Lead optimization of novel quinolone chalcone compounds by a structure-activity relationship (SAR) study to increase efficacy and metabolic stability.

TL;DR: In this article, the authors synthesized 17 quinolone-chalcone derivatives based on CTR-17 and CTR-20, and carried out a structure-activity relationship study.

Abstract: Many agents targeting the colchicine binding site in tubulin have been developed as potential anticancer agents. However, none has successfully made it to the clinic, due mainly to dose limiting toxicities and the emergence of multi-drug resistance. Chalcones targeting tubulin have been proposed as a safe and effective alternative. We have shown previously that quinolone chalcones target tubulin and maintain potent anti-proliferative activity vis-a-vis colchicine, while also having high tolerability and low toxicity in mouse models of cancer and refractivity to multi-drug resistance mechanisms. To identify the most effective anticancer chalcone compound, we synthesized 17 quinolone-chalcone derivatives based on our previously published CTR-17 and CTR-20, and then carried out a structure-activity relationship study. We identified two compounds, CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] and CTR-32 [((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] as potential leads, which contain independent moieties that play a significant role in their enhanced activities. At the nM range, CTR-21 and CTR-32 effectively kill a panel of different cancer cells originated from a variety of different tissues including breast and skin. Both compounds also effectively kill multi-drug resistant cancer cells. Most importantly, CTR-21 and CTR-32 show a high degree of selectivity against cancer cells. In silico, both of them dock near the colchicine-binding site with similar energies. Whereas both CTR-21 and CTR-32 effectively prevents tubulin polymerization, leading to the cell cycle arrest at G2/M, CTR-21 has more favorable metabolic properties. Perhaps not surprisingly, the combination of CTR-21 and ABT-737, a Bcl-2 inhibitor, showed synergistic effect in killing cancer cells, since we previously found the "parental" CTR-20 also exhibited synergism. Taken together, CTR-21 can potentially be a highly effective and relatively safe anticancer drug.

Summary

Introduction

• Microtubules, the polymers of alpha and beta tubulin proteins, are essential for a wide range of cellular functions including proliferation, intracellular tra cking, cell signaling, cell shape and migration, and even tumor angiogenesis.
• Microtubule targeting drugs are classi ed in two major groups: microtubule-stabilizing agents which promote microtubule polymerization while also inhibiting disassembly by binding to the tubulin polymer, and microtubule-destabilizing agents which bind to the tubulin dimers and block microtubule polymerization 3, 4 .
• They suffer from some common drawbacks that limit their effectiveness in many cases.
• Due to their simple chemistry which allows for an abundance of substitutions coupled with their known antiproliferative activities, the authors synthesized 19 chalcone derivatives using the Claisen-Schimdt condensation.
• The authors sought to enhance their anti-growth/proliferation properties with a structure-activity relationship (SAR) approach to design and synthetize more effective candidates that would be more potent against cancer cells while sparing normal cells at low concentrations and maintaining selectivity.

Results

• The synthesis of the 19 quinolone chalcones were carried out using acetanilides commercially available or synthesized using standard protocols from anilines 11 (Fig. 1 ).
• Examining the SAR of the methoxy group placement on the quinolone ring, the authors determined that if there is a methoxy group present, its position is vitally important to the cytotoxic activity of the compound.
• The authors previously showed that CTR-17 and CTR-20 are selective against malignant when compared their e cacies against non-malignant cells.
• The authors previously showed through in vitro studies and in silico modeling that CTR-17 and CTR-20 bind to tubulin overlapping the colchicine binding site, and inhibit tubulin polymerization 10 .
• Therefore, although CTR-32 is effective at killing cancer cells, it appears that the 2-ethoxy group makes it less stable than the 2-methoxy group seen in CTR-21.

Discussion

• In an effort to optimize the e cacy and stability of novel quinolone chalcones CTR-17 and CTR-20, the authors created a small pool of derivatives with different modi cations to the quinolone and/or phenyl ring moieties.
• The authors found that the most effective changes were moving the 6-methoxy group to 8-methoxy on the quinolone ring (CTR-21), and replacing the 2-methoxy with an ethoxy group (CTR-32).
• Both CTR-21 and CTR-32 are predicted to bind near the chalcone binding site in the α/β-tubulin dimer.
• This leads to the cell cycle arrest at G2/M and eventually cell death by apoptosis.
• In conclusion, CTR-21 shows similar molecular properties to their previously characterized CTR-17 and CTR-20.

