Synthesis of new 1,3,4-oxadiazole and benzothiazolylthioether derivatives of 4-arylmethylidene-3-substituted-isoxazol-5(4H)-one as potential antimycobacterial agents

Article abstract of DOI:10.1007/s00044-019-02420-7

A new series of 4-[(substituted benzylidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-ylthio)-methyl]isoxazol-5(4H)-one (6a–g) and 4-(substituted benzylidene)-3-((benzo[d]thiazol-2-ylthio)methyl)isoxazol-5(4H)-one (8a–g) was synthesized. All the synthesiz

Full text of DOI:10.1007/s00044-019-02420-7

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02420-7
ORIGINAL RESEARCH
Synthesis of new 1,3,4-oxadiazole and benzothiazolylthioether
derivatives of 4-arylmethylidene-3-substituted-isoxazol-5(4H)-one as
potential antimycobacterial agents
1
Abhijit P. Chavan1,2 Rujuta R. Deshpande3 Nandkumar A. Borade4 Abhijit Shinde1 Pravin C. Mhaske
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Dhiman Sarkar5 Vivek D. Bobade2
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Received: 14 March 2019 / Accepted: 5 August 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
A new series of 4-[(substituted benzylidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-ylthio)-methyl]isoxazol-5(4H)-one
(6a–g) and 4-(substituted benzylidene)-3-((benzo[d]thiazol-2-ylthio)methyl)isoxazol-5(4H)-one (8a–g) was synthesized. All
the synthesized compounds were screened for antitubercular activity against Mycobacterium tuberculosis H37Ra (ATCC
25177) and Mycobacterium bovis BCG (ATCC 35743) and antibacterial activity against Escherichia coli (NCIM 2576),
Pseudomonas flurescence (NCIM 2059), Staphylococcus aureus (NCIM 2602), Bacillus subtilis (NCIM 2162). Amongst the
synthesized 1,3,4-oxadiazole and benzothiazoyl thioether derivatives, compounds 6b and 8b showed excellent
antimycobacterial activity and compounds 6b, 8a, 8b, and 8d showed excellent antibacterial activity against all tested
antibacterial strains. The synthesized compounds were further evaluated for their cytotoxic activity against the HCT 116 and
HeLa cancer cell lines. The 1,3,4-oxadiazole and benzothiazoyl thioether derivatives 6a–g and 8a–g did not show
cytotoxicity.
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Keywords Isoxazol-5(4H)-one 1,3,4-Oxadiazole Benzothiazol Thioeteher Antitubercular activity Antibacterial activity
Introduction
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-019-02420-7) contains supplementary
material, which is available to authorized users.
Enormous numbers of antibiotics and chemotherapeutics
drugs have contributed towards controlling human ailments.
Since the last three decades, the complications of multidrug-
resistant microorganisms have reached an alarming level in
many countries around the world (Brown and Wright 2016;
Aminov 2017; Powers 2004). The proliferation in antibiotic
resistance due to numerous aspects has intensified the
search for new, safe, and more efficient drugs which can
prove to be dynamic against multidrug-resistant pathogens
(Kunin and Ellis 2000; Cannon et al. 2009; Achkar and
Fries 2010; Kathiravan et al. 2012).
* Pravin C. Mhaske
mhaskepc18@gmail.com
* Vivek D. Bobade
v_bobade31@rediffmail.com
1
Department of Chemistry, Sir Parashurambhau College, Affiliated
to Savitribai Phule Pune University, Tilak Road, Pune 411030,
India
2
Department of Chemistry, HPT Arts and RYK Science College,
Affiliated to Savitribai Phule Pune University, Nasik 422005,
The development of clubbed heterocyclic compounds is
enormously valuable due to the pharmacological potential
owned by the individual heterocycles themselves as well as
a new one due to the reciprocal influence of the incorpo-
rated heterocycles. Compounds containing 1,3,4-oxadiazole
nucleus showed broad spectrum of biological activities such
as anti-inflammatory (Sahoo et al. 2014), antibacterial (Li
et al. 2013), herbicidal (Tajik and Dadras 2011), insecticidal
(Li and He 2012), and antifungal activities (Asan et al.
India
3
Department of Zoology, Modern College of Arts, Science and
Commerce, Affiliated to Savitribai Phule Pune University,
Shivajinagar Pune, Pune 411005, India
4
Department of Chemistry, Kirti M. Doongursee College, Affiliated
to University of Mumbai, Kashinath Dhuru Road, Off. Veer
Savarkar Marg, Dadar (W), Mumbai 400028, India
5
CombiChemBio Resource Centre, CSIR-National Chemical
Laboratory, Pune 411008, India

Medicinal Chemistry Research
Fig. 1 Biologically active
isoxazole derivatives
2
3
2
3
Antifungal
Antibacterial
3
Antibacterial
Antibacterial
H3C
1
N
S
R
CONH
O
N
N
N
CH3
O
Cl
Anticancer
anslgesic and anti-inflammatory
Antitubercular
O
O
N
N
O
CF3
OH
O
CF3
N
O
N
N
R
NH
S
O
N
CH3
N
N
O
H3C
O
X
F
Anticancer
Immunosuppressant
Anticancer
2012). Commercial fungicides or herbicides, such as tria-
dimefon, triadimenol, flusilazole, and flupoxam (Guo et al.
2014) contains 1,3,4-oxadiazoles. On the other hand, the
heterocyclic scaffold comprising isoxazole shows various
biological activities, such as antibacterial (Badrey and
Gomha 2014), anticonvulsant (Kaur et al. 2010), anticancer
(Yong et al. 2014), anthelmintics (Mondal et al. 2012), anti-
inflammatory (El-Hawash et al. 2014), adenosine antagonist
(Choi et al. 2005), fungicidal (Reddy and Nagaraj 2008),
herbicidal (Zhang et al. 2008), hypoglycaemic (Kumar et al.
2009), muscle relaxant (Tatee et al. 1986), nematocidal
(Srinivas et al. 2010), insecticidal (Wang et al. 2011),
antiviral (Muratov et al. 2011), antimicrobial (Gollapalli
2015), and antitubercular (Kozikowski et al. 2009) activ-
ities. (Fig. 1) Furthermore, benzothiazole nucleus contain-
ing molecules are also reported as anti-allergic (Musser
et al. 1984), antitumor (Yoshida et al. 2005; Catriona et al.
2006), and anticancer (Bradshaw et al. 2002) and it was
declared that these molecules can be used as an Aβ plaque
imaging against for the study of patients with Alzheimer’s
disease (Kim et al. 2009).
thioether derivatives of 4-arylmethylidene-3-substituted-
isoxazol-5(4H)-one as potential antimycobacterial agents.
Material and methods
Chemistry
Experimental
The reaction progress was monitored by thin layer chro-
matography (TLC). Physical constants of the pure com-
pounds were determined in capillary tubes in silicon oil bath
using a Veego melting point apparatus and are uncorrected.
