Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone derivatives: DFT calculations and biological activities

Article abstract of DOI:10.1007/s00706-021-02764-0

The present work describes the synthesis, characterization, and biological activities of a novel series of isoxazole linked benzimidazolones. The structures of the target compounds and intermediates were characterized by ¹H NMR, ¹³C NMR, and HRMS. Furthermore, all isolated molecules were evaluated for their antioxidant and antimicrobial activities. Some of these compounds were found to be the most potent antimicrobial agents. Moreover, most compounds exhibited significant scavenging activity on DPPH assay. Besides, the compounds’ chemical reactivity was also assessed theoretically through Conceptual Density Functional Theory (CDFT) indices. The wB97X-D/cc-pVDZ calculation allowed to classify some derivatives as moderate-to-strong electrophiles and strong nucleophiles. Graphic abstract: [Figure not available: see fulltext.].

Full text of DOI:10.1007/s00706-021-02764-0

Monatshefte für Chemie - Chemical Monthly (2021) 152:523–535
https://doi.org/10.1007/s00706-021-02764-0
ORIGINAL PAPER
Microwave‑assisted synthesis of (3,5‑disubstituted isoxazole)‑linked
benzimidazolone derivatives: DFT calculations and biological activities
Sana Ibrahim · Ameni Ghabi · Nesrine Amiri · Hasan Mtiraoui · Melek Hajji · Radhouane Bel‑Hadj‑Tahar4,5
1
1
2
1
3
·
Moncef Msaddek1
Received: 20 December 2020 / Accepted: 5 April 2021 / Published online: 5 May 2021
©
Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
The present work describes the synthesis, characterization, and biological activities of a novel series of isoxazole linked
1
13
benzimidazolones. The structures of the target compounds and intermediates were characterized by H NMR, C NMR,
and HRMS. Furthermore, all isolated molecules were evaluated for their antioxidant and antimicrobial activities. Some of
these compounds were found to be the most potent antimicrobial agents. Moreover, most compounds exhibited signiꢀcant
scavenging activity on DPPH assay. Besides, the compounds’ chemical reactivity was also assessed theoretically through
Conceptual Density Functional Theory (CDFT) indices. The wB97X-D/cc-pVDZ calculation allowed to classify some
derivatives as moderate-to-strong electrophiles and strong nucleophiles.
Graphic abstract
Keywords N-Isopropenyl benzimidazolone · Click chemistry · 3,5-Disubstituted isoxazole · Terminal alkyne ·
Antimicrobial · Antioxidant · DFT calculations
*
Melek Hajji
melek.hajji@ipeik.u-kairouan.tn;
melekhajji.univk@gmail.com
Introduction
1
Among the various nitrogenic heterocycles, benzimida-
zolone derivatives are broadly distributed in many synthetic
and natural products [1, 2]. A literature survey revealed that
fused benzimidazolone is of great biological interest regard-
ing its anti-HIV [3, 4], antibacterial [5], antituberculosis [6],
antifungal [7], antimicrobial [8], and anticancer [9] activ-
ity. Droperidol [10], ꢁibanserin [11], domperidone [12],
and sumanriol [13] are examples of marketed drugs bearing
benzimidazolone ring. On the other hand, isoxazole and its
derivatives have attracted the attention of medicinal chem-
ists due to their usefulness in pharmacology and in synthetic
organic chemistry [14]. A good number of such heterocy-
clic scaꢂolds are found to possess a variety of interesting
Laboratory of Heterocyclic Chemistry, Natural Products
and Reactivity: L.C.H.P.N.R, Faculty of Sciences
of Monastir, University of Monastir, Avenue of the
Environnement, 5000 Monastir, Tunisia
2
Laboratory of Physico-Chemistry of Materials, University
of Monastir, Avenue of the Environment, 5019 Monastir,
Tunisia
3
4
5
Research Unit: Electrochemistry, Materials and Environment,
University of Kairouan, 3100 Kairouan, Tunisia
College of Science, Department of Chemistry, King Khalid
University, Abha 61413, Saudi Arabia
Photovoltaic Laboratory, Research and Technology Center
of Energy, Borj-Cedria Science and Technology Park, BP 95,
2
050 Hammem-Lif, Tunisia
Vol.:(0
1
123456789)
3

524
S. Ibrahim et al.
biological activities. Among these activities are its antibacte-
rial [15], antiviral [16], anti-inꢁammatory [17], antioxidant
large library wide range of molecular architectures contain-
ing 3,5-disubstitued isoxazole heterocycles. In many cases,
expensive and sensitive metallic catalysts or harsh reaction
conditions are needed and the corresponding building blocks
are usually prepared on a small scale and/or poor regiose-
lectivity. Nowadays, milder methods like click chemistry
are widely used in the preparation of these ꢀve heterocyclic
compounds, under mild reaction conditions and with excel-
lent properties such as high stability, excellent yield, and
higher regioselectivity [30]. Previous reports in the literature
suggest that the use of Cu-catalyzed in 1,3-dipolar cycload-
dition reaction of nitrile oxides and alkynes signiꢀcantly
[
18], anticancer [19, 20], anti-alzheimer [21], antileish-
manial, antiglycation [22], and antifungal [23] activities.
Figure 1 shows that some drugs based on isoxazole scaf-
fold contain sulꢀsoxazole [24], valdecoxib [25], cloxacillin
[
26], and leꢁunomide [27]. Organic chemists generally use
various methods for the synthesis of isoxazoles compounds.
Among these methods, one can cite 1,3-dipolar cycloaddi-
tion, condensation, cycloisomerization, and direct function-
alization [28, 29]. However, from a synthetic point of view,
these methods are usually not general enough to produce a
Fig. 1 Representative examples of biologically active drugs containing benzimidazolone and isoxazole derivatives
1
3

Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone…
525
improved particularly concerning yields and regioselectivity
only 3,5-disubstituted regioisomer is formed) [31]. Another
gives 4-methyl-1,5-benzodiazepin-1-one, but by prolonging
the reaction time and by thermal degradation of the diaz-
epinic intermediate (I) gives N-isopropenylbenzimidazol-
2-one (2) as a result of a [1, 3]-sigmatropic rearrangement
[33, 34]. In this report, a microwave-assisted technique was
developed for the preparation of 2 via condensation reaction
of the starting materials 1a and 1b in DMF. The precursor
N-isopropenylbenzimidazol-2-one was obtained with excel-
lent yield (96%). Using the microwave-irradiation technique
reduces the reaction time and increase yield. Mechanisti-
cally, the synthesis of the fragment 2 starts by condensation
reaction between the reagents 1a and 1b to give the interme-
diate I (4-methyl-1,5-benzodiazepin-1-one) with the elimi-
nation of ethanol molecule [35]. Intramolecular rearrange-
ment, type 1,3-sigmatropic, of the so formed intermediate
led to N-isopropenyl benzimidazolone (2). The structure of 2
(
way to synthesize valuable target compounds in higher
yields with shorter reaction times and under mild reaction
conditions is the use of microwave-irradiation technique.
To date, microwave-irradiation-assisted organic synthesis is
dominant among green methods, due to its higher selectiv-
ity, higher yield, and operational simplicity, as compared to
conventional synthesis routes which use minimization of a
byproduct formation and reduced energy consumption for
rapid synthesis of biologically active organic scaꢂolds [32].
The diverse biological properties of benzimidazolone and
isoxazole derivatives along with the advantages of micro-
wave-irradiation technology allowed us to synthesize new
hybrid compounds by combining of benzimidazol-2-one
moiety with isoxazole via 1,3-dipolar cycloaddition using
various nitrile oxides. A regiospeciꢀc, simple, versatile
copper(I)-catalyzed and microwave-assisted procedure was
used to prepare a series of isoxazoles linked to benzimi-
dazolone. The antimicrobial and antioxidant activities of
the prepared compounds were evaluated. Conceptual Den-
sity Functional Theory (CDFT) (wB97X-D/cc-pVDZ) was
applied to understand the chemical reactivity of the com-
pounds 5a, 5d, 5f, and 5h (Fig. 1).
1
13
was ascertained on the basis of their H and C NMR spec-
tral data and ES-HRMS. Subsequently, N-isopropenyl benzi-
midazol-2-one was propargylated by reaction with propargyl
bromide and potassium carbonate K CO at the reꢁux of
2
3
acetonitrile to give the corresponding N -propargyl N -iso-
1
3
propenyl benzimidazol-2-one (3) in excellent yield (95%).
The structure of the synthesized derivative 3 was evidenced
1
by their NMR. In fact, the H NMR spectrum of this scaꢂold
3
showed the absence of the singlet related to exchangeable
proton (-N H-) at δ=10.61 ppm and the presence of doublet
1
Results and discussion
at 4.61 ppm (J = 2.4 Hz) attributable to methylene group
protons (N -CH -) and a triplet at 2.23 ppm (J = 2.4 Hz)
1
2
1
3
Chemistry
corresponding to the acetylenic proton (–C≡C–H). The C
NMR spectra of 3 displayed in particular three new signals
at δ=30.4 ppm, 72.8 ppm, and 76.6 ppm are attributable to
carbons C , C , and C of propargyl group, respectively,
In view of the above facts, we planned to prepare 3,5-disub-
stituted isoxazole linked to benzimidazol-2-one to study the
antibacterial, antifungal, and antioxidant activities of these
privileged heterocyclic compounds. To achieve this goal, it
was necessary to ꢀrst synthesize the N-isopropenyl benzimi-
dazol-2-one. Generally, the condensation of o-phenylenedi-
amine (1a) with ethyl acetoacetate (1b), in reꢁuxed xylene
1
′
2′
3′
(Scheme 1).
Having established appropriate access to the inter-
mediate key 3, its triple-bond group made it a valuable
key precursor to obtain the desired 5a-5k hybrid com-
pounds through the 1,3-dipolar cycloaddition reaction.
Scheme 1
1
3

526
S. Ibrahim et al.
Furthermore, this type of reaction depends on the substrate
and reaction conditions (solvent, temperature, and amount
of catalyst). At the outset of our studies, we considered the
cycloaddition reaction between the N-hydroxy-4-methoxy-
benzenecarboximidoyl chloride (4c), N-hydroxy-4-chlo-
robenzenecarboximidoyl chloride (4g), and benzimidazol-
Starting by uncatalyzed 1,3-dipolar cycloaddition reac-
tion between alkyne 3 (1.4 mmol) and two equivalents of
N-hydroxy-4-chlorobenzenecarboximidoyl chloride (4g) at
100 °C with the presence of Et N resulted in a mixture of
3
two compounds: the 3,5-disubstituted isoxazole and unex-
pected product contained once 3,5-disubstituted isoxazoles
and 3,5-disubstituted isoxazoline after 2 days (ratio~78/22).
The yield and the structures of the products were determi-
2
-one-alkyne 3 as model substrates under conventional
heating and microwave irradiation in the presence or
absence of CuI catalyst. The most suitable conditions of
this study are shown in Table 1; Scheme 2.
1
nated from the H NMR spectrum of the crude cycloadduct
which showed the duplication of signals in the aromatic
Table 1 Optimization of the
Entry Equiv. CuIb Solvent
Yield/%d
Equiv. Et N Condition Time/h Formed isomer
a
3
cyclization reaction
1
2
3
4
5
6
7
8
9
0
Toluene
DCM
DCM
DCM
DMF
2
2
2
2
2
2
2
2
2
100 °C
48
4
8
20
20
4
6
0.1
0.1
Mixture
35
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
rt
rt
rt
rt
rt
rt
3,5-disubstituted 50
3,5-disubstituted 60
3,5-disubstituted 60
3,5-disubstituted 52
t-BuOH/H O
No reaction
0
2
THF/H O
3,5-disubstituted 30
3,5-disubstituted 70
3,5-disubstituted 90
2
c
t-BuOH/H O
DMF
MW
2
MWc
Bold in entry highlights the optimal reaction conditions:
a
Alkyne 3 (1.4 mmol) and 2 equiv of arylnitrile oxides 4c or 4 g
b
Referred to the starting alkyne 3
c
The reaction was performed at 300 W
Isolated yield after column chromatography based on the starting dipolarophile 3
d
Scheme 2
1
3

Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone…
527
region corresponding to the aromatic protons introduced by
the phenyl ring of arylnitoloxide 4g with the appearance of
two doublets attributed to the methylene proton of isoxazo-
line and a singlet attributable to the proton of the isoxazole
moiety (Table 1, entry 1). In this case, the Cu catalyzed reac-
tion between the dipolarophile 3 with N-hydroxy-4-meth-
oxybenzenecarboximidoyl chloride (4c) at room temperature
oꢂered specially 50% of the 3,5-rigioisomere isoxazole after
with classical heating (Table 1, entry 8). Interestingly, the
reaction under microwave conditions with the presence of
0.1 eq of catalyst CuI and triethylamine in DMF was the key
to obtain the highest yield (90%) of the desired isoxazole
3,5-disubstuted (Table 1, entry 9). The optimal condition
(Table 1, entry 9) was applied in a regiospeciꢀc approach
between 1-(prop-1-en-2-yl)-3-(prop-2-yn-1yl)-benzimida-
zol-2-one (3) and various arylnitrile oxides 4a-4k in the
4
h in DCM (Table 1, entry 2). A marked improvement of
presence of CuI catalyst and Et N resulting in the formation
3
the yield (up to 60%) was observed by prolonging reaction
time for the formation of the desired product (Table 1, entry
of isoxazole derivatives 5a-5k in excellent yields. All reac-
tions were carried out under microwave irradiation (300 W)
and completed within 4–7 min. The new compounds 5a-5k
were obtained in yields ranging from 82 to 95% (Table 2).
This method is the simplest, the most regiospeciꢀc, giving
the best yields at shorter reaction times than those reported
in the literature (Scheme 3).
3
, entry 4) at room temperature. Using the same conditions
in DMF at room temperature led to the isoxazole 3,5-disub-
stuted with a decrease in yield (Table 1, entry 5). Moreover,
when we worked in mixed-solvent systems with the same
conditions, there was no conversion even after 1 h. This
may be explained by the poor solubility of substrate 4 in
such solvent (Table 1, entry 6). Comparatively, the use of
The structures of the target compounds isoxazole-ben-
1
zimidazolone 5a-5k were evidenced by their spectral ( H
1
3
1
THF-H O as solvent with the same conditions resulted in
and C NMR) and ES-HRMS data. In fact, the H NMR
spectra showed no signal related to acetylenic proton at
2.23 ppm. However, a new signal at 6.48–6.75 ppm (1H, s),
2
the isoxazole 3,5-disubstuted with a yield of 30% (Table 1,
entry 7). To compare the classical heating methods with the
microwave-assisted synthesis method, the desired compound
corresponding to H proton because of the proximity of the
4
5
c was synthesized under microwave irradiation. It is clear
isoxazolinic oxygen, and the appearance of new character-
istic signals attributable to the aromatic protons introduced
by aromatic arylnitroloxide. The same features are appeared
that in this case, much shorter reaction time and higher yield
were achieved under microwave irradiation as compared
1
3
in C NMR spectra, showing essentially the disappearance
of the signal relating to terminal carbon of alkyne group at
δ=72.9 ppm, and the appearance of a new signal of the ter-
Table 2 One-pot synthesis of 3,5-disubstituted isoxazoles 5a-5k
Entry
Compound
R1
R2
Time /min
Yield/%
tiary carbon C at 101.2–108 ppm after cycloaddition. Fur-
4
thermore, all products exhibited a peak at 166.7–170 ppm,
1
2
3
4
5
6
7
8
9
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
OCH3
H
OH
H
Cl
H
H
H
H
6
5
4
5
7
6
5
4
4
6
7
86
89
90
86
83
82
88
92
89
84
95
which is characteristic of the quaternary carbon C of the
5
CH3
OCH3
H
OH
H
Cl
H
Br
F
NO2
isoxazole ring. These chemical values’ shift clearly conꢀrms
the regiochemistry of the cycloaddition and are in line with
the values reported in the literature [36, 37]. Further proof of
our structural assignment stemming from mass spectrometry
analysis is also in accordance with the proposed structure
for the adducts.
1
For the regioisomer 5c, the H NMR spectra showed a
new signal at δ=6.48 ppm attributable to the proton H and
4
1
1
0
1
5j
5k
signals in the aromatic region (6.90–7.65 ppm) correspond-
ing to aromatic protons introduced by the phenyl ring of
Scheme 3
1
3

528
S. Ibrahim et al.
arylnitroloxide derivative 4c. Particular characteristics are
nitro groups combined to the terminal phenyl, which may
also be responsible for higher antibacterial activity of these
species [41].
1
3
the C NMR isoxazole ring resonances with three peaks at
δ=101.2, 161.2, and 167.3 ppm for the isoxazole carbons
C , C , and C . Also, mass spectrometry analysis corrobo-
4
5
3
rates the proposed structure for the compound 5c.
Antifungal activity
In vitro antifungal activity of the novel benzimidazolone
derivatives against two fungal strains (A. brasiliensis and A.
fumigatus) and two yeast strains (C. albicans and C. neofor-
mans) were assessed using well agar diꢂusion. The diam-
eters of inhibition zones observed with the well diꢂusion
method are presented in Fig. 3. Overall, the solvent used to
prepare the compound solutions (DMSO) did not show any
inhibition against the tested organisms (negative control).
It was also observed that the strain C. neoformans was the
most resistant. In fact, all compounds exhibited low activ-
ity against this yeast strain with inhibition zones between 0
Antibacterial activity
Benzimidazolone derivatives are widely known for their sig-
niꢀcant antibacterial activity [38–40]. In the present study,
the synthesized compounds 5a-5k were evaluated for their
antibacterial activity. These compounds were tested at a con-
3
centration of 100 μg/cm against three Gram-positive (B.
subtilis, E. faecalis, and S. aureus) and three Gram-negative
(E. coli, P. aeruginosa, and S. typhi) bacteria using well agar
diꢂusion assay. The diameters of inhibition zones are shown
in Fig. 2 and compared by standard antibiotic ampicillin. All
compounds displayed strong activity against the most strains
and the data revealed that the strains E. coli, P. aeruginosa,
S. aureus, and B. subtilis were the most sensitive. Based on
zones of inhibition results and a part from the nature of the
substituents in the para- and/or ortho-positions of the ter-
minal phenyl, the lowest activity was observed for the com-
pounds 5a and 5b. The addition of the methoxy group (5c
and 5d) and halogen groups (5g, 5h, 5i, and 5j) increased the
activity. For example, 5d derivative showed a zone inhibition
3
3
mm and 2 mm . Interestingly, under identical experimental
conditions 5e, 5f, 5g, 5h, 5i, 5j, 5k showed higher inhibi-
tory eꢂect on the growth of fungi than 5a, 5b, 5c, and 5d
molecules (Fig. 3). In other words, compounds possessing
nitro, hydroxyl groups, and halogenated derivatives on the
phenyl ring showed remarkable growth inhibition. These
3
derivatives’ inhibition zone showed between 12 mm and
3
3
17 mm in diameter against A. fumigatus and 11 mm to
3
16 mm against C. albicans and A. brasiliensis. Moreover,
these compounds exhibited weaker antifungal activity than
3
diameter of 15 mm against E. coli and S. aureus bacteria,
the standard drug ꢁuconazole [42], which owns 24 mm
3
5
i have a 19 mm diameter of zone inhibition against P.
in diameter of zone inhibition against C. albicans. The lit-
erature survey revealed that the antifungal activity of these
benzimidazolone derivatives is comparable to and, in some
cases, even higher, than previously synthesized and studied
analogous benzimidazolones [45].
aeruginosa, E. coli, and S. aureus. Interestingly, 5e, 5f, and
5
k compounds showed better antibacterial activity, which
3
reached a value of 26 mm against the strain E. coli. These
values are higher than the standard drug tetracycline [39].
In addition, these compounds also contain a hydroxyl and
Fig. 2 In vitro antimicrobial
activity
1
3

Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone…
529
Fig. 3 In vitro antifungal activ-
ity
Antioxidant activity by DPPH radical scavenging
method
nitro and hydroxyl groups at diꢂerent positions in the phe-
nyl ring (compounds 5k, 5e, and 5f). The presence of nitro
group in any organic molecule in general confers signiꢀcant
biological activity [43, 44]. Moreover, the phenolic groups
were the major complementary compounds of propolis that
had beneꢀcial eꢂects as natural antioxidants and prevent
oxidative damage of DNA caused by reactive oxygen spe-
cies [45]. Among the halogenated derivatives (compounds
5g, 5h, 5i, and 5j), the ꢁuoro group produced signiꢀcant
% RSA, i.e., 60.67 as compared to the bromo and chloro
groups (% RSA 50.57 and~49, respectively). In the methoxy
derivatives, it was found that the compounds containing one
methoxy group (compounds 5c and 5d) showed low level
of antioxidant activity (% RSA 33.60 and 31.54). Similarly,
The scavenging activities of the synthesized compounds 5a-
5
k, and standard (vitamin C) against DPPH were evaluated.
The antioxidant activity results (% RSA: percentage of radi-
cal scavenging activity) and IC values are shown in Fig. 4
5
0
and Table 3, respectively. It is evident from the results that
free radical scavenging activity depends on the concentration
and basic skeleton of the molecule as well as on the nature of
substituents. % RSA values revealed that all compounds are
found to be more active than the standard (compound 5a).
Highly signiꢀcant antioxidant results (% RSA 73.08, 65.37,
and 67.66) were expressed by the derivatives containing
Fig. 4 DPPH radical scavenging
activity of the benzimidazolone
derivatives 5a-5k
1
3

530
S. Ibrahim et al.
Table 3 IC50 values of benzimidazolone derivatives for DPPH scav-
Computational analysis of chemical reactivity
properties: a conceptual DFT study
enging assay. Vitamin C was used as standard
−3
Compounds/standards
IC /mg cm
50
The chemical reactivity of isoxazol-benzimidazolone com-
pounds was investigated through conceptual density func-
tional theory (CDFT) approach, referred to as Chemical
Reactivity Theory [46, 47]. All calculations were accom-
plished at wB97X-D/cc-pVDZ level of theory. Gaussian
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
4.3185
2.4267
1.4601
1.4673
0.0244
0.0256
0.0625
0.0673
0.0609
0.0408
0.0196
0.0078
0
9, Rev D.01 software [48] was employed in conjunction
with GaussView 6.0 [49] and AVOGADRO 1.2.0 molecular
viewers [50]. The energy levels and surfaces of the high-
est occupied (HOMO) and the lowest unoccupied (LUMO)
molecular orbitals are illustrated in Figs. 5, 6. The calculated
four global reactivity descriptors are shown in Table 4.
As can be seen, the electron density distribution at HOMOs
of four derivatives was delocalized on their benzimidazolone
rings. On the other hand, LUMO isosurfaces have a relatively
uniform electron density over the isoxazole ring. The predicted
j
k
Vitamin C
compound 5b showed low level of radical scavenging activ-
ity (% RSA 27.65).
chemical potential (μ) (ꢀ ≈ 1∕2(E
+ E
)) for 5a and
LUMO
HOMO
5h was found to be −3.422 and −3.402 eV, respectively. While
5d and 5f exhibited relatively lower μ values with −3.247 and
−3.277 eV, respectively. Additionally, the calculated chemical
The results of the radical scavenging were also expressed
in terms of half-inhibition concentration (IC ) which denotes
50
the concentration required to scavenge 50% of DPPH radi-
cals, such as the higher IC values represent lower antioxi-
hardness (ꢀ) (donated as ꢀ ≈ (E
− E
)) values are
LUMO
HOMO
comparable and range from 4.412 eV (5h) to 4.611 eV (5a).
50
2
dant activity. These values are shown in Table 3 and com-
pared to that of vitamin C. The obtained results are 2.426
for 5b,~1.46 for methoxy derivatives,~0.06 for halogenated
derivatives,~0.0196 nitro group, and 0.0078 for vitamin C.
Moreover, the nitro and hydroxyl groups were found to be
more eꢂective to inhibit the formation of DPPH radicals.
The global electrophilicity index (ꢀ = ꢁ ∕2ꢂ) values
were in the range of 1.146–1.311 eV. The higher elec-
trophilicity power is shown for 5h and the lower one is
observed for 5d. According to Domingo’s electrophi-
licity scale [51], all studied molecules are classified as
moderate-to-strong electrophiles. Furthermore, the global
Fig. 5 Electron density distribu-
tion and energies of frontier
molecular orbitals calculated at
wB97X-D/cc-pVDZ level, for
compounds 5a and 5d
1
3

Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone…
531
Fig. 6 Electron density distribu-
tion and energies of frontier
molecular orbitals calculated at
wB97X-D/cc-pVDZ level, for
compounds 5f and 5h
Table 4 Chemical reactivity descriptors values (in eV) calculated at
and 5h exhibited excellent antibacterial activity particularly
against P. aeruginosa, E. coli, and S. aureus and showed the
highest antifungal activity against A. fumigatus, C. albicans,
and A. brasiliensis. Similarly, these three compounds dis-
played the most signiꢀcant antioxidant activity. Theoretical
DFT-based study has also been performed to explain the
reactivity behaviors of the products 5a, 5d, 5f, and 5h. All
studied molecules could be classiꢀed as moderate-to-strong
electrophiles.
wB97X-D/cc-pVDZ level of theory
Compound Electronic
chemical
Chemical
hardness
Global
Global
electrophilic- nucleophi-
potential (ꢀ) (ꢀ)
ity (ꢀ)
licity (N)
5
5
5
5
a
d
f
−3.422
−3.247
−3.277
−3.402
4.611
4.598
4.525
4.412
1.269
1.146
1.186
1.311
3.392
3.574
3.580
3.512
h
nucleophilicity (N) is calculated based on the HOMO ener-
gies:N = E − E , tetracyanoethylene (TCE)
Experimental
HOMO
HOMO(TCE)
was taken as reference, because it behaves lower HOMO
energy (−9.12 eV). Notably, from the nucleophilicity
index scale, it may be seen that the title species are strong
nucleophiles as they show nucleophilicity power larger than
All solvents were puriꢀed and dried using standard meth-
1
13
ods. H NMR (300 MHz) and C NMR (75 MHz) spectra
were recorded at room temperature in deuterated CDCl3
and DMSO-d6 on a Bruker AC-300 using residual non-
deuterated solvent signal as internal reference. High-res-
olution mass spectra (HRMS) were obtained with Micro-
mass LCT (ESI technique, positive mode) spectrometers.
The microwave was a Biotage AB Initiator EXP EU with
a maximum power of 800 W (2450 MHz). Coupling con-
stants are given in Hz and all chemical shifts were reported
as values (ppm). Signals multiplicities are using the fol-
lowing abbreviations: s (singlet), d (doublet), dd (double
doublet), t (triplet), m (multiplet), etc. Melting points
were determined on a Büchi-510 apparatus using capil-
lary tubes. Column chromatography puriꢀcations were
performed on silica gel (40–63 μm). All reactions were
monitored by TLC using aluminum sheets of sds silica gel
60 F254, 0.2 mm.
3
.00 eV [52] (Fig. 6).
Conclusion
In conclusion, a new series of benzimidazolone-linked
3
,5-disubstitued isoxazoles was designed and synthesized
via regiospeciꢀc 1,3-dipolar cycloaddition reaction cata-
lyzed by CuI and microwave-assisted N -propargyl N -iso-
1
2
propenylbenzimidazol-2-one with some arylnitroloxides 4a-
4
k. The easy process, short reaction time, and high yield of
isolated products make this method attractive for the synthe-
sis of potential biologically active molecules. All the newly
prepared species 5a-5k have been tested for their in vitro
antimicrobial and antioxidant activities. Compounds 5g
1
3

