Synthesis of Novel Diaziridinyl Quinone Isoxazole Hybrids and Evaluation of Their Anti-Cancer Activity as Potential Tubulin-Targeting Agents

Article abstract of DOI:10.1055/a-0810-7033

Two series of diaziridinyl quinone isoxazole derivatives were prepared and evaluated for their cytotoxic activity against MCF7, HeLa, BT549, A549 and HEK293 cell lines and interaction with tubulin. Compounds (6a-m) showed promising activity against all the 5 human cancer cell lines. Compounds 6a, 6e and 6 m were potent [IC ₅₀ ranging between 2.21 μg to 2.87 μg] on ER-positive MCF7 cell line similar to the commercially available drug molecule Doxorubicin. The results from docking models are in consistent with the experimental values which demonstrated the favourable binding modes of compounds 6a-m to the interface of α- and β-tubulin dimer.

Full text of DOI:10.1055/a-0810-7033

Thieme
Original Article
Kumar PRavi et al. Synthesis of Novel Diaziridinyl… Drug Res
2018; 00: 00–00
Synthesis of Novel Diaziridinyl Quinone Isoxazole Hybrids and
Evaluation of Their Anti-Cancer Activity as Potential
Tubulin-Targeting Agents
Authors
P. Ravi Kumar1, 2, Satyanarayana Yennam1, K. Raghavulu1, Loka Reddy Velatooru3, Siva Reddy Kotla3,
Vasudevarao Penugurthi3, Prasanta K Hota4, Manoranjan Behera1, A. Jaya Shree2
Affiliations
Correspondence
1
2
3
4
Department of Medicinal Chemistry, GVK Biosciences
Dr. Satyanarayana Yennam
Department of Medicinal Chemistry
GVK Biosciences Pvt. Ltd
Plot No’s 125(part) & 126
IDA Mallapur
Pvt. Ltd., Telangana, India
Centre for Chemical Sciences & Technology, Institute of
Science and Technology, JNT University, Telangana, India
Biochemistry Department, School of life Sciences,
University of Hyderabad, Telangana, India
Department of Chemistry, School of Sciences, HNBG
University, Uttarakhand, India
Hyderabad 500 076
Telangana
India
Tel.: + 91/40/67483 495
satya@gvkbio.com
Key words
isoxazole, quinone, aziridine, diaziridinyl quinone isoxazole,
cytotoxicity, molecular docking
ABStRAct
Two series of diaziridinyl quinone isoxazole derivatives were
prepared and evaluated for their cytotoxic activity against
MCF7, HeLa, BT549, A549 and HEK293 cell lines and interaction
with tubulin. Compounds (6a–m ) showed promising activity
against all the 5 human cancer cell lines. Compounds 6a, 6e
and 6m were potent [IC50 ranging between 2.21µg to 2.87µg]
on ER-positive MCF7 cell line similar to the commercially avail-
able drug molecule Doxorubicin. The results from docking
models are in consistent with the experimental values which
demonstrated the favourable binding modes of compounds
6a–m to the interface of α- and β-tubulin dimer.
received
accepted
04.09.2018
29.11.2018
Bibliography
DOI https://doi.org/10.1055/a-0810-7033
Published online: 2019
Drug Res
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Introduction
agent can cross-link DNA in cells, resulting in complex cellular
mechanisms that lead to cell death by apoptosis or necrosis [7, 8].
These bioreductive drugs have been developed to exploit the oxy-
gen deficiency in the hypoxic fraction of solid tumors on the prem-
ise that hypoxic cells should show a greater propensity for reduc-
tive metabolism than well-oxygenated cells [9–13].
