050 (CH aromatic); 1H NMR (DMS2O‑d6, 400 MHz) δ = 3.74 (s,
3H, OCH3), 3.85 (s, 6H, OCH3) 6.06 (s, 2H, NH2), 6.99, (s, 2H, Ar-H),
–
–
– –
N CH), 11.37 (s, 1H, CONH , D2O exchangeable),
–
8.42 (s, 1H,
12.50 (s, 1H, cyclic NH , D2O exchangeable); 13C NMR (DMSO‑d6):
(100 MHz) δ = 56.56, 60.61, 105.06, 130.38, 140.01, 144.35, 148.48,
153.70, 156.34, 161.31; EIMS m/z (%): 230.27 [M+] (29.13), 215.97
(100); Elemental analysis calculated for C13H16N6O4 (320.12) C, 48.75;
H, 5.03; N, 26.24; Found C, 48.96; H, 5.24; N, 26.45.
4.1.3.2. (4-Fluorobenzylidene)-5-methyl-7-oxo-4,7-dihydro-[1,2,4]tri-
azolo[1,5-a]pyrimidine-2-carbohydrazide 10b. White powder, yield 85%,
m.p.: 174–176 ◦C FT-IR (KBr (ʋmax/ cmꢀ 1): 3312–3258 (NH pyrimidine,
– –
NH hydrazone), 1681 (C O amide), 3019 (CH aromatic); 1H NMR
–
–
–
–
– –
(DMSO‑d , 400 MHz) δ 2.3 (s, 3H, CH3 ), 5.89 (s, 1H, Ar-H),
6
7.33,7.35 (d, J = 8.00 Hz, 2H, Ar-H), 7.80, 7.82 (d, J = 8.00 Hz, 2H,
–
4.1.1.6. 5- Amino-N′-(2,4-dichlorobenzylidene)-1H-1,2,4-triazole-3-car-
–
– – –
N CH ), 10.00 (s, 1H, cyclic NH , D2O
–
Ar-H), 8.61 (s, 1H,
exchangeable), 12.25 (s, 1H, NH , D2O exchangeable); 13C NMR
(DMSO‑d6):(100 MHz) δ = 19.55, 99.11, 115.73, 129.99, 131.25,
148.77, 151.48, 155.91, 156.02, 164.96; DEPT C135 (CDCl3, 100 MHz, δ
ppm); 19.82 (pyrimidine CH3-5), 98.96 (pyrimidine CH-6), 116.35
bohydrazide 6f. White powder, yield: 85.4%, m.p.: 215–217 ◦C; FT-IR
– –
(KBr (ʋmax/ cmꢀ 1): 3330 (forked NH ), 3426 (NH), 1672 (C
O
–
–
2
amide), 3029 (CH aromatic); 1H NMR (DMSO‑d6, 400 MHz) δ = 6.08 (s,
2H, NH2), 7.49, 7.51 (d, J = 8.00, 1H, Ar-H), 7.67 (s, 1H, Ar-H), 8.00,
–
–
–
(phenyl CH-3
+
CH-5), 116.57 (phenyl CH-2
+
CH-6), 148.66
8.02 (d, J = 8.00, 1H, Ar-H), 8.90 (s, 1H,
N CH), 11.88 (s, 1H,
–
+
–
–
N
–
– –
(
CH); EIMS m/z (%): 314.58 [M ] (31.52), 269.50 (100);
–
CONH , D2O exchangeable), 12.53 (s, 1H, cyclic
NH , D2O
exchangeable); EIMS m/z (%): 298.33 [M+] (14.54), 302.65 (4.76),
Elemental analysis calculated for C14H11FN6O2 (314.27) C, 53.50.34; H,
3.53; N, 26.74; Found C, 53.49; H,3.50; N,26.73.
303.28 (14.54), 251.87 (100); Elemental analysis calculated for
C
10H8Cl2N6O (299.12) C, 40.15; H, 2.70; N, 28.10; Found C, 40.33; H,
2.92; N, 28.31.
