Synthesis of Methoxy-, Methylenedioxy-, Hydroxy-, and Halo-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Inhibitors and Their Biological Activity for Drug-Resistant Cancer

Article abstract of DOI:10.1021/acs.jmedchem.1c00318

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to

Full text of DOI:10.1021/acs.jmedchem.1c00318

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Article
Synthesis of Methoxy‑, Methylenedioxy‑, Hydroxy‑, and Halo-
Substituted Benzophenanthridinone Derivatives as DNA
Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1
(TDP1) Inhibitors and Their Biological Activity for Drug-Resistant
Cancer
De-Xuan Hu, Wen-Lin Tang, Yu Zhang, Hao Yang, Wenjie Wang, Keli Agama, Yves Pommier,*
and Lin-Kun An*
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ABSTRACT: As a recently discovered DNA repair enzyme, tyrosyl-
DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB
(TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can
potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell
resistance to TOP1 inhibitors. On the basis of our previous study,
herein we report the synthesis of benzophenanthridinone derivatives as
TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8,
C12, and C14) showed a robust TOP1 inhibitory activity (+++ or +++
+), and four compounds (A13, C12, C13, and C26) showed a TDP1
inhibition (half-maximal inhibitory concentration values of 15 or 19 μM). We also show that the dual TOP1 and TDP1 inhibitor
C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar
concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with
topotecan in both MCF-7 and MCF-7/TDP1 cells.
TOP1-mediated DNA damage, which would result in cancer
cell resistance to therapeutic drugs, such as the TOP1 poisons
topotecan, irinotecan, 10-hydroxycamptothecin, belotecan,
enhertu, and trodelvy.6 Indeed, TDP1-overexpressing cells
are resistant to TOP1 inhibitors.18 Conversely, cells with a low
TDP1/TOP1 ratio tend to be sensitive to TOP1 inhibitors,19
as well as TDP1-deficient cells and TDP1 knockout mice,
which show hypersensitivity to camptothecin.13,20−24 Accord-
ingly, several TDP1 inhibitors have been reported to show a
synergistic activity to camptothecin derivatives both in vitro
and in vivo.25−30 Therefore, TDP1 is a rational target for the
development of anticancer drugs.16,31
INTRODUCTION
■
DNA topoisomerase IB (TOP1) plays an essential role in
DNA replication, transcription, and genomic stability by
relaxing supercoiled DNA.1−3 During this process, TOP1
cleaves a single strand of DNA to form a transient covalent
complex (TOP1cc) that allows supercoils to be dissipated.3
TOP1cc can be trapped by TOP1 poisons, such as
camptothecin (CPT) derivatives that intercalate between the
DNA base pairs at the cleavage site thereby preventing further
religation of the broken strand, generating double-strand
breaks and causing cell death.4−6 DNA damage produced by
TOP1 poisons can be repaired through several pathways,
including homologous recombination, cell cycle checkpoint
signaling, and tyrosyl-DNA phosphodiesterase 1 (TDP1)-
dependent pathways.3,7−9 TDP1 is a member of the
phospholipase D superfamily and can specifically hydrolyze
the 3′-phosphotyrosyl bond between the nicked DNA and
peptide derived from TOP1cc to generate DNA breaks with
3′-phosphates and 5′-hydroxyl ends,10−13 following its recruit-
ment by poly(ADPribose) polymerase (PARP1) activa-
tion.14,15 The resulting DNA breaks can be further repaired
by PNKP, XRCC1, DNA polymerase β, and DNA ligase
III.16,17 In this pathway, the TDP1-catalyzed hydrolysis of the
3′-phosphotyrosyl bond is a key step for initiating repair of the
In addition, TDP1 also hydrolyzes a broad range of 3′-end
blocking lesions generated by DNA oxidation and alkylation as
well as 5′-phosphotyrosyl bonds derived from DNA topo-
isomerase II (TOP2)-mediated DNA damage, implying TDP1
Received: February 20, 2021
Published: May 19, 2021
https://doi.org/10.1021/acs.jmedchem.1c00318
© 2021 American Chemical Society
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has a broader role in the maintenance of genomic
stability.20,21,31−34
TDP1 inhibitors, two chalcone derivatives (Figure 1) have
been recently identified from the roots of Isodon ternifolius
Kudo with moderate dual TOP1 and TDP1 inhibition.42 In
addition, oxynitidine derivatives have been found to have
TOP1 and TDP1 inhibitory activities.25,43 And the oxynitidine
TDP1 inhibitors exhibited a synergistic effect with CPT in
MCF-7 cells. However, the dual inhibitors showed good TOP1
inhibition but low TDP1 inhibition. For example, NTD-96
(Figure 1) showed a TOP1 inhibitory activity of +++ and only
a 12% TDP1 inhibitory activity at 100 μM concentration.25
Molecular modeling shows two hydrogen bonds between
the oxygen atoms of the methylenedioxy group of NTD-96,
and the residues of TOP1, Thr718 (3.6 Å), and Asn722 (3.7
Å) were observed, implying this methylenedioxy group as
playing a key role in the interaction between TOP1 and the
inhibitor.25 Additionally, the position and number of the
methoxy or methylenedioxy group affects the biological activity
of the natural benzo[c]phenanthridine alkaloids, such as
sanguinarine,44−46 chelerythrine,45,47 macarpine,48 fagari-
dine,49,50 and fagaronine (Figure S1, Supporting Informa-
tion).51,52 These observations encouraged us to study the effect
of the position and number of methoxy and methylenedioxy
group as well as the displacement by a hydroxy or halogen
group on TOP1 and TDP1 inhibitory activities. Therefore,
three series of benzophenanthridinone derivatives were
designed. On the basis of the structure of NTD-96, Series A
analogues was designed with or without a single substituent in
the A-, C-, or D-ring; Series B derivatives include two
substituents or one methylenedioxy group in the A- or D-ring;
and in Series C, multiple substituents were introduced in the
A- and D-rings. Herein, we report the synthesis, TOP1 and
Because of the unique physiological functions of TOP1 and
TDP1, the discovery of their inhibitors has become a focus of
attention. Despite the numerous TOP1 inhibitors and several
TDP1 inhibitors that have been described,26,27,35−37 only a few
dual inhibitors have been described in the literature.30,38−42
The indenoisoquinoline derivatives were the first reported dual
TOP1 and TDP1 inhibitors.40,41 The representative bis-
(indenoisoquinoline) inhibitor (Figure 1) shows a strong
Figure 1. Reported dual TOP1/TDP1 inhibitors.
TOP1 inhibition equal to that of camptothecin and TDP1
inhibition with a half-maximal inhibitory concentration (IC50)
value of 1.5 μM.41 In our effort to discover dual TOP1 and
a
Scheme 1. Synthesis of A1−A13
a
Reagents and conditions: (a) NH2(CH2)2NMe2, MeOH, rt. (b) (i) N2, Ni(cod)2, P(o-Tol)3, MeCN, 80 °C; (ii) CsOH, K3[Fe(CN)6], MeOH,
H2O, 80 °C. (c) (i) (COCl)2, DMSO, TEA, DCM, −60 °C; (ii) conc. hydrochloric acid, AcOH, 65 °C. (d) Na, CuI, MeOH, DMF, rt-110 °C. (e)
(i) SOCl2, reflux; (ii) TEA, DCM, rt. (f) (i) Cl(CH2)2NMe2/HCl, NaH, DMF, 85 °C; (ii) N2, Pd(OAc)2, P(o-tol)3, Ag2CO3, DMF, 150 °C.
