Synthesis and biological evaluation of imidazo[2,1-b][1,3,4]thiadiazole- linked oxindoles as potent tubulin polymerization inhibitors

Article abstract of DOI:10.1002/cmdc.201400069

A series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles composed of an A, B, C and D ring system were synthesized and investigated for anti-proliferative activity in various human cancer cell lines; test compounds were variously substituted at rings C and D. Among them, compounds 7 ((E)-5-fluoro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)-imidazo[2,1-b][1,3,4] thiadiazol-5-yl)methylene)indolin-2-one), 11 ((E)-3-((6-p-tolyl-2-(3,4,5- trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), and 15 ((E)-6-chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3, 4]thiadiazol-5-yl)methylene)indolin-2-one) exhibited potent anti-proliferative activity. Treatment with these three compounds resulted in accumulation of cells in G₂/M phase, inhibition of tubulin assembly, and increased cyclin-B1 protein levels. Compound 7 displayed potent cytotoxicity, with an IC₅₀ range of 1.1-1.6 μM, and inhibited tubulin polymerization with an IC₅₀ value (0.15 μM) lower than that of combretastatin A-4 (1.16 μM). Docking studies reveal that compounds 7 and 11 bind with αAsn101, βThr179, and βCys241 in the colchicine binding site of tubulin. Imidazzling microtubule blockers! A series of imidazo[2,1-b][1,3,4] thiadiazole-linked oxindole conjugates were synthesized and evaluated for anticancer potential. Conjugate 7 displayed promising cytotoxicity, arrested cells at the G₂/M phase, and showed potent tubulin polymerization inhibition with an IC₅₀ value of 0.15 μM.

Full text of DOI:10.1002/cmdc.201400069

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DOI: 10.1002/cmdc.201400069
Synthesis and Biological Evaluation of Imidazo[2,1-
b][1,3,4]thiadiazole-Linked Oxindoles as Potent Tubulin
Polymerization Inhibitors
Ahmed Kamal,*[a, c] M. P. Narasimha Rao,[a] Pompi Das,[b] P. Swapna,[a] Sowjanya Polepalli,[b]
Vijaykumar D. Nimbarte,[c] Kishore Mullagiri,[a] Jeshma Kovvuri,[a] and Nishant Jain[b]
A
series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles
methylene)indolin-2-one) exhibited potent anti-proliferative ac-
tivity. Treatment with these three compounds resulted in accu-
mulation of cells in G2/M phase, inhibition of tubulin assembly,
and increased cyclin-B1 protein levels. Compound 7 displayed
potent cytotoxicity, with an IC50 range of 1.1–1.6 mm, and inhib-
ited tubulin polymerization with an IC50 value (0.15 mm) lower
than that of combretastatin A-4 (1.16 mm). Docking studies
reveal that compounds 7 and 11 bind with aAsn101, bThr179,
and bCys241 in the colchicine binding site of tubulin.
composed of an A, B, C and D ring system were synthesized
and investigated for anti-proliferative activity in various human
cancer cell lines; test compounds were variously substituted at
rings C and D. Among them, compounds 7 ((E)-5-fluoro-3-((6-
p-tolyl-2-(3,4,5-trimethoxyphenyl)-imidazo[2,1-b]-
[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), 11 ((E)-3-((6-p-
tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-
yl)methylene)indolin-2-one), and 15 ((E)-6-chloro-3-((6-phenyl-
2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-
Introduction
Cancer is a disease in which normal cells dramatically alter
their behavior: the multiply uncontrollably, ignore signals to
stop, and accumulate to form tumors. Cancer has become
a major cause of morbidity throughout the world, accounting
for 7.6 million deaths (~13% of all deaths) in 2008. According
to the World Health Organization, deaths due to cancer world-
wide were projected to continue to rise to over 13.1 million in
2030. There are nearly 200 different types of cancer, each
named for the organ or type of cell from which it originates.
Cancers of the colon, stomach, lung, liver, and breast cause the
greatest number of deaths each year.
used to treat helminthic infections and in the amelioration of
various autoimmune disorders.[3,4] Furthermore, I has been
shown to have anticancer activity in combination with fluo-
rouracil (5-FU) as an adjuvant therapy for tumor-node-metasta-
sis (TNM) stage III (Dukes’ C) colon carcinoma.[5] Imidazo[2,1-
b]thiazole derivatives of levamisole have been reported as po-
tential antitumor agents.[6] Subsequently, the antitumor activity
of 5-formyl-6-arylimidazo[2,1-b]-1,3,4-thiadiazole sulfonamides
V were also reported.[7] In 2012 Andreani and co-workers[8] re-
ported that 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indoli-
nones cause G2/M phase arrest and inhibit tubulin assembly.
Tubulin is a heterodimer of two closely related and tightly
linked globular polypeptides called a- and b-tubulin, which
polymerize to form microtubules. Their function in mitosis and
cell division makes them an important target for anticancer
drugs. Microtubules and their dynamics are the targets of
a chemically diverse group of antimitotic drugs that have been
used with great success in the treatment of cancer. There has
been considerable interest in the discovery and development
of small molecules that affect tubulin polymerization.[9] Howev-
er, the success of tubulin polymerization inhibitors as anticanc-
er agents has stimulated significant interest in the identifica-
tion of new compounds that may be more potent or more se-
lective in targeted tissues or tumors.
Levamisole (I) is an immunomodulatory compound toward
various cancer cell types including colorectal and breast can-
cers, melanoma, and leukemia (Figure 1).[1] It was previously
shown to affect cell proliferation in different cancers,[2] and to
modulate the phosphorylation of proteins involved in cell-
cycle progression and apoptosis. Levamisole has also been
[a] Dr. A. Kamal, M. P. N. Rao, P. Swapna, K. Mullagiri, J. Kovvuri
Medicinal Chemistry and Pharmacology
CSIR – Indian Institute of Chemical Technology
Hyderabad 500 007 (India)
E-mail: ahmedkamal@iict.res.in
[b] P. Das, S. Polepalli, Dr. N. Jain
Chemical Biology, CSIR – Indian Institute of Chemical Technology
Hyderabad 500 007 (India)
Oxindoles are versatile moieties that display diverse biologi-
cal activities, including anticancer activity. They exhibit antitu-
mor activity by inhibiting tyrosine kinase receptors such as
PDGF-R, VEGF-R, and CDK.[10] One of the most important exam-
ples is sunitinib (II), which has been widely used in the treat-
ment of gastrointestinal stromal tumors and metastatic renal
[c] Dr. A. Kamal, V. D. Nimbarte
Department of Medicinal Chemistry
National Institute of Pharmaceutical Education and Research (NIPER)
Hyderabad 500 037 (India)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201400069.
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pared by Vilsmeier–Haack reac-
tion with the corresponding
imidazo[2,1-b][1,3,4]thiadiazoles
5a–e. Conjugates 7–32 were
prepared by single-step Knoeve-
nagel reaction between com-
pounds 6a–e and various substi-
tuted oxindoles in ethanol/piper-
idine. These conjugates were
1
characterized by H and 13C NMR
spectroscopy, mass spectrome-
try, HRMS, and IR spectroscopy.
All compounds were obtained in
pure E isomeric form and con-
firmed with those previously re-
ported.[16,17]
Figure 1. Structures of levamisole (I), sunitinib (II), combretastatin A-4 (III), colchicine (IV), 5-formyl-6-arylimidazo-
[2,1-b][1,3,4]thiadiazolesulfonamide derivatives V, and synthesized imidazo[2,1-b][1,3,4]thiadiazole-indolin-2-one
conjugates 7–32.
cell cancer.[11,12] Chen and co-workers reported that
oxindoles not only inhibit CDK, but also inhibit micro-
tubule polymerization by binding at the colchicine
binding site of tubulin.[13] On the other hand, a 3,4,5-
trimethoxyphenyl ring is present in most of the syn-
thesized and naturally occurring biologically active
compounds, including colchicine (IV), combretastatin
A-4 (CA-4; III), and its derivatives.
Our continued efforts toward the synthesis of a va-
riety of new molecules led to the development of ef-
ficient anticancer agents.[14,15] In view of the biologi-
cal importance of both imidazothiadiazoles and oxin-
doles, in this study we designed and synthesized
a series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxin-
doles. Use of the core imidazothiadiazole scaffold
functionalized with 3,4,5-trimethoxyphenyl ring A, an
indolinone as ring C, and a phenyl group as ring D re-
sulted in the generation of imidazo[2,1-b]-
[1,3,4]thiadiazolindolin-2-ones. These compounds
were evaluated for in vitro anticancer activity, cell-
cycle effects, and the capacity to inhibit tubulin poly-
merization.
Results and Discussion
Chemistry
The imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles
were prepared as shown in Scheme 1. Compound 3
was prepared by reaction of 3,4,5-trimethoxybenzoic
acid with thiosemicarbazide in POCl3 at reflux. 6-(4-
Aryl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazoles 5a–e were obtained in high yield
by treating compound 3 with various phenacyl bro-
mides 4a–e in ethanol with the addition of 3–4
drops of N,N-dimethylformamide. The imidazo[2,1-b]-
Scheme 1. Synthesis of imidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates 7–32. Re-
agents and conditions: a) POCl3, reflux, 4 h, 90%; b) EtOH, reflux, 12 h, 80–85%;
c) POCl3 +DMF, reflux, 5 h, 75–80%; d) 5- or 6-substituted oxindoles, EtOH, cat. (3–4
[1,3,4]thiadiazole-5-carbaldehydes 6a–e were pre- drops) piperidine, reflux, 3–4 h, 75–85%.
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Biology
Cytotoxicity
A sulforhodamine B assay was performed to evaluate the cyto-
toxic effects of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles
7–32 against A549, HeLa, MCF-7, and HCT116 human cancer
cell lines, and the IC50 values are listed in Table 1. Compounds
7–15 showed considerable cytotoxicity, with IC50 values rang-
ing from 1.1 to 8.9 mm against A549, HeLa, MCF-7, and HCT116
human cancer cell lines.
methoxy groups and that are devoid of substitution on indoli-
none (ring C) and that have a fluoro group on ring D. Based on
the results of sulforhodamine B assays, compounds either me-
thylated or that lack substitution on ring D showed the most
potent activities in the series; therefore, the activities of the
most potent compounds can be ranked in the order of 7 > 11
> 15 > 21. To further determine their potency in other cell
types, conjugates 7, 11, 15, and 21 were evaluated in the
sixty-cell-line panel of the US National Cancer Institute (NCI),
which includes leukemia, melanoma, renal, lung, colon, ovary,
prostate, breast, and central nervous system cancer cell lines;
compounds 11 and 21 were selected for the five-dose assay
(Table 2). These compounds displayed significant cytotoxicity
in most of the NCI panel cell lines, with GI50 values ranging
from 0.30 to 5.8 mm. In particular, compound 11 showed prom-
ising cytotoxicity, with GI50 values of 0.30 and 0.42 mm against
OVCAR-4 (ovarian cancer) and HOP-92 (lung cancer) cell lines.