4.1.3.3. (2,4-Dichlorobenzylidene)-5-methyl-7-oxo-4,7-dihydro-[1,2,4]
triazolo[1,5-a] pyrimidine-2-carbohydrazide 10c. White powder, yield
70.5%, m.p.: 189–191 ◦C FT-IR (KBr (ʋmax/ cmꢀ 1): 3413–3336 (NH
4.1.1.7. 5-Amino-N′-(4-(dimethylamino)benzylidene)-1H-1,2,4-triazole-
–
–
3-carbohydrazide 6 g. Yellow powder, yield: 70.8%, m.p.: 246–248 ◦C;
pyrimidine, NH hydrazone), 1681 (C O amide), 3020 (CH aromatic);
1
FT-IR (KBr (ʋmax/ cmꢀ 1): 3320 (forked NH ), 3331 (NH), 1689 (C
O
–
– –
H NMR (DMSO‑d6, 400 MHz) δ = 2.34 (s, 3H, CH3 ), 5.85 (s, 1H, Ar-
–
amide), 3040 (CH aromatic); 1H NMR (DMS2O‑d6, 400 MHz) δ = 2.98 (s,
H), 7.53,7.55 (d, J = 8.00 Hz, 1H, Ar-H), 7.72 (s, 1H, Ar-H), 8.03, 8.05
–
–
–
N CH ), 12.51 (s, 1H, cyclic
–
(d, J = 8.00 Hz, 1H, Ar-H), 9.01 (s, 1H,
6H, CH3), 6.05 (s, 2H, NH2), 6.75, 6.77 (d, J = 8.00, 2H, Ar-H), 7.50,
–
–
N
– –
– –
NH , D2O exchangeable), 13.36 (s, 1H, NH , D2O exchangeable);
EIMS m/z (%): 363.65 [M+ꢀ H] (18.84), 366.93 [M+2] (5.60), 276.34
(100); Elemental analysis calculated for C14H10Cl2N6O2 (365.17) C,
46.05; H, 2.76; N, 23.01; Found C, 46.02; H,2.75; N,23.00.
7.52 (d, J = 8.00, 2H, Ar-H), 8.34 (s, 1H,
CH), 11.04 (s, 1H,
–
–
–
– –
CONH , D2O exchangeable), 12.44 (s, 1H, cyclic NH , D2O
exchangeable); EIMS m/z (%): 273.69 [M+] (45.79), 174.79 (100);
Elemental analysis calculated for C12H15N7O (273.29) C, 52.74; H, 5.53;
N, 35.88; Found C,52.51;H,5.39;N,35.68.
4.1.4. 2-(5-Methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo[1,5-a]pyrimidine-2-
carbonyl)-N-phenylhydrazinecarbothioamide 11
4.1.2. Synthesis of 2-(5-amino-1H-1,2,4-triazole-3-carbonyl)-N-
phenylhydrazinecarbothioamide 7
Phenyl isothiocynate (0.0009 mol) was added to a solution of com-
pound 9 (0.0009 mol) in absolute ethanol (20 mL) and heated under
reflux for 3 h. The reaction mixture was concentrated under reduced
pressure; the product was collected and re-crystallized from ethanol to
give the corresponding compound 11.
Phenyl isothiocynate (0.002 mol) was added to a solution of com-
pound 5 (0.002 mol) in absolute ethanol (20 mL) and heated under
reflux for 3 h. The reaction mixture was concentrated under reduced
pressure; the product was collected and re-crystallized from ethanol to
give the corresponding compound 7.
