Synthesis and Antimicrobial Evaluation of Bis-3,5-disubstituted Isoxazoles Based Chalcones

Article abstract of DOI:10.1134/S1070363218090232

A series of bis-isoxazoles blended chalcones 7a–7j are synthesized in high yields. The combination of three pharmacologically active moieties in a single scaffold results in the synergistic effect in their bioactivity. All the newly synthesized compounds are characterized by IR, NMR and Mass spectroscopy. The target compounds 7a–7j are assessed for their antimicrobial activity and these demonstrate high to excellent activity against tested bacterial and fungal strains. The products 7f, 7h, 7j, and 7i demonstrate potent antimicrobial activity at concentrations 75 and 100 μg/mL.

Full text of DOI:10.1134/S1070363218090232

ISSN 1070-3632, Russian Journal of General Chemistry, 2018, Vol. 88, No. 9, pp. 1904–1911. © Pleiades Publishing, Ltd., 2018.
Synthesis and Antimicrobial Evaluation
of Bis-3,5-disubstituted Isoxazoles Based Chalcones1
V. Sunithaa*, A. Kishore Kumarb, M. Maheshc, P. Shankaraiahb,
P. Jalapathia, and Ch. Abraham Lincolnc
a Department of Chemistry, University College of Science, Osmania University,
Saifabad, Hyderabad, Telangana, 500004 India
*e-mail: vinalasunitha@gmail.com
b Department of Humanities and Sciences, Vardhaman College of Engineering,
Shamshabad, Hyderabad, Telangana, 501218 India
cDepartment of Chemistry, University College of Science, Osmania University,
Hyderabad, Telangana, 500007 India
Received July 20, 2018
Abstract—A series of bis-isoxazoles blended chalcones 7a–7j are synthesized in high yields. The combination
of three pharmacologically active moieties in a single scaffold results in the synergistic effect in their
bioactivity. All the newly synthesized compounds are characterized by IR, NMR and Mass spectroscopy. The
target compounds 7a–7j are assessed for their antimicrobial activity and these demonstrate high to excellent
activity against tested bacterial and fungal strains. The products 7f, 7h, 7j, and 7i demonstrate potent
antimicrobial activity at concentrations 75 and 100 µg/mL.
Keywords: chalcones, 1,5-disubstituted isoxazoles, antimicrobial activity
DOI: 10.1134/S1070363218090232
INTRODUCTION
anti-invasive [12], anticancer [13], antioxidant [14],
antihyperglycemic [15], anti-inflammatory [16],
analgesic [17], antiplatelet [18], and many more.
Isoxazoles, chalcones and their derivatives
demonstrate remarkable biological activity and are
used as versatile building blocks in organic and natural
compounds chemistry. Among interesting biological
activities of isoxazoles is potent and selective
antagonism of the N-methyl-D-aspartate receptor
(NMDA). These are structural blocks of well known
drugs such as valdecoxib (non-steroidal anti-inflam-
matory), leflunomide (antirheumatic), isocarboxazid
(antidepressant), sulfisoxazole (antibacterial), risperi-
done (antipsychotic), sulfamethoxazole (antibacterial),
and zonisamide (anticonvulsant). Some biologically
active chalcone and isoxazole based heterocyclic
molecules (1 [1], 2 [2], and 3 [3]) are presented in the
figure.
The above mentioned biological activities of
isoxazoles and chalcones encouraged us to make an
attempt to conjugate these two important pharma-
cophores in one molecular structure. So, bis-isoxazoles
based chalcones 7a–7j were synthesized.
RESULTS AND DISCUSSION
2,5-Disubstituted isoxazoles based chalcones 7a–7j
were synthesized by a 4-step protocol (Scheme 1).
Initially, the aromatic aldehydes 1a–1e were converted
to oximes 2a–2e using hydroxylamine hydrochloride.