Taken together, results from the cytotoxicity assays agreed re-
markably with the NCI 60-cell-line screen. We then elucidated
whether the anti-proliferative properties of 7, 11, and 15 were
due to inhibition of tubulin polymerization. To investigate this
possibility, 7, 11, and 15 were incubated at varying concentra-
tions with tubulin, and polymerization assays were performed.
Compound 7, which potently inhibited cell growth, also signifi-
cantly decreased tubulin assembly, with an IC50 value of
0.15 mm. Compounds 11 and 15 were only slightly less potent,
with IC50 values of 1.23 and 2.11 mm, respectively.
Table 1. Cytotoxicity of compounds 7–32 and levamisole on A549, HeLa,
MCF-7, and HCT116 human cancer cell lines.
Compd
IC50 [mm][a]
HeLa[c] MCF-7[d]
A549[b]
HCT116[e]
7
8
9
10
11
1.1Æ0.7
5.7Æ3.1
5.3Æ0.2
8.5Æ0.8
2.6Æ0.9
4.2Æ0.9
5.0Æ0.79
6.8Æ0.8
2.3Æ1.6
19.5Æ1.9
18.5Æ0.2
12Æ1.1
1.4Æ1.7
3.9Æ0.7
3.8Æ1.7
2.8Æ1.2
2.5Æ0.3
2.5Æ1.6
3.4Æ1.3
3.6Æ2.3
3.2Æ1.0
12.8Æ1.2
23.0Æ0.5
24Æ0.2
1.2Æ0.4
1.6Æ0.5
7.3Æ2.7
2.9Æ0.8
8.9Æ1.8
3.0Æ1.5
2.5Æ0.9
7.6Æ0.8
7.4Æ1.2
7.5Æ0.9
2.4Æ1.6
22.9Æ1.0
21.5Æ1.6
10.1Æ0.6
13.1Æ1.3
12.9Æ0.3
2.1Æ1.3
8.3Æ0.7
5.6Æ2.1
2.9Æ1.2
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
levamisole
5.8Æ4.2
8.2Æ1.0
8.4Æ2.1
3.0Æ3.0
11.7Æ0.9
13.5Æ0.7
13.1Æ1.1
11.2Æ1.4
18.6Æ0.4
15.2Æ0.3
27.3Æ4.1
13.5Æ0.9
20.2Æ0.4
13.6Æ0.4
18.9Æ1.6
26.2Æ6.2
29.2Æ1.3
18.2Æ4.1
14.7Æ2.7
24.6Æ5.3
19.5Æ1.0
18.6Æ0.3
17.4Æ1.9
3.1Æ1.3
36.9Æ1.9
33.1Æ1.8
4.2Æ1.7
27.8Æ1.0
20.5Æ1.2
29.7Æ2.3
22.4Æ0.9
15.3Æ1.5
23.2Æ1.2
19.7Æ0.9
22.5Æ1.7
15.8Æ3.1
25.9Æ1.0
28.1Æ1.1
96
13.2Æ0.1
16.4Æ1.9
31.6Æ1.5
26.9Æ1.3
28.4Æ1.8
29.1Æ1.9
16.8Æ0.2
20.7Æ2.8
18.6Æ0.7
15.3Æ2.9
24.5Æ0.5
24.6Æ3.6
21.3Æ1.1
35.2Æ2.4
17.7Æ1.7
25.3Æ1.2
29.6Æ2.0
24.9Æ2.2
21.8Æ0.7
26.7Æ1.1
20.0Æ1.8
21.4Æ1.4
>100
>100
>100
[a] 50% inhibitory concentration; values are the mean ÆSD of three indi-
vidual experiments determined after 48 h treatment. [b] NSC lung cancer.
[c] Human epithelial cervical cancer. [d] Human breast adenocarcinoma.
[e] Colon cancer.
Effect of the compounds on cell cycle
Because the compounds inhibit tubulin polymerization, we
evaluated their ability to stall cells at the G2/M phase. A549
cells were treated with test compounds at 5 mm for 24 h. Cells
were then harvested and analyzed by flow cytometry for DNA
content (Figure 2). The control cells treated with DMSO
showed 26.5% arrested in G2/M phase. In contrast, compounds
7 and 11 increased the cell population in G2/M phase by 67.3
and 64.8%, respectively. Moreover, cells treated with 15 mani-
fested 66.7% of cells in G2/M phase.
Compound 7, which is fluorinated on ring C and has
a methyl group on the ring D, exhibited potent cytotoxicity
with IC50 values in the range 1.1–1.6 mm. In comparison, conju-
gate 11, devoid of any substitution on ring C, also displayed
potent anti-proliferative activity, with IC50 values of 2.5–2.9 mm.
Moreover, compound 15, chlorinated at the 6-position on
ring C and lacking ring D substitution, and 21, with a 5-fluoro
group on ring C, and a methoxy group on ring D, both exhibit-
ed potent cytotoxicity.
Effect of compounds on microtubules
Pharmacological inhibition of microtubule formation results in
a profound disruption of the cellular microtubule network, and
as such, spindle fibers composed of microtubules are routinely
disrupted from treatments with anti-tubulin agents.[18] The
imidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates inhibit cell
However, compounds with 5-methoxy (8), 5-chloro (9), and
6-chloro (10) substituents on ring C showed moderate cytotox-
icity (IC50 values 2–8 mm). Overall, activity was decreased in
cells treated with compounds that contain chloro, fluoro, or
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exhibit a typical rounded mitotic
phenotype, and DAPI was used
to counter-stain the nuclei
(Figure 3).
Table 2. In vitro cytotoxicity of compounds 11 and 21 against the NCI panel of 60 human cancer cell lines.
Cancer panel/cell line
GI50 [mm][a]
11[b]
Cancer panel/cell line
GI50 [mm][a]
11[b]
21[c]
21[c]
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Melanoma
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
–
5.49
2.17
13.1
4.54
11.3
23.5
–
16.3
–
0.59
–
3.39
5.58
9.58
1.64
3.35
13.3
Effect of compounds on endoge-
nous tubulin in A549 cells
>100
–
–
–
Microtubules are present in a dy-
namic equilibrium with free tu-
bulin monomers. Anti-tubulin
compounds disrupt this dynamic
equilibrium. Microtubule-depoly-
merizing agents increase the
amount of free tubulin mono-
mer in cells, thereby preventing
microtubule polymerization.[19]
As compounds 7, 11, and 15 effi-
ciently arrest cells at the G2/M
phase and inhibit tubulin assem-
bly, we elucidated their ability to
block endogenous tubulin poly-
merization. A549 cells were
treated with compounds 7, 11,
and 15 at 5 mm for 24 h. We
then permeabilized cells with
UACC-257
UACC-62
2.35
5.47
Non-Small Cell Lung Cancer
A549/ATCC
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
Ovarian Cancer
IGROV 1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
2.12
4.14
0.42
22.5
4.54
10.3
1.98
10.2
2.13
1.94
2.02
16.8
4.25
2.74
3.42
5.44
4.75
2.90
0.30
3.39
2.32
1.77
6.14
5.34
21.9
2.84
>100
3.71
3.05
15.2
NCI-H522
Renal Cancer
786-0
A498
Colon Cancer
COLO 205
HCC-2998
HCT116
HCT15
HT29
KM12
SW-620
5.80
7.28
4.97
16.0
12.2
3.29
4.48
3.96
14.2
2.26
15.0
–
1.61
5.25
14.2
2.50
2.18
4.04
3.21
>100
ACHN
2.59
2.68
–
CAKI-1
RXF 393
SN 12C
TK-10
15.4
2.24
5.25
1.69
8.44
>100
–
UO-31
CNC Cancer
SF-268
SF-295
Prostate Cancer
PC-3
DU-145
detergent-containing
buffers
3.63
5.04
12.9
7.89
0.55
1.59
4.40
3.00
56.0
15.1
10.3
1.77
81.1
2.55
24.7
1.87
(details in the Experimental Sec-
tion below). The insoluble and
soluble fractions were analyzed
for tubulin (Figure 4). DMSO-
treated cells demonstrated equal
amounts of tubulin across the
soluble and insoluble fractions.
In contrast, 7 and 11 effected
a marked increase in the soluble
fraction similar to CA-4-treated
cells. In comparison, paclitaxel-
treated cells possessed increased
levels of tubulin in the insoluble
SF-539
SNB-19
SNB-75
U251
Breast Cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
–
1.66
5.25
16.2
14.9
2.01
2.53
3.51
0.50
4.69
3.13
4.84
Melanoma
LOX IMVI
MALME-3M
M14
–
0.76
–
6.88
9.15
5.51
MDA-MB-468
[a] Compound concentration required to decrease cell growth to half that of untreated cells. [b] 11
(NSC775003). [c] 21 (NSC774982).
proliferation, tubulin assembly, and arrest cells at the G2/M
phase (Table 3). To further validate their effect on microtubules,
we treated A549 cells with compounds 7, 11, and 15 for 24 h.
Immunofluorescence analyses revealed that the conjugates
profoundly alter microtubule the network. Treated cells also
fraction. Next, we analyzed whether the cells arrested at the
G2/M phase were indeed mitotic in nature. We confirmed their
state by assaying for the mitotic marker cyclin-B1. As expected,
treatments with compound 7 induced increased levels of
cyclin-B1, when compared with 11 and 15. b-actin was used as
a loading control.
Table 3. Anti-tubulin polymerization activity of compounds 7, 11, 15 and
Molecular docking studies
CA-4.
Among the synthesized compounds, 7 and 11 displayed signif-
icant cytotoxicity, tubulin polymerization inhibition with IC50
values less than or equal to that of CA-4, and arrested cells at
the G2/M phase of the cell cycle. Molecular docking studies
were performed to determine whether the active conjugates 7
and 11 exert their cytotoxicity and capacity to block tubulin
polymerization by binding to tubulin at the colchicine binding
site. Docking analyses suggest that both compounds occupy
Compd
IC50 [mm][a]
7
0.15Æ0.3
1.23Æ1.0
2.11Æ0.7
1.16Æ0.1
11
15
CA4
[a] Concentration of compounds required to inhibit tubulin assembly by
50%; values are the mean ÆSD of two independent experiments per-
formed in triplicate.
