Synthesis of 1,2,4-triazolo[1,5-a]pyrimidine derivatives: Antimicrobial activity, DNA Gyrase inhibition and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2019.103411

A series of 1,2,4-triazolo[1,5-a]pyrimidine derivatives was designed, synthesized and screened for their antibacterial and antifungal activities as well as their safety profile. Compounds 2b, 3a, 6b, 8b, 8c, 8h, 9a,b, 10b, 11a,b and 12a,b showed high activity against Gram-positive and Gram-negative bacteria with MIC values ranging from 0.25 to 2.0 μg/mL. Many compounds were safe with no cytotoxicity against human embryonic kidney and red blood cells at concentration up to 32 μg/mL. Moreover, compound 9a showed the highest inhibitory activity against DNA Gyrase with IC₅₀ = 0.68 μM compared to ciprofloxacin IC₅₀ = 0.85 μM. Molecular docking at DNA Gyrase active site revealed binding mode and docking scores comparable to that of ciprofloxacin confirming their antibacterial activity via DNA Gyrase inhibition.

Full text of DOI:10.1016/j.bioorg.2019.103411

Journal Pre-proofs
Synthesis of 1,2,4-triazolo[1,5-a]pyrimidine derivatives: Antimicrobial activi-
ty, DNA Gyrase inhibition and molecular docking
Rehab H. Abd El-Aleam, Riham F. George, Ghaneya S. Hassan, Hamdy M.
Abdel-Rahman
PII:
S0045-2068(19)31342-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.103411
YBIOO 103411
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
15 August 2019
26 October 2019
28 October 2019
Please cite this article as: R.H. Abd El-Aleam, R.F. George, G.S. Hassan, H.M. Abdel-Rahman, Synthesis of
1
,2,4-triazolo[1,5-a]pyrimidine derivatives: Antimicrobial activity, DNA Gyrase inhibition and molecular
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Synthesis of 1,2,4-triazolo[1,5-a]pyrimidine derivatives: Antimicrobial
activity, DNA Gyrase inhibition and molecular docking
a
b*
b,c
Rehab H. Abd El-Aleam , Riham F. George , Ghaneya S. Hassan , and Hamdy M.
Abdel-Rahman
d,e
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Modern University for
Technology and Information MTI, Cairo 11571, Egypt.
b
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo,
1
1562, Egypt.
c
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Badr University, Badr
City, Cairo 11829, Egypt.
d
Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526,
Egypt.
e
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Nahda University, Beni
Suef, Egypt.
Declarations of interest: none
Abstract
A series of 1,2,4-triazolo[1,5-a]pyrimidine derivatives was designed, synthesized and
screened for their antibacterial and antifungal activities as well as their safety profile.
Compounds 2b, 3a, 6b, 8b, 8c, 8h, 9a,b, 10b, 11a,b and 12a,b showed high activity
against Gram-positive and Gram-negative bacteria with MIC values ranging from 0.25-
2
.0 휇g/mL. Many compounds were safe with no cytotoxicity against human embryonic
kidney and red blood cells at concentration up to 32 휇g/mL. Moreover, compound 9a
showed the highest inhibitory activity against DNA Gyrase with IC = 0.68 µM
5
0
compared to ciprofloxacin IC = 0.85 µM. Molecular docking at DNA Gyrase active site
5
0
revealed binding mode and docking scores comparable to that of ciprofloxacin
confirming their antibacterial activity via DNA Gyrase inhibition.
Keywords:
1
,2,4-Triazolo[1,5-a]pyrimidine; antibacterial; antifungal; DNA Gyrase inhibitors
*
Corresponding author: Riham F. George, e-mail: riham.eskandar@pharma.cu.edu.eg
1

1
. Introduction
According to the World Health Organization (WHO), antibacterial drug resistance has
been recognized as one of the biggest threats to global public health, especially infections
caused by Enterococcus faecium, Methicillin Resistant Staphylococcus aureus (MRSA),
Klebsiella pneumoniae, and Acinetobacter baumannii strains that are resistant to the
available antibiotics [1]. Therefore, there is a continuous need for development of novel
antibacterial agents that act on well known and clinically validated targets and are not
liable to the existing resistance mechanisms.
DNA Gyrase is a member of the bacterial type topoisomerase IIA family and it is a well
validated target for many antibacterials such as fluoroquinolones exemplified by nalidixic
acid (I) and ciprofloxacin (II) which bind to DNA-enzyme complex and stabilize it in its
cleaved state preventing DNA replication that leads finally to bacterial cell death [2, 3].
Although, they have a proven track record for the treatment of bacterial infections for
many decades, resistance to this class has recently emerged due to mutations that affect
molecular target recognition [4]. Therefore, compounds that are active against resistant
strains, with improved pharmacokinetic and safety profiles are goals for current and
future research in this area [5]. Recently, many pharmaceutical companies have
independently discovered novel (non-fluoroquinolone) bacterial topoisomerase IIA
inhibitors (NBTIs) such as Gepotidacin and zoliflodacin that have both successfully
completed phase II clinical trials for the treatment of Neisseria gonorrhoeae infections,
and zoliflodacin is now due to enter an international phase III trial [6,7]. Moreover,
Gepotidacin is being studied for treatment of uncomplicated urinary tract infections, like
acute cystitis, respiratory infections, and skin-structure infections [8].
Furthermore, 1,2,4-triazolo[1,5-a]pyrimidines represent an important core of many
antimicrobial agents. Essramycin (III) is the first 1,2,4-triazolo[1,5-a]pyrimidine
antibiotic, that has been isolated from culture broth of marine Streptomyces species and
exhibited potent activity against Gram-positive and Gram-negative bacteria with MIC
ranging from 2.0 to 8.0 µg/mL [9].
Moreover, the triazolopyrimidine-6-carboxylic acid (IV) revealed 92% growth inhibition
against M. tuberculosis H R at concentration of 6.25 μg/mL and showed the same
3
7
V
antibacterial activity against B. cereus as well as it was non toxic to mammalian cells
2

[10]. Additionally, 6-hydrazide derivatives Va,b showed potent antibacterial activity
against Gram positive and Gram negative bacterial strains with MIC values 2 μg/mL and
they revealed IC = 61 and 40 μg/mL, respectively in the DNA displacement assay [11].
5
0
Finally, the thiosemicarbazide derivative VI showed good activity against Gram-positive
bacteria strain with DNA displacement activity (IC = 22 µg/mL) [11].
5
0
Based on the preceding facts and coupled with the well-known antimicrobial activity of
the 1,2,4-triazolo[1,5-a]pyrimidines, this work focused on the synthesis of new molecules
via keeping 1,2,4-triazolo[1,5-a]pyrimidine nucleus of essramycin (III) and IV with
retaining some of the common pharmacophore of fluoroquinolones in one frame.
Consequently, a series of 2-unsubstituted/methyl-4,7-dihydro-7-oxo-1,2,4-triazolo[1,5-
a]pyrimidine-6-carboxylic acids 3a,b were synthesized as quinolone-3-carboxylic acid
structural analogs. Furthermore, the hydrazide analogs for compounds Va,b were
prepared (i.e. compounds 6a,b and 7a,b) in addition to their bioisostere, hydroxamic acid
derivatives 4a,b. Further derivatization of 7a,b were done via preparation of their Schiff
bases 8a-h and benzohydrazides 9a,b aiming to find interesting antimicrobial profiles.
Additionally, cyclization of the hydrazide moiety in compounds 7a,b into the oxadiazole
ring in compounds 10a,b was performed to elucidate the effect of the presence of steric
moiety at position 6 on the obtained activity. Finally, compounds 12a,b were prepared as
analogs for the reported thiosemicarbazide derivative VI hoping to obtain promising
antimicrobial agents (Figure 1). They were screened for their antimicrobial activity
against different bacterial and fungal strains in addition to evaluation of their safety and
hemolysis liability. The most active compounds were tested for their mode of action as
DNA Gyrase inhibitors. Moreover, molecular docking studies for some selected
representative compounds were performed to investigate their binding modes in the
active site.
3