Synthesis of compound 4 was readily achieved by
refluxing 1-(2,4-dihydroxyphenyl)ethanone 3 with
propargyl bromide and potassium carbonate in dry
acetone for 12 h. Structure of the product was
Chalcones (1,3-diarylprop-2-en-1-one) also demon-
strate such biological activities as antileishmanial [4],
antibacterial [5], antifungal [6], antitumour [7],
antimalarial [8, 9], antiviral [10], antitubercular [11],
1
confirmed by H NMR spectrum, which demonstrated
two doublets at 4.79 and 4.74 ppm assigned to two
OCH2 protons and characteristic triplet at 2.57 ppm
attributed to two acetylenic (≡CH) protons of
dipropagylated compound 4. Dioxazolic aceto-
1 The text was submitted by the authors in English.
1904

SYNTHESIS AND ANTIMICROBIAL EVALUATION
1905
(a)
(b)
H3CO
HO
O
O
H2N
N
N
O
O
Br
Br
O
O
N
O
N
O
H
H
1
2
(c)
O
F
F
O
O
N
3
Representative bioactive chalcone based isoxazoles: (a) (E)-4-{3-[3-(4-aminophenyl)acryloyl]-4,5-dihydroisoxazol-5-yl}-6-bromo-
4-methyl-1H-benzo[d][1,3]oxazin-2(4H)-one (1), antibacterial, (b) (E)-6-bromo-4-{3-[3-(4-hydroxy-3-methoxyphenyl)acry-loyl]-
4,5-dihydroisoxazol-5-yl}-4-methyl-1H-benzo[d][1,3]oxazin-2(4H)-one (2), antibacterial, and (c) 1-{4-[(3-{3,4-difluorophenyl}-
isoxazol-5-yl)methoxy]phenyl}-3-phenylprop-2-en-1-one (3), mushroom tyrosinase and melanin synthesis in murine B16 cells.
phenones 5a–5e were synthesized with high yields by
reacting dipropargylated compound 4a with the nitrile
oxides. The letter were generated in situ by treatment
of oximes 2a–2e with the electrophilic reagent N-
chlorosuccinimide (NCS).
or group (NO2) on isoxazole molecules could stimulate
the antibacterial activity more significantly than the
electron donating group (CH3) on isoxazole. Among
all products 7a–7j, para methoxy substituted chal-
cones with the electron withdrawing atoms or group on
isoxazole (7f and 7h) demonstrated higher activity
than the other compounds.
Bis-isoxazoles based chalcones 7a–7j were syn-
thesized by treatment of dioxazolic acetophenones 5a–
5e with benzaldehydes 6a–6b in the presence of
ethanolic KOH. The structures of all the newly syn-
thesized compounds were elucidated from IR, NMR
and MASS spectra.
Antifungal activity. Antifungal activity studies
were conducted for the compounds 7a–7j in concentra-
tions 75 and 100 μg/mL against three dermatophytes
vize, Microsporum canis, Microsporum gypseum,
Epidermophyton floccosum. Nystatin was used as the
standard drug. According to the accumulated data
(Table 2) antifungal activity was determined to be the
highest for the compounds 7f, 7h, 7j, and 7i.
Antibacterial activity. The newly synthesized bis-
isoxazoles based chalcones 7a–7j were screened for
their antibacterial activity. These compounds demon-
strated significant inhibition of the tested four gram
positive and four gram negative bacterial strains
compared to the standard drug Gentamicin sulphate
(Table 1). Among the tested compounds the products
7f, 7h, 7j, and 7i were highly active at concentrations
75 and 100 µg/mL against Micrococcus luteus,
Methicillin-resistant Staphylococcus aureus, Bacillus
subtilis, Bacillus cereus, Pseudomonas aeruginosa,
Klebsiella pneumoniae, Escherichia coli, Proteus
vulgaris (Table 1). These results indicated that
presence of the electron withdrawing atoms (Br or Cl)
EXPERIMENTAL
Commercially available reagents were used as
supplied, and all solvents were distilled before use. All
reactions were performed in an oven-dried glassware.
Melting points were measured in open capillaries
and are uncorrected. IR spectra were recorded on a
Perkin−Elmer 337 IR spectrophotometer for solid
samples pelleted in KBr. NMR spectra were measured
on a Bruker AV-400 and AV-300 NMR spectrometers
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 9 2018

1906
SUNITHA et al.
