Synthesis, Antiproliferative Effect, and Topoisomerase II Inhibitory Activity of 3-Methyl-2-phenyl-1 H-indoles

Article abstract of DOI:10.1021/acsmedchemlett.9b00557

A series of 3-methyl-2-phenyl-1H-indoles was prepared and investigated for antiproliferative activity on three human tumor cell lines, HeLa, A2780, and MSTO-211H, and some structure-activity relationships were drawn up. The GI50 values of the most potent compounds (32 and 33) were lower than 5 μM in all tested cell lines. For the most biologically relevant derivatives, the effect on human DNA topoisomerase II relaxation activity was investigated, which highlighted the good correlation between the antiproliferative effect and topoisomerase II inhibition. The most potent derivative, 32, was shown to induce the apoptosis pathway. The obtained results highlight 3-methyl-2-phenyl-1H-indole as a promising scaffold for further optimization of compounds with potent antiproliferative and antitopoisomerase II activities.

Full text of DOI:10.1021/acsmedchemlett.9b00557

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Letter
Synthesis, antiproliferative effect and topoisomerase
II inhibitory activity of 3-methyl-2-phenyl-1H-indoles
Nace Zidar, Daniela Secci, Tihomir Tomaši#, Lucija Peterlin Masic, Danijel Kikelj, Daniele
Passarella, Aida Nelly Garcia Argaez, Mariafrancesca Hyeraci, and LISA DALLA VIA
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/
acsmedchemlett.9b00557 • Publication Date (Web): 24 Jan 2020
Downloaded from pubs.acs.org on January 24, 2020
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Synthesis, antiproliferative effect and
topoisomerase II inhibitory activity of 3-methyl-2-
phenyl-1H-indoles
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Nace Zidar, a Daniela Secci, a Tihomir Tomašič, a Lucija Peterlin Mašič, a Danijel Kikelj, a
Daniele Passarella, b Aida Nelly Garcia Argaez, c Mariafrancesca Hyeraci, c Lisa Dalla Via c, *
a University of Ljubljana, Faculty of Pharmacy, Aškerčeva cesta 7, 1000 Ljubljana, Slovenia
b Department of Chemistry, University of Milan, Via Golgi 19, 20133 Milan, Italy
c Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Via
Marzolo 5, I-35131 Padova, Italy
Corresponding author
*Lisa Dalla Via
Phone: +39-049-8275712. Email: lisa.dallavia@unipd.it
ORCID
Lisa Dalla Via: 0000-0002-9828-9388
ABSTRACT: A series of 3-methyl-2-phenyl-1H-indoles was prepared, investigated for
antiproliferative activity on three human tumour cell lines, HeLa, A2780 and MSTO-211H,
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and some structure-activity relationships were drawn up. The GI50 values of the most potent
compounds (32 and 33) were lower than 5 µM in all tested cell lines. For the most
biologically relevant derivatives, the effect on human DNA topoisomerase II relaxation
activity was investigated, which highlighted good correlation between the antiproliferative
effect and topoisomerase II inhibition. The most potent derivative, 32, was shown to induce
the apoptosis pathway. The obtained results highlight 3-methyl-2-phenyl-1H-indole as a
promising scaffold for further optimisation of compounds with potent antiproliferative and
anti-topoisomerase II activities.
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KEYWORDS: 3-methyl-2-phenyl-1H-indole; antiproliferative activity; topoisomerase II;
apoptosis.
ABBREVIATIONS
DMF, N,N-dimethylformamide; DIAD, diisopropylazodicarboxylate; DMSO, dimethyl
sulfoxide;
ethylenediaminetetraacetic acid; m-AMSA, meta-amsacrine; NMM, N-methylmorpholine;
TBTU, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; THF,
EDC,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;
EDTA,
tetrahydrofuran; topo II, human DNA topoisomerase II.