White powder, yield 90%, m.p.: 227–229 ◦C FT-IR (KBr (ʋmax
/
cmꢀ 1): 3280–3265 (NH pyrimidine, NH amidic), 1667 (C O amide),
–
–
White powder, yield 88%, m.p.: 288–290 ◦C FT-IR (KBr (ʋmax
/
cmꢀ 1): 3447 (forked NH2), 3367 ( NH ), 1681 (C O amide), 3020
–
–
– –
3055 (CH aromatic), 1522 (C═S); 1H NMR (DMSO‑d6, 400 MHz) 1H
1
– –
NMR (DMSO‑d6, 400 MHz) δ = 2.30 (s, 3H, CH3 ), 3.46 (s, 1H,
– –
– –
(s,2H,NH2, D2O exchangeable), 7.13–7.49 (m, 5H, Ar-H), 9.88 (s, 1H,
(CH aromatic); H NMR (DMSO‑d6, 400 MHz) δ = 4.16 (s, 1H, NH
,
– –
NH , D2O exchangeable), 5.77 (s, 1H, Ar-H), 7.10–7.33 (m,5H, Ar-
D2O exchangeable), 5.33 (s, 1H, NH , D2O exchangeable), 6.43
– –
– –
H), 7.53 (s, 1H, NH , D2O exchangeable), 9.46 (s, 2H, NH , D2O
9

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 20.94, 98.06, 98.07,
128.49, 154.99, 155.28, 155.89, 156.92, 157.05, 158.98; DEPT C135
(CDCl3, 100 MHz, δ ppm); 20.93 (pyrimidine CH3-5), 98.10 (pyrimidine
CH-6), 117.24 (phenyl CH-2 + CH-6), 121.49 (phenyl CH-3 + CH-5),
128.46 (phenyl CH-4); EIMS m/z (%): 343.70 [M+] (3.20), 131.20
(100); Elemental analysis calculated for C14H13N7O2S (343.36) C,
48.97; H, 3.82; N, 28.55; Found C,48.97; H,3.81; N,28.54.
calculated for C9H8FN5O (221.19) C, 48.87; H, 3.65; F, 8.59; N, 31.66;
Found C,48.95;H,3.38;N,31.49.
4.1.6.3. 5-
Amino-N-(4-nitrophenyl)-1H-1,2,4-triazole-3-carboxamide
13c. yellow powder, yield: 70.4%, m.p.: 261–263 ◦C; FT-IR (KBr (ʋmax
/
cmꢀ 1): 3420 (forked NH ), 3309 (NH), 1686 (C O amide); 1H NMR
–
–
2
(DMSO‑d6, 400 MHz) δ = 6.11 (s,2H,NH2), 6.60, 6.62 (d, J = 8.00 Hz,
– –
2H, Ar-H), 7.92, 7.94 (d, J = 8.00 Hz, 2H, Ar-H), 10.07 (s, 1H, NH
,
4.1.5. General procedure for the synthesis of compounds 12a,b
– –
D2O exchangeable), 12.54 (s, 1H, cyclic NH , D2O exchangeable);
Elemental analysis calculated for C9H8N6O3 (248.20) C, 43.55; H, 3.25;
N, 33.86; Found C, 43.22; H, 3.43; N, 33.57.
A mixture of compound 4 (0.007 mol), appropriate aliphatic amine
(0.0021 mol) and triethylamine (0.005 mol) was heated under reflux for
6 h. Then, triethylamine and excess aliphatic amine were distilled off in
a vacuum; 2–3 mL of water was added to the residue; the precipitate was
filtered off and re-crystallized from water to offer compounds 12a,b.
4.1.7. General procedure for the synthesis of compounds 14a,b
A solution of compounds 13a,b (0.001 mol) and ethyl acetoacetate
(0.002 mol) in glacial aceic acid (10 mL) was heated under reflux for 7 h.
Then, the excess solvent was distilled off under reduced pressure and the
residue was re-crystallized from ethanol to offer compounds 14a, b.