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the colchicine binding site of a/
b-tubulin (Figure 5). The 3,4,5-tri-
methoxyphenyl group of the
conjugates anchors the molecule
in the a/b interface of tubulin,
similar to the binding mode of
colchicines.
This binding pattern is sup-
ported by the hydrogen bond-
ing interactions between the
3,4,5-trimethoxyphenyl ring and
aGln11 and bAsn258, the distan-
ces from which are in the range
of 1.8–2.2 ꢁ. Moreover, rings B of
compounds 7 and 11 are in-
volved in hydrophobic interac-
tions with aThr179. Some hydro-
gen bonding interactions were
also observed between ring C
and bThr353, bCys241, and
aAsn101 of a/b-tubulin. The p-
tolyl groups of 7 and 11 are in-
volved in hydrogen bonding in-
teractions with aSer178. Based
on the analysis of docking re-
sults, we conclude that com-
Figure 2. Antimitotic effects of b) 7, c) 11, and d) 15 by FACS analysis. Induction of cell-cycle G2/M arrest by com-
pounds 7, 11, and 15. A549 cells were harvested after treatment at 5 mm for 24 h. Untreated cells and a) DMSO-
treated cells served as controls. Flow cytometry was used to quantify the percentage of cells in each phase of the
cell cycle.
Figure 4. Distribution of tubulin in polymerized versus soluble fractions:
A549 cells were treated with compounds 7 and 11 at 5 mm for 24 h. Tubulin
was detected by immunoblot analysis. Cyclin-B1 protein levels were measure
with specific antibodies, and b-actin was used as a loading control.
pounds 7 and 11 occupy the colchicine binding site at the a/
b interface of tubulin (Table 4).
Overall, some determinant hydrophobic interactions were
observed between ring C and aAsn101 and bCys241, and be-
Table 4. Comparisons of docking scores of synthesized compounds 7
and 11 with colchicine (IV).
Figure 3. Effect of 7, 11, and 15 on microtubule dynamics. A549 cells were
independently treated with 7, 11, and 15 at 5 mm for 24 h. Cells were then
fixed and stained for tubulin antibody (green), and DAPI was used as coun-
ter-stain (blue). The merged images of cells stained for tubulin and DAPI are
shown at right.
IV
À8.84
À10.22
À9.19
À8.84
À10.21
À9.16
À8.84
À9.91
À9.10
À8.84
À9.67
À9.08
À8.84
À9.57
À9.08
À8.84
À9.57
À7.45
7
11
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Figure 5. Superposition of the docked conformations of 11 (green sticks) and 7 (blue sticks) over the X-ray crystal structure of the colchicine binding site of
tubulin (PDB ID: 3E22).
tween ring B and bAla180 and bThr179. Hydrophobic interac-
tions between ring A and bLeu255, bLeu248, aGln11, and
bAsn249 were also observed (Figure 6). Some hydrophobic in-
teractions are similar to those
cycle arrest in the G2/M phase, disruption of microtubule net-
works, and increase levels of cyclin-B1. Tubulin disassembly by
compounds 7 and 11 was further supported by western blot
observed for colchicine (IV);
however, a few additional inter-
actions were also observed with
aGln11,
bAsn258,
aThr179,
bThr353, aAsn101, and aSer178.
Conclusions
We synthesized
a
series of
imidazo[2,1-b][1,3,4]thiadiazole–
oxindoles composed of A, B, C,
and
D ring systems. Two of
these conjugates were selected
for the NCI 60-cell-line panel,
five-dose screen. SAR analysis re-
veals that amongst the conju-
gates synthesized, those with
a methyl group and devoid of
any substitution on ring D exhib-
it significant activity. Compounds
7, 11, and 15 displayed potent
cytotoxicity, with IC50 values of
1.1–3.2 mm. These three com-
pounds exert their cytotoxic ac-
tivity by inhibition of tubulin
polymerization, with IC50 values
of 0.15, 1.23, and 2.11 mm, re-
Figure 6. Binding poses of a,b) 7 and c,d) 11 within the a/b interface of tubulin. Shown are the trimethoxyphenyl
spectively. They also induce cell- group and N atom (blue) of the oxindole ring involved in hydrophobic and hydrogen bonding interactions.
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7.73 (d, J=7.9 Hz, 2H), 7.22 (d, J=7.9 Hz, 2H), 7.08 (s, 2H), 3.96 (s,
6H), 3.92 (s, 3H), 2.38 ppm (s, 3H); MS (ESI, m/z): 382 [M+H]+.
analysis. Moreover, docking studies provided some molecular
insight into the binding mode of compounds 7 and 11, which
bind at the colchicine binding site in a/b interfaces of poly-
merized tubulin. Based on these results, it is evident that com-
pounds of this structural class are acquiescent to further modi-
fications and function as a suitable template for the design of
a new class of tubulin polymerization inhibitors for the treat-
ment of cancer.
6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (5b): Compound 5b was prepared according to
the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-
diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-phenylethanone
4b (3.92 g, 14.98 mmol) to obtain pure product 5b as pale-yellow
1
solid (4.45 g, 81% yield). H NMR (500 MHz, CDCl3): d=8.02 (s, 1H),
7.84–7.82 (m, 2H), 7.43–7.40 (m, 2H), 7.32–7.28 (s, 1H), 7.08 (s, 2H),
3.96 (s, 6H), 3.92 ppm (s, 3H); MS (ESI, m/z): 368 [M+H]+.
6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (5c): Compound 5c was prepared according to
the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-
diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-methoxyphe-
nyl)ethanone 4c (3.43 g, 14.98 mmol) to obtain pure product 5c as
Experimental Section
Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma–Al-
drich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey
Co., Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai, India),
and were used without further purification. TLC was performed on
glass plates containing silica gel 60 GF254, and visualization was
achieved by UV light or iodine indicator-monitored reactions.
Column chromatography was performed with Merck 60–120 mesh
silica gel. 1H and 13C NMR spectra were recorded on Bruker
UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR Unity (400,
500 MHz) instruments. Chemical shifts (d) are reported in ppm
downfield from an internal TMS standard. ESIMS spectra were re-
corded on a Micro-mass Quattro LC instrument equipped with an
ESI mode positive ion trap detector and running ESI+ software;
capillary voltage was 3.98 kV. High-resolution mass spectra (HRMS)
were recorded on a QSTAR XL Hybrid MS–MS mass spectrometer.
Melting points were determined with an electrothermal melting
point apparatus, and are uncorrected. 1H and 13C NMR, IR, and
HRMS spectra of final compounds 7–32 are provided in the Sup-
porting information.
1
pale-yellow solid (4.75 g, 80% yield). H NMR (300 MHz, CDCl3): d=
7.93 (s, 1H), 7.74 (d, J=8.6 Hz, 2H), 7.07 (s, 2H), 6.94 (d, J=8.6 Hz,
2H), 3.96 (s, 6H), 3.92 (s, 3H), 3.84 ppm (s, 3H); MS (ESI, m/z): 398
[M+H]+.
6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (5d): Compound 5d was prepared according to
the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-
diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-chloropheny-
l)ethanone 4d (3.46 g, 14.98 mmol) to obtain pure product 5d as
1
pale-yellow solid (5.10 g, 85% yield). H NMR (500 MHz, CDCl3): d=
8.00 (s, 1H), 7.75 (d, J=8.5 Hz, 2H), 7.39–7.37 (m, 2H), 7.08 (s, 2H),
3.96 (s, 6H), 3.93 ppm (s, 3H); MS (ESI, m/z): 402 [M+H]+.
6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole (5e): Compound 5e was prepared according to
the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-
diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-fluoropheny-
l)ethanone 4e (3.22 g, 14.98 mmol) to obtain pure product 5e as
1
pale-yellow solid (4.72 g, 82% yield). H NMR (300 MHz, CDCl3): d=
Preparation of 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-
amine (3): POCl3 (21 mL) was added to a mixture of 3,4,5-trime-
thoxybenzoic acid (1, 10 g, 47 mmol) and thiosemicarbazide (2,
4.27 g, 47 mmol), and the mixture was heated at 758C for 30 min.
After cooling to room temperature, water was added slowly. The
reaction mixture was further heated at 1108C for 4 h. After cooling,
the mixture was adjusted to pH 8 by dropwise addition of a 50%
NaOH solution under stirring. The precipitate obtained was filtered
and washed with water (2ꢂ100 mL). The pale-yellow solid ob-
tained (11.3 g, 90%) was dried and used directly for the next step.
8.40 (s, 1H), 8.01 (d, J=8.5 Hz, 2H), 7.24–7.27 (m, 2H), 7.14 (s, 2H),
3.98 (s, 6H), 3.94 ppm (s, 3H); MS (ESI, m/z): 386 [M+H]+.
General procedure for the preparation of 6-(4-substituted
phenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehydes 6a–e: The Vilsmeier reagent
was prepared at 0–58C by dropping POCl3 (5.4 equiv) into a stirred
solution of dry DMF (6.5 equiv) in CHCl3 (10 mL). Compounds 5a–
e (1 equiv) in CHCl3 (100 mL) was added dropwise to the Vilsmeier
reagent while maintaining stirring and cooling. The reaction mix-
ture was kept for 3 h at room temperature and under reflux for
24 h. Chloroform was removed under reduced pressure, and the
resulting oil was poured into crushed ice. The mixture was extract-
ed with EtOAc (2ꢂ200 mL), and the separated organic layer was
dried over anhydrous Na2SO4. The organic layer was concentrated
under reduced pressure, and the crude product was purified by
column chromatography using EtOAc/hexane (3:7) as the eluent to
afford compounds 6a–e with high purity.
General procedure for the preparation of 6-(4-substituted
phenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazoles 5a–e: A mixture of 3 (1 equiv) and 4a–
e (1 equiv) along with a few drops of DMF was held at reflux in
ethanol (30 mL) for ~20–24 h. The solvent was evaporated, and
the remainder was dissolved in EtOAc (100 mL), washed with a sa-
turated sodium bicarbonate solution (2ꢂ100 mL), and the separat-
ed organic layer was dried over anhydrous Na2SO4. The organic
layer was concentrated under reduced pressure, and the crude
product was purified by column chromatography with EtOAc/
hexane (2:8) as the eluent to afford compounds 5a–e with high
purity.