The synthesized compounds were analyzed using NMR and
the spectra were recorded on Varian mercury XL-300 or
Bruker spectrometer instruments; tetramethylsilane was
used as an internal standard and chemical shifts are reported
in δ units. Mass spectra were recorded on a Brucker LC-MS
QP MS-3200Q trap spectrometer with an ionization
potential of 70 eV.
Aryl and alkyl substituted thioethers are the backbone of
many biologically active compounds reported for antibacterial
(Macaev et al. 2005; McReynolds et al. 2004), antifungal
(Chen et al. 2000), antioxidants in polymers (Coran 2003),
anti-hyperplasia (Quaglia et al. 2002), and anticancer (Sugita
et al. 2001) activities. 1,3,4-Oxadiazolin-2-thiones have
earned a great deal of attention in heterocyclic chemistry as
versatile intermediates due to the fact that the thiol group on
the oxadiazole ring undergoes nucleophilic substitution reac-
tion readily (Mekuskiene et al. 2003).
In view of the significance of isoxazole and 1,3,4-oxa-
diazole nucleus and in prolongation of our work (Abhale
et al. 2015; Abhale et al. 2017) on hunt for bioactive het-
erocyclic compounds, in the present study, we have repor-
ted synthesis of 1,3,4-oxadiazole and benzothiazoyl
General procedure for synthesis of 4-[(substituted
benzylidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-
ylthio)-methyl]isoxazol-5(4H)-one (6a–g)
The solution of 4-[(aryl)-methylidene-]-3-chloro-methyl-5
(4H)-isoxazolone (4a–g, 1 mmol), sodium hydrogen car-
bonate (1 mmol) and 5-(pyridine-4-yl)-1,3,4-oxadiazole-2-
thiol (5, 1 mmol) in dry ethanol was refluxed for 3 h. After
the completion of the reaction (TLC) the mixture was
cooled to room temperature, the solid product was separated
then filtered and washed with cold ethanol. The product
(6a–g) thus obtained was further purified by crystallization
from aqueous ethanol. Similar procedure was used for the
synthesis of 8a–g using benzothiazole-2-thiol (7).

Medicinal Chemistry Research
4-(4-hydroxybenzylidene)-3-((5-(pyridin-4-yl)-1,3,4-oxadia-
zol-2-ylthio)methyl)isoxazol-5(4H)-one, (6a) 1H NMR
(400 MHz, DMSO-d6) δ 4.76 (s, 2H, S–CH2-isoxazole),
7.03 (d, J = 8.9 Hz, 2H, Ar–H), 7.89 (d, J = 6.0 Hz, 2H,
Py–H), 8.22 (s,1H, =CH), 8.46 (d, J = 8.9 Hz, 2H, Ar–H),
8.81(d, J = 6.0 Hz, 2H, Py–H), 11.46 (bs, 1H, –OH); 13C
NMR (100 MHz, DMSO-d6) δ 26.97 (CH2, –S–CH2-iso-
xazole), 110.96 (C, Ar C-1), 116. 42 (CH, Ar C-3 and C-5),
119.99 (CH, Py C-3 and C-5), 124.43 (C, isoxazole C-4),
129.94 (CH, =CH), 137.94 (CH, Ar C-2 and C-6), 150.89
(CH, Py C-2 and C-6), 152.73 (C, Ar C-4), 161.10 (C, Py
C-4), 163.76 (C, isoxazole C-3), 164.01 (C, oxadiazole C-
5), 164.80 (C, oxadiazole C-2), 168.59 (C, isoxazole C=O),
MS(m/z): 381.0573 (M+H)+.
NMR (100 MHz, CDCl3) δ 24.03 (CH3,-CH3), 26.75 (CH2,
S–CH2–isoxazole), 112.50 (C, Ar C-1), 114.46 (CH, Ar C-3
and C-5), 119.70 (CH, Py C-3 and C-5), 125.75 (C, iso-
xazole C-4), 129.95 (CH, =CH), 137.30 (CH, Ar C-2 and
C-6), 150.55 (CH, Py C-2 and C-6), 152.15 (C, Ar C-4),
160.44 (C, Py C-4),163.70 (C, isoxazole C-3), 163.95 (C,
oxadiazole C-5), 164.70 (C, oxadiazole C-2), 168.01 (C,
isoxazole C=O); MS(m/z): 379.0788 (M+H)+
.
4-(benzo[d][1,3]dioxol-5-ylmethylene)-3-(((5-(pyridin-4-yl)-
1,3,4-oxadiazol-2-yl)thio)methyl) isoxazol-5(4H)-one, (6e)
1H NMR (400 MHz,CDCl3) δ 4.71 (s, 2H, –S–CH2-iso-
axzole), 6.08 (s, 2H, –OCH2O–), 6.82 (d, J = 6.1 Hz, 2H,
Py–H), 7.35–7.39 (m, 1H, Ar–H), 7.73–7.51 (m, 2H,
Ar–H), 7.89 (s, 1H, =CH), 7.95 (d, 2H, J = 6.1 Hz, Py–H);
13C NMR (100 MHz, CDCl3) δ 27.31 (CH2, S–CH2-iso-
xazole), 101.28 (CH2, –OCH2O–), 112.10 (CH, Ar C-6),
116.32 (CH, Ar C-3), 119.69 (CH, Ar C-4), 121.45 (CH, Py
C-3 and C-5), 124.98 (C, isoxazole C-4), 128.53 (C, Ar C-
5), 148.13 (C, Ar C-2), 148.50 (C, oxadiazole C-2), 148.64
(C, Ar C-1), 151.16 (CH, Py C-2), 151.32 (CH, Py C-4),
161.28 (C, isoxazole C-3), 164.93 (C, oxadiazole C-5),
4-(4-methoxybenzylidene)-3-((5-(pyridin-4-yl)-1,3,4-oxadia-
zol-2-ylthio)methyl)isoxazol-5(4H)-one,
(6b): IR(KBr):
1
1760, 1610, 1580, 1568, 1179 cm−1; H NMR (400 MHz,
CDCl3) δ 3.93 (s, 3H, OCH3), 4.73 (s, 2H, S–CH2-iso-
xazole), 7.09 (d, J = 8.9 Hz, 2H, Ar–H), 7.88 (d, J =
6.1 Hz, 2H, Py–H), 8.22 (s, 1H, =CH), 8.54 (d, J = 8.9 Hz,
2H, Ar–H), 8.80 (d, J = 6.1 Hz, 2H, Py–H); 13C NMR
(100 MHz, CDCl3) δ 26.78 (CH2, S–CH2–isoxazole), 55.59
(CH3, OCH3), 112.52 (C, Ar C-1), 114.45 (CH, Ar C-3 and
C-5), 119.73 (CH, Py C-3 and C-5), 125.71 (C, isoxazole
C-4), 129.92 (CH, =CH), 137.25 (CH, Ar C-2 and C-6),
150.59 (CH, Py C-2 and C-6), 152.10 (C, Ar C-4), 160.47
(C, Py C-4), 163.72 (C, isoxazole C-3), 163.90 (C, oxa-
diazole C-5), 164.66 (C, oxadiazole C-2), 168.09 (C, iso-
xazole C=O); MS(m/z): 395.0815 (M+H)+.
