Synthesis, cytotoxicity and structure-activity relationship of indolizinoquinolinedione derivatives as DNA topoisomerase IB catalytic inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.04.040

Our previous studies reveal that indolizinoquinolinedione scaffold is a base to develop novel DNA topoisomerase IB (TOP1) catalytic inhibitors. In this work, twenty-three novel indolizinoquinolinedione derivatives were synthesized. TOP1-mediated relaxation, nicking and unwinding assays revealed that three fluorinated derivatives 26, 28 and 29, and one N,N-trans derivative 46 act as TOP1 catalytic inhibitors with higher TOP1 inhibition (++++) than camptothecin (+++) and without TOP1-mediated unwinding effect. MTT assay against five human cancer cell lines indicated that the highest cytotoxicity is 20 for CCRF-CEM cells, 25 for A549 and DU-145 cells, 26 for HCT116 cells, and 33 for Huh7 cells with GI₅₀ values at nanomolar range. The drug-resistant cell assay indicated that compound 26 may mainly act to TOP1 in cells and are less of Pgp substrates. Flow cytometric analysis showed that compounds 26, 28 and 29 can obviously induce apoptosis of HCT116 cells. Moreover, the structure-activity relationship (SAR) of indolizinoquinolinedione derivatives was analyzed.

Full text of DOI:10.1016/j.ejmech.2018.04.040

European Journal of Medicinal Chemistry 152 (2018) 195e207
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Synthesis, cytotoxicity and structure-activity relationship of
indolizinoquinolinedione derivatives as DNA topoisomerase IB
catalytic inhibitors
1
1
Qian Yu , Hui Yang , Teng-Wei Zhu, Le-Mao Yu, Jian-Wen Chen, Lian-Quan Gu,
*
Zhi-Shu Huang, Lin-Kun An
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510006, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 March 2018
Received in revised form
Our previous studies reveal that indolizinoquinolinedione scaffold is a base to develop novel DNA
topoisomerase IB (TOP1) catalytic inhibitors. In this work, twenty-three novel indolizinoquinolinedione
derivatives were synthesized. TOP1-mediated relaxation, nicking and unwinding assays revealed that
three fluorinated derivatives 26, 28 and 29, and one N,N-trans derivative 46 act as TOP1 catalytic in-
hibitors with higher TOP1 inhibition (þþþþ) than camptothecin (þþþ) and without TOP1-mediated
unwinding effect. MTT assay against five human cancer cell lines indicated that the highest cytotox-
icity is 20 for CCRF-CEM cells, 25 for A549 and DU-145 cells, 26 for HCT116 cells, and 33 for Huh7 cells
with GI50 values at nanomolar range. The drug-resistant cell assay indicated that compound 26 may
mainly act to TOP1 in cells and are less of Pgp substrates. Flow cytometric analysis showed that com-
pounds 26, 28 and 29 can obviously induce apoptosis of HCT116 cells. Moreover, the structure-activity
relationship (SAR) of indolizinoquinolinedione derivatives was analyzed.
17 April 2018
Accepted 19 April 2018
Available online 22 April 2018
Keywords:
Indolizinoquinolinedione
Topoisomerase
Inhibitor
DNA damage
Cytotoxicity
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
camptothecin (CPT, Fig. 1) derivatives, of which the only known
target in cells is TOP1 [7,15]. To date, two camptothecin derivatives,
DNA topoisomerase IB (TOP1) is an essential enzyme that reg-
ulates DNA topology structure so that it may be replicated and
transcribed [1e4]. To perform its functions, TOP1 breaks one strand
of DNA by transesterification reactions using the active site tyrosine
as the nucleophile that attacks the DNA phosphodiester backbone,
topotecan (1) and irinotecan (2) have been approved by the FDA for
cancer treatment, and several derivatives are in clinical trials [7,9].
In addition to that there are several non-camptothecin TOP1 poi-
sons in clinical trials, including indolocarbazole 3, dibenzonaph-
thyridinone 4 and indenoisoquinolines 5 and 6 [16e18]. In spite of
their effectiveness in solid tumors, camptothecin poisons suffer
from many shortcomings, including bone marrow dose-limiting
toxicity, severe gastrointestinal toxicity [19], poor solubility,
chemical instability under physiological pH, and drug efflux-
mediated resistance [7].
0
and covalently attaches to the 3 -end of the broken DNA to form a
transient enzyme-DNA covalent complex (TOP1cc) [2,5]. TOP1 is a
validated molecular target for anticancer agents [6,7]. Inhibition of
TOP1 or trapping of TOP1cc can result in DNA damage, which
triggers cell death [8e10].
TOP1 inhibitors are classified as TOP1 “poisons” and “catalytic
inhibitors” based on their molecular mechanism of action. TOP1
poisons are able to trap TOP1cc to prevent further relegation of the
DNA single-strand breaks [6,7,9]. TOP1 catalytic inhibitors inhibit
the catalytic DNA cleavage reaction of enzyme [11e14], which is
different from “poisons”. The classical TOP1 poisons are
In previous publication, we reported a new class of TOP1 cata-
lytic inhibitors, the indolizinoquinolinedione derivatives (Fig. 1,
7e9) [11,13,20]. Derivative 8 can inhibit catalytic cleavage DNA
reaction of TOP1, which prevent the formation of TOP1cc [11].
Further structural modification indicated that the ester function-
alized derivatives at position 6 with alkylamino terminus exhibited
significantly increased TOP1 inhibition and cytotoxicity, and pro-
vided a TOP1 catalytic inhibitor 9 with good cytotoxicity and higher
TOP1 inhibition than CPT [13]. The previous structure-activity
relationship (SAR) evaluation also indicated that: 1) the existence
*
Corresponding author.
E-mail address: lssalk@mail.sysu.edu.cn (L.-K. An).
These authors contributed equally to this paper.
1
https://doi.org/10.1016/j.ejmech.2018.04.040
223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
0

196
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
Fig. 1. Chemical structures of the representative TOP1 poisons (A) and our reported TOP1 catalytic inhibitors (B).
of a nitrogen atom in the A-ring is important for the cytotoxicity; 2)
N,N-syn isomers have higher TOP1 inhibitory activity and cytotox-
icity than the corresponding N,N-trans isomers; 3) the derivatives
with electron-donating substituent at position 7 show poor cyto-
toxicity [20]. To investigate the effect of introduction of an electron-
withdrawing group at D-ring, and the position and number of ni-
trogen atom in the A-ring, three kinds of novel indolizinoquinoli-
nedione derivatives were designed and synthesized based on the
previous SAR. TOP1 inhibition and cytotoxicity were evaluated and
reported here.

Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
197
2
. Results and discussion
24e39.
To assess the effects of the position and number of nitrogen
atom in the A-ring on the biological activity, compounds 46e48
were synthesized as shown in Scheme 4. Similar to the preparation
of compounds 24e39, the products 46e48 were synthesized suc-
cessively through hydrolysis, acyl chlorination and esterification
reactions of materials 40e42 prepared in our laboratory [20,25,26].
The structures of all synthesized target compounds were char-
2
.1. Chemistry
According to the reported preparation [20,21], the indolizino-
quinolinedione derivatives, for example 14 and 15 could be syn-
thesized from the reaction of 6,7-dichloroquinoline-5,8-dione with
ethyl acetoacetate and 3-halopyridine with low isolation yield in
two steps (5% for 14 and 6% for 15 from 8-hydroxyl quinoline). In
addition, the second step (cyclization reaction) gave four
regioisomers [22], which were hard to isolate. To provide these
compounds through an easier and effective method, a novel syn-
thetic pathway was designed and explored. As shown in Scheme 1,
the dibromide 11 was obtained from the bromination of 8-hydroxyl
quinoline (10) [23,24]. It was then oxidized to give 7-
bromoquinoline-5,8-dione (12) [23,24]. The cyclization of bro-
mide 12 with ethyl acetoacetate and 3-halopyridine mainly gave
the N,N-syn isomers 13 and 14, respectively, with a higher overall
yield in three steps (14% for 13 and 17% for 14). The derivative 15
with a Boc-protected amino group at position 8 was also synthe-
sized according this pathway in 31% yield.
