Magnetic silica nanoparticle-supported copper complex as an efficient catalyst for the synthesis of novel triazolopyrazinylacetamides with improved antibacterial activity

Article abstract of DOI:10.1007/s10593-020-02685-6

[Figure not available: see fulltext.] A novel superparamagnetic iron oxide modified with copper via 2-aminobenzamide is synthesized by the modification of Fe₃O₄@SiO₂ with amine, followed by the reaction with isatoic anhydride. The catalyst is fully characterized by various methods. The catalytic activity of the catalyst is evaluated in the synthesis of a series of novel N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide analogs. Antibacterial activity of the synthesized compounds is evaluated. The catalyst is recoverable and shows excellent reusability in 10 sequential runs.

Full text of DOI:10.1007/s10593-020-02685-6

DOI 10.1007/s10593-020-02685-6
Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
Magnetic silica nanoparticle-supported copper complex as an efficient
catalyst for the synthesis of novel triazolopyrazinylacetamides
with improved antibacterial activity
Mohammad Sadegh Asgari1,3, Saghi Sepehri2, Saeed Bahadorikhalili1, Parviz Rashidi Ranjbar1,
Rahmatollah Rahimi3, Ahmad Gholami4, Aboozar Kazemi4,5, Mehdi Khoshneviszadeh4,
Bagher Larijani6, Mohammad Mahdavi6*
1 School of Chemistry, College of Science, University of Tehran,
P. O. Box 14155-6455, Tehran, Iran
2 Department of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences,
Ardabil, 5618953141, Iran
3 Department of Chemistry, Iran University of Science and Technology,
Narmak, Tehran 16846-13114, Iran; e-mail: sadegh.asgari.1@gmail.com
4 Pharmaceutical Science Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran
5 Department of Pharmaceutical Biotechnology, School of Pharmacy,
Shiraz University of Medical Sciences,
Shiraz, Iran
6 Endocrinology and Metabolism Research Center,
Endocrinology and Metabolism Research Institute,
Tehran University of Medical Sciences,
Tehran, Iran; е-mail: momahdavi@sina.tums.ac.ir
Published in Khimiya Geterotsiklicheskikh Soedinenii,
2020, 56(4), 488–494
Submitted September 29, 2019
Accepted after revision January 21, 2020
Fe3O4@SiO2
H2N
Cu
O
O
O
O
H
O
Si
N
OH
R1
O
O
O
Cl
NaN3
NC
N
N
N
+
HN
R2
N
NH2
R1
R2
H2O, rt, 12 h
77–95%
HC
R1 = Ar, R2 = Alk
A novel superparamagnetic iron oxide modified with copper via 2-aminobenzamide is synthesized by the modification of Fe3O4@SiO2
with amine, followed by the reaction with isatoic anhydride. The catalyst is fully characterized by various methods. The catalytic activity
of the catalyst is evaluated in the synthesis of a series of novel N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-
yl)acetamide analogs. Antibacterial activity of the synthesized compounds is evaluated. The catalyst is recoverable and shows excellent
reusability in 10 sequential runs.
Keywords: nanoparticle-supported copper catalyst, triazole, antibacterial agents, click reaction, structure–activity relationship.
About half of all deaths in tropical countries are caused
by infectious diseases. Although the number of deaths from
bacterial infections has decreased in the developed
survival, and has added an average of 20 years to the
human lifetime.3 Gram-negative and Gram-positive
pathogens are responsible for the majority of hospital-
acquired and community-acquired infections, resulting in
wide mortality and burden on worldwide healthcare
system.4 One of the attractive scaffolds for researchers in
making new therapeutic agents is triazole structure. This
nitrogen-containing heterocycle is prominent among
pharmaceuticals approved by FDA.5 1,2,3-Triazole ring is
attractive connecting unit, since it is stable to metabolic
countries, these infections remain
a
problem in
undeveloped regions. Antibiotics are effective weapons to
fight against bacterial infections, but their overuse has put
pressure on bacteria to develop resistance.1 The global
spread of antibiotic-resistant infections is a serious problem
which threatens global healthcare.2 The discovery and
development of antibiotics has been vital to human
0009-3122/20/56(4)-0488©2020 Springer Science+Business Media, LLC
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Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
transformations, for example, oxygenation at benzylic posi-
tions,20 alkane oxidation,21 alcohol oxidation,22 click reac-
tions,23 reductive coupling reactions.24 The catalytic reduction
of nitro group is probably one of the emerging areas in
catalytic organic chemistry that might have a tremendous
impact in chemical industry in the next decades.
Among the wide range of solid supports for transition
metal catalysts, magnetic nanoparticles have displayed high
applicability and are known as effective research tools to
improve novel and operative recoverable catalysts.
