Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel anti tubulin agents: Design, synthesis, biological evaluation and molecular modeling studies

Article abstract of DOI:10.1016/j.bmc.2015.10.013

Keeping in view the limitations associated with currently available anticancer drugs, molecular hybrids of mono carbonyl curcumin and isatin tethered by triazole ring have been synthesized and evaluated for in vitro cytotoxicity against THP-1, COLO-205, HCT-116, A549, HeLa, CAKI-I, PC-3, MiaPaca-2 human cancer cell lines. The results revealed that the compounds SA-1 to SA-9, SB-2, SB-3, SB-4, SB-7 and SC-2 showed a good range of IC₅₀ values against THP-1, COLO-205, HCT-116 and PC-3 cell lines, while the other four cell lines among these were found to be almost resistant. Structure activity relationship revealed that the nature of Ring X and substitution at position R influences the activity. Methoxy substituted phenyl ring as Ring X and H as R were found to be the ideal structural features. The most potent compounds (SA-2, SA-3, SA-4, SA-7) were further tested for tubulin inhibition. Compound SA-2 was found to significantly inhibit the tubulin polymerization (IC₅₀ = 1.2 μM against HCT-116). Compound SA-2, moreover, lead to the disruption of microtubules as confirmed by immunofluorescence technique. The significant cytotoxicity and tubulin inhibition by SA-2 was streamlined by molecular modeling studies where it was docked at the curcumin binding site of tubulin.

Full text of DOI:10.1016/j.bmc.2015.10.013

Accepted Manuscript
Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel
anti tubulin agents: Design, synthesis, biological evaluation and molecular mod-
eling studies
Sahil Sharma, Manish K. Gupta, Ajit K. Saxena, Preet Mohinder S. Bedi
PII:
S0968-0896(15)30091-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.10.013
BMC 12613
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 August 2015
6 October 2015
11 October 2015
Please cite this article as: Sharma, S., Gupta, M.K., Saxena, A.K., Bedi, P.M.S., Triazole linked mono carbonyl
curcumin-isatin bifunctional hybrids as novel anti tubulin agents: Design, synthesis, biological evaluation and
molecular modeling studies, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.
2015.10.013
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Triazole linked mono carbonyl curcumin-isatin bifunctional hybrids as novel anti tubulin
agents: Design, synthesis, biological evaluation and molecular modeling studies
Sahil Sharmaa,*, Manish K. Guptab, Ajit K. Saxenac, Preet Mohinder S. Bedia,*
aDepartment of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar, Punjab 143005, India
bLloyd Institute of Management and Technology, Greater Noida, UP, India
cIndian Institute of Integrative Medicine, Jammu, India
Corresponding author: 1) Sahil Sharma
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Mob no. +918427007614
2) Dr. P.M.S. Bedi
Department of Pharmaceutical Sciences,
Guru Nanak Dev University,
Amritsar, Punjab 143005, India
Mob no. +919815698249

1. Introduction
Microtubules, the key cytoskeletal filaments, are long, hollow cylinders of tubulin dimmers that
are found to be involved in many cellular processes such as maintaining structure of the cell,
together with microfilaments and intermediate filaments.1 Microtubule continuously undergoes
the process of mounting and dwindling but it still seems to be entirely stable. More to the point,
microtubules also provide podium for intracellular transport and are involved in diverse cellular
processes, viz., movement of secretory vesicles, organelles, and intracellular substances.2 Tubulin
which is in essence a globular protein, consists of six discrete families, the alpha (α), beta (β),
gamma (γ), delta (γ), epsilon (ε) and zeta (ξ) tubulin, found chiefly in kinetoplastid protozoa.
Normally, the tubulin dimmers do not dissociate because of the well-built force that holds α -
tubulin and β -tubulin in the heterodimeric complex. The tremendous role of microtubules in cell
division, thus, makes it a captivating target for anticancer drugs.3
The molecular hybridization (MH) is a stratagem of coherent design of new ligands or prototypes
based on the recognition of pharmacophoric sub-units in the molecular structure of two or more
known bioactive derivatives which, through the adequate fusion of these sub-units, provides new
hybrid architectures.4
Hybrid drugs are basically designed to compensate the known side effects associated with the other
hybrid part or to intensify their effect through action on another biological target or to potentiate
their effect by binding to distinct sites on the same target or to interact with multiple targets as one
single molecule5, thus, lowering the risk of drug-drug interactions hybrid drugs can be designed
and minimizing the drug resistance as well.6
Curcumin (diferuloylmethane), a naturally occurring polyphenolic compound has shown
tremendous success in various forms of therapeutic activities7, among which, the chemotherapeutic
and chemopreventive effect in diverse forms of cancer is well documented and the most successful
one as well.8 Nevertheless, curcumin and some of its analogs are reported to bind tubulin 32 Å
away from the colchicine-binding site and inhibit its self assembly9, but their precise mechanism
of action in cancer cells is not so clear. Moreover, the poor solubility, absorption, bioavailability
and rapid metabolism of curcumin in human probably limits its clinical efficacy.10 To overcome
many of such limitations, mono carbonyl analogues of curcumin have been synthesized by deleting
the reactive β-diketone moiety (which was considered to be responsible for the pharmacokinetic
limitation of curcumin) (Fig. 1a) through several chemical modifications in the basic structure of

curcumin in order to increase its potential, stability and bioavailability. Although, some of the
recently developed mono carbonyl curcumin analogs, (1-5) (Fig. 1b) exhibit potent anticancer
activity11, their cytotoxicity via tubulin inhibition is not explored yet.
Isatin, an indole derivative is considered as a versatile nucleus with diverse assortment of
biological actions. This heterocycle is known to exert its anticancer effects through different
mechanism among which tubulin inhibition by binding to its novel site, being one of the highest-
flying ones. Some of the isatin hybrids with potent anticancer activity (Fig. 2) includes uracil-isatin
conjugates (6)12, triazole tethered bis-isatin conjugates (7)13, iastin-benzothiazole analogues (8)14,
isatin-4-pirazinylquinoline hybrids (9)15, isatin-thiazolidinone hybrid (10)16 and isatin-3,5-diaryl
N-acetyl pyrazoline hybrids (11).17
A sufficient number of recent reports on the cell darnage potential of isatin-based molecular
hybrids, rationalize its inclusion in the hybrids of isatin and mono carbonyl curcumin, designed by
our research group. Thus, these two privileged structures have been selected as the key building
blocks in designing and synthesizing the hybrids. Keeping in view the triumph of these
functionalities, their hybrids can give new dimensions to the molecules modulating tubulin as a
target and can thus be extremely beneficial for the society which at the moment is striving hard to
fight with this dreadful disease.
Numerous reports on the cytotoxic potential of triazoles further attracted us towards their selection
as a linker for the two functionalities.12,13,17,18
With this background, triazole linked mono carbonyl curcumin-isatin based bifunctional hybrids
were designed and synthesized. The synthetics were then evaluated for their anticancer potential
against eight cancer cell lines. The designed strategy for the synthesis of these hybrids is shown in
Fig. 3.
2. Results and discussion
2.1. Chemistry
Target hybrids were synthesized via a sequence of reactions (Scheme 1) starting from vanillin,
where it was treated with acetone in the presence of 40% KOH at room temperature. The desired
product vinyldenacetone (VDA) was thus formed, which was characterized by two doublets for
one proton each at approximately 6.5 and 7.5 ppm, having J value of more than 16 Hz in 1H NMR.
VDA was then treated with propargyl bromide in DMF as a solvent, in the presence of K2CO3 at
room temperature to get the desired product i.e. propargylated vinyldenacetone (PVDA),

characterized by two singlets at approximately 2.5 ppm (for one proton) and 4.8 ppm (for two
protons) in 1H NMR. PVDA was further treated with various substituted aldehydes in methanol as
a solvent and the presence of 5% NaOH at room temperature in order to obtain the desired product
wiz propargylated mono carbonyl curcumin analogues.
On the other hand, various substituted isatins were treated with 1,2-dibromoethane in the presence
of K2CO3, in DMF as a solvent at room temperature. The desired product (N-bromoethyl isatin)
was purified through column chromatography. N-bromoethyl isatin was further reacted with NaN3
in DMF as a solvent, at room temperature to yield N-azidoethyl isatin.
These N-azidoethyl isatins were treated with propargylated mono carbonyl curcumin analogues in
the presence of copper sulfate and sodium ascorbate, in DMF as a solvent, at room temperature to
gain the desired mono carbonyl curcumin-isatin hybrids linked through triazole.
2.2. Bioassay
All the synthetics were evaluated for in-vitro cytotoxicity against THP-1, COLO-205, HCT-116,
A549, HeLa, CAKI-I, PC-3 and MiaPaCa-2 human cancer cell lines using sulforhodamine B19.19
The cells were allowed to proliferate in the presence of test material for 48 h. All the synthesized
hybrids were screened against the cell lines at 50 µM. The molecules showing percentage
inhibition (Table 1) of greater than 60% at least against one cell line were only evaluated at
different concentrations and IC50 values (Table 2) were calculated. From Tables 1 and 2, it is clear
that among the 8 cell lines, THP-1, COLO-205, HCT-116 and PC-3 were the most sensitive
towards the designed hybrids whereas A549, HeLa, CAKI-I and MiaPaCa-2 were found to be
resistant. The clear evaluation of the Table 2 reveals that among the four cell lines, HCT-116
cancer cell line was the most sensitive one towards these compounds. Compounds SA-2, SA-3,
SA-4 and SA-7 displayed significant cytotoxicity with IC50 value ranging from 1.12-5.67 µM,
2.67-7.64 µM, 3.45-8.95 µM and 5.61-9.40 µM against HCT-116, THP-1, COLO-205 and PC-3.
The most active hybrid SA-2 with trimethoxy phenyl ring as Ring X (IC50=1.12 µM) was almost
two folds more active than SA-3 (dimethoxy phenyl ring as Ring X, IC50= 2.67 µM), the second
most potent hybrid against HCT-116 cell line. Careful observations of Table 1 and Table 2 revealed
that the methoxy substituted phenyl ring as Ring X remarkably enhances the cytotoxic potential
(compare SA-1 with SA-2, SA-3 and SA-4) whereas halo and nitro substituted phenyl ring as
Ring X diminishes the cytotoxic effect (compare SA-1 with SA-10, SA-11, SA-12, SA-13 and SA-
14). The placement of a hateroaryl ring in place of the unsubstituted phenyl ring (Ring X) such as

