Synthesis and evaluation of modified chalcone based p53 stabilizing agents

Article abstract of DOI:10.1016/j.bmcl.2017.07.042

Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to various cellular stresses thereby preventing cancer development. Activation and stabilization of p53 through small organic molecules is, therefore, an attractive approach for the treatment of cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds against human colorectal (HCT116) and breast (CAL-51) cancer cell lines revealed potent antiproliferative activities. Nine compounds displayed GI₅₀ values in the low micromolar to submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (SSE14108) showed GI₅₀ of 0.473 ± 0.043 μM against HCT116 cells. Further analysis of these compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-h post-treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control, Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2) demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide (CHX) and the results showed that the p53 stabilization was regulated at post-translational level through blockage of its degradation. These chalcones can, therefore, act as fragment leads for further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-proliferative activities.

Full text of DOI:10.1016/j.bmcl.2017.07.042

Accepted Manuscript
Synthesis and evaluation of modified chalcone based p53 stabilizing agents
Sunniya Iftikhar, Sardraz Khan, Aishah Bilal, Safia Manzoor, Muhammad
Abdullah, Abdel-Hamid Emwas, Salim Sioud, Xin Gao, Ghayoor Abbas
Chotana, Amir Faisal, Rahman Shah Zaib Saleem
PII:
S0960-894X(17)30738-2
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.042
BMCL 25155
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
17 March 2017
11 July 2017
14 July 2017
Please cite this article as: Iftikhar, S., Khan, S., Bilal, A., Manzoor, S., Abdullah, M., Emwas, A-H., Sioud, S., Gao,
X., Chotana, G.A., Faisal, A., Saleem, R.S.Z., Synthesis and evaluation of modified chalcone based p53 stabilizing
agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.042
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Synthesis and evaluation of modified chalcone
based p53 stabilizing agents
Sunniya Iftikhar,a Sardraz Khan,a Aishah Bilal,b Safia Manzoor,a Muhammad
Abdullah,a Abdel-Hamid Emwas,c Salim Sioud,c Xin Gao,c Ghayoor Abbas
Chotana,a Amir Faisal,b* Rahman Shah Zaib Saleema*
a
Department of Chemistry, SBASSE, Lahore University of Management Sciences, Lahore,
Pakistan, 54792
b
Department of Biology, SBASSE, Lahore University of Management Sciences, Lahore,
Pakistan, 54792
c King Abdullah University of Science and Technology, Thawal, Saudi Arabia.
Abstract
Tumor suppressor protein p53 induces cell cycle arrest and apoptotic cell death in response to
various cellular stresses thereby preventing cancer development. Activation and stabilization of
p53 through small organic molecules is, therefore, an attractive approach for the treatment of
cancers retaining wild-type p53. In this context, a series of nineteen chalcones with various
substitution patterns of functional groups including chloro, fluoro, methoxy, nitro, benzyloxy, 4-
methyl benzyloxy was prepared using Claisen-Schmidt condensation. The compounds were
characterized using NMR, HRMS, IR and melting points. Evaluation of synthesized compounds
against human colorectal (HCT116) and breast (Cal-51) cancer cell lines revealed potent
antiproliferative activities. Nine compounds displayed GI50 values in the low micromolar to
submicromolar range; for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one
(SSE14108) showed GI50 of 0.473 ± 0.043 µM against HCT116 cells. Further analysis of these
compounds revealed that (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one (SSE14105) and (E)-
3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) caused rapid (4 and 8-hour post-
treatment) accumulation of p53 in HCT116 cells similar to its induction by positive control,
Nutlin-3. Such activities were absent in 3-(4-methoxyphenyl)propiophenone (SSE14106H2)
demonstrating the importance of conjugated ketone for antiproliferative and p53 stabilizing
activity of the chalcones. We further evaluated p53 levels in the presence of cycloheximide
(CHX) and the results showed that the p53 stabilization was regulated at post-translational level
through blockage of its degradation. These chalcones can, therefore, act as fragment leads for
further structure optimization to obtain more potent p53 stabilizing agents with enhanced anti-
proliferative activities.
Keywords: Chalcones, Antitumor, Drug discovery,
*
Corresponding
authors:
Rahman
Shah
Zaib
Saleem
(Rahman.Saleem@lums.edu.pk) and Amir Faisal (Amir.Faisal@lums.edu.pk),