4.1.5.1. 5-
Amino-N-(4-methylpiperazin-1-yl)-1H-1,2,4-triazole-3-car-
boxamide 12a. yellow powder, yield: 30.6%, m.p.: 279–281 ◦C; FT-IR
(KBr (ʋmax/ cmꢀ 1): 3459 (forked NH ), 3433 (NH), 1651 (C
O
–
–
2
amide); 1H NMR (DMSO‑d6, 400 MHz) δ = 1.27 (s,3H,N-CH3), 3.72–3.75
(t, J = 12.00 Hz, 4H, piperazine), 4.23–4.25 (t, J = 8.00 Hz, 4H,
4.1.7.1. 5-Methyl-7-oxo-N-phenyl-4,7-dihydro-[1,2,4]triazolo[1,5-a]py-
rimidine-2-carboxamide 14a. White powder, yield 83.5%, m.p.:
178–180 ◦C FT-IR (KBr (ʋmax/ cmꢀ 1): 3356–3210 (NH pyrimidine, NH
– –
NH , D2O
piperazine), 6.13 (s,2H,NH2),12.66 (s, 1H, cyclic
exchangeable); EIMS m/z (%): 225.72 [M+] (83.20), 142.25 (100);
Elemental analysis calculated for C8H15N7O (225.25) C, 42.66; H, 6.71;
N, 43.53; Found C, 42.45; H, 6.58; N, 43.74.
1
–
amidic), 1681 (C O amide), 3035 (CH aromatic); H NMR (DMSO‑d ,
–
6
– –
400 MHz) δ = 2.38 (s, 3H, CH3 ), 5.96 (s, 1H, Ar-H), 7.11–7.83 (m,
–
5H, Ar-H), 10.19 (s, 1H, NH, D2O exchangeable), 11.73 (s, 1H, cyclic
NH , D2O exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 19.26,
– –
4.1.5.2. (5-Amino-1H-1,2,4-triazol-3-yl)(morpholino)methanone 12b.
99.27, 121.11, 129.10, 129.4, 138.63, 138.82, 151.55, 152.97, 156.70,
157.8; DEPT C135 (CDCl3, 100 MHz, δ ppm); 19.27 (pyrimidine CH3-5),
99.28 (pyrimidine CH-6), 120.78 (phenyl CH-2 + CH-6), 124.74 (phenyl
CH-4), 129.15 (phenyl CH-3 + CH-5); EIMS m/z (%): 269.36 [M+]
(29.78), 116.69 (100); Elemental analysis calculated for C13H11N5O2
(269.26) C, 57.99; H, 4.12; N, 26.01; Found C,57.98; H,4.11; N,26.01.
White powder, yield: 33.7%, m.p.: 269–271 ◦C; FT-IR (KBr (ʋmax
/
cmꢀ 1): 3328 (forked NH ), 3272 (NH), 1659 (C O amide), 3055 (CH
–
–
2
aromatic); 1H NMR (DMSO‑d6, 400 MHz) δ = 2.59–2.64 (t, J = 10.00 Hz,
4H,-morpholine), 3.17–3.22 (t, J = 10.00 Hz, 4H,-morpholine), 5.98
(s,2H,NH2), 13.5 (s, 1H, cyclic NH , D2O exchangeable); 13C NMR
– –
(DMSO‑d6):(100 MHz) δ = 42.52, 66.93, 138.94, 157.74, 158.69; EIMS
m/z (%): 197.23 [M+] (36.70), 124.88 (100); Elemental analysis
calculated for C7H11N5O2 (197.19) C, 42.64; H, 5.62; N, 35.51; Found C,
42.43; H, 5.46; N, 35.36.
4.1.7.2. N-(4-Fluorophenyl)-5-methyl-7-oxo-4,7-dihydro-[1,2,4]triazolo
[1,5-a]pyrimidine-2-carboxamide 14b. White powder, yield 86.8%, m.p.:
169–171 ◦C FT-IR (KBr (ʋmax/ cmꢀ 1): 3394–3354 (NH pyrimidine, NH
1
–
amidic), 1682 (C O amide), 3025 (CH aromatic); H NMR (DMSO‑d ,
–
6
4.1.6. General procedure for the synthesis of compounds 13a-c
A mixture of compound 4 (0.007 mol), appropriate aromatic amine
(0.05 mol) and ammonium chloride (0.005 mol) was heated under
reflux for 7 h. Then, the excess amine was distilled off under reduced
pressure; the residue was collected and re-crystallized from water to
offer compounds 13a-c.