O
N
O
O
N
O
O
O
F
OH
N
N
OH
N
N
H C
3
N
N
H
HN
C H
2
5
Essramycin (III)
Nalidixic acid (I)
Ciprofloxacin (II)
O
O
C
O
O
C
O
O
H
H
NH2
N
N
N
N
N
N
N
N
N
N
N
N
N
OH
R1
H
N
H
F3C
S
N
H
Va,b
N
H
N
H
VI
IV
O
O
Va R1=
Vb R1=
O
O
O
O
H
N
NH2
N
N
N
N
OH
N
N
N
R
R
H
S
N
N
H
a,b
N
H
O
O
NH2
N
3
N
N
1
2a,b
R
H
R= H, CH3
N
R= H, CH3
N
Bioisostere
R1
Cyclization
6
a,b & 7a,b
6
6
7
7
a R= H, R = CH C H
1 2 6 5
b
R= CH , R = CH C H
3 1 2 6 5
a R= R = H
NH
N
O
O
N
O
1
S
b R=CH , R = H
N
OH
3
1
N
O
N
N
N
N
R
R
H
N
N
H
Derivatization
Derivatization
H
a,b
R= H, CH3
1
0a,b
4
R= H, CH
3
R1
O
O
O
O
H
N
N
N
N
N
N
N
N
N
N
R
H
R
H
O
N
H
N
H
9
a,b
8
a-h
R= H, CH
3
8
a R= H , R = H
1
8
8
8
8
8
8
8
b R= CH , R = H
3
1
c R= H , R = 2-OH
1
d R= CH , R = 2-OH
3
1
e R= H , R = 4-OH
1
f R= CH , R = 4-OH
3
1
g R= H , R = 4-OCH3
1
h R= CH , R = 4-OCH
3
3
1
Figure 1. Structures of the lead and target compounds.
4

2
. Results and discussion
.1. Chemistry
2
The pathways adapted for synthesis of the target compounds were illustrated in Schemes
-3. The starting 3-amino-1,2,4-triazoles 1a,b were synthesized as reported by the
1
reaction of aminoguanidine bicarbonate with the appropriate carboxylic acid [12,13].
Cyclization of 1a,b with diethyl ethoxymethylenemalonate (DEEM) in glacial acetic acid
afforded the corresponding 1,2,4-triazole[1,5-a]pyrimidines 2a,b [10,14]. Alkaline
hydrolysis of ethyl esters 2a,b gave the corresponding carboxylic acids 3a,b. IR spectra
-1
of 3a,b showed the stretching bands at 3471-3452 and 1727-1706 cm due to NH and
-1
carboxylic C=O moiety, respectively. An additional broad band at 3000-2513 cm due to
1
carboxylic OH was also found. H NMR of 3a,b illustrated the disappearance of the
triplet-quartet signals of the ethyl protons. Furthermore, reaction of 2a,b with
hydroxylamine hydrochloride in methanol in the presence of potassium hydroxide
afforded the corresponding hydroxamic acid derivatives 4a,b. IR spectra of 4a,b showed
-1
bands at 3427-3400, 3220-3218 and 1650-1640 cm attributed to amidic NH, OH and
1
amidic carbonyl group, respectively. H NMR of 4a,b revealed an exchangeable signal at
2
.30 and 2.18 ppm assigned to OH proton.
Alkylation of esters 2a,b with benzyl bromide in dry DMF in the presence of sodium
1
hydride at room temperature gave the corresponding N-benzyl derivatives 5a,b. H NMR
spectra of 5a,b showed appearance of a multiple signal at 훿 = 7.31-7.46 ppm for aromatic
protons of phenyl ring in addition to a singlet signal at 5.31-5.47 ppm due to benzylic
CH protons. Reaction of the esters 5a,b with hydrazine hydrate gave the corresponding
2
1
hydrazides 6a,b. H NMR spectra of 6a,b revealed the disappearance of the triplet-quartet
signals corresponding to the ethyl protons and the appearance of singlet signals at 훿 =
7
.14, 4.59 and 6.77, 6.61 ppm assigned for NH and NH, respectively (Scheme 1).
2
5

O
O
b
N
N
N
OH
OH
O
O
R
N
H
a,b
N
N
N
O
N
N
NH
NH2
a
R
R
3
N
H
a,b
O
O
2
c
1
a,b
N
N
N
N
H
R
N
H
d
1
1
a, 2a, 3a, 4a, 5a, 6a: R= H
b, 2b, 3b, 4b, 5b, 6b: R= CH3
4a,b
O
O
O
O
e
NH2
N
N
N
N
N
O
R
H
R
N
N
N
N
6
a,b
5
a,b
Reagents and conditions:
o
(a) Diethyl ethoxymethylene malonate, glacial acetic acid, heat 115-120 C, 5 h then
refrigerate overnight; (b) NaOH solution, reflux, 3 h, then acidified by 4 M HCl;
(
c) hydroxylamine hydrochloride, KOH, methanol, stirring at room temperature;
d) benzyl bromide, NaH, DMF, stirring overnight, RT; (e) hydrazine hydrate, methanol,
(
reflux, 4 h.
Scheme 1. Synthesis of compounds 3a,b - 6a,b.
Similarly, hydrazinolysis of 2a,b under reflux in methanol resulted in the hydrazides 7a,b
as reported [15] (Scheme 2). Moreover, the reaction of acid hydrazides 7a,b with the
appropriate aromatic aldehyde in absolute ethanol in the presence of glacial acetic acid
yielded the corresponding hydrazones 8a-h. It was noteworthy that some of the
hydrazones were obtained as mixtures of their E and Z geometrical isomers. That,
compounds 8a,b,d and 8f were isolated as one geometrical isomer as evidenced from
1
their H NMR spectral data that revealed one singlet assigned to N=CH proton of the
1
isolated isomer at 8.44-8.73 ppm. However, the H NMR spectra of compounds 8c,e,g
and 8h revealed signals assigned for the protons of both isomers where duplication of
some signals was observed for example the signal for N=CH proton appeared as two
singlets at 8.24-8.37 and 8.41-8.66 ppm attributed to Z and E isomer, respectively (see the
6

experimental part). On the other hand, the benzoylation of the acid hydrazides 7a,b with
benzoyl chloride in dry dioxane in the presence of pyridine resulted in benzoyl
1
derivatives 9a,b. H NMR spectra of 9a,b lacked the singlet at 훿 = 5.75 and 7.02 ppm,
respectively of NH proton and displayed a singlet signal at 훿 = 10.46, 10.58 attributed to
2
NH proton in addition to a multiple signals at 훿 = 7.49-7.62 ppm corresponding to the
aromatic protons of phenyl ring (Scheme 2).
O
O
O
O
O
O
H
N
N
N
O
N
NH2
N
N
N
N
c
R
N
N
N
N
H
R
H
R
a
N
H
O
N
H
N
H
2
a,b
9
a,b
7
a,b
2
2
a, 7a, 9a: R= H
b, 7b, 9b: R= CH3
b
8a: R= H , R = H
1
8
8
b: R= CH , R = H
3 1
c: R= H , R = 2-OH
1
O
O
R1
8d: R= CH , R = 2-OH
3 1
N
N
N
8
8
e: R= H , R = 4-OH
1
f: R= CH , R = 4-OH
N
N
H
R
3
1
N
H
8g: R= H , R = 4-OCH
1
3
8
a-h
8
h: R= CH , R = 4-OCH
3 1 3
Reagents and conditions:
a) Hydrazine hydrate, methanol, reflux, 4 h; (b) aromatic aldehyde, ethanol, glacial
acetic acid, reflux, 4 h; (c) benzoyl chloride, pyridine, dioxane, stirring, RT, overnight.
(
Scheme 2. Synthesis of compounds 8a-h and 9a,b.
Further, the reaction of the acid hydrazides 7a,b with carbon disulfide in ethanolic
potassium hydroxide gave the corresponding 1,2,4-oxadiazole derivatives 10a,b
1
(
Scheme 3). H NMR of 10a,b lacked the singlet at 훿 = 5.75, 7.02 ppm attributed to NH2
protons and displayed a D O exchangeable singlet signal at 훿 = 10.66 ppm corresponding
2
to NH proton of the oxadiazole ring. Alkylation of 10a,b with benzyl bromide in dry
1
DMF in the presence of sodium carbonate yielded compounds 11a,b. H NMR spectra of
1
1a,b revealed a multiple signal at 훿 = 7.23 - 7.49 ppm for aromatic protons of the phenyl
ring and a singlet at 4.50, 4.58 ppm due to CH protons. Finally, the reaction of acid
2
hydrazides 7a,b with ammonium thiocyanate in ethanol under reflux gave the
corresponding 1,4-disubstituted thiosemicarbazides 12a,b as reported [15] (Scheme 3).
7