Methods

• All chemicals and solvents used were commercially available and were of reagent grade.
• 1H NMR spectra were recorded on a Bruker Avance II 400 spectrometer, using DMSO-d6 as solvent and tetramethylsilane (TMS) as internal standard.
• Peripheral blood mononuclear cells from healthy donors were isolated by density gradient centrifugation using Ficoll solution.
• 1 mL of pre-warmed medium was added drop-wise and the entire contents of the cryovial was transferred to a 15-mL tube.
• The cells were centrifuged, re-suspended in PBS solution, followed by centrifugation.

Figures

Figure 5

Figure 3

Figure 2

Full text

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Lead optimization of novel quinolone chalcone
compounds by a structure-activity relationship (SAR)
study to increase ecacy and metabolic stability
James Knockleby
Health Sciences North
Aicha Djigo
Health Sciences North
I. Kalhari Lindamulage
Health Sciences North
Chandrabose Karthikeyan
Indira Gandhi National Tribal University
Piyush Trivedi
Bharati Vidyapeeth Deemed University
Hoyun Lee (  hlee@hsnri.ca )
Health Sciences North
Research Article
Keywords: CTR compound, anticancer agent, SAR, quinolone, chalcone; breast cancer cell, NCI-60 cancer panel
Posted Date: February 16th, 2021
DOI: https://doi.org/10.21203/rs.3.rs-198850/v1
License:   This work is licensed under a Creative Commons Attribution 4.0 International License.  Read Full
License

Page 2/20
Abstract
Many agents targeting the colchicine binding site in tubulin have been developed as potential anticancer agents.
However, none has successfully made it to the clinic, due mainly to dose limiting toxicities and the emergence of
multi-drug resistance. Chalcones targeting tubulin have been proposed as a safe and effective alternative. To
identify the most effective anticancer chalcone compound, we synthesized 17 quinolone-chalcone derivatives based
on our previously published CTR-17 and CTR-20, and then carried out a structure-activity relationship study. We
identied two compounds, CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)]
and CTR-32 [((E)-3-(3-(2-ethoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)] as potential leads, which contain
independent moieties that play a signicant role in their enhanced activities. At the nM range, CTR-21 and CTR-32
effectively kill a panel of different cancer cells originated from a variety of different tissues including breast and
skin. Both compounds also effectively kill multi-drug resistant cancer cells. Most importantly, CTR-21 and CTR-32
show a very high degree of selectivity against cancer cells.
In silico
, both of them dock near the colchicine-binding
site with similar energies. However, only CTR-21 effectively prevents tubulin polymerization, leading to the cell cycle
arrest at G2/M and, eventually, cancer cell death by apoptosis. Perhaps not surprisingly, the combination of CTR-21
and ABT-737, a Bcl-2 inhibitor, showed synergistic effect in killing cancer cells, since we previously found the
“parental” CTR-20 also exhibited synergism. Taken together, CTR-21 can potentially be a highly effective and
relatively safe anti-cancer drug.
Introduction
Microtubules, the polymers of alpha and beta tubulin proteins, are essential for a wide range of cellular functions
including proliferation, intracellular tracking, cell signaling, cell shape and migration, and even tumor
angiogenesis. Drugs targeting microtubules have been shown effective, with their potency even exceeding their anti-
mitotic properties
1,2
. Microtubule-targeting agents (MTAs) have thus been widely used as chemotherapeutics for
several decades and remain relevant in cancer therapy today, either administrated alone or in combination with
other regimens. From colchicine, one of the oldest drugs, to more recent drugs such as paclitaxel and its analogues,
MTAs interact with tubulin at different sites and through different mechanisms of actions (MOA)
3
. Microtubule
targeting drugs are classied in two major groups: microtubule-stabilizing agents which promote microtubule
polymerization while also inhibiting disassembly by binding to the tubulin polymer, and microtubule-destabilizing
agents which bind to the tubulin dimers and block microtubule polymerization
3,4
. Although these drugs have been
on the market for decades, they suffer from some common drawbacks that limit their effectiveness in many cases.
These include acquired drug resistance and dose-limiting toxicities. The latter, in particular, prevents colchicine from
being deployed as a cancer therapeutic
5,6
.
Our interest in nding novel safe and effective anticancer agents has focused on rening natural product
compounds into lead candidates. One family of compounds that has shown great interest over the past few
decades are chalcones. Chalcones are found in avonoids in many different edible plants as secondary
metabolites
7
. Chalcones contain a three carbon α, β unsaturated carbonyl core that links two phenyl rings. The
phenyl rings offer sites of modication with a diversity of single and multiple additions in order to maximize
effectiveness, increase stability, and enhance solubility and other chemical properties. Chalcones that contain
similar trimethoxyphenyl rings as colchicine show promise as anti-proliferative agents
8,9
, suggesting that methyoxy
groups are likely important modications for structure-activity studies.