The 8-subsitituted indolizinoquinolinedione derivatives 18e21
were synthesized as depicted in Scheme 2. The de-protection of 15
gave the key intermediate 16, which was used for the next prepa-
ration immediately because it was structurally instable. The reac-
tion of intermediate 16 with excess of 3-bromine propionyl
chloride gave the bromide 17. After the amination of 17 with excess
amines, the target compounds 18e21 were obtained in 83%e99%
yield.
1
13
acterized from H and C NMR, melting point, and HRMS data.
2.2. TOP1 inhibitory activities
TOP1 inhibitory activity of the target compounds was assessed
by using TOP1-mediated relaxation assay with CPT as a positive
control (Supplementary Information, Figs. S1 and S2), and semi-
quantitatively expressed relative to CPT at 25
mM as follows: þþþþ,
more than 121% of the activity; þþþ, between 81% and 120% of the
activity; þþ, between 41% and 80% of the activity; þ, less than 40%
of the activity. The TOP1 inhibitory activity is summarized in
Table 1. Two 8-subsitituted derivatives 20 and 21 showed equi-
potent TOP1 inhibition to parent 7 (þþþ), while 18 and 19 had
decreased TOP1 inhibition of þþ, which indicated that the intro-
duction of a weak electron-withdrawing group at position 8 is
unable to improve the TOP1 inhibitory activity. Three fluorinated
derivatives 26, 28 and 29, one chlorinated derivative 36 and one
N,N-trans derivative 46 exhibited higher TOP1 inhibition than CPT
and the parent 7. One fluorinated derivative 25 and one phthalazine
derivative 48 exhibited equipotent inhibition (þþþ) to the parent
7
. The N,N-trans derivative 46 with nitrogen atom at position 4
showed equipotent TOP1 inhibition to the corresponding derivative
(þþþþ) with nitrogen atom at position 1 [13]. However, two
nitrogen atoms in the A-ring, such as in compounds 47 (þþ) and 48
The indolizinoquinolinedione derivatives with fluorine or
chlorine substituents at position 7 (24e39) were synthesized as
depicted in Scheme 3. The acids 22 and 23 were obtained by hy-
drolysis of compounds 13 and 14, respectively. Following acyl-
chlorination of acids 22 and 23, esterification gave the target esters
9
(
þþþ), and the introduction of a halogen atom at position 7 (30
of þ and 38 of þþ) reduced TOP1 inhibition.
Scheme 1. The novel synthetic pathway of indolizinoquinolinedione derivatives.
ꢀ
Reagents and conditions: (a) Br
2
, NaHCO
3
, Na
2
SO
3
, MeOH, rt. (b) concd. HNO
3
, concd. H
2
SO
4
, 0 C. (c) EtOH, pyridine derivatives, ethyl acetoacetate, reflux.

198
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
Scheme 2. Syntheses of compounds 18e21.
2 2 2 3 3 3
Reagents and conditions: (a) TFA, CH Cl , rt. (b) N , BrCH CH COCl, CHCl , Et N, rt. (c) amine materials (dimethylamine in Pressure Vessel), CHCl , K2CO3, KI, reflux.
2
2
Representative TOP1-mediated relaxation assay gels are shown
in Fig. 2A. They show that compounds 26, 28 and 29 inhibit TOP1
activity in a dose-dependent manner. At high concentration
TOP1-mediated unwinding assay with relaxed DNA as substrate
was conducted [27]. As shown in Fig. 2C (bottom), compounds 26,
28 and 29 indeed had no unwinding effect. The results for all
selected compounds are summarized in Table 1. Seven compounds
(
125 mM), these compounds showed stronger inhibitory activity
than CPT with almost 100% of supercoiled DNA remaining, under
condition where 58% supercoiled DNA remained for CPT. On the
contrary, CPT showed higher inhibitory activity at low tested con-
20, 21, 26, 28, 29, 46 and 48 have no unwinding effect up to 9 mM,
implying that they inhibit TOP1 not through the mechanism of
intercalating closed circular DNA [27,28], and are TOP1 catalytic
inhibitors similar to the compounds 8 and 9 [11,13]. Among them,
three fluorides 26, 28 and 29 exhibited high TOP1 inhibition
of þþþþ, equal to their corresponding derivatives without fluorine
atom, which showed obvious unwinding effect [13]. These results
implied that fluorine atom at position 7 is worthy to be introduced
for TOP1 inhibition. On the contrary, two selected compounds 25
centration (0.2 and 1
To explore the trapping TOP1cc ability of the synthesized
compounds, compounds 26, 28 and 29 at 25 and 50 M concen-
mM).
m
trations were tested in a TOP1-mediated nicking assay using excess
TOP1. As shown in Fig. 2B, the TOP1 poison CPT could trap TOP1cc
and increase the ratio of nicked DNA from 47% up to 49% at 50 mM
concentration. On contrary, compounds 26, 28 and 29 significantly
decreased the ratio of nicked DNA between 33% and 42%, imply
they do not have ability to trap TOP1cc and act upstream of catalytic
cleavage activity of TOP1 [11].
and 36 showed obvious unwinding effects at 9 mM.
2.4. Cytotoxicities
The cytotoxicity of the synthesized compounds was evaluated
using MTT assay against five human tumor cell lines, including
colon cancer (HCT116), leukemia (CCRF-CEM), non-small cell lung
cancer (A549), hepatocarcinoma (Huh7) and prostate cancer (DU-
145) cell lines. The compounds were incubated with cells for 72 h in
a five-dose assay ranging from 10 to 10 M concentration. At the
end of the incubation, MTT solution was added to test the per-
centage growth of tumor cells. The GI50 values, defined as the
concentration of the compound that resulted in 50% cell growth
inhibition, were plotted and summarized in Table 1.
2
.3. TOP1-mediated unwinding effect
To assess whether the synthesized compounds induce DNA
unwinding effect in the presence of excess TOP1, nine compounds
20, 21, 25, 26, 28, 29, 36, 46 and 48) with high TOP1 inhibition
ꢁ
8
ꢁ4
(
of þþþ and þþþþ were selected to be tested against TOP1-
mediated unwinding assay using the DNA intercalator ethidium
bromide (EB) as a positive control [27,28]. As shown in Fig. 2C
(
upper), EB exhibited clear unwinding effect with supercoiled
pBR322 DNA as substrate. The representative compounds 26, 28
and 29 had no unwinding effect. In order to confirm these results,
The compounds 18e21 with alkyl side chain at position 8
showed high cytotoxicity with GI50 values at micromolar

Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
199
Scheme 3. Syntheses of compounds 24e39.
2 3 3 3
Reagents and conditions: (a) 15% K CO , i-propanol, reflux, 24 h (b) i) SOCl , Et N, CHCl , reflux, 5 h; ii) alcohol materials, DMAP, CHCl , rt.
2
3
Scheme 4. Syntheses of compounds 46e48.
2 3 3 3
Reagents and conditions: (a) 15% K CO , i-propanol, reflux. (b) i) SOCl , Et N, CHCl , reflux, 5 h; ii) 2-morpholinoethanol, DMAP, CHCl , rt, 5 h.
2
3
concentration. And compound 20 showed the highest cytotoxicity
against CCRF-CEM (GI50 ¼ 0.11 M) with the equal TOP1 inhibition
þþþ) to parent 7.