Cancer cells manipulate various key signaling pathways that allow them to continue dividing by
overcoming tight controls that regulate cell death and proliferation in normal cells, and to acquire
resistance towards chemotherapy.1 TP53 tumor suppressor gene encodes for a transcription
factor that is responsible for maintaining genomic integrity.2, 3 When activated due to cellular
stress, p53 migrates from the cytoplasm into the nucleus where it binds to DNA and transcribes
its target genes leading to cell cycle arrest or apoptosis.4, 5 p53 has also been shown to induce
apoptosis independent of its transcriptional activity by directly interacting with the members of
Bcl-2 family.6-8 Stabilization and activation of p53 can lead to p53 dependent cell cycle arrest,
apoptosis or both. The strategy of activating p53 has been an attractive area of research with
example of many compounds that stabilize p53 by inhibiting its binding to MDM2, an E3
ubiquitin ligase responsible for ubiquitination mediated degradation of p53.9-13
Chalcones, the open chain flavonoids, constitute an important class of naturally occurring and
synthetic compounds.14 Various chalcones have been reported with different biological
16
activities15,
including anti-infective17, anti-inflammatory,18 antifungal,19 anti-bacterial,20
antimalarial,21 anti-oxidant22 and anti-protozoal activities.23 Modified chalcones have also been
studied for potential anti-cancer properties in various cancers including ovarian cancer,24 non-
small cell lung carcinoma,25 skin carcinogenesis,
prostate cancer,27, 28 and pulmonary and
26
mammary carcinogenesis.15, 29, 30 Structure of some of the chalcones has been decorated with
various functional groups and heterocyclic scaffolds to make them potent against various
targets.31-33 Chalcones have also been implicated in inhibition of MDM2-p53 interaction. Often
however, these studies suggest that in addition to disruption of MDM2-p53 interaction,
alternative mechanisms may also be responsible for the activities of many of these compounds.
Our analysis of these reports reveals a pattern of three-atom long chain on either one of the
phenyl rings in these compounds (shown in Figure S1 of supplementary information). In one
instance, this chain carries a carboxylic acid terminus,34 in another a boronic acid terminus35 and
in the third report, it is simply a propyl chain.36 A couple of other publications have discussed the
activity of chalcones with tri-substituted phenyl rings.37, 38 Intrigued with these reports, we have
been interested in finding out if the increase in carbon count on such chains would be favorable
and can a simple chalcone with only one substituent cause p53 stabilization and act as a lead for
future structure optimization. In this regard, we have prepared a library of 19 chalcones with
various electron donating and electron with-drawing functional groups (for example, chloro,
fluoro, methoxy, nitro, benzyloxy, and 4-methyl benzyloxy) on the two phenyl rings offering
various levels of electron richness in the rings and lengths of the molecules. Eight of these
compounds are either new or do not have any reported biological activity in Reaxys (Table S1 in
the supplementary information), furthermore, this is the first report of the cytotoxicity of ten of
the remaining eleven compounds against HCT116 and Cal-51 cells line or for p53 stabilization.
The Claisen-Schmidt condensation routes used for the syntheses of chalcones are presented in
scheme 1. Compounds SSE14101- SSE14109 and SSE14115 were prepared by reacting 4-
substituted acetophenone with appropriate aldehyde in ethanol using 15 N ethanolic solution of
sodium ethoxide (as shown in scheme 1a). For compounds SSE14110- SSE14114 and
SSE14116- SSE14119, 4-hydroxyacetophenone (1 eq) was first reacted with appropriate benzyl
bromide (1.3 eq) in the presence of K2CO3 (1.3 mmol). The mixture was stirred for 2 hours at
room temperature. The product was extracted using ethyl acetate and dried over magnesium
sulfate. Removal of solvent yielded the desired ketone, which was then reacted with appropriate
aldehyde (1 eq) in ethanol using 15 N ethanolic solution of sodium ethoxide (as shown in scheme
1b). The yields were in the range of 61-98% and the structures were established using 1H NMR,

13C NMR, IR and mass spectrometry. The values of coupling constants revealed formation of E-
isomers.
Scheme 1: General procedure for the synthesis of compounds SSE14101- SSE14119
A 3 day Sulforhodamine B (SRB) proliferation assay was carried out to identify the compounds
with significant antiproliferative activity against p53 competent HCT116 colon cancer cells.
Cells were treated in a 96-well plate with two different concentrations, 25µM and 50µM, of each
of the nineteen compounds; treatment with DMSO alone was used as a solvent control. The
experiments were performed in duplicates and percentage inhibition compared to DMSO control
was calculated as shown in figure 1 (and table S2 in supplementary information). The results
revealed that nine compounds (SSE14101-SSE14109) with smaller substituents displayed
excellent anti-proliferative activity. One the other hand, the compounds with longer benzyloxy or
4-methyl benzyloxy at R1 or R4 had lower or no activity at these concentrations suggesting that
the linear extension of the structure in this dimension beyond three-atoms may be making the