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Cancer is one of the leading causes of mortality worldwide, and its incidence is rapidly
growing.1,2 The use of conventional chemotherapeutic agents, such as alkylating cytostatics,
nucleoside analogues, anthracyclines and compounds that stabilise microtubules is often
limited because of their severe side effects. Drugs like tyrosine kinase inhibitors and
monoclonal antibodies that target specific signalling oncoproteins have been developed to
increase specificity and thus to reduce side effects, but these can also have limitations, such
as development of resistance.3 Despite the considerable progress made with new targeted
therapies, for many patients the therapeutic options remain limited. Thus, new
chemotherapeutics with improved pharmaco-toxicological profiles are urgently needed.4
One of the important targets for anticancer drug discovery is human DNA topoisomerase II
(topo II).3,5-9 Topoisomerases are enzymes that catalyse changes in DNA topology, and they
are crucial for processes such as DNA replication and transcription, and chromosomal
segregation. They introduce transient breaks in DNA molecule by cleaving one (type I
topoisomerases) or both (type II topoisomerases) strands.10,11 Topo II is a type II
topoisomerase, and two isoforms, α and β, are expressed in mammalian cells. Topo IIβ is
expressed constitutively throughout the cell cycle, whereas topo IIα predominantly in highly
proliferating cells.
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Based on their mechanism of action, compounds targeting topo II can be divided into two
large groups: poisons12 and catalytic inhibitors.6,13-16 Topo II poisons stabilise the covalent
DNA–topo II complex, leading to DNA damage and apoptotic cell death and are used in the
clinic for the treatment of breast, lung, prostate and haematological cancers and sarcomas.17
They included etoposide, doxorubicin and mitoxantrone. A common side effect of DNA
poisons is the risk of secondary malignancies and cardiotoxicity, which are mostly connected
to the effects of these drugs on topo IIβ.18 On the other hand, catalytic inhibitors affect one of
the steps in the catalytic cycle of the enzyme. In particular, they can block binding of the
DNA molecule to the enzyme, compete for binding with ATP, inhibit cleavage of the DNA
molecule, or prevent hydrolysis of ATP12 and interestingly, they do not increase DNA
cleavage, which could indicate an improved safety profile.5
We report here the design and synthesis of a series of 3-methyl-2-phenyl-1H-indole
analogues with antiproliferative and topo II inhibitory effects (Fig. 1A). The compounds were
designed based on the 3-methyl-2-phenyl-1H-indole hit I (Fig. 1A), which was identified
through screening of in-house library of compounds against three selected human tumour cell
lines: cervix adenocarcinoma (HeLa), ovarian carcinoma (A2780), and biphasic
mesothelioma (MSTO-211H). Compound I showed good antiproliferative activity, with
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growth inhibition values (GI50) in the micromolar range. Based on compound I, a series of
analogues was designed and synthesised, through varying the substituents on R1–R4 (Fig.
1A). The antiproliferative activities of compounds were evaluated in vitro against HeLa,
A2780 and MSTO-211H cells. Because of structural similarities of our compounds with
some naturally occurring flavonoids that inhibit topo II, such as quercetin and luteolin (Fig.
1B),19-21 the topo II inhibitory effects of the most potent analogues were investigated. For
compound 32, cytofluorimetric analysis was performed to investigate the mechanism of cell
death.
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A
R3
N
PhO
OH
R2
PhO
HO
N
OH
N
R1 and R2=H, OH or OCH3
R3=H, CH3, alkyl or phenoxyalkyl
R4=OH, OCH3 or arylcarboxyl
HO
R4
I
OH
R1
32
B
O
OH
O
O
OH
HO
O
OH
OH
HO
HO
OH
OH
quercetin
luteolin
Figure 1. Design of the 3-methyl-2-phenyl-1H-indoles as antiproliferative compounds, based
on screening hit I (A); structures of flavonoids quercetin and luteolin with topo II inhibitory
activities (B).