6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (6a): Compound 6a was pre-
pared according to the general procedure, using compound 5a
(3 g, 7.8 mmol), POCl3 (3.96 mL, 42.4 mmol), and DMF (3.95 mL,
50.7 mmol) to obtain pure product 6a as a yellow solid (2.41 g,
6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
1
55% yield). H NMR (300 MHz, CDCl3): d=10.09 (s, 1H), 7.78 (d, J=
[1,3,4]thiadiazole (5a): Compound 5a was prepared according to
the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-
diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-p-tolylethanone
4a (3.19 g, 14.98 mmol) to obtain pure product 5a as pale-yellow
7.9 Hz, 2H), 7.32 (d, J=7.9 Hz, 2H), 7.16 (s, 2H), 3.98 (s, 6H), 3.94
(s, 3H), 2.44 ppm (s, 3H); MS (ESI, m/z): 410 [M+H]+.
6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (6b): Compound 6b was pre-
1
solid (4.56 g, 80% yield). H NMR (500 MHz, CDCl3): d=7.99 (s, 1H),
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pared according to the general procedure, using compound 5b
(3 g, 8.17 mmol), POCl3 (4.12 mL, 44.1 mmol), and DMF (4.12 mL,
53.1 mmol) to obtain pure product 6b as a pale-yellow solid
(2.45 g, 76% yield). 1H NMR (300 MHz, CDCl3): d=10.10 (s, 1H),
7.85–7.82 (m, 2H), 7.41–7.43 (m, 2H), 7.34–7.30 (m, 1H), 7.09 (s,
2H), 3.98 (s, 6H), 3.94 ppm (s, 3H); MS (ESI, m/z): 396 [M+H]+.
(E)-5-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (9): Compound 9
was prepared according to the method described for compound 7,
employing 6a (200 mg, 0.48 mmol) and 5-chloroindolin-2-one
(81 mg, 0.48 mmol) to obtain pure product 9 as a yellow solid
(215 mg, 79% yield); mp: 292–2948C; IR (KBr): n˜ =3432, 2935,
1699, 1628, 1482, 1463, 1411, 1316, 1249, 1129, 1100, 1002, 822,
6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (6c): Compound 6c was pre-
pared according to the general procedure, using compound 5c
(3 g, 7.55 mmol), POCl3 (3.81 mL, 40.8 mmol), and DMF (3.80 mL,
49.0 mmol) to obtain pure product 6c as a pale-yellow solid
(2.51 g, 78% yield). 1H NMR (300 MHz, CDCl3): d=10.10 (s, 1H),
7.89 (d, J=8.8 Hz, 2H), 7.41–7.43 (m, 2H), 7.15 (s, 2H), 7.03 (d, J=
8.8 Hz, 2H), 3.98 (s, 6H), 3.94 (s, 3H), 3.88 ppm (s, 3H); MS (ESI, m/
z): 426 [M+H]+.
1
749, 645 cmÀ1; H NMR (300 MHz, CDCl3): d=8.13, (bs, 1H), 7.95 (s,
1H), 7.70 (d, J=7.9 Hz, 2H), 7.21 (d, J=7.7 Hz, 2H), 6.95 (s, 2H),
6.78–6.70 (m, 2H), 6.49 (s, 1H), 3.91 (s, 3H), 3.86 (s, 6H), 3.43 (s,
3H), 2.37 ppm (s, 3H); 13C NMR (75.47 MHz, CDCl3 +[D6]TFA): d=
167.9, 155.6, 153.8, 145.1, 142.8, 141.9, 140.2, 134.9, 130.6, 127.6,
123.1, 122.8, 121.1, 118.3, 117.5, 117.2, 112.5, 111.7, 104.7, 61.3, 56.3,
56.0, 21.3 ppm; MS (ESI, m/z): 559 [M+1]+; HRMS (ESI m/z) calcd
for C29H24O4N4ClS: 559.12013, found: 559.11907 [M+1]+.
(E)-6-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (10): Compound
10 was prepared according to the method described for com-
pound 7, employing 6a (200 mg, 0.48 mmol) and 6-chloroindolin-
2-one (81 mg, 0.48 mmol) to obtain pure product 10 as a yellow
solid (217 mg, 80% yield); mp: 346–3488C; IR (KBr): n˜ =3435, 3135,
2937, 1708, 1612, 1522, 1482, 1456, 1346, 1240, 1213, 1131, 1072,
6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (6d): Compound 6d was pre-
pared according to the general procedure, using compound 5d
(3 g, 7.4 mmol), POCl3 (3.76 mL, 40.3 mmol) and DMF (3.77 mL,
48.6 mmol) to obtain pure product 6d as a pale-yellow solid
(2.43 g, 76% yield). 1H NMR (300 MHz, CDCl3): d=10.15 (s, 1H),
7.92 (d, J=8.4 Hz, 2H), 7.47 (d, J=8.4 Hz, 2H), 7.15 (s, 2H), 3.98 (s,
6H), 3.94 ppm (s, 3H); MS (ESI, m/z): 430 [M+H]+.
1000, 808, 699, 575, 529, 488 cmÀ1 1H NMR (300 MHz, CDCl3 +
;
[D6]TFA): d=7.83, (bs, 1H), 7.58, (d, J=8.1 Hz, 1H), 7.36 (s, 2H),
7.03 (s, 3H), 6.88 (d, J=7.1 Hz, 1H), 6.80 (d, J=7.7 Hz, 1H), 3.96 (s,
6H), 3.88 (s, 6H), 2.42 ppm (s, 3H); 13C NMR (75.47 MHz, CDCl3 +
[D6]TFA): d=167.8, 153.8, 142.9, 142.0, 137.4, 130.6, 127.8, 126.8,
123.0, 122.8, 118.9, 117.4, 111.7, 104.7, 61.3, 56.3, 21.3 ppm; MS
(ESI, m/z): 559 [M+1]+; HRMS (ESI m/z) calcd for C29H24O4N4ClS:
559.12013, found: 559.11971 [M+1]+.
6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazole-5-carbaldehyde (6e): Compound 6e was pre-
pared according to the general procedure, using compound 5e
(3 g, 7.7 mmol), POCl3 (3.92 mL, 42.0 mmol), and DMF (3.93 mL,
50.6 mmol) to obtain pure product 6e as a pale-yellow solid
(2.56 g, 80% yield). 1H NMR (500 MHz, CDCl3): d=10.14 (s, 1H),
8.00–7.96 (m, 2H), 7.20 (t, J=8.6 Hz, 2H), 7.14 (s, 2H), 3.98 (s, 6H),
3.94 ppm (s, 3H); MS (ESI, m/z): 414 [M+H]+.
(E)-3-((6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (11): A mixture of
compound 6a (200 mg, 0.48 mmol) and indolin-2-one (64 mg,
0.48 mmol) was dissolved in ethanol (6 mL) and piperidine (3–4
drops) was added. The reaction mixture was heated at reflux for
3 h. Progress of the reaction was monitored by TLC, and the pre-
cipitate formed on cooling was collected by filtration, washed
twice a small amount of ethanol to obtain the pure product 11 as
a yellow solid (207 mg, 80% yield); mp: 294–2968C; IR (KBr): n˜ =
3427, 3169, 2936, 2832, 2358, 1697, 1616, 1488, 1465, 1368, 1348,
(E)-5-Fluoro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (7): Compound 7
was prepared according to the method described for compound 7,
employing 6a (200 mg, 0.48 mmol) and 5-fluoroindolin-2-one
(73 mg, 0.48 mmol) to obtain pure product 7 as a yellow solid
(206 mg, 78% yield); mp: 308–3108C; IR (KBr): n˜ =3427, 3173,
2936, 1709, 1613, 1520, 1474, 1432, 1414, 1362, 1337, 1292, 1241,
1
1131, 1069, 1000, 802, 771, 759, 732, 652, 620, 589 cmÀ1; H NMR
1315, 1291, 1241, 1002, 844, 820, 779, 742, 733, 623, 524, 489 cmÀ1
;
(300 MHz, CDCl3 +[D6]TFA): d=7.89, (bs, 1H), 7.57 (s, 2H), 7.35 (s
2H), 7.10 (s, 2H), 7.10–6.92 (m, 2H), 6.59 (d, J=7.5 Hz, 1H), 7.36 (s,
2H), 3.97 (s, 3H), 3.90 (s, 6H), 2.40 ppm (s, 3H); 13C NMR
(75.47 MHz, CDCl3 +[D6]TFA): d=168.1, 157.9, 153.7, 145.2, 143.2,
141.7, 140.7, 137.0, 127.7, 122.9, 118.5, 118.0, 117.8, 113.5, 111.9,
104.8, 61.3, 56.3, 21.5 ppm; MS (ESI, m/z): 543 [M+1]+; HRMS (ESI
m/z) calcd for C29H24O4N4FS: 543.14968, found: 543.14855 [M+1]+.
1H NMR (300 MHz, CDCl3 +[D6]TFA): d=9.40 (bs, 1H), 7.80 (s, 1H),
7.68–7.50 (m, 1H), 7.40–7.20 (m, 4H), 7.00 (s, 2H), 6.98 (s, 1H),
6.91–6.79 (m, 2H), 3.93 (s, 3H), 3.84 (s, 6H), 2.40 ppm (s, 3H);
13C NMR (75 MHz, CDCl3 +[D6]TFA): d=170.7, 165.5, 153.8, 141.9,
141.2, 140.8, 130.3, 130.1, 127.8, 125.9, 123.5, 122.2, 121.0, 110.4,
104.3, 61.0, 56.3, 21.3 ppm; MS (ESI, m/z): 525 [M+1]+; HRMS (ESI
m/z) calcd for C29H25O4N4S: 525.15910, found: 525.15948 [M+1]+.