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a
Scheme 2. Synthesis of B1−B8
a
Reagents and conditions: (a) NH2(CH2)2NMe2, MeOH, rt. (b) (i) N2, Ni(cod)2, P(o-Tol)3, MeCN, 80 °C; (ii) CsOH, K3[Fe(CN)6], MeOH,
H2O, 80 °C. (c) (i) (COCl)2, DMSO, TEA, DCM, −60 °C; (ii) conc. hydrochloric acid, AcOH, 65 °C. (d) (i) SOCl2, reflux; (ii) TEA, DCM, rt.
(e) (i) Cl(CH2)2NMe2/HCl, NaH, DMF, 85 °C; (ii) N2, Pd(OAc)2, P(o-tol)3, Ag2CO3, DMF, 150 °C.
a
Scheme 3. Synthesis of C1−C30
a
Reagents and conditions: (a) NH2(CH2)2NMe2, MeOH, rt. (b) (i) N2, Ni(cod)2, P(o-Tol)3, MeCN, 80 °C; (ii) CsOH, K3[Fe(CN)6], MeOH,
H2O, 80 °C. (c) (i) (COCl)2, DMSO, TEA, DCM, −60 °C; (ii) conc. hydrochloric acid, AcOH, 65 °C. (d) (i) SOCl2, reflux; (ii) TEA, DCM, rt.
(e) (i) Cl(CH2)2NMe2/HCl, NaH, DMF, 85 °C; (ii) N2, Pd(OAc)2, P(o-tol)3, Ag2CO3, DMF, 150 °C. (f) For C12, C13 and C22, concentration
hydrochloric acid, AcOH, 65 °C.
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Table 1. TOP1 and TDP1 Inhibition, and Cytotoxicity of A1−A13 and B1−B8
c
GI50 SD (μM)
a
b
Cpd
CPT
NTD-96
A1
A2
A3
A4
A5
A6
A7
TOP1 inhibition
TDP1 inhibition
HCT-116
DU145
MCF-7
A549
d
++++
+++
+
+
+
+
+
0
0
+
+
+
0
0
0/+
+
+
+
ND
0.050 0.016
0.14 0.018
1.27 0.13
6.79 0.59
3.22 0.19
5.66 1.24
6.92 0.28
94.05 1.02
12.18 1.27
0.78 0.11
2.18 0.13
6.55 0.61
13.24 2.11
9.84 0.87
6.93 0.45
2.01 0.11
6.34 0.88
49.77 6.57
12.90 0.63
11.10 0.27
4.17 1.15
1.26 0.02
9.76 0.66
0.21 0.07
0.078 0.007
25.21 0.06
10.88 0.38
15.39 0.24
24.98 0.95
27.15 0.66
3.53 1.97
60.27 0.96
15.57 0.90
3.77 0.02
12.34 0.27
18.17 0.29
6.72 0.22
1.88 0.17
9.94 0.65
17.38 0.10
68.10 18.74
30.97 0.52
2.04 0.59
20.46 0.49
1.41 0.59
4.51 0.72
0.35 0.04
0.20 0.03
14.49 1.33
13.01 3.11
83.26 5.42
13.20 0.61
11.67 0.61
91.30 5.94
54.67 4.87
63.56 13.88
9.71 2.44
2.28 0.66
7.26 1.89
21.69 4.61
2.65 0.15
8.85 1.15
10.98 0.71
5.20 1.65
10.61 2.00
81.31 2.65
2.73 0.83
0.53 0.07
2.26 0.06
0.31 0.03
0.32 0.04
10.98 0.95
11.86 2.08
2.61 0.39
4.96 0.68
11.95 0.84
65.14 8.90
12.18 1.54
2.75 0.11
0.94 0.06
6.41 0.34
17.05 0.92
17.72 1.70
6.68 0.31
0.79 0.08
12.31 0.73
13.07 2.82
8.58 0.55
65.53 7.45
3.78 0.44
1.22 0.14
2.33 0.76
12%
0
0
0
0
0
0
22%
57%
0
42%
0
A8
A9
A10
A11
A12
A13
B1
B2
B3
B4
B5
0
15 2.2
0
26 0.75
12%
0
0
++
+
+
0
0
B6
B7
B8
49%
15%
0
a
The TOP1 cleavage inhibitory activity of the compounds was semiquantitatively expressed relative to CPT at 1 μM as follows: 0, no inhibition; 0/
+, less than 20%; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++, equal activity.
b
The TDP1 inhibition, determined by using a fluorescence assay, was expressed as the percentage inhibition (%) of the compounds at 100 μM
c
concentration or the IC50 value (μM, mean SD). Every experiment was repeated at least three times independently. The cytotoxicity GI50 values
were defined as the concentrations corresponding to a 50% cell growth inhibition and obtained from an MTT assay. “ND” means “not
d
determined”.
TDP1 inhibitory activities, and the anticancer activity of these
three series of novel benzophenanthridinone derivatives.
Similarly, Series B (B1−B8) and C (C1−C30) analogues
were also synthesized through these two pathways. As shown
in Scheme 2, compounds B1−B8 with a dimethoxy, difluoro,
or methylenedioxy substituent at the A- or D-ring were
synthesized.
RESULTS AND DISCUSSION
■
Chemistry. According to our previously reported meth-
od,25,53,54 the Series A analogues were synthesized and
outlined in Pathway I (Scheme 1). The obtained Schiff’s
base 2 by the coupling reaction of 2-bromobenzaldehyde 1
with N,N-dimethylaminoethylamine reacted with alkyne 3 to
give the lactam intermediate 4 under Ni-based catalysis.
Following the Swern oxidation of the hydroxyl group of 4,
cyclization reaction under acid conditions gave the target
products A1−A3, A5, A8−A10. From the intermediate 4ab′,
the cyclization reaction gave two products, namely, A2 with a
1-methoxy substituent and A5 with a 3-methoxy substituent, at
the same time. Also, two products A8 and A9 were obtained at
the same time from the cyclization of 4ca′. The 2-bromo
derivative A3 reacted with sodium methoxide to give the target
A4 with a 2-methoxy substituent. Surprisingly, the cyclization
reaction would be hampered if the R1 group was a 3- or 6-
methoxy group or if the R2 group was a 2′-methoxy group on
the intermediate 4. As a result, target compounds A6, A7, and
A11 could not be prepared through Pathway I (Scheme 1).
Therefore, we designed another synthetic route, Pathway II in
Scheme 1. 2-Bromobenzoic acid 5 was amidated with
naphthylamine 6 to provide the amide intermediate 7.