furan and thiophene improved the activity profile (compare SA-1 with SA-8 and SA-9). Both these
heteroaryl substituted derivatives, SA-8 and SA-9 displayed almost a similar profile in terms of
their cytotoxic potential. A positive effect was observed with the increased number of methoxy
substituents on phenyl ring as Ring X such as trimethoxy phenyl > dimethoxy phenyl >
monomethoxy phenyl (compare SA-2, SA-3, SA-4, SA-5 and SA-6). However, the placement of
naphthyl ring as Ring X also proved out to be a good companion for dimethoxy substituted phenyl
ring as their IC50 values were similar to the hybrids exhibiting dimethoxy phenyl ring as Ring X
(compare SA-3 and SA-7). The cytotxic potential of 2 and 3 substituted monomethoxy phenyl ring
as Ring X was found almost similar to the heteroaryl ring (Ring X) substituted derivatives of these
hybrids (compare SA-5 and SA-8, SA-6 and SA-9). Thus, the overall preference order of Ring X
is established as follows: trimethoxy phenyl > dimethoxy phenyl = naphthyl > 4-methoxy phenyl
> 3-methoxyphenyl = furan > 2-methoxyphenyl = thiophene > phenyl > phenyl with halo and nitro
groups. On the other hand, any substitution as R significantly decreased the cytotoxic profile of
the compounds (compare SA-1 with SB-1 and SC-1). The influence of the size of halogens as R
was also observed. The preference order for substituents as R is as follows H > chloro > bromo.
The structure activity relationship has been depicted in Fig. 4.
The most active compounds (SA-2, SA-3, SA-4, SA-7) among the series were evaluated for their
inhibitory effects on tubulin polymerization as per the reported assay using a cytoskeleton tubulin
polymerization assay kit.20 Compound SA-2 (most potent hybrid) with a trimethoxy phenyl ring
(Ring X) also displayed the most potent antitubulin activity with IC50 value of 2.36 µM. SA-3 also
exhibited significant inhibition of tubulin polymerization with an IC50 value of 3.97 µM. SA-4 and
SA-7 with 4-methoxy phenyl and naphthyl ring (Ring X) were found to be endowed with weak
inhibitory potential for tubulin polymerization. The results of the in-vitro tubulin polymerization
assay clearly indicate that both SA-2 and SA-3 exert their cytotoxic effects through tubulin
inhibition (Table 3). The immunofluorescence technique was used to determine the effect of SA-
2 on microtubule structure using confocal microscopy. In this study, untreated HCT-116 cell lines
showed the normal distribution of microtubules. However, cells treated with compound SA-2
showed the disrupted microtubule organization at 5 µM concentration (Fig. 5).
2.3. Molecular docking
The docking study showed that SA-2 fits well at the interface of β1and α2 subunits of tubulin and
stabilized by H-bonds, polar and van der Walls interactions (Fig. 6a). Residues from the both

subunits (β1and α2) were involved in interactions with the inhibitor (Fig. 6b). The trimethoxy-
phenyl ring (ring A)gets positioned in a cavity formed byArg2α2, Gly73β1, Gln96β1 (polar
residues) and Pro72β1 (a nonpolar residue). The ring B was found to be sandwiched between two
polar residues namely Gln11β1 and Glu254α2. The –OCH3 group of ring B is oriented towards
the side chain carboxylate ionic head of Glu254α2. The methoxy hydrogen (H-bond donor) has
shown a weak H-bond interactions with carboxylate head of Glu254 (H-bond acceptor, d =
2.900Å). The carboxylate ionic head of Asp197β1 is involved in polar interactions with ring C
(triazole ring). The ring D/E (indoline-2,3-dione moiety) was placed in a hydrophobic cavity
formed by Val177β1, Pro222β1, Thr223β1 and Thr224β1. Here, ring E has shown face-to-face
pai-pai stacking interaction with Tyr224β1.21 The presence of three methoxy groups (-OCH3) in
the aromatic phenyl ring (ring A) provides a unique blend polarity and hydrophobicity to the
inhibitor SA-2. Such functional group significantly increases the protein binding of inhibitors via
van der Waals and dispersion interactions.22 The trimethoxyphenyl group is also present in potent
tubulin inhibitors such as colchicine, podophyllotoxin and combretastatin A-4. Therefore, this can
be suggested that the presence of trimethoxyphenyl group in SA-2 may be attributed for its superior
activity over other analogs.
Microtubules are dynamic polymers of αβ-tubulin that form various cellular structures including
mitotic spindle for cell division. The microtubule-associated proteins (MAPs) and motor proteins
regulate the growth and shrinkage of microtubule by selective binding to distinct conformations
of polymerized or unpolymerized αβ-tubulin.23,24 The αβ-tubulin can adopt distinct conformation
which contributes to the polymerization dynamics of microtubules. The diverse MAPs identify the
distinct αβ-tubulin conformations to build proper microtubule networks. This can be suggested
that binding of SA-2 at the interface of β1-α2 heterodimer restrict the tubulin to adopt MAP-
recognizable conformations and thus inhibit the tubulin polymerization into microtubule assembly.
This results in G2/M arrest within the cell cycle and eventually cell death.23-26
3. Conclusion
Mono carbonyl curcumin-isatin hybrids were designed, synthesized and evaluated for cytotoxicity
against a panel of human cancer cell lines in the present study. Tubulin inhibitory potential of the
most potent compound SA-2 (with a trimethoxy phenyl ring as ring X and R=H) was confirmed
by in vitro tubulin assay and immunofluorescence technique. The worth mentioning inhibition of
tubulin by compound SA-2 was also streamlined by molecular modelling studies.

4. Experimental section
4.1. Materials and measurements
The reagents were purchased from Sigma-Aldrich, Loba and CDH, India and used without further
purification. All yields refer to isolated products after purification. Products were characterized by
comparison with authentic samples and by spectroscopic data (1H and 13C NMR, HRMS,
Elemental Analysis). 1H NMR and 13C NMR Spectra were recorded on Avance III HD 500 MHz
Bruker Biospin Nuclear Magnetic Resonance Spectrometer. The spectra were measured in CDCl3
and DMSO-d6 relative to TMS (0.00 ppm). In 1H NMR chemical shifts were reported in δ values
using tetramethylsilane as internal standard with number of protons, multiplicities (s-singlet, d-
doublet, t-triplet, q-quartet, m-multiplet, dd-double doublet) and coupling constants (J) in Hz
(Hertz) in the solvent indicated. HRMS was recorded on micrOTOF-QII Bruker Daltonik LC-
MS/MS High Resolution Mass Spectrometer. Melting points were determined in open capillaries
and were uncorrected.
4.2. General procedure for the synthesis of vinyldenacetone (VDA)
Vanillin (20 g) was dissolved in acetone (50 ml) in a round bottom flask and 40% KOH (5 ml)
was added to this reaction mixture. It was kept on stirring at room temperature. After the
completion of reaction (monitored by TLC), reaction mixture was acidified with dilute
hydrochloric acid until pH 7 was achieved. It was then poured in ice-cold water, filtered and dried
to get the desired product i.e. vinyldenacetone (VDA). The physical data of vinyldenacetone is
given below:
1
4.2.1. (E)-4-(4-hydroxy-3-methoxyphenyl)but-en-2-one (VDA): Yield 72%; mp 71-73oC. H
NMR (CDCl3, 500 MHz, δ, TMS = 0): 2.36 (s, 3H), 3.91 (s, 3H), 6.58 (d, J = 16.5 Hz, 1H), 6.92
(d, J = 7.5 Hz, 1H), 7.04–7.08 (m, 2H), 7.45 (d, J = 16.5 Hz, 1H). 13C NMR (CDCl3, 125 MHz, δ,
TMS = 0): 27.22, 55.84, 109.56, 114.99, 123.52, 124.82, 126.79, 144.03, 147.08, 148.51, 198.72.
4.3. General procedure for the synthesis of propargylated vinyldenacetone (PVDA)
VDA (1 eq) was dissolved in DMF, propargyl bromide (1 eq) and K2CO3 (1.5 eq) were added.
Reaction mixture was kept on stirring at room temperature. After the completion of reaction
(monitored by TLC), reaction mixture was poured on crushed ice and kept aside for some time.