The designed compounds were prepared according to synthetic pathways presented in
Schemes 1 and 2. Full synthetic procedures and analytical data are available in the
Supporting Information. The synthesis of the central indole scaffold started with benzyl
protection of hydroxyl group(s) of propiophenones 1 and 2, to obtain ethers 3 and 4 (Scheme
1). These ethers were α-brominated with bromine under acidic conditions, to yield 5 and 6,
which were then reacted with 4-benzyloxyaniline in the Bischler-Möhlau indole synthesis, to
give indoles 8-9. Indole 7 was obtained by Fischer indole synthesis from ketone 3 and
phenylhydrazine under acidic conditions. Removal of the benzyl protecting groups of 7-9 to
obtain phenols 10-12 was achieved by hydrogenation, using palladium on charcoal as
catalyst. In addition, esters 13 and 14 were prepared from phenol 10: for 13, with 4-
methylthiazole-5-carboxylic acid using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) as coupling reagent, and for 14, from 1H-imidazole-4-carboxylic acid using 2-(1H-
benzotriazole-1-yl)-1,1,3,3-tetramethylaminium tetrafluoroborate (TBTU). Moreover, the
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benzyl protected indoles 7-9 were reacted with different haloalkanes using NaH as the base in
N,N-dimethylformamide (DMF), to give N-alkylated indoles 15-25, which were deprotected
by catalytic hydrogenation to obtain the final compounds 26, 27 and 29-37 (Scheme 2).
Carboxylic acid derivative 28 was obtained by alkaline hydrolysis of ester 27. In addition,
methylation of 37 with methyl iodide in the presence of caesium carbonate gave ether 38.
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Scheme 1. Synthesis of Compounds 10-14a
b
R2
O
O
O
HN
Br
a
c
d
HO
BnO
BnO
BnO
R1
R1
R1
R1
1 (R1=H)
2 (R1=OH)
3 (R1=H)
4 (R1=OBn)
5 (R1=H)
6 (R1=OBn)
7 (R1=R2=H)
8 (R1=H, R2=OBn)
9 (R1=R2=OBn)
R2
HN
HN
e
f
O
HO
R3
O
R1
10 (R1=R2=H)
11 (R1=H, R2=OH)
12 (R1=R2=OH)
13 (R3=4-methylthiazole-5-yl)
14 (R1=1H-imidazole-4-yl)
aReagents and conditions: (a) benzyl bromide, K2CO3, acetone, reflux, 18 h; (b) for 7:
phenylhydrazine, H2SO4, acetone, reflux, 24 h; (c) Br2, glacial acetic acid, 0 °C, 1 h; (d) for 8,
9: 4-benzyloxyaniline∙HCl, Et3N, DMF, 120 °C, 2 h, then 150 °C, 3 h; (e) H2, 10% Pd/C,
EtOH, r.t., 18 h; (f) for 13: 4-methylthiazole-5-carboxylic acid, EDC, 4-
dimethylaminopyridine, CH2Cl2, r.t., 15 h; for 14: 1H-imidazole-4-carboxylic acid, TBTU,
NMM, DMF, r.t., 15h.
Scheme 2. Synthesis of Compounds 26-38a
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PhO
R3
R3
R2
R2
O
N
N
N
7,
8
or
9
d
a
b
O
BnO
HO
R1
R1
38
O
15 (R1=R2=H, R3=propyl)
26 (R1=R2=H, R3=propyl)
c
16 (R1=R2=H, R3=2-ethoxy-2-oxoethyl)
17 (R1=R2=H, R3=4-phenoxybutyl)
18 (R1=H, R2=OBn, R3=propyl)
27 (R1=R2=H, R3=2-ethoxy-2-oxoethyl)
29 (R1=R2=H, R3=4-phenoxybutyl)
30 (R1=H, R2=OH, R3=propyl)
28 (R1=R2=H, R3=carboxymethyl)
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19 (R1=H, R2=OBn, R3=2-phenoxyethyl)
20 (R1=H, R2=OBn, R3=3-phenoxypropyl)
21 (R1=H, R2=OBn, R3=4-phenoxybutyl)
22 (R1=R2=OBn, R3=methyl)
23 (R1=R2=OBn, R3=propyl)
24 (R1=R2=OBn, R3=3-phenoxypropyl)
25 (R1=R2=OBn, R3=4-phenoxybutyl)
31 (R1=H, R2=OH, R3=2-phenoxyethyl)
32 (R1=H, R2=OH, R3=3-phenoxypropyl)
33 (R1=H, R2=OH, R3=4-phenoxybutyl)
34 (R1=R2=OH, R3=methyl)
35 (R1=R2=OH, R3=propyl)
36 = compd. I (R1=R2=OH, R3=3-phenoxypropyl)
37 (R1=R2=OH, R3=4-phenoxybutyl)
aReagents and conditions: (a) for 15, 18, 23: 1-bromopropane; for 16: ethyl bromoacetate; for
17, 21, 25: (4-bromobutoxy)benzene; for 20, 24: (3-bromopropoxy)benzene; for 19: (2-
bromoethoxy)benzene; for 22: methyl iodide; NaH, DMF, r.t., 3 h; (b) H2, 10% Pd/C,
THF/EtOH, r.t., 18 h; (c) 1 M NaOH; (d) methyl iodide, Cs2CO3, acetone, 60 °C, 48 h.