168.36 (C, isoxazole C=O); MS(m/z): 409.0530 (M+H)+
.
4-(4-(dimethylamino)benzylidene)-3-((5-(pyridin-4-yl)-1,3,4-
oxadiazole-2-ylthio)methyl) isoxazol-5(4H)-one, (6f) IR
(KBr): 1718, 1571, 1541, 1512, 1274, 1197 cm−1; 1H NMR
(400 MHz, CDCl3) δ 3.18 (s, 6H, –N(CH3)2) 4.58 (s, 2H,
S–CH2-isoxazole), 6.72 (d, J = 6 Hz, 2H, Ar–H), 7.61 (s,
1H, =CH), 7.87 (d, J = 5.5 Hz, 2H, Py–H), 8.43(d, J =
6 Hz, 2H, Ar–H), 8.81 (d, J = 5.5 Hz, 2H, Py–H); 13C NMR
(100 MHz, CDCl3) δ 27.46 (CH2, S–CH2-isoxazole), 40.20
(CH3,–N(CH3)2), 107.68 (C, Ar C-1), 111.76 (CH, Ar C-3
and C-5), 120.12 (CH, Py C-3 and C-5), 121.68 (C, iso-
xazole C-4), 130.42 (CH, =CH), 138.45 (CH, Ar C-2 and
C-6), 150.10 (CH, Py C-2 and C-6), 150.95 (C, Ar C-4),
154.86 (C, Py C-4), 159.86 (C, isoxazole C-3), 164.91 (C,
oxadiazole C-5), 169.84 (C, isoxazole C=O); MS(m/z):
409.1060 (M+H)+.
4-(4-hydroxy-3-methoxybenzylidene)-3-((5-(pyridin-4-yl)-
1,3,4-oxadiazole-ylthio)methyl) isoxazol-5(4H)-one, (6c)
1H NMR (400 MHz, DMSO-d6) δ 3.85 (s, 3H, OCH3), 4.73
(s, 2H, S–CH2-isoxazole), 6.98 (d, J = 8.4 Hz, 1H, Ar–H),
7.90 (d, J = 8.4 Hz, 1H, Ar–H), 7.92 (d, J = 6.1 Hz, 2H,
Py–H), 8.11 (s, 1H, =CH), 8.51 (s, 1H, Ar–H), 8.81 (d, J =
6.1 Hz, 2H, Py–H), 11.06 (bs, 1H, –OH); 13C NMR
(100 MHz, DMSO-d6) δ 26.97 (CH2, –S–CH2–isoxazole),
111.96 (C, Ar C-1), 114.42 (CH, Ar C-2), 115. 88 (CH, Ar
C-5), 119.99 (CH, Py C-3 and C-5), 120.2 (CH, Ar C-6),
124.43 (C, isoxazole C-4), 129.90 (CH, =CH),148.91 (CH,
Ar C-4), 150.89 (CH, Py C-2 and C-6), 151.35 (CH, Ar C-
3), 161.10 (C, Py C-4), 163.76 (C, isoxazole C-3), 164.01
(C, oxadiazole C-5), 164.80 (C, oxadiazole C-2), 168.59 (C,
4-((1H-indol-3-yl)methylene)-3-((5-(pyridin-4-yl)-1,3,4-oxa-
diazole-2-ylthio)methyl)isoxazol-5(4H)-one, (6g) IR(KBr):
1
3291, 1714, 1592, 1574, 1473, 1217, 1190 cm−1; H NMR
(400 MHz, DMSO-d6) δ 4.81 (s, 2H, –S–CH2-isoaxzole),
7.31–7.33 (m, 2H, Ar–H), 7.55–7.58 (m, 1H, Ar), 7.88 (d,
J = 6.1 Hz, 2H, Py–H), 8.13–8.15 (m, 1H, Ar–H), 8.52
(s,1H, Ar–H), 8.77 (d, J = 6.1 Hz, 2H, Py–H), 12.86 (bs,
1H, NH); 13C NMR (100 MHz, DMSO-d6) δ 26.78 (CH2,
S–CH2-isoxazole), 110.52 (C, Ar C-3), 113.45 (CH, Ar C-
8), 119.02 (CH, Ar C-5), 119.73 (CH, Py C-3 and C-5),
120.22 (CH, Ar C-7), 122.25 (CH, Ar C-6), 125.71 (C,
isoxazole C-4), 125.99 (C, Ar C-4), 129.92 (CH, =CH),
130.81 (CH, Ar C-2), 135.45 (C, Ar C-9), 150.59
isoxazole C=O); MS(m/z): 411.0685 (M+H)+
.
4-(4-methylbenzylidene)-3-((5-(pyridin-4-yl)-1,3,4-oxadia-
zole-2-ylthio)methyl)isoxazol-5(4H)-one, (6d) 1H NMR
(400 MHz, CDCl3) δ 2.34 (s, 3H, CH3), 4.70 (s, 2H,
S–CH2-isoxazole), 7.12 (d, J = 8.7 Hz, 2H, Ar–H), 7.90 (d,
J = 6.2 Hz, 2H, Py–H), 8.24 (s, 2H, =CH), 8.50 (d, J =
8.7 Hz, 2H, Ar–H), 8.84 (d, J = 6.2 Hz, 2H, Py–H); 13C

Medicinal Chemistry Research
(CH, Py C-2 and C-6), 160.47 (C, Py C-4), 163.72 (C,
isoxazole C-3), 163.90 (C, oxadiazole C-5), 164.66 (C,
oxadiazole C-2), 168.09 (C, isoxazole C=O); MS(m/z):
(CH3, OCH3), 112.30 (C, Ar C-1), 115.72 (CH, Ar C-5),
123.41 (C, benzothiazole C-5), 120.50 (C, benzothiazole C-
8), 125.90 (C, benzothiazole C-6), 126.15 (C, benzothiazole
C-7), 126.45 (C, isoxazole C-4), 135.71 (C, benzothiazole
C-9), 137.48 (CH, Ar C-2 and C-6), 146.48 (CH, Ar C-3),
149.62 (C, Ar C-4), 151.30 (CH, =CH), 161.30 (C, ben-
zothiazole C-4), 164.92 (C, benzothiazole C-2), 166.02 (C,
isoxazole C-3), 169.59 (C, isoxazole C=O); MS(m/z):
404.0745 (M+H)+
.