NH
N
N
O
N
O
S
S
N
N
N
N
N
O
b
N
O
R
R
N
H
O
a
N
H
O
NH2
N
N
N
N
11a,b
10a,b
R
H
N
H
c
O
O
7a,b
H
N
NH2
N
N
N
R
H
S
7
7
a, 10a, 11a: R= H
b, 10b, 11b: R= CH3
N
N
H
12a,: R= H ,
R1= H
12a,b
1
2b: R= CH , R = H
3
1
Reagents and condition:
(
a) CS , KOH, ethanol, reflux, 12h; (b) benzyl bromide, Na CO , DMF, stirring, RT, overnight;
2
2
3
(
c) NH SCN, ethanol, reflux, 3 h.
4
Scheme 3. Synthesis of compounds 10a,b, 11a,b and 12a,b.
2
.2.
.2.1. Antibacterial and antifungal activities
The antibacterial and antifungal screenings were performed at CO-ADD (The
Biological evaluation
2
Community for Antimicrobial Drug Discovery), funded by the Wellcome Trust (UK) and
The University of Queensland (Australia).
Twenty-six compounds were tested by CO-ADD at 32 휇g/mL for their antibacterial
activity against five bacterial strains: E. coli, K. pneumoniae, A. baumannii, P.
aeruginosa (Gram negative) and Methicillin Resistant S. aureus (MRSA) (Gram positive)
using ciprofloxacin as a positive control. The antifungal activity against two fungal
species: C. albicans and C. neoformans was performed using fluconazole as a positive
control. Furthermore, compounds displaying significant growth inhibition % (80-100%)
at 32 휇g/mL against any of the tested bacterial or fungal species were screened for their
minimum inhibitory concentrations (MIC) and the results were summarized in (Table 1).
From the obtained results, it was observed that thirteen compounds 2b, 3a, 6b, 8b, 8c,
8
h, 9a,b, 10b, 11a,b, and 12a,b revealed high activity against both Gram-positive and
Gram-negative bacteria expressed by their MIC ranging from 0.5-2.0 휇g/mL and 0.25-2.0
휇g/mL, respectively. The following structure activity relationships can be concluded; the
methyl derivative 2b revealed higher activity against Gram positive and Gram negative
8

bacterial strains than its unsubstituted analog 2a. Whereas, the unsubstituted carboxylic
acid derivative 3a was more active than its methyl congener 3b. Furthermore, the
hydroxamic acid derivatives 4a,b and N-benzyl esters 5a,b revealed mild activity against
all tested bacterial strains. However, the 2-methyl-N-benzyl-6-hydrazide 6b exhibited
remarkable activity compared to its unsubstituted congeners 6a and 7a,b. Moreover,
Schiff`s bases 8b,c,h showed higher activity than 8a,e,g. on the other hand, both
benzohydrazide derivatives 9a,b exerted high activity against the tested bacterial strains
with MIC values 0.25-1 μg/mL. Additionally, the oxadiazole containing compounds 10b
reported higher activity than its unsubstituted analog 10a, while, 5-benzylthio-oxadiazole
derivatives 11a,b showed higher activity than 10a. Finally, the semicarbazide derivatives
1
2a,b elicited high activity against both Gram positive and Gram negative bacteria with
MIC values 0.25-1 μg/mL. Therefore, it can be concluded that the variation of the
substitution at position 6 namely: ester, acid, hydrazide, arylidene hydrazide,
benzohydrazide, oxadiazole and substituted semicarbazide keeps the antibacterial activity
of the newly synthesized compounds. On the other hand, all the tested compounds
revealed mild antifungal activity except compound 12a that showed moderate antifungal
activity
against
C.
neoformans
with
MIC
25.62
휇g/mL
9

Table 1. The antibacterial and antifungal activities (growth inhibition %, MIC µg/mL) of the tested compounds.
Gram positive
bacteria
MRSA
GI%h MIC
Gram negative bacteria
Fungi
a
Ecb
Kpc
Abd
Pae
GI%h MIC GI%h MIC GI%h MIC
Caf
Cng
Code
GI%h
7.92
MIC
>32
GI%h MIC GI%h
MIC
>32
0.5
0.5
>32
2
a
-8.18
95.78
98.28
15.34
-0.17
11.89
3.55
11.86
-2.07
97.68
2.43
>32
0.5
1
-3.7
>32 -13.35
8.75
97.08
100.11
35.52
8.8
27.58
10.58
32.04
16.1
100.1
28.66
29.43
11.97
84.97
92.58
19.57
8.87
>32
1
2
84.94
3.85
4.89
7.02
3.85
-2.11
-2.6
3.57
4.95
7.3
6.33
8.65
3.64
6.33
2.25
6.28
9.36
3.02
1.85
32
32.55
>32
2
b
98.62
100.17
99.08
2.7
≤ 0.25 97.56
≤ 0.25 99.81
0.5
1
>32
98.77
99.81
8.62
>32 -161.88 >32
>32 -17.66
>32 -83.91
3
a
>32
>32
3
b
>32
32
NT
NT
NT
NT
NT
27.54
14.72
24.53
-10.46
10.85
9.55
>32
i
i
i
i
i
i
i
i
i
i
i
4
a
NT
NT
NT
NT
NT
NT
NT
NT
13.72
10.06
17.56
-5.05
14.82
99.68
35.96
45.92
12.06
94.26
97.21
27.43
15.12
96.12
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
-11.38
-91.08
7.01
NT
NT
NT
i
i
i
i
i
i
i
i
4
b
-0.32
0.27
3.3
i
i
i
i
i
5
a
i
i
i
i
i
5
b
NT -162.14 NT
i
i
i
i
i
NTi
>32
>32
>32
6
a
NT
1.99
NT
NT
NT
NT
-5.44
6
b
1
100.4
74.22
81.13
1.47
≤ 0.25 100.23
>32 20.2
≤ 0.25 10.88
8.66
≤ 0.25 83.84
1
0.5
>32
>32
2
>32 -20.82
>32 -53.46
>32 -48.91
7
a
>32
>32
>32
>32
>32
>32
7
b
2.94
0.09
i
i
i
i
i
i
i
8
a
NT
NT
NT
NT
NT
NT
-18.31
-89.1
-24.6
-16.19
-30.91
NT
8
b
89.77
96.68
7.71
-2.72
90.66
99.9
0.5
2
80.15
83.25
30.76
20.89
95.52
100.54
0.5
2
0.5
1
1
4
NT
>32
>32
>32
>32
NT
8
8
c
e
≤ 0.25 91.66
i
i
i
i
i
i
NT
NT
NT
NT
1.99
1.07
≤ 0.25 95.29
≤ 0.25 100.94
NT
NT
NT
NT
NT
NT
i
i
i
i
i
i
i
8
g
NT
NT
8
h
2
0.5
2
0.5
1
≤
94.49
101.09
2
1
>32 -104.45 >32
i
i
9
a
100.15
NT
-47.02
NT
0
.25
9
b
92.34
11.86
90.68
0.5
NT
1
96.16
3.3
95.15
≤ 0.25 93.47
0.5
NT
1
97.05
-5.05
95.77
0.5
NT
0.5
93.59
32.04
94.47
1
NT
2
6.4
3.57
1.35
>32 -155.2
NT -162.14 NT
>32 -203.78 >32
>32
i
i
i
i
i
i
i
1
0a
NT
10.85
1
0b
≤ 0.25 94.79
1
0

1
1a
98.2
91.26
2
0.5
99.75
94.77
≤ 0.25 99.85
≤ 0.25 94.11
2
0.5
99.65
96.3
1
≤
98.01
93.7
4
1
5.66
-0.66
>32 -72.55
NT -152.97 NT
>32
i
i
1
1b
0
.25
1
2a
99.36
90.85
0.5
0.5
99.79
95.26
Cipro.
≤ 0.25 99.94
≤ 0.25 94.79
0.5
0.5
99.95
94.91
Cipro. 1
0.5
0.5
100.37
94.62
Cipro.
1
1
43.87
3.92
>32 -88.61 25.62
>32 -156.43 >32
1
2b
Ref. Cipro. 0.25
0.007
Cipro. 0.5
0.25 Flucon. 0.13 Flucon.
8
a: Methicillin Resistant S. aureus, : E. coli, : K. pneumonia, : A. baumannii, : P. aeruginosa, : C. albicans, : C. neoforma, :
b
c
d
e
f
g
h
growth
inhibition
%,
i:
not
tested
1
1

2
.2.2. Cytotoxicity against human embryonic kidney cell line and hemolysis of human
red blood cells effects
These tests were carried out by CO-ADD (The Community for Antimicrobial Drug
Discovery), funded by the Wellcome Trust (UK) and The University of Queensland
(Australia). The compounds which exhibited significant growth inhibition% against any
of the tested bacterial or fungal strains (17 compounds) were tested for their cytotoxicity
against a human embryonic kidney cell line and for hemolysis of human red blood cells
using tamoxifen and melittin as positive control, respectively and the results were
illustrated in (Table 2).
Table 2. Cytotoxicity and hemolysis ability of the tested compounds.
a
b
Code
CC (µg/mL)
HC (µg/mL)
5
0
10
2
a
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
9
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
25.62
>32
NTc
2.7
2
b
3
a
3
6
b
b
7
a
7
8
b
b
8
c
8
h
9
a
9
b
1
0b
1a
1b
2a
2b
1
1
1
1
Tamoxifen
Melittin
NTc
a:CC50 is the concentration at 50% cytotoxicity (Cytotoxicity against a human embryonic kidney cell line).
b: HC10 is the concentration at 10% hemolysis (hemolysis of human red blood cells); : NT, Not Tested.
C
1
2