Most of fluorine substituted derivatives 24e33 displayed potent
cytotoxicity against five human tumor cells with GI50 values at
submicromolar or nanomolar levels. Compound 25 showed the
corresponding fluoride derivative 28 (þþþþ), it had decreased
cytotoxicity for these five tumor cell lines, implying the presence of
fluorine atom at position 7 is preferred. Comparing to the corre-
sponding derivatives without halogen atom at position 7 [13], the
fluorides 27e29 with pyrrolidinyl and piperidinyl terminus of side
chain showed increased cytotoxicity against A549 cells, and the
fluorides 30 and 31 with morpholinyl terminus and the chlorides
35e39 showed decreased cytotoxicity.
Although 46 (þþþþ) had higher and 48 (þþþ) had equipotent
TOP1 inhibition with parent 7, they showed decreased cytotoxicity
against these five human cancer cell lines, implying that the ni-
trogen atom at position 1 is important for cytotoxicity.
m
(
highest cytotoxicity against A549 (GI50 ¼ 0.018
GI50 ¼ 0.025 M) with equal TOP1 inhibition (þþþ) to parent 7.
Compound 26 was the most potent against HCT116 cells with GI50
of 0.023
M and TOP1 inhibition of þþþþ. Although compound 33
had lower TOP1 inhibition (þþ) than parent 7, it showed the
highest cytotoxicity against Huh7 cells (GI50 ¼ 0.042 M). Although
the chloride derivative 36 had equipotent TOP1 inhibition to its
mM) and DU-145
(
m
m
m

200
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
Table 1
TOP1 inhibitory activity, unwinding effect and cytotoxicity of the synthesized compounds.
Cpd.
Relaxation assaya
Unwinding effect
Cytotoxicity [GI50 ± SD (m
M)]b
HCT116
CCRF-CEM
A549
Huh7
DU-145
7
þþþ
þþ
No
e
0.33 ± 0.009
1.75 ± 0.18
1.90 ± 0.16
1.43 ± 0.04
0.30 ± .038
0.68 ± 0.16
0.29 ± 0.016
0.54 ± 0.054
0.11 ± 0.010
0.60 ± 0 .073
1.09 ± 0.18
0.37 ± 0.013
0.79 ± 0.064
0.51 ± 0.042
0.38 ± 0.10
0.35 ± 0.021
1.10 ± 0.23
0.49 ± 0.078
0.45 ± 0.038
0.32 ± 0.021
48.23 ± 1.22
85.26 ± 8.31
0.39 ± 0.013
5.35 ± 0.23
61.26 ± 5.09
68.56 ± 3.32
70.52 ± 6.62
1.61 ± 0.22
1.35 ± 0.17
0.002 ± 0.001
0.16 ± 0.089
19.64 ± 5.36
29.26 ± 3.32
2.65 ± 0.26
0.85 ± 0.036
1.65 ± 0.24
1.80 ± 0.24
1.15 ± 0.22
0.34 ± 0.07
0.31 ± 0.25
0.080 ± 0.020
0.12 ± 0.019
0.11 ± 0.045
0.059 ± 0.050
0.12 ± 0.016
0.57 ± 0.019
0.13 ± 0.072
0.11 ± 0.003
0.042 ± 0.012
2.25 ± 0.52
12.16 ± 0.95
0.17 ± 0.10
1.32 ± 0.16
30.26 ± 0.15
28.45 ± 0.58
14.15 ± 1.62
7.33 ± 1.10
2.92 ± 0.34
0.006 ± 0.001
0.093 ± 0.001
2.27 ± 0.21
0.70 ± 0.030
3.12 ± 0.34
16.43 ± 3.71
0.69 ± 0.12
0.025 ± 0.008
0.082 ± 0.025
0.092 ± 0.001
0.091 ± 0.002
0.21 ± 0.005
0.23 ± 0.026
0.25 ± 0.021
0.029 ± 0.006
0.073 ± 0.003
1.58 ± 0.23
c
18
19
20
21
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
46
47
48
þþ
e
þþþ
þþþ
þþ
No
No
e
9.68 ± 4.48
0.092 ± 0.041
0.066 ± 0.050
0.023 ± 0.015
0.029 ± 0.011
0.025 ± 0.013
0.027 ± 0.021
0.11 ± 0.013
0.038 ± 0.020
0.044 ± 0.030
0.044 ± 0.050
10.25 ± 1.11
38.25 ± 0.88
0.31 ± 0.039
5.15 ± 0.50
28.56 ± 5.91
19.12 ± 0.56
11.12 ± 0.38
13.15 ± 1.52
3.43 ± 0.022
0.009 ± 0.001
27.25 ± 0.23
0.018 ± 0.002
0.060 ± 0.15
0.080 ± 0.033
0.29 ± 0.004
0.072 ± 0.050
0.13 ± 0.017
0.23 ± 0.074
0.052 ± 0.012
0.021 ± 0.034
5.39 ± 0.30
24.46 ± 1.09
0.12 ± 0.002
0.89 ± 0.022
48.59 ± 0.17
37.49 ± 0.38
4.52 ± 2.42
þþþ
þþþþ
þþ
Yes
No
e
þþþþ
þþþþ
þ
No
No
e
þ
e
þþ
e
þþ
e
þ
e
þ
e
8.32 ± 0.43
2.52 ± 0.23
þþþþ
þþ
Yes
e
1.73 ± 0.081
29.49 ± 0.14
29.12 ± 0.26
1.20 ± 0.052
2.55 ± 0.21
þþ
e
þ
e
þþþþ
þþ
No
e
8.52 ± 0.15
0.86 ± 0.45
0.003 ± 0.001
þþþ
þþþ
No
e
1.22 ± 0.12
0.019 ± 0.009
CPT
a
TOP1 inhibitory activity was semiquantitatively expressed relative to CPT at 25
m
M as follows: þþþþ, more than 121% of the activity; þþþ, between 81% and 120% of the
activity; þþ, between 41% and 80% of the activity; þ, less than 40% of the activity. Every experiment was repeated at least twice independently.
b
GI50 values (means ± SD) were defined as the concentrations of compounds that resulted in 50% cell growth inhibition, and obtained from MTT assay. Every experiment
was repeated at least three times.
c
“
-” mean “ not determined”.
2.5. Cytotoxicity of compound 26 in drug-resistant cell lines
As shown in Fig. 3, after being treated with the tested compounds
1, 3, and 9 M) for 12 h, the apoptotic cells increased obviously in a
(
m
To evaluate the cytotoxicity in drug-resistant cell lines, 26 was
dose-dependent manner. Compounds 26, 28 and 29 induced the
selected and tested by using MTT assay. The results were summa-
rized in Table 2. HCT116-siTop1 subline was developed by trans-
fection of colon cancer parental cells HCT116 with short hairpin
RNA vectors expressing siRNA for TOP1 [29]. Comparing to the
parental cell line HCT116, HCT116-siTop1 subline showed 8.3-fold
resistant to CPT, of which TOP1 is the only known cellular target
major population of HCT116 cells into the late apoptotic stage
(32.10%, 38.17% and 31.59%) at 9
. Conclusion
In summary, a series of indolizinoquinolinedione derivative
mM concentration.
3
[
7,15], and about 6.5-fold to indolizinoquinolinedione 26, implying
were synthesized. TOP1-mediated assays revealed that the syn-
thesized compounds act as TOP1 catalytic inhibitors and four
compounds 26, 28, 29, and 46 exhibit higher TOP1 inhibitory ac-
tivity (þþþþ) than the parent 7 without TOP1-mediated un-
that TOP1 may be the major cellular target of 26.