The antiproliferative effect exerted by the new compounds 10-14, 26-38 and by the well-
known inhibitors of topo II m-amsacrine (m-AMSA) and quercetin was assayed on three
human tumour cell lines: HeLa, A2780 and MSTO-211H. The results are expressed as GI50
values, i.e. the concentration (M) of compound able to inhibit the growth in 50% of cells
with respect to a control culture. The obtained data are shown in Table 1.
Table 1. Chemical structures and cell growth inhibition in the presence of compounds
10-14, 26-38 and of m-AMSA and quercetin as reference.
R3
N
R4
R2
R1
GI50 (µM)a
Compd.
R1
R2
H
R3
R4
HeLa
A2780
MSTO-
211H
10
11
12
13
H
H
H
H
H
H
OH
OH
OH
O
>20
16±2
>20
>20
11±2
>20
>20
>20
>20
OH
OH
H
OH
H
2.2±0.5
13±1
>20
O
N
S
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O
14
H
H
H
>20
>20
>20
N
O
NH
7
8
9
26
H
H
H
CH2CH2CH3
CH2COOCH2CH3
CH2COOH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OCH3
>20
>20
9.0±2.2
13±1
7.1±1.4
>20
27
H
10
11
12
13
14
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28
H
H
>20
>20
>20
29
H
H
CH2CH2CH2CH2OPh
CH2CH2CH3
11±2
7.9±1.9
15±0.3
4.4±1.0
4.0±0.6
>20
3.1±0.6
2.3±0.6
4.7±0.9
2.2±0.4
2.0±0.6
14±3
3.3±0.5
2.9±0.8
7.6±1.2
2.4±0.4
2.9±0.2
>20
30
H
OH
OH
OH
OH
OH
OH
OH
OH
31
H
CH2CH2OPh
32
H
CH2CH2CH2OPh
CH2CH2CH2CH2OPh
CH3
33
H
34
OH
OH
OH
OH
35
36b
CH2CH2CH3
>20
8.4±1.3
7.4±0.7
4.3±1.3
17±4
>20
CH2CH2CH2OPh
CH2CH2CH2CH2OPh
CH2CH2CH2CH2OPh
18±1
10±1
>20
7.1±1.2
3.4±0.9
13±2
37
38
OCH3 OCH3
m-AMSAc
Quercetinc
0.031±0.016 0.027±0.005 0.019±0.001
17.7±0.9 19.7±2.0 >20
a Values are the mean ± SD of at least three independent experiments.
b Compound I
c Reference compounds
Most of the tested compounds had significant antiproliferative effect on the selected cell
lines, showing GI50 values ranging from 2.0 to 18 µM. In general, the analogues 10-14, with
the unsubstituted R3 (R3 = H), showed a rather low antiproliferative effect. Nevertheless, their
GI50 values allowed to draw some preliminary structure-activity relationships. In particular, it
can be seen that 10 (unsubstituted at R1 and R2; hydroxyl group at R4) did not have any effect
at the tested concentrations. The introduction of a hydroxyl group at R2 (11) induced a small
increase in antiproliferative activity against HeLa and A2780 cells, while the insertion of a
hydroxyl group at both R1 and R2 (12) resulted in a GI50 value of 2.2 µM on A2780 cells.
Finally, the data obtained for compounds 13 and 14 suggest that large arylcarboxy
substituents at R4 do not improve the cell effect.