From the results illustrated in (Table 2), the tested compounds showed CC > 32 휇g/mL
5
0
against the human embryonic kidney cell line, so, they are safe and non-cytotoxic
compared to tamoxifen (CC = 9 휇g/mL) . Where, samples were classified cytotoxic
5
0
when their CC ≤ 32 휇g/mL (concentration at 50% cytotoxicity) or CC ≤ 10 휇M in
5
0
50
either replicate (n=2) on different plates. In addition, samples were flagged as partial
cytotoxic if DMax ≥ 50% even with CC > the maximum tested concentration (D is the
50
Max
maximal percentage of cytotoxicity).
Furthermore, the tested compounds exhibited HC10 more than 32 휇g/mL (HC10 is
concentration at 10% hemolysis), they are considered as safe and non-toxic compounds
except compound 12a has HC = 25.62 휇g/mL. As, hemolysis samples were classified by
1
0
HC ≤ 32 휇g/mL or HC ≤ 10 휇M in either replicate. In addition samples were flagged
1
0
10
as partial hemolytic if DMax ≥ 50% even with HC > the maximum tested concentration
10
(DMax is the maximal percentage of hemolysis).
2
.2.3. DNA Gyrase inhibition activity
In order to validate that the mode of action of the tested compounds is DNA gyrase
inhibition, five compounds 2b, 8b, 9a, 9b and 11b with the best MIC values (MIC ≤ 0.25
-
1 휇g/mL) along with compounds 2a and 7b with mild activity (MIC > 32 휇g/mL against
the tested bacteria strains) were selected for screening as DNA Gyrase inhibitors. This
test was performed at Confirmatory Diagnostic Unit VACSERA – Egypt, Ciprofloxacin
was used as a standard reference and the results as IC values (µM) were listed in (Table
5
0
3
).
1
3

Table 3. IC value (µM) against E. Coli DNA Gyrase
5
0
Code.
DNA Gyrase IC (µM)
50
2
a
14.94
1.018
12.95
2.08
2
7
8
b
b
b
9
a
0.68
9
b
5.60
1
1b
1.07
Ciprofloxacin
0.85
The obtained results were consistent with the MIC values of the tested compounds
against E. coli except for compounds 7b and 9b that exhibited moderate DNA Gyrase
inhibition (12.95 and 5.60 μM, respectively) while their MIC value was less than 0.25
μg/mL. Thus, they may exert their antibacterial action through another mode of action.
On the other hand, N-benzoylhydrazide derivative 9a showed better activity (IC = 0.68
5
0
µM) than the standard drug, ciprofloxacin (IC = 0.85 µM), while ethyl carboxylate 2b
5
0
and benzylthio-oxadiazole derivative 11b were equipotent in DNA Gyrase inhibition
IC = 1.018 and 1.07 µM, respectively) in addition to 2-methyltriazolopyrimidine-N-
(
5
0
benzylidene 8b with moderate DNA Gyrase inhibition (IC = 2.08 µM) that were
5
0
correlated to their MIC value (less than 0.25 μg/mL). Furthermore, ethyl carboxylate of
2
-unsubstituted derivative, compound 2a showed the lowest activity (IC = 14.94 µM,
50
MIC value > 32 μg/mL).
-
The structure activity relationship showed that methyl substituted derivative 2b was
more active than the unsubstituted analog 2a. Furthermore, Schiff’s base 8b exhibited
promising activity higher than the hydrazide derivative 7b. On the other hand, the
unsubstituted benzohydrazide derivative 9a revealed better inhibitory activity than its
methyl substituted derivative 9b.
-
It is noteworthy that the mechanism of DNA Gyrase inhibition of these compounds
should be further investigated to validate either they act by direct DNA Gyrase inhibition
as Ciprofloxacin or indirectly via ATPase inhibition as aminocoumarin.
1
4

-
Moreover, due to structural similarity between these compounds and previously
reported human topoisomerase IIα inhibitors [16,17], these compounds may bind to
human topoisomerase IIα resulting in targeted anticancer effects. Therefore, these
compounds need further investigation as they may have clinical significance as human
topoisomerase IIα inhibitors.
2
.3.
Seven compounds which were tested as DNA Gyrase inhibitors (compounds 2a, 2b, 7b,
b, 9a, 9b and 11b) and ciprofloxacin (II) were docked at binding site of DNA Gyrase
PDB ID: 5BTC) [18] to shed light on their potential binding modes and investigate their
Molecular docking study
8
(
similarity to the native ligand. The results and the bonding interactions of the docked
compounds and the native ligand, ciprofloxacin with amino acids of the active site were
illustrated in Table 4, figure 2 and (supplementary materials, Figures 1-11).
Table 4. Results of the molecular docking study of ciprofloxacin and the tested
compounds at DNA Gyrase
Interacting
moiety
the drug
DNA
Compound
Energy
score
Type
interaction
of Bond
length
in Amino acid Gyrase
involved
IC (μM)
50
(S)
(Chain type)
Kcal/mol
H-acceptor
Metal
Metal
π- π
π- π
π- π
3.42
2.18
2.23
3.97
3.95
3.70
3.74
3.45
2.27
3.77
3.96
3.18
O
O
O
6-ring
6-ring
6-ring
6ring
N
Arg 128 (C)
Ciprofloxacin
-13.65
MG 601 (A) 0.85
MG 601 (A)
DG 11 (D)
DG 11 (D)
DA 11 (E)
π- π
DA 11 (E)
H-acceptor
Metal
π- π
π- π
H-donor
Arg 128 (C)
MG 601 (A) 14.94
DG 11 (D)
DA 11 (E)
DA 11 (E)
2
a
-10.67
O
6-ring
6-ring
N
1
5

H-acceptor
Metal
π- π
π- π
H-donor
3.15
2.03
3.78
3.44
3.53
N
O
6-ring
6-ring
N
Arg 128 (C)
MG 601 (A) 1.018
DG 11 (D)
DA 11 (E)
DA 11 (E)
2
b
-11.40
H-acceptor
Metal
π- π
3.24
1.99
3.88
3.56
O
O
6-ring
6-ring
Arg 128 (C)
MG 601 (A) 12.95
DG 11 (D)
7
8
9
9
b
b
a
b
-7.53
π- π
DA 11 (E)
H-acceptor
Metal
π- π
3.80
2.29
3.73
3.42
3.09
3.52
2.82
2.38
3.56
3.84
3.34
2.21
3.80
3.37
3.85
3.08
4.48
3.53
2.62
3.78
3.48
3.84
N
O
6-ring
6-ring
N
N
N
O
6-ring
6-ring
N
O
6-ring
N
6-ring
N
6-ring
N
Arg 128 (C)
MG 601 (A) 2.08
DG 11 (D)
DA 11 (E)
DA 11 (E)
Arg 128 (C)
MG 601 (A) 0.68
MG 601 (A)
DG 11 (D)
-12.96
-13.91
-12.92
π- π
H-donor
H-acceptor
Metal
Metal
π- π
π- π
DA 11 (E)
H-acceptor
Metal
π- π
H-donor
π- π
H-donor
π -H
H-acceptor
Metal
π- π
Arg 128 (C)
MG 601 (A) 5.60
DG 11 (D)
DG 11 (D)
DA 11 (E)
DA 11 (E)
Gly 483 (B)
Arg 128 (C)
MG 601 (A) 1.07
DG 11 (D)
DA 11 (E)
DA 11 (E)
1
1b
-13.33
O
6-ring
Oxadiazole
6-ring
π- π
π- π
1
6

(A)
(B)
(C)
(D)
(E)
(F)
Figure 2. (A) 3D interaction diagram of ciprofloxacin with DNA Gyrase (PDB: 5BTC);
(
B) 2D interaction diagram of ciprofloxacin with DNA Gyrase; (C) 3D interaction
diagram of 9a with DNA Gyrase; (D) 2D interaction diagram of 9a with DNA Gyrase;(E)
D interaction diagram of 11b with DNA Gyrase; (F) 2D interaction diagram of 11b with
DNA Gyrase.
3
1
7