Scheme 1. Synthesis of bis-3,5-disubstituted isoxazoles based chalcones 7a–7j.
R
R
N
O
NOH
NaOAc, MeOH
DMF, NCS
N
.
NH2OH HCl
DBU,
0oC−room temperature,
R
H
R
H
O
O
room temperature,
3 h
room
2a−2e
1a−1e
O
O
O
temperature,
2 h
Br
HO
OH
O
O
O
5a−5e
O
K2CO3, acetone
reflux, 12 h
ethanol, KOH
O
3
4
8 h, room temperature
R1
H
6a−6b
R
N
R
N
O
O
O
O
R1
O
7a−7j
R = Ph, 4-(Me)Ph, 4-(Cl)Ph, 4-(Br)Ph, 4-(NO2)Ph; R1 = Ph, 4-(OMe)Ph.
for CDCl3 solutions with TMS used as an internal
standard. CHN analysis was carried out on a Perkin
Elmer Model 2400 CHNS elemental analyzer. Electron
Spray Ionization (ESI) mass spectra were measured on
a QSTARXL hybrid MS system (Applied Bio
Systems). TLC was carried out on Merck TLC silica
gel 60 F254 plates. The spots were visualized in UV
light at 254 nm or by staining with aqueous basic
potassium permanganate. Column chromatography
was performed on a Merck silica gel 60A (100–
200 mesh).
layer was dried over anhydrous sodium sulphate,
filtered and concentrated under reduced pressure. The
crude product was purified by column chromatography
using 100–200 mesh silica gel, eluted by 10% ethyl
acetate/pet ether to afford 1-(2,4-dihydroxyphenyl)-
ethanone as reddish-brown needles. Yield 76%, mp
142–144°C [19].
Synthesis
of
1-(2,4-bis-(prop-2-yn-1-yloxy)
phenyl)ethanone (4). To a stirred mixture of 1-(2,4-
dihydroxyphenyl)ethanone (3) (3 g, 0.019 mol) with
K2CO3 (7.86 g, 0.057 mol) in dry acetone propargyl
bromide 80% in toluene (6.78 g, 0.057 mol) was added
dropwise, and the reaction mixture was refluxed for ca
12 h. Upon completion of the process (TLC), the
reaction mixture was cooled down to room
temperature and the excess of acetone was evaporated
under reduced pressure. Then the mixture was diluted
by water (50 mL) and extracted with ethyl acetate
(3×50 mL). The combined organic layers were washed
with brine (2×25 mL), dried over anhydrous sodium
sulphate, filtered, and concentrated in vacuum. The
crude product was purified by column chromatography
using 100–200 mesh silica gel, eluted by 10% ethyl
Synthesis of 1-(2,4-dihydroxyphenyl)ethanone
(3). A mixture of acetic acid (3 g, 0.05 mol) with
freshly fused zinc chloride (6.8 g, 0.05 mol) was
heated to 120°C for 30 min, then, upon addition of
resorcinol (5.5 g, 0.05 mol), the reaction mixture was
heated to 140°C for 30 min while monitoring it by
TLC. Upon completion of the process, the reaction
mixture was cooled down to room temperature and
poured into ice cold water (100 mL), then extracted
with ethyl acetate (3×50 mL). The combined organic
layers were washed with 20% HCl (50 mL), saturated
NaHCO3 (25 mL) and brine (2×25 mL). The organic
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 9 2018

SYNTHESIS AND ANTIMICROBIAL EVALUATION
Table 1. Antibacterial activity of the compounds 7a–7j
1907
Zone of inhibition, mm
gram positive bacteria
gram negative bacteria
Compound
7a
75
100
75
06
09
19
22
13
16
22
25
15
18
36
40
08
11
33
36
25
28
32
35
27
30
No activity No activity
No activity No activity
05
08
23
26
13
16
27
30
13
16
31
34
07
09
29
32
28
31
27
30
31
34
08
11
18
21
11
14
23
26
12
14
31
33
07
10
29
31
26
29
26
29
28
31
06
08
19
23
12
15
20
23
12
15
33
36
06
09
30
33
24
26
26
29
27
30
07
No activity
09
No activity
20
7b
22
25
14
16
26
29
14
17
35
49
20
23
12
15
24
27
12
15
36
49
23
100
75
26
22
7c
15
14
100
75
17
16
7d
26
23
100
75
29
26
7e
15
14
100
75
18
17
7f
30
28
100
75
33
31
7g
No activity No activity
No activity No activity
No activity
07
100
75
No activity
09
7h
30
33
27
31
28
32
31
33
28
31
27
30
26
28
30
33
31
34
26
30
28
31
31
33
30
100
75
33
7i
7j
25
100
75
28
27
100
75
30
Gentamicin
29
100
31
acetate in pet ether to afford 1-(2,4-bis(prop-2-yn-1-
yloxy)phenyl)ethanone (4) as a white solid. H NMR
spectrum, δ, ppm: 7.85 d (J = 8.97 Hz, 1H), 6.68–6.63
m (2H), 4.79 d (J = 2.38 Hz, 2H), 4.75 d (J = 2.39 Hz,
2H), 2.61 s (3H), 2.57 t (J = 2.29 Hz, 2H).
