Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design, synthesis, biological investigation and docking studies

Article abstract of DOI:10.1016/j.bmcl.2017.07.069

In an attempt to develop potent anti-tubulin agents against most dreadful disease cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC₅₀ value of 1.06 μM which was further confirmed by using confocal microscopy. Possible binding interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by molecular modeling studies.

Full text of DOI:10.1016/j.bmcl.2017.07.069

Accepted Manuscript
Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin
agents: Design, synthesis, biological investigation and docking studies
Harbinder Singh, Jatinder V. Singh, Manish K. Gupta, Ajit K. Saxena, Sahil
Sharma, Kunal Nepali, Preet Mohinder S. Bedi
PII:
S0960-894X(17)30776-X
http://dx.doi.org/10.1016/j.bmcl.2017.07.069
BMCL 25182
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
24 March 2017
15 July 2017
27 July 2017
Please cite this article as: Singh, H., Singh, J.V., Gupta, M.K., Saxena, A.K., Sharma, S., Nepali, K., Bedi, P.M.S.,
Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents: Design, synthesis, biological
investigation and docking studies, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/
j.bmcl.2017.07.069
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents:
Design, synthesis, biological investigation and docking studies
Harbinder Singha, Jatinder V. Singha, Manish K. Guptab, Ajit K. Saxenac, Sahil Sharmaa,*, Kunal
Nepalia,*, Preet Mohinder S. Bedia,*
aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab
143005, India
bLloyd Institute of Management and Technology, Greater Noida, UP, India
cIndian Institute of Integrative Medicine, Jammu, India
*Corresponding author: 1) Dr. Sahil Sharma
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Email id: ss.gq2009@gmail.com
2) Dr. Kunal Nepali
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
2) Dr. Preet Mohinder Singh Bedi
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India

Abstract: In an attempt to develop potent anti-tubulin agents against most dreadful disease
cancer, a library of 28 novel triazole tethered isatin-coumarin hybrids were synthesized by click
chemistry approach. Synthesized hybrids were characterized and evaluated against a panel of
human cancer cell lines viz. THP-1, COLO-205, HCT-116 and PC-3. Biological assay unveiled
that, compounds A-1 to A-6, B-1 to B-4 and C-1 to C-3 displayed significant inhibitory potential
against THP-1, COLO-205 and HCT-116 cell lines which were more sensitive towards the
designed hybrids. PC-3 among these cell lines was found to be almost resistant. Established SAR
revealed marked dependence of the cytotoxic activity on the type of substituent on isatin and the
length of carbon-bridge connecting isatin moiety with triazole ring. Unsubstituted isatin and two
carbon-bridge were found to be crucial for cytotoxicity. Three most potent hybrids (A-1, A-2 and
B-1) were further tested for tubulin polymerization inhibition. Among these three compounds, A-
1 found to be endowed with most prominent tubulin polymerization inhibition potential with IC50
value of 1.06 µM which was further confirmed by using confocal microscopy. Possible binding
interactions between the most potent hybrid molecule A-1 and tubulin were also divulged by
molecular modeling studies.
Keywords: isatin; coumarin; hybrid; antitumor; anti tubulin; docking