Magnetically separable systems have attracted a great deal
of attention as an interesting alternative to improve the
efficient separation of heterogeneous catalysts using an
external magnet thus providing improved reusability.25
Their widespread usage and versatility make magnetic
silica core-shell nanoparticles excellent and interesting
supporting materials for providing new and improved green
catalysts. Magnetic nanoparticles can offer very promising
features as catalyst supports due to their large specific
surface areas and magnetic properties, which facilitate the
separation of the catalyst upon reaction completion.26
Although magnetic silica core-shell nanoparticles,
thanks to the nanometric sizes, are well dispersed in many
solvents, they cannot provide a suitable exposure of their
catalytically active centers in the proximity of organic
substrates, especially when hydrophilic solvents are used as
the reaction medium. Therefore, we were interested in
designing and preparing a novel catalyst system by
emphasizing controlling its high dispersion in hydrophilic
environment-friendly ethylene glycol while avoiding time-
consuming recycling stages. Following our previous efforts
on extending nanoparticles as supports for homogeneous
transition metal catalysts on one hand, and synthesis of
novel biologically active compounds on the other hand,
hereby we report a copper–2-aminobenzamide (ABA)
complex covalently attached to modified magnetic silica
core-shell nanoparticles as an efficient catalyst for the
synthesis of a series of N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide derivatives.
The synthesized compounds have also been screened for
their antibacterial activity in vitro. Various functional
groups were introduced into the target compounds in order
to investigate preliminary structure–activity relationship.
[Cu@ABA-Fe3O4@SiO2] catalyst was synthesized by
the immobilization of copper on the surface of the modified
magnetic iron oxide nanoparticles. Superparamagnetic iron
oxide nanoparticles were prepared by the coprecipitation of
Fe2+ and Fe3+ in basic media. The superparamagnetic iron
oxide nanoparticles were then capsulated by silica,
followed by the reaction with 3-aminopropyltriethoxysilane
to introduce active amine groups on the surface of the
nanoparticles. The [Amine-Fe3O4@SiO2] finally reacted
with 2H-3,1-benzoxazine-2,4(1H)-dione to form amino-
benzamide-modified magnetic nanoparticles. This structure
was used as a copper support to form [Cu@ABA-
Fe3O4@SiO2] catalyst. The step by step preparation of the
catalyst is presented in Scheme 1.
Figure 1. Chemical structures of commercial triazole-containing
antibacterial drugs cefatrizine and tazobactam.
degradation, oxidative/reductive conditions and actively
participates in binding to biomolecular targets and
improves solubility of the molecule by hydrogen bonding
and dipole interactions.6 1,2,3-Triazoles are extensively
used in medicinal chemistry because of their easy synthesis
through click reaction and attractive features.7 1,2,3-
Triazole and its derivatives have attracted a great deal of
interest due to their varied biological activities such as
antifungal,8 antibacterial,9 antitubercular,10 anticancer,11
and antiviral.12 This heterocyclic moiety has gained special
attention in the discovery of new antibiotics because
numerous drug compounds containing 1,2,3-triazole group,
such as for example cefatrizine and tazobactam, are used
for the treatment of bacterial infections (Fig. 1).
Click chemistry is a good synthetic method applying
several near-perfect chemical transformations for the
synthesis and assembly of chemical scaffolds with potential
biological activity.13 These reactions are modular, exten-
sive in scope, give very high yields, they are stereospecific
and generate only harmless byproducts that can be removed
by nonchromatographic techniques.13 The click chemistry
concept has received a considerable use in many fields of
chemical science, including drug design and chemical
investigations for the discovery of biologically active com-
pounds.6,14 In addition, Ugi/alkyne-azide cycloaddition reaction
is applied for the synthesis of fused triazolo derivatives.15
The application of copper in catalytic processes has
attracted great attention in the last decade. The different
oxidation states of copper associates well with a large
number of different functional groups via Lewis acid
interactions or π-coordination. These features confer a
remarkably broad range of activities allowing copper to
catalyze the oxidation, as well as click reactions.15,16 In
recent years, Cu-promoted processes have evolved into viable
alternatives to the more widely practiced Pd-catalyzed
reactions.17 The Cu-based methods are distinguished by
their broad scope, high efficiency, and mild reaction
conditions, thus making them attractive vehicles for further
applications. Although the synthesis and catalytic activities
of copper catalysts have been successfully demonstrated,
their separation from the reaction mixture and recycling are
tedious, which limits their use for practical applications.