Then it was filtered and dried to obtain the desired product i.e. propargylated vinyldenacetone
(PVDA). The physical data of propargylated vinyldenacetone is given below:
4.3.1. (E)-4-(3-methoxy-4-(prop-2-ynyloxy)phenyl)but-3-en-2-one (PVDA): Yield 74%; mp
74-77oC. 1H NMR (CDCl3, 500 MHz, δ, TMS = 0): 2.38 (s, 3H). 2.55 (s, 1H), 3.92 (s, 3H), 4.81
(s, 2H), 6.63 (d, J = 16.5 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 7.08–7.14 (m, 2H), 7.47 (d, J = 16.5
Hz, 1H).13C NMR (CDCl3, 125 MHz, δ, TMS = 0): 27.38, 55.93, 56.62, 76.28, 77.94, 110.30,
113.75, 122.45, 125.71, 128.50, 143.28, 149.00, 149.83, 198.33.
4.4. General procedure for the synthesis of propargylated mono carbonyl curcumin
analogues
PVDA (1 eq) was dissolved in methanol, 3,4-dimethoxy benzaldehyde (1 eq) and 5% NaOH (0.5
ml) were added. Reaction mixture was kept on stirring at room temperature. After the completion
of reaction (monitored by TLC), reaction mixture was poured on crushed ice and kept aside for
some time. Then it was filtered and dried to obtain the desired product i.e. propargylated mono
carbonyl curcumin analogue, S-3. The physical data of propargylated mono carbonyl curcumin
analogue, S-3 is given below:
4.4.1 (1E, 4E)-1-(3-methoxy-4-(prop-2-ynyloxy)phenyl)-5-(3,4-dimethoxyphenyl)penta-1,4-
o
1
dien-3-one (S-3): Yield 71%, mp 76-78 C. H NMR (CDCl3, 500 MHz, δ, TMS = 0): 2.88 (s,
1H), 3.91 (s, 3H), 3.93 (s, 6H), 4.81 (s, 2H), 6.94 (d, J = 8.5 Hz, 1H), 7.03–7.08 (m, 3H), 7.23–
7.25 (m, 4H), 7.66-7.70 (m, 2H). 13C NMR (CDCl3, 125 MHz, δ, TMS = 0): 60.75, 61.38, 64.92,
81.90, 83.25, 114.94, 115.01, 115.55, 116.18, 118.70, 127.90, 128.14, 128.53, 129.01, 132.96,
133.72, 147.40, 147.81, 153.72, 154.00, 154.50, 156.12, 175.48, 193.29.
All the other propargylated mono carbonyl curcumin analogues were synthesized by following the
above mentioned procedure, using various substituted aromatic aldehydes.
4.5. General procedure for the synthesis of N-bromoethyl isatins
Isatin (1 eq) was dissolved in DMF, dibromoethane (1 eq) and K2CO3 (1.5 eq) were added.
Reaction mixture was kept on stirring at room temperature. After the completion of reaction
(evident by TLC), reaction mixture was poured on crushed ice and kept aside for some time. Then
it was filtered and dried to obtain the desired product i.e. N-bromoethyl isatin.

All the other N-bromoethyl isatins were synthesized by following the above mentioned procedure,
using various substituted isatins.
4.6. General procedure for the synthesis of N-azidoethyl isatins
N-bromoethyl isatin (1 eq) was dissolved in DMF and sodium azide (1 eq) was added. Reaction
mixture was kept on stirring at room temperature. After the completion of reaction (evident by
TLC), reaction mixture was poured on crushed ice and kept aside for some time. Then it was
filtered and dried. Crude product thus obtained was impure, therefore, subjected to column
chromatography, using chloroform as an eluent, to get the desired product i.e. N-azidoethyl isatin.
All the other N-azidoethyl isatins were synthesized by following the above mentioned procedure,
using various substituted N-bromoethyl isatins.
4.7. General procedure for the synthesis of triazole linked mono carbonyl curcumin-isatin
hybrids
Mono carbonyl curcumin (1 eq) and N-azidoethyl isatin (1 eq) were dissolved in DMF, catalytic
amount of copper sulphate and sodium ascorbate were added in it. Reaction mixture was kept aside
at room temperature. After the completion of reaction (evident by TLC), reaction mixture was
filtered on crushed ice to remove the excess of copper sulphate and sodium ascorbate and then
kept aside for some time. Then it was filtered and dried to obtain the desired product i.e. triazole
linked mono carbonyl curcumin-isatin hybrids (SA, SB, SC). The physical data of all the triazole
linked mono carbonyl curcumin-isatin hybrids (SA, SB, SC) is given below:
4.7.1. (1E,4E)-1-(4-phenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-triazol-
4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-1)
Yield 71%; mp 68-70oC; MW 534.56. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.19 (m, 4H), 7.20-7.30 (m, 4H), 7.52-7.66 (m, 3H),
13
7.79-7.83 (d, J = 8 Hz, 2H), 8.29-8.33 (m, 3H). C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
56.26, 72.62, 111.67, 115.28, 117.82, 119.73, 122.14, 123.46, 124.95, 126.41, 128.03, 128.50,
128.78, 130.12, 134.89, 135.21, 142.44, 149.03, 149.75, 152.76, 160.18, 184.47. HRMS: M+ at
m/z 534.5609. Anal. Calcd. For C31H26N4O5: C, 69.65; H, 4.90; N, 10.48. Found: C, 69.61; H, 4.86;
N, 10.52.

4.7.2. (1E,4E)-1-(3,4,5-methoxyphenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-2)
Yield 77%; mp 70-72oC; MW 708.71. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.67-3.73 (s,
6H), 3.78 (s, 3H), 3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.26 (d, J = 8.5 Hz, 2H),
7.03-7.19 (m, 3H), 7.66 (d, J = 8.5 Hz, 2H), 7.52-7.83 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 46.82, 49.84, 56.28, 56.54, 72.62, 103.96, 111.64, 117.81, 120.92,
122.15, 123.49, 124.90, 128.55, 129.56, 130.13, 134.86, 138.43, 142.45, 148.14, 149.00, 149.75,
150.79, 152.75, 160.18, 184.44, 188.69. HRMS: M+ at m/z 624.6452. Anal. Calcd. For
C41H32N4O8: C, 65.38; H, 5.16; N, 8.97. Found: C, 65.42; H, 5.20; N, 8.93.
4.7.3.
(1E,4E)-1-(3,4-methoxyphenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-3)
Yield 72%; mp 65-67oC; MW 626.61. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.82 (s, 3H) ,
3.83 (s, 3H), 3.85 (s, 3H), 4.15 (m, 2H), 4.67(m, 2H), 5.14 (s, 2H), 6.86 (d, J = 8.5Hz, 1H), 7.03
(d, J = 8.5 Hz, 1H), 7.18-7.26 (m, 4H), 7.30-7.33 (m, 2H), 7.40-7.41 (m, 2H), 7.53 (d, J = 8.5 Hz,
1H), 7.60 (m, 1H), 7.69-7.72 (m, 2H), 8.33 (s, 1H).13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
47.46, 56.11, 61.98, 111.00, 111.31, 112.14, 113.66, 115.68, 119.16, 119.24, 123.44, 123.70,
124.31, 124.47, 126.07, 128.02, 128.47, 137.40, 143.43, 149.18, 149.62, 150.13, 151.56, 158.39,
182.30, 188.59. HRMS: M+ at m/z 594.1485. Anal. Calcd. For C33H30N4O9: C, 66.66; H, 5.09; N,
9.42. Found: C, 66.62; H, 5.13; N, 9.46.
4.7.4.
(1E,4E)-1-(4-methoxyphenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-4)
Yield 72%; mp 69-72oC; MW 564.59. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.72-6.75 (d, J = 8 Hz, 2H), 7.03-7.19( m,
5H), 7.52-7.66 (m, 4H), 7.79-7.83 (m, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ,
TMS = 0): 46.82, 49.85, 55.92, 56.27, 72.68, 114.25, 115.21, 116.64, 117.88, 119.79, 120.96,
122.17, 123.44, 124.92, 127.49, 127.56, 128.53, 130.11, 134.88, 142.49, 148.10, 149.07, 149.71,
152.74, 159.98, 160.19, 184.45, 188.66. HRMS: M+ at m/z 564.5918. Anal. Calcd. For
C32H28N4O6: C, 68.01; H, 5.00; N, 9.92. Found: C, 68.05; H, 4.96; N, 9.88.