stirred at room temperature for 3 h. After completion
of the process as monitored by TLC, the reaction
mixture was quenched by adding crushed ice, and the
precipitate formed was filtered off, washed
with hexane and dried to afford substituted aldoximes
2a–2e.
1
Synthesis of aldoximes 2a–2e. To a solution of an
aldehyde (1 equiv.) in methanol was added hydroxyl-
amine hydrochloride (1 equiv.) followed by sodium
acetate (1.5 equiv.). The resulting reaction mixture was
Synthesis of isoxazoles 5a–5e. For chlorination of
aldoximes 2a–2e, the solutions of aldoximes (1 equiv.)
in DMF at room temperature were mixed with NCS
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1908
SUNITHA et al.
Table 2. Antifungal activity of the compounds 7a–7j
Zone of inhibition, mm
Concentration,
Compound
µg/mL
Microsporum canis
Microsporum gypseum
Epidermophyton floccosum
7a
7b
75
100
75
No activity
08
No activity
No activity
10
No activity
12
10
09
100
75
15
13
11
7c
No activity
12
No activity
100
75
No activity
15
No activity
7d
No activity
No activity
No activity
100
75
No activity
No activity
No activity
7e
18
21
25
28
05
08
24
27
19
22
21
24
25
28
16
18
22
26
03
06
19
22
17
20
17
20
20
24
15
17
22
24
05
07
18
21
16
19
16
18
20
23
100
75
7f
100
75
7g
100
75
7h
100
75
7i
100
75
7j
100
75
Nystatin
100
(1 equiv.) and stirred for 30 min. The reaction mixture
was cooled down to 0°C and the catalytic amount of
1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (0.2 equiv.)
was slowly added. This was followed by addition of
compound 4. After consumption of the starting
materials, as indicated by TLC, ice-cold water was
added to the reaction mixture, precipitate so obtained
was filtered off, washed with water and cold MeOH to
afford the corresponding pure product.
(1 equiv.) was added and stirring was continued for 8 h
at the same temperature. After consumption of the
starting material, as indicated by TLC, ice-cold water
was added to the reaction mixture and it was
neutralized by 0.1–0.2 N HCl, at which point
precipitation occurred. The precipitate was filtered off,
washed with water and cold MeOH to afford the
corresponding pure 3,5-disubstituted isoxazoles based
chalcones 7a–7j.