Therefore, to overcome mentioned difficulties, hetero-
genized copper catalysts on solid supports has received
increased attention. By immobilizing homogeneous catalysts
on insoluble supports, the advantages of both homogeneous
as well as heterogeneous catalysts are achieved, so that
without losing catalytic activity the catalysts can be
separated easily from the desired products by a simple
filtration. During the past years, several solid supports have
been efficiently prepared.18,19 Immobilized copper catalysts on
solid supports have been applied in several organic
The exact structure of [Cu@ABA-Fe3O4@SiO2] catalyst
was studied by several methods including field emission
scanning electron microscopy (FE-SEM), transmission
electron microscopy (TEM), inductively coupled plasma
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Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
Scheme 1. Preparation steps of [Cu@ABA-Fe3O4@SiO2] catalyst
atom emission spectroscopy (ICP-AES), and Fourier
transform infrared spectrometry (FT-IR). Electron
microscope images, including FE-SEM and TEM are
presented in Figure 2. FE-SEM results (Fig. 2a) show the
morphology of the catalyst to be spherical with the average
particle size of about 25 nm. The TEM image, presented in
Figure 2b, confirms the structure and the particle sizes of
the catalyst. Figure 2c shows the energy-dispersive X-ray
spectroscopy (EDS) analysis of [Cu@ABA-Fe3O4@SiO2]
catalyst. It could be seen that all the expected elements,
including Fe, Si, O, C, and N could be seen in EDS
spectrum. In addition, copper is also seen in EDS spectrum,
which confirm the presence of Cu in the structure of
[Cu@ABA-Fe3O4@SiO2] catalyst.
The magnetic behavior of the catalyst was studied by
vibrating-sample magnetometer (VSM) method. The VSM
curve is presented in Figure 3b. The magnetic hysteresis
measurement was done using VSM in an applied magnetic
field at room temperature with the field sweeping from
–8000 to +8000 Oe to assay the magnetic behavior of
catalyst. The S-type magnetization hysteresis loop confirmed
the superparamagnetic nature of [Cu@ABA-Fe3O4@SiO2]
catalyst. For the sake of better magnetic behavior demonstra-
To prove the strucutre of the synthesized catalyst, FT-IR
spectroscopy was applied (Fig. 3a). The FT-IR spectrum of
the catalyst shows an absorption at 579 cm–1, which
represents Fe–O vibrations. Si–O vibrations show an
intense peak at 1098 cm–1. In addition, the band at 1688 cm–1
can be assigned to the carbonyl group in the structure of the
catalyst. Vibration bands at 3441 and 3317 cm–1 can be
assigned to O–H and N–H vibrations, respectively.
Figure 2. a) FE-SEM image, b) TEM image, and c) EDS results
of [Cu@ABA-Fe3O4@SiO2] catalyst.
Figure 3. a) FT-IR spectrum, b) VSM curve, and c) magnetic
separation of [Cu@ABA-Fe3O4@SiO2] catalyst.
490

Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
Table 1. Antibacterial activity of the synthesized compounds 3a–j
(minimal inhibition concentration (MIC), μg/ml)
tion of the catalyst, the separation of [Cu@ABA-Fe3O4@SiO2]
by an external magnet is presented in Figure 3c. Moreover,
the copper content of the catalyst was determined to be
2 mmol per gram of the catalyst.
Com-
pound
E.
S.
P.
E.
R1
R2
coli aureus aeruginosa faecalis
The synthetic route for the newly synthesized com-
pounds is outlined in Scheme 2. This reaction was
performed at room temperature in H2O. Sodium azide was
added to 2-chloroacetic acid (1) at room temperature in
H2O to yield azide 2. Then, without isolation of azide 2,
aromatic aldehyde, prop-2-yn-1-amine, alkyl isocyanide,
and catalytic amount of [Cu@ABA-Fe3O4@SiO2] catalyst
were added to the reaction mixture to give the desired
products 3a–j. (Scheme 2). Chemical structures of the
20.62 165
165
10.31
3a
165 82.5
41.25 82.5
165
165
20.62
20.62
3b
3c
1
synthesized compounds were confirmed by H and 13C
NMR, FT-IR spectroscopy, and elemental analysis.
Scheme 2. Synthesis of N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro-4H-
[1,2,3]triazolo[1,5-a]pyrazin-5-yl)acetamides 3a–j
41.25 165
20.62 165
82.5
165
165
3d
3e
82.5
20.62 82.5
20.62 82.5
20.62 82.5
165
82.5
82.5
20.62
82.5
82.5
3f
3g
3h
Antibacterial activity of all synthesized compounds was
screened against Gram-negative and Gram-positive
bacterial strains including Escherichia coli, Staphylococcus
aureus, Pseudomonas aeruginosa, Enterococcus faecalis.
