2-Arylaminobenzothiazole-arylpropenone conjugates as tubulin polymerization inhibitors

Article abstract of DOI:10.1039/c6md00562d

A new series of 2-arylaminobenzothiazole-arylpropenone conjugates 5-6(a-r) was designed, synthesized and investigated for their cytotoxic potency against the various human cancer cell lines. Most of these conjugates exhibited cytotoxic activity and inhibi

Full text of DOI:10.1039/c6md00562d

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RESEARCH ARTICLE
2-Arylaminobenzothiazole-arylpropenone
conjugates as tubulin polymerization inhibitors†
Cite this: DOI: 10.1039/
A. V. Subba Rao,ab Bala Bhaskara Rao,bc Satish Sunkari,ab Siddiq Pasha Shaik,ab
c6md00562d
a
ab
*
Bajee Shaik and Ahmed Kamal
A new series of 2-arylaminobenzothiazole-arylpropenone conjugates 5–6IJa–r) was designed, synthesized
and investigated for their cytotoxic potency against the various human cancer cell lines. Most of these con-
jugates exhibited cytotoxic activity and inhibited in vitro tubulin polymerization effectively. Conjugates 5d
and 6d cause cell cycle blocks in the G2/M phase in HeLa cells and treatments with 5d and 6d manifested
increased mRNA and protein levels of the G2/M marker, cyclin B1. Immunocytochemistry revealed loss of
intact microtubule structure in cells treated with 5d and 6d. Western blot analysis revealed that these con-
jugates accumulate more tubulin in the soluble fraction. Moreover, the triggering of apoptotic cell death
after mitotic arrest was investigated by studying their effect on Hoechst staining, mitochondrial membrane
potential, ROS generation.
Received 11th October 2016,
Accepted 5th January 2017
DOI: 10.1039/c6md00562d
rsc.li/medchemcomm
bule dynamics by binding to distinct colchicine domain of tu-
1. Introduction
bulin are colchicine (1).10 Nocodazole (2) is well-known
Despite much progress in the development of antiproliferative
drugs that inhibit the function of various target proteins, we
are still faced with some significant deficits in finding effec-
tive drugs. Thus the search for new anti-tumor active agents
is of great importance and challenging of medicinal chemis-
try research. Microtubules are the key motif in the process of
cell division in eukaryotic cells makes them an important tar-
get for anticancer drugs.1 The microtubules that make up the
mitotic spindle are in a particularly delicate state of balance
between assembly and disassembling into their constituent
subunits of αβ-tubulin heterodimers. These are essential for
many cellular processes, such as maintenance of cellular
shape, intracellular transport and mitotic spindle assembly
during cell division.2 Generally, drugs that target microtu-
bules bind to one of three main sites of tubulin3 which in-
cludes the taxane-site for the microtubule stabilizing
agents,4,5 the vinca domain6 maytansine binding site7 and
the colchicine domain8 for the destabilizing agents. Interfer-
ing with the dynamic stability of microtubules, these agents
act as spindle poisons arresting the dividing cells in G2/M
phase of the cell cycle, causing mitotic catastrophy and finally
leading to apoptotic cell death.9 Some of the well-known natu-
rally occuring tubulin binding ligand that affect the microtu-
inhibitor of tubulin polymerization which inhibits cell prolif-
eration and is largely used as a pharmacological tool and pos-
itive control.11 Benzoheterocycles such as benzothiazoles,
benzimidazoles and benzoxazoles can serve as unique and
versatile scaffolds for experimental drug design. Among the
all benzoheterocycles, benzothiazole has considerable place
in research area especially in synthetic as well as in pharma-
ceutical chemistry because of its potent and significant phar-
macological activities. Benzothiazole is a privileged bicyclic
ring system and its derivatives have long been therapeutically
used for the treatment of various diseases.12–18 However, in
recent years, 2-(4 aminophenyl)benzothiazoles have emerged
as an important pharmacophore in the development of anti-
tumor agents.19 Among them (5F-203) (3)20 and its prodrug,
phortress (4)21 which is water soluble and chemically stable,
is found to rapidly and quantitatively revert to its parent
amine in mice, rats and dogs (in vivo). Thus, clinical evalua-
tion of phortress has demonstrated potent and selective anti-
tumor activity via a different mechanism of action which pro-
vides substantial scope for the development of benzothiazole-
based derivatives as anticancer agents.22 Considering the bio-
logical importance of these moieties, an attempt has been
made in the present study to synthesize new benzothiazole
derivatives. Our recent research studies have been mainly fo-
cused on the synthesis, evaluation and mechanistic aspects of
newer molecules based on different heterocyclic scaffolds as
potential anticancer agents particularly, the targeting of tubu-
lin by using new diversified ligands.23–25 In continuation to
our efforts for identifying new potent and selective anticancer
a Medicinal Chemistry and Pharmacology, India
b Academy of Scientific and Innovative Research (AcSIR), India
c Centre for Chemical Biology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500 007, India. E-mail: ahmedkamal@iict.res.in
† The authors declare no competing interests.
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Scheme 2 Reagents and conditions: (a) ethynylmagnesium bromide,
THF, 0 °C rt, 8–9 h, 72–78%; (b) 2-iodoxybenzoic acid, DMSO, rt, 5 h,
70–76%; (c) ethanol, rt, 4 h, 75–82%.
Fig.
1 Tubulin polymerization inhibitors (1, 2, 5 and 6a–r) and
antitumor agents (3 and 4).
agents, in view of considering the biologic importance of
2-(4-aminophenyl)benzothiazole derivatives, we design and
synthesis of 2-(4-aminophenyl)benzothiazole-arylpropionone
conjugates (5a–r and 6a–r) (Fig. 1). These were evaluated for
cytotoxic potential followed by structure–activity relationship
(SAR) and mode of action of mechanism is elucidated.
ethynylmagnesium bromide in dry tetrahydrofuran (THF) at
0 °C to room temperature, producing 1-aryl-2-propyn-1-ol
(18a–f). Oxidation of (18a–f) with 2-iodoxybenzoic acid (IBX)
in the presence of dimethyl sulfoxide (DMSO) gave 1-aryl-2-
propyn-1-one (19a–f). Condensation of (19a–f) with aryl
amines (15a–c and 16a–c) in ethanol at room temperature
resulted in the formation of (Z)-3-((4-(benzoijd]thiazol-2-
yl)aryl)amino)-1-arylprop-2-en-1-ones 5 and 6(a–r) in good
yields and their structures have shown in Table 1 were con-
2. Results and discussions
2.1. Chemistry
1
2.1.1. Synthesis of substituted 2-(4-amino-phenyl)-
firmed by H, 13C NMR and HRMS spectral analysis.
benzothiazoles 15–16IJa–c). The preparation of various
substituted
2-(4-aminophenyl)benzothiazole
precursors
2.2. Biology
(15a–c) and (16a–c) was achieved by the Jacobson thioanilide
radical cyclization. Thus, reaction of substituted anilines
(7a–c) with substituted p-nitrobenzoylchlorides (8a–b) in pyri-
dine gave the benzanilides (9a–c) and (10a–c) which were fur-
ther converted to their corresponding thiobenzanilides
(11a–c) and (12a–c) using Lawesson's reagent. These were
cyclized by using Jacobson method to afford nitro-
benzothiazole derivatives (13a–c) and (14a–c) using potas-
sium ferricyanide and aqueous sodium hydroxide which
upon reduction of these nitro compounds with stannous
chloride yielded the corresponding substituted 2-(4-amino-
phenyl)benzothiazoles (15a–c) and (16a–c) as illustrated in
Scheme 1.
2.2.1. Antiproliferative activity. All newly synthesized con-
jugates of arylaminobenzothiazole-arylpropenone conjugates
5–6IJa–r) were evaluated for their antiproliferative activity in a
concentration-dependent manner against a panel of four differ-
ent human cancer cell lines A549, HeLa, MDAMB-231 and MIA
PaCa-2 using the SRB assay. The results of growth inhibitory
activities (IC50 values) are presented in Table 2 in micromolar
concentrations, nocodazole used as the reference compound.
2.2.2. Structure–activity relationships (SAR). These studies
reveal that the cytotoxicity of the arylaminobenzothiazole-
arylpropenone conjugates 5–6IJa–r) was totally dependent on
the nature and position of the substituents present on the
two scaffold rings containing A, B and C-rings. The cytotoxic-
ity data (Table 2) clearly showed that the conjugates (5d, 6d
and 6f) exhibited promising cytotoxicity in HeLa cells (IC50
range 0.5–1.2 μM) and the results were comparable with posi-
tive control, nocodazole. These conjugates are more active
when benzothiazole ring (A-ring) possessing methoxy group
at 6th position with unsubstitution or monosubstituted
(methyl) group 3rd position of phenyl ring (B-ring) and with
a combination of a methoxy and a halogen atom (F) on
C-ring (5d and 6d). On the basis of this observation, addi-
tional methoxy groups were introduced on the benzothiazole
ring, while keeping a methoxy groups on the C-ring to evalu-
ate the influence of these groups on the cytotoxicity of the
molecules (5e–f and 6e–f). From the cytotoxicity data
(Table 2), it was clearly indicate that 5,7-dimethoxy substitu-
tion (5e and 6e) moderately decreased the potency of the mol-
ecules while 5,6,7-trimethoxy substitution (5f) resulted in the
loss of the cytotoxicity to several fold than 5d and wherein
case of 6f enhanced the cytotoxicity with comparable to 6d.
2.1.2. Synthesis of (Z)-3-((4-(benzoijd]thiazol-2-yl)aryl)-
amino)-1-arylprop-2-en-1-ones 5 and 6(a–r). The synthesis of
target compounds (Z)-3-((4-(benzoijd]thiazol-2-yl)aryl)amino)-1-
arylprop-2-en-1-ones
5
and 6(a–r) were illustrated in
Scheme 2. Arylaldehydes (17a–f) were treated with
Scheme 1 Reagents and conditions: (a) pyridine, reflux, 3 h, 90%; (b)
Lawesson's reagent, toluene, reflux, 8 h, 80%; (c) K3FeIJCN)6, aq. NaOH,
.
EtOH, 90 °C, 2–3 h, 60–70%; (d) SnCl2 2H2O, EtOH, 3 h, 75–80%.
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Table 1 Structures of 2-arylaminobenzothiazole-arylpropenone conju-
gates 5–6IJa–r)
the 5m–o and 6m–o conjugates, the resulted conjugates (5p–r
and 6p–r) are enhanced the cytotoxicity than counterparts.
The best activity in these conjugate series could be
obtained only when a molecule bears 6-methoxy on the A ring
and methyl group on the B-ring with combination methoxy
and fluoro atom on C-ring. After identifying the most potent
conjugates (5d and 6d) in the series towards HeLa cell lines,
these conjugates taken for further detailed studies with a
view to understand the cell growth inhibition mechanisms.
2.2.3. Effect on cell cycle. Many anticancer compounds ex-
ert their growth inhibitory effect either by arresting the cell
cycle at a particular checkpoint of cell cycle or by induction
of apoptosis or a combined effect of both cycle block and ap-
optosis.26,27 Furthermore regulation of the cell cycle and apo-
ptosis are considered to be effective cancer therapeutic
methods.28 To evaluate this possibility, HeLa cells were
treated with 5d, 6d and nocodazole used as positive control
at 3 μM for 48 h. The data obtained from the study as
presented in Fig. 2 show that treatment with 5d, 6d and
nocodazole resulted in the accumulation of cells in the G2/M
phase of the cell cycle with 88.7%, 86.1% and 87.5% respec-
tively. Whereas, DMSO treated cells showed predominant G1
phase, with 33.4% of cells in G2/M. Overall, these results sug-
gest that these conjugates efficiently stall cells at G2/M
phase.
Compound
R
R1
R2
R3
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
3-OH,4-OMe
3-OH,4-OMe
3-OH,4-OMe
3-F,4-OMe
3-F,4-OMe
3-F,4-OMe
3-NO2,4-OMe
3-NO2,4-OMe
3-NO2,4-OMe
3-NO2,4,5-diOMe
3-NO2,4,5-diOMe
3-NO2,4,5-diOMe
3,4,5-triOMe
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
H
OMe
OMe
5j
5k
5l
5m
5n
5o
5p
5q
5r
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
3,4,5-triOMe
3,4,5-triOMe
2-Br,3,4,5-triOMe
2-Br,3,4,5-triOMe
2-Br,3,4,5-triOMe
3-OH,4-OMe
3-OH,4-OMe
3-OH,4-OMe
3-F,4-OMe
H
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
3-F,4-OMe
3-F,4-OMe
3-NO2,4-OMe
3-NO2,4-OMe
3-NO2,4-OMe
3-NO2,4,5-diOMe
3-NO2,4,5-diOMe
3-NO2,4,5-diOMe
3,4,5-triOMe
3,4,5-triOMe
3,4,5-triOMe
2-Br,3,4,5-triOMe
2-Br,3,4,5-triOMe
2-Br,3,4,5-triOMe
2.2.4. Effect on cellular cyclin-B1 levels by immunoblot
analysis. Cyclin-B1 is induced at the G2/M boundary to pro-
mote cell division. This protein is one of the important regu-
latory proteins of mitosis and accumulation of cyclin-B1 is an
indication for G2/M arrest.29 Since these compounds arrest
the cells at G2/M phase, we investigated their effect on cyclin
B1 protein levels. Therefore, HeLa cells were treated with 3
6m
6n
6o
6p
6q
6r
OMe
μM concentration of these compounds for 24
h and
performed immunoblot analysis for cyclin-B1. For compari-
son, nocodazole and taxol were included as positive controls
and tubulin as loading control. Immunoblot analysis revealed
that these compounds 5d and 6d strongly induce cyclin B1
levels, a well-recognized G2/M marker (Fig. 3a). In addition,
to further validate that the increase in protein levels of cyclin
B1 was not due to increased stability of cyclin B1 protein in
the presence of compounds 5d and 6d by any cross-reactivity
of the compounds with cyclin B1 protein. Thus we performed
semi-quantitative RT-PCR analysis for mRNA levels of cyclin
B1 in control and compound treated cells. GAPDH was used
as an internal control. Notably, treatments with the conju-
gates 5d and 6d showed robustly activated cyclin B1 mRNA
levels compared to control (Fig. 3b). Thus our results support
the suggestion that conjugates cause an accumulation of cells
at the G2/M phase of the cell cycle.
2.2.5. Effect of 5d and 6d on in vitro and in vivo tubulin
polymerization. Since 5d and 6d robustly activate G2/M arrest
in cells and the compounds possess moieties that interact
with tubulin, we assessed their ability to inhibit in vitro tubu-
lin assembly. Nocodazole was used as a positive control.
Whereas, DMSO served as a vehicle control. The compounds
at five different doses were pre-incubated with tubulin
In addition we replaced the fluoro atom (F) with hydroxyl
(OH) group in C-ring and the resulted molecules are 5a–c
and 6a–c lost the activity, indicating that electronegative
group at that position is required for the activity of the
molecule.
