Target β-catenin/CD44/Nanog axis in colon cancer cells by certain N′-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides

Article abstract of DOI:10.1016/j.bmcl.2016.02.064

Cell surface molecule CD44 plays a major role in regulation of cancer stem cells CSCs on both phenotypic and functional level, however chemical inhibition approach of CD44 to targets CSCs is poorly studied. Herein, we report the discovery of certain N′-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides as a novel inhibitor of CD44. Molecular docking study showed interference of the scaffold of these compounds with β-catenin/TCF-4 complex, building a direct relationship between CD44 inhibition and observed well-fitted binding domain. Compound 11a, most potent member elicits inhibition effect on TCF/LEF reporter activity conformed the involvement of Wnt pathway inhibition as a mechanism of action. Furthermore, the treatment by the mentioned compound leads to inhibition of embryonic transcriptional factor Nanog but not Sox2 or Oct-4 suggested specific targeted effect. Moreover, the cytotoxicity and cell cycle effect of this series seems to be dependent on CD44 expression.

Full text of DOI:10.1016/j.bmcl.2016.02.064

Accepted Manuscript
Target β-catenin /CD44/Nanog Axis in Colon Cancer Cells by Certain N’-(2-
oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides
Awwad A. Radwan, F. Al-Mohanna, Fares K. Alanazi, P.S. Manogaran,
Abdullah Al-Dhfyan
PII:
S0960-894X(16)30188-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.064
BMCL 23618
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Accepted Date:
16 January 2016
21 February 2016
Please cite this article as: Radwan, A.A., Al-Mohanna, F., Alanazi, F.K., Manogaran, P.S., Al-Dhfyan, A., Target
β-catenin /CD44/Nanog Axis in Colon Cancer Cells by Certain N’-(2-oxoindolin-3-ylidene)-2-
(benzyloxy)benzohydrazides, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/
j.bmcl.2016.02.064
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Target β-catenin /CD44/Nanog Axis in Colon Cancer Cells by Certain N’-(2-
oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides
Awwad A. Radwan*a,b, Al-Mohanna Fc, Fares K.Alanazia, Manogaran PSd, Abdullah Al-
Dhfyan*d,e
akayyali Chair, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Assiut
University, Assiut, Egypt.
cDepartment of Comparative Medicine, King Faisal Specialist Hospital and Research
Centre.
dStem Cell & Tissue Reengineering Program, King Faisal Specialized Hospital and
Research Center, Riyadh, Saudi Arabia.
eDepartment of Pharmacology & Toxicology, Collage of Pharmacy, King Saud
University.
*Corresponding authors.
Awwad A. Radwan,
Kayyali Chair, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia.
E-mail: dhna_2001@hotmail.com
Abdullah Al-Dhfyan, BPharm.HonDSc
Stem Cell Therapy & Tissue Re-Engineering Program, Research Centre
King Faisal Specialist Hospital & Research Centre
MBC-03 P.O.Box 3354, Riyadh 11211, Saudi Arabia
Tel.: (+966-1) 4647272 Ext. 27862 / 37393
Fax:(+966-1) 4647272 Ext. 27858
E-mail: aaldhfyan@kfshrc.edu.sa
1

ABSTRACT
Cell surface molecule CD44 plays a major role in regulation of cancer stem
cells CSCs on both phenotypic and functional level, however chemical inhibition
approach of CD44 to targets CSCs is poorly studied. Herein, we report the
discovery of certain N’-(2-oxoindolin-3-ylidene)-2-(benzyloxy)benzohydrazides
as a novel inhibitor of CD44. Molecular docking study showed interference of the
scaffold of these compounds with β-catenin/TCF-4 complex, building a direct
relationship between CD44 inhibition and observed well-fitted binding domain.
Compound 11a, most potent member elicits inhibition effect on TCF/LEF reporter
activity conformed the involvement of Wnt pathway inhibition as a mechanism of
action. Furthermore, the treatment by the mentioned compound leads to inhibition
of embryonic transcriptional factor Nanog but not Sox2 or Oct-4 suggested
specific targeted effect. Moreover, the cytotoxicity and cell cycle effect of this
series seems to be dependent on CD44 expression.
Keywords: Synthesis; Docking; Benzohydrazides; β-catenin
2