Ampicillin was tested under the same conditions as the
reference drug. The results expressed as minimum
inhibitory concentrations (MICs, µg/ml) are summarized in
Table 1. Compound 3a showed the most potent anti-
bacterial activity against E. coli and E. faecalis strains
(10.31 and 20.62 µg/ml, respectively). But compounds 3g
and 3h were most effective against both S. aureus and
P. aeruginosa strains, with MIC value of 82.5 µg/ml. It is
noteworthy that compounds 3a and 3f both have MIC
values 20.62 µg/ml against E. coli but 10.31 and 20.62 µg/ml
against E. faecalis, respectively. This indicates that their
efficacy is comparable to clinical antibiotics such as
ampicillin (15.60 µg/ml against E. coli and 6.26 µg/ml
against E. faecalis).
However, all compounds exhibited weak to good
inhibitory effect against the growth of all strains. This may
be attributed to the poor ability of these compounds to
penetrate the outer membrane of bacteria. Compound 3g
having monomethoxyphenyl group and compound 3h with
trimethoxyphenyl group exhibited similar levels of in vitro
antibacterial activity, while compound 3e bearing
4-chlorophenyl group, displayed weaker in vitro anti-
bacterial activity compared to compounds 3g and 3h against
S. aureus and P. aeruginosa strains. In general, compounds
bearing cyclohexane moiety at the NH amide position
showed superior activity against all tested bacteria than
41.25 165
82.5
82.5
3i
41.25 165
15.60 13.12
82.5
82.5
6.25
3j
–
–
15.60
Ampicillin
compounds bearing tert-butyl moiety at the same position
(e. g., compounds 3a and 3c vs compounds 3e and 3h).
Compounds with electron-withdrawing substituents in
the phenyl ring, such as chlorine (compound 3c) exhibited
more potent activity with MICs 20.62 and 82.5 µg/ml
against E. faecalis and S. aureus, while compounds with
electron-donating substituents such as methoxy (compound
3g), methyl (compound 3d), and isopropyl (compound 3i)
groups in the para position of phenyl ring with MICs 82.5
and 165 µg/ml showed weak to moderate activity. In
addition, obviously that methyl substitution at the para
position of phenyl ring (compound 3d) decreased the
inhibitory effects compared to the corresponding com-
pound 3j substituted at meta position against three strains
of E. coli, S. aureus, and E. faecalis. Methyl functionality
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Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
Vista-Pro). Transmission electron microscopy (TEM)
analyses were performed on a Philips model CM 10
instrument. Energy-dispersive X-ray spectroscopy analyses
for the characterization of the catalyst were recorded on a
GNR Explorer machine. Melting points were determined in
open capillary tubes on a Barnstead Electrothermal 9100
BZ circulating oil melting point apparatus. The reaction
monitoring was accomplished by TLC on silica gel
PolyGram SILG/UV254 plates. Column chromatography
was carried out on columns of silica gel 60 (70–230 mesh).
Chemicals were purchased from Merck, Acros, and Aldrich
chemical companies and used without further purification.
Preparation of [Amine-Fe3O4@SiO2]. A solution of
3-aminopropyltriethoxysilane (0.1 g, 0.45 mmol) in EtOH
(30 ml) was added dropwise to a vigorously stirring
mixture of Fe3O4@SiO2 nanoparticles (100 mg), prepared
according to the reported procedure,19 in EtOH–H2O, 1:2
(30 ml) and HCl (pH 4). After vigorous stirring for 24 h,
the solution was filtered off and washed thoroughly. The
residue was dried at 100°C under reduced pressure for 12 h.
Preparation of [ABA-Fe3O4@SiO2]. 2H-3,1-Benzoxazine-
2,4(1H)-dione (450 mg, 2.76 mmol) was added to a mixture
of [Amine-Fe3O4@SiO2] (100 mg) and Et3N (0.1 g, 1.7 mmol)
in EtOH and stirred at 60°C for 24 h. Afterward, the solid
was separated by an external magnet, washed with EtOH
(3×10 ml), and dried under reduced pressure at 60°C.
Preparation of [Cu@ABA-Fe3O4@SiO2]. CuCl (495 mg,
5 mmol) was added to a mixture of [ABA-Fe3O4@SiO2]
(0.1 g) in dry CH2Cl2 (50 ml) and stirred at room temperature
under argon atmosphere for 24 h. The obtained solid was
magnetically separated and washed with CH2Cl2 (2×10 ml)
and Et2O (2×10 ml). [Cu@ABA-Fe3O4@SiO2] catalyst was
obtained after drying under reduced pressure for 12 h.