When electron withdrawing nitro group introduced in
place of fluoro atom on the C-ring resulted molecules were
(5g–i and 6g–i) lost their cytotoxic potential on cancer cells,
showed that the fluoro (F) atom at those position is critical
for the activity of the molecules with exception of 5h with 2.9
μM. Additional nitro group was introduced on the C-ring of
the molecules i.e. 5g–i and 6g–i resulted molecules were 5j–l
and 6j–l completely loss of cytotoxicity with few exceptions
on certain cell lines. Also to evaluate the importance of the of
3,4,5-trimethoxy groups on C-ring, we switched these methoxy
groups on to the benzothiazole rings (A and B) and tested the
resulted molecules (5m–o and 6m–o) for the cytotoxicity. The
loss of cytotoxicity activity observed when compare to 5d and
6d indicating that hydrophilic group at that positions are crit-
ical for activity. Among them, 5o showed significant activity
with 5.3 μM on HeLa cell line. Surprisingly, in addition to
these molecules we introduced bromo (Br) atom on C-ring of
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Table 2 Cytotoxic effects on arylaminobenzothiazole-arylpropenone conjugates 5–6IJa–r)
a
IC50
Compound
A549b
HeLac
6.9 0.04
12.1 0.03
14.1 0.05
0.5 0.02
7.3 0.1
MDAMB-231d
MIA PaCa-2e
5a
5b
5c
5d
5e
12.8 0.47
10.61 01
23.0 0.79
0.9 0.42
5.3 0.4
18.9 0.17
7.61 25
18.1 0.23
1.1 0.33
6.7 0.4
21.9 0.29
11.6 0.39
32.77 0.02
1.3 0.12
4.3 0.36
5f
15.5 58
7.3 0.06
17.6 0.03
12.9 0.04
11.4 0.7
16.1 0.03
19.01 0.5
7.8 0.02
14.1 0.13
17.8 0.14
5.3 0.26
8.02 0.15
10.3 0.41
6.6 0.23
12.01 71
16.1 0.62
11.4 0.13
0.6 0.29
14.7 0.5
1.2 0.02
17.6 0.03
9.3.9 0.3
13.4 0.6
12.1 0.05
11.4 0.03
13.1 0.09
16.1 0.43
16.4 0.69
11.09 0.72
13.4 0.13
14.7 0.55
14.3 0.41
0.84 0.05
16.5 0.33
17.6 0.09
12.9 0.07
17.1 0.7
7.61 05
19.4 0.16
23.6 0.01
2.9 0.09
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
13.6 0.06
5.8 0.06
21.2 0.2
21.61 01
13.0 0.09
11.8 0.02
12.1 0.11
21.8 0.92
15.5 0.88
6.0 0.89
12.03 0.24
13.6 0.46
18.02 66
14.3 0.11
15.4 0.48
1.4 0.46
15.3 0.3
1.69 0.05
13.6 0.06
12.1 0.5
9.1 0.7
22.06 0.01
15.4 0.08
17.4 0.06
11.06 0.35
14.4 0.07
23.29 0.37
6.4 0.04
14.03 0.43
10.3 0.24
0.82 06
15.9 0.5
25.6 0.09
12.7 0.09
15.8 0.05
31.1 0.73
26.8 0.87
29.05 0.06
14.7 0.49
19.03 0.96
23.2 0.51
17.01 0.89
22.1 0.27
15.4 0.06
2.3 0.02
3.06 0.03
9.8 0.03
19.61 0.25
15.8 0.11
16.5 0.53
11.06 0.23
16.09 0.03
15.6 0.39
14.02 06
3.1 0.49
12.4 0.57
2.0 0.09
13.3 0.7
2.9 0.01
17.6 0.09
10.8 0.6
12.4 0.7
5.05 0.09
7.4 0.03
12.8 0.09
17.01 0.62
13.8 0.43
12.9 0.21
9.4 0.73
17.3 0.37
7.3 0.18
0.71 0.05
16.3 0.17
1.8 0.07
23.6 0.01
13.9 0.8
23. 4 0.3
12.0 0.07
10.4 0.01
22.03 0.09
10.03 0.77
17.03 0.24
22.8 0.27
23.4 0.51
16.03 0.27
24.3 0.53
0.91 0.02
Nocodazole
a
b
c
IC50 = compound concentration required to inhibit tumor cell proliferation by 50% after 48 h of drug treatment. Lung cancer. Cervical
cancer. Breast cancer and. e Pancreatic cancer.
d
protein. Later the tubulin polymerization was initialed by the
addition of 4 mM GTP. After 30 min, the assays revealed that
DMSO control did not inhibit tubulin assembly. In compari-
son, nocodazole, 5d and 6d acted as tubulin inhibitors. 5d
and 6d showed an IC50 of 2.2 μM and 2.9 μM respectively
(Table 3). Next, microtubule depolymerizing drugs cause im-
proper chromosome separation by inhibiting the organiza-
tion of the mitotic spindle, and predominantly arrest chro-
mosomes in metaphase of mitosis.30 Occurrence of irregular
spindle fibers due to disrupted microtubule network is a hall-
mark of cells treated with antitubulin agents.
The inhibitors of tubulin assembly cause severe perturba-
tion in the microtubule dynamics leading to irregular mor-
phology. Since these benzothiazole derivatives exhibit pro-
found inhibition of tubulin assembly and arrest cells at G2/M
phase of cell cycle, we investigated their ability to alter
the microtubule network. Therefore, it was of interest to
examine the intracellular effect of 5d and 6d by monitor-
ing cellular microtubules, as well as nuclear condensation.
HeLa cells were treated with 3 μM of these compounds for 24
h cells upon staining and showed severe disruption in micro-
tubule organization. However, vehicle-control/DMSO treated
cells manifested normal microtubule network as shown in
Fig. 4.
2.2.6. Distribution of soluble versus polymerized tubulin
in cells. Since inhibition of tubulin polymerization disturbs
the microtubule dynamics, we evaluated the levels of soluble
(S) versus polymerized (I) forms of tubulin in HeLa cells fol-
lowing treatment with 3 μM of 5d and 6d for 24 h. In addi-
tion, cells were treated with nocodazole, as positive and taxol
as negative controls in parallel experiments. Western blot
analysis reveals that the amount of tubulin protein in both
soluble and polymerized fractions was approximately the
same in DMSO treated cells. Nocodazole treated cells
exhibited a shift of tubulin from the polymerized fraction
into the soluble fraction.
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Fig.
4
Effect of 5d and 6d on microtubule network:
immunofluorescence images of HeLa cells stained with anti-b-tubulin
antibody FITC-conjugated and then observed by confocal microscopy,
magnification at 60×. Cells were exposed to 3 μM of representative
compounds for 24 h and then fixed and analyzed by fluorescence
microscopy. The potent inhibitors (5d and 6d) of tubulin assembly
show an irregular or rounded morphology. Cells were also
counterstained with DAPI to visualize the nuclei.
Fig. 2 FACS analysis of cell cycle distribution of HeLa cells after
treatment with 5d and 6d at 3 μM concentrations and nocodazole for
24 h. Cell cycle analysis was performed employing propidium iodide as
indicated under Materials and methods.
In comparison, paclitaxel a microtubule polymerization
agent showed more amount of tubulin in the polymerized
fraction. As expected the cells treated with 5d and 6d signifi-
cantly increased the tubulin content in the soluble fraction,
with almost all tubulin present in the soluble fraction similar
to that of the positive control. Therefore, increased tubulin in
soluble fraction of cells treated by these hybrids corroborated
with the inhibition of tubulin assembly and arrest of cells in
G2/M phase as shown in Fig. 5.
2.2.7. Hoechst staining. To investigate the apoptotic in-
ducing effect of these conjugates (5d and 6d), Hoechst
staining assay was carried out on HeLa cells. Hoechst 33242
is a cell membrane permeable nuclear staining dye, which
emits blue fluorescence and stains the live cell nuclei as light
blue, whereas the apoptotic cell nuclei appear as bright blue
due to chromatin condensation. HeLa cells were treated with
5d and 6d at concentration of 1 μM for 24 h and stained with
Hoechst and nocodazole was used as a reference compound.
The results from Fig. 6 indicated that in the control group,
the untreated cells did not show obvious morphological
Fig. 3 Western blot and RT-PCR analysis of cyclin B1 in 5d and 6d
treated HeLa cells. 3a). The cells were treated with 3 μM of 5d, 6d,
nocodazole and taxol for 24 h. Tubulin was used as loading for equal
loading of protein samples and DMSO as vehicle control. 3b). Semi-
quantitative RT-PCR analysis of cyclin B1 gene expression in conju-
gates 5d, 6d and Nocodazole treated HeLa cells. GAPDH was used as
an internal control and DMSO as vehicle control.
Table 3 Tubulin polymerization inhibitory effect of compounds 5d and
6d
Compound
Anti-tubulin activity (IC50)
5d
6d
2.2 0.23
2.9 0.26
1.8 0.04
Nocodazole
Fig. 5 HeLa cells were treated with 3 μM of 5d and 6d for 24 h.
Nocodazole and taxol were used as reference standards. Levels of
tubulin was detected by Western blot analysis. S: soluble fraction. I:
polymerized fraction.
Note: Effect of conjugates on tubulin polymerization. IC50 values for
5d, 6d and nocodazole were determined from the tubulin
polymerization assays. Nocodazole was employed as positive control.
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Fig. 8 Effect of conjugates 5d and 6d on the intracellular levels of
ROS. HeLa cells were treated with 5d and 6d for 24 h and stained with
Fig. 6 Conjugates 5d and 6d induced nuclear morphological changes
of HeLa cells.
carboxy DCFH-DA. Images were captured by
a
fluorescence
microscope (Nikon). The intensity of DCF-DA fluorescence was mea-
sured by spetrofluorometer using an excitation wavelength of 485 nm
and an emission wavelength of 535 nm.
changes (all the cells exhibited uniform rounded cell mor-
phology), however in compounds (5d, 6d and nocodazole)
treated group, the cells exhibited typical apoptotic morphol-
ogy such as highly condensed nuclei (brightly stained). This
observation demonstrates that these conjugates 5d and 6d
are able to induce apoptosis in HeLa cells.
2.2.8. Mitochondrial membrane potential (ΔΨm). Previous
reports have suggested that the loss of ΔΨm leads to apopto-
sis.31 In order to further investigate the apoptosis-inducing
effect of target compounds, changes in ΔΨm was measured
after staining with Rhodamine-123. Mitochondria that main-
tain normal ΔΨm can retain the Rhodamine 123, which give
strong green fluorescence. Disruption of ΔΨm was associated
with a decrease in green fluorescence due to a lack of Rh-123
retention.32 Thus the relationship between ΔΨm and Rh-123
fluorescence is considered as linear. HeLa cells were treated
with these hybrids 5d and 6d at 1 μM concentration for 24 h
and stained with Rh-123 and the intensity of Rh-123 fluores-
cence was measured by spectrofluorometer. The results from
the Fig. 7 revealed that these conjugates induced 40–60% loss
of ΔΨm in HeLa cells. Conjugate 5d is more potent in causing
drop in ΔΨm, which showed 40% of collapse of ΔΨm followed
by the conjugate 6d that induced 30% losses of ΔΨm, respec-
tively. These results demonstrate that these compounds in-
duce apoptosis in HeLa cells through changes in ΔΨm.
2.2.9. Activation of reactive oxygen species. Loss of ΔΨm
and elevation of intracellular ROS levels are two closely re-
lated events that occur during apoptosis.33 The levels of ROS
were measured in HeLa cells by using a fluorescent probe,
2′,7′-dichlorofluorescein diacetate (H2DCFDA) to examine
whether the drop in ΔΨm was on account of the production
of ROS.34 The non-fluorescent DCFH-DA is oxidized to green
fluorescent DCFH by intracellular esterases when it diffuses
into cells. Thus, the intracellular ROS levels can be expressed
as mean fluorescence intensity (MFI) of DCF. As shown in
Fig. 8, HeLa cells treated with conjugates 5d, 6d and
nocodazole at 1 μM concentration showed increase in green
fluorescence compared to the control (untreated cells).
Hence, it could be concluded that these conjugates signifi-
cantly elevate the intracellular ROS levels.
3. Conclusions
In conclusion, we designed and synthesized a series of
2-arylaminobenzothiazole-arylpropeonones conjugates based
on the structure 3 and developed a potent conjugates 5d and
6d which displayed a strong cytotoxic activity with IC50 values
of 0.5 and 0.6 μM against the human HeLa cancer cell lines.
Flow cytometry analysis indicates that these compounds in-
duce cell cycle arrest at G2/M phase in HeLa cells. Treat-
ments with 5d and 6d manifested increased mRNA and pro-
tein levels of the G2/M marker, cyclin B1 which is
corroborated cell cycle arrest at G2/M phase. Tubulin poly-
merization assay showed that they are potent inhibitors of
Fig.
7 Conjugates 5d and 6d induced loss of mitochondrial
membrane potential (ΔΨm) in HeLa cells analyzed using Rhodamine
123 staining. The loss in intensity of fluorescence was measured by
spetrofluorometer. Data are mean
experiments.
SD from three independent
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tubulin polymerization. Increased levels of tubulin in the sol-
uble fraction of cells remarkably corroborated with inhibition
of tubulin polymerization by these conjugates 5d and 6d. In
addition, they induce apoptosis in HeLa cells by studying
their effect on Hoechst staining, collapse of ΔΨm, elevation of
ROS production. Overall, these results demonstrate that the
arylaminobenzothiazole-arylpropenone conjugates have the
potential to be developed as leads and their further amenable
structural modifications may produce promising anticancer
agents for HeLa cancer cells.
4.1.1.1.2. N-(4-Methoxyphenyl)-3-methyl-4-nitrobenzamide
(10a). Yellow solid, yield 71%; mp: 152–154 °C; 1H NMR
(300 MHz, CDCl3): δ 8.00 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.83
(s, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.52 (d, J = 8.8 Hz, 2H), 6.90
(d, J = 9.0 Hz, 2H), 3.82 (s, 3H), 2.64 (s, 3H); MS (ESI): m/z
287 (M + H)+.
4.1.1.1.3. N-(3,5-Dimethoxyphenyl)-4-nitrobenzamide
1
(9b). Yellow solid, yield 88%; mp: 211–213 °C; H NMR (300
MHz, CDCl3 + DMSO): δ 10.22 (s, 1H), 8.31 (d, J = 8.9 Hz,
2H), 8.20 (d, J = 8.9 Hz, 2H), 7.07 (d, J = 2.2 Hz, 2H), 6.25 (t, J
= 2.2 Hz, 1H), 3.80 (s, 6H); MS (ESI): m/z 303 (M + H)+.
4.1.1.1.4. N-(3,5-Dimethoxyphenyl)-3-methyl-4-nitro-
benzamide (10b). Yellow solid, yield 72%; mp: 189–192 °C;
1H NMR (500 MHz, CDCl3): δ 8.03 (s, 1H), 7.98 (d, J = 8.4 Hz,
1H), 7.81 (s, 1H), 7.75 (dd, J = 8.4, 1.5 Hz, 1H), 6.87 (d, J = 2.0
Hz, 2H), 6.29 (t, J = 2.2 Hz, 1H), 3.79 (s, 6H), 2.62 (s, 3H); MS
(ESI): m/z 317 (M + H)+.
4. Experimental section
4.1. Chemistry
4.1.1. Materials and methods. 1H NMR spectra were
recorded on Avance 300, Inova 400, Avance 500, and Bruker
600 MHz spectrometers using tetramethylsilane (TMS) as the
internal standard. Chemical shifts are reported in parts per
million (ppm) downfield from tetramethyl silane. Spin multi-
plicities are described as s (singlet), brs (broad singlet), d
(doublet), dd (double doublet), t (triplet), q (quartet), and or
m (multiplet). Coupling constants are reported in Hertz (Hz).