Cancer remains one of the most difficult health problems worldwide. The
difficulty on our opinion is origin from the development nature of these diseases and
weak activity of current available treatments. Cancers may have a hierarchical
organization in which subpopulations are arranged into tumorigenic and non-tumorigenic
cells.1 Recently, several studies demonstrated that tumorigenic potential of the tumor
cells is driving by small sub population called cancer stem cells (CSCs). The model of
CSCs had been applied in leukemia2 and extended to breast,3 brain,4 lung,5 colon,6 and
other cancers. The CSCs hypothesis is based on tumorigenic potential enabled by self-
renewal in which that tumors are maintained by a self-renewing CSC population capable
of differentiating into non-self-renewing cell populations that constitute the bulk of the
tumor.7 The self-renew/tumorigenic state of tumor cells is plastic and cells with stem
cells properties can be generated by epithelial-mesenchymal transition (EMT).8 CD44 is a
member of a large family of cell adhesion molecule. In addition to its role in cellular
adhesion, CD44 play a major role involved in growth and motility of many cancers. The
crosstalk between CD44 and CSCs had been demonstrated in both phenotypic and
functional level. Phenotypically, CD44 is used as a marker of CSCs in many cancers. On
functional level, first CD44 might regulate the CSCs metastasis through adhesion and
EMT.8,9 Second, CD44 might regulate self-renew through Nanog phosphorylation,
Hedgehog and Notch pathway.9-13 Third, CD44 through interaction with hyaluronic acid
can stimulate the upregulation of MDR-1 and IAPs. Moreover, knockdown of CD44
resulted in reduce Bcl-2 level in breast CSCs.14,15 Fourth, Wnt signaling pathways play a
major role in embryonic development and carcinogenesis. Functionally, Wnt pathway
work in the absence of secreted glycolipoprotein Wnt, cytoplasmic β-catenin protein is
3

constantly degraded by the action of the Axin complex, which is composed of the
scaffolding protein Axin, the tumor suppressor adenomatous polyposis coli gene product
(APC), casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3). CK1 and GSK3
sequentially phosphorylate the amino terminal region of β-catenin, resulting in β-catenin
recognition by β-Trcp, an E3 ubiquitin ligase subunit, and subsequent β-catenin
ubiquitination and proteasomal degradation.16 Activation of Wnt receptors blocks the
GSK3β activity and degradation of β-catenin, which is translocated to the nucleus where
it interacts with members of the T cell factor (TCF)/LEF family of HMG-domain
transcription factors to activate Wnt target gene transcription.17 Cell surface molecule
CD44 is a downstream target of Wnt/β-catenin pathway.18 Finally, a therapeutic approach
using an activating monoclonal antibody directed to the adhesion molecule CD44 had
been successfully used to eradicate human acute myeloid leukemic stem cells.19
Using the crystal structure information of β-catenin/Tcf4, 6 hot spots or cavities were
identified on its surface (figure 1). Of all these hot spots, cavity 2 is the largest one as it
has two small pockets on both sides, one containing K435 and the other near R469. As a
result, cavity 2 contains several clefts that can potentially anchor a small molecular
weight inhibitor and was, therefore selected as a hot spot for Tcf4 competitor study.20
Figure 1. β-catenin crystal structure.21 The Tcf4 is deleted and the β-catenin is shown as
solid surface. The hotspot areas are shown as solid yellow surface.
Recently, starting from both biophysical-combined virtual screening and biochemical
screening studies, new inhibitors of the β-catenin/Tcf4 interaction were discovered. In the
4