Synthesis of 3,5-disubstituted isoxazoles based
chalcones 7a–7j. To a solution of isoxazoles 5a–5e
(100 mg, 1 equiv.) in EtOH was added KOH
(1.5 equiv.), and the mixture was stirred at room
temperature for 15 min. An aldehyde 6a or 6b
(E)-1-{2,4-Bis[(3-phenylisoxazol-5-yl)methoxy]-
phenyl}-3-phenylprop-2-en-1-one (7a). White solid,
yield 90%, mp 171–173°C. IR spectrum, ν, cm–1: 1648
(C=O). H NMR spectrum, δ, ppm: 7.92–7.38 m
(18H), 7.08 d (J = 7.57 Hz, 1H), 6.83 s (1H), 6.67 s
1
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 9 2018

SYNTHESIS AND ANTIMICROBIAL EVALUATION
1909
13
(1H), 6.64 s (1H), 5.30 s (2H), 5.24 s (2H). C NMR
spectrum, δC, ppm: 187.8, 166.1, 161.6, 161.0, 160.9,
158.1, 158.1, 142.2, 130.4, 130.0, 129.5, 129.3, 129.0,
129.0, 128.8 (2C), 128.2 (2C), 127.5 (2C), 127.4 (2C),
127.2 (2C), 126.9 (2C), 124.6, 124.4, 121.9, 118.2,
118.0, 107.9, 101.0, 62.1, 61.6. ESI+ MS: m/z: 555 [M +
H]+. Found, %: C 75.54; H 4.48; N 4.74. C34H25ClN6O4.
Calculated, %: C 75.80; H 4.73; N 5.05.
4.19.C38H32N2O6. Calculated, %: C 74.49; H 5.26; N
4.57.
(E)-1-{2,4-Bis[(3-{4-chlorophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-phenylprop-2-en-1-one (7e).
White solid, yield 78%, mp 179–181°C. IR spectrum,
ν, cm–1: 1655 (C=O). 1H NMR spectrum, δ, ppm: 8.00–
7.38 m (16H), 7.14–7.03 m (1H), 6.82 s (1H), 6.69 s
(1H), 6.60 s (1H), 5.43–5.18 m (4H). 13C NMR
spectrum, δC, ppm: 187.3, 166.3, 161.6, 161.1, 160.7,
158.1, 158.0, 142.1, 132.4, 132.3, 129.5, 129.4, 129.2
(2C), 129.1, 129.0, 128.8 (2C), 128.7 (2C), 127.9 (2C),
127.7 (2C), 126.3 (2C), 124.4, 124.1, 121.5, 118.1,
118.0, 107.8, 101.3, 62.0, 61.3. ESI+ MS: m/z: 623 [M +
H]+. Found, %: C 67.14; H 3.56; N 4.17. C35H24Cl2N2O5.
Calculated, %: C 67.42; H 3.88; N 4.49.
(E)-1-{2,4-Bis[(3-phenylisoxazol-5-yl)methoxy]-
phenyl}-3-(4-methoxyphenyl)prop-2-en-1-one (7b).
White solid, yield 88%, mp 157–159°C. IR spectrum,
ν, cm–1: 1658 (C=O). 1H NMR spectrum, δ, ppm: 8.00–
7.38 m (15H), 7.02 d (J = 7.53 Hz, 1H), 7.00–6.86 m
(2H), 6.80 s (1H), 6.67–6.59 m (2H), 5.38–5.21 m
(4H), 3.81 s (3H). 13C NMR spectrum, δC, ppm: 187.8,
166.1, 161.6, 161.0, 160.9, 158.1, 158.1, 142.2, 130.4,
130.0, 129.5, 129.3, 129.0, 129.0, 128.8 (2C), 128.2
(2C), 127.5 (2C), 127.4 (2C), 127.2 (2C), 126.9 (2C),
124.6, 124.4, 121.9, 118.2, 118.0, 107.9, 101.0, 62.1,
61.6. ESI+ MS: m/z: 585 [M + H]+. Found, %: C
73.44; H 4.47; N 4.33. C36H28N2O6. Calculated, %: C
73.96; H 4.83; N 4.79.
