Discovery and Mechanistic Study of Tailor-Made Quinoline Derivatives as Topoisomerase 1 Poison with Potent Anticancer Activity

Article abstract of DOI:10.1021/acs.jmedchem.8b01938

To overcome chemical limitations of camptothecin (CPT), we report design, synthesis, and validation of a quinoline-based novel class of topoisomerase 1 (Top1) inhibitors and establish that compound 28 (N-(3-(1H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the highest potency in inhibiting human Top1 activity with an IC₅₀ value of 29 ± 0.04 nM. Compound 28 traps Top1-DNA cleavage complexes (Top1ccs) both in the in vitro cleavage assays and in live cells. Point mutation of Top1-N722S fails to trap compound 28-induced Top1cc because of its inability to form a hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability and is not a substrate of P-glycoprotein 1 (permeability glycoprotein) advancing its potential anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible Top1cc-induced DNA double-strand breaks as detected by γH2AX foci immunostaining after 5 h of drug removal.

Full text of DOI:10.1021/acs.jmedchem.8b01938

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Article
Discovery and Mechanistic Study of Tailor-Made Quinoline Derivatives
as Topoisomerase 1 Poisons with Potent Anticancer Activity
Biswajit Kundu, Subhendu Kumar Das, Srijita Paul Chowdhuri, Sourav Pal, Dipayan Sarkar, Arijit
Ghosh, Ayan Mukherjee, Debomita Bhattacharya, BENU BRATA DAS, and Arindam Talukdar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01938 • Publication Date (Web): 21 Mar 2019
Downloaded from http://pubs.acs.org on March 21, 2019
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Discovery and Mechanistic Study of Tailor-Made
Quinoline Derivatives as Topoisomerase 1 Poison with
Potent Anticancer Activity
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†#
‡#
‡#
†
†
Biswajit Kundu, Subhendu Kumar Das, Srijita Paul Chowdhuri, Sourav Pal, Dipayan Sarkar,
‡
†
†
‡*
*
Arijit Ghosh, Ayan Mukherjee, Debomita Bhattacharya, Benu Brata Das, and Arindam Talukdar†
†Department of Organic and Medicinal Chemistry, CSIR-Indian Institute of Chemical Biology, 4 Raja
S. C. Mullick Road, Kolkata 700032, WB (India).
‡
Laboratory of Molecular Biology, School of Biological Sciences; Indian Association for the
Cultivation of Science, 2A & 2B, Raja S. C. Mullick Road, Kolkata, 700032, India WB (India).
Academy of Scientific and Innovative Research, Kolkata 700032, WB, India.

#
These authors contributed equally
*
AT and BBD are corresponding authors
KEYWORDS: Topoisomerase 1, Camptothecin, poison, ADME, FRAP, H2AX
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ABSTRACT
To overcome chemical limitations of camptothecin (CPT), we report design, synthesis and validation
of quinoline based novel class of topoisomerase I (Top1) inhibitors and establish compound 28 (N-(3-
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(1H-imidazol-1-yl)propyl)-6-(4-methoxyphenyl)-3-(1,3,4-oxadiazol-2-yl)quinolin-4-amine) exhibits the
highest potency in inhibiting human Top1 activity with a IC value of 29 nM. Compound 28 traps
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Top1-DNA cleavage complexes (Top1cc) both in the in vitro cleavage assays and in live cells. Point
mutation of Top1-N722S fails to trap compound 28-induced Top1cc due to its inability to make
hydrogen bond with compound 28. Unlike CPT, compound 28 shows excellent plasma serum stability
and is not a substrate of P-glycoprotein 1 (permeability glycoprotein; Pgp) advancing its potential
anticancer activity. Finally, we provide evidence that compound 28 overcomes the chemical instability
of CPT in human breast adenocarcinoma cells through generation of persistent and less reversible
Top1cc-induced DNA double strand breaks as detected by H2AX foci immunostaining after 5h of drug
removal.
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INTRODUCTION
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Topoisomerases are ubiquitous enzymes, which are indispensable for relaxing the topological
constraints arising during the DNA metabolic processes of replication, transcription, and chromatin
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remodeling. Human topoisomerase 1 (HTop1) creates a single-stranded nick onto the DNA by a
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nucleophilic attack on the DNA phosphodiester bond to form a “cleavage complex” in which the
enzyme is covalently linked to the 3 end of the broken DNA strand (Top1 cleavage complexes;
Top1cc). This is then followed by a “controlled rotation” of the broken scissile strand around the intact
strand resulting in the relaxation of the DNA super helical tension. Eventually, the 5‟end of the scissile
strand mediates a nucleophilic attack on the phosphotyrosyl-DNA phosphodiester to religate the DNA
5,6
and release the enzyme which ends the catalytic cycle of the enzyme. Trapped Top1cc‟s generate
detrimental lesions which lead to the formation of DNA double-strand breaks (DSBs) upon collision
with ongoing replication forks and/or transcription machinery and are accountable for the killing of
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proliferating malignant cancer cells.
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Figure 1. (A) The design of tailor-made analog conceived from the structural features of known ligand
and analysis of the active site; (B) 2D binding interactions of compound 28 with the important amino
acid residues and base pairs. Red dotted lines imply hydrogen bond and green dotted lines indicate -
stacking interaction; (C) X-ray crystal structure of compound 28; (D) Hypothetical binding model of the
ternary complex of human Top1-DNA-28. The structure was generated from the crystal structure of
Top1-DNA-CPT (PDB ID: 1T8I).
2,5,10,11
There are two types of topoisomerase inhibitors, which include catalytic inhibitors and poisons.