The tumor tissue has a lower oxidative reduction (redox) poten-
tial relative to most normal tissues, which could increase the reduc-
tive activation of these quinone derivatives in tumors [8]. There-
fore, the selectivity of bioreductive drugs is governed not only by
differences in oxygen tension between tumor and normal tissue,
but also by levels of enzymes catalyzing bioreductive activation
such as DT-diaphorase [9, 10]. In many cases, the biological activ-
ity of quinones is attributed to their ability to accept electrons to
form the corresponding radical anion or dianion species. A quinone
Mitomycin C and other mitomycins are isolated from Streptomy-
ces caespitosus[1]. These mitomycin derivatives are the most im-
portant clinically relevant compounds of all the aziridinyl quinone
derivatives. Mitomycin C compound has 2 potentially active con-
stituents, quinone and unusual aziridine ring system [2]. The azir-
idine substituted benzoquinones such as Mitomycin C, Triaziquone
(TZQ), RH1 and tirapazamine (TPZ), are 4 of the principal aziridinyl
quinone (▶Fig. 1) class of hypoxia-specific cytotoxins that are being
developed for clinical use [3–5]. These agents are composed of
aziridinyl moieties on a quinone structure, and they are converted
by reductive metabolism in to a bifunctional alkylating species that
can cross-link major groove DNA by interacting predominantly at
guanine-N7 [6]. In case of di-aziridinyl substituted quinones such
as TPZ and CI-1010, the highly cytotoxic bifunctional alkylating
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

Thieme
Original Article
▶Fig. 1 Structures of the drugs containing Aziridinyl quinones.
moiety substituted with an aziridine has been shown as a potent
alkylating agent due to bioreduction either by one-electron reduc-
ing enzymes (e. g., NADPH cytochrome P450 reductase, cy-
tochrome b5 reductase) or by 2-electron reducing enzyme ((NADP)
H oxidoreductase, NQO1) to form the corresponding aziridinyl hy-
droquinones [12–14]. The hydroquinone in the corresponding
aziridinyl hydroquinone effectively changes the pK of the aziridine
ring such that it is protonated and become activated towards the
nucleophilic attack under physiological pH.
in each structure [30, 31]. The aziridinyl moiety within the ana-
logues served as an important alkylation group [30], but the cyto-
toxic effects of the synthetic analogues towards carcinoma cells
might not solely be due to the aziridinyl moiety, as the quinone
structure is common in numerous natural products that are asso-
ciated with antitumor activities [32]. The cytotoxic mechanisms
induced by quinones include redox cycling and the production of
superoxide and other reactive oxygen radicals, reactions with thi-
ols and amines, drug-uptake and DNA alkylation [33, 34]. Among
quinone, various substituted napthaquionones were synthesized
and studied extensively for their anticancer activity [35]. In our con-
tinuation endeavour to prepare novel hybrid molecules consisting
variety of natural products [36], we have developed interest in the
synthesis of diaziridinyl quinone isoxazole derivatives for evalua-
tion of their cytotoxic activities. The efficient 5 step synthesis de-
veloped herein provides speedy creation of a series of diaziridinyl
quinone isoxazole analogues. Molecular docking studies of these
diaziridinyl quinone isoxazole derivatives will predict the molecu-
lar interaction of the synthesized compounds with tubulin dimer
and to obtain the best orientation of compounds.
Tubulin polymerization is an essential for mitosis, chromosome
segregation, intracellular transport and cell division [15]. Microtu-
bule dynamics are regulated by guanosine triphosphate (GTP) and
the tubulin binding effectors protein [16–19]. Thus, small molecules
that target microtubule are widely used to study the microtubule
cytoskeleton [20] and towards the development of anti-tubulin drugs
molecule that blocks the tubulin mediated defective cell growth by
interfering the tubulin polymerization process. In recent years, many
such anti-cancer molecules are shown to inhibit cancer cell growth
by inhibiting microtubule polymerization [21–23]. Thus, drugs like
paclitaxel, colchicine, vinca alkaloids, etc stabilize or inhibit the mi-
crotubule dynamics by blocking its polymerization site [23–26] and
consequently, block the mitosis progression in defective cell.
Pharmacomodulation of biologically active compounds through
conjunctive approaches to produce a single hybrid molecule has
become an area of active research in different fields of medicinal
chemistry [27]. In this connection, Braga and da Silva Júnior have
reported the synthesis of hybrid molecules having 2 redox centre
which showed promising anti-tumour activity [28]. In the last few
years, a series of bis-type aziridinyl-quinone-isoxazole bioreduc-
tive compounds have been developed in our laboratory and their
anticancer activities evaluated [29]. These diaziridinyl-quinone-
isoxazole compounds have 5-substituted thiophene moiety at the
5-position of isoxazole ring. Cytotoxicity to various tumor cells var-
ies with the substituted pattern at the thiophene ring, but DNA
alkylation reactivity is related to the presence of aziridinyl groups
Results and Discussion
Chemistry
We have synthesized 2 series of diaziridinyl quinone isoxazole de-
rivatives 6a–m from corresponding dihydroxy benzaldehyde by the
route as describe in ▶Fig. 2 and evaluated their cytotoxic activity.