4-(4-hydroxybenzylidene)-3-((benzo[d]thiazol-2-ylthio)
methyl)isoxazol-5(4H)-one, (8a) 1H NMR (400 MHz,
DMSO-d6) δ 4.78 (s, 2H, S–CH2-isoaxzole), 7.00 (d, J =
8.8 Hz, 2H, Ar–H), 7.39 (td, J = 7.6 and 1.0 Hz, 1H, benzo
[d]thiazol-H), 7.48 (td, J = 7.6 and 1.0 Hz, 1H, benzo[d]
thiazol-H), 7.88 (d, J = 7.6 Hz, 1H, benzo[d]thiazol-H),
8.04 (d, J = 7.6 Hz, 1H, benzo[d]thiazol-H), 8.16 (s, 1H,
=CH), 8.45 (d, J = 8.8 Hz, 2H, Ar–H), 11.30 (bs, 1H,-OH);
13C NMR (75 MHz, DMSO-d6) δ 27.39 (CH2, S–CH2-iso-
xazole), 111.33 (C, Ar C-1), 116.40 (CH, Ar C-3 and C-5),
121.34 (C, benzothiazole C-5), 121.96 (C, benzothiazole C-
8), 124.42 (C, benzothiazole C-6), 124.79 (C, benzothiazole
C-7), 126.49 (C, isoxazole C-4), 135.01 (C, benzothiazole
C-9), 137.91 (CH, Ar C-2 and C-6), 152.38 (CH, =CH),
152.61 (C, Ar C-4), 161.39 (C, benzothiazole C-4), 164.60
(C, benzothiazole C-2), 164.76 (C, isoxazole C-3), 168.61
(C, isoxazole C=O); MS(m/z): 369.0289 (M+H)+.
399.0398 (M+H)+
.
4-(4-methylbenzylidene)-3-((benzo[d]thiazol-2-ylthio)
methyl)isoxazol-5(4H)-one, (8d) 1H NMR (400 MHz,
DMSO-d6) δ 2.35 (s, 3H, CH3), 4.65 (s, 2H, S–CH2-iso-
axzole), 6.90 (d, J = 8.6 Hz, 2H, Ar–H), 7.38 (td, J = 7.5
and 1.2 Hz, 1H, benzo[d]thiazol-H), 7.45 (td, J = 7.5,
1.2 Hz, 1H, benzo[d]thiazol-H), 7.75 (d, J = 7.5 Hz, 1H,
benzo[d]thiazol-H), 7.88 (s, 1H, =CH), 7.92 (d, J = 7.5 Hz,
1H, benzo[d]thiazol-H), 8.32 (d, J = 8.6 Hz, 2H, Ar–H);
13C NMR (100 MHz, DMSO-d6) δ 24.3 (C, CH3), 27.33
(CH2, S–CH2-isoxazole), 113.32 (C, Ar C-1), 114.72 (CH,
Ar C-3, C-5), 121.41 (C, benzothiazole C-5), 121.56 (C,
benzothiazole C-8), 124.98 (C, benzothiazole C-6), 125.93
(C, benzothiazole C-7), 126.46 (C, isoxazole C-4), 135.68
(C, benzothiazole C-9), 137.45 (CH, Ar C-2 and C-6),
138.35 (C, Ar C-4), 151.25 (CH, =CH), 161.24 (C, ben-
zothiazole C-4), 164.90 (C, benzothiazole C-2), 165.08 (C,
isoxazole C-3), 168.51 (C, isoxazole C=O); MS(m/z):
367.0467 (M+H)+.
4-(4-methoxybenzylidene)-3-((benzo[d]thiazol-2-ylthio)
methyl)isoxazol-5(4H)-one, (8b) IR (KBr): 1744, 1590,
1509, 1270, 1233, 1189 cm−1; 1H NMR (400 MHz, CDCl3)
δ 3.72 (s, 3H, OCH3), 4.69 (s, 2H, S–CH2-isoaxzole), 6.91
(d, J = 8.8 Hz, 2H, Ar–H), 7.35 (t, J = 7.6 and 1.1 Hz, 1H,
benzo[d]thiazol-H), 7.47 (t, J = 7.6, 1.1 Hz, 1H, benzo[d]
thiazol-H), 7.77 (d, J = 7.6 Hz, 1H, benzo[d]thiazol-H),
7.90 (s, 1H, =CH), 7.94 (d, J = 7.6 Hz, 1H, benzo[d]thia-
zol-H), 8.30 (d, J = 8.8 Hz, 2H, Ar–H); 13C NMR
(100 MHz, CDCl3) δ 27.35 (C, S–CH2-isoxazole), 55.75
(CH2, OCH3), 113.38 (C, Ar C-1), 114.70 (CH, Ar C-3 and
C-5), 121.43 (C, benzothiazole C-5), 121.54 (C, ben-
zothiazole C-8), 124.94 (C, benzothiazole C-6), 125.96 (C,
isoxazole C-4), 126.48 (C, benzothiazole C-7), 135.67 (C,
benzothiazole C-9), 137.46 (CH, Ar C-2 and C-6), 151.26
(CH, =CH), 152.481 (C, Ar C-4), 161.28 (C, benzothiazole
C-4), 164.89 (C, benzothiazole C-2), 165.02 (C, isoxazole
C-3), 168.59 (C, isoxazole C=O); MS(m/z): 383.0524 (M
+H)+.
4-((benzo[d][1,3]dioxol-5-yl)methylene)-3-((benzo[d]thia-
zol-2-ylthio)methyl)isoxazol-5(4H)-one, (8e) 1H NMR
(400 MHz, DMSO-d6) δ 4.77 (s, 2H, S–CH2-isoxazole),
6.20 (s, 2H, OCH2O), 6.90 (d, J = 8.4 Hz, 1H, Ar–H), 7.38
(td, J = 7.7 and 1.2 Hz, 1H, benzo[d]thiazol-H), 7.48 (td, J
= 7.7 and 1.2 Hz, 1H, benzo[d]thiazol-H), 7.86 (d, J =
8.4 Hz, 1H, Ar–H), 7.89 (d, J = 7.7 Hz, 1H, benzo[d]thia-
zol-H), 8.00 (d, J = 7.7 Hz, 1H, benzo[d]thiazol-H), 8.09 (s,
1H, =CH) 8.51 (s, 1H, Ar–H); 13C NMR (100 MHz,
CDCl3) δ 27.30 (CH2, -CH2-isoxazole), 110.50 (CH2,
–OCH2O–), 112.30 (CH, Ar C-6), 116.72 (CH, Ar C-3),
119.69 (CH, Ar C-4), 123.41 (C, benzothiazole C-5),
120.50 (C, benzothiazole C-8), 125.90 (C, benzothiazole C-
6), 126.15 (C, benzothiazole C-7), 126.45 (C, isoxazole C-
4), 128.53 (C, Ar C-5), 135.71 (C, benzothiazole C-9),
146.48 (CH, Ar C-3), 148.13 (C, Ar C-2), 148.64 (C, Ar C-
1), 151.30 (C, Ar C-4), 152.28 (CH, =CH), 161.24 (C,
benzothiazole C-4), 164.92 (C, benzothiazole C-2), 166.02
(C, isoxazole C-3), 169.59 (C, isoxazole C=O); MS(m/
4-(4-hydroxy-3-methoxybenzylidene)-3-((benzo[d]thiazol-2-
ylthio)methyl)isoxazol-5(4H)-one,
(8c) 1H
NMR
(400 MHz, DMSO-d6) δ 3.84 (s, 3H, OCH3), 4.77 (s, 2H,
S–CH2-isoxazole), 6.95 (d, J = 8.5 Hz, 1H, Ar–H), 7.38 (td,
J = 7.6 and 1.1 Hz, 1H, benzo[d]thiazol-H), 7.48 (td, J =
7.6 and 1.1 Hz, 1H, benzo[d]thiazol-H), 7.86 (d, J = 8.5 Hz,
1H, Ar–H), 7.89 (d, J = 7.6 Hz, 1H, benzo[d]thiazol-H),
8.00 (d, J = 7.6 Hz, 1H, benzo[d]thiazol-H), 8.09 (s, 1H,
=CH), 8.51 (s, 1H, Ar–H), 8.47 (bs, 1H, OH); 13C NMR
(100 MHz, CDCl3) δ 28.42 (CH2, S–CH2-isoxazole) 56.85
z):397.0234 (M+H)+
.