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Catalytic inhibitors directly bind with the free topoisomerase and inhibit its catalytic activity.
While,
poisons reveal their anticancer activity by selectively trapping the Top1-DNA covalent cleavage
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,14,15
complexes
(Top1cc stabilizes the Top1-DNA cleavage complex) inside cells which include
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camptothecin (CPT) and its clinical derivatives as well as several non-CPT Top1 poisons including
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indenoisoquinolines,
indolocarbazoles, and thiohydantoin derivatives. Clinical derivatives of
camptothecin like topotecan and irinotecan are used for advanced colorectal carcinomas and ovarian
cancers. However, CPT and its derivatives suffer from dose-limiting toxicity resulting in severe diarrhea
and neutropenia rapidly inactivated in plasma due to hydrolysis of lactone E-ring and binding of the
ensuing hydroxyl acid to plasma proteins and finally accumulate resistance in the Topoisomerase I
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(
Top1) gene. CPT was also found to be a substrate of Pgp. Moreover, irinotecan is a prodrug, which
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requires a carboxylesterase enzyme to convert into an active metabolite. Recent advances in
developing non-CPT Top1-targeted drug leads to the discovery of indenoisoquinolines (indotecan and
15,22,23
indimitecan) which are in clinical trials for treatment in adults with solid tumors and lymphomas.
Here we report design, synthesis, mechanism study and validation of a novel class of Top1 poison
based on quinoline core, identified through the structural features of known ligands (CPT, topotecan,
irinotecan and indenoisoquinolines) binding through network of interactions in the active site of human
18,24–29
Top1 enzyme as revealed by cocrystal structures
(Figure 1A) for potent anticancer activity.
Compound 28 mediated induction of Top1cc formation in live cells was substantiated by fluorescence
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recovery after photobleaching (FRAP) assays. We also show Top1-N722
is critical for the
compound‟s in vivo trapping of Top1cc in cancer cells treated with compound 28. We provide
compelling in vitro ADME, biochemical and cellular evidence that advocate for compound 28 as a
potential anticancer agent.
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a
Scheme 1
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Reagents and conditions: (a) 2-Morpholinoethanamine (for 2) or 3-morpholinopropan-1-amine (for 3),
DIPEA, 1,4-dioxane, rt, 12 h; (b) Hydrazine monohydrate, EtOH, rt, 8-12 h; (c) Triethyl orthoformate,
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10 ºC, 12 h (for 6 and 12) or triethyl orthoacetate, EtOH, reflux, 8 h (for 7); (d) 2-(4-Methoxyphenyl)-
,4,5,5-tetramethyl-1,3,2-dioxaborolane (for 8, 10 and 13), (3,4-dimethoxyphenyl)boronic acid (for 9,
11 and 14), p-tolylboronic acid (for 15), Pd(PPh ) , 2M Na CO , 1,4-dioxane, 100 ºC, 8-12 h.
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CHEMISTRY
Compound 1 was treated with suitable aminoalkyl amines in the presence of a base (DIPEA) in 1,4-
dioxane solvent to afford compound 2 and 3 in excellent yield. Compound 2 or 3 was dissolved in
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ethanol and treated with the solution of hydrazine monohydrate to obtain compound 4 and 5
respectively. Compound 6 was prepared by heating compound 4 with triethyl orthoformate at 110 ºC for
12 hours. Similarly, compound 4 was refluxed with triethyl orthoacetate in ethanol for 8 hours to obtain
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. Compound 4 was subjected to Suzuki coupling reaction with suitable boronic acids to get compound
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). Similar reaction sequence (Scheme 2) was followed for imidazole analogs. Finally, the Suzuki
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coupling was performed to produce a series of compounds (22-34).
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for compound 17), DIPEA, 1,4-dioxane, rt (for 17) or 100 ºC (for 16), 12 h; (b) Hydrazine
monohydrate, EtOH, rt, 8-12 h; (c) Triethyl orthoformate, 100 ºC, 12 h; (d) Various boronic acids,
Pd(PPh ) , 2M Na CO , 1,4-dioxane, 100 ºC, 7-24 h.
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In scheme 3, compound 19 was converted to oxadiazol-2-amine derivative 35 and oxadiazol-5-methyl
derivative 37 by treating with cyanogen bromide in methanol and triethyl orthoacetate in ethanol
respectively under refluxing condition. Suzuki reaction of 35 and 37 with 2-(4-methoxyphenyl)boronic
ester provided compound 36 and 38 respectively. Similar Suzuki reaction of 17 with the respective
boronic acid provided compound 39 and 40. By following the similar reaction sequence in scheme 1,
compound 1 was converted to methylamine derivative 41 followed by hydrazine derivative 42.
Subsequent oxadiazole formation with triethyl orthoformate leads to compound 43, which on Suzuki
coupling with 2-(4-methoxyphenyl)boronic ester provided compound 44. All compounds subjected to
assay were checked for > 95% purity by HPLC.
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Scheme 3
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tetramethyl-1,3,2-dioxaborolane (for 36, 38, 39 and 44) or (3,4-dimethoxyphenyl)boronic acid (for 40),
Pd(PPh ) , 2M Na CO , 1,4-dioxane, 100 ºC, 6-12 h. (c) Triethyl orthoacetate, EtOH, reflux, 6 h; (d)
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Methylamine, DIPEA, THF, 60 ºC, 16 h; (e) Hydrazine monohydrate, EtOH, rt, 12 h; (f) Triethyl
orthoformate, 140 ºC, 14 h.