Synthesis of N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro-4H-
[1,2,3]triazolo[1,5-a]pyrazin-5-yl)acetamides 3a–j (General
method). 2-Chloroacetic acid (1) (1 mmol) and NaN3
(1 mmol) were added into a round-bottom flask containing
H2O (5 ml) and stirred for 30 min at room temperature.
Then aromatic aldehyde (2 mmol), prop-2-yn-1-amine
(1 mmol), alkyl isocyanide (1 mmol), and [Cu@ABA-
Fe3O4@SiO2] (10 mol %) were added, and the reaction
mixture was stirred at room temperature for 12 h. After the
completion of the reaction, the catalyst was separated by an
external magnet. H2O was added to the reaction mixture,
the resulting precipitate was filtered off.
Figure 4. Recovery results of [Cu@ABA-Fe3O4@SiO2] catalyst
in 10 sequential reactions of the compound 3a synthesis.
in ortho position showed to be much more active against
the studied bacteria than that in para position (Table 1).
Compounds 3i,j both with isopropyl group in the para
position of phenyl ring and with either cyclohexane at NH
amide position (compound 3i) or tert-butyl group at NH
amide position (compound 3j) exhibited similar levels of in
vitro antibacterial activity against four strains.
An important feature of [Cu@ABA-Fe3O4@SiO2] catalyst
is its reusability. For the reusability study, the catalyst was
isolated after the reaction completion, washed, and used in
the next reaction. This study was performed for 10 sequential
runs. The recovery results are presented in Figure 4. It can
be observed that, the desired products are obtained in high
isolated yields even after 10 runs, without essential
decrease in the catalyst activity. For the stability studying,
the catalyst was stirred in the reaction conditions for 24 h.
The catalyst was separated using a magnet and the solution
was analyzed by ICP method. The result showed that the
copper content in the solution was less than 1 ppm, which
proves the stability of the catalyst in the reaction conditions.
In this research, a novel catalyst based on the immobi-
lization of copper onto modified superparamagnetic iron
oxide via aminobenzamide is introduced. The catalyst is
synthesized by the modification of Fe3O4@SiO2 with
propylamine, followed by the reaction with 2H-3,1-
benzoxazine-2,4(1H)-dione. The successful formation of
the catalyst was proved by various characterization
methods. Catalyst proved to be recoverable and
demonstrated excellent reusability in 10 sequential runs.
The catalytic activity of the catalyst was evaluated in the
synthesis of novel N-alkyl-2-aryl-2-(6-oxo-6,7-dihydro-
4H-[1,2,3]triazolo[1,5-a]pyrazin-5-yl)acetamides. Several
products, bearing wide range of functional groups were
synthesized. The antibacterial activity of the synthesized
compounds was evaluated against E. coli, S. aureus,
P. aeruginosa, and E. faecalis, and the results showed good
activity of the compounds against the tested bacteria.
For studying the reusability of [Cu@ABA-Fe3O4@SiO2]
catalyst, the reaction of 2-chloroacetic acid, 3,4,5-tri-
methoxybenzaldehyde, propargylamine, and cyclohexyl
isocyanide was selected as the model reaction. The reaction
was performed according to the conditions, mentioned
above. After the completion of the reaction, the catalyst
was separated from the reaction mixture, using an external
magnet and was used directly in the next reaction. The
reusability study was evaluated for 10 sequential runs.
Results are presented in Figure 4.
N-Cyclohexyl-2-(6-oxo-6,7-dihydro[1,2,3]triazolo[1,5-a]-
pyrazin-5(4H)-yl)-2-(3,4,5-trimethoxyphenyl)acetamide
(3a). Yield 0.420 g (95%), white solid, mp 220–222°C.
IR spectrum, ν, cm–1: 3120, 1661, 1625, 1434, 1065.
1H NMR spectrum, δ, ppm (J, Hz): 8.32 (1H, d, J = 7.8, NH);
7.63 (1H, s, H triazole); 6.54 (2H, s, H Ar); 6.20 (1H, s,
Experimental
IR spectra were recorded on a Shimadzu FT-IR 550
spectrometer in KBr pellets. 1H and 13C NMR spectra were
recorded on a Bruker Avance spectrometer (500 and 125 MHz,
respectively) in DMSO-d6 solution with TMS as internal
standard. Elemental analysis was performed on a VarioEL
system in CHNS mode. ICP analysis was performed using
an inductively coupled plasma (ICP) analyzer (Varian,
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Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
CH); 5.21 (2H, dd, J = 17.4, J = 17.4, CH2); 4.91 (1H, d,
N-Cyclohexyl-2-(3-methylphenyl)-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3f).