As regards compounds 26-38, characterised by alkyl substituents at R3, they appeared
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generally more potent than 10-14. This can be seen by comparing the cell growth inhibition
data of 10 (GI50 > 20 µM, on all tested cell lines) with those of 26, 27 and 29. Interestingly,
28 with a carboxymethylene substituent at R3 was not active (GI50 > 20 µM), which indicated
that introduction of groups with low pKa is detrimental for cellular activity. Similarly as for
10, 26, 27 and 29, also the activity of 12 (R1, R2 and R4 = OH) on HeLa and MSTO-211H
cells increased when alkyl substituents were introduced at R3 (12, GI50 > 20µM; 34-37, GI50
from 3.4 to 18 µM). On the other hand, the activity of 12 on A2780 cells (GI50 = 2.2 µM) was
higher compared to that of 34 (methyl group at R3; GI50 = 14 µM), 35 (propyl group at R3;
GI50 = 8.4 µM), 36 (phenoxypropyl group at R3; GI50 = 7.4 µM) and 37 (phenoxybutyl group
at R3; GI50 = 4.3 µM).
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The compounds with a hydroxyl group at R2 (11, 12, 30-37) were generally more potent than
those with unsubstituted R2 (10, 13, 26-29), as demonstrated by the comparison between 26
(unsubstituted R2) and 30 (hydroxyl group at R2). A similar behaviour can also be observed
for 29 (unsubstituted R2) with respect to 33, even if the differences in GI50 values appeared
notably reduced.
The presence of an OH group at R1 had unfavourable effects on the antiproliferative activity
as demonstrated by the higher potencies of 30 (R1 = H) versus 35 (R1 = OH), 32 (R1 = H)
versus 36 (R1 = OH) and 33 (R1 = H) versus 37 (R1 = OH). The good antiproliferative profile
of 30 (GI50 = 7.9 µM, 2.3 µM and 2.9 µM for HeLa, A2780 and MSTO-211H, respectively)
was further improved by the addition of a phenoxy group to the propyl side chain of R3, to
obtain the 3-phenoxypropyl derivative 32. Promising activity was maintained even when the
length of the alkyl linker was increased from propylene to butylene, as for 33. The GI50
values of 32 and 33 were in the low micromolar range for all cell lines tested, and these
values were the lowest among all of the studied analogues, thus highlighting 32 and 33 as the
most promising compounds of the series. The comparison between the antiproliferative
activities of 32 (3-phenoxypropyl substituent at R3) and 31 (3-phenoxyethyl substituent at R3)
showed a significant decrease in activity for the latter in all tested cell lines. This result
indicates that the appropriate length of the side chain at R3 is crucial for the biological effects.
Finally, alkylation of the hydroxyl groups of 37 to obtain the trimethoxy derivative 38
resulted in a significant decrease in the antiproliferative effect, which indicates the possibility
of hydrogen bonding between the OH groups and the biological target.
Based on the structural similarities of the prepared compounds with some flavonoid inhibitors
of topo II (Fig. 1B) and with the aim to investigate the molecular mechanism responsible for
the antiproliferative activity, we evaluated the effect of the most interesting analogues on the
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relaxation of supercoiled plasmid DNA catalysed by topo II.22,23
In the series of compounds with unsubstituted R3 (10-14), the activities of the most potent
derivatives, 11 and 12, were analysed and m-AMSA (Fig. 2) and quercetin (Fig. 1 SI) were
used as references.
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Figure 2. Effect of compounds 11 and 12 on relaxation of supercoiled pBR322 DNA by
human recombinant topoisomerase II. Supercoiled DNA (DNA) was incubated with the
enzyme in the absence (topo II) and in the presence of 11 and 12 at 100 µM (A) or 25 µM
and 50 µM (B). 20 µM m-AMSA was used as reference.
Both 11 and 12 are able to inhibit topo II relaxation activity at 100 µM concentration (Fig.
