Design, Synthesis, and Anticancer Activity of Bis-isoxazole Incorporated Benzothiazole Derivatives

Article abstract of DOI:10.1134/S1070363220100229

Abstract: A novel series of bis-isoxazole incorporated benzothiazole derivatives hasbeen designed and synthesized. Molecular structures of the compounds have beenconfirmed by ¹H and ¹³CNMR, and mass spectra. All products have been tested for their in vitro anticancer activity against breast MCF-7and MDA MB-231, lungs A549, and prostate DU-145 cancer cell lines using the MTTassay and etoposide as a standard drug. Most of the compounds have demonstratedgood to moderate activity, and some of those have exhibited more potent activitythan etoposide.

Full text of DOI:10.1134/S1070363220100229

ISSN 1070-3632, Russian Journal of General Chemistry, 2020, Vol. 90, No. 10, pp. 1981–1989. © Pleiades Publishing, Ltd., 2020.
Design, Synthesis, and Anticancer Activity of Bis-isoxazole
Incorporated Benzothiazole Derivatives
Purna Koteswara Rao Cherukumallia, Bhaskara Rao Tadiboinaa,*, Kali Charan Gulipallia,
Srinu Bodigea, Kiran Gangarapub, and Gattu Sridharc
a Department of Chemistry, Koneru Lakshmaiah Education Foundation, Green Fields,
Vaddeswaram, Guntur, 522502 India
b School of Pharmacy, Anurag Group of Institutions, Venkatapur, Ghatkaser, Medchal,
Telangana, 500088 India
c Department of Chemistry, Kakatiya Institute of Technology and Science, Warangal, 506015 India
*e-mail: bhaskararaotadiboina1org@gmail.com
Received August 1, 2020; revised September 10, 2020; accepted October 26, 2020
Abstract—Anovel series of bis-isoxazole incorporated benzothiazole derivatives has been designed and synthesized.
Molecular structures of the compounds have been confirmed by 1H and 13C NMR, and mass spectra. All products
have been tested for their in vitro anticancer activity against breast MCF-7 and MDA MB-231, lungs A549, and
prostate DU-145 cancer cell lines using the MTT assay and etoposide as a standard drug. Most of the compounds
have demonstrated good to moderate activity, and some of those have exhibited more potent activity than etoposide.
Keywords: benzothiazole, isoxazole, anticancer activity
DOI: 10.1134/S1070363220100229
INTRODUCTION
Similarly, 1,2-isoxazoles demonstrated antitumor
[11], anti-oxidant [12], antifungal [13], and a range of
other activities. One of the anticancer drug candidates
Luminespib (3, NVP-AUY922, Fig. 1) contains a
synthetic 1,2-isoxazole scaffold [14].
Heterocyclic compounds, and specifically for the
current study benzothiazoles, demonstrate a broad
range of biological activities including anticancer [1],
antimicrobial [2], antiviral [3], analgesic [4], antimalarial
[5], fungicidal [6], antihelmintic [7], antitubercular [8],
and many more. For example, molecules of compounds
NSC-710305 (1, Fig. 1) and violatinctamine (2, Fig. 1)
[9] contain benzothiazole moieties in their structures.
Compound 1 demonstrated anticancer activity and has
been processed to phase-1 clinical trial [10].
In view of the above and in continuation of the ongoing
studies, we have designed and synthesized a new series
of bis-isoxazole incorporated benzothiazole derivatives
(14a–14j) and confirmed their structures by 1H and 13
C
NMR, and mass spectra. The products were screened
for their anticancer activity towards four human cancer
cell lines.
Fig. 1. Structures of anticancer drugs: (a) NSC-710305, 1, (b) Violatinctamine, 2, and (c) NVP-AUY922, 3.
1981

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PURNA KOTESWARA RAO CHERUKUMALLI et al.