(E)-1-{2,4-Bis[(3-{4-chlorophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-(4-methoxyphenyl)prop-2-en-1-
one (7f). White solid, yield 75%, mp 156–158°C. IR
1
spectrum, ν, cm–1: 1656 (C=O). H NMR spectrum, δ,
ppm: 8.11–7.25 m (13H), 7.07–7.02 m (1H), 7.01–6.93
s (1H), 6.83 d (J = 7.26 Hz, 1H), 6.78–6.59 m (2H),
5.38–5.21 m (4H), 3.81 s (3H). 13C NMR spectrum, δC,
ppm: 189.1, 166.2, 161.7, 161.0, 160.7, 159.3, 158.1,
158.0, 143.4, 131.9, 130.4, 130.0, 129.2 (2C), 129.0,
128.7, 128.1 (2C), 127.5 (2C), 127.3 (2C), 127.2 (2C),
127.0 (2C), 124.9, 124.3, 121.0, 118.0, 117.7, 108.3,
101.2, 62.5, 61.3, 56.3. ESI+ MS: m/z: 653 [M + H]+.
Found, %: C 65.89; H 3.88; N 4.07.C36H26Cl2N2O6.
Calculated, %: C 66.16; H 4.01; N 4.29.
(E)-1-{2,4-Bis[(3-{p-tolyl}isoxazol-5-yl)methoxy]-
phenyl}-3-phenylprop-2-en-1-one (7c). White solid,
yield 85%, mp 203–205°C. IR spectrum, ν, cm–1: 1659
1
(C=O). H NMR spectrum, δ, ppm: 8.00–7.22 m
(16H), 7.04 d (J = 7.32 Hz, 1H), 6.80 s (1H), 6.71 s
(1H), 6.59 s (1H), 5.39–5.19 m (4H), 2.41–2.19 m
(6H). 13C NMR spectrum, δC, ppm: 187.6, 166.2,
161.7, 161.0, 160.9, 158.2, 158.0, 142.3, 131.7, 131.6,
129.4, 129.3, 129.1 (2C), 129.0, 128.7, 128.4 (2C),
127.9 (2C), 127.5 (2C), 127.3 (2C), 127.0 (2C), 124.5,
124.2, 121.8, 118.2, 118.0, 107.9, 101.1, 62.2, 61.1,
21.5, 21.3. ESI+ MS: m/z: 583 [M + H]+. Found, %: C
76.01; H 4.89; N 4.62. C37H30N2O5. Calculated, %: C
76.27; H 5.19; N 4.81.
(E)-1-{2,4-Bis[(3-{4-bromophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-phenylprop-2-en-1-one (7g).
White solid, yield 75%, mp 207–209°C. IR spectrum,
ν, cm–1: 1656 (C=O). 1H NMR spectrum, δ, ppm: 7.99–
7.28 m (16H), 7.15–7.05 m (1H), 6.83 s (1H), 6.68 s
(1H), 6.59 s (1H), 5.44–5.17 m (4H). 13C NMR
spectrum, δC, ppm: 187.2, 166.2, 161.7, 161.3, 160.4,
158.3, 158.1, 142.2, 132.7, 132.2, 129.9, 129.7, 129.6
(2C), 129.3, 129.1, 128.9 (2C), 128.6 (2C), 127.8 (2C),
127.6 (2C), 126.4 (2C), 124.0, 123.2, 121.7, 118.3,
118.1, 107.9, 101.5, 62.1, 61.6. ESI+ MS: m/z: 711
[M + H]+. Found, %: C 58.81; H 3.18; N 3.71.
C35H24Br2N2O5. Calculated, %: C 59.01; H 3.40; N
3.93.
(E)-1-{2,4-Bis[(3-{p-tolyl}isoxazol-5-yl)methoxy]-
phenyl}-3-(4-methoxyphenyl)prop-2-en-1-one (7d).