532
S. Ibrahim et al.
1
,3‑Dihydro‑1‑isopropenyl‑2H‑benzo[d]imidazol‑2‑one
removal of solvent in vacuum, the resulting residue was
puriꢀed by column chromatography on silica gel and eluted
with cyclohexane: ethyl acetate (from 9:1 to 7:3) to give the
pure new cycloadducts 5a-5k in 82–95% yield.
(
2, C H N O) A mixture of 10 g o-phenylenediamine (1a,
10 10 2
0
.092 mol) and 12 g ethyl acetoacetate (1b, 0.092 mol) was
3
stirred in 15 cm dry DMF. The reaction mixture was stirred
under irradiation (300 W) for 5 min. The water and ethanol
formed were removed progressively by extraction with ether
1,3‑Dihydro‑1‑[(3‑phenylisooxazol‑5‑yl)methyl]‑3‑(pr
diethylic. The organic phase was dried with MgSO and con-
op‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one (5a, C H
4
20 17
1
centrated. The mixture was then cooled, and the precipitated
brown solid was collected by ꢀltration, washed two times
with ether, dried in the oven to give the N-isopropenylben-
N O ) White solid; m.p.: 138–140 °C; yield: 200 mg (86%); H
3
2
NMR (300 MHz, CDCl ): δ=2.18 (d, 3H, J=3 Hz, CH ), 5.15 (m,
3
3
3H, H , H ), 5.30 (d, 1H, J=3 Hz,H ), 6.48 (s, 1H, CH ),
6
7a
7b
isoxazol
zimidazolone 2. Yield: 15 g (96%); brown solid; m.p.: 122–
7.02–7.06 (m, 4H, H ), 7.33–7.36 (m, 4H, H ), 7.66–7.69 (m,
arom arom
1
13
1
5
24 °C; H NMR (300 MHz, CDCl ): δ=2.18 (s, 3H, CH ),
1H, H ) ppm; C NMR (75 MHz, CDCl ): δ=20.4 (CH ), 36.7
arom 3 3
3
3
.24 (s, 1H, CH ), 5.40 (d, 1H, J=1.2 Hz, CH ), 7.04–7.13
(CH ), 101.5 (C ), 108.5 (CH ), 109.5 (CH ), 113.6 (CH ),
2 4 arom arom 2
2
2
13
(
m, 4H, H ), 10.67 (s, 1H, NH) ppm; C NMR (75 MHz,
122.2 (CH ), 122.2 (CH ), 122.3 (CH ), 127 (CH ), 127
arom arom arom arom
arom
CDCl ): δ = 20.4 (CH ), 109.2 (CH
), 110.1 (CH
),
(CH ), 128.8 (Cq), 129.1 (Cq), 129.2 (CH ), 129.2 (CH ),
arom arom arom
3
3
arom
arom
1
1
14.1 (CH ), 121.5 (CH ), 122.1 (CH ), 128.6 (Cq),
130.4(Cq), 138 (Cq), 152.5 (C=O), 163 (C ), 167.6(C ) ppm;
5 3
2
arom
arom
+
30.1 (Cq), 137.9 (Cq), 154.9 (C=O) ppm; HRMS (ESI):
HRMS (ESI): m/z calcd for C H N NaO ([M+Na] ) 354.1208,
20 17 3 2
+
m/z calcd for C H N NaO ([M + Na] ) 197.0703, found
found 354.1213.
1
0
10
2
1
97.0704.
1
,3‑Dihydro‑1‑(prop‑1‑en‑2‑yl)‑3‑[[3‑(p‑tolyl)isoxa‑
1
,3‑Dihydro‑1‑(prop‑1‑en‑2‑yl)‑3‑(prop‑2‑yn‑1‑yl)‑2H
zol‑5‑yl]methyl]‑2H‑benzo[d]imidazol‑2‑one (5b, C21
H N O ) Yellow solid; m.p.: 145–147 °C; yield: 215 mg
‑
benzo[d]imidazol‑2‑one (3, C H N O) To a solution
1
3
12
2
19
3
2
1
of 4 g N-isopropenylbenzimidazol-2-one (2, 23 mmol) in
(89%); H NMR (300 MHz, CDCl ): δ=2.25 (s, 3H, CH ),
3 3
3
3
1
00 cm acetonitrile at 80 °C was added 1.53 cm 3-bromo-
2.38 (s, 3H, CH ), 5.23 (m, 3H, H , H ), 5.38 (d, 1H,
3
6
7a
propyne (1.1 mmol) and K CO (46 mmol) for 12 h. The
J = 3 Hz,H ), 6.53 (s, 1H, CH
), 7.12–7.13 (m, 4H,
2
3
7b
isoxazol
crude reaction was ꢀltered through Celite and the ꢀltrate
was concentrate under reduced pressure to give a residue.
The mixture obtained was purified by recrystallization
H
H
), 7.21 (d, 2H, J=9 Hz, H ), 7.64 (d, 2H, J=6 Hz,
arom
arom
1
3
) ppm; C NMR (75 MHz, CDCl ): δ = 20.4 (CH ),
arom
3
3
21.6 (CH ), 36.7 (CH ), 101.4 (C ), 108.5 (CH ), 109.5
3
2
4
arom
from ether diethylic to obtain colorless crystals. Yield:
(CH ), 113.6 (CH ), 122.2 (CH ), 122.3 (CH ), 126
arom 2 arom arom
1
4
.6 g (95%); brown solid; m.p.: 123–125 °C; H NMR
(CH ), 126.9 (CH ), 126.9 (CH ), 129 (Cq), 129.2
arom arom arom
(
(
300 MHz, CDCl ): δ=2.14 (t, 3H, J=0.6 Hz, CH ), 2.22
(Cq), 129.8 (CH
), 129.8 (Cq), 138 (Cq), 140.5 (Cq),
arom
3
3
t, 1H, J=2.7 Hz, CH C≡CH), 4.61 (d, 2H, J=2.7 Hz, N-
152.5 (C= O), 162.9 (C ), 167.4 (C ) ppm; HRMS (ESI):
5 3
2
+
CH ), 5.12 (d, J = 0.6 Hz, 1H, CH ), 5.27 (m, 1H, CH ),
m/z calcd for C H N NaO ([M+Na] ) 368.1369, found
21 19 3 2
2
2
2
13
7
.03–7.14 (m, 4H, H ) ppm; C NMR (75 MHz, CDCl ):
368.1369.
arom
3
δ=20.4 (CH ), 30.4 (CH ), 72.8 (C≡CH), 76.6 (CH), 108.7
3
2
(
(
CH ), 109.3 (CH ), 113.4 (CH ), 121.9 (CH ), 122
1,3‑Dihydro‑1‑[[3‑(4‑methoxyphenyl)isoxazol‑5‑yl]
methyl]‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(5c, C H N O ) Yellow solid; m.p.: 142–144 °C; yield:
arom
arom
2
arom
CH ), 128.8 (Cq), 129.1. (Cq), 137.9 (Cq), 152.1 (C=O)
arom
+
ppm; HRMS (ESI): m/z calcd for C H N NaO ([M+Na] )
13
12
2
21 19
3
3
1
2
35.0802, found 235.0804.
230 mg (90%); H NMR (300 MHz, CDCl ): δ = 2.23 (s,
3
3
H, CH ), 3.81 (s, 3H, OCH ), 5.18–5.21 (m, 3H, H ,
3
3
6
H ), 5.38 (d, 1H, J = 3 Hz,H ), 6.48 (s, 1H, CH
),
7
a
7b
isoxazol
General procedure for the synthesis
of compounds 5a‑5k
6.90 (d, 2H, J = 9 Hz, H
), 7.07–7.11 (m, 4H, H
),
arom
arom
1
3
7.65 (d, 2H, J=9 Hz, 2H, H ) ppm; C NMR (75 MHz,
arom
CDCl ): δ = 20.4 (CH ), 36.7 (CH ), 55.5 (OCH ), 101.2
3
3
2
3
At room temperature and stirring, to a mixture of dipo-
(C ), 108.5 (CH ), 109.5 (CH ), 113.6 (CH ), 114.5
4 arom arom 2
larophile 3 (0.7 mmol, 1 equiv), triethylamine (2 equiv,
(CH ), 114.5 (CH ), 121.3 (CHarom), 122.2 (CH ),
arom arom arom
2
.8 mmol), and 0.1 equiv copper(I) iodide (CuI) in DMF, the
122.2 (CH
), 122.2 (CH
), 128.4 (Cq), 128.4 (Cq),
arom
arom
appropriate hydroximyl chlorides 4a-4k (2 equiv, 2.8 mmol)
were added and the mixture was stirred under microwave
irradiations at 300 W for 4–7 min. The crude reaction was
diluted with water and then extracted with CH Cl (3×20
129.2 (Cq), 138 (Cq), 152.6 (C=O), 161.2 (Cq), 162.5 (C ),
5
167.3 (C ) ppm; HRMS (ESI): m/z calcd for C H N NaO
3
21 19
3
3
+
([M+Na] ) 384.1302, found 384.1304.
2
2
3
cm ). The organic layer was dried over Na SO . After
1,3‑Dihydro‑1‑[[3‑(2‑methoxyphenyl)isoxazol‑5‑yl]
methyl]‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
2
4
1
3