Table 1. In vitro cytotoxicity of the synthesized compounds 14a–14j (IC50, μM)a
Compound
14a
MCF-7
3.45±2.67
0.45±0.059
1.29±0.94
7.10±4.56
12.4±6.20
15.3±7.34
0.041±0.0078
0.32±0.061
0.76±0.083
1.52±0.56
2.11 ± 0.024
A549
4.11±2.10
0.10±0.012
1.98±0.33
3.77±2.88
5.69±3.14
Not determined
0.023±0.0056
0.95±0.033
0.33±0.099
1.61±0.48
DU-145
MDA MB-231
13.4±6.45
0.11±0.087
2.34±1.23
2.86±1.66
Not determined
5.04±3.77
0.07±0.0091
0.05±0.0039
0.90±0.083
0.18±0.066
1.91 ± 0.84
ND
14b
14c
14d
14e
14f
14g
14h
14i
1.22±0.72
1.56±0.27
9.94±5.34
4.71±2.18
Not determined
0.65±0.064
0.49±0.077
0.85±0.018
2.08±1.89
14j
Etoposide
3.08 ± 0.135
1.97 ± 0.45
a Each data is presented as mean ±S.D value.
RESULTS AND DISCUSSION
activity. The compounds 14e and 14f with 3-bromophenyl
and 4-nitrophenyl rings exhibited moderate activity.
The compound 14g containing 4-pyridyl heterocyclic
ring demonstrated excellent activity on all cell lines.
Replacement of 4-pyridyl ring with 2-pyrrolyl ring
resulted in slightly lower activity of compound 14h.
Compound 14i containing 2-thiophenyl ring exhibited
lower activity than 14h. 5-Bromothiophenyl substituent
in derivative 14j caused its slightly decreased activity
than that of 14i.
Synthesis of new series of bis-isoxazole incorporated
benzothiazole derivatives 14a–14j is outlined in Scheme 1.
The 3,4,5-trimethoxyphenyl oxime (4) was reacted with
1-ethynyl-4-nitrobenzene (5) in presence aq NaOCl in
anhydrous media of CH2Cl2 at room temperature to give
pure isoxazole derivative 6.
Reduction of the nitro group in the intermediate 6 by
Pd/C, H2 in ethanol yielded pure amino intermediate 7.
Its reaction with 4-isothiocyanatophenol (8) in ethanol
led to pure intermediate 9, cyclization of which in the
presence of [bbim][Br3] ionic liquid at 70°C afforded pure
benzothiazole intermediate 10. Reaction of compound
10 with propargyl bromide 11 in presence of K2CO3 in
anhydrous DMF resulted in formation of the intermediate
12, following cyclization of which with different types
of aryl oximes (4, 13a–13i) in presence of aq. NaOCl
and TEA gave the corresponding pure target compounds
14a–14j.
Molecular docking studies. Molecular docking
studies were carried out for the synthesized compounds
14a–14j to identify the molecular interactions between
anticancer target human topoisomerase II beta in complex
with DNA ligands bound with inhibitor etoposide (PDB
ID:3QX3) by using AutoDock4.2.4.
All the docked ligands demonstrated interactions with
Glu477, Asp479, Arg503, Met555, and Gln778 of Topo-
II enzyme and DT9, DC8, DA12, and DG13 of DNA
nucleotide residues (Table 2).
Biological activity of the products. The newly
synthesized bis-isoxazole incorporated benzothiazole
derivatives 14a–14j were tested for their in vitro
anticancer activity against breast MCF-7 and MDAMB-
231, lungsA549, and prostate DU-145 cancer cell lines by
using the MTT assay and etoposide as the standard drug
(Table 1). All the compounds exhibited low to moderate
activity. Among those the products 14b, 14c, 14g–14j
were determined to be more potent than etoposide. The
structure-activity relationship (SAR) analysis indicated
that the compound with 3,4,5-trimethoxyphenyl ring (14b)
exhibited high anticancer activity on all cell lines, whereas
the one with 2,4-dimethoxyphenyl ring (14c) displayed
somewhat lower activity, and the 2,3-dimethoxyphenyl
substituted analogue (14d) was characterized by very poor
Binding affinity of the docked compounds was
expressed as negative energy in kcal/mol (dock score).
The ligands with more negative value of dock score
had higher affinity with Topo-II and DNA binding.
As an example, pyridinyl substituted compound 14g
formed H-bond with Gln778 (2.71 Å) and Arg503
(3.01 Å) residues, as well as hydrophobic and electrostatic
interactions with Arg503 residue (Table 2, Fig. 2).
Similarly, bromothiophene substituted compound 14j
demonstrated H-bond interactions with Glu777(2.73),
DG10(2.09), DT9(2.82), DA12(2.41), and electrostatic
interactions with Arg503, DC8, DT9, and hydrophobic
interactions with DC8, DA12, DG10, Arg503 active
residues (Table 2, Fig. 3).