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RESULTS AND DISCUSSION
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Design of Top1 Inhibitor: The basis of design has been depicted in figure 1B or 1A. As CPT and its
clinically approved derivatives topotecan and irinotecan suffer from several limitations, we embarked
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on the discovery of „non-CPT‟ Top1 poisons
with improved efficacy. We initiated our design from
the quinoline core, which is also shared by CPT and its clinically approved derivatives topotecan and
irinotecan (Figure 1A). The substitution pattern on quinoline core was strategically positioned at C-3, C-
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and C-6 to avail the conserved hydrogen bond interactions, hydrophobic interactions such as -
stacking and to attain the requisite geometry to be able to stabilize the Top1-DNA cleavage complex
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(Figure 1B). In the indenoisoquinoline series, the importance of suitable alkyl amino chain containing a
heteroatom has been previously validated as it is capable of serving as a hydrogen-bond acceptor into
33–35
the major groove of the ternary complex.
Also, the dimethylamine group in topotecan is predicted to
project into the major groove to attain similar interactions. We presumed that similar alkyl amino chain
at the C-4 position of quinoline core will project in the major groove towards interacting residue
Asn352. The alkyl chain attached heterocycle installed at C-4 can serve a similar purpose and attain
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similar interactions as observed in indenoisoquinoline and CPT derivatives. The crystal structures of
the ternary complex of CPT and indenoisoquinoline revealed the importance of polycyclic rings with
flat planar geometry that can accommodate in the interface of the Top1-DNA cleavage complexes and
block the relegation reaction. We envisioned that the strategically placed C-6 substituted aromatic ring
along with a heterocycle at C-3 position would impart requisite geometry and suitable curvature
essential to attain the conserved hydrogen bond interactions as well as hydrophobic interactions such as
- stacking for stabilizing Top1-DNA cleavage complex (Figure 1B). As a next logical step, we
explored the nature of substitution on the aromatic ring at C-6 and on heterocycles at C-3 that can
modulate top1 inhibition to identify potent Top1 poison.
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Related to Top1 inhibition, half maximal inhibitory concentration (IC ) values for the designed
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analogs were investigated in plasmid DNA relaxation inhibition assays by using either recombinant
Top1 or MCF7 cellular lysates as a source of Top1 (Table 1). We used both biochemical as well as
cellular studies to validate the mechanism of Top1 inhibition. Further insight into the ability of the
potent top1 inhibitor (compound 28) to trap mutant Top1 at residue Asn722 justified by FRAP assays in
live cells. The accumulation and disappearance of DNA damage by the identified compounds were
measured by immunological staining with phosphorylated histone H2AX (H2AX) under confocal
microscopy. Subsequently, in vitro ADME studies were conducted to determine the metabolic stability
and efflux property of these analogs.
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Morpholine analogs. We synthesized the first batch of compounds keeping alkyl substituted
morpholine group at the C-4 position, 4-methoxy and 3,4-dimethoxy phenyl groups at the C-6 position
and oxadiazole group at C-3 (Scheme 1). The choice of substitution pattern at C-4 and C-6 position was
inspired from the structural features present in CPT derivatives and indenoisoquinoline derivatives
(indotecan and indimitecan) (Figure 1A) established as Top1 poison and are currently under clinical
1
5,23,34,37
trials.
Previous reports have suggested that installation of such moiety resulted in improved top1
3
3,34
inhibitory activity.
Compound 8 with two carbon morpholine group at C-4 and the 4-methoxy group
at C-6 and compound 9 with two carbon morpholine group at C-4 and 3,4-dimethoxy phenyl group at C-
6 did not show significant Top1 inhibitory activity in the plasmid DNA relaxation assays at 10 µM
concentration (Table 1). Although 4-methoxy derivative 8 and 3,4-dimethoxy phenyl derivative 9 did
not show significant inhibition at 10 µM but at higher concentration (100 µM) compound 8 showed
5
0% inhibition as compared to 9 with 20% inhibition. Installation of a methyl group at C-5” position of
oxadiazole ring in 8 and 9 resulted in compound 10 and 11 (Scheme 1) respectively which also failed to
show 50% Top1 inhibition (Table 1) at 10 µM concentration. Thereafter, we increased the chain length
1
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connected to the morpholine group at C-4 from two carbons to three carbons. Indeed, increasing the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chain length in compound 13 with the 4-methoxyphenyl group at C-6 showed marked increase in Top1
2
0,38–40
inhibitory activity with a IC value of 1.79 µM against recombinant human Top1.
However, 14
5
0
with 3,4-dimethoxy phenyl group failed to inhibit at least 50% Top1 relaxation activity (Table 1) at 10
µM concentration. An initial comparison between 8, 9, 13 and 14 suggests that combination of three
carbon chain length at C-4 with the mono substitution of 4-methoxyphenyl group at C-6 reinforces Top1
inhibitory activity. The importance of 4-methoxyphenyl group at C-6 was further substantiated by
replacement of the –OCH group present at the C-6 substituted phenyl ring by a methyl group (-CH ) in
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
3
compound 15 leading to loss of activity. Taken together, the initial studies indicate that morpholine
substituted propylamine at C-4 and 4-methoxyphenyl group at the C-6 position are two important
features that can trigger Top1 inhibition in quinoline core.