Following the nucleophilic substitution with 2-dimethylami-
noethyl chloride, a Heck cyclization reaction under Pd-based
catalysis gave the target compounds A6, A7, and A11-A13.55
The syntheses of compounds C1−C30 were shown in
Scheme 3, and their structures were outlined in Table 2. For
the synthesis of the target compounds (C12, C13, C18, C19,
C20, C22, C28, C29, and C30) with a hydroxyl substituent,
the benzyl-protected materials (7qe′, 7re′, 4fd′, 4gd′, 4hd′,
7ue′, 4ef′, 4eg′, and 4eh′) were used. In Pathway I (Scheme
3), the benzyl groups were removed during the cyclization step
in an acid condition to give the products C18−C20, and C28−
C30. In Pathway II, following the Pd-catalyzed cyclization, the
benzyl groups were removed with concentrated hydrochloric
acid. From compounds 7re′ and 7ue′, the deprotection
reaction gave the target products C13 and C22, respectively.
However, we did not get the deprotection product using
compound 7qe′; the methyl group at the 7-position was
removed along with the benzyl group to give compound C12.
Finally, altogether, fifty-one novel methoxy-, methylene-
dioxy-, hydroxy-, halo-, and nitro-substituted benzophenan-
thridinone derivatives were synthesized. Their structures were
characterized through high-resolution mass spectrometry
(HRMS) and NMR spectra.
TOP1 Inhibition. All synthesized compounds were tested
for TOP1 inhibition at 100, 10, 1, and 0.1 μM concentrations
with the TOP1-mediated DNA cleavage assay.56 The TOP1
inhibitory activity, summarized in Tables 1 and 2, was
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Table 2. Structures of C1−C30 and Their TOP1 and TDP1 Inhibition, and Cytotoxicity
a
GI50 SD (μM)
b
c
Cpd
CPT
NTD-96
C1
C2
C3
C4
C5
C6
C7
R, R′, R′′
TOP1 inhibition
TDP1 inhibition
HCT-116
DU145
MCF-7
A549
d
f
/
++++
+++
+
+++
0
+++
++++
++
+++
+++
++
+
0
+++
+
+++
+
+
0
+
+
+
0
0
+
0
ND
0.050 0.016
0.14 0.018
1.13 0.03
0.93 0.13
10.12 0.17
0.24 0.07
1.18 0.11
4.35 0.78
2.92 0.78
0.14 0.02
5.21 0.28
8.41 0.21
13.41 0.73
1.44 0.28
4.38 0.65
2.79 0.14
4.21 0.87
0.90 0.02
41.56 4.86
9.35 0.63
0.63 0.15
8.48 0.33
26.17 0.98
11.31 0.44
11.56 1.67
>100
0.21 0.07
0.078 0.007
0.86 0.05
0.28 0.04
11.68 0.83
0.91 0.08
0.32 0.01
3.82 0.95
0.52 0.04
0.32 0.01
1.00 0.03
4.13 0.89
40.21 0.42
0.80 0.07
0.44 0.08
1.22 0.01
3.27 0.84
6.61 0.30
7.88 1.18
1.14 0.45
3.11 2.70
>100
71.73 0.83
11.25 0.71
15.78 4.46
2.90 0.83
0.26 0.05
25.43 0.39
>100
41.67 0.75
0.29 0.09
15.40 0.52
0.35 0.04
0.20 0.03
1.51 0.22
1.44 0.21
5.44 0.50
0.57 0.01
2.99 0.08
10.30 2.05
10.18 0.32
0.20 0.04
12.74 0.62
8.54 0.91
21.76 7.69
0.77 0.10
0.53 0.08
3.24 0.10
6.95 1.92
2.79 0.20
42.20 2.06
>100
6.26 3.01
93.35 3.04
24.40 6.28
>100
24.63 5.96
7.22 2.37
10.72 0.35
0.35 0.02
>100
0.31 0.03
0.32 0.04
6.66 0.08
7.95 0.37
6.87 1.11
2.40 0.36
3.13 0.82
9.27 0.32
2.28 0.88
1.61 0.06
1.49 0.14
12.21 0.59
12.81 0.83
1.11 0.02
3.09 1.17
10.35 0.17
1.86 0.93
3.35 0.40
34.71 2.26
>100
3.93 0.73
84.94 1.82
40.45 0.95
2.79 0.35
23.74 1.89
12.71 3.61
40.35 1.96
1.53 1.10
66.61 1.92
10.81 0.81
1.76 0.39
2,3-OCH2O-8, 9-diOMe
2,3-OCH2O-7-OMe
2,3-OCH2O-8-F
2,3-OCH2O-8-Br
2,3-OCH2O-8-NO2
2,3-OCH2O-8-OMe
2,3-OCH2O-9-F
2,3-OCH2O-9-Cl
2,3-OCH2O-9-OMe
12%
0
0
0
0
0
0
0
C8
C9
0
0
2,3-OCH2O-9-CF3
C10
C11
C12
C13
C14
C15
C16
C17
C18
C19
C20
C21
C22
C23
C24
C25
C26
C27
C28
C29
C30
2,3-OCH2O-7,8-OCH2O
2,3-OCH2O-7,8-diOMe
2,3-OCH2O-7,8-diOH
2,3-OCH2O-7-OH, 8-OMe
2,3-OCH2O-8, 9-diF
2,3-OCH2O-8,9-OCH2O
2,3-OCH2O-8-OMe, 9-Oi-Pr
2,3-OCH2O-8-Oi-Pr, 9-OMe
2,3-OCH2O-8-OMe, 9-OH
2,3-OCH2O-8-OH, 9-OMe
2,3-OCH2O-8, 9-diOH
2,3-OCH2O-9, 10-diOMe
2,3-OCH2O-9-OMe, 10-OH
2,3-diOMe 7,8-OCH2O-
2,3,7,8-tetraOMe
27 0.51
0
19 0.91
15 0.31
0
49%
0
0
0
53 1.5
0
0
0
0
0
54 1.0
19 0.35
0
0
44%
0
2,3-diOMe 8,9-OCH2O-
2,3,8,9-tetraOMe
2,3,9,10-tetraOMe
2-OMe, 3-OH 8,9-diOMe
2-OH, 3-OMe 8,9-diOMe
2,3-diOH 8,9-diOMe
0/+
0/+
0
0/+
+
10.15 0.35
9.93 0.33
>100
12.88 0.42
1.21 0.64
26.19 2.70
2.70 0.72
1.66 0.23
3.62 0.38
++
8.47 0.76
a
b
The cytotoxicity GI50 values were defined as the concentrations corresponding to 50% cell growth inhibition and obtained from MTT assay. The
TOP1 cleavage inhibitory activity of the compounds was semiquantitatively expressed relative to CPT at 1 μM as follows: 0, no inhibition; 0/+, less
c
than 20%; +, between 20% and 50% activity; ++, between 50% and 75% activity; +++, between 75% and 95% activity; ++++, equal activity. The
TDP1 inhibition, determined by using a fluorescence assay, was expressed as the percentage inhibition (%) of the compounds at 100 μM
d
concentration or the IC50 value (μM, mean SD). Every experiment was repeated at least three times independently. “/” means “inapplicable”.
f
ND means not determined.
semiquantitatively expressed relative to the TOP1 inhibition of
camptothecin (CPT) at 1 μM: 0, no inhibition; 0/+, less than
20%; +, between 20% and 50% activity; ++, between 50% and
75% activity; +++, between 75% and 95% activity; ++++, equal
activity to camptothecin at 1 μM concentration. Compound
A1, the benzophenanthridinone scaffold with a 5-dimethyla-
minoethyl substituent, showed a low TOP1 inhibitory activity
of + (Table 1). The addition of a single methoxy group in the
A-, C-, and D-rings did not increase the TOP1 inhibition.