4.7.5. (1E,4E)-1-(3-methoxyphenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-5)
Yield 75%; mp 65-67oC; MW 564.59.1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-6.86 (m, 4H), 7.03-7.19 (m, 5H), 7.52-
7.83 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.84, 49.82,
56.26, 55.98, 72.67, 110.65, 111.67, 113.53, 115.24, 117.80, 118.71, 119.76, 120.98, 122.15,
123.42, 124.94, 128.59, 129.70, 130.17, 134.84, 136.21, 142.42, 148.10, 149.00, 149.75, 152.73,
160.10, 160.65 184.41, 188.63. HRMS: M+ at m/z 564.5920. Anal. Calcd. For C32H28N4O6: C,
68.07; H, 5.00; N, 9.72. Found: C, 68.03; H, 5.04; N, 9.76.
4.7.6. (1E,4E)-1-(2-methoxyphenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-6)
Yield 70%; mp 68-70oC; MW 564.59. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-6.86 (m, 3H), 7.03-7.19 (m, 3H), 7.52-
7.66 (m, 3H), 7.79-7.93 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS =
0): 46.86, 49.81, 56.25, 56.38, 72.60, 111.64, 114.28, 115.07, 115.22, 117.88, 119.70, 120.99,
121.00, 122.15, 123.46, 124.92, 127.40, 128.55, 129.06, 130.12, 134.84, 142.43, 148.18, 149.09,
149.70, 152.72, 157.71, 160.16, 184.48, 188.63. HRMS: M+ at m/z 564.5918. Anal. Calcd. For
C32H28N4O6: C, 68.07; H, 5.00; N, 9.92. Found: C, 68.11; H, 4.96; N, 9.96.
4.7.7.
(1E,4E)-5-(napthalen-1-yl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-7)
Yield 71%; mp 69-71oC; MW 584.62.1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.84 (s, 3H),
4.15 (m, 2H), 4.68 (m, 2H), 5.15 (s, 2H), 6.90 (d, J = 7.5 Hz, 1H), 7.09 (m, 1H), 7.20 (m, 1H),
7.36-7.66 (m, 4H), 7.82 (d, J = 16.5Hz, 1H), 8.01-8.07 (m, 3H), 8.35 (m, 2H), 8.55 (d, J = 16.5 Hz,
1H).13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.77, 55.97, 62.43, 109.77, 110.90, 113.65,
117.41, 123.08, 123.37, 123.76, 124.33, 125.04, 125.20, 125.14, 126.35, 127.02, 128.00, 128.35,
128.83, 130.69, 131.60, 132.10, 133.68, 138.44, 139.26, 143.47, 149.65, 150.05, 150.24, 158.44,
182.80, 188.41. HRMS: M+ at m/z 585.2059. Anal. Calcd. For C35H28N4O5: C, 71.91; H, 4.83; N,
9.58. Found: C, 71.87; H, 4.86; N, 9.54.

4.7.8.
(1E,4E)-1-(furan-2-yl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-8)
Yield 71% mp 68-70oC; MW 524.52. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.82 (s, 3H),
4.15 (m, 2H), 4.68 (m, 2H), 5.13 (s, 2H), 6.68 (s, 1H), 6.90 (d, J = 7.5 Hz, 1H), 6.98-7.01 (m, 2H),
7.08-7.11 (m, 2H), 7.17 (d, J = 7.5 Hz, 1H), 7.25 (d, J = 16.5 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H),
7.43 (m, 1H), 7.53-7.59 (m, 3H), 7.66 (d, J =16.5 Hz, 1H), 7.90 (s, 1H). 13C NMR (d6-DMSO, 125
MHz, δ, TMS = 0): 47.67, 55.96, 62.31, 110.02, 110.88, 112.94, 113.63, 116.12, 117.45, 123.07,
123.16, 123.69, 124.12, 124.94, 128.35, 129.13, 138.46, 142.98, 145.36, 149.64, 150.03, 150.35,
151.43, 158.44, 182.94, 188.07. HRMS: M+ at m/z 524.5245. Anal. Calcd. For C29H24N4O6: C,
66.41; H, 4.61; N, 10.08. Found: C, 66.37; H, 4.65; N, 10.12.
4.7.9.
(1E,4E)-5-(thiophen-2-yl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-9)
Yield 70%; mp 72-74oC; MW 540.59. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.82 (s, 3H),
4.15 (m, 2H), 4.68 (m, 2H), 5.13 (s, 2H), 6.73-6.91 (m, 1H), 6.98 (d, J = 16.5 Hz, 1H), 7.09 (m,
1H), 7.18-7.30 (m, 4H), 7.42 (m, 1H), 7.53-7.59 (m, 3H), 7.68 (d, J = 16.5 Hz, 1H), 7.76 (s, 1H),
13
7.92 (d, J = 16.5 Hz, 1H). C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.43, 56.10, 62.00,
110.71, 111.34, 113.66, 117.81, 123.76, 124.21, 124.97, 125.11, 128.40, 129.21, 130.44, 132.89,
135.54, 138.60, 140.71, 143.24, 149.63, 150.65, 158.59, 184.00, 188.00. HRMS: M+ at m/z
540.5717. Anal. Calcd. For C29H24N4O5S: C, 64.43; H, 4.47; N, 10.36; S, 5.93. Found: C, 64.59;
H, 4.51; N, 10.32; S, 5.89.
4.7.10. (1E,4E)-1-(4-chlorophenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-10)
Yield 79%; mp 64-66oC; MW 569.01. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.22-7.24 (m, 4H), 7.03-7.19 (m, 4H), 7.52-7.66 (m, 3H),
7.79-7.83 (d, J = 8.5 Hz, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
46.85, 49.82, 56.29, 72.65, 111.61, 115.27, 117.83, 119.74, 120.90, 122.13, 123.48, 124.95,
127.86, 128.59, 128.82, 130.10, 133.34, 134.88, 133.55, 142.43, 148.12, 149.01, 149.79, 152.70,
160.14, 184.43, 188.68. HRMS: M+ at m/z 569.0186. Anal. Calcd. For C31H25ClN4O5: C, 65.44;
H, 4.43; Cl, 6.23; N, 9.85. Found: C, 65.40; H, 4.39; Cl, 6.19; N, 9.89.

4.7.11. (1E,4E)-1-(4-bromophenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-11)
Yield 71%; mp 70-72oC; MW 613.46. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.83 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 6.86 (d, J = 7.5 Hz, 1H), 7.20-7.23 (m, 2H), 7.32 (d, J = 7.5 Hz, 1H),
13
7.40-7.43 (m, 2H), 7.54 (d, J = 7.5 Hz, 1H), 7.60 (m, 1H), 7.66-7.79 (m, 7H), 8.33 (s, 1H). C
NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.34, 56.03, 62.05, 111.31, 112.42, 113.61, 119.16,
123.41, 124.07, 124.50, 126.04, 126.73, 127.87, 128.05, 130.74, 132.31, 134.54, 137.39, 141.23,
143.88, 149.26, 149.71, 150.36, 158.41, 182.29, 188.79. HRMS: M+ at m/z 613.1881. Anal. Calcd.
For C31H25BrN4O5: C, 60.69; H, 4.11; Br, 13.03; N, 9.13. Found: C, 60.73; H, 4.15; Br, 13.07; N,
9.17.
4.7.12. (1E,4E)-1-(3-bromophenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-12)
Yield 75%; mp 68-70oC; MW 613.46. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.10 (m, 3H), 7.19-7.47 (m, 5H), 7.52-7.83 (m, 5H),
8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.85, 49.82, 56.29, 72.61,
111.62, 115.27, 117.83, 119.70, 120.95, 122.17, 123.09, 123.45, 124.94, 125.43, 128.59, 129.90,
130.15, 130.91, 134.84, 137.46, 142.45, 148.12, 149.03, 149.70, 152.79, 160.16, 184.45, 188.66.
HRMS: M+ at m/z 613.4607. Anal. Calcd. For C31H25BrN4O5: C, 60.69; H, 4.11; Br, 13.03; N,
9.13. Found: C, 60.65; H, 4.15; Br, 13.07; N, 9.09.
4.7.13. (1E,4E)-1-(4-nitrophenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-13)
Yield 71% mp 73-75oC; MW 611.56. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.84 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.86 (d, J = 8.5 Hz, 1H), 7.20-7.25 (m, 3H), 7.34 (d, J =
8.5Hz, 1H), 7.43 (m, 1H), 7.53-7.60 (m, 3H), 7.79-7.84 (m, 2H), 8.06-8.07 (m, 2H), 8.29-8.33 (m,
13
3H). C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):47.47, 56.10, 61.98, 111.42, 112.40, 113.66,
115.68, 119.16, 123.74, 124.48, 126.11, 127.58, 127.98, 129.66, 129.92, 131.28, 137.40, 139.80,
144.55, 148.43, 149.20, 149.63, 150.45, 158.39, 182.29, 188.65. HRMS: M+ at m/z 579.5632.
Anal. Calcd. For C31H25N5O9: C, 64.24; H, 4.35; N, 12.08. Found: C, 64.20; H, 4.31; N, 12.12.