Melting points were determined in a capillary tube using an
electrothermal apparatus (Model IA9200) and are
uncorrected. The IR spectra were recorded by employing a Ni-
colet FTIR model MX-1spectrophotometer. Analytical thin
layer chromatography (TLC) was performed on MERCK
precoated silica gel 60- F254 (0.5 mm) glass plates. Visualiza-
tion of the spots on TLC plates was achieved either by expo-
sure to iodine vapour or UV light or by dipping the plates
into methanolic sulphuric acid-β-naphthol or to ethanolic
anisaldehyde-sulphuric acid–acetic acid or to ethanolic nin-
hydrin solution and heating the plates to 120 °C. Column
chromatography was performed using silica gel 60–120 and
100–200 mesh. Moisture sensitive reactions were carried out
using standard syringe septum Techniques and under inert
atmosphere of nitrogen. All solvents and reagents were puri-
fied by standard techniques. All evaporation of solvents was
carried out under reduced pressure on Laborota-4000 rotary
evaporator below 45 °C. The names of all the compounds
given in the experimental section were taken from Chem Ul-
tra, Version 11.0.
4.1.1.1.5. 4-Nitro-N-(3,4,5-trimethoxyphenyl)benzamide
1
(9c). Yellow solid, yield 88%; mp: 220–224 °C; H NMR (300
MHz, CDCl3 + DMSO): δ 10.12 (s, 1H), 8.32 (d, J = 8.8 Hz,
2H), 8.20 (d, J = 8.8 Hz, 2H), 7.19 (s, 2H), 3.88 (s, 6H), 3.81 (s,
3H); MS (ESI): m/z 333 (M + H)+.
4.1.1.1.6. 3-Methyl-4-nitro-N-(3,4,5-trimethoxyphenyl)-
benzamide (10c). Yellow solid, yield 76%; mp: 210–214 °C;
1H NMR (500 MHz, CDCl3): δ 8.23 (s, 1H), 8.03 (s, 1H), 8.02
(s, 1H), 7.51 (s, 1H), 6.85–6.58 (m, 2H), 3.84–3.82 (m, 6H),
3.81–3.80 (m, 3H), 2.44–2.42 (m, 3H); MS (ESI): m/z 347 (M +
H)+.
4.1.1.2. General method B for the synthesis of substituted
thiobenzanilides 11–12IJa–c). To a solution of substituted
benzanilides 9–10IJa–c) (6 g, 0.018 mol) in toluene (40 mL),
Lawesson's reagent (0.9 eq.) was added and reflux it for 8 h.
After completion of the reaction, cool to rt and solvent was
removed in vacuo and work up with ethylacetate and water.
Separation of the organic layer and evaporation followed by
column chromatography gave yellow solids 11–12IJa–c)
4.1.1.2.1. N-(4-Methoxyphenyl)-4-nitrobenzothioamide
(11a). Yellow solid, yield 84%; mp: 173–175 °C; MS (ESI):
m/z 289 (M + H)+.
4.1.1.2.2. N-(4-Methoxyphenyl)-3-methyl-4-nitro-
benzothioamide (12a). Yellow solid, yield 72%; mp: 160–162
°C; MS (ESI): m/z 303 (M + H)+.
4.1.1.1. General method a for the synthesis of substituted
benzanilides 9–10IJa–c). Substituted 4-nitrobenzoyl chlorides
(8a–b) (0.029 mol) were added slowly to a solution of the
appropriately substituted anilines (7a–c) (0.04 mol) in
pyridine (110 mL). The resulting solution was stirred under
reflux for 3 h and then poured into ice water (400 mL). The
precipitate formed was collected and washed with 2 N HCl
(100 mL), followed by water and methanol, to afford the
respective benzanilides 9–10IJa–c) as a yellow solids.
4.1.1.2.3. N-(3,5-Dimethoxyphenyl)-4-nitrobenzothioamide
(11b). Yellow solid, yield 90%; mp: 138–140 °C; MS (ESI):
m/z 319 (M + H)+.
4.1.1.2.4. N-(3,5-Dimethoxyphenyl)-3-methyl-4-nitro-
benzothioamide (12b). Yellow solid, yield 74%; mp: 178–180
°C; MS (ESI): m/z 333 (M + H)+.
4.1. 1.2. 5. 4-Nitro-N-(3,4,5-trimethoxyphenyl)-
benzothioamide (11c). Yellow solid, yield 84%; mp: 198–200
°C; MS (ESI): m/z 349 (M + H)+.
4.1.1.1.1. N-(4-Methoxyphenyl)-4-nitrobenzamide (9a).
Yellow solid, yield 85%; mp: 199–202 °C; H NMR (300 MHz,
DMSO): δ 10.47 (s, 1H), 8.36 (d, J = 8.8 Hz, 2H), 8.17 (d, J =
8.8 Hz, 2H), 7.68 (d, J = 9.0 Hz, 2H), 6.95 (d, J = 9.0 Hz, 2H),
3.75 (s, 3H); MS (ESI): m/z 273 (M + H)+.
4.1.1.2.6. 3-Methyl-4-nitro-N-(3,4,5-trimethoxyphenyl)-
benzothioamide (12c). Yellow solid, yield 72%; mp: 136–138
°C; MS (ESI): m/z 363 (M + H)+.
1
4.1.1.3. General method C for the Jacobson synthesis of
substituted 2-(4-nitrophenyl)benzothiazoles 13–14IJa–c).
A
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solution of the substituted thiobenzanilides 11–12IJa–c) (0.017
mol) in aqueous sodium hydroxide (8 eq. in 50 mL of water)
containing ethanol (3 mL) was added dropwise to a pre-
heating solution of potassium ferricyanide (4 eq.) in water
(30 mL) taken in a 250 mL RB flask at 90 °C over a period of
1 h. The resulting solution was stirred at 90 °C for a further 2
h and then cooled to room temperature. The precipitate
formed was filtered and washed with water. Products were
purified by column chromatography (ethylacetate/hexane)
and to furnish the 4-nitrophenyl-benzothiazoles 13–14IJa–c) as
yellow solids.
4.1.1.3.1. 6-Methoxy-2-(4-nitrophenyl)benzoijd]thiazole
(13a). Yellow solid, yield 62%; mp: 216–217 °C; 1H NMR
(300 MHz, CDCl3): δ 8.34 (d, J = 8.9 Hz, 2H), 8.21 (d, J = 8.9
Hz, 2H), 8.00 (d, J = 9.0 Hz, 1H), 7.39 (d, J = 2.5 Hz, 1H), 7.15
(dd, J = 9.0, 2.5 Hz, 1H), 3.92 (s, 3H); MS (ESI): m/z 287 (M +
H)+.
4.1.1.4.1. 4-(6-Methoxybenzoijd]thiazol-2-yl)aniline
(15a). Yellow solid, yield 92%; mp 191–193 °C; 1H NMR
(300 MHz, CDCl3): δ 7.88 (t, J = 8.6 Hz, 3H), 7.34 (d, J = 2.4
Hz, 1H), 7.07 (dd, J 8.9, 2.5 Hz, 1H), 6.75 (d, J = 8.6 Hz, 2H),
3.99 (s, 1H), 3.90 (s, 3H); MS (ESI): m/z 257 (M + H)+.
4.1.1.4.2. 4-(6-Methoxybenzoijd]thiazol-2-yl)-2-methyl-
aniline (16a). Yellow solid, yield 95%; mp 151–153 °C; 1H
NMR (300 MHz, CDCl3): δ 7.87 (d, J = 8.9 Hz, 1H), 7.78 (d, J =
1.2 Hz, 1H), 7.69 (dd, J = 8.2, 1.9 Hz, 1H), 7.31 (d, J = 2.5 Hz,
1H), 7.04 (dd, J = 8.9, 2.6 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H),
3.91 (s, 1H), 3.87 (s, 3H), 2.23 (s, 3H); MS (ESI): m/z 271 (M +
H)+.
4.1.1.4.3. 4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)aniline
(15b). Yellow solid, yield 90%; mp 150–152 °C; 1H NMR
(500 MHz, CDCl3): δ 7.90–7.85 (m, 2H), 7.14 (d, J = 2.0 Hz,
1H), 6.75–6.69 (m, 2H), 6.45 (d, J = 2.0 Hz, 1H), 3.98 (s, 1H),
3.95 (s, 3H), 3.88 (s, 3H); MS (ESI): m/z 286 (M)+.
4.1.1.3.2. 6-Methoxy-2-(3-methyl-4-nitrophenyl)benzoijd]-
thiazole (14a). Yellow solid, yield 64%; mp: 195–197 °C; H
NMR (300 MHz, CDCl3): δ 8.10 (d, J = 8.5 Hz, 1H), 8.04 (s,
1H), 7.99 (t, J = 8.4 Hz, 2H), 7.39 (d, J = 2.4 Hz, 1H), 7.15 (dd,
J = 9.0, 2.5 Hz, 1H), 3.93 (s, 3H), 2.72 (s, 3H); MS (ESI): m/z
301 (M + H)+.
4.1.1.4.4. 4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)-2-methyl-
aniline (16b). Yellow solid, yield 91%; mp: 142–145 °C; 1H
NMR (300 MHz, CDCl3): δ 7.84 (s, 1H), 7.75 (dd, J = 8.3, 2.0
Hz, 1H), 7.16 (d, J = 2.0 Hz, 1H), 6.73 (d, J = 8.3 Hz, 1H), 6.47
(d, J = 2.0 Hz, 1H), 3.97 (s, 3H), 3.96–3.93 (m, 1H), 3.91 (s,
3H), 2.26 (s, 3H); MS (ESI): m/z 301 (M + H)+.
1
4.1.1.3.3. 5,7-Dimethoxy-2-(4-nitrophenyl)benzoijd]thiazole
(13b). Yellow solid, yield 60%; mp: 238–239 °C; 1H NMR
(300 MHz, CDCl3): δ 8.37–8.32 (m, 2H), 8.24 (d, J = 8.9 Hz,
2H), 7.22 (d, J = 1.9 Hz, 1H), 6.56 (d, J = 1.9 Hz, 1H), 3.99 (s,
3H), 3.92 (s, 3H); MS (ESI): m/z 317 (M + H)+.
4.1.1.4.5. 4-(5,6,7-Trimethoxybenzoijd]thiazol-2-yl)aniline
(15c). Yellow solid, yield 89%; mp: 145–147 °C; 1H NMR
(300 MHz, CDCl3): δ 7.85 (d, J = 8.6 Hz, 2H), 7.30 (s, 1H), 6.73
(d, J = 8.6 Hz, 2H), 4.09 (s, 3H), 3.99 (s, 2H), 3.94 (d, J = 4.4
Hz, 6H); MS (ESI): m/z 317 (M + H)+.
4 . 1 . 1 . 3 . 4 . 5 , 7 - D i m e t h o x y - 2 - ( 3 - m e t h y l - 4 -
nitrophenyl)benzoijd]thiazole (14b). Yellow solid, yield 65%;
mp: 223–225 °C; 1H NMR (500 MHz, CDCl3): δ 8.00 (dd, J =
1.9, 1.2 Hz, 1H), 7.82 (d, J = 8.4 Hz, 1H), 7.06–7.02 (m, 2H),
6.83 (d, J = 2.4 Hz, 1H), 3.93 (s, 3H), 3.80 (s, 3H), 2.65 (s, 3H);
MS (ESI): m/z 330 (M + H)+.
4.1.1.4.6. 2-Methyl-4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)-
aniline (16c). Yellow solid, yield 90%; mp: 150–152 °C; 1H
NMR (500 MHz, CDCl3): δ 7.79 (s, 1H), 7.71 (dd, J = 8.2, 2.0
Hz, 1H), 7.30 (s, 1H), 6.71 (d, J = 8.2 Hz, 1H), 4.09 (s, 3H),
3.94 (d, J = 7.0 Hz, 8H). MS (ESI): m/z 331 (M + H)+.
4.1.1.5. General procedure for the synthesis of 1-aryl-2-
propyn-1-ol (18a–f). A solution of aldehyde 17a–f (5 mmol) in
dry tetrahydrofuran (THF) was added to a stirred solution of
ethynylmagnesium bromide in THF (0.5 M solution, 7.5
mmol) at 0 °C. The solution was stirred at 0 °C for 2 h and
then warmed to room temperature and stirred for another 6–
7 h. Saturated aqueous ammonium chloride solution 5 mL
was added, and the mixture was evaporated in vacuo and
partitioned between ethyl acetate and saturated ammonium
chloride solution. The organic layer was washed with brine,
dried over anhydrous Na2SO4, and evaporated in vacuo to get
pure compounds and were used for next step without further
purification.
4.1.1.6. General procedure of 1-arylprop-2-yn-1-one (19a–f).
To the stirred solution of 1-arylprop-2-yn-1-ol (1 mmol) in
dimethylsulfoxide (DMSO), a solution of 2-iodoxy-benzoic
acid (IBX) (1.1 mmol) in dimethyl sulfoxide (DMSO) (10 mL)
was added at 10–15 °C. Then, the reaction mixture was slowly
rise the temperature to RT and allowed to stir for 3–4 h. the
reaction was monitored by TLC using ethyl acetate/hexane
(3 : 7) as a solvent system. Appropriate amount of ice water
was added, and the reaction mixture was filtered through
celite, and the aqueous layer was extracted with ethyl acetate.
4 . 1 . 1 . 3 . 5 . 5 , 6 , 7 - T r i m e t h o x y - 2 - ( 4 -
nitrophenyl)benzoijd]thiazole (13c). Yellow solid, Yield 66%;
1
mp 160–168 °C; H NMR (300 MHz, CDCl3): δ 8.36 (d, J = 8.8
Hz, 2H), 8.23 (d, J = 8.8 Hz, 2H), 7.40 (s, 1H), 4.14 (s, 3H),
3.99 (d, J = 6.4 Hz, 6H); MS (ESI): m/z 347 (M + H)+.
4 . 1 . 1 . 3 . 6 . 5 , 6 , 7 - T r i m e t h o x y - 2 - ( 3 - m e t h y l - 4 -
nitrophenyl)benzoijd]thiazole (14c). Yellow solid, yield 69%;
1
mp 176–179 °C; H NMR (300 MHz, CDCl3): δ 7.96 (d, J = 8.4
Hz, 1H), 7.86 (s, 1H), 7.61 (d, J = 8.3 Hz, 1H), 7.63–7.38 (m,
1H), 7.37 (s, 1H), 4.11 (d, J = 5.7 Hz, 6H), 3.97 (d, J = 5.6 Hz,
6H); MS (ESI): m/z 361 (M + H)+.
4.1.1.4. General method D for the reduction of substituted
2-(4-nitrophenyl)benzothiazoles 15–16IJa–c). To a solution of
substituted 2-(4-nitrophenyl)benzothiazoles 13–14IJa–c) (3 g,
8.66 mmol) in ethanol, tinIJII) chloride dihydrate (3 eq., 25.98
mmol) was added and refluxed it for 3 h. The solvent was
removed under vacuum and the resulting oil taken up in
chloroform (75 mL) was quenched with aq. NaHCO3 solution.
The resulting organic layer was separated and evaporated to
leave a residue of the amine which was purified by column
chromatography (eluent: ethylacetate/hexane) to furnish the
4-aminophenyl-benzothiazoles 15–16IJa–c) as yellow solids.
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The organic layer was washed with water, brine, dried over
anhydrous Na2SO4, and evaporated by using vacuum to get
crude compounds. The compound was purified by column
chromatography and the compound was eluted in ethyl
acetate/hexane (3 : 7) as solvent system.