Microwave-assisted synthesis of (3,5-disubstituted isoxazole)-linked benzimidazolone…
533
(
2
5d, C H N O ) Yellow solid; m.p.: 140–142 °C; yield:
36.4 (CH ), 101.2 (C ), 108.2 (CH ), 109.4 (CH ), 109.4
2
1
19
3
3
2
4
arom
), 122 (CH
2
1
20 mg (86%); H NMR (300 MHz, CDCl ): δ=2.22 (d, 3H,
(CH
), 113.4 (CH
), 122.1 (CH
),
arom
3
arom
arom
arom
J=0.6 Hz, CH ), 3.82 (s, 3H, OCH ), 5.20 (m, 3H, H , H ),
122.1 (CH ), 127.1 (CH ), 128.1 (CH ), 128.7 (Cq),
arom arom arom
3
3
6
7a
5
.35 (d, 1H, J=1.5 Hz, H ), 6.75 (s, 1H, CH ), 6.86
129 (Cq), 129.2 (Cq), 136.2 (Cq), 137.7 (Cq), 152.3 (C=O),
7
b
isoxazol
(
d, J = 9 Hz, 1H, H ), 7.09–7.10 (m, 5H, H ), 7.30–
161.8 (C ), 167.7 (C ) ppm; HRMS (ESI): m/z calcd for
arom
arom
5
3
1
3
+
7
.33 (m, 1H, H
), 7.80–7.81 (m, 1H, H
) ppm;
C
C H ClN NaO ([M+Na] ) 388.0822, found 388.0823.
arom
arom
20 16 3 2
NMR (75 MHz, CDCl ): δ=20.4 (CH ), 36.6 (CH ), 56.1
3
3
2
(
(
OCH ), 108.6 (C ), 109.4 (CH ), 109.4 (CH ), 112.9
1‑[[3‑(2‑Chlorophenyl)isoxazol‑5‑yl]methyl]‑1,3‑di‑
hydro‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(6h, C H ClN O ) White solid; m.p.: 155–157 °C; yield:
3
4
arom
arom
CH ), 113.6 (CH
), 119.1 (CHarom), 122.1 (CH
),
arom
2
arom
1
1
1
22.1 (CH ), 122.2 (CH ), 122.2 (CH ), 126 (Cq),
arom arom arom
20 16
3
2
1
29.1 (Cq), 129.2 (Cq), 131.1 (Cq), 138 (Cq), 155.8 (C=O),
235 mg (92%); H NMR (300 MHz, CDCl ): δ=2.22 (s, 3H,
3
66.4 (C ), 170 (C ) ppm; HRMS (ESI): m/z calcd for
CH ), 5.20–5.22 (s, 3H, H , H ), 5.35 (d, 1H, J=1.2 Hz,
5
3
3
6
7b
+
C H N NaO ([M+Na] ) 384.1302, found 384.1303.
H ), 6.70 (s, 1H, CH
), 7.09–7.14 (m, 4H, H
),
arom
2
1
19
3
3
7b
isoxazol
7
.29–7.45 (m, 3H, H ), 7.63–7.67 (m, 1H, H ) ppm;
arom
arom
1
3
1
,3‑Dihydro‑1‑[[3‑(4‑hydroxyphenyl)isoxazol‑5‑yl]met
C NMR (75 MHz, CDCl ): δ = 20.3 (CH ), 36.6 (CH ),
3 3 2
hyl]‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(
104.7 (C ), 108.4 (CH ), 109.5 (CH ), 113.6 (CH ),
4
arom
arom
), 127.2 (CH
2
5e, C H N O ) Brown solid; m.p.: 148–150 °C; yield:
122.1 (CH
), 122.2 (CH
), 128.1
2
0
17
3
3
arom
arom
arom
1
2
20 mg (84%); H NMR (300 MHz, CDCl ): δ=2.22 (s, 3H,
(CH ), 129 (CH ), 129.1 (CH ), 130.5 (Cq), 131.1
arom arom arom
3
CH ), 5.18–5.21 (m, 3H, H , H ), 5.36 (d, 1H, J=1.2 Hz,
(Cq), 131.1 (Cq), 131.1 (Cq), 138 (Cq), 152.4 (C = O),
3
6
7a
H ), 6.47 (s, 1H, CH
), 6.91 (d, 2H, J=9 Hz, H ),
161.5 (C ), 166.7 (C ) ppm; HRMS (ESI): m/z calcd for
7
b
isoxazol
arom
5
3
+
7
.07–7.11 (m, 4H, H ), 7.65 (d, 2H, J=9 Hz, H ) ppm;
C H ClN NaO ([M+Na] ) 388.0822, found 388.0823.
arom
arom
20 16 3 2
1
3
C NMR (75 MHz, CDCl ): δ = 20.4 (CH ), 36.7 (CH ),
3
3
2
1
1
02.2 (C ), 108.5 (CH ), 109.5 (CH ), 113.6 (CH ),
1‑[[3‑(4‑Bromophenyl)isoxazol‑5‑yl]methyl]‑1,3‑di‑
hydro‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(5i, C H BrN O ) White solid; m.p.: 150–152 °C; yield:
4
arom
arom
2
14.5 (CH
), 114.5 (CHarom), 121.3 (CHarom), 122.2
arom
(
CH
), 122.2 (CH
), 128.4 (CH
), 128.4 (Cq),
arom
arom
arom
20 16
3
2
1
1