4.7.14
(1E,4E)-1-(3-nitrophenyl)-5-(3-methoxy-(4((1-(2-(2,3-dioxoindolyl)ethyl)-1H-1,2,3-
triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SA-14)
Yield 67%; mp 69-72oC; MW 579.56. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.28 (m, 4H), 7.47-7.69 (m, 4H), 7.77-7.83 (m, 3H),
8.07-8.23 (d, J = 8.5 Hz, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
46.82, 49.84, 56.26, 72.62, 111.68, 115.24, 117.83, 119.77, 120.30, 120.99, 121.34, 122.12,
123.45, 124.95, 128.54, 129.61, 130.16, 132.57, 134.83, 136.11, 142.42, 148.15, 148.37, 149.08,
149.9, 152.74, 160.11, 184.44, 188.65. HRMS: M+ at m/z 579.5678. Anal. Calcd. For C31H25N5O7:
C, 64.24; H, 4.35; N, 12.08. Found: C, 64.28; H, 4.38; N, 12.04.
4.7.15
(1E,4E)-1-(4-phenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-1)
Yield 77%; mp 75-77oC; MW 570.01. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.21 (m, 5H), 7.30-7.53 (m, 4H), 7.66-7.80 (m, 4H),
8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.81, 49.83, 56.27, 72.61,
111.63, 115.28, 119.20, 119.71, 120.96, 123.45, 123.58, 126.46, 128.03, 128.51, 128.73, 130.23,
130.41, 134.98, 135.25, 142.48, 146.29, 149.03, 149.71, 152.75, 160.19, 184.46, 188.67. HRMS:
M+ at m/z 569.0183. Anal. Calcd. For C31H26ClN4O5: C, 65.44; H, 4.43; Cl, 6.23; N, 9.85. Found:
C, 65.48; H, 4.47; Cl, 6.19; N, 9.81.
4.7.16.
(1E,4E)-1-(3,4,5-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
chloroindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-2)
Yield 74%; mp 69-71oC; MW 659.08. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.69-3.72 (s,
6H), 3.74 (s, 3H), 3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.70-7.03 (m, 5H), 7.53-
13
7.80 (m, 5H), 8.29-8.33 (m, 3H). C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.82, 49.85,
56.21, 56.59, 72.62, 103.98, 111.60, 115.21, 119.73, 120.97, 123.41, 123.50, 128.58, 129.53,
130.28, 134.45, 134.91, 138.46, 142.47, 146.28, 149.01, 149.77, 150.76, 152.78, 160.13, 184.41,
188.67. HRMS: M+ at m/z 659.0812. Anal. Calcd. For C34H31ClN4O8: C, 61.96; H, 4.74; Cl, 5.38;
N, 8.50. Found: C, 61.92; H, 4.78; Cl, 5.34N, 8.46.
4.7.17.
(1E,4E)-1-(3,4-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
chloroindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-3)

Yield 78%; mp 74-76oC; MW 602.01. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.70-3.73 (s,
6H), 3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.72-6.75 (m, 3H), 7.03-7.66 (m, 5H),
7.53-7.80 (m, 3H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.84, 49.81,
56.25, 72.61, 111.68, 115.29, 119.25, 119.71, 120.94, 123.48, 123.51, 128.59, 130.21, 130.47,
134.98, 142.43, 146.21, 149.08, 149.73, 152.75, 160.19, 184.45, 188.61. HRMS: M+ at m/z
629.0674. Anal. Calcd. For C31H26ClN4O7: C, 63.01; H, 4.65; Cl, 5.64; N, 8.91. Found: C, 62.97;
H, 4.69; Cl, 5.68; N, 8.87.
4.7.18.
(1E,4E)-1-(4-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
chloroindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-4)
Yield 70%; mp 65-67oC; MW 599.03. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.61 (d, J = 8.5 Hz, 1H), 7.03-7.19 (m, 5H),
7.66 (d, J = 8.5 Hz, 2H), 7.53-7.80 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ,
TMS = 0): 46.81, 49.83, 55.90, 56.26, 72.68, 111.61, 114.23, 115.26, 119.21, 119.77, 120.90,
123.43, 123.56, 127.49, 127.51, 128.57, 130.22, 130.41, 134.98, 142.40, 146.22, 149.01, 149.79,
152.72, 159.91, 160.17, 184.43, 188.61. HRMS: M+ at m/z 599.0346. Anal. Calcd. For
C32H27ClN4O6: C, 64.16; H, 4.54; Cl, 5.92; N, 9.35. Found: C, 64.12; H, 4.58; Cl, 5.88; N, 9.39.
4.7.19.
(1E,4E)-1-(3-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
chloroindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-5)
Yield 67%; mp 60-62oC; MW 599.03. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-7.03 (m, 5H), 7.53-7.66 (m, 4H), 7.77-
7.80 (m, 3H), 8.29-8.33 (m, 3H).13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.81, 49.82, 56.27,
55.90, 72.64, 110.67, 111.66, 113.51, 115.24, 118.70, 119.21, 119.78, 120.95, 123.42, 123.54,
128.55, 129.71, 130.26, 130.48, 134.92, 136.23, 142.41, 146.27, 149.08, 149.72, 152.78, 160.15,
160.66, 184.43, 188.65. HRMS: M+ at m/z 599.0318. Anal. Calcd. For C32H27ClN4O6: C, 64.16;
H, 4.54; Cl, 5.92; N, 9.35. Found: C, 64.12; H, 4.50; Cl, 5.96; N, 9.39.
4.7.20.
(1E,4E)-1-(2-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
chloroindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-6)
Yield 65%; mp 67-69oC; MW 599.03 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-6.86 (m, 3H), 7.03-7.19 (m, 4H), 7.53-

13
7.93 (m, 5H), 8.29-8.33 (m, 3H). C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.81, 49.88,
56.28, 56.35, 72.67 111.62, 114.07, 115.04 115.29, 119.21, 119.77, 120.95, 121.07, 123.41,
123.59, 127.43, 128.56, 129.07, 130.46, 134.98, 142.43, 146.21, 149.01, 149.73, 152.72, 157.77,
160.12, 184.41, 188.62. HRMS: M+ at m/z 599.0389. Anal. Calcd. For C32H27ClN4O6: C, 64.16;
H, 4.54; Cl, 5.92; N, 9.35. Found: C, 64.20; H, 4.58; Cl, 5.88; N, 9.31.
4.7.21. (1E,4E)-1-(napthalen-1-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-7)
Yield 70%; mp 70-72oC; MW 619.07.1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03 (d, J = 8 Hz, 2H), 7.27-7.53 (m, 5H), 7.55-7.66 (m,
4H), 7.67-7.80 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.82,
49.85, 56.27, 72.63, 111.64, 115.21, 119.28, 119.73, 120.91, 123.15, 123.49, 123.51, 124.08,
125.84, 126.02, 128.31, 128.57, 128.80, 130.25 130.47, 131.08, 133.58, 134.95, 135.62, 142.43,
146.21, 149.09, 149.75, 152.70, 160.16, 184.45, 188.67. HRMS: M+ at m/z 619.0712. Anal. Calcd.
For C35H27ClN4O5: C, 67.90; H, 4.40; Cl, 5.73; N, 9.05. Found: C, 67.94; H, 4.36; Cl, 5.76; N,
9.01.
4.7.22. (1E,4E)-1-(furan-2-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-8)
Yield 74%; mp 65-67oC; MW 523.52. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.61 (d, J = 7.5 Hz, 1H), 7.00-7.03 (m, 3H), 7.53-7.66
(m, 4H), 7.77-7.80 (m, 3H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0):
46.86, 49.89, 56.27 72.61, 111.44, 111.63, 112.71, 115.28, 119.22, 119.79, 120.93, 123.45, 123.59,
128.52, 129.40, 130.26, 130.41, 134.97, 139.05, 142.48, 145.94, 146.28, 149.09, 149.76, 151.65,
152.78, 160.19, 184.46, 188.67. HRMS: M+ at m/z 558.7742. Anal. Calcd. For C29H23N4O6: C,
62.31; H, 4.15; Cl, 6.34; N, 10.02. Found: C, 62.35; H, 4.19; Cl, 6.30; N, 9.98.
4.7.23. (1E,4E)-1-(thiophen-2-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-9)
Yield 69%; mp 74-76oC; MW 539.58.1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.31 (m, 3H), 7.33-7.62 (m, 4H), 7.66-7.80 (m, 4H),
8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.83, 49.82, 56.21, 72.64,