2A). In detail, compound 12 showed an electrophoretic pattern with almost solely supercoiled
DNA, which suggests that it completely inhibited topo II activity. For compound 11, as well
as the presence of supercoiled DNA, some topoisomers were formed, thus suggesting a
slightly lower inhibitory activity. To further confirm these effects, topo II was incubated in
the presence of 11 and 12 at 25 µM and 50 µM (Fig. 2B). At the lower concentration, only 12
inhibited the activity of topo II, while 11 had no measurable effect. Interestingly, these results
correlate with the antiproliferative effects on A2780 cells, where 11 showed a GI50 value
(GI50 = 11 µM) about 5 times higher with respect to 12 (GI50 = 2.2 µM) (Table 1).
As regard the compounds having alkyl substituents at the R3 position (26-38), the effects on
topo II activity of 100 µM 26-29 (unsubstituted at R1 and R2) are shown in Fig. 3. For 26-28,
the electrophoretic patterns appeared comparable to that of topo II alone, with a series of
topoisomers as a result of the enzyme relaxation activity. On the other hand, as well as a
weak formation of topoisomers, in the presence of 29, a strong band corresponding to
supercoiled DNA also appears, indicative of an inhibitory effect, and in agreement with the
antiproliferative data shown in Table 1.
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Figure 3. Effect of compounds 26-29 on relaxation of supercoiled pBR322 DNA by human
recombinant topoisomerase II. Supercoiled DNA (DNA) was incubated with topo II in the
absence (topo II) and in the presence of 26-29 at 100 µM.
Compounds 30-33 (hydroxyl substituent at R2) were characterised by low micromolar GI50
values (Table 1), and actually they showed significant topo II inhibitory activity at 100 µM
concentration (Fig. 4A). In particular, in these experimental conditions, for 30, 32 and 33 a
marked band of supercoiled DNA was observed. For 31, a slightly lower inhibitory activity
was detected, in agreement with its lower antiproliferative effect with respect to 30, 32 and
33 (Table 1). In addition, incubation of the enzyme with 25 µM and 50 µM concentrations
(Fig. 4B) highlighted 32 as the most effective and 31 as the least effective of this group of
compounds. Indeed, in the presence of 32 a complete block of DNA relaxation can be
observed at 50 µM, while no effect was obtained in the presence of 31 at the same
concentration.
Figure 4. Effect of compounds 30-33 on the relaxation of supercoiled pBR322 DNA by
human recombinant topoisomerase II. Supercoiled DNA (DNA) was incubated with topo II
in the absence (topo II) and in the presence of 30-33 at 100 µM (A), or at 25 µM and 50 µM
(B).
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Finally, topo II inhibition by 34-38, which showed intermediate antiproliferative profiles, was
also investigated (Fig. 5). Incubation of topo II in the presence of 100 M 34-38 indicates
compounds 34 and 38 as the less active derivatives of this series, in agreement with their
relatively low antiproliferative effect, while a more pronounced inhibitory activity is exerted
by 35-37, according with their more significant inhibition of cell proliferation (Fig. 5; Table
1).
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Figure 5. Effect of compounds 34-38 on the relaxation of supercoiled pBR322 DNA by
human recombinant topoisomerase II. Supercoiled DNA (DNA) was incubated with topo II
in the absence (topo II) and in the presence of 34-38 at 100 µM.
The interesting biological profile of 32 prompted us to investigate more in depth its effects on
cells, and in particular the mechanism involved in cell death. For this purpose, the most
sensitive A2780 cells were treated with increasing concentrations of 32, incubated with the
fluorescent probe JC-1, and then analysed by flow cytometry, as previously described.24 The
green monomeric fluorescent dye JC-1 accumulates in the mitochondrial matrix driven by the
membrane potential, negative inside. This uptake leads to an increase in JC-1 concentration
in mitochondria that promotes the formation of aggregates, which are characterised by high
red fluorescence. The activation of the apoptosis pathway can result in collapse of
mitochondrial transmembrane potential and release of mitochondrial proapoptotic factors in
the cytoplasm. This collapse allows the leakage of JC-1, with a consequent decrease in red
fluorescence. Fig. 6 shows that A2780 cells incubated in the presence of 32 underwent a
concentration-dependent decrease of mitochondrial transmembrane potential, with about 40%
of cells showing depolarised mitochondria at 50 µM concentration.