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DESIGN, SYNTHESIS, AND ANTICANCER ACTIVITY
1983
Scheme 1. Synthetic approach to the target bis-isoxazole incorporated benzothiazole derivatives 14a–14j.
All ligands demonstrated the same interactions with
Topo-II protein and DNAnucleotides and accordingly the
compounds had potential of inhibiting the Topo-II protein
and intercalating with the base pairs of DNAnucleotides.
EXPERIMENTAL
All chemicals and reagents were obtained fromAldrich
(Sigma–Aldrich, St. Louis, MO, USA) and Lancaster
(AlfaAesar, Johnson Matthey Company, Ward Hill, MA,
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PURNA KOTESWARA RAO CHERUKUMALLI et al.
Table 2. Molecular docking data for the synthesized compounds against the DNA topoisomerase II beta in complex with DNA
(PDB ID: 3QX3)
Interactions
elecrostatic
Dock
score
Compound
LE Ki, pM
H-bond (distance, Å)
Lys505(3.54)
hydrophobic
DC8, His775, DG7,
Lys505
14a
–9.99 –0.23 4769
Glu477, DC8
14b
14c
–11.78 –0.34 2721 DC8 (3.14), DT9 (3.99), Met555 (3.14)
DC8, DT9
Ala481, Arg503,
Leu484, DA12
Leu484, DA12,
DG13
–8.87 –0.28 2940
–11.64 –0.23 2840
–12.27 –0.28 189.78
Glu477 (3.09), Gln778 (2.25), DT9
(3.84)
Asp503, DC8
14d
DT9 (3.68), Asp848 (2.96), Gln778
(3.64)
DT9
Ala772, DT9, DC8,
Arg503
14e
14f
14g
14h
14i
Arg503 (3.05), DT9 (3.43), DG10
(3.36), Gln778 (3.74)
Asp557, DT9
DA12, His774, DC8,
DG13
DC8, DT9, Met555,
Leu484
Phe500, DC8, DA12,
DG13, DC8, Arg503
DA12, DG13, DT9,
Leu484
Ala481, DA12,
Met555, Leu484,
DT9
–12.92 –0.26 338.78 Ala481 (3.13), Arg503 (3.03), Gln778
Arg503, Glu477,
Asp557, DT9
Arg503
(3.02)
–12.44 –0.27 764.14 Arg503 (3.01), Gln778 (2.71), Pro501
(2.88), DC8 (3.28)
–11.91 –0.26 1850
Ser480 (2.65), Asp557 (3.55), Tyr643
(3.30)
Glu477
–12.71 –0.28 481.83 DT9 (3.34), DC8 (2.88), Met555 (2.99),
Gln778 (2.90)
Glu477, Asp557,
DT9
14j
–12.97 –0.28 313.5
Glu777 (2.73), DG10 (2.09), DT9
(2.82), DA12 (2.41)
Arg503, DC8, DT9 DC8, DA12, DG10,
Arg503
Etoposide –13.54 –0.20 158.36
Asp479 (2.51), Gln778 (3.11), DC8
(3.21), DG13 (2.71), Gly478 (3.02),
DT9 (3.30)
Arg503, Met782
DG13, DA12,
Met782
USA), and used without further purification. Reactions
were monitored by TLC, performed on silica gel plates
covered by 60 F-254 and visualized under UV light or
by iodine indicator. Melting points were determined
with an electro thermal melting point apparatus and are
uncorrected.
1H and 13C NMR spectra were measured on a
BRUKER NMR 300 MHz and 400 MHz spectrometers
using TMS as the internal standard. ESI spectra were
measured on a Micro mass, Quattro LC using ESI+
software with capillary voltage 3.98 kV and ESI mode
positive ion trap detector.
with dichloromethane. The combined organic layers were
dried with Na2SO4 and concentrated. The crude product
was purified by column chromatography with ethyl
acetate/hexanes (4 : 6) to afford pure compound 6, yield
1
66%. H NMR spectrum, δ, ppm: 3.71 s (3H), 3.92 s
(6H), 7.23 s (2H), 7.91 s (1H), 8.21 d (2H, J = 8.7 Hz),
8.42 d (2H, J = 8.7 Hz). MS (ESI): 357 [M + H]+. Found,
%: C 60.62; H 4.49; N 7.91. C18H16N2O6.Calculated, %:
C 60.67; H 4.53; N 7.86.