To start with 2,5-dimethoxy-benzaldehyde (2a) was prepared by
treating with commercially available 2,5-dihydroxy benzaldehyde
(1a) and 2,5-dimethoxy-4-methylbenzaldehyde (2b) was prepared
by treating with commercially available 2,5-dihydroxy-4-methylb-
enzaldehyde (1b) with MeI/K2CO3 in acetone for 18h at room tem-
perature. Then the compound 2aand 2b were reacted with NH2OH.
HCl to produce the oxime derivatives 3a and 3b. Subsequent one
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

pot reaction of oxime derivatives (3a, 3b) with phenyl acetylene in
the presence of NaOCl/Et3N in DCM as solvent gave the isoxazole
compounds 4a and 4g. These compounds 4a was oxidized with ce-
rium ammonium nitrate (CAN) to give the quinone isoxazole hy-
brids 5a[37]. The compound 5a was characterized by 1HNMR,
13CNMR and HRMS. The characteristic isoxazole proton at 7.14 δ
and quinone proton at 6.88 δ in 1HNMR confirms the oxidation of
compound 4a to generate compound 5a. IR spectra of the com-
pound 5a showed the carbonyl peak at 1655cm− 1. The 2 carbonyl
peaks at 186.7 & 185.1 δ in 13CNMR spectra validate the benzoqui-
none moiety. All the benzoquinones are characteristic yellowish
colored solids. The quinone compound 5a was treated with fresh-
ly prepared aziridine [38] in the presence of Cu(OAc)2 in methanol
at room temperature to obtain the target diaziridinyl quinone isox-
azole hybrid 6a. The characteristic aziridinyl protons at δ 2.35 and
2.29 confirms the aziridine insertion in the compound 5a to gen-
erate compound 6a. After successfully establishing the method to
prepare compound 6a, various substituted diaziridinyl quinone
isoxazole derivatives (6b–m) were prepared by following the same
protocol (▶Fig. 2). All the substrates reacted well under these con-
ditions and did not produce any by-products. All the diaziridinyl
quinone isoxazole hybrids were fully characterized by spectral data
and obtained in good yields (▶table 1).
ences (NCCS), Pune, India. Cells were cultured in DMEM media, sup-
plemented with 10 % heat-inactivated fetal bovine serum (FBS),
1mM NaHCO3, 2mM glutamine, 100units/ml penicillin and 100μg/
ml streptomycin. All cell lines were maintained in culture at 37 °C
in an atmosphere of 5 % CO2.
Test concentrations
Initially, stock solutions of each test substances were prepared in
100 % Dimethyl Sulfoxide (DMSO, Sigma Chemical Co., St. Louis,
MO) with a final concentration of 8 mg/ml. Exactly 25 μl of stock
was diluted to 1ml in culture medium to obtain experimental stock
concentration of 200 μg/ml. This solution was further serially di-
luted with media to generate a dilution series of 1µg to 100µg/ml.
Exactly 100 µl of each diluent was added to 100 µl of cell suspen-
sion (total assay volume of 200 µl) and incubated for 24 h at 37 °C
in 5 % CO2. Respected volume of DMSO used as a control.
Cytotoxicity
Cytotoxicity was measured using the MTT [3-(4, 5-dimethylthiazol-
2-yl)-2, 5-diphenyl tetrazolium bromide] assay, according to the
method of Mossman (1983). Briefly, the cells (3 × 103) were seed-
ed in each well containing 0.1ml of medium in 96 well plates. After
overnight incubation at 37°C in 5% CO2, the cells were treated with
100 µl of different test concentrations of test compounds at iden-
tical conditions with 5 replicates each. The final test concentrations
were equivalent to 1 – 100 µg/ml or 1 – 100 ppm. The cell viability
was assessed after 24h, by adding 10μl of MTT (5mg/ml) per well.