Cancer is the uncontrolled, rapid and pathological proliferation of abnormal cells, a major
problematic affliction world-wide, causing 2-3% annual deaths.1-3 Its 100% prevention is a major
challenge for researchers. Most of the currently available anti-tumor drugs are unable to
overcome primary or secondary resistance mechanisms in the abnormal cells and does not have
ability to differentiate the normal and abnormal cells.4-6 Present chemotherapy interfering with a
single biological molecule or pathway has been successfully utilized. However, a general thought
is that the drugs affecting more than single target or multiple sites on solitary target could have
prominent efficacy as compared to solo target agents.
Molecular hybridization is a stratagem to design new drug molecules based on recognition of
pharmacophoric sub-units in the molecular skeleton of two or more known biologically active
derivatives. The revived interest among the researchers in the discovery of hybrids that can
concomitantly address more than one biological target is increasing day by day.7-9 Hybrid
molecules thus give new dimensions by lowering the risk of drug-drug interactions and
minimizing drug resistance as well,10 hence are extremely beneficial for the society fighting with
this dreadful disease.
Tubulin is one of the most useful strategic molecular target for antitumor drugs as it is involved
in several cellular processes (maintaining cell structure, cell motility, intracellular transport and
chromosome segregation during cell division).11 Its inhibition by various anti tubulin agents by
binding to its distinct sites can block these processes and ultimately leads to cell death by both
apoptosis and necrosis.12
A newly focused area for developing new anti-cancer agents is triazole tethered bi-functional
hybrids. Several published reports attracted researchers towards this architecture for selection as
a linker to joint two bioactive functionalities.13-16
Isatin, an indole derivative is considered as a versatile nucleus with diverse assortment of
biological actions. This heterocycle is known to exert its anticancer effects through different
mechanism among which tubulin inhibition by binding to its novel site, being one of the highest-
flying ones. Number of recent reports on cell killing potential of isatin based hybrids includes,
curcumin-isatin bi-functional hybrids (1), uracil-isatin conjugates (2), triazole tethered bis-isatin
conjugates (3), iastin-benzothiazole analogs (4), isatin-4-pirazinylquinoline hybrids (5), isatin-
thiazolidinone hybrids (6) and isatin-3,5-diaryl N-acetylpyrazoline hybrids (7) (Fig. 1) which

clearly indicates the success of isatin-based molecular hybrids and justifies its inclusion in the
conjugates.14,17-22
Furthermore, promising therapeutic perspectives about an elite class of naturally occurring
moiety coumarin, rationalize its inclusion to develop potent hybrid molecules. Recently reported
coumarin based derivatives (8 & 9) exerting cytotoxic potential via tubulin inhibition are given
in Fig. 1.23-25
O
O
F
H3CO
O
OCH3
OCH3
O
O
N
N
O
N
O
N
N
N
N
N
O
OCH3
N
N
N
N
N
N
Cl
O
Cl
1
2
O
N
Br
CH3
N
O
Cl
N
S
O
O
O
N
N
N
N
N
Cl
N
N
O
O
N
N
N
N
O
O
N
3
4
5
O
O
O
OCH3
O
N
N
N
N
O
O
H3CO
H3CO
N
N
HN
S
N
N
HN
O
NO2
OCH3
6
7
O
H3CO
O
OCH3
OCH3
O
O
OCH3
Cl
N
N
O
N
O
8
9
O
O
Fig. 1 Isatin (1-7) and coumarin (8 & 9) based derivatives with cytotoxic potential
Therefore, in our continuous quest of hybridization to fight against cancer, we designed (Fig. 2)
and synthesized triazole tethered isatin-coumarin bifunctional hybrids via simple synthetic
approach and evaluated them against four human cancer cell lines by using sulforhodamine B

assay. Several recent reports concluded that isatin and coumarin based derivatives17, 25 exhibiting
potential cytotoxicity by inhibiting the tubulin, as one of their primary target. Therefore, hybrid
molecules with significant cytotoxicity were further evaluated for their in vitro tubulin
polymerization inhibition. Microtubules disruption by the most active anti tubulin hybrid was
further confirmed by confocal microscopy and rationalized by molecular docking studies.
O
OH
O
R
O
N
O
H
HYBRIDIZATION
Coumarin
Triazole as Linker
Isatin
O
O
O
N
O
O
N
n
N
N
R
Isatin-coumarin hybrids
Fig. 2 Design strategy
Target hybrids were synthesized via a sequence of reactions (Scheme 1) starting from isatin,
where it was stirred with diverse 1,2-dibromoalkanes (1 eq) in DMF as a solvent, in the presence
of K2CO3 (1.5 eq) at room temperature. Resulting product then reacted with NaN3 (1 eq) in DMF
at room temperature to yield 1-(4-azidoalkyl)indoline-2,3-dione.