Imidazole analogs. As both morpholine and imidazole group are present in indotecan, indimitecan and
indenoisoquinolines derivatives, therefore, we replaced the C-4 substituents containing morpholine
moiety with imidazole in our newly synthesized analogs and investigated in Top1 inhibition. A series of
compounds was prepared bearing imidazole group with varying chain length at C-4, substituted phenyl
group at C-6 and oxadiazole ring at C-3 (Scheme 2). Based on our previous observation which showed
the importance of morpholine substituted propylamine at C-4, it seemed prudent to keep the chain
length of the flexible aminoalkyl group to three carbon linker. A series of compounds was prepared by
substituting the C-4’ position of the phenyl group at C-6 with cyano (24), fluoro (25), hydroxyl (26),
methyl (27) and methoxy (28) group (Scheme 2). Compounds with cyano (24), fluoro (25) and hydroxyl
(26) modifications failed to show significant Top1 inhibitory activity in plasmid DNA relaxation assays
at 10 µM (Table 1). Remarkably, compound 28 bearing 4-methoxy group at C-4‟ showed the highest
ability to inhibit recombinant Top1 activity with the IC of 29 nM in the plasmid DNA relaxation assay
5
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Journal of Medicinal Chemistry
1
2
(
Table 1). Notably, compound 27 with 4-methyl substitution at C-4‟ position of the phenyl group also
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
showed Top1 inhibitory activity with an IC50 value of 1.06 µM in Top1 inhibition. Installing the
unsubstituted phenyl group at C-6 in 23 resulted in a sharp decrease in Top1 inhibitory activity (40%
inhibition at 10µM). A direct comparison of the activities of morpholine (13 and 15) and imidazole
series (23 and 27) shows that the imidazole substituted propylamine at C-4 along with the 4-
methoxyphenyl group at C-6 position confers increased Top1 inhibitory activity.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
For a better understanding, the activity of compound 28, hypothetical binding models of 28-
Top1-DNA ternary complexes were constructed with the human Top1 crystal structure (PDB code
1
T8I). The oxygen atom of the methoxy group makes hydrogen bonding interaction with Asn722 in the
C-terminal domain of Top1. Asn722 is an important amino acid responsible for binding of the ligands
41
with Top1 cleavage complexes. The nitrogen atom in the quinoline ring also forms a hydrogen bond
with Arg364 and the three carbon alkyl chain length is ideal for imidazole ring to attain hydrogen bond
interaction with Asn352. The aminopropylamino chain at C-4 and 4-methoxyphenyl group substitution
at the C-6 position of quinoline ring make contacts with the Top1cc and DNA may attribute to Top1
inhibition (Figure 1B and 1D). In keeping with the docking analysis (Figure 1B and 1D), the X-ray
crystal structure of compound 28 (Figure 1C) reveals that it has favorable geometry to fit into the 3′ end
of the broken DNA strand covalently linked with Top1 and the free 5’-OH end to inhibit Top1 religation
42
activity.
Table 1. Recombinant HTop1 Plasmid DNA Relaxation Inhibition Assays in MCF7 Cell Line
Top1 inhibition IC50 (µM)
a
Activity index
Comp
R
1
R
2
R
3
In vitro
0.025
Ex vivo
2.5
CPT
Topotecan
+
+++
0.021
1.8
++++
1
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b
8
9
>10
>10
>10
>10
NT
+
+
+
+
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
NT
NT
NT
10
1
1
1
1
2
1
3
4
5
2
1.79 ± 0.62 2.51 ± 0.39
+++
+
>10
>10
>10
NT
NT
NT
NT
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
+
2
2
2
2
2
3
4
5
6
7
>10
>10
NT
NT
NT
+
+
+
>10
>10
1.06±0.417 3.75±0.102
0.029±0.004 2.74±0.314
+++
28
29
++++
1.054±0.792 4.11±0.339
+++
+
3
3
0
1
>10
>10
NT
NT
+
32
33
>10
NT
3±0.98
NT
+
++
+
2.36±0.884
34
>10
>10
3
3
6
8
NT
NT
NT
NT
NT
+
+
+
+
+
>10
>10
39
40
>10
>10
44
a
Compound-induced in vitro inhibition of Top1 with scores given according to the following system
based on the activity of camptothecin: + = IC > 10 µM; ++ = IC in between 2 µM and 10 µM; +++ =
5
0
50
b
IC in between 100 nM and 2 µM; ++++ = IC < 100 nM. NT = cell line not tested.
5
0
50
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Journal of Medicinal Chemistry
1
2
3
4
To further demonstrate the importance of substitution at C-4‟ in compound 28, we synthesized
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
positional analogs of 28 having –OCH group at C-2‟ (30) and at C-3‟ (29) position of the phenyl ring
3
(
Scheme 2) and were tested for Top1 inhibitory activity. Interestingly, changing the position of –OCH3
group from C-4‟ to C-3‟ results in a slight decrease in Top1 inhibition as compound 29 shows the IC50
value of 1.05 µM (Table 1). These findings were noteworthy as 29 showed that suitable substitution at
C-3‟ position can also confer Top1 inhibition. However, compound 30 failed to show any significant
Top1 inhibition at 10µM. These results underline the importance of suitable substitution at C-4‟ and C-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
‟of the phenyl group at the C-6 position of quinoline core required for Top1 inhibition. The result
prompted us to revisit the di-substitution pattern on the phenyl group at the C-6 position. In the next
series of di-substituted compounds (31, 32, 33 and 34), the position of –OCH group was fixed at C-4‟
3
and another –OCH or –CH was introduced at C-2‟ and C-3‟ position (Scheme 2). Compound 31 and
3
3
32 with dimethoxy substitution on the phenyl group at C-6 failed to show significant Top1 inhibition at
0 µM (Table 1). A similar result was obtained for morpholine analog 14. Interestingly, replacing one of
the –OCH groups at 3‟ position with –CH in 33 provided best top1 inhibition (IC value 2.36 µM)
1
3
3
50
among di-substituted phenyl group at C-6 (Table 1).