The prostate cancer cell RC0.1 has a R364H mutation in TOP1
relating to the wild-type parental cell DU-145 [30]. The TOP1 with
R364H mutation is catalytically active, but lead to RC0.1 cells
resistance to CPT because the R364 residue is close to the catalytic
tyrosine and can stabilize the open form of TOP1cc [31,32]. The
RC0.1 cells were highly resistant to TOP1 poison CPT (396.3-fold)
and less resistant to compound 26 (36.8-fold), implying that the
binding site of 26 on TOP1 is different from that of CPT.
P-glycoprotein (Pgp) mediated drug efflux is generally respon-
sible for classical multiple drug resistance [33]. The chemothera-
peutic agents doxorubicin (DOX) is substrate of Pgp, and has been
found highly resistant for breast cancer MCF-7/ADR (77.8-fold) and
hepatocellular HepG2/ADR sublines (47.6-fold), both with overex-
pressed Pgp [34]. However, compound 26 appeared to be less of
Pgp substrates (Table 2).
winding effect up to 9 mM. The 8-subsitituted derivative 20 shows
the highest cytotoxicity against CCRF-CEM cells with TOP1 inhibi-
tion of þþþ. The 7-fluorine substituted derivative 26 is the highest
cytotoxicity against HCT116 cells with high TOP1 inhibition
of þþþþ. Compound 33 shows the highest cytotoxicity against
Huh7 cells with TOP1 inhibition of þþ. Compound 25 shows the
highest cytotoxicity against A549 and DU-145 with TOP1 inhibition
of þþþ. The drug-resistant cell assays indicated that 26 may mainly
act to TOP1 in tumor cells and are less of Pgp substrates. Com-
pounds 26, 28 and 29 can effectively induce the apoptosis of
HCT116 cells in a dose-dependent manner. This study indicates that
the modification of indolizinoquinolinedione could provide novel
TOP1 catalytic inhibitors with high cytotoxicity.
2.6. Apoptosis analysis of compounds 26, 28 and 29
4. Methods and materials
The effect of compounds 26, 28 and 29 on apoptosis was esti-
4.1. General experiments
mated because they are TOP1 catalytic inhibitors with high TOP1
inhibition and cytotoxicity. Flow cytometry analysis using double
staining with annexin VeFITC/PI was carried out in HCT116 cell line.
All starting materials and reagents for synthesis were
commercially available and purchased from Sigma Aldrich Co,

Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
201
Fig. 2. (A) TOP1-mediated relaxation assay. Lane 1, supercoiled pBR322 DNA alone; Lane 2, DNA and TOP1; Lanes 3e7, DNA, TOP1 and tested compound at 0.2, 1, 5, 25, 125 mM,
respectively. (B) TOP1-mediated nicking assay. Lane 1, supercoiled pBR322 DNA alone; Lane 2, DNA and excess TOP1; Lanes 3e10, DNA, excess TOP1 and tested compound at 25,
50
m
M, respectively. (C) TOP1-mediated unwinding assay using supercoiled pBR322 DNA (upper) or relaxed pBR322 DNA (bottom) as substrate, respectively. Lane 1, DNA alone; Lane
2
, DNA and excess of TOP1; Lanes 3e5, DNA, excess TOP1 and EB at 0.3, 0.6 and 1.2 mg/L; Lanes 6e14, DNA and excess TOP1 and tested compounds at 1, 3 and 9 M, respectively. R,
m
relaxed DNA; Sc, supercoiled DNA; N, nicked DNA.

202
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
Table 2
Cytotoxicity of the compound 26 in drug-resistant human cancer cell lines.
Factory, Qingdao, China) was used for column chromatography. All
compounds tested for biological activities were measured using
HPLC and their purities were more than 95%.
Cpd.
GI50 ± SD (
m
M)a
Resistance Ratiob
Plasmid pBR322 DNA and calf thymus DNA TOP1 were pur-
Parental cell line
Resistant subline
chased from Takara Biotechnology (Dalian). One unit of TOP1 was
HCT116
HCT116-siTop1
0.15 ± 0.029
0.075 ± 0.014
RC0.1
2.94 ± 0.74
7.53 ± 1.88
MCF-7/ADR
0.097 ± 0.003
11.67 ± 1.94
HepG2/ADR
0.38 ± 0.16
9.04 ± 0.14
ꢀ
defined as the amount that relaxes 0.5
m
g of pBR322 DNA at 37 C
26
0.023 ± 0.005
0.009 ± 0.001
DU-145
0.082 ± 0.025
0.019 ± 0.009
MCF-7
0.089 ± 0.15
0.15 ± 0.003
HepG2
0.051 ± 0.027
0.19 ± 0.048
6.5
8.3
for 30 min. The human wild-type cancer cell lines A549 and Huh7
were obtained from Laboratory Animal Center, Sun Yat-sen Uni-
versity. The human wild-type cancer cell lines HCT116, CCRF-CEM
and DU-145, and the resistant cell lines HCT116-siTop1 and RC0.1
were a kind gift from Dr. Y. Pommier (Laboratory of Molecular
Pharmacology, Center for Cancer Research, NCI, NIH). The human
wild-type cancer cell lines MCF-7 and HepG2, and the resistant cell
lines MCF-7/ADR and HepG2/ADR were a kind gift from Dr. X. Z. Bu
CPT
26
36.8
396.3
CPT
26
1.1
77.8
DOX
26
7.5
47.6
DOX
(
School of Pharmaceutical Sciences, Sun Yat-sen University).
a
GI50 values (means ± SD) were defined as the concentrations of compounds that
resulted in 50% cell growth inhibition and obtained from MTT assay. Every experi-
ment was repeated at least three times.
4.2. Synthesis of compound 11
b
Resistance ratio was calculated by dividing the GI50 of the mutant cell line by the
GI50 of the corresponding parental cell line.
Compound 11 was prepared according to the reported method
[
24]. The solution of bromine (3.1 mL, 60 mmol) in MeOH (30 mL)
was added dropwise into the mixture 8-hydroxyquinoline (2.9 g,
0 mmol) and NaHCO (3.36 g, 40 mmol) in MeOH (30 mL). After
stirring for 5 min at room temperature, Na SO (2.5 g, 20 mmol)
Aladdin Reagent Database Inc, and Alfa Aesar. High performance
liquid chromatography (HPLC) was taken on a Shimadzu LC-10 A B.
Mass spectra were analyzed on an Agilent 6120. HRMS were
measured using an SHIMADZU LCMS-IT-TOF mass spectrometer.
NMR spectra were performed on Bruker Avance III-400 spectrom-
eter with TMS as an internal standard. Unless otherwise specified,
2
3
2
3
and water (100 mL) were added and then the mixture was filtered
and washed with water and dried in vacuo to give a white solid 11
1
(
1
1
5.88 g, 97%). H NMR (400 MHz, CDCl
3
)
d
8.82 (dd, J ¼ 4.2, 1.4 Hz,
H), 8.46 (dd, J ¼ 8.5,1.5 Hz,1H), 7.91 (s, 1H), 7.59 (dd, J ¼ 8.5, 4.3 Hz,
þ
chemical shifts (d) were expressed in ppm with reference to the
H). APCI-MS m/z: 301.9 (50%), 303.9 (100%), 305.9 (50%) [MþH] .
.3. Synthesis of compound 12
signals, and coupling constant (J) values were reported in Hz.