O
R
O
N
H
a
O
R
OH
O
O
N
n
O
Br
O
b
c
R
O
O
O
N
n
O
N3
d
O
O
O
N
O
O
N
n
N
N
R
n = 1, 2, 3, 4
R = H, F, Cl, Br, I, NO2, OCH3
Scheme 1 Synthesis of triazole linked isatin-coumarin hybrids. Reagents and conditions: (a)
Dibromoalkane, K2CO3, DMF, 2 hrs, stir, rt; (b) NaN3, DMF, 1 hr, stir, rt; (c) Propargyl bromide,
K2CO3, DMF, 2 hrs, stir, rt; (d) Sodium ascorbate, CuSO4, DMF, 15 mins, rt.

Simultaneously, 4-hydroxycoumarin was stirred with propargyl bromide (1eq) in DMF as a
solvent, in the presence of K2CO3 (1.5 eq) at room temperature to get the desired product i.e.4-
(prop-2-ynyloxy)-2H-chromen-2-one (PHC).
This 4-(prop-2-ynyloxy)-2H-chromen-2-one (PHC) was then treated with various 1-(4-
azidoalkyl)indoline-2,3-dione analogues in the presence of catalytic amount of copper sulfate
and sodium ascorbate, in DMF as a solvent, at room temperature to gain the desired triazole
linked isatin-coumarin hybrids.26 The structures of the synthesized compounds were elucidated
by 1H NMR and13C NMR. All spectral data were in accordance with assumed structures.
All the synthetics were investigated for in vitro cytotoxicity against four human cancer cell lines
(THP-1, COLO-205, HCT-116 and PC-3) using sulforhodamine B19.27,28 The cells were allowed
to proliferate in the presence of test material for 48 h. All the synthesized hybrids were screened
against the cell lines at 50 µM concentration. The hybrids showing percentage inhibition (Table
1) of greater than 70% at least against one cell line were only evaluated at different
concentrations and IC50 values were calculated (Table 2). Alike to our previous report,25a among
the four cancer cell lines, THP-1, COLO-205 and HCT-116 were sensitive towards the designed
hybrids whereas PC-3 was found to be resistant and among the three sensitive cell lines, THP-1
cancer cell line was the most sensitive one towards these hybrids. Cytotoxicity results in Table 1
revealed an interesting structure activity relationship for the designed hybrids: (i) type of
substituent on isatin and the length of carbon-bridge connecting isatin moiety with triazole ring
considerably influences cytotoxic potential of hybrids; (ii) presence of unsubstitued isatin
remarkably enhances the cytotoxic potential (compare A-1 with A-2 to A-7); (ii) enhanced
cytotoxicity for hybrids having isatin with electron withdrawing substituent in comparison to
electron donating substituent (compare A-2 to A-6 with A-7); (iii) cytotoxicity increases with
increase in electronegativity of substituent on isatin (compare A-2 with A-3 to A-5). Thus, the
overall preference order of R is as follows: H > F > Cl > Br > I > NO2 > OCH3; (iv) cytotoxicity
decreases significantly with the increase in chain length of carbon-bridge (compare A with B, C
and D). Thus, the overall preference order of n (chain length) is as follows: 1 > 2 > 3 > 4.

Table 1 Various substituted isatin-coumarin hybrids with their %age inhibition against four human cancer cell
lines at 50 µM
O
O
N
N
O
O
O
N
n
N
R
CODE
R
n
Percentage growth inhibition (50 µM)
HST-116 COLO-205
THP-1
PC-3
(Prostate)
28
(Colon)
87
80
70
72
70
68
65
79
75
68
66
65
60
52
72
67
62
59
57
53
48
(Colon)
80
76
76
65
62
59
53
75
69
67
61
56
52
48
68
64
60
56
50
45
41
(Leukemia)
98
A-1
A-2
A-3
A-4
A-5
A-6
A-7
B-1
B-2
B-3
B-4
B-5
B-6
B-7
C-1
C-2
C-3
C-4
C-5
C-6
C-7
H
1
1
1
1
1
1
1
2
2
2
2
2
2
2
3
3
3
3
3
3
3
F
92
26
Cl
87
22
Br
80
21
I
74
17
NO2
OCH3
H
71
15
68
14
93
25
F
86
20
Cl
78
18
Br
72
14
I
67
14
NO2
OCH3
H
60
12
56
09
85
19
F
77
17
Cl
71
14
Br
63
13
I
59
10
NO2
OCH3
55
08
52
NA