4.1.1.6.1. 1-(4-((tert-Butyldimethylsilyl)oxy)-3-
methoxyphenyl)prop-2-yn-1-one (19a). Compound 13a was
prepared according to the method described, employing
1-(3-((tert-butyldimethylsilyl)oxy)-4-methoxyphenyl)prop-2-yn-1-
ol (18a, 750 mg, 2.57 mmol) and IBX (790 mg, 2.82 mmol) to
obtain the pure product 19a as a pale yellow solid. (620 mg,
83% yield) mp 125–126 °C; 1H NMR (CDCl3, 300 MHz) δ
(ppm):7.67 (dd, J = 6.41 Hz, 0.92 Hz, 1H), 7.43 (dd, J = 0.92
Hz, 1.52 Hz), 6.73 (d, J = 8.54 Hz, 1H), 3.71 (s, 3H), 3.19 (s,
1H), 0.83 (s, 9H); MS (ESI) m/z 291 [M + H]+.
4.1.1.6.2. 1-(3-Fluoro-4-methoxyphenyl)prop-2-yn-1-one
(19b). Compound 19b was prepared according to the
method described, employing 1-(3-fluoro-4-methoxyphenyl)-
prop-2-yn-1-ol (18b, 750 mg, 2.57 mmol) and IBX (790 mg,
2.82 mmol) to obtain the pure product 19b as a yellow colour
solid. (620 mg, 83% yield) mp 125–126 °C; 1H NMR (300
MHz, CDCl3 + DMSO): δ 7.79 (d, J = 8.6 Hz, 1H), 7.63 (dd, J =
11.5, 2.1 Hz, 1H), 6.90 (dd, J = 11.3, 5.3 Hz, 1H), 3.80 (s, 3H),
3.54 (s, 1H).
4.1.1.6.3. 1-(3-Methoxy-4-nitrophenyl)prop-2-yn-1-one
(19c). Compound 19c was prepared according to the
method described employing 1-(4-methoxy-3-nitrophenyl)-
prop-2-yn-1-ol (18c, 750 mg, 3.62 mmol) and IBX (1.12 g, 3.98
mmol) to obtain the pure product 19c as a pale yellow solid.
(500 mg, 74% yield) mp 125–126 °C; 1H NMR (CDCl3, 500
MHz) δ (ppm): 8.61 (d, J = 2.0 Hz, 1H), 8.31 (dd, J = 2.0 Hz,
6.99 Hz, 1H), 7.19 (d, J = 7.99 Hz, 1H), 4.06 (s, 3H), 3.51 (s,
1H). MS (ESI) m/z 206 [M + H]+.
(s, 3H), 3.89 (s, 3H), 3.50 (s, 1H) ppm; MS (ESI) m/z 298 [M +
H]+.
4.1.1.1.7. General procedure for the synthesis of substituted
(Z)-3-((4-(benzoijd]thiazol-2-yl)phenyl)amino)-1-phenylprop-2-en-
1-ones 5a–r and 6a–r. To the stirred solution of Aryl
propynones 19IJa–f) (5 mmol) in absolute ethanol,
4-aminophenyl-benzothiazoles 15–16IJa–c) (5 mmol) was
added. The reaction was stirred for 4 h at room temperature.
After the completion of reaction (checked by TLC), the
reaction mixture was diluted with water and the crude
product was filtered. The crude product was recrystallized
from methanol to get pure yellow coloured compounds 5a–r
and 6a–r.
4.1.1.7.1. (Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5a). Compound 5a was prepared according to the method
described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline (15a)
(100 mg, 0.39 mmol) and 1-(3-((tert-butyldimethylsilyl)oxy)-4-
methoxyphenyl)prop-2-yn-1-one (19a) (113 mg, 0.39 mmol) to
obtain the pure product 5a1 as a yellow colour solid. 1 M
TBAF in THF (0.87 mL, 3.3 mmol) was added to a stirred
solution of 5a1 (160 mg, 0.29 mmol) in THF (15 mL) at 10–15
°C. Then, the temperature of the mixture was slowly
increased to RT, and the mixture was stirred for 6 h. The
progress of the reaction was monitored by TLC. Upon
completion of the reaction, THF was evaporated, and the
mixture was partitioned between water and EtOAc. The
compound was purified by column chromatography to obtain
pure product 5a as a yellow colour solid. 143 mg, 85% yield;
mp: 180–182 °C; 1H NMR (300 MHz, CDCl3): δ 12.12 (d, J =
12.7 Hz, 1H), 7.54 (d, J = 5.8 Hz, 3H), 7.41 (dd, J = 12.4, 7.8
Hz, 1H), 7.06 (d, J = 8.9 Hz, 3H), 6.91 (dd, J = 8.9, 3.2 Hz,
4H), 5.93 (d, J = 7.8 Hz, 1H), 5.69 (s, 1H), 3.97 (s, 3H), 3.82 (s,
3H). 13C NMR (75 MHz, CDCl3 + TFA): δ 191.24, 168.89,
151.12, 145.98, 145.38, 142.62, 136.28, 131.68, 130.76, 128.62,
127.34, 122.87, 119.86, 118.67, 114.39, 110.65, 105.10, 104.86,
95.68, 56.47, 56.19. MS (ESI): m/z 433 [M + H]+; HRMS calcd
for C24H21O4N2SijM + H]+ 433.12202, found 447.12175.
4.1.1.6.4. 1-(3,4-Dimethoxy-5-nitrophenyl)prop-2-yn-1-one
(19d). Compound 19d was prepared according to the
method described by employing 1-(3,4-dimethoxy-5-
nitrophenyl)prop-2-yn-1-ol (18d, 750 mg, 3.16 mmol) and IBX
(973 mg, 3.48 mmol) to obtain the pure product 19d as a pale
1
yellow solid. (617 mg, 83% yield); mp: 103–104 °C; H NMR
4.1.1.7.2. (Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-en-1-
one (6a). Compound 6a was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (16a) (100 mg, 0.37 mmol) and 1-(3-((tert-
butyldimethylsilyl)oxy)-4-methoxyphenyl)prop-2-yn-1-one (19a)
(100 mg, 0.37 mmol) to obtain the pure product 6a1 as a
yellow colour solid. 1 M TBAF in THF (0.88 mL, 3.3 mmol)
was added to a stirred solution of 6a1 (165 mg, 0.247 mmol)
in THF (15 mL) at 10–15 °C. Then, the temperature of the
mixture was slowly increased to RT, and the mixture was
stirred for 6 h. The progress of the reaction was monitored
by TLC. Upon completion of the reaction, THF was
evaporated, and the mixture was partitioned between water
and EtOAc. The compound was purified by column
chromatography to obtain pure product 6a as a yellow colour
solid. 137 mg, 83% yield; mp: 230–232 °C; 1H NMR (500
MHz, DMSO): δ 12.37 (d, J = 11.7 Hz, 1H), 9.32 (s, 1H), 8.00
(CDCl3, 300 MHz): δ 8.18 (s, 1H), 7.81 (s, 1H), 4.08 (s, 3H),
4.00 (s, 3H), 3.54 (s, 1H) ppm; MS (ESI) m/z 236 [M + H]+.
4.1.1.6.5. 1-(3,4,5-Trimethoxyphenyl)prop-2-yn-1-one
(19e). Compound 19e was prepared according to the
method described by employing 1-(3,4,5-trimethoxyphenyl)-
prop-2-yn-1-ol (18e, 750 mg, 3.38 mmol) and IBX (1.04 g, 3.72
mmol) to obtain the pure product 19e as a light yellow colour
solid. (654 mg, 88% yield) mp: 123–126 °C; H NMR (CDCl3,
300 MHz): δ 7.43 (s, 2H), 3.95 (s, 3H), 3.93 (s, 6H), 3.43 (s,
1H) ppm; MS (ESI) m/z 221 [M + H]+.
1
4.1.1.6.6. 1-(2-Bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-
one (19f). Compound 19f was prepared according to the
method
described
by
employing
1-(2-bromo-3,4,5-
trimethoxyphenyl)prop-2-yn-1-ol (18f, 750 mg, 2.50 mmol) and
IBX (770 mg, 2.75 mmol) to obtain the pure product 19f as a
pale yellow colour solid. (638 mg, 86% yield); mp: 80–81 °C;
1H NMR (CDCl3, 300 MHz): δ 7.46 (s, 1H), 3.98 (s, 3H), 3.93
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(dd, J = 11.6, 7.9 Hz, 1H), 7.92 (s, 1H), 7.88 (dd, J = 8.9, 3.4
Hz, 2H), 7.65 (d, J = 2.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.50
(dd, J = 8.5, 2.0 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.11 (dd, J =
8.9, 2.5 Hz, 1H), 7.00 (d, J = 8.5 Hz, 1H), 6.20 (d, J = 7.9 Hz,
= 4.0 Hz, 1H), 7.98 (t, J = 12.7 Hz, 1H), 7.86 (dd, J = 8.5, 5.4
Hz, 2H), 7.75 (dd, J = 12.1, 8.2 Hz, 1H), 7.33 (t, J = 7.2 Hz,
1H), 7.25 (dd, J = 9.8, 5.7 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H),
7.06–7.02 (m, 1H), 6.86 (dd, J = 8.4, 3.9 Hz, 1H), 6.51 (t, J =
2.2 Hz, 1H), 6.36 (d, J = 12.6 Hz, 1H), 3.80 (s, 3H), 3.70 (d, J =
7.1 Hz, 6H). 13C NMR (75 MHz, CDCl3 + TFA): δ 174.34,
149.49, 148.87, 139.22, 135.85, 135.12, 133.18, 132.03, 129.97,
128.59, 125.46, 124.57, 123.00, 120.27, 120.06, 119.17, 117.67,
109.66, 109.27, 60.92, 60.72, 60.24, 21.50. MS (ESI): m/z 477
[M + H]+; HRMS calcd for C26H25O5N2SijM + H]+ 477.14787,
found 477.14906.
1H), 3.86 (s, 6H), 2.45 (s, 3H). 13C NMR (75 MHz, CDCl3
+
TFA): δ 193.01, 169.73, 151.12, 144.98, 144.47, 144.23, 134.88,
131.38, 130.52, 128.40, 127.60, 122.27, 119.86, 118.67, 114.39,
113.99, 110.65, 105.10, 104.86, 56.29, 56.24, 17.11. MS (ESI):
m/z 447 [M + H]+; HRMS calcd for C25H23O4N2SijM + H]+
447.13005, found 447.13737.
4.1.1.7.3. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-1-one
(5b). Compound 5b was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)aniline (15b)
(100 mg, 0.37 mmol) and 1-(3-((tert-butyldimethylsilyl)oxy)-4-
methoxyphenyl)prop-2-yn-1-one (19a) (103 mg, 0.37 mmol) to
obtain the pure product 5b1 as a yellow colour solid. 1 M
TBAF in THF (0.85 mL, 3.2 mmol) was added to a stirred
solution of 5b1 (163 mg, 0.282 mmol) in THF (15 mL) at 10–
15 °C. Then, the temperature of the mixture was slowly
increased to RT, and the mixture was stirred for 6 h. The
progress of the reaction was monitored by TLC. Upon
completion of the reaction, THF was evaporated, and the
mixture was partitioned between water and EtOAc. The
compound was purified by column chromatography to obtain
pure product 5b as a yellow colour solid. Yield: 138 mg, 86%;
4.1.1.7.5. (Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-((4-(5,6,7-
trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5c). Compound 5c was prepared according to the method
described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline
(15c) (100 mg, 0.31 mmol) and 1-(3-((tert-butyldimethylsilyl)-
oxy)-4-methoxyphenyl)prop-2-yn-1-one (19a) (91 mg, 0.31
mmol) to obtain the pure product 5c1 as a yellow colour
solid. 1 M TBAF in THF (0.79 mL, 2.9 mmol) was added to a
stirred solution of 5c1 (158 mg, 0.260 mmol) in THF (15 mL)
at 10–15 °C. Then, the temperature of the mixture was slowly
increased to RT, and the mixture was stirred for 6 h. The
progress of the reaction was monitored by TLC. Upon
completion of the reaction, THF was evaporated, and the
mixture was partitioned between water and EtOAc. The
compound was purified by column chromatography to obtain
pure product 5c as a yellow colour solid. (135 mg, 87% yield);
1
m.p: 222–224 °C; H NMR (300 MHz, CDCl3 + DMSO): δ 10.17
1
(d, J = 12.7 Hz, 1H), 9.20 (d, J = 4.0 Hz, 1H), 7.98 (t, J = 12.7
Hz, 1H), 7.86 (dd, J = 8.5, 5.4 Hz, 2H), 7.75 (dd, J = 12.1, 8.2
Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H), 7.25 (dd, J = 9.8, 5.7 Hz, 1H),
7.14 (d, J = 8.7 Hz, 1H), 7.06–7.02 (m, 1H), 6.86 (dd, J = 8.4,
3.9 Hz, 1H), 6.51 (t, J = 2.2 Hz, 1H), 6.36 (d, J = 12.6 Hz, 1H),
3.80 (s, 3H), 3.70 (d, J = 7.1 Hz, 6H). 13C NMR (75 MHz,
CDCl3 + TFA): δ 192.05, 171.51, 163.94, 154.78, 151.48,
144.87, 142.60, 130.23, 124.84, 122.65, 120.39, 119.57, 117.71,
114.95, 113.98, 111.21, 110.80, 100.00, 91.45, 56.58, 56.39,
mp: 216–218 °C; H NMR (300 MHz, CDCl3 + DMSO): δ 8.54
(s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.63–7.44 (m, 5H), 7.21 (d, J =
8.5 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 6.08 (d, J = 8.0 Hz, 1H),
4.10 (s, 3H), 3.98–3.91 (m, 9H). 13C NMR (75 MHz, DMSO): δ
189.16, 166.34, 153.85, 151.23, 149.99, 146.39, 143.72, 143.55,
142.66, 142.09, 139.20, 139.13, 132.09, 131.44, 128.52, 128.38,
127.26, 126.26, 119.81, 119.68, 118.90, 118.76, 116.33, 115.52,
114.28, 114.06, 111.28, 100.88, 99.24, 94.53, 60.98, 60.31,
56.23, 55.61. MS (ESI): m/z 493 [M + H]+; HRMS calcd for
C26H25O6N2SijM + H]+ 493.14278, found 493.14189.
56.22. MS (ESI): m/z 463 [M
+
H]+; HRMS calcd for
C25H23O5N2SijM + H]+ 463.13222, found 463.13149.