4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]ben-
zenamine (7). Compound 6 (22 g, 0.0617 mmol) was
dissolved in ethanol and mixed with 10% Pd/C (656 mg
0.00617 mmol). The reaction mixture was stirred under
the atmosphere of H2 (1 atm) at room temperature for
6 h. After completion of reaction (TLC), the catalyst was
filtered off over celite and the filtrate was concentrated
in vacuo to provide the product 7 (yield 91%) which was
used further without purification. 1H NMR spectrum, δ,
ppm: 3.71 s (3H), 3.92 s (6H), 7.23 s (2H), 7.31 s (2H),
7.89 s (1H), 8.15 d (2H, J = 8.4 Hz), 8.29 d (2H, J =
3-(3,4,5-Trimethoxyphenyl)-5-(4-nitrophenyl)-
isoxazole (6). The compound 4 (33.7 g, 0.158 mmol)
and 1-ethynyl-4-nitrobenzene 5 (15 g, 0.101 mmol) were
mixed with dichloromethane (80 mL), and then cooled
down to 0°C. Aqueous NaOCl (11 mL, 0.158 mmol) was
added drop wise to the above mixture for over 30 min, and
then stirred vigorously for 8 h at room temperature. The
layers were separated, and the aqueous layer was extracted
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DESIGN, SYNTHESIS, AND ANTICANCER ACTIVITY
1985
Fig. 2. (a) 2D and (b) 3D interactions of compound 14g with DNA Topoisomerase II beta in complex with DNA (PDB ID: 3QX3).
8.4 Hz). MS (ESI): 327 [M + H]+. Found, %: C 66.21;
H 5.52; N 8.63. C18H18N2O4. Calculated, %: C 66.25; H
5.56; N 8.58.
completion of the reaction (TLC), chloroform was added
to the mixture and the product was separated as crude
residue. The mixture was filtered and the residue was
recrystallized from ethanol to afford pure compound
10, yield 87%. 1H NMR spectrum, δ, ppm: 3.72 s (3H),
3.93 s (6H), 7.24 s (2H), 7.41 s (1H), 7.48 d (1H, J =
8.6 Hz), 7.62 d (1H, J = 8.6 Hz), 7.95 s (1H), 8.16 d (2H,
J = 8.3 Hz), 8.27 d (2H, J = 8.3 Hz), 10.36 bs (1H), 12.54
s (1H). MS (ESI): 476 [M + H]+. Found, %: C 63.11; H
4.42; N 8.89. C25H21N3O5S. Calculated, %: C 63.15; H
4.45; N 8.84.
1-(4-Hydroxyphenyl)-3-{4-[3-(3,4,5-trimethoxy-
phenyl)isoxazol-5-yl]phenyl}thiourea (9). To a
solution of compound 7 (17 g, 0.0521 mmol) in absolute
ethanol (5 mL), 4-isothiocyanatophenol (8) (8.45 mL,
0.0782 mmol) was added at 0oC, and stirring of the
mixture was continued for 6 h at room temperature. The
formed solid was filtered off and washed thoroughly with
1
ethanol to afford the pure compound 9, yield 87%. H
NMR spectrum, δ, ppm: 3.71 s (3H), 3.92 s (6H), 7.23 s
(2H), 7.34 d (2H, J = 7.56 Hz), 7.65 d (2H, J = 7.56 Hz),
7.93 s (1H), 8.13 d (2H, J = 8.1 Hz), 8.25 d (2H, J =
8.1 Hz), 9.41 s (1H), 10.32 bs (1H), 11.40 s (1H). MS
(ESI): 478 [M + H]+. Found, %: C 62.84; H 4.82; N 8.84.
C25H23N3O5S. Calculated, %: C 62.88; H 4.85; N 8.80.
N-{4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]-
phenyl}-6-(prop-2-ynyloxy)benzo[d]thiazol-2-amine
(12). Compound 10 (16 g, 0.0336 mmol) was dissolved in
dry N,N-dimethyl formamide (50 mL), and K2CO3 (9.3 g,
0.0673 mmol) was added to the mixture which was stirred
for 15 min at room temperature. Propargyl bromide 11
(2.6 mL, 0.0336 mmol) was slowly added drop wise to
the above mixture over a period of 15 min and stirring
was continued for 4 h. The reaction was quenched with
water and extracted with ethyl acetate (3×20 mL). The
combined extracts were washed with water (3×25 mL)
2-{4-[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]-
phenylamino}benzo[d]thiazol-6-ol (10). Compound 9
(20g, 0.0419mol)wasaddedto1,3-di-n-butylimidazolium
bromide ([bbim][Br]) (11 g, 0.0419 mmol), and the
reaction mixture was stirred at 70°C for 40 min. After
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PURNA KOTESWARA RAO CHERUKUMALLI et al.