D-1
D-2
D-3
D-4
D-5
D-6
D-7
H
4
4
4
4
4
4
4
65
59
52
43
36
29
25
62
56
48
39
30
23
19
69
63
59
52
45
37
30
09
F
NA
NA
NA
NA
NA
NA
Cl
Br
I
NO2
OCH3
NA: Not Applicable
Table 2 IC50 values of active hybrids against sensitive human cancer cell lines
Code
IC50 (µM)
COLO-205
(Colon)
THP-1
HCT-116
(Leukemia)
(Colon)
A-1
A-2
A-3
A-4
A-5
A-6
B-1
B-2
B-3
B-4
C-1
C-2
C-3
0.73
1.99
5.47
6.43
7.01
8.57
5.03
5.76
6.99
7.14
5.12
7.10
7.56
3.45
6.67
3.04
5.41
5.77
8.09
8.99
9.54
5.18
7.26
9.66
11.46
8.05
10.44
12.76
8.87
10.53
12.01
12.97
5.71
8.88
10.66
12.99
7.67
9.11
13.14

Three most active compounds (A-1, A-2 and B-1) among the series were evaluated for their
inhibitory effects on tubulin polymerization using a cytoskeleton tubulin polymerization assay
kit.29,30 Compound A-1 (most cytotoxic hybrid) also displayed the most potent anti tubulin
activity with IC50 value of 1.06 µM. A-2 and B-1 also exhibited significant inhibition of tubulin
polymerization with IC50 values of 3.55 and 6.32 µM, respectively. The results of in vitro tubulin
polymerization assay clearly indicate that hybrid molecules exert their cytotoxic effect through
tubulin inhibition (Table 3).
Table 3 Tubulin polymerization inhibition potential of most active hybrids
Compound
Inhibition of tubulin
polymerization IC50 (µM)
1.06
A-1
A-2
3.55
6.32
1.2a
B-1
CA-4 (Combrtastatin A-4)
aLiterature value27
Confocal microscopy was used further to rationalize the anti tubulin effect of most potent
compound A-1. In this study, untreated THP-1 cell lines showed the normal distribution of
microtubules. However, cells treated with 5 µM of compound A-1 showed disrupted microtubule
organization even more than the positive control (1 µM of Paclitaxel) (Fig. 3).

Fig. 3 Effect of compound on the microtubule network of THP-1 cells untreated (control), 5 µM
A-1 and 1 µM Paclitaxel for 48 h. Microtubules and unassembled tubulin are shown ingreen.
DNA, stained with 4,6-diamidino-2-phenylindole (DAPI) shown in blue.
To rationalize our experimental findings, molecular docking simulations on tubulin were
performed. Primarily colchicine binding site was selected according to Dong et al.31 and A-1 was
docked several times at the colchicine binding site but in each docking simulation, the top scored
conformation of A-1 was found to be different (RMSD > 2.3). Simultaneously, A-1 was also
docked at the interface of β1-α2 heterodimer of `tubulin (same as the vinblastine binding site)
Chakrabortiet al. (2011). The docking procedure was repeated five times. In each docking run,
the top scored conformations of A-1 were found to be similar (RMSD < 0.75) and hence showed
similar drug-receptor interactions. The docking results revealed that A-1 fits well at the interface
of β1and α2 subunits of tubulin and stabilized by polar and van der Walls interactions. Residues