Furthermore, the analogues (A6, A7, A11, and A12) with a
methoxy group at the 4-, 7-, 10-, and 11-positions lost TOP1
inhibition. Similarly, the introduction of two methoxy groups
or a methylenedioxy group in the A- and D-rings (Series B)
did not increase the TOP1 inhibition (Table 1). The analogue
B5 with 8,9-difluoro substituents showed an increased TOP1
inhibition of ++.
For the Series C analogues (Table 2), the introduction of a
methylenedioxy group at the 2,3-position and simultaneously
one or two substituents in the A-ring increased the TOP1
inhibition significantly. The most potent inhibitor C5 with 2,3-
methylenedioxy and 8-methoxy groups exhibited an equal
potency (++++) to that of CPT. With the presence of a 2,3-
methylenedioxy group, the compounds with 8-fluoro (C2), 8-
nitro(C4), 9-chloro (C7), or 9-methoxy (C8) groups as well as
with 7,8-dihydroxy (C12) or 8,9-difluoro groups (C14)
exhibited the strong TOP1 inhibitory activity of +++, implying
the important role of the 2,3-methylenedioxy group for TOP1
inhibition. Conversely, the compounds with a methoxy or
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Figure 2. Representative gels of the TOP1-mediated DNA cleavage assay. Lane 1, DNA alone; lane 2, DNA and TOP1; lane 3, DNA and TOP1
with CPT (1 μM); lane 4, DNA and TOP1 with LMP744 (1 μM); lanes 5−28, DNA and TOP1 with the tested compounds at 0.1, 1.0, 10, and 100
μM concentrations, respectively. The arrows and numbers at left indicate the cleavage site positions.
Figure 3. (A) Induction of TOP1−DNA covalent cleavage complexes by in vivo complex of enzyme assay in MCF-7 cells. Lane 1, untreated
control; lanes 2 and 3, cells treated with TPT at 25 and 50 μM, respectively; lanes 4−6, cells treated with C12 at 25, 50, and 100 μM, respectively.
(B) Hypothetical binding mode of C12 in the ternary TOP1−DNA−drug cleavage complex (PDB: 1K4T). (C) Induction of TDP1-DNA covalent
complex by ICE assay in MCF-7 and MCF-7/TDP1 cells. Lane 1, untreated control; lanes 2−4, cells treated with TPT (10 μM) and C12 at 10 and
100 μM, respectively; lanes 5 and 6, cells cotreated with TPT (10 μM) and C12 (10 and 100 μM).
hydroxy group substituted at the 2- and 3-positions (C23−
C29) showed the limited TOP1 inhibitory potency of 0 to +,
except for compound C30 with a moderate TOP1 inhibition of
++. Bigger substituents at the 8- or 9-positions were
unfavorable for TOP1 potency. The compounds with 9-
isopropoxy (C16) and 8-isopropoxy (C17) showed a low
TOP1 inhibition. Surprisingly, the presence of a methoxy
group at the 7- or 10-positon strongly reduced the TOP1
inhibitory activity, and compounds A7, A11, B4, B8, C11,
C21, C24, and C27 had no detectable TOP1 inhibitory
activity. The representative cleavage gels of C2, C4, C5, C7,
C8, and C12 (Figure 2) show that the cleavage sites induced
by the novel TOP1 inhibitors are similar to that of the
indenoisoquinoline control LMP744 (Figure S2, Supporting
Information) but not to that of CPT, which is consistent with
the effects of NTD-96.25
To evaluate the ability of the synthesized novel inhibitor to
induce cellular TOP1cc, C12 was selected for the immuno-
complex of enzyme (ICE) to DNA assay in MCF-7 cancer
cells. As shown in Figure 3A, C12 induced cellular TOP1cc in
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a dose-dependent manner, similar to the positive control
topotecan (TPT), implying that TOP1 is the cellular target of
C12.
The TOP2 relaxing assay (Figure S3, Supporting Informa-
tion) indicated that C12 did not have an inhibitory activity to
TOP2 at a 25 μM concentration.
alone at low concentration (10 μM), TDP1cc significantly
increased after being coincubated with TPT and C12 (both at
10 and 100 μM) in MCF-7 cells. Similarly, in MCF-7/TDP1
cells, C12 could induce the formation of cellular TDP1cc, even
at a low concentration (10 μM). After being coincubated with
TPT and C12, cellular TDP1cc significantly increased. These
results indicated that the benzophenanthridinone TDP1
inhibitor C12 could induce the formation of cellular TDP1cc
in both MCF-7 and MCF-7/TDP1 cells.
Cytotoxicity Assays. The cytotoxicity of the synthesized
compounds was assessed by the 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) assay against four
human tumor cell lines, including colon cancer (HCT116),
prostate cancer (DU-145), breast cancer (MCF-7), and
nonsmall cell lung cancer (A549) cell lines. The cells were
treated for 72 h in a five-dose assay at concentrations ranging
from 0.01 to 100 μM. The GI50 values, defined as the
concentrations corresponding to 50% cell growth inhibition,
were summarized in Tables 1 and 2.
As shown in Table 1, Series A and B analogues, which
showed a TOP1 inhibitory activity ranging from 0 to ++,
showed cytotoxicity with GI50 values at high and low
micromolar levels. Only two Series A analogues showed a
high cytotoxicity at a sub-micromolar level (A8: 0.78 μM for
HCT-116 and A9: 0.94 μM for A549) as well as two Series B
analogues (B1: 0.79 μM for A549 and B7: 0.53 μM for MCF-
7). Compared with Series A and B analogues, the multialkoxy-
substituted derivatives (Series C) showed a marked cytotox-
icity against the four human cancer cells, which is consistent
with their high TOP1 inhibition potency. The dual TOP1/
TDP1 inhibitor C12 (TOP1: +++, TDP1:19 μM) showed
good cytotoxicity against the four human cancer cell lines with
GI50 values of 1.44 μM for HCT-116, 1.11 μM for A549, 0.80
μM for DU145, and 0.77 μM for MCF-7. The dual inhibitor
C13 (TOP1: +, TDP1:15 μM) also showed good cytotoxicity
with GI50 values between 0.44 and 4.38 μM. Although the
most potent TOP1 inhibitor C5 (++++) exhibited high
cytotoxicity against DU145 cells (GI50 = 0.32 0.01 μM), it
had a lower cytotoxicity than NTD-96 (+++), which might be
due to the low cellular permeability of C5. Other analogues
C2, C4, C7, and C8 with good TOP1 inhibition (+++) also
showed a high cytotoxicity with GI50 values at a sub-
micromolar range. Although C1, C19, C25, C26, and C29
showed a low TOP1 potency, they also showed a high
cytotoxicity possibly due to their effect on other cellular
targets. Among them, C8 showed the highest cytotoxicity for
HCT-116 (0.14 μM) and MCF-7 (0.20 μM) as well as C25 for
DU145 (0.26 μM) and B1 for A549 (0.79 μM).