(E)-5-Methoxy-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (8): Com-
pound 8 was prepared according to the method described for
compound 7, employing 6a (200 mg, 0.48 mmol) and 5-methox-
yindolin-2-one (79 mg, 0.48 mmol) to obtain pure product 8 as
a red solid (210 mg, 78% yield); mp: 286–2888C; IR (KBr): n˜ =3432,
2935, 1699, 1628, 1482, 1463, 1411, 1316, 1249, 1129, 1100, 1002,
(E)-3-((6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-
[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (12): Compound
12 was prepared according to the method described for com-
pound 7, employing 6b (200 mg, 0.5 mmol) and indolin-2-one
(67 mg, 0.5 mmol) to obtain pure product 12 as a yellow solid
(206 mg, 80% yield); mp: 292–2948C; IR (KBr): n˜ =3431, 3017,
3077, 3017, 2938, 2832, 1709, 1611, 1585, 1522, 1486, 1464, 1431,
822, 749, 645 cmÀ1 1H NMR (300 MHz, CDCl3): d=8.13, (bs, 1H),
;
1
7.95 (s, 1H), 7.70 (d, J=7.9 Hz, 2H), 7.21 (d, J=7.7 Hz, 2H), 6.95 (s,
2H), 6.78–6.70 (m,2H), 6.49 (s, 1H), 3.91 (s, 3H), 3.86 (s, 6H), 3.43
(s, 3H), 2.37 (s, 3H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=167.9,
155.6, 153.8, 145.1, 142.8, 141.9, 140.2, 134.9, 130.6, 127.6, 123.1,
122.8, 121.1, 118.3, 117.5, 117.2, 112.5, 111.7, 104.7, 61.3, 56.3, 56.0,
21.3 ppm; MS (ESI, m/z): 555 [M+1]+; HRMS (ESI m/z) calcd for
C30H27O5N4S: 555.16967, found: 555.16907 [M+1]+.
1414, 1349, 1297, 1238, 1129, 997, 779, 734, 692, 625 cmÀ1; H NMR
(300 MHz, CDCl3): d=8.80, (bs, 1H), 7.94 (s, 1H), 7.83 (d, J=7.1 Hz,
2H), 7.52–7.33 (m, 3H), 7.15 (t, J=7.5, 7.3 Hz, 1H), 6.94–6.85 (m,
4H), 6.79 (t, J=7.5 Hz, 1H), 3.90 (s, 3H), 3.82 ppm (s, 6H); 13C NMR
(75.47 MHz, CDCl3 +[D6]TFA): d=168.1, 157.9, 153.7, 145.3, 143.2,
141.7, 140.7, 137.0, 130.7, 127.7, 122.9, 121.3, 118.5, 118.0, 117.8,
113.5, 113.3, 111.9, 104.8, 61.3, 56.3, 21.2 ppm; MS (ESI, m/z): 511
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[M+1]+; HRMS (ESI, m/z) calcd for C28H23O4N4S: 511.14345, found:
511.14215 [M+1]+.
8.3 Hz, 1H), 4.01 (s, 6H), 3.92 ppm (s, 3H); 13C NMR (75 MHz,
CDCl3 +[D6]TFA): d=168.6, 168.2, 153.8, 142.0, 137.0, 132.3, 131.9,
130.0, 129.8, 129.3, 127.8, 123.2, 122.8, 118.4, 118.1, 117.7, 113.5,
111.8, 104.9, 104.8, 61.3, 56.5, 56.3 ppm; MS (ESI, m/z): 529 [M+1]+
; HRMS (ESI, m/z): calcd for C28H22O4N4FS: 529.13403, found:
529.13312 [M+1]+.
(E)-5-Methoxy-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (13): Com-
pound 13 was prepared according to the method described for
compound 7, employing 6b (200 mg, 0.5 mmol) and 5-methoxyin-
dolin-2-one (82 mg, 0.5 mmol) to obtain pure product 13 as
a pale-red solid (229 mg, 84% yield); mp: 289–2918C; IR (KBr): n˜ =
3418, 3159, 2998, 2937, 2833, 1710, 1628, 1583, 1515, 1483, 1460,
1358, 1327, 1250, 1197, 1128, 1107, 1093, 1070, 1035, 1000, 845,
(E)-3-((6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (17): Com-
pound 17 was prepared according to the method described for
compound 7, employing 6c (200 mg, 0.47 mmol) and indolin-2-
one (62 mg, 0.47 mmol) to obtain pure product 17 as a yellow
solid (215 mg, 85% yield); mp: 304–3068C; IR (KBr): n˜ =3419, 3181,
2936, 2832, 1711, 1613, 1588, 1519, 1504, 1487, 1465, 1414, 1432,
1368, 1346, 1302, 1252, 1213, 1175, 1131, 1095, 1032, 1004, 829,
1
805, 787, 703, 651, 610 cmÀ1; H NMR (300 MHz, CDCl3): d=8.39 (s,
1H), 7.98 (s, 1H), 7.82 (d, J=7.3 Hz, 2H), 7.41 (t, J=7.3, 7.6 Hz, 2H),
7.35 (t, J=7.3 Hz, 1H), 6.96 (s, 2H), 6.77 (d, J=8.3 Hz, 1H), 6.73 (dd,
J=8.5, 2.4 Hz, 1H), 6.47 (d, J=2.2 Hz, 1H), 3.91 (s, 3H), 3.87 (s, 6H),
3.42 ppm (s, 3H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=154.9,
147.7, 147.4, 128.5, 126.0, 125.2, 122.4, 111.5, 109.9, 56.0, 55.8, 32.7,
28.4 ppm; MS (ESI, m/z): 541 [M+1]+; HRMS (ESI, m/z): calcd for
C29H25O5N4S: 541.15402, found: 541.15342 [M+1]+.
1
807, 778, 757, 749, 735, 640, 622, 590, 524 cmÀ1; H NMR (300 MHz,
CDCl3 +[D6]TFA): d=7.78 (s, 1H), 7.62 (d, J=8.6 Hz, 2H), 7.28 (d,
J=7.5 Hz, 1H), 7.04–6.98 (m, 5H), 6.92–6.82 (m, 2H), 3.93 (s, 3H),
3.84 ppm (s, 6H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=171.1,
167.1, 162.3, 153.7, 144.7, 141.6, 141.0, 139.8, 131.5, 129.5, 125.9,
123.2, 122.9, 120.2, 118.1, 116.9, 115.4, 111.3, 104.8, 61.3, 56.3,
55.5 ppm; MS (ESI, m/z): 541 [M+1]+; HRMS (ESI, m/z): calcd for
C29H25O5N4S: 541.15402, found: 541.15329 [M+1]+.
(E)-5-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (14): Compound
14 was prepared according to the method described for com-
pound 7, employing 6b (200 mg, 0.5 mmol) and 5-chloroindolin-2-
one (84 mg, 0.5 mmol) to obtain pure product 14 as a yellow solid
(223 mg, 81% yield); mp: 303–3058C; IR (KBr): n˜ =3418, 3078,
2999, 2938, 2834, 1714, 1623, 1605, 1581, 1514, 1481, 1463, 1410,
1359, 1329, 1282, 1234, 1152, 1128, 1107, 1002, 927, 855, 845, 802,
(E)-5-Methoxy-3-((6-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (18): Compound 18 was prepared according to
the method described for compound 7, employing 6c (200 mg,
0.47 mmol) and 5-methoxyindolin-2-one (76 mg, 0.47 mmol) to
obtain pure product 18 as a yellow solid (214 mg, 80% yield); mp:
292–2948C; IR (KBr): n˜ =3428, 2935, 2836, 1701, 1633, 1607, 1586,
1485, 1464, 1414, 1358, 1302, 1251, 1205, 1175, 1130, 1069, 1035,
1
780, 717, 703, 593, 581, 550, 471 cmÀ1; H NMR (300 MHz, CDCl3):
d=9.45 (s, 1H), 7.93 (s,1H), 7.72–7.61 (m, 2H), 7.57–7.51 (m, 3H),
7.21 (d, J=8.1 Hz, 1H), 7.11 (s, 2H), 6.90 (d, J=8.3 Hz, 1H), 6.78 (s,
1H), 3.96 (s, 3H), 3.91 ppm (s, 6H); 13C NMR (75 MHz, CDCl3 +
[D6]TFA): d=170.8, 168.2, 153.8, 145.7, 142.0, 140.9, 139.3, 131.8,
130.9, 128.5, 127.8, 126.4, 126.0, 122.8, 121.4, 118.7, 112.0, 104.8,
61.3, 56.3 ppm; MS (ESI, m/z): 545 [M+1]+; HRMS (ESI, m/z): calcd
for C28H22O4N4ClS: 545.10448, found: 545.10397 [M+1]+.
1
835, 819, 780, 755, 734, 646, 588 cmÀ1; H NMR (300 MHz, CDCl3 +
[D6]TFA): d=7.82 (s, 1H), 7.61 (d, J=8.4 Hz, 2H), 7.10 (s, 2H), 7.05–
6.94 (m, 3H), 6.88 (dd, J=8.4, 2.0 Hz, 1H), 6.49 (s, 1H), 3.99 (s, 3H),
3.90 (d, J=7.1 Hz, 9H), 3.58 ppm (s, 3H); 13C NMR (75 MHz, CDCl3 +
[D6]TFA): d=171.1, 167.7, 155.5, 153.7, 144.8, 141.6, 139.8, 135.1,
129.4, 123.0, 121.1, 118.1, 117.9, 117.7, 117.1, 115.5, 112.9, 111.9,
104.8, 61.4, 56.3, 56.2, 55.5 ppm; MS (ESI, m/z): 571 [M+1]+; HRMS
(ESI, m/z): calcd for C30H27O6N4S: 571.16458, found: 571.16467 [M+
1]+.
(E)-6-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (15): Compound
15 was prepared according to the method described for com-
pound 7, employing 6b (200 mg, 0.5 mmol) and 6-chloroindolin-2-
one (84 mg, 0.5 mmol) to obtain pure product 15 as a yellow solid
(223 mg, 81% yield); mp: 322–3248C; IR (KBr): n˜ =3428, 3127,
2939, 2836, 1711, 1658, 1632, 1585, 1481, 1443, 1456, 1432, 1414,
1298, 1279, 1171, 1155, 1128, 1105, 1072, 996, 918, 840, 804, 761,
(E)-5-Chloro-3-((6-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (19): Compound 19 was prepared according to
the method described for compound 7, employing 6c (200 mg,
0.47 mmol) and 5-chloroindolin-2-one (78 mg, 0.47 mmol) to
obtain pure product 19 as a yellow solid (205 mg, 76% yield); mp:
297–2998C; IR (KBr): n˜ =3418, 3165, 2939, 2836, 1701, 1625, 1598,
1482, 1465, 1414, 1364, 1336, 1252, 1305, 1252, 1222, 1174, 1149,
1129, 1106, 1066, 1034, 1005, 915, 837, 805, 730, 704, 651, 586,
1
733, 695, 669, 652, 626, 599, 534 cmÀ1; H NMR (300 MHz, CDCl3 +
[D6]TFA): d=7.82 (s, 1H), 7.75–7.66 (m, 3H), 7.56 (s, 2H), 7.29 (s,
1H), 7.09 (d, J=8.8 Hz, 2H), 6.87 (d, J=7.9 Hz, 1H), 6.76 (d, J=
8.3 Hz,1H), 3.96 (s, 3H), 3.91 ppm (s, 6H); 13C NMR (75 MHz,
CDCl3 +[D6]TFA): d=171.0, 167.8, 153.8, 142.1, 137.4, 132.0, 131.7,
129.9, 129.7, 129.3, 127.8, 126.6, 123.3, 122.9, 122.8, 118.7, 117.3,
111.7, 104.8, 104.7, 61.2, 56.4, 56.3 ppm; MS (ESI, m/z): 545 [M+1]+
; HRMS (ESI, m/z): calcd for C28H22O4N4ClS: 545.10448, found:
545.10416 [M+1]+.