from the both subunits (β1and α2) were involved in interactions with the inhibitor (Fig. 4). The
coumarin moiety (ring A and B) gets positioned in a cavity formed by Asn249α2, Glu254α2,
Glu258α2 and Lys352α2 (polar residues) and Val250α2, Val353α2 (nonpolar residues) and gets
stabilized by van der Waals interactions. The electron rich 1,2,3-triazol-1-yl ring (ring C) was
positioned between Gln11β1, and Asp179β1 (acidic residues). Here, 2-N and 3-N of ring C have
shown polar interactions with free carbonyl function of Gln11β1, and Asp179β1, respectively
(d = 3.025Å and d = 2.738 Å). The distance of ring C with catalytic Mg++ ion was found to be
3.701Å. The isatin moiety (ring D and E) was placed in a hydrophobic cavity formed by
Val177β1, Pro222β1, Thr223β1 and Thr224β1. Here, ring E has shown face-to-face pai-pai
stacking interaction with Tyr224β1 (d = 3.199Å).31 This arrangement of energetically favorable
pai-pai stacking interactions plays an important role in stabilizing the binding positions of
aromatic substrates and might well represent one of the key features of the substrate recognition
(Fig. 4b). Microtubules are dynamic polymers of αβ-tubulin that form various cellular structures
including mitotic spindle for cell division. The microtubule-associated proteins (MAPs)
selectively bind with distinct conformations of polymerized or unpolymerized αβ-tubulin and
thereby regulate the growth/shrinkage of microtubule assemblies.17,32,33 This can be suggested
that binding of A-1 at the interface of β1-α2 heterodimer restrict the tubulin to adopt MAP-
recognizable conformations and thus inhibit the tubulin polymerization into microtubule
assembly.33-35 Incorporation of more carbons in the linker of A-1 showed remarkable decrease in
cytotoxicity, most probably due to increase in size of the molecule which may not be perfectly fit
to the active site of tubulin.

Fig. 4 (a) Docked conformation of compound A-1 (cyan) at the interface of β1-α2 subunits of
tubulin complexed with colchicine (red) (PDB id 1SA0); (b) Residues involved in drug-receptor
interactions between A-1 and tubulin (A-1: carbon atoms are shown in green)
In, conclusion, our continuous efforts to search for novel anti tubulin bi-functional hybrids,
isatin-coumarin hybrids were designed, synthesized and characterized. All the synthetics were

evaluated for cytotoxicity against a panel of four human cancer cell lines. Tubulin inhibitory
potential of the most cytotoxic compound A-1 (with R = H and n = 1) was confirmed by in vitro
tubulin polymerization inhibition assay and confocal microscopy which clearly indicates that A-
1 exerts its cytotoxic effect via tubulin inhibition mechanism. Moreover, various binding
interactions of A-1 with various amino acids of tubulin were also unveiled by docking studies.
Conflict of interest
The authors confirm that this article content has no conflict of interest.
Acknowledgement
Authors are grateful to University Grants Commission for providing funds under UPE Scheme to
carry out the research work.
References
1. Rostom SAF. Bioorg. Med. Chem. 2006;14:6475-6489.
2. Fadeyi OO, AdamsonST, MylesEL, Okoro CO. Bioorg. Med. Chem. Lett. 2008;18:4172–
4186.
3. Parkash O, Kumar P, Ajeet. Am. J. Pharmacol. Sci. 2013;6:104-115.
4. Kaur R, Kaur P, Sharma S, Singh G, Mehndiratta S, Nepali K. Rec. Pat. Anticanc. Drug
Discov. 2015;10:23-71.
5. Sharma S, Mehndiratta S, Kumar S, Singh J, Bedi PMS, Nepali K. Rec. Pat. Anticanc. Drug
Discov. 2015;10:308-341.
6. Singh H, Singh H, Sharma S, Bedi PMS. Heterocycles 2015;91:2043-2085.
7. Sharma S, Singh J, Ojha R, Singh H, Kaur M, Bedi PMS, Nepali K. Eur. J. Med. Chem.
2016;112:298-346.
8. Nepali K, Sharma S, Sharma M, Bedi PMS, Dhar KL. Eur. J. Med. Chem. 2014;77:422-487.
9. Nepali K, Sharma S, Kumar D, Budhiraja A, Dhar KL. Recent Pat. Anti Cancer Drug
Discov. 2014;9:303-339.
10. Contelles JM, Soriano E. Curr. Top. Med. Chem. 2011;11:2714-2715.