In the next development, keeping the optimized features at C-4 and C-6 position constant, a
limited survey of C-5” position of oxadiazole ring was performed by incorporating small hydrophilic
and hydrophobic groups to determine the nature of substituents that can be accommodated for
modulation of Top1 inhibition. Compound 36 with hydrophilic –NH group resulted in a drastic fall in
2
the inhibitory activity. Installation of small hydrophobic –CH group at C-5” resulted in a similar drop
3
in Top1 activity (compound 38). On a similar note, substituting aminopropylamino chain at C-4 with
methylamine in compound 44 fails to show any Top1 inhibitory activity at 10 µM. The result validated
1
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the importance of aminopropylamino side chain at C-4 position of quinoline core for providing
significant impact on Top1 inhibition.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Our efforts to develop an acceptable hypothetical binding model that rationalize the structure-
activity relationships of all the molecules resulting from minor changes in the structure proved difficult.
However, molecular modeling indicated that depending on the nature of substituents at C-3, C-4 and C-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
position of the quinoline ring, it might be capable of forming hydrogen bond interactions with
important residues such as Arg364, Asn722 and Asn352 as well as stacking interactions with DA113,
DC112, TGP11 and DT10 in the ternary complex. In representative compound 22 with the flexible two-
carbon linker, the molecular modeling indicated that the heteroatom present in the heterocycles were
unable to form a hydrogen bond with Asn352 in the major groove (Figure S2). The result signifies the
importance of the length of the flexible chain containing heterocycles. Analysis of the various poses of
disubstituted compound 31 and 33 within the ternary complex revealed that the 3,4-dimethoxy phenyl
group is not orientated in the same plane as observed in compound 28. As a result compound 31 was
unable to form hydrogen bond with Arg364 and Asn722 along with the loss in - stacking interactions.
Interestingly, compound 33 was found to attain the stacking interactions with DA113, DC112, TGP11,
and DT10 along with hydrogen bond with Arg364, which signifies the importance of specific
substitutions at the C-6 position of quinoline ring.
The structure-activity relationship signifies the importance of a perfect balance of substitution
patterns at C-3, C-4 and C-6 position in our design. All these results together vindicate the robustness of
our design and rationalize the highest activity of compound 28 against Top1 in plasmid relaxation
activity (Table 1). Compound 28 was selected for further lead assessment and mechanistic insight.
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Journal of Medicinal Chemistry
Table 2. Solubility, plasma stability and Log D results of potent compounds at pH 7.4
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Aq. Sol
Plasma
Stability
15.70
LogD @ pH
Comp.
a
(
µg/mL)
2.5
7.40*
CPT
1.74
1
2
3
8
28.08±0.11
30.09±0.66
83.38
93.34
3.26±0.01
2.48±0.04
a
Mean % remaining at 2 hrs in human Plasma, *Log P value
Table 3. Caco-2 permeability of compound 13 and 28
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-
6
Papp (10 cm/sec)
Apical to Basal to
Efflux
Ratio
Comp.
Basal
10.89
7.42
Apical
10.98
11.55
1
3
1.02
1.60
28
In vitro ADME study. Compounds 13 and 28 with similar structural features except for the nature of
weak basic group present at the C-4 position were evaluated in a panel of in vitro ADME assays (Table
2
and 3). Both these compounds were found to have moderate aqueous solubility as compared to CPT. It
is noteworthy to mention that both 13 and 28 demonstrated admirable human plasma stability even after
hours, whereas CPT due to its unstable lactone was not stable. The most active compound 28 was
found to be highly stable in human plasma with 93% present after 2 hours. The in vitro LogD values of
2
4
3-45
13 and 28 are 3.26 and 2.48 respectively, which are considered to be ideal for oral absorption.
As
CPT is known to be a substrate of Pgp, we examined the Caco-2 permeability for these compounds as
46,47
well as the efflux ratio to evaluate whether 13 and 28 are Pgp substrates.
To our great satisfaction
the efflux ratio of 13 and 28 are found to be < 2, which signify that these compounds are not Pgp
substrates. We conclude that unlike CPT, our „tailor-made‟ compounds 13 and 28 are highly plasma
48,49
stable with ideal LogD value for oral absorption and are not Pgp substrates. Based on ADME study
(Table 2 and 3) and human Top1 plasmid DNA relaxation inhibition assays (Table 1), we further extend
our study with compound 28 to get mechanistic insight into Top1 inhibition and evaluate its role in the
anticancer activity.
1
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Inhibition of Topoisomerase 1-induced plasmid DNA relaxation activity by compound 28.
(A) Most active compound 28. (B) Relaxation assay of supercoiled plasmid DNA using recombinant
HTop1 at 3:1 molar ratio. Lanes 1 and 9, pBS (SK+) DNA (90 fmol); lanes 2, pBS (SK+) DNA (90
fmol) incubated with 30 fmol of recombinant Top1; lane 3, same as lane 2 additionally Top1 was
incubated with 2% DMSO; lane 4, same as lane 2 but incubated simultaneously with 2 μM CPT; lanes
5
−9, same as lane 2 but was incubated with variable concentrations of compound 28 (as per indication)
at 37 °C for 30 min. (C) Schematic representation for MCF7 whole cell lysate preparation used as the
source of endogenous human Top1 for ex vivo Top1 relaxation assays. (D) Relaxation of supercoiled
pBS (SK+) DNA by Top1 activity from MCF7 cellular extract (each reaction volume contains 0.1 μg
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Journal of Medicinal Chemistry
protein). Lanes 1 and 10, pBS (SK+) DNA (0.3 μg); lane 2, same as lane 1 but pBS (SK+) DNA (0.3
μg) was incubated with MCF7 cell lysates; lanes 3 and 12, same as lane 2 but incubated with 2%
DMSO; lanes 4−9, same as lane 2 but MCF7 whole cell lysates were incubated simultaneously with 5
μM CPT or with varying concentrations of 28 (as indicated) together with plasmid DNA at 37 °C for 30
min. Positions of supercoiled monomer (SM) and nicked and relaxed monomer (NM/RL) are indicated.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(E and F) Quantitative representation of Top1 DNA relaxation inhibition (%) at variable concentrations
of 28 as showed in panel B and D. Recombinant Top1 (E) or endogenous Top1 from cellular extracts
(F). All the experiments were performed in triplicate and expressed as the mean ± SD.