To ensure the validity of the docking protocol, re-docking of the co-crystallized
ciprofloxacin into the active site was performed. The coordinates of the best scoring
docking pose of ciprofloxacin was compared with its coordinates in the co-crystallized
PDB file based on the binding mood and root mean square deviation (rmsd). The docking
validation results were presented in (Figure 1, supplementary materials) which showed a
perfect alignment with the original ligand obtained from the X-ray resolved PDB file with
rmsd = 0.875 and energy score (S) = -13.65 kcal/mol. Additionally, it was able to
reproduce the key interactions accomplished by the co-crystallized ligand with the hot
spots in the active site Arg 128(C), MG601, DG 11 (D) and DA 11 (E) (Figure 2).
Generally, all the tested compounds exhibited the same pattern of binding as
ciprofloxacin through interaction with Arg 128 (hydrogen bond), MG (metal bond), DG
1
1 and DA 11 (π-π interaction). In addition, compounds 2a, 2b, 8b and 9a interact with
DA 11 by hydrogen bonds, as H-donors. It is clear that, compound 9a which is the most
potent compound as DNA Gyrase inhibitor (IC = 0.68 μM) binds with MG 601 through
5
0
two metal bonds one with N-3 and the second with O of triazolopyrmidinone moiety in a
chelate structure. The same pattern was exhibited by the reference drug, ciprofloxacin
(
IC = 0.85 μM). Additionally, the energy score of compound 9a was comparable to that
50
of ciprofloxacin (-13.91 and -13.65 kcal/mol, respectively).
3
. Conclusion
Twenty-six compounds were screened for their antimicrobial activity against five
bacterial strains and two fungal species. Thirteen compounds (2b, 3a, 6b, 8b,c,h, 9a,b,
1
0b, 11a,b and 12a,b) revealed promising antibacterial activity with MIC 0.25-2 휇g/mL.
They were safe with no cytotoxicity against human embryonic kidney cell line at
concentration up to 32 휇g/mL. Additionally, these compounds don’t cause 10% red blood
cell hemolysis at concentration up to 32 휇g/mL. Compound 9a exhibited potent DNA
Gyrase inhibition higher than ciprofloxacin. Therefore, it can be considered as a hit for
further optimization to obtain more active antibacterial agents.
1
8

4
. Experimental
.1. Chemistry
4
Chemical reagents and solvents were 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 apparatus and were uncorrected. IR spectra were recorded as
1
KBr disks on a Bruker spectrophotometer (Nahda University, Beni-Suef, Egypt). H
NMR spectra were carried out on Bruker apparatus 400 MHz, (Beni-Suef University,
Beni-Suef, Egypt) using TMS as internal reference, Chemical shifts (values are given in
parts per million (ppm) using DMSO-d or CDCl as solvents. Some spectra were
6
3
performed at Main Defense Chemical Laboratory on Oxford 300 MHz, Varian Gemini
1
3
2
00, 5MM Burker and Oxford 400 MHz, Mercury plus, Varian 5 MM. C NMR were
recorded using Bruker 100 MHz NMR (Beni-Suef University, Beni-Suef, Egypt).
Elemental analyses (C, H and N) were performed on VARIO-Elementer apparatus at The
Regional Center for Mycology and Biotechonology, Al-Azhar University, Egypt.
Compounds 1a,b [12,13], 2a,b [10,14], 7a,b [15] and 12a,b [15] were prepared
according to their reported procedures.
4
.1.1. General procedure for the synthesis of 3a,b
The appropriate ethyl ester 2a,b (0.01 mol) was dissolved in a solution of sodium
hydroxide (0.8 g, 0.02 mol) in water (15 mL). The mixture was heated under reflux for 3
h. After cooling, the reaction mixture was filtered and acidified by drop wise addition of
4
M hydrochloric acid. The precipitated carboxylic acid was collected by filtration,
washed with water, air dried, and recrystallized from methanol/water.
4
.1.1.1.
7-Oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carboxylic acid (3a)
-1
Pale yellow powder, yield: 60 %, m.p. 275-276°C; FT-IR (KBr) (ʋmax/ cm ): 3452
(NH), 3003 (CH aromatic), 3000-2653 (broad band carboxylic OH), 1706 (C=O ), 1660
1
(
C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 4.95 (br s, 1H, NH, D O exchangeable),
6
2
1
3
8
1
.40 (s, 1H, C -H), 8.67 (s, 1H, C -H); C NMR (DMSO-d , 100 MHz, δ, ppm): 102.9,
5 2 6
49.3, 151.2, 151.9 (aromatic carbons), 155.6 (C=O), 164.8 (C=O); MS, m/z [%]: 180
1
9

+
(
M , 22), (44 (100); Elemental analysis calculated for C H N O (180.12): C, 40.01; H,
6 4 4 3
2
.24; N, 31.11; Found C, 40.16; H, 2.22; N, 31.39.
4
.1.1.2.
2-Methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carboxylic
acid (3b)
-1
White powder, yield: 65 %, m.p. 247-249°C; FT-IR (KBr) (ʋmax/ cm ): 3471 (NH),
3
090 (CH aromatic), 3000-2513 (broad band OH acidic), 2985 (CH aliphatic), 1727
1
(
C=O acid), 1663 (C=O); H NMR (DMSO-d , 400 MHz, δ, ppm): 2.42 (s, 3H, CH ),
6
3
1
3
8
1
9
2
.66 (s, 1H, C -H); C NMR (DMSO-d , 100 MHz, δ, ppm): 13.9 (CH ), 103.1, 151.0,
5 6 3
+
51.7, 156.1 (aromatic carbons), 159.5 (C=O), 164.9 (C=O); MS, m/z [%]: 194 (M , 12),
8 (100); Elemental analysis calculated for C H N O (194.15): C, 43.30; H, 3.11; N,
7
6
4
3
8.86; Found C, 43.54; H, 3.14; N, 29.04.
4
.1.2. General procedure for the synthesis of 4a,b
To a freshly prepared solution of hydroxylamine [prepared from a solution of
hydroxylamine hydrochloride (7.0 g, 0.1 mol) in methanol (30 mL) by addition while
cooling to a solution of potassium hydroxide (7.0 g, 0.125 mol) in methanol (30 mL)], a
solution of compound 2a,b (0.005 mol) in methanol (20 mL) was added portion-wise
with stirring at room temperature. After complete addition the mixture was left at room
temperature overnight. The formed precipitate was filtered off and the potassium salt of
hydroxamic acid was dissolved in mixture of water (15 mL), dilute acetic acid (5 mL).
The mixture was allowed to stand for three hours at room temperature; the separated solid
was filtered, dried and recrystallized from methanol.
4
.1.2.1.
N-Hydroxy-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-hydroxamic
acid (4a)
-1
White powder, yield: 70 %, m.p. 280-282°C; FT-IR (KBr) (ʋmax/ cm ): 3430 (NH),
1
3
400 (NH), 3218 (OH), 3053 (CH aromatic), 1657 (C=O), 1640 (C=O); H NMR
(
DMSO-d , 400 MHz, δ, ppm): 2.30 (s, H, OH), 8.56 (s, 1H, C -H), 8.60 (s, 1H, C -H);
6
5
2
Elemental analysis calculated for C H N O (195.14): C, 36.93; H, 2.58; N, 35.89; Found
6
5
5
3
C, 37.20; H, 4.13; N, 29.72.
2
0