11. (a) Honore S, Pasquier E, Braguer D. Cell. Mol. Life Sci. 2005;62:3039-3056; (b) Pellegrini
F, Budman D R. Cancer. Invest. 2005;23:264-273; (c) Nepali K, Ojha R, Sharma S, Bedi
PMS, Dhar KL. Recent Pat. Anti-cancer Drug Discov. 2014;9:176-220.
12. Mehndiratta S, Sharma S, Kumar S, Nepali K. A. Rahman, K. Zaman (Eds.), Topics in Anti-
Cancer Research, Patents EBook Series, vol. 4, Bentham Science Publishers Ltd. 2015, pp.
383-454.
13. Sharma S, Kaur C, Budhiraja A, Nepali K, Gupta MK, SaxenaAK, Bedi PMS. Eur. J. Med.
Chem. 2014;85:648-660.
14. Singh J, Sharma S, Saxena AK, Nepali K, Bedi PMS. Med. Chem. Res. 2013;22:3160-3169.
15. Sharma M, Sharma S, Buddhiraja A, Saxena A.K, Nepali K, Bedi PMS. Med. Chem. Res.
2014;23:4337-4344.
16. Kandi SK, Manohar S, Gerena CEV, Zayas B, MalhotraSV, RawatDS. New J. Chem.
2015;39:224-234.
17. Sharma S, Gupta MK, Saxena AK, Bedi PMS. Bioorg. Med. Chem. 2015;23:7165-7180.
18. Kumar K, Sagar S, Esau L, Kaur M, Kumar V. Eur. J. Med. Chem. 2012;58:153-159.
19. Singh P, Sharma P, Anand A, Bedi PMS, Kaur T, Saxena AK, Kumar V. Eur. J. Med.
Chem. 2012;55:455-461.
20. Solomo VR, Hua C, Lee H. Bioorg. Med. Chem. 2009;17;7585-7592.
21. Solomon VR, Hua C, Lee H. Bioorg. Med. Chem. 2010;18;1563-1572.
22. Ramshid PK, Jagadeeshan S, Krishnan A, Mathew M, Nair SA, Pillai MR. Med. Chem.
2010;6:306-312.
23. Thakur A, Singla R, Jaitak V. Eur. J. Med. Chem. 2015;101:476-495.
24. a) Pingaew R, Saekee A, Mandi P, Nantasenamat C, Prachayasittikul S, Ruchirawat S,
Prachayasittikul V. Eur. J. Med. Chem. 2014;85:65-76; b) Ma Yi, Zhou Y, Xie C, Chen D,
Li J. Acta Pharmacologica Sinica 2012;33:407-417.
25. a) Singh H, Kumar M, Nepali K, Gupta MK, Saxena AK, Sharma S, Bedi PMS. Eur. J.
Med. Chem. 2016;116:102-115; b) Parthasarathy K, Chandrasekar P, Saranraj K,
Balachandran C, Senthil Kumar P. Med. Chem. Res. 2016;25:2155-2170; c) Parthasarathy
K, Chandrasekar P, Jeyaveeran JC, Prince AAM. Bioorg. Med. Chem. Lett. 2016;26:4310-
4317.