molecule sterically challenged to fit inside the cavity of its target protein and adding additional
cost of solvent interaction for the protruding aromatic ring.
100
90
80
70
60
50
40
30
20
10
0
%age inhibitionat 25 µM
%age inhibitionat 50 µM
Figure 1: Antiproliferative activities of chalcone derivatives at 25 µM and 50 µM against
HCT116 human colon cancer cell line
We next determined the GI50 values of these compounds in same 3-day SRB proliferation assay
in order to rank these compounds according to their potency. Cells were treated in duplicates in
96-well plates with three-fold dilutions of the compounds starting from 50µM using DMSO as a
solvent control and Nutlin-3 as a positive control. Compounds SSE14101-SSE14109 had GI50
values in the range of 0.473-16.54 µM in HCT116 and 0.78-5.85 µM in Cal-51 cell lines (Figure
2 and table S3 in supplementary information). Nutlin-3, a positive control for p53 induction, had
GI50 value of 1.52 µM in HCT116 cells, indicating that we had good hits in our compounds. The
activities of compounds SSE14110-SSE14119 ranged from 14.86 to >50 µM (Table S3 in the
supplementary information). A closer look at the GI50 values suggested that the chalcones with
electron-donating methoxy group displayed superior activities, for example SSE14108 with three
methoxy groups had GI50 values of 0.473 µM in HCT116 and 0.78 µM against Cal-51 cell lines
while the SSE14106 with one methoxy group had GI50 values of 3.86 µM in HCT116 and 2.69
µM in Cal-51 cell lines. On the other hand, compounds with electron-withdrawing groups had
GI50 values on the higher side. Since the chalcones are smaller in size with potential to bind to
various cellular targets, these biological activities could be a result of their cumulative effect. In
order to identify the mechanism of action of these chalcones, we evaluated their ability to
stabilize p53.

18
16
14
12
10
8
6
4
2
0
HCT116
Cal51
Figure 2: GI50 values of chalcone derivatives in a 3 day SRB proliferation assay in HCT116 and
Cal-51 cells.
In the next phase, all the nineteen chalcones were investigated for their ability to stabilize and
accumulate p53 in HCT116 cells. Cells were treated with two concentrations (25 µM and 50
µM) of the compounds for 8 hours and analyzed for p53 levels by immunoblotting. Nutlin-3 (a
known inhibitor of p53-MDM2 interaction)39 was used as a positive control whereas DMSO was
used as a solvent control. Treatment of cells with two of these compounds, SSE14105 and
SSE14016 for 8 hours, resulted inp53 accumulation. We then analyzed these two compounds for
rapid p53 accumulation at different concentrations (6.25 µM, 12.5 µM and 25 µM) following 4
and 8 hour treatment of cells. Both compounds increased p53 expression at 4 and 8 hours in a
dose dependent manner compared to the DMSO control (Figure 3 A and B). The levels of house-
keeping protein Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading
control. Such rapid accumulation at 4 and 8 hours could possibly be a result of inhibition of p53
degradation pathways. Presence of more electron withdrawing fluoro group at this position, a
nitro-group at any position or presence of 3,4,5-trimethoxy substitution led to no p53
accumulation suggesting electronic and steric limitations of substituents. The antiproliferative
activity of some of these compounds without stabilization of p53 suggests alternative targets
responsible for their effect on proliferation. In order to determine the importance of double bond
in the structure of chalcone,we evaluated compound SSE14106H2 (Figure 3C) for p53
stabilization . As shown in Figure 3D, the p53 stabilizing activity was completely lost when the

double bond was removed. The compound SSE14106H2, with no double bond at 2-position had
a GI50 value of >100 µM (n=2) and was unable to induce p53 stabilization following 4h and 8h
treatment in HCT116 cells. This shows that the presence of conjugated ketone structure is vital
for the activity of the chalcone.
A
B
C
D