4-(4-(dimethylamino)benzylidene)-3-((benzo[d]thiazol-2-
ylthio)methyl)isoxazol-5(4H)-one, (8f) IR (KBr): 1706,
1
1588, 1549, 1525, 1376, 1199 cm−1; H NMR (400 MHz,

Medicinal Chemistry Research
DMSO-d6) δ 3.29 (s, 6H, N(CH3)2), 4.72 (s, 2H, S–CH2-
isoaxzole), 6.78 (d, J = 9.3 Hz, 2H, Ar–H), 7.37 (td, J = 7.6
and 1.1 Hz, 1H, benzo[d]thiazol-H), 7.47 (td, J = 7.6 and
1.1 Hz, 1H, benzo[d]thiazol-H), 7.87 (s, 1H, =CH), 7.92
(m, 2H, benzo[d]thiazol-H), 8.40 (d, J = 8.6 Hz, 2H,
Ar–H); 13C NMR (100 MHz, DMSO-d6) δ 27.30 (CH3, –N
(CH3)2), 45.49 (CH2, S–CH2-isoxazole), 106.55 (C, Ar C-
1), 111.55 (CH, Ar C-3 and C-5), 121.06 (C, benzothiazole
C-5), 121.15 (C, benzothiazole C-8), 121.42 (C, ben-
zothiazole C-6), 124.49 (C, benzothiazole C-7), 126.14 (C,
isoxazole C-4), 134.97 (C, benzothiazole C-9), 137.86 (CH,
Ar C-2,6), 150.72 (CH, =CH), 152.28 (C, Ar C-4), 154.50
(C, benzothiazole C-4), 160.94 (C, benzothiazole C-2),
164.70 (C, isoxazole C-3), 169.52 (C, isoxazole C=O); MS
using the following formula:
% inhibition ¼
2
3
ꢀ
ꢁ
activity of mycobacteria without compounds
=
6
7
5
 100
4
Àactivity of mycobacteria in the presence of compounds
ðactivity of mycobacteria without compounds À blankÞ
Antibacterial activity
All bacterial cultures were first grown in Luria Burtony
media at 37 °C at 180 rpm. Once the culture reaches 1
Optical Density (O.D.), it is used for an antibacterial assay.
The Gram-negative and Gram-positive bacterial strains
were obtained from NCIM, NCL, Pune. All the bacterial
strains were grown in Luria Burtony medium from Hi
Media, India. The antibacterial screening was performed in
96-well plates after 8 and 12 h for Gram-negative and
Gram-positive bacteria, respectively (Singh et al. 2011;
Khan and Sarkar 2008). Overall 0.1 % of 1 O.D. culture at
620 nm was used for screening inoculated culture was
added into each well of 96-well plate containing the com-
pounds to be tested and the optical density for each plate
was measured at 620 nm.
(m/z): 396.0762 (M+H)+
.
4-((1H-indol-3-yl)methylene)-3-((benzo[d]thiazol-2-ylthio)
methyl)isoxazol-5(4H)-one, (8g) IR (KBr): 3297, 1716,
1
1592, 1574, 1473, 1370, 1343, 1217, 1195 cm−1; H NMR
(400 MHz, DMSO-d6) δ 4.81 (s, 2H, –S–CH2-isoaxzole),
7.31–7.33 (m, 2H, Ar–H), 7.38 (td, J = 7.7 and 1.2 Hz, 1H,
benzo[d]thiazol-H), 7.48 (td, J = 7.7 and 1.2 Hz, 1H, benzo
[d]thiazol-H), 7.55–7.58 (m, 1H, Ar), 7.77 (d, J = 6.1 Hz,
2H, Py–H), 7.89 (d, J = 7.7 Hz, 1H, benzo[d]thiazol-H),
8.00 (d, J = 7.7 Hz, 1H, benzo[d]thiazol-H), 8.09 (s, 1H,
=CH), 8.13–8.15 (m, 1H, Ar–H), 8.52 (s, 1H, Ar–H), 8.77
(d, J = 6.1 Hz, 2H, Py–H), 12.86 (bs, 1H, NH); 13C NMR
(100 MHz, DMSO-d6) δ 56.85 (CH2, S–CH2-isoxazole),
111.52 (C, Ar C-3), 114.45 (CH, Ar C-8), 118.88 (CH, Ar
C-5), 120.18 (CH, Ar C-7), 120.50 (C, benzothiazole C-8),
122.25 (CH, Ar C-6), 123.41 (C, benzothiazole C-5),
124.70 (C, benzothiazole C-6), 125.60 (C, Ar C-4), 126.15
(C, benzothiazole C-7), 131.81 (CH, Ar C-2), 132.28 (C,
isoxazole C-4), 134.45 (C, Ar C-9), 135.71 (C, benzothia-
zole C-9), 151.30 (CH, =CH), 154.50 (C, benzothiazole
C-4), 164.92 (C, benzothiazole C-2), 166.02 (C, isoxazole
C-3), 169.59 (C, isoxazole C=O); MS(m/z): 392.0448
To determine the minimum inhibitory concentration
(MIC), the compounds were screened at 3, 10, and 30 μg/
mL concentration. Each concentration was tested in dupli-
cates in a single experiment and MIC90 values were calcu-
lated using Origin Pro Software.
Cytotoxic activity
Human Cancer cell lines Cervix adenocarcinoma Hela and
colorectal carcinoma HCT 116 were obtained from NCCS,
Pune and cell cultures were maintained under standard
conditions. The cytotoxic activity in growth inhibition was
determined at 30, 10, 3 μg mL−1 concentrations. The
appropriate culture medium of 0.05% trypsin and 0.02%
ethylene diamine tetra-acetic acid in PBS was diluted to
obtain a cell density of 105 cells/mL. An aliquot of 100 µL
of each suspension was seeded in 96-well cell culture plates
and was incubated at 37 °C for 24 h in an atmosphere of 5%
CO2 and 95% relative humidity in a CO2 incubator. The
synthesized compounds at 3, 10, and 30 µg/mL concentra-
tions were added to the wells containing cells (1 µL/well).
Rifampicin and dimethyl sulphoxide were used as positive
and negative control, respectively. The plates were further
incubated for 48 h, then the solution containing the unat-
tached cells was discarded and the wells were washed three
times with 1 mL of PBS followed by addition of 10 µL of
MTT (5 mg/mL in PBS) to adherent cells in growth medium.