Fig. 3. (a) 2D and (b) 3D interactions of 14j with DNA topoisomerase II beta in complex with DNA (PDB ID: 3QX3).
and brine (20 mL). The organic layer was dried over
Na2SO4, filtered, evaporated under reduced pressure, and
purified by silica gel chromatography with ethyl acetate/
hexane (1 : 1) to afford pure intermediate 12, yield 80%.
1H NMR spectrum, δ, ppm: 3.10 s (1H), 3.72 s (3H),
3.93 s (6H), 4.90 s (2H), 7.24 s (2H), 7.43 s (1H), 7.50 d
(1H, J = 8.6 Hz), 7.63 d (1H, J = 8.6 Hz), 7.95 s (1H),
8.15 d (2H, J = 8.4 Hz), 8.26 d (2H, J = 8.4 Hz), 12.55 s
(1H). MS (ESI): 514 [M + H]+. Found, %: C 65.44; H
4.47; N 8.23. C28H23N3O5S. Calculated, %: C 65.48; H
4.51; N 8.18.
afford pure compound 14a. Yield 50%, mp 212–214°C
NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.12 s
(2H), 7.23 s (2H), 7.39–7.56 m (5H), 7.64 d (1H, J = 8.7
Hz), 7.71 d (2H, J = 7.65 Hz), 7.81 s (1H), 7.96 s (1H),
8.15 d (2H, J = 8.5 Hz), 8.27 d (2H, J = 8.5 Hz), 12.56 s
(1H). 13C NMR spectrum, δ, ppm: 57.4, 61.8, 65.3, 96.3,
96.9, 107.3, 108.5, 115.3, 119.6, 120.5, 124.5, 125.6,
126.2, 126.8, 127.5, 128.2, 130.4, 134.5, 137.3, 142.5,
149.6, 154.7, 155.4, 157.6, 160.5, 164.7, 165.8, 171.8. MS
(ESI): 633 [M + H]+. Found, %: C 66.40; H 4.41; N 8.90.
C35H28N4O6S. Calculated, %: C 66.44; H 4.46; N 8.86.
The compounds 14b–14j were synthesized according
to the method developed for 14a using the appropriate
benzaldoximes 4, 13b–13i.
6-[(3-Phenylisoxazol-5-yl)methoxy]-N-{4-[3-(3,4,5-
trimethoxyphenyl)isoxazol-5-yl]phenyl}benzo[d]-
thiazol-2-amine (14a). Benzaldoxime (13a) (0.17 mL,
15.5 mmol) and compound 12 (500 mg, 9.7 mmol)
were dissolved in dichloromethane (10 mL), and the
solution was cooled to 0°C. Aqueous NaOCl (0.1 mL,
15.5 mmol) was added dropwise over 30 min, and
the reaction mixture was stirred vigorously for 8 h at
room temperature. The layers were separated, and the
aqueous layer was extracted with dichloromethane. The
combined organic layers were dried with Na2SO4 and
concentrated. The crude product was purified by column
chromatography with ethyl acetate/hexanes (6 : 4) to
6-{[3-(3,4,5-Trimethoxyphenyl)isoxazol-5-yl]-
methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl) isoxazol-
5-yl]phenyl}benzo[d]thiazol-2-amine (14b). Yield 52%,
mp 222–224°C NMR spectrum, δ, ppm: 3.72 s (3H),
3.86 s (3H), 3.92 s (6H), 3.96 s (6H), 5.09 s (2H), 7.24 s
(2H), 7.43 s (1H), 7.52–7.60 m (3H), 7.64 d (1H, J =
8.5 Hz), 7.80 s (1H), 7.95 s (1H), 8.14 d (2H, J = 8.4 Hz),
8.26 d (2H, J = 8.4 Hz), 12.54 s (1H). 13C NMR spectrum,
δ, ppm: 57.5, 58.3, 61.7, 62.4, 65.4, 96.4, 96.8, 106.5,
107.4, 107.7, 115.6, 119.6, 120.4, 126.4, 127.6, 128.7,
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DESIGN, SYNTHESIS, AND ANTICANCER ACTIVITY
1987
6-{[3-(4-Nitrophenyl)isoxazol-5-yl]methoxy}-N-
{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}-
benzo[d]thiazol-2-amine (14f). Yield 60%, mp 230–
232°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H),
5.13 s (2H), 7.24 s (2H), 7.44 s (1H), 7.56 d (1H, J = 8.4
Hz), 7.66 d (1H, J = 8.4 Hz), 7.86 s (1H), 7.96 s (1H),
8.15 d (2H, J = 8.5 Hz), 8.22 d (2H, J = 7.67 Hz), 8.27 d
(2H, J = 8.5 Hz), 8.34 d (2H, J = 7.67 Hz), 12.56 s (1H).