Melting points were recorded on open capillary tubes on a MPA100
Optimelt Automated Melting Point System without being cor-
rected. Silica gel (200e300 mesh, Qingdao Marine Chemical
4
The synthesis of compound 12 was carried out as the reported
Fig. 3. (A) Flow cytometry histograms and (B) the quantification of the apoptosis of HCT116 cells treated with compounds 26, 28 and 29 at the concentration of 1, 3, and 9
respectively for 12 h (n ¼ 3).
mM,

Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
203
method [23,24]. Concentrated HNO
to a solution of compound 11 (270 mg, 0.85 mmol) in concentrated
SO (1 mL) in an ice bath. The reaction solution was stirred for
0 min and added with ice water (10 mL), and extracted with
3
(0.15 mL) was added dropwise
solvent was evaporated under reduced pressure. The resulting
residue was purified by using silica gel column chromatography to
give the target product.
H
3
2
4
dichloromethane (5 mL x 3). The organic layer was concentrated
under reduced pressure purified by silica gel column chromatog-
4
.6.1. Ethyl-8-(3-(dimethylamino)propanamido)-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (18)
raphy (dichloromethane/ethyl acetate: 30/1) to give a yellow solid
1
Using the mixture of dichloromethane and methanol (10:1) as
(
12, 136 mg), yield 67%. H NMR (400 MHz, CDCl3)
d
9.08 (dd,
ꢀ
1
eluent to give orange powder, yield 88%, mp ¼ 250.9e251.5 C. H
J ¼ 4.7, 1.7 Hz, 1H), 8.44 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.74 (dd, J ¼ 7.9,
NMR (400 MHz, CD OD)
3
d
11.74 (s, 1H), 9.76 (d, J ¼ 7.5 Hz, 1H), 8.92
4
.7 Hz, 1H), 7.62 (s, 1H). APCI-MS m/z: 237.9 (100%), 239.9 (100%)
þ
(d, J ¼ 4.6, 1.6 Hz, 1H), 8.49 (s, 1H), 8.46 (d, J ¼ 7.9 Hz, 1H), 7.54 (dd,
J ¼ 7.9, 4.7 Hz, 1H), 7.37 (d, J ¼ 7.5 Hz, 1H), 4.44 (q, J ¼ 7.1 Hz, 2H),
[MþH] .
2
.64 (t, J ¼ 6.0 Hz, 2H), 2.50 (t, J ¼ 6.0 Hz, 2H), 2.37 (s, 6H), 1.46 (t,
4.4. General procedure for synthesis of compounds 13e15
13
J ¼ 7.5 Hz, 3H). C NMR (100 MHz, CD
3
OD) d 178.8, 171.7, 171.3,
162.4, 153.7, 149.1, 140.4, 139.5, 134.5, 130.3, 128.8, 127.9, 127.8,
To a solution of 12 (1.0 g, 4.2 mmol) in EtOH (50 mL) was added
122.5, 112.1, 105.1, 103.5, 60.2, 54.7, 44.4, 34.5, 14.1. HRMS (ESI) m/z:
dropwise into ethyl acetoacetate (1.64 g, 12.6 mmol). The reaction
mixture was stirred at room temperature for 5 min. Pyridine de-
rivatives (50 mmol) was added dropwise, and the reaction mixture
was stirred at reflux for 16 h and extracted with dichloromethane
þ
4
35.1653 [MþH] , calcd for C23
23 4 5
H N O 435.1668.
4
.6.2. Ethyl 5,12-dioxo-8-(3-(pyrrolidin-1-yl)propanamido)-5,12-
(
50 mL x 3). The organic layer was concentrated under reduced
dihydroindolizino[2,3-g]quinoline-6-carboxylate (19)
pressure purified by silica gel column chromatography (petroleum
ether/ethyl acetate: 1/2) to give the target product.
Using the mixture of dichloromethane and methanol (10:1) as
ꢀ
1
eluent to give orange powder, yield 85%, mp ¼ 265.5e266.8 C. H
Using 3-fluoropyridine or 3-chloropyridine as material, an or-
ange red solid (22, 314 mg, 22%) or a red solid (23, 388 mg, 26%) was
obtained, respectively. Their structures were characterized through
NMR (400 MHz, CD OD)
3
d
10.95 (s, 1H), 9.69 (d, J ¼ 7.3 Hz, 1H), 9.00
(
s, 1H), 8.80 (s, 1H), 8.43 (d, J ¼ 8.0 Hz, 1H), 7.81 (dd, J ¼ 7.5, 4.7 Hz,
1
2
4
H), 7.48 (d, J ¼ 7.5 Hz, 1H), 4.33 (q, J ¼ 7.0 Hz, 2H), 3.47 (t, J ¼ 7.0 Hz,
1
13
H and C NMR spectra, similar to our reported data [20].
H), 3.10 (t, J ¼ 7.0 Hz, 2H), 2.95 (t, J ¼ 7.0 Hz, 4H), 1.41 (t, J ¼ 7.0 Hz,
13
3
H), 1.23 (s, 3H). C NMR (100 MHz, CD OD) d 179.6, 172.4, 169.6,
4.4.1Ethyl 8-((tert-butoxycarbonyl)amino)-5,12-dioxo-5,12-
163.0, 154.2, 149.5, 140.8, 139.7, 135.2, 130.9, 128.5, 127.8, 127.3,
dihydroindolizino[2,3-g]quinoline-6-carboxylate (15)
123.1, 112.6, 105.8, 104.0, 60.8, 53.8, 53.8, 49.7, 32.8, 22.8, 22.8, 14.3.
Using the N-Boc protected 4-animopyridine as material, a red
þ
HRMS (ESI) m/z: 461.1823 [MþH] , calcd for C25
25 4 5
H N O 461.1825.
1
solid 15 (0.57 g) was obtained in 31% yield. H NMR (400 MHz,
CDCl
3
)
d
9.76 (d, J ¼ 7.2 Hz, 1H), 8.93 (dd, J ¼ 4.8, 1.6 Hz, 1H), 8.46
4
.6.3. Ethyl 5,12-dioxo-8-(3-(piperidin-1-yl)propanamido)-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (20)
Using the mixture of dichloromethane and methanol (10:1) as
(
dd, J ¼ 7.6, 1.6 Hz, 1H), 8.39 (d, J ¼ 1.6 Hz, 1H), 7.55 (dd, J ¼ 7.8,
4
2
d
.6 Hz, 1H), 7.29 (d, J ¼ 7.6 Hz, 1H), 6.83 (s, 1H), 4.44 (q, J ¼ 7.1 Hz,
13
H), 1.49 (s, 9H), 1.44 (t, J ¼ 7.1 Hz, 3H). C NMR (100 MHz, CDCl
179.2, 163.2, 153.9, 151.7, 149.6, 149.1, 141.6, 139.8, 135.3, 130.9,
29.4, 126.7, 122.6, 111.4, 105.4, 104.8, 100.0, 82.2, 61.1, 28.2, 14.3.
3
)
ꢀ
1
eluent to give purple powder, yield 99%, mp ¼ 259.3e261.2 C. H
NMR (400 MHz, CD OD)
12.08 (s, 1H), 9.82 (d, J ¼ 7.2 Hz, 1H), 8.99
d, J ¼ 3.7 Hz, 1H), 8.70 (s, 1H), 8.53 (d, J ¼ 7.5 Hz, 1H), 7.74e7.56 (m,
1H), 7.40 (d, J ¼ 6.6 Hz, 1H), 4.50 (dd, J ¼ 13.8, 6.8 Hz, 2H), 2.72e2.80
m, 6H), 1.77e1.84 (m, 4H), 1.60e1.68 (m, 2H). 1.51 (t, J ¼ 7.0 Hz, 2H),
3
d
1
(
The structure of 15 was also confirmed through 2D NMR spectrum.
ESI-MS m/z: 436.1 [MþH] .