4
.1.2.2.
N-Hydroxy-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-
hydroxamic acid (4b)
-1
Pale orange powder, yield: 80 %, m.p. 297-299°C; FT-IR (KBr) (ʋmax/ cm ):
3
460 (NH), 3427 (NH), 3220 (OH), 3045 (CH aromatic), 2964 (CH aliphatic), 1659
1
(
C=O), 1650 (C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 2.18 (s, H, OH), 2.31 (s,
6
1
3
3
1
2
3
H, CH ), 8.54 (s, 1H, C -H); C NMR (DMSO-d , 100 MHz, δ, ppm): 15.2 (CH ),
3 5 6 3
22.0, 149.5, 152.0, 156.2 (aromatic carbons), 160.9 (C=O), 163.6 (C=O); MS, m/z [%]:
+
09 (M , 4), 57 (100); Elemental analysis calculated for C H N O (209.17): C, 40.20; H,
7
7
5
3
.37; N, 33.48; Found C, 40.35; H, 3.35; N, 33.72.
4
.1.3. General procedure for the synthesis of 5a,b
A solution of compound 2a,b (0.01mol) in dry DMF (25 mL) was treated with
sodium hydride (0.4 g, 0.0167 mol) and stirred at room temperature for 10 minutes.
Benzyl bromide (5.13 g, 3.60 mL, 0.03 mol) was added and stirring was continued
overnight. The reaction mixture was poured onto ice-water (100 mL), the resulting
precipitate was filtered, washed with water and air dried. The product was recrystallized
from methanol/water.
4
.1.3.1.
4-Benzyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carboxylic
acid ethyl ester (5a)
-1
Pale yellow powder, yield: 40 %, m.p. 121-123°C; FT-IR (KBr) (ʋmax/ cm ): 3076
1
(
CH aromatic), 2976 (CH aliphatic), 1717 (C=O), 1695 (C=O); H NMR (DMSO-d , 400
6
MHz, δ, ppm): 1.29 (t, 3H, J = 7.00 Hz, -CH CH ), 4.26 (q, 2H, J = 7.06 Hz, -CH CH ),
2
3
2
3
1
3
5
.31 (s, 2H, CH ), 7.31-7.45 (m, 5H, Ar-H), 8.30 (s, 1H, C -H), 9.01 (s, 1H, C -H); C
2 5 2
NMR (DMSO-d , 100 MHz, δ, ppm): 14.6 (CH CH ), 55.1 (CH ), 61.1 (CH CH ), 103.9,
6
3
2
2
3
2
1
28.4, 128.7, 129.2, 135.2, 149.5, 151.0, 152.6 (aromatic carbons), 152.7 (C=O), 163.2
+
(
C=O); MS, m/z [%]: 298 (M , 8), 91 (100); Elemental analysis calculated for
C H N O (298.30): C, 60.40; H, 4.73; N, 18.78; Found C, 60.31; H, 4.86; N, 18.97.
1
5
14
4
3
4
.1.3.2.
4-Benzyl-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-
carboxylic acid ethyl ester (5b)
2
1

-1
Pale orange powder, yield: 45 %, m.p. 125-127°C; FT-IR (KBr) (ʋmax/ cm ):
1
3
070 (CH aromatic), 2984 (CH aliphatic), 1733 (C=O), 1686 (C=O); H NMR (DMSO-
d , 300 MHz, δ, ppm): 1.30 (t, 3H, J = 7.20 Hz, -CH CH ), 2.37 (s, 3H, CH ), 4.24 (q,
6
2
3
3
2
H, J = 7.20 Hz, -CH CH ), 5.47 (s, 2H, CH ), 7.31-7.46 (m, 5H, Ar-H), 8.89 (s, 1H, C -
2 3 2 5
H); Elemental analysis calculated for C H N O ( 312.32): C, 61.53; H, 5.16; N, 17.94;
1
6
16
4
3
Found C, 61.70; H, 5.24; N, 18.21.
4
.1.4. General procedure for the synthesis of 6a,b
A mixture of the ester 5a,b (0.1 mol) and 100% hydrazine hydrate (0.43 mL) in
methanol (10 mL) was heated under reflux for 4 h. The excess of hydrazine hydrate was
removed under vacuum and the residue was filtered off, dried and recrystallized from
ethanol.
4
.1.4.1. 4-Benzyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carbohydrazide
(6a)
-1
White powder, yield: 45 %, m.p. 201-203°C; FT-IR (KBr) (ʋmax/ cm ): 3469, 3458
1
(
forked NH ), 3292 (NH), 3061 (CH aromatic), 1730 (C=O), 1667 (C=O); H NMR
2
(
DMSO-d , 400 MHz, δ, ppm): 4.47 (s, 2H, CH ), 6.77 (s, 1H, NH, exchangeable with
6
2
D O), 7.14-7.30 (m, 7H, Ar-H + NH ), 7.53 (s, 1H, C -H), 8.09 (s, 1H, C -H); Elemental
2
2
5
2
analysis calculated for C H N O (284.27): C, 54.93; H, 4.25; N, 29.56; Found C,
1
3
12
6
2
5
5.19; H, 4.41; N, 29.45.
4
.1.4.2.
4-Benzyl-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6
carbohydrazide (6b)
-1
Off white powder, yield: 47 %, m.p. 196-197°C; FT-IR (KBr) (ʋmax/ cm ): 3475,
3
1
461 (forked NH ), 3298 (NH), 3031 (CH aromatic), 2890 (CH aliphatic), 1730 (C=O),
2
1
661 (C=O); H NMR (DMSO-d , 400 MHz, δ, ppm): 2.08 (s, 3H, CH ), 4.28 (s, 2H,
6
3
CH ), 4.59 (br s, 2H, NH , D O exchangeable), 6.61 (s, 1H, NH), 7.20-7.33 (m, 6H, Ar-H
2
2
2
1
3
+
C -H); C NMR (DMSO-d , 100 MHz, δ, ppm): 14.1 (CH ), 51.6 (CH ), 104.0, 127.0,
5 6 3 2
1
28.5, 128.9, 136.4, 141.1, 153.2, 158.7 (aromatic carbons), 163.0 (C=O), 168.6 (C=O);
2
2

+
MS, m/z [%]: 298 (M , 6), 77 (100); Elemental analysis calculated for C H N O
2
1
4
14
6
(
298.30): C, 56.37; H, 4.73; N, 28.17; Found C, 56.50; H, 4.89; N, 28.40.
.1.5. General procedure for the synthesis of 8a-h
A mixture of 7a,b (0.72 mmol) and the appropriate aldehyde (0.76 mmol) in
4
absolute ethanol (20 mL) and glacial acetic acid (1 mL) was heated under reflux for 4 h.
The obtained precipitate from the hot ethanolic solution or after cooling was filtered and
recrystallized from ethanol.
4
.1.5.1.
N'-Benzylidene-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-
carbohydrazide (8a)
-1
Pale yellow powder, yield: 85 %, m.p. 274-275°C; FT-IR (KBr) (ʋmax/ cm ): 3338
1
(
NH), 3248 (NH), 3061 (CH aromatic), 1682 (C=O), 1654 (C=O); H NMR (DMSO-d ,
6
4
00 MHz, δ, ppm): 7.43-7.45 (m, 3H, Ar-H), 7.72-7.75 (m, 2H, Ar-H), 8.44 (s, 2H, C -H
5
+
N=CH), 8.77 (s, 1H, C -H), 11.89 (s, 2H, 2NH, exchangeable with D O); Elemental
2 2
analysis calculated for C H N O (282.26): C,55.32; H, 3.57; N, 29.77; Found C, 54.98;
1
3
10
6
2
H, 3.64; N, 30.04.
4
.1.5.2.
N'-Benzylidene-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]
pyrimidine-6-carbohydrazide(8b)
-1
Pale yellow powder, yield: 88 %, m.p. 285-287 °C; FT-IR (KBr) (ʋmax/ cm ): 3341
NH), 3245 (NH), 3080 (CH aromatic), 2896 (CH aliphatic), 1679 (C=O), 1662 (C=O);
H NMR (DMSO-d , 400 MHz, δ, ppm): 2.42 (s, 3H, CH ), 7.40-7.45 (m, 3H, Ar-H),
(
1
6
3
7
.73-7.75 (m, 2H, Ar-H), 8.43 (s, 1H, C5-H), 8.73 (s, 1H, N=CH), 11.93 (s, 2H, 2NH,
exchangeable with D O); Elemental analysis calculated for C H N O (296.28): C,
2
14 12
6
2
5
6.75; H, 4.08; N, 28.36; Found C, 57.01; H, 4.21; N, 28.64.
4
6
.1.5.3. N'-(2-Hydroxybenzylidene)-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-
-carbohydrazide (8c)
-1
Yellow powder, yield: 88 %, m.p. 288-289°C; FT-IR (KBr) (ʋmax/ cm ): 3356 (NH),
1
3
252 (NH), 3230 (OH), 3045 (CH aromatic), 1692 (C=O), 1659 (C=O); H NMR
(
DMSO-d , 300 MHz, δ, ppm): 6.92-6.99 (m, 2H, Ar-H), 7.30, 7.36 (2t, 1H, J = 8.00 Hz,
6
2
3