Yield 0.349 g (95%), white solid, mp >250°C. IR spectrum,
ν, cm–1: 3121, 1659, 1625, 1432, 1058. 1H NMR spectrum,
δ, ppm (J, Hz): 8.31 (1H, d, J = 8.0, NH); 7.61 (1H, s,
H triazole); 7.30 (1H, t, J = 7.4, H Ar); 7.19 (1H, d, J = 7.3,
H Ar); 7.08–7.06 (2H, m, H Ar); 6.27 (1H, s, CH); 5.20
(2H, dd, J = 18.5, J = 18.4, CH2); 4.93 (1H, d, J = 16.3,
CH2 pyrazine); 4.05 (1H, d, J = 16.9, CH2 pyrazine); 3.62
(1H, br. s, CH cyclohexyl); 2.31 (3H, s, CH3); 1.79–1.52
(5H, m, CH2 cyclohexyl) ; 1.27–1.11 (5H, m, CH2 cyclohexyl).
13C NMR spectrum, δ, ppm: 167.6; 163.1; 138.0; 134.6;
129.5; 129.0 (2C); 128.7; 125.9; 58.7; 48.8; 47.8; 32.2;
25.1; 24.5; 24.4; 21.0. Found, %: C 65.43; H 6.80; N 19.03.
C20H25N5O2. Calculated, %: C 65.37; H 6.86; N 19.06.
N-tert-Butyl-2-(4-methoxyphenyl)-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3g).
Yield 0.343 g (96%), white solid, mp 192–194°C. IR spectrum,
ν, cm–1: 3118, 1667, 1621, 1438, 1066. 1H NMR spectrum,
δ, ppm (J, Hz): 8.03 (1H, s, NH); 7.60 (1H, s, H triazole);
7.22 (2H, d, J = 8.3, H Ar); 6.99 (2H, d, J =8.3, H Ar); 6.25
(1H, s, CH); 5.21 (2H, dd, J = 17.5, J = 17.2, CH2); 4.92
(1H, d, J = 16.0, CH2 pyrazine); 4.00 (1H, d, J = 16.2, CH2
pyrazine); 3.77 (3H, s, OCH3); 1.28 (9H, s, C(CH3)3).
13C NMR spectrum, δ, ppm: 169.0; 163.5; 159.6; 130.8;
129.5; 129.5; 127.1; 114.7; 58.9 (2C); 55.6; 51.0; 49.2;
40.0; 28.9. Found, %: C 60.43; H 6.46; N 19.65. C18H23N5O3.
Calculated, %: C 60.49; H 6.49; N 19.59.
N-tert-Butyl-2-(6-oxo-6,7-dihydro-4H-[1,2,3]triazolo-
[1,5-a]pyrazin-5-yl)-2-(3,4,5-trimethoxyphenyl)acetamide
(3h). Yield 384 g (92%), white solid, mp 190–192°C.
IR spectrum, ν, cm–1: 3115, 1670, 1621, 1437, 1060.
1H NMR spectrum, δ, ppm (J, Hz): 8.10 (1H, s, NH); 7.62
(1H, s, H triazole); 6.54 (1H, s, H Ar); 6.21 (1H, s, CH);
5.22 (2H, dd, J = 17.5, J = 17.3, CH2); 4.93 (1H, d,
J = 16.3, CH2 pyrazine); 4.08 (1H, d, J = 16.3, CH2 pyrazine);
3.74 (6H, s, 2OCH3); 3.68 (3H, s, OCH3); 1.29 (9H, s,
C(CH3)3). 13C NMR spectrum, δ, ppm: 168.7; 163.5; 153.4;
137.8; 130.8; 129.7; 129.5; 106.7; 60.5; 59.5; 56.3 (2C);
51.1; 49.2; 28.8. Found, %: C 57.58; H 6.58; N 16.81.
C20H27N5O5. Calculated, %: C 57.54; H 6.52; N 16.78.
N-Cyclohexyl-2-(4-isopropylphenyl)-2-(6-oxo-6,7-di-
hydro[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3i).
Yield 0.371 g (94%), white solid, mp 192–194°C. IR spectrum,
ν, cm–1: 3118, 1670, 1629, 1429, 1057. 1H NMR spectrum,
δ, ppm (J, Hz): 8.30 (1H, s, NH); 7.61 (1H, s, H triazole);
7.29–7.21 (4H, m, H Ar); 6.29 (1H, s, CH); 5.29–5.13 (2H,
m, CH2); 4.95 (1H, d, J = 16.0, CH2 pyrazine); 4.05 (1H, d,
J = 16.4, CH2 pyrazine); 3.63 (1H, br. s, CH cyclohexyl);
3.22 (1H, s, CH); 2.95–2.83 (1H, m, CH(CH3)2); 1.81–1.53
(5H, m, CH213cyclohexyl); 1.24–1.13 (11H, m, CH2 cyclohexyl,
CH(CH3)2). C NMR spectrum, δ, ppm: 168.1; 163.6; 149.0;
132.6; 129.5; 129.4; 127.2; 126.4; 58.9; 53.0; 49.2; 48.3; 33.6;
32.7; 25.6; 25.0 (2C); 24.3. Found, %: C 68.87; H 7.40; N 17.75.