13C NMR spectrum, δ, ppm: 57.6, 61.8, 65.6, 96.4, 97.6,
107.6, 108.3, 115.4, 119.5, 120.7, 126.4, 127.3, 127.8,
128.6, 130.5, 131.3, 134.7, 137.6, 142.4, 149.6, 150.4,
154.6, 155.5, 157.4, 160.6, 164.5, 165.8, 171.8. MS (ESI):
678 [M + H]+. Found, %: C 62.99; H 3.97; N 10.37.
C35H27N5O8S. Calculated, %: C 62.03; H 4.02; N 10.33.
130.5, 134.2, 137.6, 142.3, 149.6, 154.3, 155.5, 157.3,
158.1, 159.5, 164.6, 165.7, 171.8. MS (ESI): 723 [M +
H]+. Found, %: C 63.11; H 4.70; N 7.80. C38H34N4O9S.
Calculated, %: C 63.15; H 4.74; N 7.75.
6-{[3-(2,4-Dimethoxyphenyl)isoxazol-5-yl]meth-
oxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]-
phenyl}benzo[d]thiazol-2-amine (14c). Yield 47%, mp
227–229°C NMR spectrum, δ, ppm: 3.70 s (3H), 3.72 s
(3H), 3.86 s (3H), 3.92 s (6H), 4.97 s (2H), 6.85 s (1H),
7.09 d (1H, J = 7.34 Hz), 7.24 s (2H), 7.43 s (1H), 7.55 d
(1H, J = 8.3 Hz), 7.61–7.68 m (2H), 7.81 s (1H), 7.95 s
(1H), 8.15 d (2H, J = 8.5 Hz), 8.26 d (2H, J = 8.5 Hz),
12.55 s (1H). 13C NMR spectrum, δ, ppm: 56.4, 57.6,
58.5, 61.7, 65.7, 96.4, 97.6, 101.7, 107.4, 107.8, 109.5,
110.3, 115.4, 119.6, 120.3, 126.4, 126.7, 127.5, 130.5,
134.2, 137.5, 142.3, 149.5, 154.3, 157.6, 158.2, 160.4,
162.4, 164.5, 164.7, 165.6, 171.7. MS (ESI): 693 [M +
H]+. Found, %: C 64.10; H 4.62; N 8.14. C37H32N4O8S.
Calculated, %: C 64.15; H 4.66; N 8.09.
6-{[3-(Pyridin-4-yl)isoxazol-5-yl]methoxy}-N-{4-
[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}-
benzo[d]thiazol-2-amine (14g). Yield 56%, mp 255–
257°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H),
5.15 s (2H), 7.24 s (2H), 7.45 s (1H), 7.56 d (1H, J =
8.5 Hz), 7.65 d (1H, J = 8.5 Hz), 7.87 s (1H), 7.96 s (1H),
8.15 d (2H, J = 8.6 Hz), 8.27 d (2H, J = 8.6 Hz), 8.52 d
(2H, J = 8.01 Hz), 8.78 d (2H, J = 8.01 Hz), 12.56 s
(1H). 13C NMR spectrum, δ, ppm: 57.4, 61.8, 65.7, 96.4,
97.8, 107.5, 108.3, 115.3, 119.6, 120.5, 121.8, 126.5,
127.4, 130.5, 134.6, 137.3, 138.5, 142.6, 149.7, 152.4,
154.6, 155.7, 157.4, 160.5, 164.5, 165.8, 171.8. MS (ESI):
634 [M + H]+. Found, %: C 64.39; H 4.25; N 11.10.