Ar-H), 7.39, 7.40 (2d, 1H, J = 8.00 Hz, Ar-H), 8.30, 8.66 (2s, 1H, CH=N), 8.78 (s, 1H,
C -H), 8.99 (s, 1H, C -H), 11.12 (s, 1H, NH, exchangeable D O), 11.29 (s, 1H, NH,
5
2
2
1
3
exchangeable D O), 12.08 (s, 1H, OH, exchangeable D O); C NMR (DMSO-d , 100
2
2
6
MHz, δ, ppm): 103.3, 116.9, 117.0, 118.7, 119.4, 120.0, 130.3, 131.3, 131.9, 133.7,
1
49.4, 149.8, 152.4, 152.6, 157.0, 158.0, 160.1 (aromatic carbons), 159.1 (C=O), 163.2
+
(
C=O); MS, m/z [%]: 298 (M , 6), 69 (100); Elemental analysis calculated for
C H N O (298.26): C, 52.35; H, 3.38; N, 28.18; Found C, 52.49; H, 3.36; N, 28.42.
1
3
10
6
3
4
.1.5.4.
N'-(2-Hydroxybenzylidene)-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-
a]pyrimidine-6-carbohydrazide (8d)
-1
White powder, yield: 90 %, m.p. 298-299°C; FT-IR (KBr) (ʋmax/ cm ): 3345 (NH),
3
320 (OH), 3241 (NH), 3069 (CH aromatic), 2862 (CH aliphatic), 1684 (C=O), 1665
1
(
C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 2.42 (s, 3H, CH ), 6.89-6.94 (m, 2H,
6
3
Ar-H), 7.29 (t, 1H, J = 8.10 Hz, Ar-H), 7.50 (d, 1H, J = 7.50 Hz, Ar-H), 8.65 (s, 1H, C5-
H), 8.73 (s, 1H, N=CH ), 11.32 (s, 2H, 2NH, D O exchangeable), 12.15 (s, 1H, OH,
2
1
3
exchangeable with D O); C NMR (DMSO-d , 100 MHz, δ, ppm): 14.4 (CH ), 103.4,
2
6
3
1
16.8, 119.0, 119.8, 130.3, 131.8, 149.2, 151.2, 155.0 (aromatic carbons), 157.9 (C=O),
60.5 (C=O); Elemental analysis calculated for C H N O (312.28): C, 53.85; H, 3.87;
1
1
4
12
6
3
N, 26.91; Found C, 54.12; H, 3.91; N, 27.09.
4
.1.5.5.
N'-(4-Hydroxybenzylidene)-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]
pyrimidine-6-carbohydrazide (8e)
-1
Yellow powder, yield: 85 %, m.p. 270-272°C; FT-IR (KBr) (ʋmax/ cm ): 3321 (NH),
1
3
248 (NH), 3234 (OH), 3032 (CH aromatic), 1696 (C=O), 1660 (C=O); H NMR
(
DMSO-d , 300 MHz, δ, ppm): 6.84, 6.86 (2d, 2H, J = 7.40 Hz, Ar-H), 7.58, 7.68 (2d,
6
2
H, J = 7.70 Hz, Ar-H), 8.16, 8.18 (2s, 1H, C -H), 8.24, 8.54 (2s, 1H, N=CH), 8.66, 8.74
5
(
2s, 1H, C -H8), 9.85, 10.0 (2s, 1H, NH exchangeable with D O), 10.39 (s, 1H, NH,
2
2
exchangeable with D O), 12.21 (s, 1H, OH, exchangeable with D O); Elemental analysis
2
2
calculated for C H N O (298.26): C, 52.35; H, 3.38; N, 28.18; Found C, 52.51; H,
1
3
10
6
3
3
.44; N, 28.42.
2
4

4
.1.5.6.
N'-(4-Hydroxybenzylidene)-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo
[1,5-a]pyrimidine-6-carbohydrazide (8f)
-1
Yellow powder, yield: 92 %, m.p. 274-275°C; FT-IR (KBr) (ʋmax/ cm ): 3350 (NH),
3
245 (NH), 3169 (OH), 3022 (CH aromatic), 2895 (Aliphatic-H), 1681 (C=O), 1667
1
(
C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 2.40 (s, 3H, CH ), 6.83 (d, 2H, J = 8.70,
6
3
Ar-H), 7.58 (d, 2H, J = 8.70, Ar-H), 8.65 (s, 1H, C -H), 8.71 (s, 1H, N=CH), 9.89 (s, 1H,
5
NH, D O exchangeable), 10.74 (s, 1H, NH, exchangeable D O), 11.91 (s, 1H, OH, D O
2
2
2
exchangeable); 13C NMR (DMSO-d , 100 MHz, δ, ppm): 14.4 (CH ), 103.8, 116.1,
6
3
1
25.8, 127.5, 129.4, 148.5, 150.6, 153.4, 158.0 (aromatic carbons), 156.9 (C=O), 159.9
(
C=O); Elemental analysis calculated for C H N O (312.28): C, 53.85; H, 3.87; N,
1
4
12
6
3
2
6.91; Found C, 54.11; H, 3.93; N, 27.12.
4
.1.5.7.
N'-(4-Methoxybenzylidene)-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]
pyrimidine-6-carbohydrazide (8g)
-1
Yellow powder, yield: 84 %, m.p. 258-260°C; FT-IR (KBr) (ʋmax/ cm ): 3348 (NH),
1
3
257 (NH), 3087 (CH aromatic), 2890 (Aliphatic-H), 1691 (C=O), 1663 (C=O); H NMR
(
DMSO-d , 400 MHz, δ, ppm): 3.82, 3.83 (2s, 3H, OCH ), 7.04, 7.07 (2d, 2H, J = 9.20
6
3
Hz, Ar-H), 7.71, 7.82 (2d, 2H, J = 8.80 Hz, Ar-H), 8.37, 8.41 (2s, 1H, N=CH), 8.64 (s,
H, C -H), 8.78 (s, 1H, C -H), 10.60 (s, 1H, NH, exchangeable with D O), 11.93 (s, 1H,
1
5
2
2
1
3
NH, exchangeable with D O); C NMR (DMSO-d , 100 MHz, δ, ppm): 55.9 (OCH ),
2
6
3
1
02.9, 114.8, 127.1, 127.1, 129.2, 130.4, 147.8, 150.8, 152.7, 157.5, 160.8 (aromatic
+
carbons), 161.3 (C=O), 162.2 (C=O); MS, m/z [%]: 312 (M , 11), 122 (100); Elemental
analysis calculated for C H N O (312.28): C, 53.85; H, 3.87; N, 26.91; Found C,
1
4
12
6
3
5
4.12; H, 3.91; N, 27.13.
4
.1.5.8.
N'-(4-Methoxybenzylidene)-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo
[1,5-a]pyrimidine-6-carbohydrazide (8h)
-1
Pale yellow powder, yield: 89 %, m.p. 295-296°C; FT-IR (KBr) (ʋmax/ cm ): 3352
(NH), 3255 (NH), 3061 (CH aromatic), 2852 (CH aliphatic), 1685 (C=O), 1660 (C=O);
1
H NMR (DMSO-d , 400 MHz, δ, ppm): 3.81, 3.83 (2s, 3H, OCH ), 7.02, 7.05 (2d, 2H, J
6
3
=
8.40 Hz, Ar-H), 7.70, 7.81 (2d, 2H, J = 8.80 Hz, Ar-H), 8.31, 8.63 (2s, 1H, N=CH),
2
5

8
.71 (s, 1H, C -H), 10.35 (s, 1H, NH, exchangeable with D O), 12.21 (s, 1H, NH,
5 2
1
3
exchangeable with D O); C NMR (DMSO-d , 100 MHz, δ, ppm): 14.7 (CH ), 55.9
2
6
3
(
OCH ), 101.9, 114.9, 127.0, 127.5, 129.3, 130.5, 147.3, 152.9, 157.5, 161.2 (aromatic
3
+
carbons), 161.5 (C=O), 162.2 (C=O); MS, m/z [%]: 326 (M , 25), 174 (100); Elemental
analysis calculated for C H N O (326.31): C, 55.21; H, 4.32; N, 25.75; Found C,
1
5
14
6
3
5
5.49; H, 4.40; N, 25.98.
4
.1.6. General procedure for the synthesis of 9a,b
A solution of benzoyl chloride (1.4 g, 1.2 mL, 0.01 mol) in dry dioxane (5 mL) was
added drop wise to a mixture of compound 7a,b (0.01 mol), pyridine (1.2 mL, 0.015
o
mol) and dry dioxane (10 mL) with constant stirring at 0-5 C. The reaction mixture was
stirred for 30 min at this temperature, then for 4 h at room temperature. The mixture was
then poured onto ice-water (50 mL) and the formed precipitate was filtered, washed with
water and recrystallized from methanol.
4
.1.6.1. N'-Benzoyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-carbohydrazide
(9a)
-1
White powder, yield: 78 %, m.p. 244-245°C; FT-IR (KBr) (ʋmax/ cm ): 3335
(NH), 3265 (NH), 3207 (NH), 3054 (CH aromatic), 1687 (C=O),1682 (C=O), 1666
1
(
C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 7.49-7.59 (m, 5H, Ar-H), 7.90 (s, 1H,
6
1
3
C -H), 7.93 (s, 1H, C -H), 10.46 (s, 2H, 2NH, D O exchangeable); C NMR (DMSO-d ,
2
5
2
6
1
00 MHz, δ, ppm): 102.5, 127.2, 127.9, 128.9, 132.3, 133.1, 142.6, 149.3, 157.0
+
(
aromatic carbons), 160.4 (C=O), 166.3 (2 C=O); MS, m/z [%]: 299 (M +1, 13), 298
+
(
M , 66), 83 (100); Elemental analysis calculated for C H N O (298.26): C, 52.35; H,
1
3
10
6
3
3
.38; N, 28.18; Found C, 52.43; H, 3.41; N, 28.40.
4
.1.6.2.
N'-Benzoyl-2-methyl-7-oxo-4,7-dihydro-1,2,4-triazolo[1,5-a]pyrimidine-6-
carbohydrazide (9b)
-1
White powder, yield: 78 %, m.p. 220-225°C; FT-IR (KBr) (ʋmax/ cm ): 3349
(NH), 3274 (NH), 3216 (NH), 3075 (CH aromatic), 2875 (Aliphatic-H), 1691
1
(
C=O),1689 (C=O), 1669 (C=O); H NMR (DMSO-d , 300 MHz, δ, ppm): 2.41 (s, 3H,
6
2
6