1
545, 525 cmÀ1; H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.89 (s, 1H),
7.56 (d, J=7.9 Hz, 2H), 7.24 (s, 1H), 7.12 (s, 2H), 7.04 (d, J=7.7 Hz,
2H), 6.95 (d, J=8.3 Hz, 1H), 6.80 (s, 1H), 3.98 (s, 3H), 3.92 (s, 6H),
3.87 ppm (s, 3H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=168.0,
162.5, 153.8, 141.7, 139.3, 131.1, 129.5, 128.7, 126.3, 122.9, 121.5,
118.4, 118.2, 115.7, 112.2, 104.9, 61.4, 56.3, 55.5 ppm; MS (ESI, m/z):
575 [M+1]+; HRMS (ESI, m/z): calcd for C29H24O5N4ClS: 575.11504,
found: 575.11527 [M+1]+.
(E)-5-Fluoro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (16): Compound
16 was prepared according to the method described for com-
pound 7, employing 6b (200 mg, 0.5 mmol) and 5-fluoroindolin-2-
one (76 mg, 0.5 mmol) to obtain pure product 16 as a yellow solid
(218 mg, 82% yield); mp: 276–2788C; IR (KBr): n˜ =3417, 3174,
2938, 2835, 1710, 1658, 1639, 1620, 1566, 1519, 1443, 1475, 1415,
1393, 1338, 1239, 1180, 1128, 997, 840, 733, 708, 694, 651,
(E)-6-Chloro-3-((6-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (20): Compound 20 was prepared according to
the method described for compound 7, employing 6c (200 mg,
0.47 mmol) and 6-chloroindolin-2-one (78 mg, 0.47 mmol) to
632 cmÀ1 1H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.90 (s, 1H),
;
7.68–7.54 (m, 5H), 7.29 (s, 1H), 7.13 (s, 2H), 6.99 (s, 1H), 6.59 (d, J=
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obtain pure product 19 as a yellow solid (205 mg, 76% yield); mp:
352–3548C; IR (KBr): n˜ =3408, 3120, 2940, 2836, 1711, 1611, 1487,
1461, 1416, 1303, 1283, 1256, 1216, 1175, 1154, 1133, 1105, 1072,
(24): Compound 24 was prepared according to the method de-
scribed for compound 7, employing 6d (200 mg, 0.46 mmol) and
5-methoxyindolin-2-one (75 mg, 0.46 mmol) to obtain pure prod-
uct 24 as red solid (217 mg, 81% yield); mp: 284–2868C; IR (KBr):
n˜ =3418, 3164, 2941, 2830, 1700, 1631, 1519, 1483, 1464, 1438,
1408, 1359, 1289, 1255, 1171, 1128, 1091, 1036, 1012, 921, 893,
1
995, 918, 828, 804, 728, 706, 650, 620, 599, 523, 504 cmÀ1; H NMR
(300 MHz, CDCl3 +[D6]TFA): d=7.78 (s, 1H), 7.61 (d, J=8.5 Hz, 2H),
7.06–7.04 (m, 5H), 6.91 (d, J=8.2 Hz, 1H), 6.81 (d, J=8.2 Hz,1H),
3.97 (s, 3H), 3.88 ppm (s, 9H); 13C NMR (75 MHz, CDCl3 +[D6]TFA):
d=171.2, 167.6, 162.4, 153.8, 145.1, 142.0, 137.3, 129.6, 126.8,
122.9, 122.7, 118.9, 118.3, 117.5, 115.5, 111.7, 104.7, 61.2, 56.3,
55.5 ppm; MS (ESI, m/z): 575 [M+1]+; HRMS (ESI, m/z): calcd for
C29H24O5N4ClS: 575.11504, found: 575.11586 [M+1]+.
832, 788, 746, 634, 623, 565 cmÀ1 1H NMR (300 MHz, CDCl3 +
;
[D6]TFA): d=7.80 (s, 1H), 7.59 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.4 Hz,
2H), 7.11 (s, 2H), 6.98 (d, J=8.4 Hz, 1H), 6.87 (dd, J=8.6, 2.2 Hz,
1H), 6.44 (d, J=2.0 Hz, 1H), 3.99 (s, 3H), 3.90 (s, 6H), 3.58 ppm (s,
3H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=170.9, 167.3, 155.6,
153.8, 146.1, 142.1, 140.4, 137.6, 134.8, 131.0, 130.0, 128.9, 126.0,
122.9, 121.1, 118.9, 117.6, 116.7, 112.9, 112.4, 111.5, 104.6, 61.1, 56.3,
55.8 ppm; MS (ESI, m/z): 575 [M+1]+; HRMS (ESI, m/z): calcd for
C29H24O5N4ClS: 575.11502, found: 575.11529 [M+1]+.
(E)-5-Fluoro-3-((6-(4-methoxyphenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (21): Compound 21 was prepared according to
the method described for compound 7, employing 6c (200 mg,
0.47 mmol) and 5-fluoroindolin-2-one (70 mg, 0.47 mmol) to obtain
pure product 21 as a yellow solid (204 mg, 78% yield); mp: 297–
2998C; IR (KBr): n˜ =3408, 2937, 2835, 1713, 1608, 1585, 1519, 1475,
1414, 1361, 1249, 1175, 1127, 1005, 861, 840, 757, 735, 705, 590,
(E)-5-Chloro-3-((6-(4-chlorophenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (25): Compound 25 was prepared according to
the method described for compound 7, employing 6d (200 mg,
0.46 mmol) and 5-chloroindolin-2-one (77 mg, 0.46 mmol) to
obtain pure product 25 as a yellow solid (210 mg, 78% yield); mp:
286–2888C; IR (KBr): n˜ =3427, 3160, 2939, 2835, 1706, 1629, 1606,
1585, 1514, 1480, 1464, 1431, 1414, 1340, 1297, 1238, 1171, 1128,
1
565, 547 cmÀ1; H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.87 (s, 1H),
7.59 (d, J=7.9 Hz, 2H), 7.10 (s, 2H), 7.06–6.92 (m, 4H), 6.60 (d, J=
7.4 Hz, 1H), 3.97 (s, 3H), 3.88 ppm (s, 9H); 13C NMR (75 MHz,
CDCl3 +[D6]TFA): d=171.2, 167.6, 162.4, 153.8, 145.1, 142.0, 137.3,
129.6, 126.8, 122.9, 122.7, 118.9, 118.3, 117.5, 115.5, 111.7, 104.7,
61.2, 56.3, 55.5 ppm; MS (ESI, m/z): 559 [M+1]+; HRMS (ESI, m/z):
calcd for C29H24O5N4FS: 559.14460, found: 559.14456 [M+1]+.
1090, 1066, 1002, 914, 895, 835, 759, 719, 647, 588, 577, 552 cmÀ1
;
1H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.86 (s, 1H), 7.60 (d, J=
8.4 Hz, 2H), 7.49 (d, J=8.1 Hz, 2H), 7.29 (d, J=8.4 Hz, 1H), 7.13 (s,
2H), 7.00 (d, J=8.3 Hz, 1H), 6.87 (s,1H), 3.99 (s, 3H), 3.92 ppm (s,
6H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d=170.7, 168.1, 153.8,
141.9, 139.4, 138.3, 131.2, 130.3, 129.0, 128.6, 126.0, 122.9, 121.4,
118.2, 112.1, 104.8, 61.3, 56.3 ppm; MS (ESI, m/z): 579 [M+1]+;
HRMS (ESI, m/z): calcd for C28H21O4N4 Cl2S: 579.06551, found:
579.06610 [M+1]+.
(E)-3-((6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-nitroindolin-2-one (22):
Compound 22 was prepared according to the method described
for compound 7, employing 6c (200 mg, 0.47 mmol) and 5-nitroin-
dolin-2-one (83 mg, 0.47 mmol) to obtain pure product 22 as
a yellow solid (211 mg, 77% yield); mp: 298–3008C; IR (KBr): n˜ =
3412, 3088, 2937, 2837, 1716, 1607, 1519, 1485, 1465, 1412, 1368,
1335, 1313, 1254, 1193, 1149, 1128, 1107, 1074, 1003, 904, 843, 777,
(E)-6-Chloro-3-((6-(4-chlorophenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (26): Compound 26 was prepared according to
the method described for compound 7, employing 6d (200 mg,
0.46 mmol) and 6-chloroindolin-2-one (77 mg, 0.46 mmol) to
obtain pure product 26 as a yellow solid (210 mg, 78% yield); mp:
336–3388C; IR (KBr): n˜ =3428, 3138, 2939, 2836, 1713, 1613, 1585,
1519, 1479, 1459, 1431, 1414, 1346, 1307, 1284, 1239, 1214, 1172,
1154, 1129, 1071, 994, 920, 829, 761, 739, 721, 671, 642, 599,
750, 738, 708, 686, 650, 599, 584, 547 cmÀ1 1H NMR (300 MHz,
;
CDCl3 +[D6]TFA): d=8.20 (dd, J=8.6, 2.0 Hz, 1H), 8.09 (s, 1), 7.67
(d, J=1.8 Hz, 1H), 7.59 (d, J=8.6 Hz, 2H), 7.16 (s, 1H), 7.13 (s, 2H),
6.94 (d, J=8.4 Hz, 2H), 3.98 (s, 3H), 3.92 (s, 6H), 3.82 ppm (s, 3H);
13C NMR (75 MHz, CDCl3 +[D6]TFA): d=171.1, 168.3, 162.4, 153.8,
145.8, 143.2, 142.0, 129.5, 127.0, 123.1, 122.8, 121.3, 120.5, 118.4,
117.7, 115.4, 110.8, 104.8, 61.3, 56.4, 55.5 ppm; MS (ESI, m/z): 586
[M+1]+; HRMS (ESI, m/z): calcd for C29H24O7N5S: 586.13910, found:
586.13965 [M+1]+.