The cytotoxicity of C12 was further evaluated in MCF-7/
TDP1 cells. As shown in Table 3, compared with the parental
cells MCF-7, the MCF-7/TDP1 cells exhibited a significant
resistance to the well-known TOP1 inhibitors CPT (9.2-fold)
and TPT (21.2-fold), as well as to our oxynitidine TOP1
inhibitor NTD-96 (19.9-fold), implying TDP1 overexpression
results in a cellular resistance to TOP1 inhibitors. However,
MCF-7/TDP1 cells showed a decreased resistance to C12
(3.4-fold), indicating that the TDP1 inhibition might reverse
the resistance of MCF-7/TDP1 cells to TOP1 inhibitors.
Synergistic Effects of C12 with TPT. In our previous
work, the oxynitidine TDP1 inhibitors were shown to have
synergistic effects with TOP1 inhibitors in MCF-7 cancer
cells.25 To assess whether a dual TOP1 and TDP1 inhibition
could act synergistically with TOP1 inhibitors, the combined
To evaluate the rationale for TOP1 poisoning and the
molecular binding mode of the synthesized novel inhibitors
within the TOP1-DNA complex, molecular modeling was
conducted on compound C12. A hypothetical binding mode
was built by using in silico docking from the X-ray crystal of
TOP1-DNA-ligand (PDB ID: 1K4T)57 according to our
reported method.25 As shown in Figure 3B, the benzophenan-
thridinone scaffold of C12 intercalated in the DNA break and
stacked with the +1 and −1 base pairs flanking the DNA
cleavage site. Similar to the binding mode of NTD-96 with the
TOP1-DNA complex,25 the 5-dimethylaminoethyl substituent
extended into the minor groove of the DNA. Two hydrogen
bonds (3.1 and 3.2 Å) between the lactam oxygen atom and
the side-chain nitrogen of Arg364 were observed, implying the
important role of hydrogen-bond receptor to the TOP1
inhibition potency. Also, two hydrogen bonds between the 7-
hydroxy group and a base pair (2.9 Å) and between an oxygen
atom in the dioxole ring and Asn722 (3.2 Å) might contribute
to the trapping of TOP1cc, implying the important role of the
dioxole ring for TOP1 inhibition. For the 2,3-dimethoxy-
substituted derivative, there is no hydrogen bond observed
between the compound and TOP1. The hypothetical binding
mode of C26 (Figure S4, Supporting Information) indicated
that the distance between the oxygen atom of the 3-methoxy
group and Asn722 was 4.0 Å and that the distance between the
lactam oxygen atom and Arg364 was 4.3 Å, possibly due to the
steric bulk of 2,3-dimethoxy groups. This observation is
consistent with the structure−activity relationship.
TDP1 Inhibition. The TDP1 inhibition of the synthesized
compounds was evaluated through a fluorescence assay, using a
quenched fluorescent single-stranded oligonucleotide (5′-
FAM-AGGATCTAAAAGACTT-BHQ-3′) as substrate.58
The compounds were first tested at 100 μM concentration,
and the percentage inhibition was calculated. Compounds
A13, B2, C10, C12, C13, C19, C25, and C26, with over 50%
percentage inhibition, were further tested to determine their
IC50 values, defined as the concentration of compound that
inhibits 50% of enzyme activity. As shown in Tables 1 and 2,
compound C12 showed both TOP1 (+++) and TDP1
inhibitory activity (IC50 = 19
0.91 μM). A13 and C13
showed the most potent TDP1 inhibition, both with the IC50
value of 15 μM. However, their TOP1 inhibitory activities
were low (0/+ and +). Similarly, C26 also showed a good
TDP1 inhibition (19 0.35 μM) and a low TOP1 inhibition
(0/+). The most potent TOP1 inhibitor was C5 (++++) as
well as C2, C4, C7, C8, and C14, which show a good TOP1
inhibition (+++) lacking a detectable TDP1 inhibitory activity.
It has been proposed that a transient enzyme-DNA covalent
complex intermediate (TDP1cc) forms during the TDP1
function.21,59 To assess whether C12 can induce the formation
of cellular TDP1cc, ICE assays were conducted in MCF-7 and
TDP1-overexpressing MCF-7 (MCF-7/TDP1) cancer cells,
established by the transfection of parental MCF-7 cells. As
shown in Figure 3C, C12 induced the formation of cellular
TDP1cc in MCF-7 cells at a high concentration (100 μM).
Although there was no obvious TDP1cc formation observed
after being incubated with the TOP1 inhibitor TPT or C12
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and TDP1 inhibitor also showed a synergistic effect with the
TOP1 inhibitor TPT in both MCF-7 and MCF-7/TDP1 cells.
DNA Damage and Apoptosis Induced by C12. To
evaluate the DNA damage in response to the dual TOP1 and
TDP1 inhibitor C12 in cancer cells, drug-induced γH2AX foci
were tested by an immunofluorescence assay in both MCF-7
and MCF-7/TDP1 cells using TPT as a positive control.
Similar to TPT, C12 induced γH2AX foci in MCF-7 cells in a
dose-dependent manner (Figure 5A,B), implying the ability of
C12 to induce cellular DNA damage due to the trapping of
cellular TOP1cc. In MCF-7/TDP1 cells, the number of
γH2AX foci induced by TPT decreased by 34% relative to
wild-type (WT) MCF-7 cells, implying the ability of TDP1 to
process TOP1cc.8,9 Although the number of γH2AX foci
induced by C12 in MCF-7/TDP1 cells was also reduced, the
decrease (22% and 28% at 1 and 2 μM, respectively) was less
when compared with TPT. In addition, a combined treatment
with C12 and TPT significantly increased the number of
γH2AX foci in both MCF-7 and MCF-7/TDP1 cells (Figure
5A,B). In addition, C12 reduced the number of γH2AX foci
induced by TPT (14% and 18% vs 34% for TPT alone),
consistent with the TDP1 inhibition by C12. These results
indicated that C12 effectively induced cellular DNA damage
but also increased the ability of TPT to induce DNA damage
in both MCF-7 and MCF-7/TDP1 cells, possibly due to its
dual TOP1 and TDP1 inhibitory activities.