CH ), 7.50-7.62 (m, 3H, Ar-H), 7.91 (d, 2H, J = 7.20 Hz, Ar-H), 8.69 (s, 1H, C -H),
3
5
1
3
1
0.48 (s, 2H, 2NH), 10.58 (s,1H, NH); C NMR (DMSO-d , 100 MHz, δ, ppm): 14.2
6
(
CH ), 103.9, 127.9, 128.9, 132.3, 133.1, 139.1, 153.0,157.0 (aromatic carbons), 162.7
3
+
+
(
C=O), 165.6 (C=O), 166.3 (C=O); MS, m/z [%]: 314 (M +2, 13), 313 (M +1, 7), 312
+
(
M , 24), 239 (100); Elemental analysis calculated for C H N O (312.29): C, 53.85; H,
1
4
12
6
3
3
.87; N, 26.91; Found C, 54.08; H, 3.90; N, 27.14.
4
.1.7. General procedure for the synthesis of 10a,b
A mixture of hydrazide 7a,b (0.05 mol), potassium hydroxide (3.0 g, 0.05 mol)
and carbon disulfide (10 mL, 0.17 mol) in ethanol (70 mL) was heated under reflux with
stirring until the evolution of hydrogen sulfide ceased (12 h). Ethanol was distilled off
under reduced pressure; the residue was dissolved in water and then acidified with dilute
hydrochloric acid (10%). The resulting precipitate was filtered, washed with water, dried,
and recrystallized from ethanol.
4
7
.1.7.1.
6-(5-Thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-1,2,4-triazolo[1,5-a]pyrimidin-
(4H)-one (10a)
-1
Canary yellow powder, yield: 80 %, m.p. 184-186°C; FT-IR (KBr) (ʋmax/ cm ):
1
3
362 (NH), 3251 (NH), 3065 (CH aromatic), 1683 (C=O), 1247 (C=S); H NMR
(
DMSO-d , 300 MHz, δ, ppm): 8.47 (s, 1H, C -H), 8.74 (s, 1H, C -H), 10.60 (s, 1H, NH,
6 2 5
1
3
exchangeable with D O), 10.66 (s, 1H, NH oxadiazole, exchangeable with D O); C
2
2
NMR (DMSO-d , 100 MHz, δ, ppm): 97.5, 146.3, 150.9, 152.7 (aromatic carbons), 157.8
6
+
(
C=O), 177.2 (C=S); MS, m/z [%]: 236 (M , 3), 59 (100); Elemental analysis calculated
for C H N O S (236.21): C, 35.59; H, 1.71; N, 35.58; Found C, 35.82; H, 1.69; N, 35.84.
7
4
6
2
4
.1.7.2.
2-Methyl-6-(5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)-1,2,4-triazolo[1,5-
a]pyrimidin-7(4H)-one (10b)
-1
Canary yellow powder, yield: 87 %, m.p. 269-270°C; FT-IR (KBr) (ʋmax/ cm ):
3
376 (NH), 3245 (NH), 3090 (CH aromatic), 2882 (CH aliphatic), 1692 (C=O), 1212
1
(
C=S); H NMR (DMSO-d , 400 MHz, δ, ppm): 2.43 (s, 3H, CH ) 8.55 (s, 1H, C -H);
6
3
5
1
3
C NMR (DMSO-d , 100 MHz, δ, ppm): 13.7 (CH ), 98.0, 146.4, 151.5, 152.3 (aromatic
6
3
2
7

+
carbons), 158.1 (C=O), 177.3 (C=S); MS, m/z [%]: 250 (M , 16), 43 (100); Elemental
analysis calculated for C H N O S (250.24): C, 38.40; H, 2.42; N, 33.58; Found C,
8
6
6
2
3
8.67; H, 2.40; N, 33.81.
4
.1.8. General procedure for the synthesis of 11a,b
A solution of compound 10a,b (0.01 mol) in dry DMF (25 mL) was treated with
sodium carbonate (0.0167 mol) and stirred at room temperature for 10 minutes. Benzyl
bromide (5.13 g, 3.6 mL, 0.03 mol) was added and stirring was continued overnight. The
reaction mixture was poured onto ice-water (100 mL), the resulting precipitate was
filtered, washed with water and dried. The product was recrystallized from
methanol/water.
4
.1.8.1.
6-[5-(Benzylthio)-1,3,4-oxadiazol-2-yl]-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-
one (11a)
-1
Yellow powder, yield: 65 %, m.p. 176-177°C; FT-IR (KBr) (ʋmax/ cm ): 3367 (NH),
1
3
087 (CH aromatic), 1672 (C=O); H NMR (DMSO-d , 400 MHz, δ, ppm): 4.60 (s, 2H,
6
CH ), 7.23-7.49 (m, 7H, 5Ar-H + C -H + C -H), 8.39 (s, 1H, NH, exchangeable with
2
5
2
1
3
D O); C NMR (DMSO-d , 100 MHz, δ, ppm): 38.3 (CH ), 102.1, 127.2, 127.9, 128.2,
2
6
2
1
28.6, 129.5, 136.8, 145.5, 149.6, 152.7, 153.0, 160.3 (aromatic carbons), 156.0 (C=O);
+
MS, m/z [%]: 326 (M , 17), 42 (100); Elemental analysis calculated for C H N O
1
4
10
6
(326.33): C, 51.53; H, 309; N, 25.75; Found C, 51.79; H, 3.13; N, 26.02.
4
7
.1.8.2. 6-[5-(Benzylthio)-1,3,4-oxadiazol-2-yl]-2-methyl-1,2,4-triazolo[1,5-a]pyrimidin-
(4H)-one (11b)
-1
Pale yellow powder, yield: 73 %, m.p. 193-194°C; FT-IR (KBr) (ʋmax/ cm ): 3377
1
(
NH), 3085 (CH aromatic), 2853 (CH aliphatic), 1679 (C=O); H NMR (DMSO-d , 300
6
MHz, δ, ppm): 2.32 (s, 3H, CH ), 4.50 (s, 2H, CH ), 7.27-7.48 (m, 5H, Ar-H), 8.45 (s,
3
2,
1
3
1
H, C -H), 9.03 (s, 1H, NH, exchangeable with D O); C NMR (DMSO-d , 100 MHz, δ,
5 2 6
ppm): 14.8 (CH ), 38.2 (CH ), 98.5, 127.9, 128.2, 128.8, 129.2, 129.5, 135.3, 145.3,
3
2
1
51.2, 152.0, 154.0, 160.6 (aromatic carbons), 157.6 (C=O); Elemental analysis
2
8

calculated for C H N O S (340.36): C, 52.93; H, 3.55; N, 24.69; Found C, 53.26; H,
1
5
12
6
2
3
.59; N 24.97.
4
4
.2.
Biological evaluation
.2.1. Antibacterial assay
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 OD ), then added to each well of the compound containing plates, giving a
6
00
5
cell density of 5x10 CFU/mL and a total volume of 50 µL. All the plates were covered
and incubated at 37 °C for 18 h without shaking [19].
4
.2.2. Antifungal assay
Fungi strains were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD) agar at
6
6
3
0 °C. A yeast suspension of 1 x 10 to 5 x 10 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 suspension of
3
2
3
.5 x10 CFU/mL and a total volume of 50 µL. All plates were covered and incubated at
5 °C for 24 h without shaking [19].
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 supplemented with 10% FBS was used as growth media
and the cells were incubated together with the compounds for 20 h at 37 °C in 5% CO2
[19].
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
8
suspended in same to a concentration of 0.5 x 10 cells/mL, as determined by manual cell
count in Neubauer hemocytometer. The washed cells were then added to the 384-well
2
9