4.1.1.7.6. (Z)-1-(3-Hydroxy-4-methoxyphenyl)-3-((2-methyl-
4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-
1-one (6c). Compound 6c was prepared according to the
method described by 2-methyl-4-(5,6,7-trimethoxybenzoijd]-
thiazol-2-yl)aniline (16c) (100 mg, 0.3 mmol) and 1-(3-((tert-
butyldimethylsilyl)oxy)-4-methoxyphenyl)prop-2-yn-1-one (19a)
(87 mg, 0.3 mmol) to obtain the pure product 6c1 as a yellow
colour solid. 1 M TBAF in THF (0.72 mL, 2.7 mmol) was
added to a stirred solution of 6c1 (148 mg, 0.238 mmol) in
THF (15 mL) at 10–15 °C. Then, the temperature of the
mixture was slowly increased to RT, and the mixture was
stirred for 6 h. The progress of the reaction was monitored
by TLC. Upon completion of the reaction, THF was
evaporated, and the mixture was partitioned between water
and EtOAc. The compound was purified by column
chromatography to obtain pure product 6c as a yellow colour
solid. 127 mg, 83% yield; mp: 190–192 °C; 1H NMR (500
MHz, CDCl3): δ 12.34 (d, J = 11.8 Hz, 1H), 7.92 (d, J = 1.2 Hz,
1H), 7.89 (dd, J = 8.4, 2.0 Hz, 1H), 7.62–7.58 (m, 1H), 7.58–
4.1.1.7.4. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)-2-
methylphenyl)amino)-1-(3-hydroxy-4-methoxyphenyl)prop-2-en-
1-one (6b). Compound 6b was prepared according to the
method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-
methylaniline (16b) (100 mg, 0.34 mmol) and 1-(3-((tert-
butyldimethylsilyl)oxy)-4-methoxyphenyl)prop-2-yn-1-one (19a)
(96 mg, 0.33 mmol) to obtain the pure product 6b1 as a
yellow colour solid. 1 M TBAF in THF (0.79 mL, 3.0 mmol)
was added to a stirred solution of 6b1 (156 mg, 0.264 mmol)
in THF (15 mL) at 10–15 °C. Then, the temperature of the
mixture was slowly increased to RT, and the mixture was
stirred for 6 h. The progress of the reaction was monitored
by TLC. Upon completion of the reaction, THF was
evaporated, and the mixture was partitioned between water
and EtOAc. The compound was purified by column
chromatography to obtain pure product 6b as a yellow colour
solid. (134 mg, 85% yield); mp: 242–244 °C; 1H NMR (300
MHz, CDCl3 + DMSO): δ 10.17 (d, J = 12.7 Hz, 1H), 9.20 (d, J
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7.54 (m, 2H), 7.33 (s, 1H), 7.24 (d, J = 7.8 Hz, 1H), 6.92–6.90
(m, 1H), 6.09 (d, J = 7.9 Hz, 1H), 5.70 (s, 1H), 4.10 (s, 3H),
3.95 (d, J = 7.8 Hz, 10H), 3.94 (s, 4H), 2.52 (s, 3H). 13C NMR
(75 MHz, TFA): δ 192.77, 171.22, 157.51, 150.94, 146.70,
145.23, 144.51, 143.54, 140.92, 137.48, 131.24, 131.07, 128.10,
127.61, 121.87, 119.86, 114.90, 114.11, 113.92, 110.48, 94.51,
61.84, 61.38, 56.72, 56.14, 17.20. MS (ESI): m/z 507 [M + H]+;
HRMS calcd for C27H27O6N2SijM + H]+ 507.15843, found
507.15696.
[M + H]+; HRMS calcd for C25H22O4N2FSijM + H]+ 465.12788,
found 465.12727.
4.1.1.7.10. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)-2-
methylphenyl)amino)-1-(3-fluoro-4-methoxyphenyl)prop-2-en-1-
one (6e). Compound 6e was prepared according to the
method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10b) (100 mg, 0.33 mmol) and 1-(3-fluoro-4-
methoxyphenyl)prop-2-yn-1-one (13b) (62 mg, 0.33 mmol) to
obtain the pure product 6e as a yellow colour solid. (128 mg,
80% yield); mp: 229–231 °C; 1H NMR (500 MHz, CDCl3): δ
12.34 (d, J = 11.9 Hz, 1H), 7.93 (dd, J = 14.0, 5.6 Hz, 1H), 7.73
(dd, J = 16.1, 5.1 Hz, 2H), 7.62 (dd, J = 11.8, 8.0 Hz, 1H), 7.25
(d, J = 6.4 Hz, 2H), 7.17 (d, J = 2.0 Hz, 1H), 7.01 (t, J = 8.5 Hz,
1H), 6.49 (d, J = 2.0 Hz, 1H), 6.07 (d, J = 7.9 Hz, 1H), 3.96 (d,
J = 2.8 Hz, 6H), 3.90 (s, 3H), 2.52 (s, 3H). 13C NMR (75 MHz,
CDCl3 + TFA): δ 192.04, 172.29, 163.92, 154.79, 152.18,
152.04, 144.92, 144.45, 142.57, 131.33, 130.52, 128.61, 127.59,
125.50, 120.15, 115.80, 115.55, 114.60, 113.14, 110.84, 99.92,
91.45, 56.56, 56.42, 56.21, 17.04. MS (ESI): m/z 479 [M + H]+;
HRMS calcd for C26H23O4N2FSijM + H]+ 479.1362, found
479.1432.
4.1.1.7.11. (Z)-1-(3-Fluoro-4-methoxyphenyl)-3-((4-(5,6,7-
trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5f). Compound 5f was prepared according to the method
described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline
(9c) (100 mg, 0.31 mmol) and 1-(3-fluoro-4-methoxyphenyl)-
prop-2-yn-1-one (13b) (56 mg, 0.31 mmol) to obtain the pure
product 5f as a yellow colour solid. (132 mg, 85% yield); mp:
143–145 °C; 1H NMR (400 MHz, CDCl3): δ 12.17 (d, J = 12.1
Hz, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 7.3 Hz, 1H), 7.69
(d, J = 1.9 Hz, 1H), 7.53 (dd, J = 12.1, 8.0 Hz, 1H), 7.33 (s,
1H), 7.17 (d, J = 8.7 Hz, 2H), 7.01 (t, J = 8.5 Hz, 1H), 6.01 (d, J
= 8.0 Hz, 1H), 4.10 (s, 3H), 3.96 (d, J = 1.6 Hz, 6H), 3.94 (s,
3H). 13C NMR (100 MHz, CDCl3): δ 189.04, 167.10, 154.03,
150.85, 150.56, 146.77, 143.47, 142.21, 139.69, 132.13, 132.08,
128.85, 124.25, 120.05, 116.19, 115.32, 115.13, 112.46, 100.59,
94.41, 61.51, 60.57, 56.29. MS (ESI): m/z 495 [M + H]+; HRMS
calcd for C26H24O5N2FSijM + H]+ 495.13845, found 495.13760.
4.1.1.7.12. (Z)-1-(3-Fluoro-4-methoxyphenyl)-3-((2-methyl-4-
(5,6,7-trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-
one (6f). Compound 6f was prepared according to the
method described by 2-methyl-4-(5,6,7-trimethoxybenzoijd]-
thiazol-2-yl)aniline (10c) (100 mg, 0.30 mmol) and
1-(3-fluoro-4-methoxyphenyl)prop-2-yn-1-one (13b) (59 mg,
0.30 mmol) to obtain the pure product 6f as a yellow colour
solid. (126 mg, 82% yield); mp: 190–191 °C; 1H NMR (400
MHz, CDCl3): δ 12.34 (d, J = 11.8 Hz, 1H), 7.94–7.88 (m,
2H), 7.77–7.70 (m, 2H), 7.61 (dd, J = 11.8, 8.0 Hz, 1H), 7.33
(s, 1H), 7.24 (s, 1H), 7.01 (t, J = 8.5 Hz, 1H), 6.07 (d, J = 7.9
Hz, 1H), 4.10 (s, 3H), 3.96 (s, 6H), 3.94 (s, 3H), 2.52 (s, 3H).
13C NMR (75 MHz, CDCl3 + DMSO) δ 188.20, 166.47, 153.23,
149.74, 145.93, 143.10, 140.11, 138.80, 131.36, 129.02,
127.48, 125.73, 125.56, 123.63, 119.07, 114.43, 114.18,
112.67, 111.81, 99.80, 94.04, 60.69, 59.76, 55.53, 16.94. MS
(ESI): m/z 509 [M + H]+; HRMS calcd for C27H26O5N2FSijM +
H]+ 509.15410, found 509.15216.
4.1.1.7.7. (Z)-1-(3-Fluoro-4-methoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5d). Compound 15a was prepared according to the method
described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline (9a) (100
mg, 0.39 mmol) and 1-(3-fluoro-4-methoxyphenyl)prop-2-yn-1-
one (13b) (70 mg, 0.39 mmol) to obtain the pure product 5d
as a yellow colour solid (149 mg, 88% yield); mp: 189–191 °C;
1H NMR (400 MHz, CDCl3): δ 12.11 (d, J = 12.3 Hz, 1H), 8.40
(d, J = 1.5 Hz, 1H), 8.09–8.05 (m, 1H), 7.94 (d, J = 9.0 Hz, 1H),
7.73–7.68 (m, 2H), 7.41 (dd, J = 12.4, 7.7 Hz, 1H), 7.06–7.03
(m, 2H), 6.99 (t, J = 8.5 Hz, 1H), 6.92–6.87 (m, 2H), 5.89 (d, J
= 7.8 Hz, 1H), 3.95 (s, 3H), 3.80 (s, 3H). 13C NMR (75 MHz,
CDCl3 + TFA): δ 190.09, 169.87, 155.87, 151.12, 146.78,
146.47, 138.98, 134.88, 131.64, 130.52, 128.26, 127.61, 122.53,
119.86, 118.67, 114.54, 113.99, 105.10, 104.86, 94.93, 56.38,
56.29. MS (ESI): m/z 435 [M
+
H]+; HRMS calcd for
C24H20O3N2SFijM + H]+ 435.1100, found 435.1172.
4.1.1.7.8. (Z)-1-(3-Fluoro-4-methoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-en-1-
one (6d). Compound 6d was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10a) (100 mg, 0.37 mmol) and 1-(3-fluoro-4-
methoxyphenyl)prop-2-yn-1-one (13b) (65 mg, 0.37 mmol) to
obtain the pure product 6d as a yellow colour solid. (135 mg,
82% yield); mp: 132–135 °C; 1H NMR (500 MHz, DMSO): δ
12.31 (d, J = 12.27 Hz, 1H), 9.33 (s, 1H), 8.01 (dd, J = 12.31,
7.9 Hz, 1H), 7.93 (s, 1H), 7.89 (dd, J = 8.8, 3.4 Hz, 2H), 7.65
(d, J = 2.5 Hz, 1H), 7.59 (d, J = 8.7 Hz, 1H), 7.50 (dd, J = 8.5,
2.0 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.11 (dd, J = 8.8, 2.5 Hz,
1H), 7.00 (d, J = 8.5 Hz, 1H), 6.20 (d, J = 7.9 Hz, 1H), 3.86 (s,
6H), 2.45 (s, 3H). MS (ESI): m/z 449 [M + H]+; HRMS calcd for
C25H22O3N2SFijM + H]+ 449.13320, found 449.13378.
4.1.1.7.9. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(3-fluoro-4-methoxyphenyl)prop-2-en-1-one
(5e). Compound 5e was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)aniline (9b)
(100 mg, 0.35 mmol) and 1-(3-fluoro-4-methoxyphenyl)prop-2-
yn-1-one (13b) (62 mg, 0.35 mmol) to obtain the pure product
5e as a yellow colour solid. (134 mg, 83% yield); mp: 190–192
1
°C; H NMR (400 MHz, CDCl3): δ 12.17 (d, J = 12.1 Hz, 1H),
8.05 (d, J = 8.7 Hz, 1H), 7.75–7.68 (m, 1H), 7.53 (dd, J = 12.1,
8.0 Hz, 1H), 7.20–7.15 (m, 1H), 7.01 (dd, J = 10.8, 6.2 Hz, 1H),
6.49 (d, J = 2.0 Hz, 1H), 6.02 (d, J = 8.0 Hz, 1H), 3.96 (d, J =
2.5 Hz, 6H), 3.90 (s, 3H). 13C NMR (75 MHz, CDCl3 + TFA): δ
191.20, 185.30, 172.58, 171.80, 164.69, 155.36, 146.62, 145.87,
143.20, 130.44, 128.00, 125.97, 124.22, 120.22, 118.36, 112.84,
108.99, 100.75, 100.20, 91.46, 56.65, 56.28. MS (ESI): m/z 465
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4.1.1.7.13. (Z)-1-(4-methoxy-3-nitrophenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5g). Compound 5g was prepared according to the method
described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline (9a) (100
mg, 0.39 mmol) and 1-(4-methoxy-3-nitrophenyl)prop-2-yn-1-
one (13c) (80 mg, 0.39 mmol) to obtain the pure product 5g
as a yellow colour solid. (142 mg, 79% yield); mp: 240–243
methylaniline (10b) (100 mg, 0.33 mmol) and 1-(4-methoxy-3-
nitrophenyl)prop-2-yn-1-one (13c) (68 mg, 0.33 mmol) to
obtain the pure product 6h as a yellow colour solid. (131 mg,
78% yield); mp: 272–274 °C; 1H NMR (500 MHz, CDCl3): δ
12.24 (d, J = 11.9 Hz, 1H), 7.91 (dd, J = 14.0, 5.6 Hz, 1H), 7.77
(dd, J = 16.1, 5.1 Hz, 2H), 7.61 (dd, J = 11.9, 7.9 Hz, 1H), 7.23
(d, J = 6.4 Hz, 2H), 7.14 (d, J = 2.0 Hz, 1H), 7.00 (t, J = 8.5 Hz,
1H), 6.45 (d, J = 2.0 Hz, 1H), 6.05 (d, J = 7.9 Hz, 1H), 3.96 (d,
J = 2.8 Hz, 6H), 3.90 (s, 3H), 2.52 (s, 3H). 13C NMR (75 MHz,
CDCl3 + TFA): δ 190.58, 172.34, 163.91, 156.60, 154.79,
145.36, 144.32, 142.60, 138.99, 134.18, 131.34, 130.18, 128.70,
127.66, 125.97, 120.38, 114.77, 114.05, 110.88, 99.94, 91.53,
57.00, 56.59, 56.22, 17.27. MS (ESI): m/z 506 [M + H]+; HRMS
calcd for C26H24O6N3SijM + H]+ 506.13832, found 506.13793.
4.1.1.7.17. (Z)-1-(4-Methoxy-3-nitrophenyl)-3-((4-(5,6,7-
trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5i). Compound 5i was prepared according to the method
described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline
(9c) (100 mg, 0.31 mmol) and 1-(4-methoxy-3-nitrophenyl)-
prop-2-yn-1-one (13c) (64 mg, 0.31 mmol) to obtain the pure
product 5i as a yellow colour solid. (136 mg, 83% yield); mp:
194–196 °C; 1H NMR (500 MHz, CDCl3): δ 12.20 (d, J = 12.2
Hz, 1H), 8.44 (d, J = 2.0 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.03
(d, J = 8.5 Hz, 2H), 7.58 (dd, J = 12.2, 7.9 Hz, 1H), 7.33 (s,
1H), 7.19 (d, J = 8.5 Hz, 2H), 7.16 (d, J = 8.8 Hz, 1H), 6.03 (d,
J = 7.9 Hz, 1H), 4.10 (s, 3H), 4.03 (s, 3H), 3.96 (s, 3H), 3.94 (s,
3H). 13C NMR (101 MHz, CDCl3): δ 187.55, 166.95, 155.37,
154.07, 150.52, 146.75, 144.38, 141.87, 139.73, 139.30, 133.19,
131.29, 129.21, 128.87, 125.16, 120.07, 116.41, 113.26, 100.59,
93.91, 61.51, 60.58, 56.83, 56.31. MS (ESI): m/z 522 [M + H]+;
HRMS calcd for C26H24O7N3SijM + H]+ 522.13295, found
522.13245.