C22H29N5O2. Calculated, %: C 66.81; H 7.39; N 17.71.
N-tert-Butyl-2-(4-isopropylphenyl)-2-(6-oxo-6,7-dihydro-
4H-[1,2,3]triazolo[1,5-a]pyrazin-5-yl)acetamide (3j). Yield
0.354 g (96%), white solid, mp 188–190°C. IR spectrum,
ν, cm–1: 3119, 1662, 1616, 1431, 1064. 1H NMR spectrum,
δ, ppm (J, Hz): 8.08 (1H, s, NH); 7.61 (1H, s, H triazole);
7.31 (2H, d, J = 8.1, H Ar); 7.22 (2H, d, J = 8.1, H Ar);
J = 16.1, CH2 pyrazine); 4.13 (1H, d, J = 16.2, CH2 pyrazine);
3.74 (6H, s, 2OCH3); 3.67–3.57 (7H, m, OCH3, CH cyclohexyl);
1.84–1.53 (5H, m, CH2 cyclohexyl); 1.28–1.10 (5H, m, CH2
cyclohexyl). 13C NMR spectrum, δ, ppm: 167.9; 163.6;
153.5; 137.8; 130.5; 129.6; 129.5; 106.7; 60.5; 59.4; 56.4; 49.2;
48.2; 32.7; 32.4; 25.6; 24.9. Found, %: C 59.57; H 6.64; N 15.77.
C22H29N5O5. Calculated, %: C 59.58; H 6.59; N 15.79.
N-tert-Butyl-2-(2-methylphenyl)-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3b).
Yield 0.330 g (97%), white solid, mp 235–237°C. IR spectrum,
ν, cm–1: 3122, 1661, 1617, 1429, 1061. 1H NMR spectrum,
δ, ppm (J, Hz): 8.06 (1H, s, NH); 7.60 (1H, s, H triazole);
7.24–7.17 (4H, m, H Ar); 6.26 (1H, s, CH); 5.22 (2H, dd,
J = 15.2, J = 15.1, CH2); 4.91 (1H, d, J = 16.2, CH2 pyrazine);
3.98 (1H, d, J = 16.2, CH2 pyrazine); 2.31 (3H, s, CH3); 1.27
(9H, s, C(CH3)3). 13C NMR spectrum, δ, ppm: 168.8; 163.5;
138.1; 132.4; 129.8; 129.5; 129.4 (2C); 129.3; 59.2; 51.0;
49.2; 28.9; 21.2; 5.2. Found, %: C 63.25; H 6.74; N 20.45.
C18H23N5O2. Calculated, %: C 63.32; H 6.79; N 20.51.
2-(4-Chlorophenyl)-N-cyclohexyl-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide
(3c).
Yield 0.368 g (95%), white solid, mp 215–217°C. IR spectrum,
ν, cm–1: 3118, 1669, 1623, 1441, 1062. 1H NMR spectrum,
δ, ppm (J, Hz): 8.35 (1H, d, J = 7.8, NH); 7.61 (1H, s,
H triazole); 7.50 (2H, d, J = 8.0, H Ar); 7.31 (2H, d,
J = 8.2, H Ar); 6.30 (1H, s, CH); 5.22 (2H, dd, J = 17.7,
J = 17.3, CH2); 4.93 (1H, d, J = 16.1, CH2 pyrazine); 4.11 (1H,
d, J = 16.1, CH2 pyrazine); 3.63–3.61 (1H, m, CH cyclohexyl);
1.80–1.53 (5H, m, CH2 cyclohexyl); 1.30–1.06 (5H, m, CH2
cyclohexyl). 13C NMR spectrum, δ, ppm: 167.6; 163.7;
134.2; 133.5; 131.3; 129.5; 129.4; 129.3; 58.6; 49.3; 48.3;
32.6; 25.6; 25.0; 24.9. Found, %: C 58.78; H 5.65; N 18.09.
C19H22ClN5O2. Calculated, %: C 58.84; H 5.72; N 18.06.
N-Cyclohexyl-2-(4-methylphenyl)-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3d).