Page 3/20
Due to their simple chemistry which allows for an abundance of substitutions coupled with their known anti-
proliferative activities, we synthesized 19 chalcone derivatives using the Claisen-Schimdt condensation. The
quinolone chalcone compounds (named CTRs) microtubule-destabilizing agents, two of which (CTR-17 and CTR-
20) showed a selective and potent anti-tubulin activity and caused a prolonged mitotic arrest at the spindle
assembly checkpoint (SAC), eventually lead to cell death
10
. We sought to enhance their anti-growth/proliferation
properties with a structure–activity relationship (SAR) approach to design and synthetize more effective candidates
that would be more potent against cancer cells while sparing normal cells at low concentrations and maintaining
selectivity. We identied that CTR-21 [((E)-8-Methoxy-3-(3-(2-methoxyphenyl)-3-oxoprop-1-enyl) quinolin-2(1H)-one)]
is the most desirable anticancer agent among this series of chalcone compounds.
Results
Chemistry. The synthesis of the 19 quinolone chalcones were carried out using acetanilides commercially available
or synthesized using standard protocols from anilines
11
(Fig.1). 2-chloroquinoline 3-carboxaldehydes were
synthesized by treating acetanilides with DMF and POCl
3
under Vilsmeier Haack conditions
12
. We then synthesized
3-(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones utilizing Claisen-Schmidt condensation of the 2-chloroquinoline-3-
carbaldehydes under basic conditions (sodium methoxide or NaOH) with the appropriate acetophenones
13,14
The 3-
(2-chloroquinolin-3-yl)-1-phenylprop-2-en-1-ones were reuxed with aqueous glacial acetic acid to induce O-
nucleophilic substitution at the 2-chloro group of the quinoline ring to derive the corresponding quinolone chalcones.
Compounds were conrmed with a combination of their infrared (IR) spectroscopic
1
H NMR and mass spectral data
(see Methods).
CTR-21 and CTR-32 are the most effective anti-proliferative quinolone chalcones examined. We had initially
determined that quinolone chalcones were promising anti-proliferative through a rst round screening and identied
CTR-17 and CTR-20 as favorable structures for anticancer activity
10
. CTR-17 and CTR-20 have been modelled to
bind to the colchicine binding pocket on β–tubulin and cause a prolonged mitotic arrest at the spindle assembly
checkpoint, eventually leading to apoptosis. We carried out a comparative SAR study to determine if we could
further optimize the quinolone chalcone to maximize ecacy. The Sulforhodamine B (SRB) assay was used to
measure the anti-proliferative/cell killing activities of CTRs, for which several different cell lines were use: the
cervical cancer HeLa, the breast cancer cell lines MDA-MB231, MDA-MB468, MDA-MB231TaxR (an induced
paclitaxel-resistant sub-cell line of the MDA-MB231
10
) and MCF7. In addition, a subset was tested against the
melanoma cell lines MZ-MEL-3.1, Mel-SOE, UKRV-Mel-38, UKRV-Mel-17 and MDA-MB-435 (Table1, Supplemental
Table S1). CTR-21 and CTR-32 were highly potent with GI
50
ranging from 5 nM to 91 nM. Notably, the ability for
CTR-21 and CTR-32 to maintain effectiveness against MDA-MB231TaxR suggests that these two compounds
remain refractive to multi-drug resistance mechanisms, as previously shown by CTR-20
10
. CTR-21 and CTR-32 were
also highly effective against the NCI-60 panel of representative cancer cell lines (Supplemental Figs. S1-S8).