Table 3. Cytotoxicity of C12 in Drug-Resistant MCF-7 Cells
a
GI50 SD (μM)
b
Cpd
CPT
TPT
NTD-96
C12
MCF-7
MCF-7/TDP1
resistance index
0.35 0.04
0.35 0.01
0.20 0.03
0.77 0.10
3.21 0.17
7.42 0.24
3.98 0.9
2.59 0.31
9.2
21.2
19.9
3.4
a
GI50 values (mean
SD) were defined as the concentrations of
compounds that resulted in a 50% cell growth inhibition and obtained
from an MTT assay. Every experiment was repeated at least three
b
times. The resistance index was calculated by dividing the GI50 of
MCF-7/TDP1 cells by the GI50 of MCF-7 cells.
effects of C12 with TPT were tested in MCF-7 and MCF-7/
TDP1 cells. As shown in Figure 4A, after an incubation for 96
h at 37 °C, the cytotoxicity of TPT against MCF-7 cells
significantly increased in the presence of C12 in a dose-
dependent manner. At a 50 nM concentration, ∼23% of cells
were killed by TPT alone, and ∼87% of cells were killed after
being coincubated with 2 μM of C12. The combination index
(CI) values were calculated and are shown in Figure 4B. The
CI values of the combination treatment of TPT and C12 fell in
the range of 0.1−0.9, and four doses showed strong synergistic
effects (CI < 0.3). Similarly, C12 also showed a synergistic
effect with TPT in MCF-7/TDP1 cells (Figure 4C). After a 96
h incubation, ∼19% of MCF-7/TDP1 cells were killed by 100
nM TPT alone, and ∼86% of cells were killed when being
coincubated with 2 μM of C12. Figure 4D indicated that the
CI values of the combination treatment of TPT and C12 for
MCF-7/TDP1 cells also fell in the range of 0.1−0.9, and six
doses showed strong synergistic effects (CI < 0.3). These
results indicated that the benzophenanthridinone dual TOP1
To evaluate the induction of cell apoptosis by C12, flow
cytometry assays were conducted. After an incubation with
C12 for 24 h, apoptotic MCF-7 cells were detected, and C12
significantly induced MCF-7 cell apoptosis in a dose-
dependent manner (Figure 5C). Approximately 74.99%
(26.53% early apoptotic cells and 48.46% late apoptotic
cells) were scored as apoptotic after a 24 h incubation with
C12 at 4 μM concentration.
Figure 4. Synergistic effect and CI of C12 with TPT in the MCF-7 (A, B) and MCF-7/TDP1 (C, D) cells. The cells were coincubated for 96 h.
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Figure 5. (A) Histone γH2AX foci (red) induced by TPT and C12 alone or combined treatment in the MCF-7 and MCF-7/TDP1 cells. The cells
were treated with drugs for 6 h at the indicated concentrations. DNA was stained with DAPI (blue). (B) Quantification of the number of γH2AX
foci on the figure (A). Every experiment was repeated at least three times independently. (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, and (****)
P < 0.0001. (C) Flow cytometry histograms of apoptosis in MCF-7 cells induced by C12 at 1, 2, and 4 μM, respectively.
and MCF-7/TDP1 cells, resulting in cancer cell apoptosis.
This work suggests that the benzophenanthridinone derivatives
deserve to be further investigated and potentially developed as
dual TOP1 and TDP1 inhibitors.
CONCLUSION
■
Herein three series of benzophenanthridinone derivatives
(Series A, B, and C) with methoxy, methylenedioxy, hydroxy,
and halo substituents in A-, C-, and D-rings were synthesized
and evaluated for TOP1 and TDP1 inhibitory activities and
cytotoxicity against four human cancer cell lines (HCT116,
MCF-7, DU-145, and A549 cell lines). Enzyme-based assays
indicated that C5 with an 8-methoxy substituent showed the
highest TOP1 inhibitory potency (++++) equal to CPT, and
six compounds C2, C4, C7, C8, C12, and C14 showed the
strong TOP1 inhibitory activity of +++. Four compounds A13
(15 μM), C12 (19 μM), C13 (15 μM), and C26 (19 μM) also
showed TDP1 inhibitory activity. Several compounds showed
a high cytotoxicity in human cancer cells with GI50 values at a
sub-micromolar concentration range. C8 with good TOP1
inhibition (+++) showed the highest cytotoxicity for HCT-116
(0.14 μM) and MCF-7 (0.20 μM). The dual TOP1 and TDP1
inhibitor C12 showed good cytotoxicity against these four
human cancer cell lines with GI50 values ranging from 0.77 to
1.44 μM and enhanced activity in the drug-resistant MCF-7/
TDP1 cancer cells, possibly due to the dual TOP1 and TDP1
inhibition. C12 also showed synergy in combination with
topotecan in both MCF-7 and MCF-7/TDP1 cells. C12
induced both cellular TOP1cc and TDP1cc in ICE assays.
Additionally, C12 not only induced DNA damage but also
enhanced topotecan-induced DNA damage in both MCF-7
EXPERIMENTAL SECTION
■
General Experiments. All chemicals were purchased from local
commercial suppliers and used without further purification unless
otherwise specified. All the solvents were of an analytical reagent
grade. Nuclear magnetic resonance spectra were recorded on a Bruker
BioSpin GmbH spectrometer at 400 or 500 MHz using
tetramethylsilane (TMS) as the internal reference. Melting points
were determined in slides on an X-5 micro melting point apparatus
without being corrected. Mass spectra were recorded on a Shimadzu
LCMS-2020 instrument with an electrospray ionization (ESI) mass
selective detector, and HRMS were recorded on a Shimadzu LCMS-
IT-TOF mass spectrometer. The synthesized compounds were
purified through column chromatography with silica gel (200−300
mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. All
compounds tested for biological activities were analyzed by high-
performance liquid chromatography (HPLC), and their purities were
more than 95%. The analysis condition was detection at 220 nm, 1.0
mL/min flow rate, and a linear gradient from 55% to 15% phosphate-
buffered solution (PBS) buffer (pH 3) and 45%−85% methanol
(MeOH) in 30 min.
General Procedure for the Synthesis of Schiff’s Base Inter-
mediates (2). The Schiff’s base intermediates were prepared from the
reaction of 2-bromobenzaldehyde derivatives and N,N-dimethylami-
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noethylamine according to our reported method.25 The intermediates
were used for the next synthesis without further purification.
General Procedure for the Synthesis of Lactam Intermediates
(4). The intermediate 4 was prepared from the reaction of the Schiff’s
base 2 and the acetylene material 3 according to our reported
method.25
temperature, saturated saline (20 mL) was added to the reaction
solution. The resulting solution was extracted with ethyl acetate (3 ×
20 mL). The combined organic layer was washed with saturated saline
(3 × 20 mL), dried (MgSO4), and then concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy using dichloromethane/methanol (30:1) as eluent to give the
target products and the benzyl-protected compounds of C12, C13,
and C22.
Procedure for the Synthesis of Compounds C12, C13, and C22.
The benzyl-protected compounds of C12, C13, and C22 were
deprotected as follows: The corresponding residue was added to the
solution of concentrated hydrochloric acid (0.2 mL) and acetic acid
(2 mL) in a 25 mL round-bottomed flask. The reaction solution was
stirred at 65 °C overnight and then adjusted to pH 7 with a saturated
NaHCO3 aqueous solution. The resulting solution was extracted with
CH2Cl2 (3 × 20 mL). The combined organic layer was washed with
saturated saline (3 × 30 mL), dried (MgSO4), and then concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (20:1) as eluent to
give the target products.
General Procedure for the Synthesis of Compounds of Series A,
B, and C from Lactams 4 (Pathway I). The target products were
prepared from the intermediate 4 according to our reported
method.25 Briefly, the intermediate 4 (0.5 mmol) in a solution of
freshly distilled CH2Cl2 (8 mL) was oxidized by dimethyl sulfoxide
(DMSO) (365 μL, 5 mmol) and (COCl)2 (215 μL, 2.5 mmol) at
−60 °C for 30 min. And then Et3N (1.5 mL, 10 mmol) was added
dropwise to the reaction solution. The reaction solution was brought
to room temperature (rt) and stirred for 2 h. The reaction was
quenched by the addition of water (1 mL) at 0 °C. The organic layer
was concentrated under reduced pressure, and the resulting residue
was purified by silica gel column chromatography using dichloro-
methane (DCM)/methanol (40:1) as eluent. The obtained
intermediate was added to a solution of acetic acid (AcOH) (10
mL) and concentrated hydrochloric acid (1.0 mL) in a 50 mL round-
bottomed flask. The reaction solution was stirred at 65 °C overnight.