– –
= 8.00 Hz, 2H, Ar-H), 7.86, 7.88 (d, J = 8.00 Hz, 2H, Ar-H), 10.32 (s, 1H,
400 MHz) δ = 2.38 (s, 3H, CH3 ), 5.96 (s, 1H, Ar-H), 7.19,7.21 (d, J
– –
– –
NH , D2O
NH
,
D2O exchangeable), 11.72 (s, 1H, cyclic
exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 19.26, 99.27,
120.78, 129.10, 129.14, 138.63, 138.82, 151.55, 152.97, 156.70,
157.87; DEPT C135 (CDCl3, 100 MHz, δ ppm); 19.28 (pyrimidine CH3-5),
99.28 (pyrimidine CH-6), 115.55 (phenyl CH-3 + CH-5), 122.64 (phenyl
CH-2 + CH-6); EIMS m/z (%): 287.39 [M+] (30.57), 96.68 (100);
Elemental analysis calculated for C13H10FN5O2 (287.25) C, 54.36; H,
3.51; N, 24.38; Found C,54.35; H,3.50; N,24.37.
4.1.6.1. 5-
Amino-N-phenyl-1H-1,2,4-triazole-3-carboxamide
13a.
Brown powder, yield: 42.5%, m.p.: 254–256 ◦C; FT-IR (KBr (ʋmax
/
cmꢀ 1): 3479 (forked NH ), 3447 (NH), 1693 (C O amide); 1H NMR
–
–
2
(DMSO‑d , 4–00 M–Hz) δ = 6.20 (s,2H,NH ), 7.09–7.79 (m, 5H–, Ar-H–),
6
9.90 (s, 1H, NH , D2O exchangeable),212.59 (s, 1H, cyclic NH
,
D2O exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 120.56,
128.80, 129.26, 138.94, 154.87, 157.74, 158.69; DEPT C135 (CDCl3,
100 MHz, δ ppm); 120.54 (phenyl CH-2 + CH-6), 128.08 (phenyl CH-4),
124.09 (phenyl CH-3 + CH-5); EIMS m/z (%): 203.00 [M+] (34.85),
173.46 (100); Elemental analysis calculated for C9H9N5O (203.20)
C,53.20;H,4.46;N,34.47; Found C,53.42;H,4.66;N,34.68.
4.2. Biological evaluation
The antimicrobial screening and cytotoxicity studies were performed
at CO-ADD (The Community for Antimicrobial Drug Discovery), funded
by the Wellcome Trust (UK) and The University of Queensland, Australia
(Supplementary M1).
4.2.1. Antibacterial assay
4.1.6.2. 5- Amino-N-(4-fluorophenyl)-1H-1,2,4-triazole-3-carboxamide
All bacteria were cultured in cation-adjusted Mueller Hinton broth
(CAMHB) at 37 ◦c overnight. A sample of each culture was then diluted
40-fold in fresh broth and incubated at 37 ◦c for 1.5–3 h. The resultant
mid-log phase cultures were diluted (CFU/mL measured by OD600), then
added to each well of the compound containing plates, giving a cell
density of 5x105 CFU/mL and a total volume of 50 µL. All the plates were
covered and incubated at 37 ◦C for 18 h without shaking [27].
13b. Reddish brown powder, yield: 74.4%, m.p.: 222–224 ◦C; FT-IR
(KBr (ʋmax/ cmꢀ 1): 3225 (forked NH ), 3353 (NH), 1692 (C
O
–
–
2
amide); 1H NMR (DMSO‑d6, 400 MHz) δ = 6.13 (s,2H,NH2), 7.16, 7.18
(d, J = 8.00 Hz, 2H, Ar-H), 7.81, 7.83 (d, J = 8.00 Hz, 2H, Ar-H), 10.03
– –
– –
(s, 1H, NH , D2O exchangeable), 12.56 (s, 1H, cyclic NH , D2O
exchangeable); 13C NMR (DMSO‑d6):(100 MHz) δ = 115.71, 122.46,
135.42, 154.99, 157.63, 158.76, 159.91; DEPT C135 (CDCl3, 100 MHz, δ
ppm); 115.71 (phenyl CH-3 + CH-5), 122.45 (phenyl CH-2 + CH-6);
EIMS m/z (%): 221.01 [M+] (52.06), 106.24 (100); Elemental analysis
4.2.2. Antifungal assay
Fungi strains were cultured for 3 days on Yeast Extract-Peptone
10

N.H. Amin et al.