former study, Trosset screened a library of 17,000 compounds and selected 22
compounds for biophysical screening, of which three compounds confirmed as binders
and Tcf4 competitors. PNU74654 (Figure 2) was the most active hit.20 The latter study
was done by Lipourcelet using biochemical screening of a library of 7000 natural
compounds. Lipourcelet succeeded to identify 6 compounds (Figure 2) as antagonists of
β-catenin/Tcf4 complex with in vitro IC50 values <10 µM.22
Figure 2. Chemical structures of small- and large- size inhibitors of Tcf4/β-catenin
interaction.20,22
Trosset et al predicted the binding mode of PNU74654 in cavity 2 as it binds one small
pocket near K435 and another small pocket near R469 on both sides of cavity2 (Figure
3). On the other hand, the larger lipourcelet’s compounds don’t show binding to the two
adjacent pockets of K435/R469 hot spot or cavity 2 as deeply as PNU74654. The large
central ring systems make more extensive hydrophobic contact with the floor of the hot
spot, which may explain their potency (Figure 3).20,22
The present study is aimed to report the discovery of small molecule inhibitor of CD44
and its viability to target cancer stem cells in preclinical setting of colon cancer. The
compounds were designed to combine the central large hydrophobic moiety aimed to
make hydrophobic contact with the floor of cavity2 as in lipourcelet’s compounds and
small moieties that bind to the small pockets or clefts in cavity 2 as with Trosset’s
compounds (Figure 3).
5

Figure 3. β-catenin crystal structure.21 Cavity 2 shows small binding pockets near
Lys435/Arg469 referred to number 1, binding cleft between Lys508 and Glu571 referred
to number 2; the floor area refers to number 3.
A series of related analogs, aimed to combine the two binding modes, were
designed, and synthesized in multi-steps reaction as shown in Scheme 1. At first, the 2-
benzyloxy/chlorobenzyloxy benzohydrazides were prepared via known two-step
synthesis starting with the reaction of methyl salicylate with the appropriate benzyl
chloride in alkaline hydromethanolic solution.23 Hydrazinolysis of the resulting methyl 2-
benzyloxy/chlorobenzyloxy benzoates 1a,b resulted in the corresponding
benozhydrazides 2a,b.24 Synthetic entry to the designed compounds 3a,b-11a,b
(Supplementary information) was readily undertaken in a two-step sequence reaction. At
first, compounds 3a,b is converted into hydrazide hydrazone upon condensation of 2a,b
with isatin followed by installation of alkylaminomethyl or aralkylaminomethyl group via
a Mannich reaction resulting in compounds 4a,b-11a,b.
Scheme 1. Synthesis of target compounds. Reagents and conditions: (i) appropriate
benzylchlordie, CH3ONa (ii) NH2NH2.H2O/C2H5OH, reflux; (iii) isatin/C2H5OH, reflux;
(iv) appropriate secondary amine, HCHO, DMSO, rt.
Recently the main stream of cancer drug discovery shifted to target CSCs, be
caused of tumor initiation, chemoresistance and metastasis potential. Cell surface
6

molecule CD44 play a major role in regulation of CSCs in functional level, therefore we
used a flow cytometry to evaluate the inhibitory effect of the targeted derivatives 3a,b-
11a,b on CD44 compared with untreated cells (Table 1). The inhibitory results against
the cell surface CD44 were reported in Table1 indicate that we have produced some new
potent
CD44
inhibitors
belonging
to
the
2-oxoindolin-3-ylidene)-2-
(benzyloxy)benzohydrazides series 3a,b-11a,b. The majority of the compounds showed
high CD44 inhibition percent (> 50%) at 10 μM (Compounds 4a, 5a,b–10a,b, 9a, 11a,b).
The most active member in this series is 11a with 76.18% inhibition effect. Focusing on
the results reported in Table 1, we can observe that elimination of p-chloro substituent is
unfavourable for ligand-binding site interaction (compare 3a-11a and 3b-11b). Also, the
elimination of the N-Mannich base fragment is unfavourable for CD44 inhibition activity
(compare 3a,b and 4a,b-11a,b). At the same time, increasing the length of the carbon
chain of the secondary amine of the Mannich base substituent results in a significant
increase in the inhibition activity (compare 4a,b, 5a,b, 6a,b and 7a,b). While, the
morpholino and piperdino Mannich bases show comparable CD44 inhibition activity with
that of the diethylamino Mannich bases (compare 5a,b with 8a,b and 9a,b). The Mannich
base derivatives bearing 2-hydroxy-N-methylethylamino or the N-methylbenzylamino
fragment explored highest CD44 inhibition activity (compare 10a,b, 11a,b). Indicating
the bulk effect on potency has a ceiling and the bulkier p-chlorobenzyl group such as 3b-
11b showed lower activity (Inhibition %, 18.48-59.52) than the corresponding
analougues 3a-11a (Inhibition %, 31.25-76.18). This is supportive of the notion of
binding to a hydrophobic pocket where a size limit of the benzyl side chain is imposed
that the chlorobenzyl group is oriented in different position than that of the benzyl group.
7