Yield 0.345 g (94%), white solid, mp 163–165°C. IR spectrum,
ν, cm–1: 3119, 1664, 1628, 1439, 1071. 1H NMR spectrum,
δ, ppm (J, Hz): 8.49 (1H, d, J = 10.0, NH); 7.79 (1H, s,
H triazole); 7.42 (2H, d, J = 8.0, H Ar); 7.37 (2H, d,
J = 8.0, H Ar); 6.47 (1H, s, CH); 5.20 (2H, dd, J = 17.6,
J = 17.5, CH2); 5.12 (1H, d, J = 15.0, CH2 pyrazine); 4.24
(1H, d, J = 16.1, CH2 pyrazine); 3.81–3.79 (1H, m, CH
cyclohexyl); 3.55 (3H, s, CH3); 1.94–1.71 (5H, m, CH2
cyclohexyl); 1.43–1.28 (5H, m, CH2 cyclohexyl). 13C NMR
spectrum, δ, ppm: 168.1; 163.6; 138.2; 132.1; 129.9; 129.4;
129.3; 59.0 (2C); 49.2; 48.3; 32.7; 32.6; 25.6; 25.0; 24.9;
21.1. Found, %: C 65.43; H 6.90; N 18.99. C20H25N5O2.
Calculated, %: C 65.37; H 6.86; N 19.06.
N-tert-Butyl-2-(4-chlorophenyl)-2-(6-oxo-6,7-dihydro-
[1,2,3]triazolo[1,5-a]pyrazin-5(4H)-yl)acetamide (3e).
Yield 0.347 g (96%), white solid, mp 221–223°C. IR spectrum,
ν, cm–1: 3126, 1665, 1619, 1442, 1069. 1H NMR spectrum,
δ, ppm (J, Hz): 8.14 (1H, s, NH); 7.61 (1H, s, H triazole); 7.51
(2H, d, J = 8.2, H Ar); 7.29 (2H, d, J = 8.1, H Ar); 6.29 (1H, s,
CH); 5.23 (2H, dd, J = 17.5, J = 17.3, CH2); 4.93 (1H, d,
J = 16.0, CH2 pyrazine); 4.03 (1H, d, J = 16.2, CH2 pyrazine);
1.27 (9H, s, C(CH3)3). 13C NMR spectrum, δ, ppm: 168.3;
163.7; 134.5; 133.5; 131.3; 129.5; 129.4; 129.3; 58.7; 58.6;
51.2; 49.3; 28.8. Found, %: C 56.47; H 5.63; N 19.27.
C17H20ClN5O2. Calculated, %: C 56.43; H 5.57; N 19.36.
493

Chemistry of Heterocyclic Compounds 2020, 56(4), 488–494
11. Ma, L.-Y.; Pang, L.-P.; Wang, B.; Zhang, M.; Hu, B.; Xue, D.-Q.;
6.30 (1H, s, CH); 5.23 (2H, dd, J = 17.4, J = 17.4, CH2);
4.96 (1H, d, J = 16.2, CH2 pyrazine); 4.00 (1H, d, J = 16.1,
CH2 pyrazine); 2.90 (1H, septet, J = 13.6, CH(CH3)2); 1.29
(9H, s, C(CH3)3); 1.21 (6H, d, J = 8.7, CH(CH3)2).
13C NMR spectrum, δ, ppm: 168.8; 163.5; 148.9; 132.9;
129.5 (2C); 129.3; 127.2; 59.0; 51.1; 49.2; 33.6; 28.9; 24.3;
24.2. Found, %: C 65.07; H 7.29; N 18.93. C20H27N5O2.
Calculated, %: C 65.02; H 7.37; N 18.96.
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Evaluation of in vitro antibacterial activity. The
minimum inhibitory concentration (MIC) of all samples
was evaluated by microdilution method according to the
guidelines presented by Clinical and Laboratory Standards
Institute.27 To achieve this purpose, a serial dilution of each
sample was performed in a 96-well microplate using
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To prepare bacterial suspensions for inoculation, this
suspension was diluted to yield 5 × 106 CFU/ml. Finally,
10 µl of the prepared suspension was inoculated into each
microplate well. After an overnight incubation at 37°C, the
optical density of the wells was obtained by an ELISA
reader apparatus (BioTek, Power Wave XS2) at wave-
length 600 nm. In this assay, ampicillin and culture media
served as positive and negative controls, respectively. The
MIC was defined as lowest concentration of antimicrobial
agents that inhibited 90% of the bacterial growth after an
overnight incubation in comparison with negative control.
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13C NMR spectra of all syntesized compounds is available at
the journal website at http://link.springer.com/journal/10593.
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