The plates were incubated at 37°C for additional 3 h. The medium
was discarded and the formazan blue, which formed in the cells,
was dissolved with 100μl of DMSO. The rate of color formation was
Biology
Cell lines and cell culture
The cell lines MCF7 (human breast adenocarcinoma), HeLa (human
cervical cancer), BT549 (human ductal adenocarcinoma), A549
(human lung carcinoma) and HEK 293 (human embryonic kidney)
cell lines were obtained from the National Centre for Cellular Sci-
▶Fig. 2 The synthesis route of target compounds 6a–m. Reagents and conditions: (a) MeI, K2CO3, acetone, RT, 18 h (2a–85 %; 2b–80 %).
(b) NH2OH.HCl NaOAc in MeOH, RT, 18 h (3a–95 %; 3b–92 %) (c) corresponding alkyne, NaOCI (9–12 % in H2O), TEA, DCM, RT, 20 h, 65–85 %.
(d) Ceric ammonium nitrate, CH3CN/H2O (8:2), RT, 1 h, 70–85 %. (e) Aziridine, Cu(OAc)2, MeOH, RT, 1 h, 52–85 %.
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

Thieme
Original Article
▶table 1 List of Synthesized Quinone isoxazoles and Diaziridinyl quinone isoxazoles.
measured at 570 nm in a spectrophotometer (Spectra MAX Plus;
Molecular Devices; supported by SOFT max PRO-5.4). The percent
inhibition of cell viability was determined with reference to the con-
trol values (without test compound). The data were subjected to
linear regression analysis and the regression lines were plotted for
the best straight-line fit. The IC50 (inhibition of cell viability) concen-
trations were calculated using the respective regression equation.
The compounds (5a–m and 6a–m) were screened for cytotox-
icity against 5 human cancer cell lines, namely human breast ade-
nocarcinoma cells (MCF7-ER-positive), human cervical cancer cells
(HeLa), human ductal adenocarcinoma cells (BT549), human lung
carcinoma cells (A549) and one normal human embryonic kidney
cells (HEK293) by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide] assay, according to the method of
Mossman [39] (1983). IC50 values of the test compounds for 24 hrs
on each cell line were calculated and presented in ▶table 2.
It is an evident from the results that most of the prepared com-
pounds have shown significant cytotoxic activity on all the cancer
cell lines tested in a concentration dependent manner (▶table 2)
but less active on normal cell line (HEK 293). This data indicated that
diaziridinyl quinone isoxazole derivatives may selectively inhibit the
proliferation of cancer cells which make it as a potential anticancer
agents. Compare to aziridine containing derivatives (6a–m), non
aziridine derivatives (5a–m) were shown less cytotoxic activity
against all cell lines. Most of the diaziridinyl quinone isoxazole de-
rivatives (6a–m) were exhibited cytotoxic properties against all
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

▶table 2 In vitro cytotoxic evaluation of diaziridinyl quinone-isoxazole derivatives against MCF7, HeLa, BT549, A549 and HEK 293 cell lines by MTT assay.