Here it can be seen that increasing the dialkylamino from dimethylamino (4a, 4b
No
H-bond donor
H-bond
H-bond
distance
Score
in
Inhibition
%
MFI* SD
acceptor
Energy
Inhibition % 29.77, 50.65 respectively) to the corresponding dibutylamino (7a, 7b
Inhibition % 53.50, 66.34 respectively) leads to an increase of activity. This indicates that
hydrophobicity is an important parameter at this position. However, cyclisation of the
dibutylamino group (7a, 7b Inhibition % 53.50, 66.34 respectively) into the
corresponding morpholinyl group (8a, 8b Inhibition % 50.55, 59.54 respectively) or
piperidinyl moiety (9a, 9b Inhibition % 45.63, 51.95 respectively) leads to decrease of
activity. In addition, the piperidinomethyl derivative 9a,b is less active than the
morpholino methyl analogue 8a,b. The N-methyl benzylamine compounds are the most
active (11a, 11b Inhibition % 59.52, 76.18 respectively). This is supportive of the notion
of preferred binding of the planar aromatic ring to a hydrophobic pocket.
Table 1. Measured in Vitro CD44 inhibition % and docking results of compounds 3a,b-
11a,b
8

ResName.No.Atom
ResName.No.At
om
rec ASN.516.NH2
rec ASN.516.NH2
rec ARG.471.NH2
4a 4a.N1
3a 3a.N3
3a 3a.O3
4a 4a.O2
rec
3.282
3.422
3.269
2.897
-36.557
-42.34
3a
4a
31.25
50.65
3142 747
2276.66 893
ASN.516.OD1
5a 5a.O2
5a 5a.O1
6a 6a.O2
7a 7a.O2
7a 7a.O3
8a 8a.N2
8a 8a.O3
9a 9a.O1
9a 9a.O2
rec LYS.508.O
10a 10a.O2
11a 11a.O1
11a 11a.O2
3b 3a.N3
3b 3a.O1
4b 4b.O1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.515.NH1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.515.NH2
rec ARG.474.NH1
rec ARG.474.NH2
10a 10a.O4
3.132
2.927
3.222
3.398
3.541
3.553
3.751
3.124
3.081
3.137
3.008
3.055
3.017
3.367
3.617
4.063
-43.243
5a
52.65
2164 226
-45.139
-44.916
6a
7a
58.96
66.34
1875.33 411
1538 543
-43.087
-42.307
-45.825
-46.023
-39.684
-38.586
8a
59.54
51.95
65.46
76.18
18.48
29.77
1849 421
2195.66 904
1578.33 36
1088.33 47
3725.66 564
9a
10a
11a
3b
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.515.NH2
rec ASN.516.NH2
ASN.516.NH2
4b
3209.66 986.
8
rec ARG.515.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.474.NH1
5b 5b.O1
6b 6b.N2
6b 6b.O2
7b 7b.N2
2.897
3.382
2.868
3.323
-42.153
-43.747
5b
6b
51.13
52.25
2233.33 171
2182 226.3
2125 225.3
-44.792
7b
53.50
9

rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.515.NH2
10b 10b.O4
7b 7b.O2
8b 7b.O1
8b 7b.O2
9b 9b.O2
10b. 10b.O1
10b 10b.O3
Rec
2.932
3.482
3.231
3.215
3.086
2.991
3.459
-41.511
8b
50.55
2260 713
-40.505
-45.181
9b
45.63
54.92
2484.66 183
2060 256
10b
Glu571.OE1
11b 6a.O2
rec ARG.474.NH2
2.992
-46.399
11b
59.52
1850 192.6
* Mean fluorescence intensity
**The expression of treated cells by the different compounds is normalized to untreated cells.
Based on the fact that cell surface molecule CD44 is a downstream target of
Wnt/β-catenin pathway,18 docking studies were carried out in order to investigate a
possible binding mode of the synthesized compounds, using the available crystal
structure of β-catenin, pdb code 1jdh.25 Compound 11a was docked into the allosteric
site, cavity 2 of β-catenin and possible binding modes were analysed based on their
Gridscore fitness. The docking poses show four key interactions with the receptor: one
nitrogen of the hydrazine group, imine nitrogen, act as H-bond acceptor for the Arg515
while the other nitrogen, amide nitrogen, of the hydrazine group acts as H-bond donors
for the side chain of a glutamic acid Glu571; the benzene ring of the salicylic acid moiety
binds to a small cleft between Lys508 and Glu571; the benzyl group binds to a small
pocket adjacent to Lys435 while the N-methylbenzylamine moiety binds to a small
pocket adjacent to Arg469; the isatin scaffold shows hydrophobic stack with the floor of
the binding site. In addition, a –cation interaction was suggested between a charged
10

lysine and arginine and the aromatic rings of the compound. In addition, the lysine,
Lys508 and arginine, Arg571 residues present in the site could show –cation
interactions with the aromatic rings N-methylbenzylamine and benzyloxy groups
respectively (Figure 4).
Figure 4. Suggested binding mode of 11a (magenta) to the cavity 2 of 1jdh.pdb. It binds
to both the floor of the cavity and the Lys435/Arg469 hot spot. Hydrogen bonds are
colored green.
To confirm the docking study on a real setting, the most active compound 11a
was investigated whether it could reduce the activity of Wnt pathway reporter. In this
study we used colon cancer cells transfected with TCF/LEF reporter plasmid. The
activity is measured by the expression of green fluorescence GFP by flow cytometer. The
transfected cells were cultured in 6 well and challenged by 2.5,5 and 10 for 48hrs.
The flow cytometric analysis showed consistence inhibition effect as seen in figure 5.
Figure 5. a) Show representative dot plot of Wnt reporter-GFP of untreated and treated
cells with 11a 2.5,5 and 10 μM
b) Bar graphs showing mean±s.d. Wnt repoter-GFP. Data represents the average ± SD
(error bar) of three independent experiments. P-value <0.05 (t-test)
11

Embryonic transcriptional factors Nanog, Sox2 and Oct-4 play critical roles in
maintaining the pluripotence and self-renewal characteristics of CSCs. Therefore, it will
be very interested for us to address the effect of N’-(2-oxoindolin-3-ylidene)-2-
(benzyloxy)benzohydrazide derivatives represented by 11a on these transcriptional
factors. Practically, LOVO colon cancer cells were cultured in 6 well and treated by 11a
in 2.5, 5 and 10 for 48hrs. The RNA was extracted and the genes expression was
measured by RT-PCR. The treatment by different doses showed decreased in the
expression of Nanog in dose dependent manner as shown in figure 6. However, no
change in the expression of Sox2 and Oct-4 was detected at 10 , suggested specificity
in the effect and supported by the fact that CD44 increased the expression of Nanog
through the interaction with Hyaluronan (HA).26
Figure 6. Endogenous levels of embryonic transcriptional factors Oct-4,Sox-2 and Nanog
mRNA were examined by real-time RT-PCR. Nanog transcript was notably decreased in
10 μM and no changes were detected in the transcript of Oct-4 or Sox-2.
The endpoint in cancer treatment is killing cancer cells and inhibiting the highly
proliferative nature of tumor cells. To test whether targeted derivatives induction of
cytotoxicity is dependent on CD44 expression or not, we used highly CD44 expressed
colon cancer cell line LOVO and low CD44 expressed colon cancer cell line HT-29. The
target derivatives with more than 50% inhibition effect on CD44 were used for the
screening. To this end, by the used of WST-1 cytotoxic/cell proliferation assay, a
selective cytotoxic effect of these targeted derivatives on LOVO cells but not HT-29 cells
were found and IC50 for the selected derivatives was shown on table 2. To confirmed
12