Compound 28 poisons human Top1-DNA cleavage complexes. To examine the specificity of
compound 28 (Figure 2A) for Top1, we used both recombinant Top1 enzyme (Figure 2B) and
12,20
endogenous Top1 from the whole cell extracts of human breast adenocarcinoma (MCF7) cells
(Figure 2C and 2D). When recombinant Top1 and compound 28 were added simultaneously in the
plasmid DNA relaxation assays (Figure 2B, lanes 5-9), 85-90 % inhibition of Top1 could be achieved at
0
.1 µM concentration of compound 28 (Figure 2B, lane 7). Next, the cellular extracts were utilized as a
source of Top1 (Figure 2B) for the plasmid DNA relaxation assays (ex-vivo) (Figure 2C). The utility of
using whole cell extract lies in the fact that the Top1 enzyme in the extract is conserved in its native
structure amongst a plethora of other cellular proteins. The ex-vivo relaxation inhibition assays with
compound 28 efficiently inhibited Top1 activity when the cellular extracts were incubated with
compound 28 (Figure 2D, lanes 5-9) and the quantification in figure 2E-F. Taken together, all these
results indicate that compound 28 selectively inhibits human Top1, both as a recombinant enzyme
(Figure 2B) and as an endogenous protein (Figure 2D), without being impaired by the pool of proteins
contained in the whole cell extracts.
1
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1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Compound 28 mediated trapping of Top1-DNA cleavage complexes. (A) Representative gel
image depicting plasmid DNA cleavage mediated by Top1 in the presence of CPT or compound 28.
Lane 1, 60 fmol of pBS (SK+) supercoiled DNA. Lanes 2−9, same as lane 1 but incubated with equal
amounts of recombinant human Top1 (100 fmol) at the indicated concentrations of CPT or compound
2
8 or only DMSO at 37 °C for 30 min. Positions of the supercoiled substrate (form I) and nicked
monomers (form II) are indicated. Schematic representation of Top1 mediated nicked DNA formation.
(B) Representative gel showing Top1-mediated 25 mer duplex oligonucleotide cleavage in the presence
32
of CPT and compound 28. Lane 1, 15nM 5‟- P-end labeled 25-mer duplex oligo as indicated above.
Lane 2, same as lane 1 but incubated with recombinant Top1 (0.2 μM). Lanes 3−5, same as lane 2 but
2
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Journal of Medicinal Chemistry
incubated with an indicated concentration of CPT. Lanes 6−8, same as lane 2 but incubated with an
indicated concentration of compound 28. Positions of uncleaved oligonucleotide (25-mer) and the
cleavage product (12-mer oligonucleotide complexed with residual Top1) are indicated. Schematic
representation of the formation of 12-mer oligonucleotide attached with Top1 in presence of Top1
poison. Quantitative measurement of cleavage complex (Top1cc) formation (%) by CPT and compound
1
2
3
4
5
6
7
8
9
1
1
1
1
1
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8 either by supercoiled DNA (C) or through oligo cleavage assay (D). All the experiments were
performed three times and expressed as the mean ± SD. (E) Compound 28 is not a DNA intercalator.
Compound 28-DNA interaction as investigated by agarose gel electrophoresis in Top1 unwinding
+
+
assays. Lane 1, 50 fmol of pBS (SK ) DNA. Lane 2, relaxed pBS (SK ) DNA generated by an excess of
Top1. Lanes 3–6, same as lane 2, but incubated with 50 and 200 µM of m-AMSA and etoposide,
respectively. Lanes 7–10, same as lane 2, but incubated with 20, 50, 100, 200 µM of compound 28 as
indicated. (F) Fluorescence-based ethidium bromide displacement assay. All Samples contained 1 µM
of EtBr and 5 nM CT DNA. Graphical representation of EtBr bound (%) of increasing concentration (0–
3
00 µM) of compound 28, m-AMSA and etoposide were added as indicated. EtBr fluorescence was
monitored with excitation wavelength at 510 nm and emission at 590 nm.
As both CPT and indenoisoquinolines stabilize Top1-DNA cleavable complexes (Top1cc) to
5,9,14,15,38,39,50
inhibit Top1 activity,
the mechanism of Top1 inhibition with compound 28 was
investigated in the plasmid DNA cleavage assays. Closed circular DNA (form I) get converted to nicked
circular DNA (form II) by Top1 in the presence of specific inhibitors and are referred to as “cleavage
complex” (Figure 3A, right panel). Figure 3A shows that compound 28 also stabilizes Top1cc formation
like CPT, suggesting that compound 28 acts as a Top1 poison. We further confirmed that compound 28
is capable of stabilizing Top1cc in single turnover equilibrium cleavage assays (Figure 3B, lanes 3-8) by
allowing recombinant Top1 to react with 25-mer duplex oligonucleotides modified to harbor preferred
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Page 22 of 72
Top1 cleavage sites in the presence of indicated concentrations of compound 28 or CPT consistent with
plasmid DNA cleavage assays (Figure 3A). In addition, quantification of cleavage assays (Figure 3C-D)
suggests that the extent or rate of Top1-DNA cleavage complex formation (% cleavage) with compound
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8 at indicated concentration is similar to CPT (Figure 3C and 3D). Cumulatively, our data are
indicative that compound 28 is able to stabilize Top1 cleavage complexes and inhibit the religation
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activity with similar efficacy as that of CPT.