White solid, yield 90%, mp 183–185°C. IR spectrum,
ν, cm–1: 1657 (C=O). 1H NMR spectrum, δ, ppm: 8.00–
7.21 m (15H), 7.07–7.01 m (2H), 6.98 s (1H), 6.78 d
(J = 7.29 Hz, 1H), 6.77–6.62 m (2H), 5.39–5.20 m
(4H), 3.82 s (3H), 2.41-2.20 m (6H). 13C NMR
spectrum, δC, ppm: 189.1, 166.1, 161.6, 161.0, 160.8,
159.2, 158.2, 158.0, 143.3, 131.8, 130.3, 130.1, 129.3
(2C), 129.1, 128.8, 128.5 (2C), 127.5 (2C), 127.4 (2C),
127.3 (2C), 127.1 (2C), 124.6, 124.3, 121.9, 118.1,
118.0, 108.0, 101.2, 62.4, 61.2, 56.1, 21.4, 21.2. ESI+
MS: m/z: 613 [M + H]+. Found, %: C 74.08; H 5.01; N
(E)-1-{2,4-Bis[(3-{4-bromophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-(4-methoxyphenyl)prop-2-en-1-
one (7h). White solid, yield 80%, mp 186–188°C. IR
1
spectrum, ν, cm–1: 1657 (C=O). H NMR spectrum, δ,
ppm: 8.08–7.21 m (13H), 7.08–7.01 m (1H), 6.95 s
(1H), 6.89 d (J = 7.34 Hz, 1H), 6.76–6.58 m ( H), 5.37–
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 9 2018

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SUNITHA et al.
5.21 m (4H), 3.80 s (3H). 13C NMR spectrum, δC,
ppm: 189.4, 166.4, 161.4, 161.2, 160.5, 159.4, 158.2,
158.1, 142.6, 132.8, 130.4, 130.1, 129.4 (2C), 128.9,
128.4, 128.3 (2C), 127.8 (2C), 127.6 (2C), 127.4 (2C),
127.2 (2C), 124.7, 124.3, 121.3, 118.1, 117.9, 108.4,
101.0, 62.1, 61.6, 56.4. ESI+ MS: m/z: 741 [M + H]+.
Found, %: C 58.01; H 3.30; N 3.48. C36H26Br2N2O6.
Calculated, %: C 58.24; H 3.53; N 3.77.
sporum gypseum (ATCC-24102), Epidermophyton
floccosum (ATCC-15694) were collected from
department of biotechnology, Chaitanya postgraduate
college (autonomous), Kishanpura, Hanamkonda,
Warangal-506001, Telangana, India. All microbial
strains were stored at ‒80°C, streaked on Luria‒Bertani
(LB) agar plates (Hi-media Laboratories, Mumbai,
India) and incubated at 37°C for 20−24 h. A few
isolated colonies were selected from each plate and
suspended in 5 mL of LB broth in a sterile culture
vessel. The vessel was plugged with cotton and
incubated with gentle shaking (140 rpm) at 37°C for
20 h.
(E)-1-{2,4-Bis[(3-{4-nitrophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-phenylprop-2-en-1-one (7i).
Yellow solid, yield 88%, mp >300°C. IR spectrum, ν,
1
cm–1: 1646 (C=O). H NMR spectrum, δ, ppm: 8.40–
8.22 m (4H), 7.95–7.35 m (12H), 7.06 d (J = 7.33 Hz,
1H), 6.82 s (1H), 6.70 s (1H), 6.61 s (1H), 5.34 s (2H),
5.25 s (2H). 13C NMR spectrum, δC, ppm: 187.0,
166.4, 162.0, 161.6, 160.6, 158.5, 158.4, 148.2, 147.9,
133.3 (2C), 133.2 (2C), 130.1, 129.9, 129.7, 129.6
(2C), 129.3, 129.1, 127.9 (2C), 127.8 (2C), 126.6 (2C),
124.2, 123.3, 121.8, 118.2, 118.0, 108.1, 101.5, 62.0,
61.2. ESI+ MS: m/z: 645 [M + H]+. Found, %: C
64.95; H 3.48; N 8.40. C35H24N4O9. Calculated, %: C
65.22; H 3.75; N 8.69.
Preparation of inoculums. Following the protocol
of the Kirby–Bauer disk diffusion assay [20], four or
five well-isolated colonies of the same morphological
type were picked with an inoculating loop, transferred
into 5 mL of nutrient broth, and incubated at 37°C for
24 h until a slightly visible turbidity appeared. The
turbidity of the actively growing broth cultures was
then adjusted with broth to a density equivalent to that
of a 0.5 McFarland standard, and the resulting
suspensions were used as the initial inocula in the
assay.