þ
(
13
1
.26 (t, J ¼ 7.1 Hz, 3H). C NMR (100 MHz, CD
3
OD) d 179.3, 172.3,
4.5. Synthesis of compound 17
170.5, 163.0, 154.2, 149.7, 140.9, 139.9, 135.1, 130.9, 129.5, 128.4,
1
27.7, 123.0,112.6, 105.7, 104.0, 60.7, 53.4, 53.4, 52.6, 34.5, 24.2, 24.2,
To the solution of compound 15 (435 mg, 1 mmol) in dichloro-
þ
2
2.8, 14.1. HRMS (ESI) m/z: 475.1967 [MþH] , calcd for C26
27 4 5
H N O
methane (20 mL), trifluoro acetic acid (4 mL) was added dropwise
at room temperature. The reaction solution was stirred for 2 h and
concentrated under reduced pressure. The red residue gel (crude
compound 16) was dissolved in dried chloroform (20 mL). Under
475.1981.
4
.6.4. Ethyl 8-(3-morpholinopropanamido)-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (21)
Using the mixture of dichloromethane and methanol (10:1) as
nitrogen, the solution was stirred and successively added Et
3
N
(
1.4 mL) and a solution of pre-prepared 3-bromo propionyl chloride
in chloroform (15 mL). The reaction solution was stirred for 8 h at
room temperature and concentrated under reduced pressure. The
residue was purified by silica gel column chromatography
ꢀ
1
eluent to give orange powder, yield 83%, mp ¼ 267.9e269.7 C. H
NMR (400 MHz, CD OD)
11.60 (s, 1H), 9.84 (d, J ¼ 7.5 Hz, 1H), 8.99
d, J ¼ 3.0 Hz, 1H), 8.62 (s, 1H), 8.49 (d, J ¼ 7.5 Hz, 1H), 7.62 (dd,
3
d
(
(
(
dichloromethane/methanol: 50/1) to give an orange solid 17
J ¼ 7.5, 4.7 Hz, 1H), 7.45 (d, J ¼ 6.0 Hz, 1H), 4.51 (q, J ¼ 7.0 Hz, 2H),
240 mg, 51%), which was used for the next preparation immedi-
3
2
.89 (s, 4H), 2.80 (d, J ¼ 5.5 Hz, 2H), 2.70 (s, 4H), 2.63 (d, J ¼ 5.7 Hz,
13
þ
ately. ESI-MS m/z: 470.1 (100%), 470.1 (100%) [MþH] .
H), 1.52 (t, J ¼ 7.1 Hz, 3H). C NMR (100 MHz, CD
3
OD) d 178.8,
171.8, 171.5, 162.4, 153.8, 149.0, 140.4, 139.4, 134.4, 130.2, 128.8,
4
.6. General procedure for the synthesis of compounds 18e21
127.9, 127.1, 122.4, 112.1, 105.0, 103.3, 66.1, 60.1, 53.7, 52.9, 34.1, 14.1.
þ
HRMS (ESI) m/z: 477.1756 [MþH] , calcd for C25
25 4 6
H N O 477.1774.
The suspension of compound 17 (235 mg, 0.5 mmol), K
2
CO
3
(
134 mg, 1 mmol), KI (83 mg, 0.5 mmol), and amines (in Pressure
Vessel for dimethylamine) in chloroform (50 mL) was stirred and
heated under reflux overnight. The solvent was evaporated under
reduced pressure. The residue was dissolved in TCM (50 mL). The
organic layer was washed with water (10 mL x 2) and saturated
4.7. Hydrolysis of compounds 13 and 14
The ester analogues 13 or 14 (1 mmol) were hydrolyzed with
CO (15%) in isopropanol solution (100 mL) to give the acid an-
alogues 22 or 23, respectively.
K
2
3
4
aqueous saline (10 mL), and dried with anhydrous MgSO . The

204
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
4
6
.7.1. 7-Fluoro-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-
2.11e2.02 (m, 2H), 1.84e1.77 (m, 4H). 13C NMR (100 MHz, CDCl
179.2, 172.7, 163.2, 154.4, 154.4, 149.9, 135.2, 130.4, 128.8, 127.0,
3
)
-carboxylic acid (22)
d
Amaranth solid, yield 53%, mp ¼ 281e282 C. 1H NMR
ꢀ
126.5, 124.7, 124.6, 122.5, 117.1, 110.5, 64.9, 54.1, 52.9, 28.1, 23.4.
þ
(
400 MHz, DMSO)
d
13.50 (s, 1H), 9.48 (d, J ¼ 6.8 Hz, 1H), 9.03 (d,
HRMS (ESI) m/z: 422.1502 [MþH] , calcd for C23
3 4
H21FN O 422.1511.
J ¼ 4.4 Hz, 1H), 8.46 (d, J ¼ 7.8 Hz, 1H), 7.83 (dd, J ¼ 7.6, 4.8 Hz, 1H),
þ
7.47e7.33 (m, 2H). ESI-MS m/z: 311.0 [MþH] .
4.8.5. 2-(Piperidin-1-yl)ethyl-7-fluoro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g] quinoline-6-carboxylate (28)
4
6
.7.2. 7-Chloro-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quinoline-
-carboxylic acid (23)
Using the mixture of dichloromethane and methanol (12:1) as
ꢀ
1
eluent to give red powder, yield 29%, mp ¼ 162.8e163.5 C. H NMR
Amaranth solid, yield 30%, mp ¼ 289.5e291.2 C. 1H NMR
ꢀ
(400 MHz, CDCl
)
d
9.64 (d, J ¼ 6.7 Hz, 1H), 9.05 (dd, J ¼ 4.7, 1.7 Hz,
3
(
(
400 MHz, DMSO)
d, J ¼ 8.4 Hz, 1H), 8.44 (d, J ¼ 8.4 Hz, 1H), 7.87 (s, br, 1H), 7.74 (d,
d
11.97 (s,1H), 9.81 (s, br,1H), 9.07 (s, br,1H), 8.53
1H), 8.53 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.66 (dd, J ¼ 7.9, 4.7 Hz, 1H),
7.17e7.07 (m, 2H), 4.63 (t, J ¼ 6.1 Hz, 2H), 2.85 (t, J ¼ 6.0 Hz, 2H),
þ
13
J ¼ 12.0 Hz, 1H). ESI-MS m/z: 327.1 (100%), 329.1 (33%) [MþH] .
2.60e2.49 (m, 4H), 1.65e1.59 (m, 4H), 1.48e1.45 (m, 2H). C NMR
(
3
100 MHz, CDCl ) d 179.3, 172.8, 163.3, 154.5, 153.1, 149.9, 135.0,
4
4
.8. General procedure for the synthesis of compounds 24e39 and
6e48
130.4, 127.3, 124.8, 122.4, 117.3, 117.2, 110.8, 110.6, 107.2, 63.3, 56.9,
þ
54.6, 25.6, 24.0. HRMS (ESI) m/z: 422.1511 [MþH] , calcd for
23 3 4
C H21FN O 422.1511.
The target compounds 24e39 and 46e48 were prepared ac-
cording to our reported method [13]. Following acylchlorination of
acid analogues 22, 23 and 43e45, esterification gave the target
products.
4.8.6. 3-(Piperidin-1-yl)propyl-7-fluoro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (29)
Using the mixture of dichloromethane and methanol (12:1) as
ꢀ
1
eluent to give red powder, yield 26%, mp ¼ 139.1e141.1 C. H NMR
4.8.1. 2-(Dimethylamino)ethyl-7-fluoro-5,12-dioxo-5,12-
(400 MHz, CDCl
3
)
d
9.65 (dd, J ¼ 6.7, 0.9 Hz, 1H), 9.06 (dd, J ¼ 4.7,
dihydroindolizino[2,3-g]quinoline-6-carboxylate (24)
1.7 Hz, 1H), 8.53 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.68 (dd, J ¼ 7.9, 4.7 Hz, 1H),
7.17e7.08 (m, 2H), 4.54 (t, J ¼ 6.4 Hz, 2H), 2.68e2.58 (m, 6H),
Using the mixture of dichloromethane and methanol (10:1) as
ꢀ
13
eluent to give red brown powder, yield 58%, mp ¼ 199.3e200.7 C.