Page 4/20
Table 1
GI
50
values of quinolone chalcone analogs on breast cancer, melanoma and cervical cancer cells.*

Melanoma cell lines Breast cancer cell lines Cervical
cancer
MZ-
Mel-3
Mel-
SOE
UKRV-
Mel-
38
UKRV-
Mel-17
MDA-
MB435
MDA-
MB231
MDA-
MB231-
TaxR
MDA-
MB468
MCF7 HeLa
CTR17
(nM)
227 ±
30
786 ±
52
626 ±
154
504 ±
33
290 ±
66
657 ±
72
1,299 ±
83
320 ±
26
485 ±
97
280 ±
78
CTR18
(nM)
239 ±
13
817 ±
34
746 ±
177
596 ±
162
307 ± 7 530 ±
119
1,214 ±
179
304 ± 2 510 ±
38
443 ±
112
CTR19
(nM)
6,832
± 957
10,095
± 64
3,766
± 206
11,166
± 1,660
4,723
± 2,761
6,506
± 1,388
7,089 ±
1,059
3,538
± 60
11,910
± 564
6,823 ±
1,389
CTR20
(nM)
98 ±
21
338 ±
78
338 ±
33
283 ±
57
90 ± 10 216 ±
21
966 ±
163
408 ±
12
194 ±
21
124 ±
36
CTR21
(nM)
6 ± 1 27 ± 5 13 ± 3 9 ± 2 7 ± 2 17 ± 3 32 ± 8 29 ± 10 16 ± 5 8 ± 3
CTR32
(nM)
6 ± 2 33 ± 9 22 ± 3 18 ± 3 13 ± 5 20 ± 2 22 ± 4 26 ± 0 27 ± 6 11 ± 2
CTR40
(nM)
11 ± 0 53 ± 4 42 ±
11
28 ± 5 23 ± 0 30 ± 4 54 ± 13 77 ± 13 41 ± 5 19 ± 1
Tax
(nM)
§
58 ±
15
60 ± 16 6 ± 1 14 ± 4 4 ± 1 3 ± 1 131 ±
35
9 ± 3 6 ± 1 8 ± 1
Noco
(nM)
§
23 ± 2 51 ± 11 34 ± 3 39 ± 1 18 ± 2 35 ± 4 47 ± 11 48 ± 7 33 ± 7 25 ± 1
* GI
50
values were derived from a non-linear sigmoidal dose-response (variable slope) curve tted by GraphPad
Prism v.4.03 software.
§
Tax and Noco denote paclitaxel and Nocodazole, respectively.

SAR analysis reveals two separate moieties that synergistically increase anti-growth/proliferative effects. The
diversity of chemical groups in our library allowed us to determine which moieties found on the two ring structure
might best enhance cytotoxicity (Fig.1). CTR-17 shows the simplest design with an unsubstituted quinolone ring
linked to 2-methoxy phenyl moiety through an ‘enone’ group and it displays an anti-proliferative activity in the
medium range (GI
50
= 464 nM; Table1). The 2-methoxy group on the phenyl ring is critically important to the ecacy
of the CTRs
15,16
. Introducing a 6-methyl group on the quinolone ring does not lead to any change in ecacy (CTR-
18, GI
50
= 499 nM; Fig.1) with all 9 cell lines showing similar GI
50
to that of CTR-17. The introduction of a 5-methoxy
to CTR-17 (i.e., CTR-26) does not seem to have an effect either in terms of anti-growth/proliferative activity (GI
50
=
443 nM), whereas CTR-29 (5-uorophenyl) has a lower GI
50
of 118 nM. In contrast, the addition of a 6-methoxy
group on the phenyl ring (CTR-25, GI
50
= 1.6 µM, Supplemental Table1) leads to a 4-fold increase in GI
50
. This
suggests the possibility that the presence of a second methoxy group may induce steric hindrance or adverse
interactions if a methoxy group is near the quinolone group. In contrast, a single methoxy group on the phenyl ring