111.63, 115.21, 119.25, 119.78, 120.92, 123.44, 123.51, 127.16, 128.24, 128.52, 129.43, 130.27,
130.48, 130.54, 134.98, 137.81, 141.79, 142.42, 146.2, 149.07, 149.73, 152.71, 160.15, 184.46,
188.63. HRMS: M+ at m/z 575.0356. Anal. Calcd. For C29H23N4O5S: C, 60.57; H, 4.03; Cl, 6.17;
N, 9.74; S, 5.68. Found: C, 60.52; H, 4.07; Cl, 6.13; N, 9.78; S, 5.64.
4.7.24. (1E,4E)-1-(4-chlorophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-10)
Yield 80%; mp 66-68oC; MW 603.45. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.83 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.86 (d, J = 8.5 Hz, 1H), 7.19-7.23 (m, 2H), 7.31-7.41
(m, 4H), 7.52-7.59 (m, 3H), 7.71 (d, J = 16.5 Hz, 1H), 7.77 (d, J = 16.5 Hz, 1H), 7.82 (d, J = 8.5
Hz, 2H), 8.33 (s, 1H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 47.47, 56.09, 61.98, 111.39,
112.39, 113.66, 114.66, 115.68, 119.16, 123.54, 124.47, 126.10, 126.68, 127.98, 128.34, 129.47,
130.61, 131.28, 134.25, 135.31, 137.40, 141.14, 143.85, 149.20, 149.61, 150.29, 158.38, 162.78,
182.29, 188.71. HRMS: M+ at m/z 603.4536. Anal. Calcd. For C31H24Cl2N4O5: C, 61.70; H, 4.01;
Cl, 11.75; N, 9.28. Found: C, 61.74; H, 4.05; Cl, 11.71; N, 9.24.
4.7.25. (1E,4E)-1-(4-bromophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-11)
1
1
Yield 78%; mp 65-66oC; MW 647.9. H NMR (CDCl3, 500 MHz, δ, TMS = 0): H NMR (d6-
DMSO, 500 MHz, TMS = 0): 3.84 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (m, 2H), 6.86 (d, J =
8.5 Hz, 1H), 7.20-7.25 (m, 1H), 7.33 (d, J = 8.5 Hz, 1H), 7.43 (s, 1H), 7.52-7.61 (m, 3H), 7.80-
7.85 (m, 2H), 7.96 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 8.29-8.34 (m, 3H). 13C NMR (d6-DMSO, 125
MHz, TMS = 0): 14.43, 22.53, 31.42, 31.65, 47.47, 56.08, 61.97, 112.39, 119.17, 123.75, 128.49,
126.12, 127.97, 129.93, 137.39, 148.43, 149.20, 149.61, 158.39, 162.77. HRMS: M+ at m/z
647.1313. Anal. Calcd. For C31H24BrClN4O5: C, 57.47; H, 3.73; Br, 12.33; Cl, 5.47; N, 8.65.
Found: C, 57.51; H, 3.69; Br, 12.29; Cl, 5.51; N, 8.69.
4.7.26 (1E,4E)-1-(3-bromophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-12)
Yield 72%; mp 77-79oC; MW 647.9. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.10 (m, 3H), 7.24-7.41 (m, 4H), 7.53-7.80 (m, 5H),
8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.81, 49.87, 56.29, 72.60,

111.66, 115.23, 119.21, 119.74, 120.98, 123.04, 123.47, 123.53 125.42, 128.51, 129.97, 130.26,
130.90, 134.93, 137.41, 142.48, 146.23, 149.07, 149.74, 152.71, 160.12, 184.43, 188.67. HRMS:
M+ at m/z 647.9047. Anal. Calcd. For C31H24BrClN4O5: C, 57.47; H, 3.73; Br, 12.33; Cl, 5.47; N,
8.65. Found: C, 57.43; H, 3.76; Br, 12.29; Cl, 5.51; N, 8.61.
4.7.27. (1E,4E)-1-(4-nitrophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-13)
1
Yield 80%; mp 80-82oC; MW 614. H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.83 (m, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.14 (m, 2H), 6.84 (m, 2H), 7.19-7.22 (m, 2H), 7.33 (m, 1H), 7.39-
13
7.42 (m, 2H), 7.53 (m, 1H), 7.59 (s, 1H), 7.65-7.76 (m, 6H), 8.33 (s, 1H). C NMR (d6-DMSO,
125 MHz, δ, TMS = 0): 47.48, 56.07, 61.97, 111.33, 112.33, 113.61, 119.15, 123.55, 124.15,
124.45, 126.10, 126.70, 127.99, 128.32, 130.82, 132.39, 134.57, 137.39, 141.22, 143.87, 149.19,
149.59, 150.28, 158.38, 182.29, 188.69. HRMS: M+ at m/z 614.1453. Anal. Calcd. For
C31H24ClN5O7: C, 60.64; H, 3.94; Cl, 5.77; N, 11.41. Found: C, 60.68; H, 3.90; Cl, 5.73; N, 11.45.
4.7.28. (1E,4E)-1-(3-nitrophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-chloroindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SB-14)
Yield 72%; mp 61-63oC; MW 614. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H), 4.15
(m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.28 (d, J = 8.5 Hz, 2H), 7.47-7.69 (m, 4H), 7.77-7.80
(m, 3H), 8.07-8.23 (d, J = 8.5 Hz, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS
= 0): 43.34, 47.16, 56.23, 66.17, 111.68, 115.24, 119.23, 119.79, 120.34, 121.39, 123.48, 123.53,
128.52, 128.77, 129.09, 129.61, 130.26, 130.42, 132.50, 134.94, 136.17, 146.29, 148.37, 149.09,
149.71, 152.74, 160.18, 184.44, 188.67. HRMS: M+ at m/z 613.1447. Anal. Calcd. For
C31H24ClN5O7: C, 60.64; H, 3.94; Cl, 5.77; N, 11.41. Found: C, 60.68; H, 3.98; Cl, 5.81; N, 11.37.
4.7.29.
(1E,4E)-1-(4-phenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-1)
Yield 70%; mp 68-70oC; MW 613.46. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.14 (m, 3H), 7.21-7.30 (m, 4H), 7.66-7.69 (m, 3H),
7.72 (d, J = 8 Hz, 2H), 7.96 (s, 1H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS =
0): 46.82, 49.81, 56.26, 72.64, 111.61, 115.26, 119.21, 119.77, 120.03, 120.98, 123.49, 124.31,
126.46, 128.55, 133.62, 135.25, 137.72, 142.46, 147.13, 149.08, 149.74, 152.78, 160.15, 184.47,

188.69. HRMS: M+ at m/z 613.4684. Anal. Calcd. For C31H25BrN4O5: C, 60.69; H, 4.11; Br, 13.03;
N, 9.13. Found: C, 60.73; H, 4.15; Br, 12.97; N, 9.09.
4.7.30.
(1E,4E)-1-(3,4,5-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
bromoindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-2)
Yield 74%; mp 77-79oC; MW 703.54. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.69-3.71 (s,
6H), 3.73 (s, 3H), 3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.26 (m, 2H), 7.03 (d, J
= 8 Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 7.69-7.96 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO,
125 MHz, δ, TMS = 0): 46.81, 49.86, 56.24, 56.59, 72.65, 103.91, 111.66, 115.29, 119.24, 119.79,
120.07, 120.98, 123.44, 124.39, 128.56, 129.50, 133.67, 137.73, 142.44, 147.10, 149.05, 149.77,
150.74 152.79, 160.15, 184.42, 188.68. HRMS: M+ at m/z 703.5410. Anal. Calcd. For
C34H31BrN4O8: C, 58.04; H, 4.44; Br, 11.36; N, 7.96. Found: C, 58.08; H, 4.40; Br, 11.32; N, 7.80.
4.7.31.
(1E,4E)-1-(3,4-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
bromoindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-3)
1
Yield 72%; mp 62-64oC; MW 673.51. H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.71-3.73 (s,
6H), 3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.70 (d, J = 8.5 Hz, 1H), 6.75-7.03 (m,
4H), 7.66-7.69 (m, 4H), 7.96 (s, 1H), 7.72 (d, J = 8.5 Hz, 1H), 8.29-8.33 (m, 3H). 13C NMR (d6-
DMSO, 125 MHz, δ, TMS = 0): 46.89, 49.83, 56.22, 72.68 111.61, 115.24, 119.70, 120.07, 123.47,
124.39, 128.51, 133.67, 137.94, 142.42, 147.19, 149.08, 149.71, 152.77, 160.19, 184.42, 188.69.
HRMS: M+ at m/z 673.5108. Anal. Calcd. For C33H29BrN4O7: C, 58.85; H, 4.34; Br, 11.86; N,
8.32. Found: C, 58.89; H, 4.38; Br, 11.82; N, 8.28.
4.7.32.
(1E,4E)-1-(4-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
bromoindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-4)
Yield 80%; mp 67-69oC; MW 655.49. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.61 (d, J = 8 Hz, 1H), 7.03-7.19 (m, 5H),
7.66-7.69 (m, 4H), 7.72 (d, J = 8.5 Hz, 1H), 7.96 (s, 1H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO,
125 MHz, δ, TMS = 0): 46.80, 49.87, 56.22, 55.94, 72.67, 111.69, 114.26, 115.24, 119.22, 119.71,
120.05, 120.93, 124.39, 127.42, 127.51, 123.47, 128.55, 133.63, 137.78, 142.41, 147.15, 149.09,
149.74, 152.72, 159.90, 160.16, 184.42, 188.69. HRMS: M+ at m/z 643.4846. Anal. Calcd. For
C33H27BrN4O6: C, 59.73; H, 4.23; Br, 12.42; N, 8.71. Found: C, 59.69; H, 4.26; Br, 12.38; N, 8.75.