26. (PHC) was treated with various 1-(4-azidoalkyl)indoline-2,3-dione analogues in the
presence of catalytic amount of copper sulfate and sodium ascorbate, in DMF as a solvent,
at room temperature to gain the desired triazole linked isatin-coumarin hybrids. 1-(2-(4-((2-
oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)ethyl)indoline-2,3-dione
(A-1):
Yield 78%, mp 104-108oC. 1H NMR (d6-DMSO, 300 MHz, δ, TMS = 0): 4.21 (s, 2H), 4.77
(s, 2H), 5.38 (s, 2H), 6.11 (s, 1H), 6.91 (d, 1H, J = 7.8 Hz), 7.08 (t, 1H), 7.41-7.46 (m, 2H),
7.54-7.56 (m, 2H), 7.68-7.71 (m, 2H), 8.49 (s, 1H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS
= 0): 47.64, 62.93, 91.50, 110.52, 115.43, 116.84, 117.64, 123.29, 123.79, 124.64, 125.00,
133.33, 138.59, 150.54, 153.11, 158.55, 162.14, 164.73, 183.32. Anal.Calcd for
C22H16N4O5: C, 63.46; H, 3.87; N, 13.46 Found: C, 63.26; H, 3.98; N, 13.15.
27. Monks A, Scudiero AD, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J,
Cronise P, Vaigro-Wolff A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M. J. Natl.
Cancer Inst. 1991;83:757-766.
28. Skehan P, Storeng R, Scudiero D, Monks A, Mc Mohan J, Vistica D, Warren JT, Bokesch
H, Kenney S, Boyd MR. J. Natl. Cancer Inst. 1990;82:1107-1112.
29. Pettit GR, Toki B, Herald DL, Verdier-Pinard P, Boyd MR, Hamel H, Pettit RK. J. Med.
Chem. 1998;41:1688-1695.
30. a) Shelanski ML, Gaskin F, Cantor CR, Proc. Natl. Acad. Sci. 1973;70:765-768;
b) Lee JC, Timassheff SN. Biochem. 1977;16:1754-1764.
31. a) Dong C, Yibin L, Wei Y, Chunyu W, Peng B, Taijin W, Minghai T, Xiaoyan W, Zhuang
Y, Buyun M, Liang M, Lei L, Fang W, Bixue X, Yuanyuan Z, Tao Y, Lijuan C. J. Med.
Chem. 2016;59:5721−5739; b) Burley SK, Petsko GA. Science. 1985;229:23-38.
32. Belmont LD, Mitchison TJ.Cell. 1996;84:623-631.
33. Ayaz P, Ye X, Huddleston P, Brautigam CA, Rice LM. Science. 2012;337:857-60.
34. Lodish H, Berk A, Zipursky SL, Paul M, David B, James D. Molecular Cell Biology. Fourth
ed., W.H. Freeman, New York, 2000.
35. Brouhard GJ, Rice LM. J. Cell Biol. 2014;207:323-334.
36. Ravelli RB, Gigant B, Curmi PA, Jourdain I, Lachkar S, Sobel A, Knossow M. Nature.
2004;428:198-202.

37. Chakraborti S, Das L,Kapoor N, Das A, Dwivedi V, Poddar A, Chakraborti G, Janik M,
Basu G, Panda D, Chakrabarti P, Surolia A, Bhattacharyya B. J. Med. Chem. 2011;54:6183-
6196.
38. GOLD 5.3.0. Cambridge Crystallographic Data Centre: Cambridge, U.K. 2015.

 Design of isatin-coumarin hybrids based on the cytotoxicity of each pharmacophore
 Synthesis of 28 novel isatin-coumarin hybrids using click chemistry approach
 Biological evaluation of hybrids against a panel of four human cancer cell lines
 Tubulin polymerization inhibition by the most cytotoxic hybrids
 Docking of the most potent hybrid at curcumin binding site of tubulin

GRAPHICAL ABSTRACT
Triazole tethered isatin-coumarin based molecular hybrids as novel antitubulin agents:
Design, synthesis, biological investigation and docking studies
Harbinder Singha, Jatinder V. Singha, Manish K. Guptab, Ajit K. Saxenac, Sahil Sharmaa,*, Kunal
Nepalia,*, Preet Mohinder S. Bedia,*
aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab
143005, India
bLloyd Institute of Management and Technology, Greater Noida, UP, India
cIndian Institute of Integrative Medicine, Jammu, India
Coumarin
O
O
Triazole as Linker
Isatin
O
O
O
O
O
O
O
N
O
N
n
O
O
O
N
N
HYBRIDIZATION
N
N
N
N
Most potent compound
IC50 = 0.73 µM (THP-1)
IC50 = 1.06 µM (Tubulin)
O
R
R
n = 1, 2, 3, 4
Isatin-coumarin hybrids
O
N
H