547 cmÀ1 1H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.55 (s, 1H),
;
7.61–7.52 (m, 4H), 7.13 (s, 1H), 7.05 (s, 2H), 6.92 (d, J=7.5 Hz, 1H),
6.79 (d, J=8.1 Hz, 1H), 4.00 (s, 3H), 3.90 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3 +[D6]TFA): d=171.0, 168.0, 153.8, 145.6, 142.2,
141.8, 138.6, 137.9, 130.3, 129.1, 126.7, 124.4, 123.2, 122.9, 119.2,
118.6, 116.7, 112.1, 104.8, 61.4, 56.3 ppm; MS (ESI, m/z): 579 [M+
1]+; HRMS (ESI, m/z): calcd for C28H21O4N4 Cl2S: 579.06551, found:
579.06673 [M+1]+.
(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (23): Com-
pound 23 was prepared according to the method described for
compound 7, employing 6d (200 mg, 0.46 mmol) and indolin-2-
one (62 mg, 0.46 mmol) to obtain pure product 23 as a yellow
solid (203 mg, 80% yield); mp: 318–3208C; IR (KBr): n˜ =3152, 3077,
2937, 2833, 1709, 1612, 1586, 1520, 1483, 1464, 1432, 1414, 1349,
1307, 1286, 1238, 1173, 1130, 1093, 1071, 996, 829, 779, 758, 737,
(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-fluoroindolin-2-one
(27): Compound 27 was prepared according to the method de-
scribed for compound 7, employing 6d (200 mg, 0.46 mmol) and
5-fluoroindolin-2-one (70 mg, 0.46 mmol) to obtain pure product
27 as a yellow solid (209 mg, 80% yield); mp: 315–3178C; IR (KBr):
n˜ =3433, 3174, 3077, 2939, 2836, 1709, 1637, 1619, 1587, 1519,
1475, 1432, 1414, 1363, 1339, 1304, 1286, 1239, 1182, 1129, 1093,
1
718, 635, 621 cmÀ1; H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.76 (s,
1H), 7.59 (d, J=8.3 Hz, 2H), 7.49 (d, J=8.3 Hz, 2H), 7.34 (t, J=
7.5 Hz, 1H), 7.06 (s, 3H), 6.92 (t, J=7.5 Hz, 1H), 6.81 (d, J=7.7 Hz,
1H), 3.98 (s, 3H), 3.87 ppm (s, 6H); 13C NMR (75 MHz, CDCl3 +
[D6]TFA): d=171.0, 167.9, 153.7, 145.3, 141.8, 141.2, 138.4, 131.9,
130.2, 129.0, 125.8, 124.5, 123.2, 122.9, 120.1, 116.3, 111.4, 104.8,
61.3, 56.3 ppm; MS (ESI, m/z): 545 [M+1]+; HRMS (ESI, m/z): calcd
for C28H22O4N4ClS: 545.10448, found: 545.10440 [M+1]+.
1
1070, 1000, 830, 763, 713 cmÀ1; H NMR (300 MHz, CDCl3 +[D6]TFA):
d=7.83 (s, 1H), 7.64–7.48 (m, 4H), 7.11 (s, 2H), 7.03 (d, J=4.9 Hz,
2H), 6.59 (d, J=8.3 Hz, 1H), 4.00 (s, 3H), 3.91 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3 +[D6]TFA): d=170.9, 168.3, 157.3, 153.8, 145.7,
141.8, 139.3, 138.7, 137.2, 130.3, 129.0, 124.4, 122.9, 121.0, 118.9,
(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-methoxyindolin-2-one
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118.5, 118.1, 117.8, 113.4, 113.0, 112.2, 112.1, 104.9, 61.4, 56.3 ppm;
MS (ESI, m/z): 563 [M+1]+; HRMS (ESI, m/z): calcd for C28H21O4N4 Cl
FS: 563.09506, found: 563.09582 [M+1]+.
1413, 1432, 1363, 1339, 1173, 1157, 1128, 1090, 1070, 1000, 924,
1
903, 863, 837, 806, 761, 735, 655, 617, 587 cmÀ1; H NMR (300 MHz,
CDCl3 +[D6]TFA): d=7.85 (s, 1H), 7.65–7.59 (m, 2H), 7.20 (d, J=
7.9 Hz, 2H), 7.12 (s, 2H), 7.04–6.98 (m, 2H), 6.58 (d, J=8.4 Hz, 1H),
4.00 (s, 3H), 3.92 ppm (s, 6H); 13C NMR (75 MHz, CDCl3 +[D6]TFA):
d=171.0, 167.7, 163.3, 157.8, 153.8, 142.0, 137.1, 130.0, 122.9,
121.2, 118.8, 117.8, 117.6, 117.3, 117.1, 113.3, 113.1, 111.7, 104.6,
61.2, 56.3 ppm; MS (ESI, m/z): 547 [M+1]+; HRMS (ESI, m/z): calcd
for C28H21O4N4F2S: 547.12461, found: 547.12525 [M+1]+.
(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-
[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-nitroindolin-2-one (28):
Compound 28 was prepared according to the method described
for compound 7, employing 6d (200 mg, 0.46 mmol) and 5-nitroin-
dolin-2-one (82 mg, 0.46 mmol) to obtain pure product 28 as
a yellow solid (209 mg, 76% yield); mp: 287–2898C; IR (KBr): n˜ =
3418, 3129, 3088, 2939, 2837, 1708, 1627, 1604, 1522, 1481, 1465,
1431, 1414, 1338, 1242, 1224, 1193, 1146, 1130, 1091, 1075, 1000,
(E)-6-Chloro-3-((6-(4-fluorophenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (32): Compound 32 was prepared according to
the method described for compound 7, employing 6e (200 mg,
0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtain
pure product 32 as a yellow solid (218 mg, 80% yield); mp: 338–
3408C; IR (KBr): n˜ =3409, 3139, 2944, 2839, 1712, 1633, 1613, 1503,
1484, 1459, 1431, 1414, 1345, 1307, 1230, 1155, 1128, 1103, 1072,
833, 749, 735, 649 cmÀ1 1H NMR (300 MHz, CDCl3 +[D6]TFA): d=
;
8.23 (dd, J=8.6, 1.8 Hz, 1H), 8.07 (s, 1H), 7.70 (d, J=1.7 Hz, 1H),
7.58 (d, J=8.3 Hz, 2H), 7.44 (d, J=8.1 Hz, 2H), 7.17 (d, J=8.8 Hz,
1H), 7.12 (s, 2H), 3.99 (s, 3H), 3.91 ppm (s, 6H); 13C NMR (75 MHz,
CDCl3 +[D6]TFA): d=170.9, 168.4, 153.7, 146.1, 143.1, 141.8, 138.5,
130.3, 129.0, 127.7, 127.4 125.1, 122.9, 121.1, 120.3, 117.4, 111.4,
111.1, 104.8, 61.3, 56.3 ppm; MS (ESI, m/z): 590 [M+1]+; HRMS (ESI,
m/z): calcd for C28H21O6N5ClS: 590.08956, found: 590.09038 [M+1]+
.
994, 903, 842, 803, 725, 705, 694, 599, 576, 522 cmÀ1 1H NMR
;
(300 MHz, CDCl3 +[D6]TFA): d=7.76 (s, 1H), 7.66–7.61 (m, 2H),
7.28–7.23 (m, 2H), 7.14 (s, 1H), 7.06 (s, 2H), 6.92 (d, J=8.3 Hz, 1H),
6.83 (d, J=8.3 Hz, 1 h), 4.01 (s, 3H), 3.91 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3 +[D6]TFA): d=171.2, 168.0, 166.5, 163.1, 153.8,
142.2, 141.7, 138.0, 130.3, 130.1, 126.7, 123.3, 122.9, 118.6, 117.6,
117.3, 116.7, 112.1, 104.9, 61.4, 56.3 ppm; MS (ESI, m/z): 563 [M+
1]+; HRMS (ESI, m/z): calcd for C28H21O4N4ClFS: 563.09506, found:
563.09601 [M+1]+.
(E)-5-Chloro-3-((6-(4-fluorophenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (29): Compound 29 was prepared according to
the method described for compound 7, employing 6e (200 mg,
0.48 mmol) and 5-chloroindolin-2-one (80 mg, 0.46 mmol) to
obtain pure product 29 as a yellow solid (218 mg, 80% yield); mp:
322–3248C; IR (KBr): n˜ =3407, 3139, 3005, 2941, 2837, 1714, 1624,
1605, 1536, 1500, 1483, 1458, 1432, 1357, 1234, 1221, 1206, 1175,
1156, 1128, 1107, 1064, 999, 907, 871, 802, 754, 728, 636, 622, 583,
Biology
1
572, 538 cmÀ1; H NMR (300 MHz, CDCl3 +[D6]TFA): d=7.88 (s, 1H),
Cell culture, maintenance, and anti-proliferative evaluation: All cell
lines used in this study were purchased from the American Type
Culture Collection (ATCC, USA). A549, MCF-7, HCT116 and HeLa
cells were grown in Dulbecco’s modified Eagle’s medium (DMEM,
containing 10% fetal bovine serum (FBS) in a humidified atmos-
phere of 5% CO2 at 378C). Cells were trypsinized when sub-conflu-
ent from T25 flasks/60 mm dishes and seeded in 96-well plates.
The test compounds were evaluated for their in vitro anti-prolifera-
tive activity against four different human cancer cell lines, using
a protocol of 48 h continuous drug exposure; a sulforhodamine B
cell proliferation assay was used to estimate cell viability or
growth. The cell lines were grown in their respective media con-
taining 10% FBS and were seeded into 96-well microtiter plates in
200 mL aliquots at plating densities according to the doubling time
of the given cell line. The microtiter plates were incubated at 378C,
5% CO2, 95% air, and 100% relative humidity for 24 h prior to the
addition of test compounds. Aliquots (2 mL) of the test compounds
were added to the wells already containing cells in 198 mL, result-
ing in the required final drug concentrations. For each compound,
five concentrations (0.01, 0.1, 1, 10, and 100 mm) were evaluated,
and each was done in triplicate wells. Plates were incubated fur-
ther for 48 h, and the assay was terminated by the addition of
10% TCA and incubated for 60 min at 48C. Later, the plates were
air-dried and washed thrice with water. The cells were then incu-
bated with 0.57% sulforhodamine B dye for 1 h at 378C, and
plates were washed thrice with 1% acetic acid. The air-dried plates
were reconstituted with 50 mL 10 mm Tris buffer (pH 8.0), and the
absorbance was read at 510 nm in EnSpire, multimode reader.