4.7.33.
(1E,4E)-1-(3-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
bromoindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-5)
Yield 70%; mp 65-67oC; MW 643.48. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-6.86 (m, 3H), 7.03-7.10 (m, 3H), 7.66-
7.69 (m, 4H), 7.72 (d, J = 8.5 Hz, 1H), 7.96 (s, 1H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125
MHz, δ, TMS = 0): 46.85, 49.88 56.27, 55.91, 72.68, 110.69, 111.66, 113.51, 115.29, 118.76,
119.21, 119.77, 120.08, 120.91, 123.44, 124.32, 128.58, 129.70, 133.61, 136.29, 137.70, 142.48,
147.17, 149.09, 149.70, 152.76, 160.10, 160.61, 184.45, 188.69. HRMS: M+ at m/z 643.4809.
Anal. Calcd. For C32H27BrN4O6: C, 59.73; H, 4.23; Br, 12.42; N, 8.71. Found: C, 59.76; H, 4.27;
Br, 12.38; N, 8.67.
4.7.34.
(1E,4E)-1-(2-methoxyphenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-
bromoindolyl)ethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-6)
Yield 61%; mp 65-67oC; MW 655.49. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.73 (s, 3H),
3.90 (s, 3H), 4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-6.86 (m, 4H), 7.03-7.19 (m, 3H), 7.66-
769 (d, J = 8.5 Hz, 2H), 7.72-7.93 (m, 3H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ,
TMS = 0): 56.29, 56.31, 46.84, 49.80, 72.66 111.67, 114.23, 115.09, 115.23, 119.21, 119.72,
120.07, 120.90, 121.02, 123.47, 124.32, 128.59, 129.05, 133.62, 137.74, 142.48, 147.16, 149.04,
149.78, 152.77, 157.74, 160.17, 184.45, 188.67. HRMS: M+ at m/z 643.4892. Anal. Calcd. For
C33H27BrN4O6: C, 59.73; H, 4.23; Br, 12.42; N, 8.71. Found: C, 59.69; H, 4.26; Br, 12.38; N, 8.75.
4.7.35. (1E,4E)-1-(napthalen-1-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-7)
Yield 70%; mp 70-72oC; MW 663.52. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03 (d, J = 8.5 Hz, 2H), 7.27-7.36 (m, 5H), 7.55 (d, J =
8 Hz, 1H), 7.66-7.67 (m, 4H), 7.69-7.96 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125
MHz, δ, TMS = 0): 46.84, 49.88, 56.26, 72.62, 111.69, 115.25, 119.29, 119.75, 120.07, 120.98,
123.14, 123.45, 124.08, 124.34, 125.85, 126.09, 126.37, 128.39, 128.52, 128.83, 131.09, 133.55,
133.65, 135.69, 137.75, 142.42, 147.17, 149.03, 149.79, 152.77, 160.11, 184.49, 188.60. HRMS:
M+ at m/z 663.5236. Anal. Calcd. For C35H27BrN4O5: C, 63.36; H, 4.10; Br, 12.04; N, 8.44. Found:
C, 63.40; H, 4.06; Br, 12.00; N, 8.48.

4.7.36. (1E,4E)-1-(furan-2-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-1H-
1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-8)
Yield 64%; mp 69-71oC; MW 603.42. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 6.75-7.03 (m, 4H), 7.66-7.69 (m, 3H), 7.72-7.96 (m, 4H),
8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.83, 49.89, 56.26, 72.64,
111.49, 111.62, 112.78, 115.22, 119.25, 119.71, 120.08, 120.96, 123.44, 124.31, 137.70, 128.56,
129.41, 133.67, 139.08, 142.44, 145.91, 147.17, 149.08, 149.74, 151.60, 152.73, 160.11, 184.49,
188.65. HRMS: M+ at m/z 603.4236. Anal. Calcd. For C29H23BrN4O6: C, 57.72; H, 3.84; Br, 13.24;
N, 9.28. Found: C, 57.68; H, 3.82; Br, 13.28; N, 9.32.
4.7.37. (1E,4E)-1-(thiophen-2-yl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-9)
Yield 71%; mp 75-77oC; MW 619.49. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03 (d, J = 8.5 Hz, 2H), 7.31 (m, 1H), 7.62-7.66 (m, 4H),
7.69-7.96 (m, 4H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125MHz, δ, TMS = 0): 46.82, 49.86,
56.29, 72.65, 111.66, 115.23, 119.26, 119.79, 120.01, 120.94, 123.47, 124.38, 127.17, 128.27,
128.59, 129.44, 130.52, 133.67, 137.74, 137.81, 141.78, 142.49, 147.10, 149.08, 149.73, 152.77,
160.17, 184.45, 188.60. HRMS: M+ at m/z 619.4983. Anal. Calcd. For C29H23BrN4O5S: C, 56.23;
H, 3.74; Br, 12.90; N, 9.04; S, 5.18. Found: C, 56.27; H, 3.78; Br, 12.86; N, 9.08; S, 5.14.
4.7.38. (1E,4E)-1-(4-chlorophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-10)
Yield 76%; mp 62-64oC; MW 647.9. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03 (d, J = 8.5 Hz, 2H), 7.22-7.26 (m, 5H), 7.66-7.72
13
(m, 4H), 7.96 (s, 1H), 8.29-8.33 (m, 3H). C NMR (d6-DMSO, 125MHz, δ, TMS = 0): 46.80,
49.89, 56.28, 72.67, 111.66, 115.25, 119.24, 119.73, 120.02, 120.91, 123.47, 124.36, 127.85,
128.59 128.80, 133.38, 133.67, 137.71, 142.45, 147.13, 149.02, 149.77, 152.76, 160.14, 184.43,
188.66. HRMS: M+ at m/z 648.0681. Anal. Calcd. For C31H24BrClN4O5: C, 57.47; H, 3.73; Br,
12.33; Cl, 5.47; N, 8.65. Found: C, 57.43; H, 3.69; Br, 12.37; Cl, 5.43; N, 8.69.
4.7.39. (1E,4E)-1-(4-bromophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-11)

Yield 66%; mp 75-77oC; MW 692.35. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.19 (m, 4H), 7.26-7.38 (m, 3H), 7.66-7.72 (m, 4H),
7.96 (s, 1H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125MHz, δ, TMS = 0): 46.87, 49.80, 56.24,
72.67, 111.63, 115.21, 119.24, 119.70, 120.02, 120.98, 123.49, 124.33, 128.51, 128.64, 131.69,
133.64, 134.20, 137.76, 142.43, 147.16, 149.01, 149.79, 152.77, 160.13, 184.49, 188.68. HRMS:
M+ at m/z 692.0178. Anal. Calcd. For C31H24Br2N4O5: C, 53.78; H, 3.49; Br, 23.08; N, 8.09. Found:
C, 53.74; H, 3.53; Br, 23.12; N, 8.05.
4.7.40. (1E,4E)-1-(3-bromophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-12)
Yield 60%; mp 70-72oC; MW 692.35. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.10 (m, 3H), 7.24-7.31 (m, 3H), 7.47-7.66 (m, 3H),
7.69-7.96 (m, 3H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125 MHz, δ, TMS = 0): 46.87, 49.86,
56.24, 72.68, 111.64, 115.29, 119.27, 119.76, 120.03, 120.92, 123.07, 123.43, 124.35, 125.47,
128.55, 129.96, 130.94, 133.67, 137.42, 137.73, 142.41, 147.12, 149.05, 149.74, 152.76, 160.14,
184.46, 188.66. HRMS: M+ at m/z 692.0146. Anal. Calcd. For C31H24Br2N4O5: C, 53.78; H, 3.49;
Br, 23.08; N, 8.09. Found: C, 53.82; H, 3.53; Br, 23.04; N, 8.05.
4.7.41. (1E,4E)-1-(4-nitrophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-13)
Yield 79%; mp 64-66oC; MW 658.46. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03 (d, J = 8.5 Hz, 1H), 7.26 (s, 1H), 7.32 (d, J = 8.5 Hz,
1H), 7.56 (d, J = 8.5 Hz, 2H), 7.66-7.72 (m, 3H), 7.96-7.80 (d, J = 8.5 Hz, 2H), 8.14 (d, J = 8.5
Hz, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO, 125MHz, δ, TMS = 0): 46.88, 49.82, 56.24,
72.65, 111.68, 115.25, 119.27, 119.79, 120.04, 120.91, 121.08, 123.49, 124.32, 127.39, 128.55,
133.62, 137.71, 141.30, 142.41, 147.18, 147.69, 149.05, 149.79, 152.77, 160.10, 184.43, 188.61.
HRMS: M+ at m/z 657.0984. Anal. Calcd. For C31H24BrN5O7: C, 56.55; H, 3.67; Br, 12.14; N,
10.64. Found: C, 56.59; H, 6.63; Br, 12.18; N, 10.60.
4.7.42. (1E,4E)-1-(3-nitrophenyl)-5-(3-methoxy-(4-((1-(2-(2,3-dioxo-5-bromoindolyl)ethyl)-
1H-1,2,3-triazol-4-yl)methoxy)phenyl)penta-1,4-dien-3-one (SC-14)