compd
§Ic50ꢀ±ꢀSDꢀ(μg/ml)
McF7
2.21 ± 0.42
HeLa
15.12 ± 1.09
Bt549
A549
HEK 293
39.52 ± 1.63
42.32 ± 3.01
NA
6a
13.46 ± 1.21
18.88 ± 3.21
55.50 ± 5.01
51.99 ± 2.89
16.81 ± 1.79
69.58 ± 6.6
38.45 ± 2.60
47.03 ± 5.33
56.71 ± 4.98
19.01 ± 2.56
50.30 ± 4.04
38.81 ± 3.03
22.20 ± 1.01
NA
62.01 ± 5.19
28.33 ± 4.10
33.7 ± 2.09
42.31 ± 5.02
8.03 ± 1.67
64.60 ± 5.21
45.17 ± 3.54
29.07 ± 1.98
39.60 ± 4.64
15.8 ± 2.37
35.19 ± 4.04
19.2 ± 2.49
13.09 ± 3.41
49.34 ± 2.98
NA
6b
3.12 ± 0.89
16.02 ± 2.31
18.43 ± 3.63
2.32 ± 1.11
18.16 ± 2.67
12.12 ± 1.76
14.16 ± 1.90
10.34 ± 2.01
6.35 ± 0.91
21.14 ± 4.23
21.93 ± 3.33
2.87 ± 0.86
51.02 ± 7.1
88.99 ± 6.06
NA
8.18 ± 2.22
41.22 ± 5.53
21.98 ± 1.98
11.01 ± 2.12
56.26 ± 4.62
21.96 ± 0.98
35.66 ± 4.07
26.57 ± 3.03
11.35 ± 1.19
32.07 ± 2.78
25.65 ± 4.12
13.81 ± 1.45
83.66 ± 7.48
NA
6c
6d
NA
6e
56.01 ± 4.19
NA
6f
6g
36.44 ± 2.89
NA
6h
6i
41.14 ± 4.09
39.60 ± 4.64
NA
6j
6k
6l
NA
6m
24.36 ± 4.19
NA
5a
5b
94.09 ± 5.03
98.77 ± 4.06
NA
59.02 ± 4.56
86.36 ± 7.77
NA
5c
NA
NA
5d
NA
NA
NA
5e
NA
NA
NA
56.69 ± 2.9
87.65 ± 6.01
36.06 ± 4.01
NA
NA
5f
38.34 ± 4.40
33.11 ± 2.85
27.90 ± 3.98
48.83 ± 3.09
26.71 ± 1.54
NA
NA
NA
NA
5g
45.84 ± 2.79
91.26 ± 7.81
NA
55.62 ± 2.75
NA
NA
5h
72.80 ± 5.06
NA
5i
98.71 ± 8.03
NA
NA
5j
NA
NA
NA
5k
NA
NA
NA
NA
5l
85.17 ± 7.10
50.43 ± 3.82
2.03 ± 0.22
NA
NA
27. 46 ± 3.18
43.51 ± 1.87
2.64 ± 0.67
NA
5m
NA
99.62 ± 8.51
2.16 ± 0.31
76.75 ± 6.32
1.13 ± 0.13
aDoxorubicin
2.23 ± 0.81
Exponentially growing cells were treated with different concentrations of diaziridinyl quinone-isoxazole derivatives for 24h and cell growth inhibition was
analyzed through MTT assay.; §IC50is defined as the concentration, which results in a 50% decrease in cell number as compared with that of the control
cultures in the absence of an test compound and were calculated using the respective regression analysis. The values represent the mean±SE of 3
individual observations.; aDoxorubicin was employed as positive control. NA indicates that the derivatives are not active at 100µg/ml concentration.
cancer cell lines (MCF7, HeLa, BT549 and A549) at below 100 µg/
ml concentration. It clearly indicating that aziridine moiety is im-
portant for the cytotoxic property of the diaziridinyl quinone isox-
azole derivatives. (▶table 3)
similar to compound 6a against MCF7. Compound 6j which has F
on aryl ring show good cytotoxicity against all the 5 tested human
cancer cell lines. Among the compounds (6a–m), compounds hav-
ing halogen on aromatic ring of (6b, 6e, 6j) showed good cytotox-
icity. Whereas electron donating groups on the compound (6 l)
having substituted aryl on the isoxazole ring showed less cytotox-
icity. In particular, the compound 6m having thiophene ring on
isoxazole showed more cytotoxicity compared to phenoxy (6l) sub-
stituted ring. In general, compounds having thiophene and aryl
with fluorine as substituent on isoxazole ring exhibited more cyto-
toxicity.
It is an apparent from the results that diaziridinyl quinone isox-
azole derivatives (6a–m) are exhibited very good cytotoxicity on
breast cancer cell line (MCF7) than the cervical (HeLa) and lung can-
cer (A549) cell lines. Compounds 6a, 6b, 6e and 6m were found to
be most active compounds among the diaziridinyl quinone isoxa-
zole derivatives with IC50 concentration < 3.5 µg/ml on ER-positive
MCF7 cell line. Compare to triple negative breast cancer cells
(BT549), ER-positive breast cancer cells (MCF7) are more sensitive
to diaziridinyl quinone isoxazole derivatives. Also compounds 6b,
6e, 6j and 6m showed significant cytotoxicity against HeLa cell line.