After 4 h at 37 °C for MTT cleavage, the formazan product
was solubilised by addition of 100 µL of 0.04 N HCl in
isopropanol. Absorbance was measured on a SpectraMax®
(M+H)+
.
Biological assay
Antitubercular activity
In vitro antitubercular activity of synthesized compounds
against M. tuberculosis H37Ra (ATCC 25177) was deter-
mined through the XTT reduction menadione assay
(XRMA), reading absorbance at 470 nm as per the protocol
described by (Singh et al. 2011; Khan and Sarkar 2008). In
vitro antitubercular activity against M. bovis BCG (ATCC
35743) was performed using the nitrate reductase (NR)
assay (Khan and Sarkar 2008) to estimate inhibition of M.
bovis BCG by compounds absorbance for the NR assay was
measured at 540 nm. The % inhibition was determined

Medicinal Chemistry Research
PLUS 384 plate reader (Molecular Devices, Sunnyvale, CA)
at a wavelength of 570 nm. Each concentration was tested in
triplicate in a single experiment. Percentage cytotoxicity was
calculated using the following formula:
formation of 4-[(aryl)-methylidene]-3-chloro-methyl-5(4H)-
isoxazolone (4a–g) derivatives (Chavan et al. 2015). 5-
(Pyridine-4-yl)-1,3,4-oxadiazole-2-thiol (5) was achieved
by a ring closure reaction of analogous acid hydrazide with
carbon disulfide in the presence of potassium hydroxide. 5-
(Pyridine-4-yl)-1,3,4-oxadiazole-2-thiol (5) and benzothia-
zole (7) retorton compounds 4a–g in the presence of sodium
hydrogen carbonate in dry ethanol furnished 4-[(substituted
benzylidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-
ylthio)-methyl]isoxazol-5(4H)-one (6a–g) and 4-(sub-
stituted benzylidene)-3-((benzo[d]thiazol-2-ylthio)methyl)
isoxazol-5(4H)-one, (8a–g) respectively, in good yield.
(Table 1).
The reaction of 4-[(4-methoxyphenyl)-methylidene-]-3-
chloro-methyl-5(4H)-isoxazolone (4b) with 5-(pyridine-4-
yl)-1,3,4-oxadiazole-2-thiol (5) in the presence of sodium
hydrogen carbonate in dry ethanol gave 4-[(4-methox-
ybenzylidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-
ylthio)-methyl]isoxazol-5(4H)-one (6b) in 92% yield. The
% cytotoxicity ¼ ðaverage of control À average of compoundÞ=
ðaverage of control À average of blankÞ Â 100Þ
Result and discussion
A general route for the synthesis of 4-[(substituted benzy-
lidene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-ylthio)-
methyl]isoxazol-5(4H)-one (6a–g) and 4-(substituted ben-
zylidene)-3-((benzo[d]thiazol-2-ylthio)methyl)isoxazol-5
(4H)-one, (8a–g) is described in Scheme 1. Ethyl-4-chloro-
acetoacetate (1), hydroxylamine hydrochloride (2), and
substituted aldehydes (3a–g) were agitated at 0 °C to room
temperature in an aqueous condition which resulted the
Scheme 1 Synthesis of
compounds 6a–g and 8a–g
2
2
o
2
2
2 5
3
3
Table 1 Yields and physical constant of compound 6a–g and 8a–g
Comp.
Ar
Yielda (%)
M.P. (°C)
Comp.
Ar
Yielda (%)
M.P. (°C)
6a
6b
6c
6d
6e
6f
4-OH C6H4
88
92
98
90
90
96
78
202
186
168
179
198
202
192
8a
8b
8c
8d
8e
8f
4-OH C6H4
96
88
89
86
89
88
85
200
175
199
178
199
225
248
4-OCH3 C6H4
3-OCH3,4-OH C6H3
4-CH3 C6H4
4-OCH3 C6H4
3-OCH3,4-OH C6H3
4-CH3 C6H4
3,4-OCH2O–C6H3
4-N(CH3)2 C6H4
3-Indole
3,4-OCH2O– C6H3
4-N(CH3)2 C6H4
3-Indole
6g
8g
aIsolated Yield

Medicinal Chemistry Research
infrared spectrum of compound 6b displayed an absorption
band at 1749 cm−1, indicating the presence of lactone C=O.
The absorbance band at 1182 cm−1 represents the thioether
(C–S–C) linkage, this observation clearly indicating the
formation of desired product 6b. The 1H NMR spectrum of
6b exhibited a sharp signal at δ 7.88, which was assigned to
one olefin proton. The two singlets at δ 3.94 and 4.73 are
attributed to –OCH3 and –CH2S, respectively. Two doublets
at δ 7.09 and 8.55 with J = 8.9 Hz corresponds to ortho and
meta protons of 4-methoxyphenyl ring and two doublets at
δ 7.88 and δ 8.80 with J = 6.08 Hz corresponds to the
protons of 4-substituted pyridine. The 13C NMR of com-
pound 6b showed two peaks in the aliphatic region at δ
26.78 and 55.59 due to –SCH2– and –OCH3, respectively.
The isoxazolone carbons resonated at δ 129.92 and 168.09
due to olefinic and isoxazole carbonyl group carbons,
respectively. The mass spectrum of compound 6b displayed
a signals at m/z 395.0815 corresponding to the [M+H]+
ions which are in accordance with the predicted values.
The reaction of 4-[(4-methoxyphenyl)-methylidene-]-3-
chloro-methyl-5(4H)-isoxazolone (4b)with benzothiazole
(7) in the presence of sodium hydrogen carbonate in dry
ethanol gives 4-(4-methoxybenzylidene)-3-((benzo[d]thia-
zol-2-ylthio)methyl)isoxazol-5(4H)-one (8b) in 88% yield,
(Table 2). The structure of compounds was confirmed by
spectroscopic methods.
8.8 Hz was assigned to 4-methoxyphenyl protons. The
aromatic protons of benzothiazole resonated at δ 7.35–7.94.
The 13C NMR spectrum of compound 8b showed peaks at δ
27.35 and δ 55.75 due to –SCH2– and –OCH3, respectively.
The isoxazolone carbons showed two peaks at δ 126.48 and
168.59 due to olefinic carbon and isoxazole carbonyl group.
The structure was further confirmed by mass spectrum
that displayed a signal at m/z 383.0524 corresponding to
[M+H]+.
All the synthesized compounds were characterized by
spectroscopic analysis and screened for their anti-
mycobacterial and antimicrobial activity.
Biological screening
Antimycobacterial activity
The antimycobacterial activity for the synthesized compounds
were evaluated by measuring % inhibition of growth against
M. tuberculosis H37Ra and M. bovis (BCG) in liquid medium.
In vitro activity studies against M. tuberculosis and M. bovis
BCG were performed using the XRMA (Singh et al. 2011;
Khan and Sarkar 2008) and NR assays (Sarkar and Sarkar
2012; Liu and Hou 2012), respectively.