To investigate the ability of the compound 28 to intercalate into DNA, we carried out both Top I
12,40
unwinding assays and EtBr displacement assays.
Topoisomerase unwinding assay is based on the
ability of the intercalating compounds to unwind the DNA duplex and thereby change the DNA twist.
Figure 3E clearly shows that in the presence of strong intercalative drug such as m-AMSA, a net
negative supercoiling of the relaxed substrate DNA was induced at 50 and 200 µM concentration
(Figure 3E, lanes 3 and 4). However, under similar conditions, non-intercalative compounds like
etoposide failed to show such effect at 50 and 200 µM concentrations (Figure 3E, lanes 5 and 6).
Compound 28 had no effect on the topological state of the relaxed plasmid DNA at the indicated
concentrations (Figure 3E, lanes 7-10), suggesting compound 28 is not a DNA intercalator (Figure 3).
Next, we further confirm that compound 28 is not a DNA intercalator by performing EtBr displacement
assays. Figure 3F shows that the intercalative drug m-AMSA has the capability to dislodge the bound
fluorophore (EtBr) at 50 µM concentration. However, non-intercalative drug such as etoposide was
unable to do so. Under similar condition compound 28 induces no displacement of fluorophore even at
high concentration (200 µM) compared with the very low IC50 (29 nM) of topoisomerase I inhibition.
Taken altogether, our data suggest that compound 28 is not a DNA intercalator.
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Figure 4. Compound 28 mediated stabilization of Top1-DNA cleavage complex in live cells. (A)
Space-filling model showing conservative H-bonding interaction between Asn722 and 28. (B)
Compound 28 accumulates immobile/bound Top1 in the nucleus. Representative images depicting the
fluorescence recovery after photobleaching (FRAP) of enhanced green fluorescence tagged HTop1
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(
EGFP-Top1WT) transiently expressed in MCF7 cells. Cells were treated with indicated concentration
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of 28 for 10 min and were analyzed by live cell spinning disk confocal microscopy and photobleaching.
A sub‐nuclear spot (ROI) indicated by a circle was bleached (BLH) for 30 ms and photographed at
regular intervals of 3 ms thereafter. Successive images taken for ~90 s after bleaching illustrate
fluorescence return into the bleached areas. (D) Top1N722 residue is critical for 28-induced nuclear
dynamics. Representative images showing the FRAP of EGFP–Top1N722S transiently expressed in
MCF7 cells. Cells were treated with 28 (indicated concentration) for 10 min and were analyzed by live
cell spinning disk confocal microscopy and FRAP experiments were carried out in a similar way as with
the EGFP-Top1WT). Right panels (C and E) quantification of FRAP data showing mean curves of Top1
variants in the presence and absence of 28. Error bars represent mean ± S.E. (n = 15).
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Compound 28 traps Top1 cleavage complexes in live cancer cells. To obtain direct evidence for
compound 28 mediated trapping of Top1cc (Figure 4A) in live cells, we ectopically expressed a green
fluorescent tagged human Top1 (EGFP-Top1) in MCF7 cells and tested the nuclear mobility of Top1
under live cell confocal microscopy combined with fluorescence recovery after photobleaching (FRAP)
1
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technology as described previously.
In absence of compound 28, FRAP recovery of EGFP-Top1
was fast (∼85–90% suggesting a large mobile population and a smaller (∼10–15%) immobile
population of EGFP-Top1 (Figure 4B, and the quantification in 4C; untreated). This data implies that
Top1 is mostly mobile and binds transiently with the DNA (reversible Top1cc) and are freely
exchanged in the nuclear compartments.
In the presence of compound 28, the fluorescence recovery of EGFP-Top1 was markedly impeded
(∼55–65%) with increasing concentration of compound 28 (Figure 4B, and the quantification in 4C; (+)
compound 28; 1 and 5 μM). These data suggest that compound 28 traps Top1cc on DNA in live cells
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like CPT,
leading to a subsequent increase in bound/immobile fraction of EGFP-Top1 (Figure 4B-
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Journal of Medicinal Chemistry
C), which is consistent with 28-mediated stabilization Top1cc in the in vitro cleavage assays (Figure 3).
The molecular modeling indicates compound 28 possibly makes hydrogen bonding interaction with
Asn722 located in the catalytic domain of HTop1 may contribute to the stabilization of Top1 cleavage
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complexes with the nicked DNA (Figure 4A). To investigate this possibility, we tested the ability of
N722S
compound 28 to trap mutant Top1 at residue Asn722 (EGFP-Top1
) in live cells by using FRAP
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kinetic analysis (see Figure 4D and E, panel (+) 28). Figure 4E suggests that the FRAP recovery of
N722S
EGFP–Top1
was unaffected in the presence of increasing dosage of 28 (Figure 4D-4E) and was
similar to no drug treatment condition (Figure 4E), suggesting 28 failed to trap Top1cc when Asn722 is
mutated to Ser. Taken together, this data indicates that Asn722 is critical for compound 28 mediated
trapping of human Top1cc.
Compound 28 accumulates persistent DNA double-strand breaks (DSBs) compared to CPT. As
compound 28 stabilizes Top1cc in vitro (Figure 3) and in live cells (Figure 4), we investigated the
accumulation and disappearance of DSBs in MCF7 cells treated with compound 28 by measuring

H2AX foci formation under confocal microscopy (Figure 5), as H2AX is a well-defined marker for
52–57
Top1‐mediated DSBs.