Figure 3: Compounds SSE14105 and SSE14106 induce p53 levels in HCT116 cells. Cells were
exposed to various concentrations of SSE14106 (A) or SSE14105 (B) for 4 and 8 hours. Samples
were analyzed by immunoblotting using p53 and GAPDH antibodies. Treatment with 10M
Nutlin-3 was used as positive control, while treatment with DMSO alone was used as a solvent
control. Blots were quantified using Image J and levels of p53 were normalized with GAPDH.
Fold change compared to DMSO control is shown at the bottom of A and B (C) Structure of
SSE14106H2 (D) Compound SSE14106H2 is unable to induce p53 levels in HCT116 cells.
Cells were exposed to various concentrations of SSE14106H2 for 4 and 8 hours. Samples were
analysed by immunoblotting using p53 and GAPDH antibodies. Treatment with 10M Nutlin-3
was used as positive control, while treatment with DMSO alone was used as a solvent control.
The accumulation of p53 could be a result of transcriptional activation or inhibition of its
degradation pathways. In order to determine whether p53 induction in response to these
compounds was regulated at the transcriptional or post-translational level, we evaluated their
effect on the p53 levels in the presence or absence of cycloheximide (CHX), which is an
inhibitor of protein translation. As shown in figure 4A, treatment with 12.5M SSE14106
resulted in increased stability of p53 compared to the DMSO control in the presence of
cycloheximide. Quantification of the blots in Figure 4B shows that the relative p53 levels in
SSE14106 treated cells remain stable for longer compared to the DMSO control in the presence
of cycloheximide. This clearly demonstrates that these compounds upregulate p53 levels by
increasing its stability independent of transcriptional or translational activation. This also point to
them being inhibitors of a cellular pathway responsible for negative regulation and subsequent
degradation of p53.

Figure 4: Compound SSE14106 stabilized p53 through post-translational mechanism. (A)
HCT116 cells were treated with cycloheximide in the presence or absence of 12.5M SSE14106
and samples were collected at times indicated. Samples were analyzed by immunoblotting using
p53 and alpha-tubulin antibodies. (B) Blots were quantified and p53 levels normalized to alpha-
tubulin.
Overall this study shows that simple chalcone derivatives exhibit significant antiproliferative
activities accompanied by rapid p53 induction in both colon and breast cancer cell lines. The
presence of an electron donating group was common in both these compounds. Presence of more
electron with-drawing fluoro group at this position, a nitro-group at any position or presence of
3,4,5-trimethoxy substitution leads to no p53 accumulation suggesting electronic and steric
limitations of substituents on this ring. Similarly, presence of a bigger substituent at positions R1
or R4 (compound SSE14110- SSE14119) or lack of conjugation (SSE14106H2) also leads to
significant decrease or complete loss of the activity. This decrease in the activity may be a
consequence of the elongation of the molecule making it sterically challenging to fit inside the
cavity of its target as the protruding aromatic ring would have to pay the cost of solvent
interaction making it unfit to bind or lack of structural rigidity in case of last. It has been
demonstrated that boronic acid derivatives and carboxylic acid derivatives can stabilize p53. In
the current work, we show that chalocnes with simpler substituents like chloro (SSE14105) and
methoxy (SSE14106) can impart p53 stabilizing features to the chalcones with better or
comparable GI50 values. Chalcones are small in size and can probably fit into the binding cavities
of various cellular targets, so their measured activity could be a result of a combination of

different interactions. The fact that these compounds are small in size (mol. wt. of SSE14106:
238) and stabilize p53 make them good leads for fragment based approach to develop novel drug
candidates through further structure optimization.
In summary, we have presented the synthesis of a series of chalcones and have evaluated them
for their biological activities. The in vitro effects on HCT116 colon cancer and Cal-51 breast
cancer cell lines led to identification of nine compounds with very good GI50 values in the low
micromolar to submicromolar range for example (E)-1-phenyl-3-(3,4,5-trimethoxyphenyl)prop-
2-en-1-one (SSE14108) showed GI50 of 0.473 ± 0.043 µM against HCT116. We have
demonstrated the antiproliferative effects of (E)-3-(4-chlorophenyl)-1-phenylprop-2-en-1-one
(SSE14105) and (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (SSE14106) and their
ability to accumulate p53. Evaluation of p53 accumulation by these compounds in the presence
of cycloheximide (CHX) rules out any transcriptional or translational activation of p53. The
chalcones synthesized in this report suggest that extension of structure to cause linear expansion
of the molecule in the form of extra-phenyl ring deteriorates their activity and the simpler
compounds with one substituent (in our study chloro or methoxy) could serve as leads for
fragment based discovery projects to develop novel and potent p53 stabilizing agents.
Acknowledgement
Authors acknowledge the support by LUMS in the form of faculty initiative fund to enable this
research work and extend our acknowledgement to KAUST for NMR and HRMS analysis.
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