CD44 dependent effect of these derivatives, the DNA cell cycle effect of the most active
derivative 11a based on CD44 expression on the same type of cells in culture was
evaluated. By using combination of DCV and CD44-APC antibody, the DNA cell cycle
in CD44 high and CD44 low expressed cells on same cells with and without treatment
can be analyzed. Interestingly, in untreated cells the majority of CD44 high cells were
found to be at G0 checkpoint in non-proliferative phase. On the other hand CD44 low
expressed cells from the same culture cells were found to be in proliferative stages at S,
G2 checkpoint and M phase. By challenging the HT-15 cells with different doses of 11a
for 48hrs, we found that 11a block the cell cycle inhibitory effect of CD44 and induced
cell accumulation of CD44 high cells in cycling phase in S, G2 and M phase as seen in
figure 7. The effect on cell cycle was seen in 5 and 10 M on CD44 High cells and no
effect on DNA cell cycle was seen in CD44 Low cells in 5μM treatment. In 10 μM
treatment, CD44 high cells showed more sensitivity than CD44 Low cells and more
accumulation were seen at S phase. One of the mechanisms of how CSCs are resistance
to chemotherapy is slow cycling nature of these cells and therefore insensitive to toxic
drug.
Taken
together,
it
is
clear
that
N’-(2-oxoindolin-3-ylidene)-2-
(benzyloxy)benzohydrazides blocks CD44 effect on cell cycle and push CD44 high cells
toward cycling phases. The general cytotoxic effect seems to be dependent on CD44
expression in which these derivatives showed no toxicity on CD44 negative HT-29 cells
as shown in table2.
13

Figure 7. a) Histogram of CD44 expression showed gated CD44 low and CD44 high
subpopulation.
b) DNA cell cycle analysis of CD44 low and CD44 high subpopulation of untreated and
treated cells with 11a in 5 and 10μM treatment.
Table 2. In vitro cytotoxic activity of 4a-11a, 11b against cancer cell lines.
Compound
IC50 a (µM)
CD44 High
CD44 Negative
HT-29
LOVO
48.77±0.01
84.78±0.02
95.54±0.7
12.91±0.03
14.76±0.26
19.77±0.17
27.22±0.36
16.30±0.08
22.40±0.59
22.42±0.38
24.87±0.47
17.19±0.25
4a
5a
6a
7a
8a
202.81±0.4
Inb
In
In
In
In
9a
10a
11a
11b
a IC50: concentration of the compound (µM) producing 50% cell growth inhibition after
48 h of compound exposure.
b Inactive within 300 µM concentration range.
In summary, we focused on the evaluation of N’-(2-oxoindolin-3-ylidene)-2-
(benzyloxy)benzohydrazide derivatives as potent and novel CD44 inhibitors. The
docking and reporter activity demonstrated the involvement of Wnt/β-catenin as a main
pathway target of these derivatives. The in vitro antitumor effect of this series including
cytotoxicity and cell cycle effect seems to be dependent on CD44 expression.
Acknowledgements
The authors would like to extend their sincere appreciation to the deanship of
Scientific Research at King Saud University for its funding of this research through the
14

research Group Project no. RGP-1435-080. The authors (A. F, M.P.S. and A. A.) would
like to thank research centre administration at King Faisal Specialized Hospital KFSHRC
for their support.
Supplementary data
Supplementary data associated with this article can be found, in:
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17

Captions:
Scheme 1. Synthesis of target compounds. Reagents and conditions: (i) appropriate
benzylchlordie, CH3ONa (ii) NH2NH2.H2O/C2H5OH, reflux; (iii) isatin/C2H5OH, reflux;
(iv) appropriate secondary amine, HCHO, DMSO, rt.
Figure 1. β-catenin crystal structure.19 The Tcf4 is deleted and the β-catenin is shown as
solid surface. The hotspot areas are shown as solid yellow surface.
Figure 2. Chemical structures of small- and large- size inhibitors of Tcf4/b-catenin
interaction.18,20
Figure 3. β-catenin crystal structure.19 Cavity 2 shows small binding pockets near
Lys435/Arg469 referred to number 1, binding cleft between Lys508 and Glu571 referred
to number 2; the floor area refers to number 3.
Figure 4. Suggested binding mode of 11a (magenta) to the cavity 2 of 1jdh.pdb. It binds
to both the floor of the cavity and the Lys435/Arg469 hot spot. Hydrogen bonds are
colored green.
Figure 5. a) Show representative dot plot of Wnt reporter-GFP of untreated and treated
cells with 11a 2.5,5 and 10 μM
b) Bar graphs showing mean±s.d. Wnt repoter-GFP. Data represents the average ± SD
(error bar) of three independent experiments.
Figure 6. Endogenous levels of embryonic transcriptional factors Oct-4,Sox-2 and Nanog
mRNA were examined by real-time RT-PCR. Nanog transcript was notably decreased in
10 μM and no changes were detected in the transcript of Oct-4 or Sox-2.
Figure 7. a) Histogram of CD44 expression showed gated CD44 low and CD44 high
subpopulation.
b) DNA cell cycle analysis of CD44 low and CD44 high subpopulation of untreated and
treated cells with 11a in 5 and 10μM treatment.
18