1
29(Cq), 129.1 (Cq), 138 (Cq), 152.4 (C=O), 161.3 (Cq),
256 mg (89%); H NMR (300 MHz, CDCl ): δ=2.22 (s, 3H,
3
62.6 (C ), 167.3 (C ) ppm; HRMS (ESI): m/z calcd for
CH ), 5.20–5.21 (m, 3H, H , H ), 5.36 (d, 2H, J=2.4 Hz,
5
3
3
6
7b
+
C H N NaO ([M+Na] ) 370.1163, found 370.1164.
H ), 6.51 (s, 1H, CH
), 7.09–7.12 (m, 4H, H
),
2
0
17
3
3
7b
isoxazol
arom
13
7
.52–7.62 (m, 4H, H ) ppm; C NMR (75 MHz, CDCl ):
arom
3
),
1
,3‑Dihydro‑1‑[[3‑(2‑hydroxyphenyl)isoxazol‑5‑yl]met
δ = 20.4 (CH ), 36.6 (CH ), 101.3 (C ), 108.4 (CH
3
2
4
arom
hyl]‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one (5f,
C H N O ) White solid; m.p.: 144–146 °C; yield: 200 mg
109.6 (CH
), 113.6 (CH ), 122.2 (CH
), 122.3
arom
2
arom
(CHarom), 124.7 (CHarom), 127.7 (CH ), 128.5 (CH ),
2
0
17
3
3
arom
arom
1
(
82%); H NMR (300 MHz, CDCl ): δ=2.23 (s, 3H, CH ),
128.5 (CH ), 128.9 (Cq), 129.2 (Cq), 132.3 (Cq), 132.3
3
3
arom
5
6
7
1
.22–5.23 (m, 3H, H , H ), 5.37 (d, 1H, J=1.2 Hz, H ),
(Cq), 138 (Cq), 152.5 (C = O), 162 (C ), 168 (C ) ppm;
6
7a
7b
5
3
+
.65 (s, 1H, CH
.13 (m, 5H, H
), 6.89–7.01 (m, 1H, H
), 7.02–
HRMS (ESI): m/z calcd for C H BrN NaO ([M+Na] )
isoxazol
arom
20 16 3 2
), 7.28–7.42 (m, 2H, H
), 9.31 (s,
arom
432.0314, found 432.0318.
arom
1
3
H, OH
) ppm; C NMR (75 MHz, CDCl ): δ = 20.2
alcool
3
(
CH ), 36.3 (CH ), 108 (C ), 109.5 (CH ), 112.8 (CH ),
1‑[[3‑(4‑Fluorophenyl)isoxazol‑5‑yl]methyl]‑1,3‑di‑
hydro‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(5j, C H FN O ) White solid; m.p.: 153–155 °C; yield:
3
2
4
arom
2
1
13.5 (CH
), 116.8 (CH
), 119.8 (CHarom), 122.1
arom
arom
(
CH
), 122.2 (CH
), 122.3 (CH
), 128 (CH
),
arom
arom
arom
arom
20 16
3
2
1
1
1
28.6 (Cq), 129 (Cq), 131.7 (Cq), 137.7 (Cq), 144.6 (Cq),
205 mg (84%); H NMR (300 MHz, CDCl ): δ=2.15 (s, 3H,
3
54.7 (C= O), 166.1 (C ), 166.7 (C ) ppm; HRMS (ESI):
CH ), 5.19–5.20 (m, 3H, H , H ), 5.36 (d, 1H, J=2.4 Hz
5
3
3
6
7a
+
m/z calcd for C H N NaO ([M+Na] ) 370.1163, found
H ), 6.50 (s, 1H, CH
), 7.06–7.12 (m, 6H, H
),
2
0
17
3
3
7a
isoxazol
arom
1
3
3
70.1162.
7.68–7.74 (m, 2H, H ) ppm; C NMR (75 MHz, CDCl ):
arom
3
),
δ = 20.2 (CH ), 36.4 (CH ), 101.9 (C ), 108.2 (CH
3
2
4
arom
1
‑[[3‑(4‑Chlorophenyl)isoxazol‑5‑yl]methyl]‑1,3‑di‑
109.3 (CH
), 113.4 (CH ), 115.9 (CH
), 121.9
arom
arom
2
hydro‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(
(CHarom), 122.4 (CHarom), 124.8 (CH ), 127.6 (CH ),
arom arom
5g, C H ClN O ) White solid; m.p.: 153–155 °C; yield:
128.7 (CH ), 129 (Cq), 129.5 (Cq), 137.8 (Cq), 161.8
2
0
16
3
2
arom
1
2
25 mg (88%); H NMR (300 MHz, CDCl ): δ=2.22 (s, 3H,
(C = O), 162.2 (Cq), 165.5 (C ), 167.6 (C ) ppm; HRMS
3
5
3
+
CH ), 5.20–5.21 (m, 3H, H , H ), 5.36 (d, 1H, J=1.2 Hz,
(ESI): m/z calcd for C H FN NaO ([M+Na] ) 372.1117,
3
6
7a
20 16 4 4
H ), 6.51 (s, 1H, CH
), 7.09–7.11 (m, 4H, H
),
found 372.1123.
7
b
isoxazol
arom
7
.37 (d, 2H, J = 8.7 Hz, H
), 7.67 (d, 2H, J = 8.7 Hz,
arom
1
3
H
) ppm; C NMR (75 MHz, CDCl ): δ = 20.2 (CH ),
3 3
arom
1
3