Yield 70%; mp 65-67oC; MW 658.46. 1H NMR (d6-DMSO, 500 MHz, δ, TMS = 0): 3.90 (s, 3H),
4.15 (m, 2H), 4.67 (m, 2H), 5.15 (s, 2H), 7.03-7.28 (m, 3H), 7.47-7.69 (m, 4H), 7.72-7.77 (d, J =
8 Hz, 2H), 7.96 (s, 1H), 8.07-8.23 (d, J = 8.5 Hz, 2H), 8.29-8.33 (m, 3H). 13C NMR (d6-DMSO,
125 MHz, δ, TMS = 0): 46.81, 49.80, 56.22, 72.69, 111.63, 115.28, 119.24, 119.77, 120.05, 120.36,
120.95, 121.37, 123.44, 124.38, 128.53, 129.69, 132.52, 133.60, 136.11, 137.76, 142.43, 147.10,
148.35, 149.03, 149.79, 152.78, 160.17, 184.44, 188.66. HRMS: M+ at m/z 657.0946. Anal. Calcd.
For C31H24BrN5O7: C, 56.55; H, 3.67; Br, 12.14; N, 10.64. Found: C, 56.51; H, 3.63; Br, 12.18; N,
10.68.
4.8. In-vitro cytotoxic assay
In vitro cytotoxicity against four human cancer cell lines was determined using 96-well tissue
culture plate. The cells were allowed to grow in carbon dioxide incubator (37ºC) for 24 h. Test
materials in complete growth medium (100 ml) were added after 24 h of incubation to the wells
containing cell suspension. The plates were stained with sulforhodamine B dye (0.4% in 1% acetic
acid, 100 ml) for 30 min. The plates were washed five times with 1% trichloroacetic acid and then
air-dried. The adsorbed dye was dissolved in Tris-HCl buffer (100 mL, 0.01 M, pH 10.4), and the
plates were gently stirred for 10 min. The plates were further incubated for 48 h in a carbon dioxide
incubator. The cell growth was stopped by gentle layering trichloroacetic acid (50%, 50 ml) on top
of the medium in all the wells. The plates were incubated at 4ºC for 1 h to fix the cells attached to
the bottom of the wells. The liquid of all the wells was gently pipette out and discarded. The plates
were washed five times with distilled water to remove trichloroacetic acid, growth medium low
molecular weight metabolites, serum on a mechanical stirrer. The optical density (OD) was
recorded on ELISA reader at 540 nm. The cell growth was determined by subtracting the mean
OD value of respective blank from the mean OD value of the experimental set. Percent growth in
presence of test material was calculated considering the growth in the absence of any test material
as 100%, and in turn, percent growth inhibition in presence of test material was calculated.19
4.9. In-vitro tubulin polymerization assay
A Cytoskeleton tubulin polymerization assay kit was purchased from Labex India. For assessing
the tubulin polymerization inhibition potential, 100 mM stock solution of each compound was
made in molecular biology grade DMSO. Further dilutions (50 mM, 25 mM, 10 mM) of each
compound were made by diluting the stock solution. General tubulin buffer was prepared by

reconstituting the constituents (available in kit) in 10 mL distilled water. General Tubulin Buffer
(1 mL) was supplemented with 10 mL of 100 mM GTP solution and 10 mg Tubulinwas suspended
in it and mixture was stored at 4ºC until used. Before starting the experiment 96 well plate and
Elisa plate reader were warmed at 37ºC as this temperature is required to achieve the proper
polymerization of tubulin. Tubulin polymerization buffer was prepared by mixing General tubulin
buffer (750 mL), Tubulin glycerol buffer (250 mL) and 100 mM GTP (10 mL). 10 mL General
tubulin buffer was added in the wells, where first two wells were kept as control (without any test
compound), and different concentrations of compounds were subsequently added in previously
marked wells and plate was kept in plate reader for 2 min at 37ºC. Meanwhile tubulin suspension
was diluted with Tubulin polymerization buffer and 100 mL of diluted tubulin was added in each
well. Plate was again kept in plate reader at 37ºC for 1 h and absorbance was measured at 340 nm
(Lee and Timasheff, 1977). Percentage inhibition and IC50 values were calculated according to the
absorbance values obtained after 1 h.20
4.10. Immunofluorescence microscopic studies
Cultured HCT116 cells were seeded on cover slips at a 35 mm culture dishes, and incubated in the
presence of 50, 30, 15, 10, 5 and 2.5 μM of compound for 24 h . Cells were washed twice in PBS
and fixed in 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized in
PBS with 0.1% TritonX-100 at room temperature for 15 min. Nonspecific binding sites were
blocked by incubating the cells in 5% BSA. Cells were then incubated with monoclonal α-tubulin
primary antibody (Sigma) diluted 1:100 in 0.1% Triton X-100 in PBS followed by Alexa flour
488 conjugated secondary antibody (Invitrogen) diluted 1:500. Cells were then washed three times
in PBS and stained with 4',6-diamidino-2-phenylindole (DAPI 1µg/ml in PBS). The cover slips
were mounted over glass slides and cells were imaged by a laser scanning confocal microscope
(Olympus Fluoview FV1000). Cells were treated with different concentrations of compound
compared to control for the disruption of microtubules. The data are representative of three
separate sets of experiments.
4.11. Docking simulations
The X-ray structure coordinates of tubulin bound with colchicine was obtained from protein data
bank (PDB entry: 1SA0; Resolution 3.5Å).27 The X-ray structure of tubulin consists of two tubulin
heterodimers in a curved complex capped by the stathmin-likedomain (SLD). In each tubulin

heterodimer, a colchicine molecule was found in complexed form at the interface of α1 and β1
subunits. Chakraborti et al. investigated the tubulin-binding mode of few curcumin analogs.9 The
study reported that curcumin binds at the interface of β1-α2 heterodimer which is approximately
32 Å away from the colchicine-binding site.9
To study the binding mode of the synthesized compounds, we have docked the most potent
compound SA-2 at the curcumin binding site of tubulin as reported by Chakraborti et al. The
docking study was carried out using the GOLD 5.3.0 software.28 ChemScore was applied as fitness
function to rank various docked conformations. ChemScore estimates the total free energy change
that occurs on ligand binding by considering parameters like hydrogen bond energies, atom radii
and polarisabilities, torsion potentials, hydrogen bond directionalities, lipophilicity, etc.29 The
Structure of SA-2 was drawn in ChemDraw Ultra (2010) and subjected to energy minimization
using the MM2 force field as implemented in Chem3D Ultra software. The compound was docked
ten times and conformation associated with highest scoring value was considered to analyze
variousdrug-receptor interactions.
Acknowledgements
Authors are grateful to Indian Council of Medical Research for providing funds to carry out the
research work (ICMR funded research project no. 58/23/2013-BMS dated 19-03-2015).
Figure captions
Figure 1. a) Chemical structure of curcumin, where the α,β-unsaturated β-diketone (heptadiene-
dione) moiety, undergoes keto-enol tautomerism and forms a hydrogen bond-containing 6-
membered ring (substrate for liver aldoketo reductases leads to rapid in vivo metabolism); b) Mono
carbonyl curcumin derivatives having anticancer potential (1-5).
Figure 2. Isatin hybrids exhibiting anticancer potential
Figure 3. Design strategy for mono carbonyl curcumin-isatin hybrids
Scheme 1. Synthesis of triazole linked mono carbonyl curcumin-isatin hybrids. Reagents and
conditions: (a) Acetone, 40% KOH, 2 hrs, stir, rt; (b) Propargyl bromide, K2CO3, DMF, 2 hrs, stir,
rt; (c) Aldehydes, 5% NaOH, MeOH, 2 hrs, stir, rt; (d) Dibromoethane, K2CO3, DMF, 2 hrs, stir,
rt; (e) NaN3, DMF, 1 hr, stir, rt; (f) Sodium ascorbate, CuSO4, DMF, 15 mins, rt.

Table 1 Various substituted bifunctional hybrids with their %age inhibition against eight human
cancer cell lines at 50 µM
Table 2 IC50 values of active hybrids against sensitive human cancer cell lines
Table 3 Tubulin polymerization inhibition potential of most active hybrids
Figure 4. Structure activity relationship
Figure 5. Effect of compound on microtubule network of HCT116 cells untreated (control), 5µM
SA-2 for 48 h. Microtubules and unassembled tubulin are shown in green. DNA, stained with 4,6-
diamidino-2-phenylindole (DAPI) shown in blue. Scale bar: 10 µm. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article)
Figure 6. (a) Docked conformation of compound SA-2 (cyan) at the interface of β1-α2 subunits
of tubulin complexed with colchicine (red) (PDB id 1SA0); (b) Residues involved in drug-receptor
interactions between SA-2 and tubulin (SA-2: carbon atoms are shown in green; only hydrogen
which is involved in H-bond interaction is are shown)
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