The antibacterial activity (Singh et al. 2011; Khan and
Sarkar 2008) of synthesized compounds was evaluated
against the standard Gram-negative bacteria, E. coli, P.
flurescence and Gram-positive bacteria, S. aureus B. sub-
tilis. In a preliminary antimycobacterial evaluation, all the
compounds were screened at 30 μg/mL concentration. The
The 1H NMR spectrum of 8b exhibited a signal at δ 7.90
which was assigned to one olefinic proton. Two singlets at δ
3.72 and 4.69 were attributed to –OCH3 and –CH2S–,
respectively. Two doublets at δ 6.91 and 8.30 with J =
Table 2 Antimycobacterial
Compound M. tuberculosis H37Ra M. bovis BCG E. coli P. flurescence S. aureus B. subtilis
activity in % inhibition at 30 μg/
mL of compounds 6a–g and
8a–g
6a
6b
6c
6d
6e
6f
4.2
96.9
–
3.71
93.39
4.29
65.68
97.7
64.55
97.73
53.39
65.32
31.09
89.42
64.38
93.98
91.24
40.73
92.77
32.49
81.65
23.47
76.56
97.15
60.62
61.24
31.54
82.15
66.4
66.38
99.27
53.6
55.13
62.02
35.32
80.26
61.08
92.23
91.13
40.78
90.4
40.2
89.9
13.4
43.4
99.4
96.5
80
–
67.17
35.92
87.21
62.66
91.94
92.51
38.63
94.68
36.47
84.04
23.07
82.33
–
6g
8a
8b
8c
8d
8e
8f
3.59
94.49
96.36
87.15
95.27
30.02
48.99
50.16
99.00
92.87
90.87
39.73
95.27
36.29
87.4
92.3
26.1
49.1
16.1
30.01
84.73
22.75
8g
25.35
Rifampicin 99.5
Ampicillin
97.00
96.6
95.6
98.5
– = Inactive
Bold values indicates good activity

Medicinal Chemistry Research
Table 3 Antimycobacterial
activities in MIC90 (µg/mL) of
compounds 6a–g and 8a–g
Compound
M. tuberculosis H37Ra M. bovis BCG E. coli P. flurescence S. aureus B. subtilis
6a
>30
7.46
nd
>30
>30
2.93
>30
>30
>30
>30
>30
24.1
11.25
>30
23.5
>30
>30
>30
1.45
1.62
>30
7.17
>30
>30
>30
>30
>30
20.13
19.5
>30
20.97
>30
>30
>30
4.36
0.49
>30
6.63
>30
>30
>30
>30
>30
11.25
11.23
>30
16.84
>30
>30
>30
1.0
>30
6b
15.01
>30
3.2
6c
>30
6d
>30
>30
>30
>30
25.42
7.9
>30
>30
6e
>30
>30
6f
>30
>30
6g
>30
>30
8a
12.33
12.22
>30
11.58
12.19
>30
8b
8c
>30
29.15
>30
>30
>30
8d
9.34
>30
21.88
>30
8e
8f
>30
>30
8g
>30
>30
Ampicillin
Kanamycin
10.32
1.35
>30
Rifampicin 0.75
0.81
nd = not determined
Bold values indicates good activity
in vitro screening (% inhibition) results against micro-
organisms tested are summarized in Table 2.
good activity against M. tuberculosis H37Ra with MIC
25.42 µg/mL and excellent activity against M. bovis with
the MIC 12.33 µg/mL concentration. It was noted that when
4-OH group was substituted by 4-OCH3 in compound 8b
(Ar = 4-OCH3–C6H4) there was threefold increase in the
activity against M. tuberculosis H37Ra with MIC 7.9 µg/
mL and the activity was retained against M. bovis with MIC
12.22 µg/mL. The 4-OH group of compound 8a when
substituted by 3-OCH3, 4-OH in compound 8c (Ar = 3-
OCH3-4-OH–C6H3) showed moderate activity against
both mycobacterium strains, whereas, the 4-OH group of
compound 8a was substituted by 4-CH3 in compound 8d
(Ar = 4-CH3–C6H4) showed decrease in activity against
M. tuberculosis H37Ra with MIC 29.15 µg/mL
and increase in activity against M. bovis with MIC 9.34 µg/
mL. Compounds 8e (Ar = 4-OCH2O–C6H3), 8f (Ar = 4-N
(CH3)2–C6H4), and 8g (Ar = 3-indol) were found less active
against both mycobacterium strains.
It is necessary to mention that the 4-methoxybenzylidene
group on isoxazol-5(4H)-one in compounds 6b and 8b were
found effective against the M. tuberculosis H37Ra and M.
bovis.
From the antibacterial activity results, it was also noticed
that most of the compounds showed excellent to moderate
activity. Among the compounds 4-(substituted benzyli-
dene)-3-[(5-(pyridine-4-yl)-1,3,4-oxadiazole-2-ylthio)-
methyl]isoxazol-5(4H)-one (6a–g), compounds 6a (Ar = 4-
OH-C6H4) showed moderate activity against all the
screened antibacterial strains. The 4-OH group of
Active compounds were further screened for MIC. The
best potential hits in the series were further evaluated for
cytotoxicity. Rifampicin was used as positive control for
antimycobacterial. Ampicillin and Kanamycin were used as
positive control for antibacterial activity. The MIC in µg/mL
is summarized in Table 3.
The antimycobacterial activity result analysis provided
some lead molecules with good to excellent anti-
mycobacterial activity. The results revealed that, among the
compounds 4-[(substituted benzylidene)-3-[(5-(pyridine-4-
yl)-1,3,4-oxadiazole-2-ylthio)-methyl]isoxazol-5(4H)-one
(6a–g), compound 6a (Ar = 4-OH-C6H4) was found inac-
tive against both mycobacterium strains, while the 4-OH
group when substituted by 4-OCH3 in compound 6b (Ar =
4-OCH3–C6H4) registered excellent activity against M.
tuberculosis H37Ra with MIC 7.46 µg/mL concentration.
Compound 6b also reported good activity against M. bovis
with MIC 15.01 µg/mL concentration. It was noticed that
the 4-OCH3 group in compound 6b (Ar = 4-OCH3–C6H4)
when substituted by the 3,4-O–CH2–O– group in compound
6e (Ar = 3,4-OCH2O–C6H3) activity decreases against
both mycobacterium strains. Whereas the compounds 6c
(Ar = 3-OCH3-4-OH–C6H3), 6f (Ar = 4-N(CH3)2–C6H4)
and 6g (Ar = 3-indol) were found less active against both
strains. Among the compounds 4-(substituted benzylidene)-
3-((benzo[d]thiazol-2-ylthio)methyl)isoxazol-5(4H)-one
(8a–g), compound 8a (Ar = 4-OH-C6H4) registered

Medicinal Chemistry Research
compound 6a, when substituted by 4-OCH3 in compound
6b (Ar = 4-OCH3-C6H4), reported excellent activity against
all bacterial strains. 4-(4-Methoxybenzylidene)-3-((5-(pyr-
idin-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)isoxazol-5(4H)-
one, (6b) registered excellent activity against Gram-
negative bacterial strains E. coli (MIC 2.93 µg/mL) and P.
flurescence (MIC 7.17 µg/mL) which are only twofold less
than the standard drug Ampicillin. Compound 6b (Ar = 4-
OCH3–C6H4) also showed excellent activity against Gram-
positive bacterial strains S. aureus (MIC 6.63 µg/mL) and B.
subtilis (MIC 3.2 µg/mL) which is threefold more potent
than the reference drug Ampicillin. Compounds 6c (Ar = 3-
OCH3-4-OH–C6H3), 6d (Ar = 4-CH3–C6H3), 6e (Ar = 3,4-
OCH2O–C6H3) 6f (Ar = 4-N(CH3)2–C6H4) and 6g (Ar = 3-
indol) reported moderate activity against bacterial strains.