Page 5/20
may not hinder the quinolone group, as the phenyl ring can rotate on the axis on its bond with the carbonyl of the
enone group which may affect how the CTR binds to its target.
Examining the SAR of the methoxy group placement on the quinolone ring, we determined that if there is a methoxy
group present, its position is vitally important to the cytotoxic activity of the compound. CTR-19, which has a 7-
methoxy on the quinolone ring is the least effective analog out of the CTRs in terms of anti-proliferative activity with
an average GI
50
of 7.3 µM (Table1), whereas the average GI
50
of CTR-20 (6-methoxy) is 232 nM (Table1) and CTR-
21 (8-methoxy) is 16.4nM (Table1). The enhanced activity of CTR-20 is likely due to the presence of the methoxy on
the C-6 since it is the only difference (i.e., methyl
vs
methoxy) from CTR-18 which shows much lower activity
(Table1). The activity of CTR-24 (6,7-dimethoxy quinolone) is between those of the 6-isomer and the 7-isomer (1.5
µM), which along with the lower toxicity potency of CTR-19 and CTR-23 (compared to CTR-17) suggesting that the
7- position should remain free for maximum ecacy, and that the 8-position is more favorable for cytotoxic activity
than the 6-position.
The introduction of a 5-uoro on CTR-20 (i.e., CTR-37) does not affect its activity contrary to what we observed
between CTR-17 and CTR-29. However, when the uorine group is added on the 4th carbon (i.e., CTR-38), the
average value of the GI
50
drops to 98 nM which - while not making it as potent as CTR-21 – still makes it one of the
most active compounds. The addition of a methoxy group has differential effect on the potency of CTR-20 as well,
depending on the position, as indicated by CTR-33 (6-methoxy, GI
50
= 1.1 µM), CTR-34 (5-methoxy, GI
50
= 648 nM),
CTR-35 (4-methoxy, GI
50
= 2 µM), and CTR-36 (4-triuoromethoxy, GI
50
= 805 nM). This evidence suggests that,
although the 2-methoxy group on the phenyl ring is important for activity, we can enhance the activity of the
compound by adding may impede activity.
Finally, substituting the 2-methoxyphenyl group for a 2-ethoxyphenyl improved the activity of the compounds.
Indeed CTR-32 which, similarly to CTR-17, possesses no additional group on the quinolone but has a 2-ethoxy group
on the phenyl ring (Fig.1) displays one of the lowest average GI
50
values of the entire collection (20 nM; Table1).
Furthermore, another compound with a 2-ethoxygroup on the phenyl ring (in combination with a 6-methoxy on the
quinolone ring), CTR-40 exhibited the third strongest activity (GI
50
= 36 nM; Table1). It is highly notable that
replacing the 2-methoxy group of the phenyl group (CTR-17) by a 2-ethoxy group (CTR-32; Table1) resulted in a 23-
fold decrease in GI
50
. It is also highly notable that introducing an 8-methoxy on the quinolone group (CTR-21;
Table1) with keeping the 2-methoxyphenyl renders a 31-fold decrease. One concern of enhancing the activity of the
CTR compounds is that toxicity is increased towards non-malignant cells. We previously showed that CTR-17 and
CTR-20 are selective against malignant when compared their ecacies against non-malignant cells. Therefore, we
next sought to determine the cytotoxicity of the most promising CTR compounds against primary melanocytes and
peripheral blood mononuclear cells (PBMCs). We found that CTR-21 and CTR-32 are far less cytotoxic toward
primary cells compared to cancer cell lines (Table2). Comparing the selectivity index (SI) of the primary
melanocytes versus the average GI
50
of cancer cells, both CTR-21 and 32 have similar SIs (CTR-21: 157 fold; CTR-
32: 158 fold; Table2). There is a slightly smaller SI between cancer cells and PBMCs (CTR-21: 106 fold; CTR-32: 67
fold; Table2), but still a substantial difference between normal and cancer cells. Taken together, these data indicate
that we have identied two independent moieties on the CTR backbone that can enhance activity without sacricing
selectivity.

Frequently asked questions

Q1. What is the role of chalcones in the development of anticancer drugs?

Novel quinolone chalcones targeting colchicine-binding pocket kill multidrugresistant cancer cells by inhibiting tubulin activity and MRP1 function. 

Q2. What is the color code of the compounds?

Data from Western blotting with whole cell extracts of asynchronously growing population show that CTR-21 causes apoptosis, which is manifested by PARP cleavage. 

Q3. What is the role of microtubules in cancer treatment?

J. J., Kanakkanthara, A. & Miller, J. H. Microtubule-targeting agents are clinically successful due to both mitotic and interphase impairment of microtubule function. 

Q4. What is the patent for a chalcone?

Canada patent (2017).17        Venghateri, J. B., Gupta, T. K., Verma, P. J., Kunwar, A. & Panda, D. Ansamitocin P3 depolymerizes microtubules and induces apoptosis by binding to tubulin at the vinblastine site.