(E)-1-{2,4-Bis[(3-{4-nitrophenyl}isoxazol-5-yl)-
methoxy]phenyl}-3-(4-methoxyphenyl)prop-2-en-1-
one (7j). Yellow solid, yield 90%, mp >300°C. IR spec-
trum, ν, cm–1: 1649 (C=O). 1H NMR spectrum, δ, ppm:
8.38–8.20 m (4H), 7.93–7.36 m (9H), 7.08–6.92 s
(3H), 6.78 d (J = 7.30 Hz, 1H), 6.72–6.59 m (2H),
5.32 s (2H), 5.24 s (2H), 3.81 s (3H). 13C NMR
spectrum, δC, ppm: 189.3, 166.2, 161.3, 161.1, 160.6,
159.3, 158.3, 158.1, 148.2, 147.9, 143.2, 133.2 (2C),
133.0 (2C), 131.8, 129.7 (2C), 129.6 (2C), 127.9 (2C),
127.8, 124.2, 123.3, 121.8, 118.2, 118.0, 116.3 (2C),
108.1, 101.5, 62.0, 61.7, 56.2. ESI+ MS: m/z: 675
[M + H]+. Found, %: C 63.82; H 3.61; N 8.08.
C36H26N4O10. Calculated, %: C 64.09; H 3.88; N 8.31.
Antimicrobial assay. The initial inocula of the test
organisms, 100 µL, were swabbed over the surface of
the agar media (20 mL) in Petri dishes and let to be
absorbed for 15 min. Wells, 8 mm in diameter, were
made with a sterile cork borer in the seeded agar
plates. Solutions of the test compounds in DMSO
(100 µL; c 75 and 100 mg/mL) were then loaded into
the wells and incubated in the air at 37°C for 24 h.
Inhibition zone diameters were measured with a zone
reader (HiAntibiotic Zone Scale). The standard drug
Gentamicin was used as the positive control.
CONCLUSIONS
Antimicrobial activity. Bacterial and fungal
strains. The gram-positive strains Micrococcus luteus
(ATCC 10240), methicillin-resistant Staphylococcus
aureus (MRSA, NCTC 13616), Bacillus subtilis
(ATCC 6633), and Bacillus cereus (ATCC 14579),
and the gram-negative strains Pseudomonas
aeruginosa (ATCC 27853), Klebsiella pneumoniae
(ATCC 43816), Escherichia coli (ATCC 8739), and
Proteus vulgaris (ATCC 13315) were purchased from
the American Type Culture Collection. Methicillin-
resistant Staphylococcus aureus was purchased from
the Public Health England Culture Collections. Fungal
strains Microsporum canis (ATCC-36299), Micro-
Bis-isoxazoles based chalcones 7a–7j are
synthesized with high yields and characterized by
different spectroscopic methods. The synthesized
compounds are tested for their antimicrobial activity.
The highest activities are determined for compounds
(7f, 7h, 7j, and 7i).
ACKNOWLEDGMENTS
V. Sunitha thanks the Head of the Department,
UCS, Osmania University, Saifabad, Hyderabad and
Central Facilities for Research and Development,
RUSSIAN JOURNAL OF GENERAL CHEMISTRY Vol. 88 No. 9 2018

SYNTHESIS AND ANTIMICROBIAL EVALUATION
1911
10.1016/j.ejmech.2007.09.014
Osmania University, Hyderabad, India for providing
analytical support.
11. Cheenpracha, S., Karalai, C., Ponglimanont, C.,
Subhadhirasakul, S., and Tewtrakul, S., Bioorganic
Med. Chem., 2006, vol. 14, no. 6, p. 1710. doi 10.1016/
j.bmc.2005.10.019
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Vernal, J., Cordeiro, M.N.S., Zenteno, M.E., Villarino, A.,
Nunes, R.J., Yunes, R.A., and Terenzi, H., Bioorganic
Med. Chem. Lett., 2008, vol. 18, no. 23, p. 622. doi
10.1016/j.bmcl.2008.09.105
13. Mukherjee, S., Kumar, V., Prasad, A.K., Raj, H.G.,
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CONFLICT OF INTERESTS
No conflict of interests was declared by the authors.
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