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Figure 6. Mitochondrial membrane potential assessed by flow cytometry in A2780 cells after
JC-1 staining. Cells were incubated for 28 h in the absence (control) and in the presence of
compound 32 at the indicated concentrations. The percentages of cells with polarized (above)
and depolarized (below) mitochondrial membranes are shown.
The ability of 32 to induce apoptosis was further confirmed by the analysis of hypodiploid
cells stained with propidium iodide. Indeed, an important hallmark of apoptosis is the
activation of endonucleases that cleave DNA at internucleosomal sites, thus creating large
number of small fragments of nucleic acid. Then, on DNA content histograms, apoptotic cells
show DNA content as sub-G0 or hypodiploid peak.25,26
Fig. 6 shows the results obtained in A2780 cells incubated with 10 µM and 50 µM 32, stained
with propidium iodide and analysed for DNA/cell content. Compound 32 induced a
significant increase in the sub-G0 phase, thus indicating apoptotic events in about 30% of
cells treated with the higher concentration (Fig. 7).
Figure 7. Flow cytometry analysis of A2780 cells treated for 28 h with compound 32 at the
indicated concentrations and stained with propidium iodide.
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A series of 3-methyl-2-phenyl-1H-indoles (10-14 and 26-38) was prepared and evaluated for
the antiproliferative effect on three human tumour cell lines, HeLa, A2780 and MSTO-211H.
The role of different substituents on the two aromatic moieties of these compounds was
discussed. In particular, compounds with an unsubstituted R1 were generally more potent than
those with a hydroxyl or a methoxy group at R1. On the other hand, at R2 and R4, hydroxyl
groups were generally preferred. The most active compounds contained alkyl or
phenoxyalkyl substituents at R3, with the size of these substituents important for the activity.
The most interesting compound was 32, showing GI50 values of 4.4 µM, 2.2 µM and 2.4 µM
on HeLa, A2780 and MSTO-211H cell lines, respectively. Notably, for six compounds (12,
29-31, 33, 37), GI50 values lower than 5 µM against at least one cell line, were obtained.
For selected compounds (11, 12, 26-38), the effect on the relaxation of supercoiled plasmid
DNA catalysed by topo II was also measured. The obtained results highlighted a good
correlation between the inhibition of enzyme and the antiproliferative effect. For the most
promising compound 32, cytofluorimetric analysis showed the ability to induce a
concentration-dependent collapse of mitochondrial transmembrane potential. Moreover, a
significant increase in the sub-G0 phase was measured in cells incubated with 32, confirming
the occurrence of apoptosis. Overall, these data highlight 3-methyl-2-phenyl-1H-indole as a
promising scaffold for the design of compounds with significant antiproliferative and anti-
topo II activities, and provide new ideas for further optimisation.
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Author Contributions
The manuscript was written using contributions from all authors. All authors have given
approval to the final version of the manuscript.
Conflict of Interest
The authors declare no conflict of interest including any financial, personal or other
relationships with other people or organizations.
Funding
The work was funded by the Slovenian Research Agency (Grant No. P1-0208) and by Italian
MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca).
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Acknowledgments
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This work was supported by the Slovenian Research Agency (Grant No. P1-0208) and by
Italian MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca). The authors thank
Dr. Žiga Hodnik for the help with chemical synthesis and Dr. Dušan Žigon (Mass
Spectrometry Center, Jožef Stefan Institute, Ljubljana, Slovenia) for recording mass spectra.
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Supporting Information
General Information – Chemistry; Synthetic Procedures and Analytical Data; 1H and 13C
Spectra of the Representative Compounds; Materials and methods - Biological studies;
Figure 1 SI Effect of quercetin on the relaxation of supercoiled pBR322 DNA by human
recombinant topoisomerase II (topo II); References.
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R3
N
PhO
HO
R2
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N
R4
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GI50 (HeLa) = 4.4 µM
GI50 (A2780) = 2.2 µM
GI50 (MSTO-211H) = 2.4 µM
Inhibitory effect on topo II relaxation
Induction of apoptosis
R1 and R2=H, OH or OCH3
R3=H, CH3, alkyl or phenoxyalkyl
R4=OH, OCH3 or arylcarboxyl
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