Then, the reaction solution was adjusted to pH 7 with a saturated
sodium bicarbonate solution and extracted with CH2Cl2 (3 × 20 mL).
The combined organic layer was washed with saturated saline (3 × 30
mL), dried (MgSO4), and then concentrated under reduced pressure.
The residue was purified by silica gel column chromatography using
chloroform/acetone (5:1) as eluent to give the target products.
Procedure for the Synthesis of Compound A4. Under a nitrogen
atmosphere, Na (280 mg, 12.2 mmol) was carefully added to absolute
methanol (5 mL). The reaction solution was stirred at room
temperature for 1 h. Then, the mixture of A3 (20 mg, 0.05 mmol) and
CuI (3.8 mg, 0.02 mmol) in dimethylformamide (DMF) (2 mL) was
added to the reaction solution. The resulting solution was stirred and
heated under reflux overnight. The reaction was quenched by the
addition of water (10 mL). The resulting solution was extracted with
CH2Cl2 (3 × 20 mL). The combined organic layer was washed with
saturated saline (3 × 30 mL), dried (MgSO4), and then concentrated
under reduced pressure. The residue was purified by silica gel column
chromatography using dichloromethane/methanol (40:1) as eluent to
give A4.
General Procedure for the Synthesis of Amide Intermediates (7).
The solution of SOCl2 (5 mL) and 2-bromobenzoic acid (1 mmol)
was stirred in a 25 mL round-bottomed flask at 65 °C for 2 h. The
excess SOCl2 was removed by reduced pressure. The solution of the
resulting residue in freshly distilled CH2Cl2 (10 mL) was added
dropwise to a solution of naphthylamine (0.8 mmol) and trimethyl-
amine (0.1 mL) in freshly distilled CH2Cl2 (10 mL). The reaction
solution was stirred at room temperature for 12 h, and then the
reaction was quenched by the addition of a saturated sodium
bicarbonate solution (1 mL). The resulting solution was washed with
saturated saline (3 × 20 mL). The organic layer was dried (MgSO4)
and concentrated under reduced pressure. The residue was purified by
silica gel column chromatography using ethyl acetate (EtOAc)/
hexane (1:1) as eluent to give the amide intermediates 7.
TOP1-Mediated DNA Cleavage Assay. A 3′-[32P]-labeled 117
base pair (bp) DNA oligonucleotide was prepared as previously
described.56 A radiolabeled DNA substrate (∼2 nM) was incubated
with recombinant TOP1 in 20 μL of reaction buffer (10 mM Tris-
HCl pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.1 mM ethyl-
enediaminetetraacetic acid (EDTA), and 15 mg/mL bovine serum
albumin (BSA)) at 25 °C for 20 min in the presence of various
concentrations of the tested compounds. The reactions were
terminated by adding sodium dodecyl sulfate (SDS) (0.5% final
concentration), followed by the addition of two volumes of loading
dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium
EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue). Aliquots of
each reaction mixture were subjected to 20% denaturing poly-
acrylamide gel electrophoresis (PAGE). Gels were dried and
visualized by using a phosphoimager and ImageQuant software
(Molecular Dynamics). Cleavage sites are numbered to reflect actual
sites on the 117 bp oligonucleotide.
TDP1 Inhibition Assay. The TDP1 fluorescence assay was
conducted according to the reported method.58 Briefly, a linear
oligonucleotide labeled with the donor fluorophore 6-carboxyfluor-
escein (FAM) and Black Hole Quencher (BHQ), 5′-FAM-
AGGATCTAAAAGACTT-BHQ-3′, was designed as a linear
quenched fluorescent substrate. TDP1 solution (20 μL/well of
purified TDP1 (100 nM) in 10 mM Tris-HCl, pH 7.5, 50 mM KCl, 1
mM EDTA, 1 mM dithiothreitol (DTT)) was dispensed into wells of
a white 384-well plate (NEST). The tested compound solution in
DMSO (5 μL) was pinned into assay plates and incubated at room
temperature for 30 min. During this time, the plates were read by a
Flash multimode reader (Molecular Devices) at Ex485/Em510 nm to
identify false-positive compounds that had autofluorescence. The
linear oligonucleotide substrate (25 μL, 35 nM) was dispensed into
the wells to start the reaction. The whole plate was immediately read
five times using a kinetic read on the Flash multimode reader
(Molecular Devices) (Ex485/Em510 nm). The TDP1 percentage
inhibition of the tested compounds was calculated by comparing
the rate of increase in fluorescence throughout the time for the
compound-treated wells to that of DMSO control wells.
General Procedure for the Synthesis of Compounds of Series A,
B, and C from Amide 7 (Pathway II). To the solution of intermediate
7 (0.5 mmol) in dry DMF (2 mL), NaH (60% dispersion in mineral
oil, 80 mg, 2 mmol) was added. The reaction solution was stirred at
room temperature for 1 h. Then, a solution of 2-dimethylaminoethyl
chloride in dry DMF (2 mL) was added dropwise. The reaction
solution was stirred at 85 °C overnight. The reaction was quenched
by an addition of icy water. The resulting solution was extracted with
ethyl acetate (3 × 20 mL). The combined organic layer was washed
with saturated saline (3 × 20 mL), dried (MgSO4), and then
concentrated under reduced pressure. Pd(OAc)2 (9 mg, 0.04 mmol),
P(o-tol)3 (24 mg, 0.08 mmol; o-tol = ortho-toluene), and Ag2CO3
(220 mg, 0.8 mmol) were added to a solution of the resulting residue
in dry DMF (10 mL). The reaction solution was stirred at 150 °C for
3 h under nitrogen atmosphere. After the mixture cooled to room
The inhibition of TDP1 was also conducted with gel-based assays
as described.60 Briefly, 1 nM of the DNA substrate (5′-Cy5-
GATCTAAAAGACTT-pY-3′) was incubated with 10 pM recombi-
nant TDP1 with a serial dilution of compound for 15 min at room
temperature in a TDP1 reaction buffer (50 mM Tris HCl, pH 7.5, 80
mM KCl, 2 mM EDTA, 1 mM DTT, 40 μg/mL BSA, and 0.01%
Tween-20). Reactions were stopped by adding an equal volume of
loading buffer (99.5% formamide, 5 mM EDTA). Samples were then
subjected to a 20% denaturing PAGE gel followed by gel scanning
using a Typhoon FLA 9500 scanner (GE Healthcare). The IC50 of
TDP1 inhibitors was calculated by comparing the percentage of
cleavage product (5′Cy5-GATCTAAAAGACTT-p-3′) to DMSO
control.