4.1.1.7.18. (Z)-1-(4-Methoxy-3-nitrophenyl)-3-((2-methyl-4-
(5,6,7-trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-
one (6i). Compound 6i was prepared according to the
method described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-
yl)aniline (10c) (100 mg, 0.30 mmol) and 1-(4-methoxy-3-
nitrophenyl)prop-2-yn-1-one (13c) (62 mg, 0.3 mmol) to obtain
the pure product 6i as a yellow colour solid. (128 mg, 79%
yield); mp: 210–211 °C; 1H NMR (400 MHz, CDCl3): δ 12.38
(d, J = 12.0 Hz, 1H), 8.46 (d, J = 2.2 Hz, 1H), 8.18 (dd, J = 8.8,
2.2 Hz, 1H), 7.91 (dd, J = 12.5, 4.0 Hz, 1H), 7.66 (dd, J = 12.0,
7.9 Hz, 1H), 7.33 (s, 1H), 7.27 (d, J = 6.9 Hz, 1H), 7.16 (d, J =
8.9 Hz, 1H), 6.09 (d, J = 7.8 Hz, 1H), 4.10 (s, 3H), 4.03 (s, 3H),
3.96 (s, 3H), 3.94 (s, 3H), 2.52 (s, 3H). 13C NMR (100 MHz,
CDCl3): δ 187.60, 167.26, 155.33, 154.04, 150.54, 146.76,
144.53, 140.53, 139.68, 139.33, 133.17, 131.37, 129.95, 128.83,
126.75, 126.49, 125.16, 120.03, 113.59, 113.23, 100.56, 94.23,
61.51, 60.57, 56.82, 56.31, 17.70. MS (ESI): m/z 536 [M + H]+;
HRMS calcd for C27H26O7N3SijM + H]+ 536.14860, found
536.14724.
1
°C; H NMR (400 MHz, CDCl3): δ 12.11 (d, J = 12.1 Hz, 1H),
8.40 (d, J = 1.5 Hz, 1H), 8.08–8.04 (m, 1H), 7.96 (d, J = 9.0 Hz,
1H), 7.72–7.66 (m, 2H), 7.42 (dd, J = 12.1, 7.7 Hz, 1H), 7.03–
7.01 (m, 2H), 6.97 (t, J = 8.5 Hz, 1H), 6.91–6.86 (m, 2H), 5.85
(d, J = 7.7 Hz, 1H), 3.94 (s, 3H), 3.81 (s, 3H). 13C NMR (75
MHz, TFA): δ 191.22, 170.05, 146.12, 138.90, 137.28, 134.76,
134.61, 130.96, 130.28, 126.27, 121.27, 120.65, 118.47, 118.21,
114.40, 105.34, 100.35, 57.04, 56.27. MS (ESI): m/z 462 [M +
H]+; HRMS calcd for C24H20O5N3SijM + H]+ 462.11285, found
462.11243.
4.1.1.7.14. (Z)-1-(4-methoxy-3-nitrophenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-en-1-
one (6g). Compound 6g was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10a) (100 mg, 0.37 mmol) and 1-(4-methoxy-3-
nitrophenyl)prop-2-yn-1-one (13c) (76 mg, 0.37 mmol) to
obtain the pure product 6g as a yellow colour solid. (143 mg,
82% yield); mp: 270–272 °C; 1H NMR (500 MHz, DMSO): δ
12.21 (d, J = 12.11 Hz, 1H), 9.29 (s, 1H), 8.00 (dd, J = 12.11,
7.9 Hz, 1H), 7.94 (s, 1H), 7.84 (dd, J = 8.8, 3.4 Hz, 2H), 7.64
(d, J = 2.5 Hz, 1H), 7.57 (d, J = 8.7 Hz, 1H), 7.49 (dd, J = 8.5,
2.0 Hz, 1H), 7.41 (d, J = 2.0 Hz, 1H), 7.10 (dd, J = 8.8, 2.5 Hz,
1H), 7.00 (d, J = 8.5 Hz, 1H), 6.20 (d, J = 7.9 Hz, 1H), 3.86 (s,
6H). 13C NMR (75 MHz, TFA): δ 190.97, 170.12, 146.07,
144.31, 138.86, 134.61, 131.32, 130.91, 130.27, 129.12, 127.71,
126.25, 120.48, 118.44, 115.07, 114.30, 105.28, 98.40, 57.01,
56.24, 16.96. MS (ESI): m/z 476 [M + H]+; HRMS calcd for
C25H22O5N3SijM + H]+ 476.12834, found 476.12784.
4.1.1.7.15. (Z)-3-((4-(5,7-dimethoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(4-methoxy-3-nitrophenyl)prop-2-en-1-one
(5h). Compound 5h was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)aniline (9b)
(100 mg, 0.35 mmol) and 1-(4-methoxy-3-nitrophenyl)prop-2-
yn-1-one (13c) (71 mg, 0.35 mmol) to obtain the pure product
5h as a yellow colour solid. (133 mg, 78% yield); mp: 229–232
1
°C; H NMR (400 MHz, CDCl3): δ 12.20 (d, J = 12.1 Hz, 1H),
8.44 (d, J = 2.2 Hz, 1H), 8.18 (dd, J = 8.8, 2.2 Hz, 1H), 8.07 (d,
J = 8.7 Hz, 2H), 7.60 (dd, J = 12.3, 7.9 Hz, 1H), 7.20 (d, J = 8.7
Hz, 2H), 7.17 (dd, J = 5.4, 3.4 Hz, 2H), 6.50 (d, J = 2.0 Hz,
1H), 6.04 (d, J = 7.9 Hz, 1H), 4.04 (s, 3H), 3.97 (s, 3H), 3.91 (s,
3H). 13C NMR (75 MHz, CDCl3 + TFA): δ 172.41, 164.10,
155.00, 145.77, 142.56, 138.93, 134.37, 130.27, 126.08, 120.82,
118.08, 114.28, 111.11, 100.10, 91.49, 57.04, 56.63, 56.23. MS
(ESI): m/z 514 [M + Na]+; HRMS calcd for C25H21O6N3SNaijM +
H]+ 514.10433, found 514.10371.
4.1.1.7.16. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)-2-
methylphenyl)amino)-1-(4-methoxy-3-nitrophenyl)prop-2-en-1-
one (6h). Compound 6h was prepared according to the
method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-
4.1.1.7.19. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5j). Compound 5j was prepared according to the method
described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline (9a) (100
mg, 0.39 mmol) and 1-(3,4-dimethoxy-5-nitrophenyl)prop-2-yn-
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1-one (13d) (91 mg, 0.39 mmol) to obtain the pure product 5j
as a yellow colour solid. (157 mg, 82% yield); mp: 238–241
°C; H NMR (400 MHz, CDCl3): δ 12.21 (d, J = 12.3 Hz, 1H),
4.1.1.7.23. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((4-(5,6,7-
trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5l). Compound 5l was prepared according to the method
described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline
(9c) (100 mg, 0.31 mmol) and 1-(3,4-dimethoxy-5-
nitrophenyl)prop-2-yn-1-one (13d) (74 mg, 2.75 mmol) to
obtain the pure product 5l as a yellow colour solid. (144 mg,
83% yield); mp: 175–178 °C; 1H NMR (400 MHz, CDCl3): δ
12.22 (d, J = 12.3 Hz, 1H), 8.05 (d, J = 8.5 Hz, 2H), 7.88 (d, J =
1.6 Hz, 1H), 7.77 (d, J = 1.6 Hz, 1H), 7.62 (dd, J = 12.3, 7.9 Hz,
1H), 7.34 (s, 1H), 7.21 (d, J = 8.6 Hz, 2H), 6.05 (d, J = 7.9 Hz,
1H), 4.11 (s, 3H), 4.05 (s, 3H), 4.02 (s, 3H), 3.96 (d, J = 7.9 Hz,
6H). 13C NMR (100 MHz, CDCl3): δ 187.65, 166.89, 154.22,
154.10, 150.52, 146.76, 145.67, 144.75, 144.36, 141.73, 139.76,
134.25, 129.42, 128.90, 120.10, 116.51, 115.43, 114.31, 100.60,
93.97, 62.17, 61.51, 60.60, 56.60, 56.31. MS (ESI): m/z 552 [M
+ H]+; HRMS calcd for C27H26O8N3SijM + H]+ 552.14351, found
552.14277.
1
8.04 (d, J = 8.7 Hz, 2H), 7.92 (d, J = 8.9 Hz, 1H), 7.87 (d, J =
1.9 Hz, 1H), 7.76 (d, J = 1.9 Hz, 1H), 7.60 (dd, J = 12.3, 7.9 Hz,
1H), 7.35 (d, J = 2.5 Hz, 1H), 7.20 (d, J = 8.7 Hz, 2H), 7.09 (dd,
J = 8.9, 2.5 Hz, 1H), 6.03 (d, J = 7.9 Hz, 1H), 4.05 (s, 3H), 4.01
(s, 3H), 3.89 (s, 3H). MS (ESI): m/z 492 [M + H]+; HRMS calcd
for C25H22O6N3SijM + H]+ 492.1151, found 492.1229.
4.1.1.7.20. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-en-1-
one (6j). Compound 6j was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10a) (100 mg, 0.37 mmol) and 1-(3,4-
dimethoxy-5-nitrophenyl)prop-2-yn-1-one (13d) (187 mg, 0.37
mmol) to obtain the pure product 6j as a yellow colour solid.
(157 mg, 84% yield); mp: 220–223 °C; 1H NMR (400 MHz,
CDCl3): δ 12.35 (d, J = 12.1 Hz, 1H), 7.94–7.91 (m, 2H), 7.89 (t,
J = 2.4 Hz, 2H), 7.78 (d, J = 1.9 Hz, 1H), 7.70–7.64 (m, 1H), 7.35
(d, J = 2.5 Hz, 1H), 7.28 (d, J = 8.8 Hz, 1H), 7.09 (dd, J = 8.9, 2.6
Hz, 1H), 6.08 (d, J = 7.8 Hz, 1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.89
(s, 3H), 2.53 (s, 3H). MS (ESI): m/z 506 [M + H]+; HRMS calcd
for C26H24O6N3SijM + H]+ 506.13803, found 506.13987.
4.1.1.7.24. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((2-
m e t h y l - 4 - ( 5 , 6 , 7 - t r i m e t h o x y b e n z o ij d ] t h i a z o l - 2 -
yl)phenyl)amino)prop-2-en-1-one (6l). Compound 6l was
prepared according to the method described by 2-methyl-4-
(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline (10c) (100 mg, 0.3
mmol) and 1-(3,4-dimethoxy-5-nitrophenyl)prop-2-yn-1-one
(13d) (71 mg, 0.3 mmol) to obtain the pure product 6l as a
4.1.1.7.21. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((4-(5,7-
dimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5k). Compound 5k was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)aniline (9b)
(100 mg, 0.35 mmol) and 1-(3,4-dimethoxy-5-nitrophenyl)prop-
2-yn-1-one (13d) (82 mg, 0.35 mmol) to obtain the pure
product 5k as a yellow colour solid. (147 mg, 81% yield); mp:
1
yellow colour solid. (143 mg, 84% yield); mp: 232–234 °C; H
NMR (500 MHz, CDCl3): δ 12.36 (d, J = 12.0 Hz, 1H), 7.95 (s,
1H), 7.92 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.79 (d,
J = 1.7 Hz, 1H), 7.69 (dd, J = 12.0, 7.8 Hz, 1H), 7.34 (s, 1H),
7.29 (d, J = 8.5 Hz, 1H), 6.10 (d, J = 7.8 Hz, 1H), 4.11 (s, 3H),
4.05 (s, 3H), 4.02 (s, 3H), 3.96 (d, J = 9.4 Hz, 6H), 2.53 (s, 3H).
13C NMR (100 MHz, CDCl3): δ 187.72, 167.22, 154.24, 154.07,
146.77, 145.61, 144.87, 144.38, 140.43, 134.40, 129.96, 129.04,
126.83, 126.54, 123.47, 120.08, 115.62, 115.44, 114.37, 113.79,
104.25, 100.58, 94.33, 62.17, 61.52, 60.59, 56.65, 56.32, 17.71.
MS (ESI): m/z 566 [M + H]+; HRMS calcd for C26H24O7N3SijM +
H]+ 566.15968, found 566.15829.
1
174–175 °C; H NMR (500 MHz, CDCl3 + TFA): δ 12.18–12.12
(m, 1H), 8.13 (s, 2H), 7.90 (s, 1H), 7.77 (s, 1H), 7.65 (s, 1H),
7.49 (s, 2H), 7.08 (s, 1H), 6.72 (s, 1H), 6.22 (s, 1H), 4.10 (s,
3H), 4.06 (s, 3H), 4.02 (s, 3H), 3.93 (s, 3H). 13C NMR (75
MHz, CDCl3 + TFA): δ 190.34, 172.12, 163.91, 154.81, 154.46,
146.13, 145.44, 143.82, 142.37, 133.19, 130.13, 120.82, 117.93,
116.34, 114.80, 111.00, 99.99, 91.32, 62.40, 56.43, 56.06. MS
(ESI): m/z 522 [M + H]+; HRMS calcd for C26H24O7N3SijM + H]+
522.13295, found 522.13227.
4.1.1.7.22. (Z)-1-(3,4-Dimethoxy-5-nitrophenyl)-3-((4-(5,7-
dimethoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-
en-1-one (6k). Compound 6k was prepared according to the
method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10b) (100 mg, 0.33 mmol) and 1-(3,4-
dimethoxy-5-nitrophenyl)prop-2-yn-1-one (13d) (78 mg, 2.75
mmol) to obtain the pure product 6k as a yellow colour solid.
(142 mg, 80% yield); mp: 246–248 °C; 1H NMR (500 MHz,
CDCl3): δ 12.37–12.29 (m, 1H), 8.05 (s, 1H), 7.92 (d, J = 1.6
Hz, 2H), 7.76 (d, J = 1.6 Hz, 2H), 7.49 (s, 1H), 7.31 (d, J = 11.0
Hz, 1H), 7.10 (s, 1H), 6.69 (s, 1H), 6.30–6.23 (m, 1H), 4.05 (d,
J = 17.7 Hz, 9H), 3.93 (s, 3H), 2.55 (s, 3H). 13C NMR (100
MHz, CDCl3): δ 189.22, 163.61, 154.56, 154.30, 146.07, 144.34,
144.26, 143.84, 133.42, 131.11, 127.97, 127.86, 123.07, 120.03,
115.71, 114.43, 114.00, 110.66, 99.51, 62.10, 56.47, 56.44,
56.11, 17.25. MS (ESI): m/z 536 [M + H]+; HRMS calcd for
C27H26O7N3SijM + H]+ 536.14132, found 536.1498.
4.1.1.7.25. (Z)-3-((4-(6-Methoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(5m). Compound 5m was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline
(9a) (100 mg, 0.39 mmol) and 1-(3,4,5-trimethoxyphenyl)prop-
2-yn-1-one (13e) (85 mg, 0.85 mmol) to obtain the pure
product 5m as a yellow colour solid. (159 mg, 86% yield);
mp: 208–210 °C; 1H NMR (300 MHz, CDCl3): δ 12.23 (d, J =
12.1 Hz, 1H), 8.02 (d, J = 8.6 Hz, 2H), 7.92 (d, J = 8.9 Hz, 1H),
7.54 (dd, J = 12.1, 8.0 Hz, 1H), 7.34 (d, J = 2.4 Hz, 1H), 7.21
(s, 2H), 7.17 (d, J = 8.7 Hz, 2H), 7.08 (dd, J = 8.9, 2.5 Hz, 1H),
6.05 (d, J = 8.0 Hz, 1H), 3.94 (d, J = 6.7 Hz, 9H), 3.89 (s, 4H).