2.16e2.14 (m, 2H), 1.72e1.69 (m, 4H), 1.54e1.45 (m, 2H). C NMR
1
H NMR (400 MHz, CDCl
3
)
d
9.64 (d, J ¼ 6.4 Hz, 1H), 9.05 (d,
3
(100 MHz, CD OD) d 178.9, 171.9, 163.1, 153.5, 152.6, 149.1, 134.9,
130.0, 127.4, 126.1, 124.4, 122.2, 117.6, 111.1, 111.0, 106.0, 63.2, 54.8,
J ¼ 3.4 Hz, 1H), 8.54 (d, J ¼ 7.7 Hz, 1H), 7.67 (dd, J ¼ 7.0, 4.9 Hz, 1H),
.22e7.04 (m, 2H), 4.59 (t, J ¼ 5.5 Hz, 2H), 2.80 (t, J ¼ 5.4 Hz, 2H),
.34 (s, 6H). 13C NMR (100 MHz, CDCl
179.3, 172.8, 163.3, 155.8,
54.4, 153.2, 150.0, 135.2, 130.4, 127.1, 124.8, 124.7, 117.1, 117.1, 110.7,
þ
7
53.6, 24.3, 23.9, 22.5. HRMS (ESI) m/z: 436.1662 [MþH] , calcd for
2
3
)
d
24 3 4
C H23FN O 436.1667.
1
1
þ
10.5, 63.6, 57.4, 45.5. HRMS (ESI) m/z: 382.1177 [MþH] , calcd for
4.8.7. 2-Morpholinoethyl-7-fluoro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (30)
Using the mixture of dichloromethane and methanol (20:1) as
C
20
H17FN
3
O
4
382.1198.
ꢀ
1
4.8.2. 3-(Dimethylamino)propyl-7-fluoro-5,12-dioxo-5,12-
eluent to give red powder, yield 21%, mp ¼ 174.2e175.1 C. H NMR
dihydroindolizino[2,3-g]quinoline-6-carboxylate (25)
(400 MHz, CDCl
3
)
d
9.63 (t, J ¼ 10.3 Hz, 1H), 9.06 (d, J ¼ 4.6 Hz, 1H),
Using the mixture of dichloromethane and methanol (10:1) as
8.53 (d, J ¼ 7.8 Hz, 1H), 7.68 (dd, J ¼ 7.8, 4.7 Hz, 1H), 7.20e7.06 (m,
ꢀ
eluent to give red brown powder, yield 58%, mp ¼ 203.8e204.9 C.
2H), 4.62 (t, J ¼ 5.8 Hz, 2H), 3.79e3.68 (m, 4H), 2.84 (t, J ¼ 5.8 Hz,
1
13
H NMR (400 MHz, MeOD)
d
9.57 (d, J ¼ 6.0 Hz, 1H), 9.01 (d,
2H), 2.59 (t, J ¼ 3.6 Hz, 4H). C NMR (100 MHz, CDCl
3
) d 179.3,172.7,
J ¼ 4.1 Hz, 1H), 8.55 (d, J ¼ 7.8 Hz, 1H), 7.84 (dd, J ¼ 8.1, 4.1 Hz, 1H),
.33 (m, 2H), 4.61 (t, J ¼ 7.7 Hz, 2H), 3.41 (t, J ¼ 7.7 Hz, 2H), 2.97 (s,
H), 2.35 (m, 2H). 13C NMR (100 MHz, MeOD)
179.5, 172.1, 163.3,
55.3,153.9,152.8,149.3,135.1,130.2,127.6,126.3,124.7,122.5,117.7,
163.1, 154.4, 154.4, 149.9, 135.1, 130.4, 127.1, 126.6, 124.8, 122.5, 117.1,
7
110.6, 110.3, 107.0, 66.8, 63.0, 56.7, 53.6. HRMS (ESI) m/z: 424.1304
þ
6
d
[MþH] , calcd for C22
3 5
H19FN O 424.1303.
1
1
11.4, 111.2, 62.5, 55.2, 42.8, 24.3. HRMS (ESI) m/z: 396.1343
4.8.8. 3-Morpholinopropyl-7-fluoro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (31)
Using the mixture of dichloromethane and methanol (20:1) as
þ
[MþH] , calcd for C21
3 4
H19FN O 396.1354.
ꢀ
1
4.8.3. 2-(Pyrrolidin-1-yl)ethyl-7-fluoro-5,12-dioxo-5,12-
eluent to give red powder, yield 30%, mp ¼ 130.6e131.3 C. H NMR
dihydroindolizino[2,3-g]quinoline-6-carboxylate (26)
(400 MHz, CDCl
3
)
d
9.65 (d, J ¼ 6.7 Hz, 1H), 9.05 (d, J ¼ 4.5 Hz, 1H),
Using the mixture of dichloromethane and methanol (10:1) as
8.53 (d, J ¼ 7.8 Hz, 1H), 7.67 (dd, J ¼ 7.8, 4.7 Hz, 1H), 7.19e7.05 (m,
ꢀ
1
eluent to give red powder, yield 63%, mp ¼ 154.7e156.2 C. H NMR
2H), 4.56 (t, J ¼ 6.4 Hz, 2H), 3.77e3.70 (m, 4H), 2.58e2.45 (m, 6H),
1
3
(
8
4
1
1
400 MHz, CDCl
3
)
d
9.63e9.62 (m, 1H), 9.04 (dd, J ¼ 4.6, 1.5 Hz, 1H),
.53 (dd, J ¼ 7.8, 1.5 Hz, 1H), 7.69e7.67 (m, 1H), 7.17e7.07 (m, 2H),
.62 (t, J ¼ 6.1 Hz, 2H), 2.97 (t, J ¼ 6.1 Hz, 2H), 2.65e2.60 (m, 4H),
179.3, 172.7, 163.2,
54.4, 153.1150.0, 135.2, 130.3, 127.0, 126.7, 124.8, 122.6, 117.2, 110.7,
3
2.08e1.99 (m, 2H). C NMR (100 MHz, CDCl ) d 179.2, 172.7, 163.2,
154.4,154.4,149.9,135.1, 130.3,128.7, 127.0, 126.5,124.8, 122.5,117.1,
110.5, 107.3, 66.9, 64.6, 55.3, 53.6, 25.6. HRMS (ESI) m/z: 438.1455
.88e1.77 (m, 4H). 13C NMR (100 MHz, CDCl
)
d
[MþH] , calcd for C23
þ
3
3 5
H21FN O 438.1460.
þ
1
07.3, 64.8, 54.4, 54.2, 23.4. HRMS (ESI) m/z: 408.1353 [MþH] ,
4.8.9. 2-(4-Methylpiperazin-1-yl)ethyl-7-fluoro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (32)
calcd for C22 408.1354.