Among the compounds 4-(substituted benzylidene)-3-
((benzo[d]thiazol-2-ylthio)methyl)isoxazol-5(4H)-one
activity decreased against S. aureus (MIC 16.84 µg/mL) and
B. subtilis (MIC 21.88 µg/mL). Compounds 8c (Ar = 3-
OCH3-4-OH–C6H3), 8e (Ar = 4-OCH2–O–C6H3), 8f (Ar =
4-N(CH3)2–C6H4) and 8g (Ar = 3-indol) were found active
against all tested bacterial strains.
It is worth mentioning that compounds 4-(4-methox-
ybenzylidene)-3-((5-(pyridin-4-yl)-1,3,4-oxadiazol-2-
ylthio)methyl)isoxazol-5(4H)-one, (6b) and 4-(4-methox-
ybenzylidene)-3-((benzo[d]thiazol-2-ylthio)methyl)iso-
xazol-5(4H)-one, (8b) reported excellent antimycobacterial
activity and compound 6b also showed excellent anti-
bacterial activity against all tested strains. The structure
activity relationship revealed that isoxazolone pharmaco-
phore substituted by 4-methoxybenzylidene at 4 position
and 5-(pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl at 3
position, respectively showed excellent antimycobacterial
and antibacterial activity.
(8a–g), compound 8a (Ar = 4-OH-C6H4), reported good
activity against Gram-negative bacterial strains E. coli (MIC
24.1 µg/mL) and P. flurescence (MIC 20.13 µg/mL) and
excellent activity against Gram-positive bacterial strains S.
aureus (MIC 11.25 µg/mL) and B. subtilis (MIC 11.58 µg/
mL). It was observed that, when 4-OH group was sub-
stituted by 4-OCH3 in compound 8b (Ar = 4-OCH3-C6H4),
the activity against E. coli increased by twofolds with MIC
11.25 µg/mL while the activity was retained against P.
flurescence (MIC 19.5 µg/mL), S. aureus (MIC 11.23 µg/
mL) and B. subtilis (MIC 12.19 µg/mL). Whereas the 4-OH
group of compound 8a when substituted by 4-CH3 in
compound 8d, the activity was retained against E. coli (MIC
23.5 µg/mL) and P. flurescence (MIC 20.97 µg/mL) while
Cytotoxic activity
The synthesized compounds were further evaluated against
two human cancer cell lines (cervix adenocarcinoma HeLa
and colorectal carcinoma HCT 116) to check the toxicity of
these compounds using the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazoliumbromide MTT assay with 48 h
exposure time of the tested compounds. Rifampicin was
used as a positive control. From the cytotoxicity activity
result analysis (Table 4), it was worth to mention that all
screened thioether derivatives are found less cytotoxic
against HeLa and HCT 116 cell lines and the active com-
pounds are leads as antimicrobials.
Table 4 Cytotoxic activity of
compounds 6a–g and 8a–g
Compound
HeLa
HCT 116
30 µg/mL
10 µg/mL
3 µg/mL
30 µg/mL
10 µg/mL
3 µg/mL
6a
49.77
–
–
37.58
12.95
–
17.20
4.16
–
2.31
6b
–
–
–
–
6c
–
–
–
–
6d
–
–
–
–
–
–
–
16.90
–
6e
–
–
34.89
–
17.34
–
6f
–
–
–
6g
–
–
–
40.73
51.97
33.56
–
27.19
25.88
–
16.45
8.46
–
8a
70.97
41.27
–
–
–
8b
–
–
–
8c
–
–
–
8d
80.80
75.80
–
0.33
–
–
65.55
22.81
–
20.92
–
10.91
–
8e
–
–
8f
–
–
–
8g
–
–
–
39.98
22.15
17.60
16.50
–
Rifampicin
25.50
20.10
15.80
10.12
– = Inactive

Medicinal Chemistry Research
Table 5 ADME prediction of compounds 6a–g and 8a–g
Comp. MW TPSA iLOGP GI absorption BBB
permeant
CYP1A2
inhibitor
CYP2C19
inhibitor
CYP2C9
inhibitor
CYP3A4
inhibitor
Lipinski
#violations
6a
6b
6c
6d
6e
6f
380.4 136
394.4 125
2.44
2.83
High
High
High
High
High
High
High
High
High
High
High
High
High
High
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Yes
Yes
No
410.4 134.23 2.99
378.4 115.77 3.03
408.4 134.23 2.99
407.4 119.01 3.03
Yes
Yes
No
6g
8a
8b
8c
8d
6e
8f
403.4 136
2.44
368.4 125.32 2.59
382.5 114.32 3.44
398.5 119.45 2.98
366.5 105.09 3.19
396.4 123.55 3.27
395.5 108.33 3.24
391.5 120.88 2.86
Yes
Yes
Yes
Yes
Yes
Yes
Yes
8g
ADME Prediction
antibacterial activities. Also, benzothiazolyl thioether at
3 position of isoxazolone and –OH or –CH3 group on 4
position of benzyledene showed good to excellent
activity against all the tested strains. It is noteworthy
that, compounds 6b and 8b could serve as lead mole-
cules, as they reveal good antimycobacterial as well as
antibacterial activity. Thus, these results will be enligh-
tening to researchers for further study.
ADME of the newly synthesized compounds were studied
via using online free portal www. swissadme.ch to check
the possible violation of any drug-like properties (Swis-
sADME 2017). All compounds following the Lipinski’s
rule of 5 so have potential to be developed as drug. The
synthesized compounds are showed high absorption at GI
tract, not crossing the blood brain barrier. Solubility of
compounds in water and lipid plays a very important role in
the distribution of drug. All compounds are lipid soluble
and poorly soluble in water except 6a, 8a, and 8f which are
moderately soluble in water. But theses have high absorp-
tion by GI. The dissolution in vivo will be rate limiting step
in drug absorption. Drug metabolism is dependent on
cytochrome p450 enzymes. Those drugs that are metabo-
lized by CYP2D6 to inactive metabolites, CYP2D6 inhi-
bitors may result in toxicity. These compounds are not
showing inhibitory action to CYP2D6 but these are the
inhibitors of rest of the isozymes of cytochrome p450
family (Table 5).
Acknowledgements The authors would like to express sincere thanks
to Savitribai Phule Pune University, Pune and DST, New Delhi for
providing financial assistance. CIF Savitribai Phule Pune University is
also acknowledged for the spectral analysis.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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