The compounds (6g–m) which has CH3 group on quinone ring
showed less cytotoxicity compared to compounds (6a–f) where
there is no methyl group on quinone ring. Similarly, when there is
CF3 group on aryl ring of isoxazole 6e, the cytotoxic activity was
Molecular docking studies
Molecular docking studies was performed to understand whether
the synthesized compounds have any role for tubulin binding and
regulation of tubulin polymerization process. The docking studies
were performed for the binding activity of 5a–m and 6a–m com-
pounds with tubulin dimer using Autodock vina [40]. The docking
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

Thieme
Original Article
▶table 3 Selectivity index (SI) for the diaziridinyl quinone-isoxazole derivatives [Selectivity index, represented by the ratio of cytotoxicities between
normal cell and different lines of cancer cells.]
compound
Selectivity index (SI) Vs HEK293
McF7
HeLa
Bt549
A549
6a
17.88235
13.5641
> 6.2422
> 5.42594
24.14224
> 5.50661
3.0066
2.61376
5.17359
> 2.42601
> 4.54959
5.08719
> 1.77746
1.65938
> 2.80426
1.54836
3.48899
> 3.11818
> 3.89864
1.76394
> 1.19531
< 0.5902
0.8636
1
2.93611
2.24153
> 1.8018
> 1.92345
3.33195
> 1.43719
0.94772
> 2.1263
0.72545
2.08311
> 1.98807
> 2.57666
1.0973
> 1
0.63732
1.49382
> 2.96736
> 2.36351
6.97509
> 1.54799
0.80673
> 3.43997
1.03889
2.50633
> 2.84172
> 5.20833
1.86096
> 2.02675
< 0.5902
0.8636
1
6b
6c
6d
6e
6f
6g
6h
> 7.06215
3.97872
6.23622
> 4.73037
> 4.55996
8.4878
6i
6j
6k
6l
6m
5a
> 1.96002
0.66322
0.8636
5b
0.62727
< 0.87435
1
5c
5d
1
5e
1
1
1
1.76398
1.1409
2.77316
< 0.728
1
5f
2.60824
3.02024
2.60932
> 2.04792
> 3.74392
1
1
1
5g
2.1815
0.79772
1
1.79791
< 0.728
> 1.01307
1
5h
5i
5j
1
1
5k
1
1
1
5l
> 1.17412
1.52191
0.55
1
1
> 3.64166
1.76396
0.428
5m
0.7675
0.461
> 0.77043
0.523
Doxorubicin
▶table 4 Ligand-tubulin Docking score obtained through Autodock vina [40].]
Ligand
ΔG(Kcal/mol)
− 8.5
K (106 Mꢀ−ꢀ1
)
Binding side
GTP
Ligand
6a
ΔG(Kcal/mol)
K(106 Mꢀ−ꢀ1
2.4
)
Binding side
5a
1.7
4.7
3.3
2.4
− 8.7
− 8.7
− 8.5
− 8.7
− 9.5
− 7.8
− 8.6
− 9.0
− 9.2
− 9.1
− 9.7
− 9.8
− 8.2
GTP
5b
− 9.1
GTP
6b
6c
2.4
GTP
5c
− 8.9
GTP
1.7
STK-C
STK
5d
− 8.7
GTP
6d
6e
2.4
5e
− 9.7
12.9
GTP
9.2
STK-C
GTP
5f
− 8.1
0.8
GTP
6f
0.5
5g
− 8.6
2.0
GTP
6g
6h
6i
2.0
GTP
5h
− 8.7
2.4
GTP
3.9
STK-C
STK-C
STK-C
STK-C
STK-C
GTP
5i
− 9.1
4.7
GTP
5.5
5j
− 9.1
4.7
GTP
6j
4.7
5k
− 9.5
9.2
STK
6k
12.9
5l
− 9.4
7.8
STK
6l
15.3
1.0
5m
− 8.1
0.8 × 106
GTP
6m
Doxorubicin
StK
− 9.9
18.1
30.0
STK-C
STK
− 10.2
− 8.6
GTP
GTP
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

results of the synthesized compounds with tubulin dimer are pre-
sented in ▶table 4. The globular protein tubulin dimer consist of 2
units of α and β tubulin monomer shown in green and blue color re-
spectively (▶Fig. 3). Both of these monomer units held to each other
through their head to tail and tail to head side chain interaction. The
tubulin polymerization occurs through combination of several such
tubulin dimer through their head to tail interaction. Hence, devel-
opment of molecules that regulates tubulin polymerization process
is urgently needed. For docking calculation, we used the pdb struc-
ture of tubulin dimer (protein PDB ID: 1SA0) deposited in RCSB (Re-
search Collaboratory for Structural Bioinformatics) protein data
bank. The pdb files of 5a–m and 6a–m series are created and opti-
mized to their minimum energy using chemBiodraw ultra12. The
docking scores are summarized in ▶table 4. The purpose is to char-
acterize the binding interface and how efficiently 5a–m and 6a–m