C34H27N5O6S. Calculated, %: C 64.44; H 4.29; N 11.05.
6-{[3-(2,3-Dimethoxyphenyl)isoxazol-5-yl]meth-
oxy}-N-{4-[3-(3,4,5-trimethoxyphenyl) isoxazol-5-yl]-
phenyl}benzo[d]thiazol-2-amine (14d). Yield 51%, mp
225–227°C NMR spectrum, δ, ppm: 3.71 s (3H), 3.73 s
(3H), 3.87 s (3H), 3.92 s (6H), 4.97 s (2H), 6.86–6.90 m
(1H), 7.12 t (1H), 7.23 s (2H), 7.44 s (1H), 7.56 d (1H,
J = 8.2 Hz), 7.60–7.69 m (2H), 7.82 s (1H), 7.96 s (1H),
8.15 d (2H, J = 8.3 Hz), 8.27 d (2H, J = 8.3 Hz), 12.56 s
(1H). 13C NMR spectrum, δ, ppm: 56.4, 57.7, 61.5, 61.8,
65.7, 96.4, 97.5, 107.3, 107.8, 115.4, 118.2, 119.5, 120.2,
120.6, 121.5, 123.4, 126.5, 127.6, 130.4, 134.2, 137.2,
142.4, 147.5, 149.7, 154.3, 154.7, 157.4, 157.8, 162.4,
164.7, 165.3, 171.8. MS (ESI): 693 [M + H]+. Found, %:
C 64.11; H 4.63; N 8.14. C37H32N4O8S. Calculated, %:
C 64.15; H 4.66; N 8.09.
6-{[3-(1H-Pyrrol-2-yl)isoxazol-5-yl]methoxy}-N-
{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}-
benzo[d]thiazol-2-amine (14h). Yield 60%, mp 258–
260°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H),
5.10 s (2H), 7.24 s (2H), 7.32 d (1H, J = 8.10 Hz), 7.39
t (1H), 7.44 s (1H), 7.52–7.59 m (2H), 7.66 d (1H, J =
8.4 Hz), 7.86 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.6 Hz),
8.27 d (2H, J = 8.6 Hz), 12.56 s (1H). 13C NMR spectrum,
δ, ppm: 57.6, 61.8, 65.7, 94.5, 96.7, 107.3, 107.8, 113.4,
115.4, 116.7, 119.5, 120.4, 122.3, 126.5, 127.3, 129.5,
130.4, 134.2, 137.5, 142.4, 149.6, 150.3, 154.5, 157.3,
157.8, 164.5, 165.2, 171.7. MS (ESI): 622 [M + H]+.
Found, %: C 63.72; H 4.33; N 11.32. C33H27N5O6S.
Calculated, %: C 63.76; H 4.38; N 11.27.
6-{[3-(3-Bromophenyl)isoxazol-5-yl]methoxy}-N-
{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}-
benzo[d]thiazol-2-amine (14e). Yield 58%, mp 229–231
NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H), 5.08 s
(2H), 7.24 s (2H), 7.39–7.51 m (3H), 7.56 d (1H, J = 8.3
Hz), 7.59–7.70 m (2H), 7.84 s (1H), 7.96 s (1H), 8.10 s
(1H), 8.15 d (2H, J = 8.5 Hz), 8.27 d (2H, J = 8.5 Hz),
12.56 s (1H). 13C NMR spectrum, δ, ppm: 57.4, 61.7,
65.8, 96.4, 97.2, 107.3, 107.7, 115.3, 119.5, 120.5, 125.4,
126.4, 127.5, 128.2, 130.4, 131.3, 133.4, 133.6, 134.7,
137.4, 142.5, 149.7, 154.6, 155.6, 157.4, 160.5, 164.5,
165.3, 171.8. MS (ESI): 713 [M + H]+. Found, %: C
59.04; H 3.77; N 7.92. C35H27N4O6BrS. Calculated, %:
C 59.08; H 3.82; N 7.87.
6-{[3-(Thiophen-2-yl)isoxazol-5-yl]methoxy}-N-
{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-5-yl]phenyl}-
benzo[d]thiazol-2-amine (14i). Yield 61%, mp 263–
265°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s (6H),
5.11 s (2H), 7.24 s (2H), 7.32 t (1H), 7.38 d (1H, J =
8.3 Hz), 7.44 s (1H), 7.54–7.59 m (2H), 7.66 d (1H, J =
8.4 Hz), 7.86 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.6 Hz),
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1988
PURNA KOTESWARA RAO CHERUKUMALLI et al.