Bioorganic Chemistry 111 (2021) 104841
Dextrose (YPD) agar at 30 ◦C. A yeast suspension of 1 × 106 to 5 × 106
CFU/mL (as determined by OD530) was prepared from five colonies.
The suspension was subsequently diluted and added to each well of the
compound-containing plates giving a final cell density of fungi suspen-
sion of 2.5 x103 CFU/mL and a total volume of 50 µL. All plates were
covered and incubated at 35 ◦C for 24 h without shaking [9,10].
refinement with rigid receptor and finally the best scoring pose of the
docked compound, receptor-ligand interactions of the complexes were
examined, Figs. 2 and 3.
4.3.2. Computational analysis: Prediction of physicochemical properties,
pharmacokinetics and drug-likeness profile
Computational studies for predicted pharmacokinetics/ pharmaco-
dynamics properties and toxicity of the most active compounds (9, 13a
and 13b) have been performed online to investigate their ADMET (ab-
sorption, distribution, metabolism, elimination, and toxicity) profiles
[35–42]. The present research tools included pre-ADMET (https:
//preadmet.bmdrc.kr) [35,36] Molinspiration (https://www.molinspir
ation.com) [37,38], Molsoft (https://molsoft.com) [39] and Swis-
sADME, (http://www.swissadme.ch) [40–42].
4.2.3. Cytotoxicity against human embryonic kidney cell line
HEK293 cells were counted manually in a Neubauer hemocytometer
and then plated in the 384-well plates containing the compounds to give
a density of 5000 cells/well in a final volume of 50 µL. DMEM supple-
mented with 10% FBS was used as growth media and the cells were
incubated together with compounds for 20 h at 37 ◦C in 5% CO2 [9,10].
4.2.4. Hemolysis of human red blood cells
Human whole blood was washed three times with 3 volumes of 0.9%
NaCl and then suspended in the same concentration of 0.5 × 108 cells/
mL, as determined by manual cell count in Neubauer hemocytometer.
The washed cells were then added to the 384-well compound-containing
plates for a final volume of 50 µL. After a 10 min shake on a plate shaker
the plates were then incubated for 1 h at 37 ◦C. After incubation, the
plates were centrifuged at 1000 g for 10 min to pellet cells and debris,
25 µL of the supermatant was then transferred to polystyrene 384-well
assay plate [9,10].
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
Authors are grateful to CO-ADD (The Community for Antimicrobial
Drug Discovery) Australia, funded by the Wellcome Trust (UK) and The
University of Queensland (Australia) for performing the antimicrobial
screening.
4.2.5. Inhibition of lanosterol 14
Compounds 9, 13a and 13b were screened for their inhibitiory ac-
tivity of lanosterol 14 -demethylase (CYP51) at Confirmatory Diag-
α-demethylase (CYP51) activity
α
nostic Unit, VACSERA, Cairo, Egypt (Supplementary M2). Since
resorufin and 7-ethoxyresorufin are light sensitive, the experiment was
carried out under yellow light in order to protect the integrity of the
stock solutions. Incubations were set up in a black 96-well plate and
consisted of substrate (7ER) and CYP1A2 Bactosomes in phosphate
buffer containing magnesium chloride. Reactions were initiated by
adding 40 µL of 5x NADPH generating system (this can be omitted from
wells containing blanks and standards). Metabolite (resorufin) forma-
tion was measured fluorometrically, using detection wavelengths cho-
sen to minimize interference from NADPH and 7ER. The substrate, 7-
ethoxyresorufin and metabolite (resorufin) were available from Cypex
[9].
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104841.
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