Legend:
Table 1. Measured in Vitro CD44 inhibition % and docking results of compounds 3a,b-
11a,b
Table 2. In vitro cytotoxic activity of 4a-11a, 11b against cancer cell lines.
19

20

21

22

23

24

25

26

Scheme 1. Synthesis of target compounds. Reagents and conditions: (i) appropriate benzylchlordie,
CH3ONa (ii) NH2NH2.H2O/C2H5OH, reflux; (iii) isatin/C2H5OH, reflux; (iv) appropriate secondary
amine, HCHO, DMSO, rt.

Table 1. Measured in Vitro CD44 inhibition % and docking results of compounds 3a,b-
11a,b
No
H-bond donor
H-bond
H-bond
distance
Å
Score
in
Inhibition
%
MFI* SD
acceptor
Energy
(Kcal/mol-1)
-36.557
ResName.No.Atom
ResName.No.At
om
rec ASN.516.NH2
rec ASN.516.NH2
rec ARG.471.NH2
4a 4a.N1
3a 3a.N3
3a 3a.O3
4a 4a.O2
rec
3.282
3.422
3.269
2.897
3a
4a
31.25
50.65
3142 747
-42.34
2276.66 893
ASN.516.OD1
5a 5a.O2
5a 5a.O1
6a 6a.O2
7a 7a.O2
7a 7a.O3
8a 8a.N2
8a 8a.O3
9a 9a.O1
9a 9a.O2
rec LYS.508.O
10a 10a.O2
11a 11a.O1
11a 11a.O2
3b 3a.N3
3b 3a.O1
4b 4b.O1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.515.NH1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.515.NH2
rec ARG.474.NH1
rec ARG.474.NH2
10a 10a.O4
3.132
2.927
3.222
3.398
3.541
3.553
3.751
3.124
3.081
3.137
3.008
3.055
3.017
3.367
3.617
4.063
-43.243
5a
52.65
2164 226
-45.139
-44.916
6a
7a
58.96
66.34
1875.33 411
1538 543
-43.087
-42.307
-45.825
-46.023
-39.684
8a
59.54
51.95
65.46
76.18
18.48
1849 421
2195.66 904
1578.33 36
1088.33 47
3725.66 564
9a
10a
11a
3b
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.515.NH2
rec ASN.516.NH2
ASN.516.NH2
-38.586
-42.153
4b
5b
29.77
51.13
3209.66 986.
8
2233.33 171
rec ARG.515.NH2
5b 5b.O1
2.897
27

rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.474.NH2
rec ARG.474.NH2
rec ARG.474.NH1
rec ARG.515.NH2
10b 10b.O4
6b 6b.N2
6b 6b.O2
7b 7b.N2
7b 7b.O2
8b 7b.O1
8b 7b.O2
9b 9b.O2
10b. 10b.O1
10b 10b.O3
Rec
3.382
2.868
3.323
2.932
3.482
3.231
3.215
3.086
2.991
3.459
-43.747
-44.792
-41.511
6b
7b
8b
52.25
53.50
50.55
2182 226.3
2125 225.3
2260 713
-40.505
-45.181
9b
45.63
54.92
2484.66 183
2060 256
10b
Glu571.OE1
11b 6a.O2
rec ARG.474.NH2
2.992
-46.399
11b
59.52
1850 192.6
* Mean fluorescence intensity
**The expression of treated cells by the different compounds is normalized to untreated cells.
28