Under similar condition, we detected a time-dependent increase in H2AX foci formation in cells
treated with compound 28 for 3h and 5h comparable with CPT-induced H2AX foci at similar time
periods, suggesting that both compound 28 and CPT generate similar levels DSBs at indicated time
periods in MCF7 cells (Figure 5B and 5C).
52,54
CPT-induced H2AX have a short half-life as they are reversible immediately after a wash,
which
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is consistent with the reversal of Top1cc intermediates within minutes after washing out CPT. Next to
test the ability of the compound 28 to induce more persistent and less reversible DNA breaks, we
investigated the disappearance of H2AX in cells treated with compound 28 and compared it with CPT
after subsequent wash and culture in the drug-free medium for indicated time periods (Figure 5A). In
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Figure 5B, the right panel shows faster disappearance of H2AX foci after washing out CPT from media
at indicated time periods (see the quantification in Figure 5C). In contrast, cells showed (3-4 fold) more
persistent H2AX foci (Figure 5B, right panel and the quantification in Figure 5C) even after washing
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out compound 28 at the indicated time. Figure 5C further indicates that H2AX foci were persistent for
5h after drug removal in compound 28 treated cells. Taken together our data suggest that compound 28
generates more persistent and less reversible Top1cc-induced DSBs compared to CPT.
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Figure 5. Compound 28 generates persistent and less reversible DNA double-strand breaks as detected
by H2AX staining (A) Overview for the protocol of drug treatment and reversal in MCF7 cells. (B)
Time-dependent accumulation of H2AX foci formation in MCF7 cells treated with compound 28 or
CPT for 3h and 5h and reversal of H2AX foci after drug removal for indicated times. (C)
Quantification of H2AX intensity per nucleus after treatment and post the removal of indicated
inhibitors (Compound 28 or CPT) obtained from immunofluorescence confocal microscopy was
calculated for 35–40 cells (mean ± S.E.M.) and plotted as a function of time (h).
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Compound 28 exhibits potent anticancer activity. Compound 28 was evaluated for its cytotoxicity
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in the cancer cell lines from different tissue origin. Cytotoxicity assays were performed in human
breast adenocarcinoma cell lines (MCF7), human cervical cancer cell lines (HeLa), human colon
carcinoma cell lines (HCT116), human ovarian adenocarcinoma cell lines (NIH:OVCAR-3) as well as
non-cancerous human embryonic kidney (HEK293) cells with variable concentrations of compound 28.
Table S2 (Supporting Information) indicates that compound 28 revealed cytotoxicity in cancerous cells
including MCF7 (IC : 2.74 µM), HeLa (IC : 2.61 µM), HCT116 (IC : 2.34 µM), NIH:OVCAR-3
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50
(
IC : 2.35 µM) cells compared to the non-cancerous cells like HEK293 (IC : 8.34 µM).
50 50
TDP1 hydrolyzes the phosphodiester bond at a DNA 3'-end linked to a tyrosyl moiety of stalled Top1-
5
1–54,59,60
DNA covalent complexes, therefore, TDP1-/- cells are hypersensitive towards Top1 poisons.
Further evidence for the poisoning of Top1cc with compound 28 and hypersensitivity in DNA repair-
deficient cells, we have performed cytotoxicity assays with TDP1-/- (IC : 1.02 µM) and TDP1+/+
50
MEFs (IC : 2.91 µM) cells in the presence of compound 28. Table S2 indicates that TDP1-/- MEFs
5
0
cells were hypersensitive to compound 28, further providing evidence that compound 28 induces
cytotoxicity by stabilizing Top1cc in cells. Taken together all data confirms that compound 28 is a
potent Top1 poison and is a potential candidate as an anticancer chemotherapeutic agent.
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CONCLUSION
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As CPT and its clinically approved drugs suffer from several limitations, there is a great interest in the
development of „non-CPT‟ Top1 poisons as anticancer agents. In the present study, we successfully
report a novel class of Top1 poisons based on quinoline core through an understanding of the structural
features of ligands essential for binding in the active site. The design signifies the importance of a
perfect balance of substitution patterns at C-3, C-4 and C-6 position of quinoline core. Most potent
compound 28 stabilizes Top1cc in vitro and in live cells to inhibit the religation activity without
intercalating to the DNA with similar efficacy as that of CPT. The mechanistic insight through FRAP
assay of compound 28 mediated trapping of human Top1cc revealed the significance of Asn722, an
important amino acid residue at the active site. Unlike CPT, compound 28 generates less reversible
DNA double-strand breaks due to the accumulation of irreversible Top1cc-induced DSBs. In vitro
ADME study revealed that unlike CPT and its derivatives, compound 28 is highly plasma stable with
ideal LogD value for oral absorption and is not a Pgp substrate. Summarily, we provide compelling
evidence that advocate for compound 28 as a potential anticancer agent.
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EXPERIMENTAL SECTION
Chemistry. General Methods. All starting materials, reagents, and solvents were purchased from
commercial suppliers and used without further purification. Air-sensitive reactions were carried out
under dry nitrogen or argon atmosphere. Solvents were distilled before use and also dried using standard
methods. TLC was performed on silica gel plates (Merck silica gel 60, F ) and the spots were
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visualized under UV light (254 nm and 365nm) or by charring the plate dipped in ninhydrin or KMnO4
or vanillin solution. For purification of the compounds along with manual column flash chromatography
was also performed with RediSepRf silica gel columns on the Teledyne ISCO CombiFlashRf system
1
using 230-400 mesh silica gel. H NMR was recorded at 300 MHz (Bruker-DPX), 400 MHz (Jeol) and
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