13C NMR (75 MHz, CDCl3): δ 190.20, 164.64, 157.64, 153.06,
148.67, 143.54, 141.91, 141.47, 136.21, 134.25, 128.96, 128.80,
123.43, 116.10, 115.59, 104.68, 104.13, 94.58, 60.97, 56.23,
55.77. MS (ESI): m/z 477 [M
+
H]+; HRMS calcd for
C26H25O5N2SijM + H]+ 477.14787, found 477.14705.
4.1.1.7.26. (Z)-3-((4-(6-Methoxybenzoijd]thiazol-2-yl)-2-
methylphenyl)amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
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(6m). Compound 6m was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10a) (100 mg, 0.37 mmol) and 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one (13e) (81 mg, 0.37 mmol) to
obtain the pure product 6m as a yellow colour solid. (159 mg,
88% yield); mp: 207–209 °C; 1H NMR (300 MHz, CDCl3): δ
12.33 (d, J = 11.9 Hz, 1H), 7.91 (t, J = 7.8 Hz, 3H), 7.62 (dd, J
= 11.7, 8.0 Hz, 1H), 7.35 (d, J = 2.3 Hz, 1H), 7.29–7.21 (m,
3H), 7.08 (dd, J = 8.9, 2.4 Hz, 1H), 6.10 (d, J = 7.9 Hz, 1H),
3.96 (s, 6H), 3.93 (s, 3H), 3.89 (s, 3H), 2.53 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ 190.38, 164.99, 157.65, 153.10, 148.73,
143.66, 141.53, 140.67, 136.22, 134.47, 129.89, 128.61, 126.55,
126.44, 123.39, 115.53, 113.34, 104.85, 104.23, 95.04, 60.97,
56.30, 55.80, 17.75. MS (ESI): m/z 491 [M + H]+; HRMS calcd
for C27H27O5N2SijM + H]+ 491.16352, found 491.16282.
142–144 °C; 1H NMR (500 MHz, CDCl3): δ 12.24 (d, J = 12.1
Hz, 1H), 8.04 (d, J = 8.6 Hz, 2H), 7.56 (dd, J = 12.1, 8.0 Hz,
1H), 7.33 (s, 1H), 7.22 (s, 2H), 7.19 (d, J = 8.7 Hz, 2H), 6.07
(d, J = 8.0 Hz, 1H), 4.11 (s, 3H), 3.96 (s, 3H), 3.95 (d, J = 4.1
Hz, 9H), 3.93 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 190.31,
167.06, 154.03, 153.10, 150.54, 146.74, 143.51, 142.17, 141.62,
139.70, 134.27, 128.86, 120.05, 116.18, 104.83, 100.58, 94.73,
61.49, 60.96, 60.56, 56.28. MS (ESI): m/z 537 [M + H]+; HRMS
calcd for C28H29O7N2SijM + H]+ 537.16900, found 537.16716.
4 . 1 . 1 . 7 . 3 0 . ( Z ) - 3 - ( ( 2 - M e t h y l - 4 - ( 5 , 6 , 7 -
trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)-1-(3,4,5-
trimethoxyphenyl)prop-2-en-1-one (6o). Compound 6o was
prepared according to the method described by 2-methyl-4-
(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline (10c) (100 mg, 0.3
mmol) and 1-(3,4,5-trimethoxyphenyl)prop-2-yn-1-one (13e) (66
mg, 0.3 mmol) to obtain the pure product 6o as a yellow
4.1.1.7.27. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(5n). Compound 5n was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)aniline (9b)
(100 mg, 0.35 mmol) and 1-(3,4,5-trimethoxyphenyl)prop-2-yn-
1-one (13e) (76 mg, 0.35 mmol) to obtain the pure product 5n
as a yellow colour solid. (153 mg, 87% yield); mp: 175–177
1
colour solid. (139 mg, 84% yield); mp: 173–175 °C; H NMR
(300 MHz, CDCl3): δ 12.33 (d, J = 11.9 Hz, 1H), 7.92 (d, J =
11.2 Hz, 2H), 7.63 (dd, J = 11.8, 8.0 Hz, 1H), 7.34 (s, 1H), 7.27
(t, J = 4.2 Hz, 1H), 7.23 (s, 2H), 6.11 (d, J = 7.9 Hz, 1H), 4.11
(s, 3H), 3.98–3.92 (m, 16H), 2.53 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 190.44, 167.38, 154.01, 153.12, 150.57, 146.76,
143.62, 141.58, 140.87, 139.66, 134.46, 129.91, 128.48, 126.59,
126.47, 120.02, 113.37, 104.88, 100.56, 95.15, 61.49, 60.96,
60.55, 56.31, 17.74. MS (ESI): m/z 551 [M + H]+; HRMS calcd
for C29H31O7N2SijM + H]+ 551.18465, found 551.18456.
4.1.1.7.31. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-one
(5p). Compound 5p was prepared according to the method
described by 4-(6-methoxybenzoijd]thiazol-2-yl)aniline (9a) (100
mg, 0.38 mmol) and 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-
2-yn-1-one (13f) (116 mg, 0.38 mmol) to obtain the pure
product 5p as a yellow colour solid. (194 mg, 90% yield); mp:
202–204 °C; 1H NMR (300 MHz, CDCl3): δ 11.97 (d, J = 12.4
Hz, 1H), 8.04 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 8.9 Hz, 1H), 7.54
(dd, J = 12.3, 7.9 Hz, 1H), 7.36 (d, J = 2.4 Hz, 1H), 7.21 (d, J =
8.6 Hz, 2H), 7.09 (dd, J = 8.9, 2.5 Hz, 1H), 6.87 (s, 1H), 5.80
(d, J = 7.8 Hz, 1H), 3.93 (d, J = 2.4 Hz, 6H), 3.90 (d, J = 3.5 Hz,
6H). 13C NMR (125 MHz, CDCl3): δ 193.10, 164.60, 157.73,
152.83, 151.10, 148.72, 144.47, 143.57, 141.79, 137.89, 136.27,
129.35, 128.85, 123.52, 116.45, 115.66, 108.08, 106.48, 104.23,
99.08, 61.21, 61.11, 56.25, 55.84. MS (ESI): m/z 577 [M + Na]+;
HRMS calcd for C26H23O5N2SBrijM + Na]+ 577.04033, found
577.03935.
4.1.1.7.32. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((4-(6-
methoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-2-en-1-
one (6p). Compound 6p was prepared according to the
method described by 4-(6-methoxybenzoijd]thiazol-2-yl)-2-
methylaniline (10a) (100 mg, 0.37 mmol) and 1-(2-bromo-
3,4,5-trimethoxyphenyl)prop-2-yn-1-one (13f) (110 mg, 0.37
mmol) to obtain the pure product 6p as a yellow colour solid.
(180 mg, 86% yield); mp: 187–189 °C; 1H NMR (500 MHz,
CDCl3): δ 12.15 (d, J = 12.1 Hz, 1H), 7.94–7.91 (m, 2H), 7.91–
7.88 (m, 1H), 7.60 (dd, J = 12.1, 7.8 Hz, 1H), 7.35 (d, J = 2.5
Hz, 1H), 7.28–7.25 (m, 1H), 7.09 (dd, J = 8.9, 2.5 Hz, 1H), 6.90
(s, 1H), 5.86 (d, J = 7.6 Hz, 1H), 3.93 (d, J = 5.4 Hz, 6H), 3.89
1
°C; H NMR (500 MHz, CDCl3): δ 12.23 (d, J = 12.0 Hz, 1H),
8.06 (d, J = 8.3 Hz, 2H), 7.55 (dd, J = 12.1, 8.0 Hz, 1H), 7.26
(s, 1H), 7.23–7.15 (m, 4H), 6.49 (s, 1H), 6.06 (d, J = 7.8 Hz,
1H), 3.96 (d, J = 6.8 Hz, 9H), 3.93 (s, 3H), 3.90 (s, 3H). 13C
NMR (75 MHz, CDCl3): δ 190.24, 168.34, 160.41, 155.85,
154.30, 153.09, 143.48, 142.21, 141.58, 134.26, 128.95, 116.11,
104.79, 97.43, 96.77, 94.69, 60.97, 56.26, 55.93, 55.77. MS
(ESI): m/z 507[M + H]+; HRMS calcd for C27H27O6N2SijM + H]+
507.15843, found 507.15661.
4.1.1.7.28. (Z)-3-((4-(5,7-Dimethoxybenzoijd]thiazol-2-yl)-2-
methylphenyl)amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(6n). Compound 6n was prepared according to the method
described by 4-(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-methyl-
aniline (10b) (100 mg, 0.33 mmol) and 1-(3,4,5-
trimethoxyphenyl)prop-2-yn-1-one (13e) (73 mg, 0.35 mmol) to
obtain the pure product 6n as a yellow colour solid. (152 mg,
88% yield); mp: 180–182 °C; 1H NMR (500 MHz, CDCl3): δ
12.33 (d, J = 11.8 Hz, 1H), 7.96 (s, 1H), 7.93 (d, J = 8.5 Hz,
1H), 7.63 (dd, J = 11.8, 8.0 Hz, 1H), 7.26 (t, J = 4.2 Hz, 1H),
7.23 (s, 2H), 7.17 (d, J = 1.9 Hz, 1H), 6.49 (d, J = 1.9 Hz, 1H),
6.11 (d, J = 7.9 Hz, 1H), 3.96 (d, J = 4.6 Hz, 9H), 3.93 (s, 3H),
3.90 (s, 3H), 2.53 (s, 3H). 13C NMR (75 MHz, CDCl3): δ
190.37, 168.66, 160.40, 155.87, 154.30, 153.10, 143.57, 143.25,
141.55, 140.90, 134.45, 129.98, 128.53, 126.56, 115.93, 113.28,
104.86, 97.43, 96.71, 95.10, 60.96, 56.30, 55.93, 55.77, 17.73.
MS (ESI): m/z 521 [M + H]+; HRMS calcd for C28H29O6N2SijM +
H]+ 521.17408, found 521.17337.
4.1.1.7.29. (Z)-3-((4-(5,6,7-Trimethoxybenzoijd]thiazol-2-
yl)phenyl)amino)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(5o). Compound 5o was prepared according to the method
described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-yl)aniline
(9c) (100 mg, 0.31 mmol) and 1-(3,4,5-trimethoxyphenyl)prop-
2-yn-1-one (13e) (69 mg, 0.31 mmol) to obtain the pure
product 5o as a yellow colour solid. (148 mg, 88% yield); mp:
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(s, 6H), 2.52 (s, 3H). 13C NMR (125 MHz, CDCl3): δ 193.06,
164.94, 157.68, 152.85, 151.05, 148.72, 144.44, 143.66, 140.46,
137.99, 136.24, 129.95, 128.93, 126.78, 126.48, 123.44, 115.60,
113.62, 108.16, 106.47, 104.24, 99.51, 61.22, 61.12, 56.26,
55.84, 17.70. MS (ESI): m/z 591 [M + Na]+; HRMS calcd for
C27H25O5N2SBrijM + Na]+ 591.05598, found 591.05626.
108.10, 107.96, 106.45, 100.59, 100.52, 100.05, 99.23, 99.08,
98.25, 61.59, 61.45, 56.37, 56.32, 56.24, 56.17. MS (ESI): m/z
637 [M + Na]+; HRMS calcd for C28H27O7N2SBrijM + Na]+
637.06144, found 637.06012.
4.1.1.7.36. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((2-
m e t h y l - 4 - ( 5 , 6 , 7 - t r i m e t h o x y b e n z o ij d ] t h i a z o l - 2 -
yl)phenyl)amino)prop-2-en-1-one (6r). Compound 6r was
prepared according to the method described by 4-(5,6,7-
trimethoxybenzoijd]thiazol-2-yl)aniline (10c) (100 mg, 0.30
mmol) and 1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one
(13f) (90 mg, 0.30 mmol) to obtain the pure product 6r as a
4.1.1.7.33. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((4-
(5,7-dimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-
one (5q). Compound 5q was prepared according to the
method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-
yl)aniline (9b) (100 mg, 0.35 mmol) and 1-(2-bromo-3,4,5-
trimethoxyphenyl)prop-2-yn-1-one (13f) (104 mg, 0.35 mmol)
to obtain the pure product 5q as a yellow colour solid. (179
1
yellow colour solid. (171 mg, 90% yield); mp: 178–179 °C; H
NMR (300 MHz, CDCl3): δ 12.14 (d, J = 11.8 Hz, 1H), 7.96–
7.87 (m, 2H), 7.61 (dd, J = 11.9, 7.9 Hz, 1H), 7.35–7.24 (m,
2H), 6.89 (s, 1H), 5.87 (d, J = 7.6 Hz, 1H), 4.11 (s, 3H), 3.99–
3.87 (m, 15H), 2.53 (s, 3H). 13C NMR (100 MHz, CDCl3): δ
193.07, 167.34, 154.01, 152.80, 151.01, 150.41, 146.71, 144.41,
143.53, 140.64, 139.65, 137.91, 129.93, 126.78, 126.46, 119.97,
113.60, 108.10, 106.41, 100.48, 99.56, 61.46, 61.15, 61.05,
60.54, 56.27, 17.63. MS (ESI): m/z 651 [M + Na]+; HRMS calcd
for C29H29O7N2SBrijM + Na]+ 651.07711, found 651.07834.
1
mg, 88% yield); mp: 193–194 °C; H NMR (300 MHz, CDCl3):
δ 11.98 (d, J = 12.5 Hz, 1H), 8.08 (d, J = 8.5 Hz, 2H), 7.55 (dd,
J = 12.3, 7.9 Hz, 1H), 7.26–7.16 (m, 3H), 6.89 (s, 1H), 6.52 (s,
1H), 5.81 (d, J = 7.8 Hz, 1H), 3.98 (s, 3H), 3.94 (d, J = 2.3 Hz,
6H), 3.91 (d, J = 4.8 Hz, 6H). 13C NMR (125 MHz, DMSO): δ
188.37, 163.53, 155.69, 151.11, 149.58, 148.08, 146.35, 139.73,
138.75, 137.28, 133.13, 124.53, 124.22, 111.67, 111.26, 103.33,
101.72, 94.40, 92.70, 92.09, 56.46, 56.36, 51.50, 51.23, 51.06.
MS (ESI): m/z 585 [M + H]+; HRMS calcd for C27H26O6N2SBrijM
+ H]+ 585.06895, found 585.06795.