series compounds can bind to tubulin dimer. Further, if molecule
binds, then there is a possibility of regulating the tubulin polymer-
ization process. From docking calculation, it is shown that these
molecules bind to tubulin dimer with 2 type of binding sides, (a) in-
terface of dimer, which overlap the STK [41] and colchicine binding
side and (b) α unit of tubulin which overlap the GTP binding side,
whose major part is on the α unit of tubulin monomer (▶Fig. 4). It
is reported that GTP bound tubulin somehow unable to participate
in polymerization process. However, hydrolysis of GTP to GDP
bound tubulin, initiate the polymerization in tubulin.
overlap GTP binding side is situated on the α tubulin monomer sur-
rounded by the amino acids Gln11, Asp71, Ser140, Asn205 and
Tyr224 of α tubulin and Lys254 of β tubulin provide the cavity for
binding site. Similarly, for other binding side which overlap the GTP
and colchicine binding side is situated on the interface of both α
and β tubulin surrounded by amino acids Gln11, Asp71, Thr73 of
α tubulin and Asn249, Lys254 of β tubulin that provide binding cav-
ity for ligand. Based on the binding energy (score value) that ob-
tained through Autodock vina, it is suggested that compound 5b,
5e, 5i, 5j strongly bind to the GTP binding side, whereas 5k and 5l
strongly bind to the dimer interface of α and β tubulin which over-
lap the colchicine binding side. Similarly, compound 6e, 6 h, 6i, 6j,
6k, 6l strongly bind to the dimer interface of α and β tubulin, which
overlap the colchicine binding side (▶Fig. 4). So these molecules
can be used to regulate the tubulin mediated biological activities.
Conclusion
In summary, we have synthesized 2 series of diaziridinyl quinone
isoxazole hybrids and were screened for cytotoxic activity against
5 human cancer cell lines. The order of sensitivity of human cancer
and normal cell lines towards diaziridinyl quinone isoxazole deriva-
tives are MCF7 > HeLa > A549 > BT549 > HEK293. Compounds 6a,
6b, 6e and 6m were potent on ER-positive MCF cell line similar to
the commercially available drug molecule, Doxorubicin. Based on
docking study on tubulin crystal structure (PDB code: 1SA0), com-
pound 6l exhibited the most potent affinity for tubulin. The above
results indicates this new compounds might be useful in designing
and developing new drugs for cancer treatment.
As the binding sides are situated between the interface of α and
β monomer units, particularly to the GTP binding side, it is our hy-
pothesis that these compounds have higher possibility of regulat-
ing the tubulin polymerization processes. The binding side which
▶Fig. 3 Docked structure of tubulin dimer (PDB: 1SA0) with 5k (magenta). The binding site present at the interface of alpha (green) and beta
(blue) tubulin monomer.
Kumar R et al. Diaziridinyl Quinone Isoxazole Hybrids … Drug Res

Thieme
Original Article
▶Fig. 4 Docked structure of tubulin dimer (PDB: 1SA0) with 6l (magenta). The binding side present at the interface of alpha (green) and beta
(blue) tubulin monomer.
[6] Mattes WB, Hartly JA, Kohn KW. Mechanism of DNA strands breakage
Acknowledgements
Authors are grateful to GVK Biosciences Pvt. Ltd., for the financial
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868: 71–76
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for the analytical data is appreciated. PKH is gratefully acknowl-
edged UGC, New Delhi for providing UGC-FRPS research start-up
grant. We thank Dr. Sudhir Kumar Singh for his invaluable support
and motivation.
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