534
S. Ibrahim et al.
1
,3‑Dihydro‑1‑[[3‑(4‑nitrophenyl)isoxazol‑5‑yl]met
incubator for 5 days to allow fungal growth. After 5 days,
the diameters of the clear zone of inhibition surrounding
the sample were measured in centimeters by digital caliper.
Yeasts strains: two yeasts strains, C. neoformans and C.
albicans, were cultured on Sabouraud agar at 37 °C (opti-
mal temperature for bacteria growth) for 48 h. Then, pure
hyl]‑3‑(prop‑1‑en‑2‑yl)‑2H‑benzo[d]imidazol‑2‑one
(
5k, C H N O ) Brown solid; m.p.: 158–160 °C; yield:
2
0
16
4
4
1
2
50 mg (95%); H NMR (300 MHz, CDCl ): δ = 2.22 (s,
3
3
H), 5.21–5.23 (m, 3H, H , H ), 5.36 (d, 1H, J = 1.2 Hz,
6
7a
H ), 6.62 (s, 1H, CH
l), 7.10–7.13 (m, 4H, H ), 7.92
7b
isoxazo
arom
3
(
d, 2H, J=9 Hz, H ), 8.27 (d, 2H, J=9 Hz, H ) ppm;
colonies were suspended in 10 cm of physiological medium
arom
arom
1
3
C NMR (75 MHz, CDCl ): δ = 20.4 (CH ), 36.6 (CH ),
and mixed well for 5 min, and suspensions were adjusted to
0.5 McFarland standard turbidity. One centimeter of yeast
suspension was spread over Sabouraud agar medium plates
3
3
2
1
1
01.6 (C ), 108.2 (CH ), 109.7 (CH ), 113.7 (CH ),
4
arom
arom
2
22.3 (CH
), 122.4 (CHarom), 122.4 (CH
), 122.4
arom
arom
3
(
CH ), 127.9 (CH ), 127.9 (CH ), 128.8 (Cq), 129.2
and incubated for 30 min at 37 °C. Then, 6 mm -diameter
arom
arom
arom
(
Cq), 134.8 (Cq), 137.9 (Cq), 148.9 (Cq), 152.4 (C = O),
wells were dug in agar medium using sterile glassy borer.
1
61.1 (C ), 168.8 (C ) ppm; HRMS (ESI): m/z calcd for
The synthesized compounds 5a-5k were prepared in DMSO
5
3
+
3
C H N NaO ([M+Na] ) 399.1065, found 399.1064.
(1 mg/cm ) and introduced into the respective wells, and
2
0
16
5
4
one of the wells was supplemented with DMSO as control.
These plates were placed in a 37 °C incubator for 48 h to
allow yeast growth. After 48 h, the diameters of the clear
zone of inhibition surrounding the sample were measured
in centimeters by digital caliper.
Antibacterial screening
The antibacterial activities of the synthesized compounds
5
a-5k were evaluated by the agar well-diꢂusion assay tech-
nique against three Gram-positive bacteria, i.e., B. subtilis,
E. faecalis, and S. aureus, and three Gram-negative bacteria,
i.e., E. coli, P. aeruginosa, and S. typhi. These pathogenic
strains were cultivated on nutrient agar at 37 °C for 24 h.
Antioxidant activity by DPPH method
The antioxidant activity of the benzimidazolone derivatives
5
a-5k was evaluated in vitro via their ability to scavenge
3
Colonies were suspended in 10 cm of physiological medium
DPPH radicals. In the experiments, diꢂerent concentrations
of our compounds were taken (prepared in DMSO) and 4.0
and mixed for 5 min, and suspensions were adjusted to 0.5
McFarland standard turbidity. One centimeter of bacterial
suspension was spread over Muller Hinton Agar medium
plates and incubated for 30 min at 37 °C. Using a punch, ≈
3
cm of DPPH (0.004%) in methanol solution was added to
our compounds’ mixture. After 30 min incubation at room
temperature, the absorbance was read against a blank at
6
mm-diameter wells were bored in the seeded agar plates
5
17 nm. DPPH radical scavenging activity was calculated
and each test compound reconstituted in DMSO was added
into the wells. DMSO was used as the control for all the test
compounds. After holding the plates at room temperature
for 2 h to allow diꢂusion of the compounds into the agar,
the plates were incubated at 37 °C for 24 h. After 24 h, the
diameters of the clear zone of inhibition surrounding the
sample were measured in millimeters by digital caliper.
using the following formula:
% Inhibition of DPPH = (A − A ∕A )x100,
1
0
0
where A was the absorbance of the control and A was the
0
1
absorbance of the presence of compounds. Vitamin C has
been used as standards and their scavenging ability was com-
pared with our results. IC (50% inhibition concentration)
5
0
values of benzimidazolone derivatives and standard (vitamin
Antifungal activity
C) were also calculated for comparison. IC value of the
5
0
sample is the required concentration to scavenge 50% of
Fungal strains: two fungi strains, A. fumigatus and A. brasil-
iensis, were cultured on inclined Sabouraud agar in Falcon
DPPH free radicals.
3
tube 15 cm at 25 °C for 7 days (optimal temperature for
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00706-021-02764-0.
their growing). The spore of fungal strain was suspended in
3
peptone water and counted to have 106 spores/cm . Then,
Acknowledgements The authors extend their appreciation to the Dean-
ship of Scientiꢀc Research at King Khalid University for funding this
work through Research Groups Project under grant number (197-40,
2019).
1
ml of fungal suspension was spread over Sabouraud agar
medium plates and incubated for 30 min at 25 °C. After
that, 6 mm-diameter wells were dug in agar medium using
sterile glassy borer. The synthesized compounds 5a-5k
3
were prepared in DMSO (1 mg/cm ) and introduced into
the respective wells, and one of the wells was supplemented
with DMSO as control. These plates were placed in a 25 °C
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