4.2. Biology
4.1.1.7.34. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((4-
(5,7-dimethoxybenzoijd]thiazol-2-yl)-2-methylphenyl)amino)prop-
2-en-1-one (6q). Compound 6q was prepared according to
the method described by 4-(5,7-dimethoxybenzoijd]thiazol-2-
yl)-2-methylaniline (10b) (100 mg, 0.33 mmol) and
1-(2-bromo-3,4,5-trimethoxyphenyl)prop-2-yn-1-one (13f) (99
mg, 0.33 mmol) to obtain the pure product 6q as a yellow
4.2.1. Cell cultures, maintenance and anti-proliferative
evaluation. The cell lines, A549, HeLa, MDAMB-231 and MIA
PaCa-2 (lung, cervical, breast, and pancreatic) used in this
study were procured from American Type Culture Collection
(ATCC), USA. The synthesized test compounds were evaluated
for their in vitro anti-proliferative activity in these four differ-
ent human cancer cell lines. A protocol of 48 h continuous
drug exposure was used, and an SRB cell proliferation assay
was used to estimate cell viability or growth. All the cell lines
were grown in Dulbecco's modified Eagle's medium
(containing 10% FBS in a humidified atmosphere of 5% CO2
at 37 °C). Cells were trypsinized when sub-confluent from
T25 flasks/60 mm dishes and seeded in 96-well plates in 100
μL aliquots at plating densities depending on the doubling
time of individual cell lines. The microtitre plates were incu-
bated at 37 °C, 5% CO2, 95% air, and 100% relative humidity
for 24 h prior to the addition of experimental drugs and were
incubated for 48 h with different doses (0.01, 0.1, 1, 10, 100
μM) of the prepared derivatives. After incubation at 37 °C for
48 h, the cell monolayers were fixed by the addition of 10%
(wt/vol) cold trichloroacetic acid and incubated at 4 °C for 1
h and were then stained with 0.057% SRB dissolved in 1%
acetic acid for 30 min at room temperature. Unbound SRB
was washed with 1% acetic acid. The protein-bound dye was
dissolved in 10 mM Tris base solution for OD determination
at 510 nm using a microplate reader (Enspire, Perkin Elmer,
USA). Using the seven absorbance measurements [time zero
(Tz), control growth (C), and test growth in the presence of
drug at the five concentration levels (Ti)], the percentage
growth was calculated at each of the drug concentration
levels. Percentage growth inhibition was calculated as
follows: [(Ti − Tz)/Tz] × 100 for concentrations for which
1
colour solid. (165 mg, 83% yield); mp: 222–223 °C; H NMR
(300 MHz, CDCl3): δ 12.16 (d, J = 12.0 Hz, 1H), 7.98 (s, 1H),
7.95 (d, J = 8.5 Hz, 1H), 7.62 (dd, J = 12.0, 7.8 Hz, 1H), 7.29
(d, J = 6.8 Hz, 1H), 7.19 (d, J = 1.7 Hz, 1H), 6.91 (s, 1H), 6.51
(d, J = 1.7 Hz, 1H), 5.88 (d, J = 7.7 Hz, 1H), 3.98 (s, 3H), 3.94
(d, J = 3.3 Hz, 6H), 3.92 (d, J = 1.5 Hz, 6H), 2.54 (s, 3H). 13C
NMR (125 MHz, CDCl3): δ 193.10, 168.64, 160.43, 155.85,
154.33, 152.85, 151.06, 144.44, 143.61, 140.71, 137.99, 130.06,
128.86, 126.78, 126.61, 115.97, 113.59, 108.15, 106.46, 99.58,
97.41, 96.79, 61.22, 61.12, 56.26, 55.98, 55.82, 17.69. MS (ESI):
m/z 601 [M + H]+; HRMS calcd for C28H28O6N2SBrijM + H]+
601.0773, found 601.0832.
4.1.1.7.35. (Z)-1-(2-Bromo-3,4,5-trimethoxyphenyl)-3-((4-
(5,6,7-trimethoxybenzoijd]thiazol-2-yl)phenyl)amino)prop-2-en-1-
one (5r). Compound 5r was prepared according to the
method described by 4-(5,6,7-trimethoxybenzoijd]thiazol-2-
yl)aniline (9c) (100 mg, 0.31 mmol) and 1-(2-bromo-3,4,5-
trimethoxyphenyl)prop-2-yn-1-one (13f) (94 mg, 0.31 mmol) to
obtain the pure product 5r as a yellow colour solid. (172 mg,
89% yield); mp: 230–233 °C; 1H NMR (500 MHz, CDCl3): δ
11.96 (d, J = 12.3 Hz, 1H), 8.05 (d, J = 8.6 Hz, 2H), 7.53 (dd, J
= 12.4, 7.8 Hz, 1H), 7.34 (s, 1H), 7.21 (d, J = 8.7 Hz, 2H), 6.87
(s, 1H), 4.11 (s, 2H), 3.97 (s, 3H), 3.95 (s, 3H), 3.93 (d, J = 4.0
Hz, 6H), 3.89 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 193.15,
167.01, 154.04, 152.82, 151.08, 150.53, 146.76, 144.45, 143.56,
143.43, 141.96, 139.70, 137.86, 129.18, 128.92, 128.79, 116.46,
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Ti < Tz. The dose-response parameter, growth inhibition
of 50% (GI50), was calculated from [(Ti − Tz)/(IJC − Tz)]
× 100 = 50, which is the drug concentration resulting in
a 50% reduction in the net protein increase (as measured by
SRB staining) in control cells during the drug incubation.
Values were calculated for this parameter if the level of activ-
ity is reached; however, if the effect is not reached or is
exceeded, the value for that parameter was expressed as
greater or less than the maximum or minimum concentra-
tion tested.
4.2.2. Analysis of cell cycle. HeLa cells in 60 mm dishes
were incubated for 24 h in the presence or absence of test
compounds 5d, 6d and nocodazole (3 μM). Cells were
harvested with trypsin–EDTA, fixed with ice-cold 70% ethanol
at 4 °C for 30 min, ethanol was removed by centrifugation
and cells were stained with 1 mL of DNA staining solution
[0.2 mg of propidium iodide (PI), and 2 mg RNase A] for 30
min as described earlier. The DNA contents of 20 000 events
were measured by flow cytometer (BD FACSC anto II). Histo-
grams were analyzed using FCS express 4 plus.35
4.2.4. Tubulin polymerization assay. An in vitro assay for
monitoring the time-dependent polymerization of tubulin to
microtubules was performed employing a fluorescence-based
tubulin polymerization assay kit (BK011, Cytoskeleton, Inc.)
according to the manufacturer's protocol. The reaction mix-
ture in a final volume of 10 μl in PEM buffer (80 mM PIPES,
0.5 mM EGTA, 2 mM MgCl2, pH 6.9) in 384 well plates
contained 2 mg mL−1 bovine brain tubulin, 10 μM fluorescent
reporter, 1 mM GTP in the presence or absence of test com-
pounds at 37 °C. Tubulin polymerization was followed by
monitoring the fluorescence enhancement due to the incor-
poration of a fluorescence reporter into microtubules as poly-
merization proceeds. Fluorescence emission at 420 nm (exci-
tation wavelength is 360 nm) was measured for 1 h at 1 min
intervals in
a multimode plate reader (Tecan M200).
Nocodazole was used as positive control under similar experi-
mental conditions. To determine the IC50 values of the conju-
gates against tubulin polymerization, the conjugates were
pre-incubated with tubulin at varying concentrations (1, 2, 3,
4 and 5 μM).
4.2.3. Dot-blot assay. Cells were trypsinized when sub-
confluent from T25 flasks/60 mm dishes and seeded in 6-well
plates. These conjugates (5d and 6d) were evaluated for their
activity against cyclin B1. HeLa cells were treated with 3 μM
concentrations of 5d, 6d and nocodazole for 24 h. Subse-
quently, cells were harvested and proteins were quantified
using Amido Black followed by densitomerty analysis. Equal
amount of protein were blotted on nitrocellulose membrane
using Bio-Dot SF microfiltration apparatus (Bio-Rad). Briefly,
nitrocellulose membrane and 3 filters papers (Whatmann 3)
were soaked in IX TBS solution for 10 min. Later, the filter
papers, membrane were arranged in the apparatus and
connected to vacuum pump (Millipore). The membranes
were rehydrated using 100 μl of 1× TBS by vacuum filtration.
Subsequently, 50 μl volumes of equal protein samples were
blotted on the membrane and washed with 200 μl of 1× TBS
through application of vacuum . The blot was blocked with
5% blotto for 1 h at room temperature. Immunoblot analysis
was performed using UVP, biospectrum 810 imaging system.
Semi-quantitative reverse transcription PCR (RT-PCR)-Total
RNA was extracted using RNase mini kit (Qiagen) and reverse
transcribed into cDNA using superscript II reverse transcrip-
tase (Invitrogen life technologies). The PCR primers for cyclin
B1 were 5′-CTACCTTTGCACTTCCTTCGG-3′ as forward and 5′-
CCTGCTGCAATTTGAGAAGG-3′ as reverse primers. For
GAPDH, the forward primer was 5′-CAAGGTCATCCATGACA-
ACTTTG-3′ and the reverse was 5′-CTTCACCACCTTCTTGAT-
GTCATC-3′. The PCR conditions for cyclin B1 and GAPDH
were, 95 °C 2 minutes, followed by 21 cycles of 95 °C-15
s, 60 °C-30 s, and 72 °C-30 s. GAPDH was used as an
internal control. The products were electrophoresed on
agarose gel (2.5%) followed by staining with ethidium bro-
mide and visualized under U.V. light. The signal intensity
of respective bands was measured by means of the quantity
one version 4.1.1 software using Bio-Rad image analysis
system.25
4.2.5. Immunohistochemistry of tubulin and analysis of
nuclear morphology. HeLa cells were seeded on glass cover
slip, incubated for 24 h in the presence or absence of test
conjugates 5d and 6d (3 μM). Cells grown on cover slips were
fixed in 3.5% formaldehyde in phosphate-buffered saline
(PBS) pH 7.4 for 10 minutes at room temperature. Cells were
permeabilized for 6 minutes in PBS containing 0.5% Triton
X-100 (Sigma) and 0.05% Tween-20 (Sigma). The perme-
abilized cells were blocked with 2% BSA (Sigma) in PBS for 1
h. Later, the cells were incubated with primary antibody for
tubulin from (sigma) at (1 : 200) diluted in blocking solution
for 4 h at room temperature. Subsequently the antibodies
were removed and the cells were washed thrice with PBS.
Cells were then incubated with FITC labeled anti-mouse sec-
ondary antibody (1 : 500) for 1 h at room temperature. Cells
were washed thrice with PBS and mounted in medium
containing DAPI. Images were captured using the Olympus
confocal microscope and analyzed with Provision software.
4.2.6. Western blot analysis of soluble versus polymerized
tubulin. Cells were seeded in 12-well plates at 1 × 105 cells per
well in complete growth medium. Following treatment of cells
with respective compounds (5d, 6d, nocodazole and pacli-
taxel) for a duration of 24 h, cells were washed with PBS and
subsequently soluble and insoluble tubulin fractions were col-
lected. To collect the soluble tubulin fractions, cells were
permeabilized with 200 μL of pre-warmed lysis buffer [80 mM
Pipes-KOH (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 0.2% Triton
X-100, 10% glycerol, 0.1% protease inhibitor cocktail (Sigma-
Aldrich)] and incubated for 3 min at 30 °C. Lysis buffer was
gently removed, and mixed with 100 μL of 3 × Laemmli's sam-
ple buffer (180 mM Tris-Cl pH 6.8, 6% SDS, 15% glycerol,
7.5% β-mercaptoethanol and 0.01% bromophenol blue). Sam-
ples were immediately heated to 95 °C for 3 min. To collect
the insoluble tubulin fraction, 300 μL of 1 × Laemmli's sam-
ple buffer was added to the remaining cells in each well, and
the samples were heated to 95 °C for 3 min. Equal volumes of
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samples were run on an SDS-10% polyacrylamide gel and were
transferred to a nitrocellulose membrane employing semidry
transfer at 50 mA for 1 h. Blots were probed with mouse anti-
human α-tubulin diluted 1 : 2000 ml (Sigma) and stained with
rabbit anti-mouse secondary antibody coupled with horserad-
ish peroxidase, diluted 1 : 5000 ml (Sigma). Bands were visual-
ized using an enhanced chemiluminescence protocol (Pierce)
and radiographic film (Kodak).
thankful to CSIR for financial support under the 12th Five
Year plan project “Affordable Cancer Therapeutics (ACT)”
(CSC0301).
Notes and references
1 M. A. Jordan and L. Wilson, Nat. Rev. Cancer, 2004, 4,
253–265.
4.2.7. Hoechst staining. HeLa cells (5 × 104 cells per well)
at their growth phase were seeded in 12 well plates and
allowed to adhere for 24 h. The culture medium containing
the test conjugates 5d and 6d at 1 μM concentration were
added to the cells and incubated for 24 h at 37 °C. After 24 h,
culture medium was removed; cells were washed with PBS
and fixed with 4% para formaldehyde solution at 4 °C for 10
min. The cells were washed twice with PBS and stained with
Hoechst 33 242 (5 μg mL−1) for 30 min at room temp. The ex-
cess dye was removed by washing twice with PBS and cells
were examined for morphological changes under fluores-
cence microscope using 350 nm excitation and 460 nm emis-
sion (Nikon, magnification 10×).
2 M. Jordan, Curr. Med. Chem.: Anti-Cancer Agents, 2002, 2,
1–17.
3 H. P. Hsieh, J. P. Liou, Y. T. Lin, N. Mahindroo, J. Y. Chang,
Y. N. Yang, S. S. Chern, U. K. Tan, C. W. Chang, T. W. Chen,
C. H. Lin, Y. Y. Chang and C. C. Wang, Bioorg. Med. Chem.
Lett., 2003, 13, 101–105.
4 D. Belotti, V. Vergani, T. Drudis, P. Borsotti, M. R. Pitelli, G.
Viale, R. Giavazzi and G. Taraboletti, Clin. Cancer Res.,
1996, 2, 1843–1849.
5 T. M. Mekhail and M. Markman, Expert Opin. Pharmacother.,
2002, 3, 755–766.
6 E. K. Rowinsky and R. C. Donehower, Pharmacol. Ther.,
1991, 52, 35–84.
4.2.8. Measurement of mitochondrial membrane potential
(MMP). HeLa cells were cultured in 6 well plates at a density
of 5 × 105 cells per mL and allowed to grow over night. The
cells were treated with the test conjugates 5d and 6d with 1
μM concentration for 24 h. After 24 h treatment, the adherent
cells were collected by trypsinisation, washed with PBS and
resuspended in solution of PBS containing Rhodamine-123
(10 μg mL−1). After incubation for 30 min at room temp, cells
were washed twice with PBS and resuspended in PBS. The
7 A. E. Prota, K. Bargsten, P. T. Northcote, M. Marsh, K. H.
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samples
were
analyzed
for
fluorescence
using
spectroflourimeter.
4.2.9. Measurement of reactive oxygen species (ROS)
levels. Intracellular ROS levels were measured by using
carboxy-2′,7′-dichlorofluorescin diacetate (carboxy-DCFH-DA).
U87MG cells were plated in 6 well plates at a density of 5×105
cells per mL and allowed to grow overnight. The cells were in-
cubated with the at 1 μM concentration of the conjugates 5d
and 6d for 24 h. After treatment, the medium was replaced
with the culture medium containing DCFH-DA (10 μM) and
further incubated for 30 min at room temperature in dark.
Cells were washed with PBS, collected from the plate and
resuspended in PBS at 5 × 104 cells per mL. The fluorescence
intensity of the 2′,7′-dichlorofluorscein formed by the hydro-
lysis of carboxy-DCFH-DA from each sample was analyzed by
spectroflourimeter at an excitation and emission wavelength
of 488 and 525 nm, respectively. Qualitative cellular fluores-
cence images were captured by using Nikon ECLIPSETE2000-
S fluorescence microscope.
Acknowledgements
The authors A. V. S. R., S. P. S., B. S. would like to thank to
CSIR, New Delhi, India for the award of research fellowships
and S. S. acknowledge the CSIR-UGC, New Delhi (India), for
the award of senior research fellowships. The authors are also
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