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Cell Culture and MTT Assay. The cancer cells were grown in
GIBCO RPMI 1640 medium or Dulbecco’s Modified Eagle’s Medium
(DMEM) complemented with 10% fetal bovine serum (FBS) at 37
°C with 5% CO2. For the drug treatment experiments, cells were
treated with compounds and collected for various times in different
experiments.
pocket. The compounds were constructed and optimized using
ChemDraw and saved in SDF file formats and were corrected and
minimized using MOE. The top 30 docking poses per ligand were
inspected visually following the docking runs. Energy minimizations
were performed for selected ligand poses. The AMBER force field was
utilized within the MOE for energy minimization.
For the cytotoxicity measurements by MTT assay, the cancer cells
were treated with the compounds (predissolved in DMSO) at a five-
dose assay ranging from 0.01 to 100 μM concentration as reported
previously.25 The percent inhibition of viability for each concentration
of the compounds was calculated relative to the control, and the GI50
values were estimated by nonlinear regression analysis (GraphPad
Prism).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00318.
■
sı
Molecular formula strings (CSV)
For the drug combination experiments, human breast cancer MCF-
7 cells and MCF-7/TDP1 cells were incubated with topotecan and
the tested compounds for 96 h at 37 °C, and then they were measured
by an MTT assay.
Characterization data for the intermediates and the
target products, the structures of benzo[c]-
phenanthridine alkaloid, and LMP744 (PDF)
Immunodetection of Cellular TOP1cc and TDP1cc. The ICE
assays for the cellular enzyme−DNA adduct were performed
according to the reported method.25,33 Briefly, mid log phase MCF-
7 or MCF-7/TDP1 cells were incubated with drugs at the indicated
concentration for 1 h, and then the cells were lysed with DNAzol
reagent (1 mL) at 25 °C for 30 min. Ethanol (0.5 mL, 100%) was
subsequently added and mixed with the lysate. The solution was
incubated at −20 °C overnight. The genomic DNA was collected by
centrifugation (12 000 rpm) at 25 °C for 10 min and washed with
75% ethanol. The precipitated DNA was dissolved in NaOH (8 mM,
0.2 mL). The pH value was adjusted to 7.2 by adding 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1 M). After
centrifugation, the supernatant was used to quantify the DNA
concentration. DNA (2 μg) was dissolved in 30 μL of a NaH2PO4
buffer (25 mM, pH 6.5) and then loaded onto nitrocellulose
membranes. Membranes were incubated with rabbit monoclonal to
human TOP1 (Abcam, 1:1000) or TDP1 (Abcam, 1:1000) at 4 °C
overnight, and then they were incubated with the appropriate
horseradish peroxidase (HRP) conjugated secondary antibodies (Cell
Signaling Technology, 1:3000) at room temperature for 1 h. Reactive
dots were detected using an Immobilon Western Chemiluminescent
HRP substrate (Millipore).
Immunofluorescence Assay. Immunofluorescence staining and
confocal microscopy were performed. After being incubated with the
tested compounds for 6 h at 37 °C, the cells (MCF-7 and MCF-7/
TDP1 cells) were fixed with 4% paraformaldehyde for 10 min, then
permeabilized with 0.5% Triton X-100/PBS at 37 °C for 30 min, and
finally blocked with 5% goat serum/PBS at 37 °C for 3 h. The
protocol was followed with the γH2AX antibody (No. 9718, Cell
Signaling Technology) at 4 °C overnight. The glass coverslips were
washed six times with blocking buffer and were then incubated with
antirabbit Alexa 488-conjugated antibody (A21206, Life Technology),
and 2 μg/mL of 4,6-diamidino-2-phenylindole (DAPI, Invitrogen)
was diluted in 5% goat serum/PBS at 37 °C for 2 h. The dishes were
again washed six times with blocking buffer, and then, digital images
were recorded using an FV3000 microscope (Olympus) and analyzed
with FV31S-SW software.
AUTHOR INFORMATION
Corresponding Authors
■
Lin-Kun An − School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, China; Guangdong
Provincial Key Laboratory of New Drug Design and
Evaluation, Guangzhou 510006, China; orcid.org/0000-
0002-6088-1503; Phone: +86-20-39943413;
Email: lssalk@mail.sysu.edu.cn; Fax: +86-20-39943413
Yves Pommier − Developmental Therapeutics Branch and
Laboratory of Molecular Pharmacology, Center for Cancer
Research, National Cancer Institute, National Institutes of
Health, Bethesda 20892, Maryland, United States;
orcid.org/0000-0002-3108-0758; Phone: +1 (240) 760-
6142; Email: pommier@nih.gov; Fax: +1 (240) 541-4475
Authors
De-Xuan Hu − School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, China
Wen-Lin Tang − School of Pharmaceutical Sciences, Sun Yat-
sen University, Guangzhou 510006, China
Yu Zhang − School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, China
Hao Yang − School of Pharmaceutical Sciences, Sun Yat-sen
University, Guangzhou 510006, China
Wenjie Wang − Developmental Therapeutics Branch and
Laboratory of Molecular Pharmacology, Center for Cancer
Research, National Cancer Institute, National Institutes of
Health, Bethesda 20892, Maryland, United States
Keli Agama − Developmental Therapeutics Branch and
Laboratory of Molecular Pharmacology, Center for Cancer
Research, National Cancer Institute, National Institutes of
Health, Bethesda 20892, Maryland, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00318
Apoptosis Detection. MCF-7 cells (2 × 105) were planted in a
culture dish and treated with C12 at the indicated concentration for
24 h. The apoptosis detection was applied using the fluorescein
isothiocyanate (FITC) Annexin V/PI apoptosis Detection Kit
(KeyGEN). The collected cells were resuspended in 500 μL of
binding buffer and added with 5 μL of FITC Annexin V and
propidium iodide (PI) dyes. The samples were slowly blended and
incubated at 25 °C for 15 min away from light. Finally, the
fluorescence-positive cells were quantified by flow cytometry (EPICS
XL).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We wish to thank Dr. H.-B. Chen, Sun Yat-Sen University
(Shenzhen), for the gift of MCF-7/TDP1 cells. This work was
supported by the Guangdong Basic and Applied Basic
Research Foundation (No. 2019A1515011317), Guangzhou
Basic and Applied Basic Research Project (No.
202002030312), and the Guangdong Provincial Key Labo-
ratory of Construction Foundation (No. 2017B030314030).
W.W., K.A., and Y.P. are supported by the Intramural Program
Molecular Modeling. The PDB file (PDB ID: 1K4T)57 for the X-
ray crystal structure of the TOP1-DNA-ligand complex was obtained,
cleaned, and inspected for errors and missing residues. Hydrogens
were added, and the water molecules and the ligand were deleted. The
ternary complex ligand centroid coordinates for docking were defined
using the ligand in the complex structure as the center of the binding
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Article
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ABBREVIATIONS USED
■
TOP1, topoisomerase IB; TOP1cc, TOP1−DNA cleavage
complex; TDP1, tyrosyl DNA−phosphodiesterase 1; TDP1cc,
TDP1-DNA covalent complex; TOP2, topoisomerase II; CPT,
camptothecin; TPT, topotecan; ICE, immunocomplex of
enzyme to DNA; NMR, nuclear magnetic resonance; HRMS,
high-resolution mass spectra; HPLC, high performance liquid
chromatography; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazolium bromide; γH2AX, phosphorylated histone
H2AX
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