Before the start of treatments, a plate was terminated at 24 h,
which served as time zero (t0). The plates were then terminated
after 48 h, containing DMSO-treated control (tc) and five-dose-treat-
ed cells (ti). The IC50 values were calculated by linear interpolation
from plots of percent growth versus log concentrations of com-
7.65–7.64 (m, 2H), 7.29–7.20 (m, 3H), 7.14 (s, 2H), 6.96 (d, J=
8.4 Hz, 1H), 6.79 (s, 1H), 3.99 (s, 3H), 3.93 ppm (s, 6H); 13C NMR
(75 MHz, CDCl3 +[D6]TFA): d=170.7, 167.9, 165.4, 163.4, 153.8,
142.0, 139.4, 130.9, 130.0, 128.4, 126.0, 122.9, 121.4, 118.5, 117.3,
117.2, 111.9, 104.7, 61.2, 56.3 ppm; MS (ESI, m/z): 563 [M+1]+;
HRMS (ESI, m/z): calcd for C28H21O4N4ClFS: 563.09506, found:
563.09539 [M+1]+.
(E)-3-((6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-
b][1,3,4]thiadiazol-5-yl)methylene)-5-methoxyindolin-2-one (30):
Compound 30 was prepared according to the method described
for compound 7, employing 6e (200 mg, 0.48 mmol) and 5-me-
thoxyindolin-2-one (78 mg, 0.46 mmol) to obtain pure product 30
as a yellow solid (218 mg, 81% yield); mp: 288–2908C; IR (KBr): n˜ =
3400, 3158, 2943, 2834, 1711, 1631, 1596, 1503, 1484, 1461, 1433,
1357, 1299, 1217, 1197, 1169, 1129, 1107, 1092, 1067, 1035, 998,
1
841, 806, 756, 721, 706, 694, 641 cmÀ1; H NMR (300 MHz, CDCl3 +
[D6]TFA): d=7.81 (s, 1H), 7.70–7.66 (m, 2H), 7.21 (t, J=8.3 Hz, 2H),
7.11 (s, 2H), 6.93 (d, J=8.4 Hz, 1H), 6.85 (dd, J=8.6, 2.2 Hz, 1H),
6.41 (d, J=2.0 Hz, 1H), 3.98 (s, 3H), 3.90 (s, 6H), 3.56 ppm (s, 3H);
13C NMR (75 MHz, CDCl3 +[D6]TFA): d=170.9, 167.6, 166.0, 162.6,
155.6, 153.8, 145.7, 142.0, 134.9, 134.8, 129.9, 123.2, 122.8, 121.0,
117.3, 117.0, 116.7, 112.9, 112.4, 111.9, 111.6, 104.6, 61.3, 56.3, 56.2,
55.8 ppm; MS (ESI, m/z): 559 [M+1]+; HRMS (ESI, m/z): calcd for
C29H24O5N4FS: 559.14460, found: 559.14501 [M+1]+.
(E)-5-Fluoro-3-((6-(4-fluorophenyl)-2-(3,4,5-
trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-
ne)indolin-2-one (31): Compound 31 was prepared according to
the method described for compound 7, employing 6e (200 mg,
0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtain
pure product 31 as a yellow solid (216 mg, 82% yield); mp: 330–
3328C; IR (KBr): n˜ =3429, 3177, 2940, 2846, 1710, 1619, 1524, 1475,
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pounds in mm. IC50 values are indicated as means ÆSD of three in-
Immunohistochemistry and nuclear morphology analysis: A549 cells
were seeded on a glass cover slip, incubated for 24 h in the pres-
ence or absence of test compounds 7, 11, and 15 at 5 mm. Cells
grown on cover slips were fixed in 3.5% formaldehyde in PBS
pH 7.4 for 10 min at room temperature. Cells were permeabilized
for 6 min in PBS containing 0.5% Triton X-100 (Sigma) and 0.05%
Tween-20 (Sigma). The permeabilized cells were blocked with 2%
BSA (Sigma) in PBS for 1 h. Later, the cells were incubated with pri-
mary antibody for tubulin (Sigma) at 1:200 diluted in blocking solu-
tion for 4 h at room temperature. The antibodies were then re-
moved, and the cells were washed thrice with PBS. Cells were then
incubated with FITC-labeled anti-mouse secondary antibody
(1:500) for 1 h at room temperature. Cells were washed thrice with
PBS and mounted in medium containing DAPI. Images were cap-
tured using an Olympus confocal microscope and analyzed with
Provision software (instrument: FLOW VIEW-FV 1000 Series; soft-
ware: FV 10 ASW 1.7 Series).[19]
dependent experiments.[18]
Cell-cycle analysis: A549 cells in 60 mm dishes were incubated for
24 h in the presence or absence of test compounds 7, 11, and 15
at 5 or 10 mm. Cells were harvested with Trypsin-EDTA, and then
fixed with ice-cold 70% ethanol at 48C for 30 min. Ethanol was re-
moved by centrifugation (400 g, 10 min), and cells were stained
with 1 mL DNA staining solution [0.2 mg propidium iodide (PI),
2 mg RNase A] for 30 min as described earlier.[18] The DNA contents
of 20000 events were measured by flow cytometry (BD FACSCan-
to II). Histograms were analyzed using FCS express 4 plus.[18]
Tubulin polymerization assays: An in vitro assay for monitoring the
time-dependent polymerization of tubulin to microtubules was
performed by using a fluorescence-based tubulin polymerization
assay kit (BK011, Cytoskeleton Inc.) according to the manufacturer’s
protocol. The reaction mixture in a final volume of 10 mL in PEM
buffer (80 mm PIPES, 0.5 mm EGTA, 2 mm MgCl2, pH 6.9) in 384-
well plates contained 2 mgmLÀ1 bovine brain tubulin, 10 mm fluo-
rescent reporter, and 1 mm GTP in the presence or absence of test
compounds at 378C. Tubulin polymerization was followed by mon-
itoring the fluorescence enhancement due to the incorporation of
a fluorescence reporter into microtubules as polymerization pro-
ceeds. Fluorescence emission at 420 nm (excitation wavelength
360 nm) was measured for 1 h at 1-min intervals in a multimode
plate reader (Tecan M200). To determine the IC50 values of the com-
pounds against tubulin polymerization, the compounds were pre-
incubated with tubulin at varying concentrations (1, 5, 10 and
20 mm). Assays were performed under similar conditions as used
for the polymerization assays described above.[18]
Docking: All geometries were optimized in Gaussian 09 using PM3
semi-empirical method.[20] The protein structure was downloaded
from RCSB Protein Data Bank (PDB ID: 3E22).[21] Docking studies
were performed using AutoDock 4.2 software.[22] The analysis of in-
termolecular interactions was performed with PyMOL v. 0.99.[23]
Acknowledgements
M.P.N.R., P.S., K.M., and J.K. acknowledge the Council of Scientific
& Industrial Research/University Grants Commission (CSIR-UGC),
New Delhi (India), for the award of senior research fellowships;
they are thankful to DST (India) for the award of Inspire fellow-
ships. The authors also acknowledge the Council of Scientific &
Industrial Research (CSIR), India, for financial support under the
12th Five-Year Plan project “Affordable Cancer Therapeutics
(ACT)” (CSC0301) and Small Molecule in Lead Exploration (SMiLE-
CSC-0111).
Western blot analysis of soluble versus polymerized tubulin and
cyclin-B1: Cells were seeded in 12-well plates at 1ꢂ105 cells per
well in complete growth medium. Cells were treated with com-
pounds 7 or 11 for 24 h. Subsequently, cells were washed with
phosphate-buffered saline (PBS), and soluble and insoluble tubulin
fractions were collected. To collect the soluble tubulin fractions,
cells were permeabilized with 200 mL pre-warmed lysis buffer
[80 mm PIPES-KOH (pH 6.8), 1 mm MgCl2, 1 mm EGTA, 0.2% Triton
X-100, 10% glycerol, AND 0.1% protease inhibitor cocktail (Sigma–
Aldrich)] and incubated for 3 min at 308C. The lysis buffer was
gently removed, and mixed with 100 mL 3ꢂ Laemmli sample buffer
(180 mm Tris·Cl pH 6.8, 6% SDS, 15% glycerol, 7.5% b-mercaptoe-
thanol, and 0.01% bromophenol blue). Samples were immediately
heated at 958C for 3 min. To collect the insoluble tubulin fraction,
300 mL 1ꢂ Laemmli sample buffer was added to the remaining
cells in each well, and the samples were heated at 958C for 3 min.
Equal volumes of samples were separated by 10% SDS-PAGE and
then transferred to a nitrocellulose membrane using semidry trans-
fer at 50 mA for 1 h. Blots were probed with mouse anti-human a-
tubulin diluted 1:2000 mL (Sigma) and stained with rabbit anti-
mouse secondary antibody coupled with horseradish peroxidase,
diluted 1:5000 mL (Sigma). Bands were visualized using an en-
hanced Chemiluminescence protocol (Pierce) and radiographic film
(Kodak). For cyclin-B1 immunoblots, cells were seeded in 12-well
plates at 1ꢂ105 cells per well in complete medium and treated
with various concentrations of 7, 11, and 15 for 24 h. After treat-
ment, cells were washed twice with PBS and lysed in 1ꢂ SDS
sample buffer. Proteins were separated, transferred, probed, and
analyzed similarly to tubulin. The primary anti-cyclin-B1 antibody
was employed at 1:1500 and b-actin (Sigma) and horseradish per-
oxidase coupled goat anti-rabbit secondary antibody diluted
1:5000 (Sigma).[20]
Keywords: antitumor agents · docking · imidazothiadiazole-
linked oxindoles · tubulin
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Received: January 29, 2014
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Published online on && &&, 0000
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A. Kamal,* M. P. N. Rao, P. Das,
P. Swapna, S. Polepalli, V. D. Nimbarte,
K. Mullagiri, J. Kovvuri, N. Jain
Imidazzling microtubule blockers! A
series of imidazo[2,1-b][1,3,4]thiadiazole-
linked oxindole conjugates were synthe-
sized and evaluated for anticancer po-
tential. Conjugate 7 displayed promising
cytotoxicity, arrested cells at the G2/M
phase, and showed potent tubulin poly-
merization inhibition with an IC50 value
of 0.15 mm.
&& – &&
Synthesis and Biological Evaluation of
Imidazo[2,1-b][1,3,4]thiadiazole-Linked
Oxindoles as Potent Tubulin
Polymerization Inhibitors
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ChemMedChem 0000, 00, 1 – 14
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