8.27 d (2H, J = 8.6 Hz), 12.56 s (1H). 13C NMR spectrum,
δ, ppm: 57.5, 61.8, 65.7, 96.4, 97.3, 107.4, 107.8, 115.6,
119.4, 120.4, 125.2, 126.4, 127.5, 128.2, 130.2, 130.8,
134.5, 137.6, 139.6, 142.4, 149.6, 151.5, 154.3, 157.3,
157.6, 164.5, 165.2, 171.8. MS (ESI): 639 [M + H]+.
Found, %: C 62.01; H 4.05; N 8.82. C33H26N4O6S2.
Calculated, %: C 62.05; H 4.10; N 8.77.
4.2. Analysis of docking results was done using MGL
Tools-1.5.6. Top scoring molecule in the largest cluster
was analysed for its interactions with the protein. The
2D interactions were generated from Proteinplus online
pose viewer. The 3D interactions were obtained with
Discovery studio Visualizer. The synthesized compounds
were docked into the X-ray crystal structure of human
topoisomerase IIbeta domain (PDB ID: 3QX3) for
considering the possible target mechanism of action.
6-{[3-(5-Bromothiophen-2-yl)isoxazol-5-yl]-
methoxy}-N-{4-[3-(3,4,5-trimethoxyphenyl)isoxazol-
5-yl]phenyl}benzo[d]thiazol-2-amine (14j). Yield 59%,
mp 270–272°C NMR spectrum, δ, ppm: 3.72 s (3H), 3.92 s
(6H), 5.13 s (2H), 7.24 s (2H), 7.42–7.50 m (2H), 7.56 d
(1H, J = 7.95 Hz), 7.66 d (1H, J = 8.5 Hz), 7.69 d (1H, J =
8.2 Hz), 7.87 s (1H), 7.96 s (1H), 8.15 d (2H, J = 8.7 Hz),
8.27 d (2H, J = 8.7 Hz), 12.56 s (1H). 13C NMR spectrum,
δ, ppm: 57.6, 61.8, 65.8, 96.5, 97.2, 107.5, 107.9, 115.2,
115.7, 119.5, 120.7, 126.4, 127.4, 130.3, 130.7, 131.5,
134.5, 137.3, 138.6, 141.4, 149.7, 152.4, 154.5, 157.3,
157.8, 164.5, 165.6, 171.9. MS (ESI): 719 [M + H]+.
Found, %: C 55.19; H 3.47; N 7.86. C33H25N4O6S2Br.
Calculated, %: C 55.23; H 3.51; N 7.81.
CONCLUSIONS
In summary, we have designed and synthesized a
novel series of bis-isoxazole incorporated benzothiazole
derivatives and tested those in vitro against human cancer
cell lines such as breast MCF-7 and MDAMB-231, lungs
A549, and prostate DU-145 using the MTT assay and
etoposide as a standard drug. Most of the compounds
have demonstrated good to moderate activity, and some of
those have exhibited more potent activity than etoposide.
According to molecular docking, the compounds can be
characterized by high free energy of binding interactions
with human topoisomerase II beta in complex with DNA.
MTT assay. Individual wells of a 96-well tissue
culture micro titer plate were inoculated with 100 μL
of complete medium containing 1×104 cells. The plates
were incubated at 37°C in a humidified 5% CO2 incubator
for 18 h prior to the experiment. After medium removal,
100 μL of fresh medium containing the test compounds
and etoposide at concentrations 0.5, 1 and 2 μM were
added to each well and incubated at 37°C for 24 h. Then
the medium was discarded and replaced with 10 μLMTT
dye. Plates were incubated at 37°C for 2 h. The resulting
formazan crystals were solubilized in 100 μL extraction
buffer. The optical density was recorded at 570 nm with
a micro plate reader (Multi-mode Varioskan Instrument-
Themo Scientific). Percentage of DMSO in the medium
never exceeded 0.25%.
ACKNOWLEDGMENTS
The authors would like to thank the management of the
AMRI Hyderabad research Centre for giving an opportunity
to carry out this research. The authors are also thankful to
Department of Chemistry, Koneru Lakshmaiah Education
Foundation for constant encouragement during this research
program.
CONFLICT OF INTEREST
No conflict of interest was declared by the authors.
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