3 4
H19FN O
Using the mixture of dichloromethane and methanol (10:1) as
ꢀ
1
4
.8.4. 3-(Pyrrolidin-1-yl)propyl-7-fluoro-5,12-dioxo-5,12-
eluent to give red powder, yield 29%, mp ¼ 136.7e138.1 C. H NMR
dihydroindolizino[2,3-g]quinoline-6-carboxylate (27)
(400 MHz, CDCl
3
)
d
9.65 (d, J ¼ 6.7 Hz, 1H), 9.05 (dd, J ¼ 4.6, 1.5 Hz,
Using the mixture of dichloromethane and methanol (12:1) as
1H), 8.53 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.67 (dd, J ¼ 7.8, 4.7 Hz, 1H),
7.18e7.05 (m, 2H), 4.61 (t, J ¼ 6.0 Hz, 2H), 2.85 (t, J ¼ 6.0 Hz, 2H),
ꢀ
1
eluent to give red powder, yield 27%, mp ¼ 132.8e133.2 C. H NMR
400 MHz, CDCl
9.65 (dd, J ¼ 6.7, 0.9 Hz, 1H), 9.05 (dd, J ¼ 4.7,
.7 Hz, 1H), 8.54 (dd, J ¼ 7.9, 1.7 Hz, 1H), 7.67 (dd, J ¼ 7.9, 4.7 Hz, 1H),
.20e7.05 (m, 2H), 4.55 (t, J ¼ 6.6 Hz, 2H), 2.68e2.52 (m, 6H),
13
(
1
7
3
)
d
3
2.75e2.40 (m, 8H), 2.30 (s, 3H). C NMR (100 MHz, CDCl ) d 179.2,
172.7, 163.2, 154.4, 154.4, 149.9, 135.1, 130.3, 128.8, 127.0, 126.6,
124.7, 122.5, 117.1, 110.6, 107.1, 63.4, 56.2, 54.9, 45.8. HRMS (ESI) m/z:

Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
205
þ
4
37.1622 [MþH] , calcd for C23
H22FN
4
O
4
437.1620.
4.8.15. 2-Morpholinoethyl-7-chloro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (38)
Using the mixture of chloroform and methanol (30:1) as eluent
4
.8.10. 3-(4-Methylpiperazin-1-yl)propyl-7-fluoro-5,12-dioxo-5,12-
ꢀ
1
to give red brown powder, yield: 50%, mp ¼ 191.7e193.4 C.
H
dihydroindolizino[2,3-g]quinoline-6-carboxylate (33)
NMR (400 MHz, CDCl
3
)
d
9.54 (d, J ¼ 6.9 Hz, 1H), 8.93 (dd, J ¼ 4.6,
Using the mixture of dichloromethane and methanol (10:1) as
ꢀ
1
1.5 Hz, 1H), 8.52 (dd, J ¼ 7.9, 1.4 Hz, 1H), 7.62 (dd, J ¼ 7.9, 4.7 Hz, 1H),
eluent to give red powder, yield 38%, mp ¼ 127.2e128.1 C. H NMR
7
3
(
.31 (d, J ¼ 7.4 Hz, 1H), 7.02 (t, J ¼ 7.2 Hz, 1H), 4.59 (t, J ¼ 5.9 Hz, 2H),
(
400 MHz, CDCl
3
)
d
9.65 (d, J ¼ 6.7 Hz, 1H), 9.05 (d, J ¼ 4.6 Hz, 1H),
.53 (d, J ¼ 7.8 Hz, 1H), 7.67 (dd, J ¼ 7.8, 4.7 Hz, 1H), 7.18e7.06 (m,
H), 4.54 (t, J ¼ 6.5 Hz, 2H), 2.71e2.40 (m, 10H), 2.31 (s, 3H),
.09e1.97 (m, 2H). 13C NMR (100 MHz, CDCl
179.2, 172.7, 163.3,
54.4,154.4,150.0,135.2,130.3,128.7,127.0,126.5,124.7,122.5,117.1,
13
.70e3.62 (m, 4H), 2.88e2.79 (m, 2H), 2.53 (m, 4H). C NMR
178.6, 173.1, 164.2, 153.8, 149.1, 134.8, 133.1,
8
2
2
100 MHz, CDCl
3
)
d
131.5, 127.5, 127.3, 126.6, 126.5, 126.4, 120.6, 117.0, 109.9, 66.7, 63.1,
3
)
d
þ
5
C
6.5, 53.6. HRMS (ESI) m/z: 440.1000 [MþH] , calcd for
1
22
3 5
H19ClN O 440.1008.
110.5, 107.3, 64.7, 55.0, 54.8, 45.9, 25.9. HRMS (ESI) m/z: 451.1771
þ
[MþH] , calcd for C24
4 4
H24FN O 451.1776.
4
.8.16. 3-Morpholinopropyl-7-chloro-5,12-dioxo-5,12-
dihydroindolizino[2,3-g]quinoline-6-carboxylate (39)
4
.8.11. 2-(Pyrrolidin-1-yl)ethyl-7-chloro-5,12-dioxo-5,12-
Using the mixture of chloroform and methanol (10:1) as eluent
dihydroindolizino[2,3-g]quinoline-6-carboxylate (34)
ꢀ
1
to give orange powder, yield: 40%, mp ¼ 207.2e209.7 C. H NMR
400 MHz, CDCl
9.55 (d, J ¼ 6.9 Hz, 1H), 8.95 (dd, J ¼ 4.6, 1.5 Hz,
H), 8.55 (dd, J ¼ 7.9,1.5 Hz,1H), 7.65 (dd, J ¼ 7.9, 4.7 Hz,1H), 7.35 (d,
J ¼ 7.5 Hz, 1H), 7.06 (t, J ¼ 7.2 Hz, 1H), 4.86 (s, 2H), 3.52e2.83 (m,
Using the mixture of chloroform and methanol (10:1) as eluent
(
3
) d
ꢀ
1
to give orange powder, yield: 40%, mp ¼ 207.2e209.7 C, H NMR
400 MHz, CDCl
9.55 (d, J ¼ 6.9 Hz, 1H), 8.95 (dd, J ¼ 4.6, 1.5 Hz,
H), 8.55 (dd, J ¼ 7.9,1.5 Hz,1H), 7.65 (dd, J ¼ 7.9, 4.7 Hz,1H), 7.35 (d,
J ¼ 7.5 Hz, 1H), 7.06 (t, J ¼ 7.2 Hz, 1H), 4.86 (s, 2H), 3.52e2.83 (m,
H), 1.95 (s, 4H). 13C NMR (100 MHz, CDCl
178.7, 173.1, 164.2,
53.7, 149.0, 134.8, 133.2, 131.5, 127.5, 127.4, 126.7, 126.5, 126.4,
1
(
1
3
) d
13
6
H), 2.58e2.45 (m, 4H), 1.95e1.75 (m, 2H). C NMR (100 MHz,
CDCl 178.7, 173.1, 164.2, 153.7, 149.0, 134.8, 133.2, 131.5, 127.5,
27.4, 126.7, 126.5, 126.4, 120.5, 117.0, 110.0, 66.9, 54.3, 53.3, 23.4.
3
) d
6
1
3
) d
1
þ
HRMS (ESI) m/z: 454.1091 [MþH] , calcd for C23
3 5
H21ClN O 454.1157.
120.5, 117.0, 110.0, 64.3, 54.3, 53.3, 23.4. HRMS (ESI) m/z: 424.1032
þ
[MþH] , calcd for C22
3 4
H19ClN O 424.1059.
4.8.17. 2-Morpholinoethyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]
quinoline-11-carboxylate (46)
Using the mixture of dichloromethane and methanol (50:1) as
4
.8.12. 3-(Pyrrolidin-1-yl)propyl-7-chloro-5,12-dioxo-5,12-
ꢀ
1
eluent to give orange powder, yield: 48%, mp ¼ 167.2e169.5 C. H
dihydroindolizino[2,3-g]quinoline-6-carboxylate (35)
NMR (400 MHz, CDCl
3
) d
9.76 (d, J ¼ 7.0 Hz, 1H), 8.95 (dd, J ¼ 4.6,
Using the mixture of chloroform and methanol (10:1) as eluent
ꢀ
1
1.6 Hz, 1H), 8.51 (dd, J ¼ 7.8, 1.6 Hz, 1H), 8.41 (d, J ¼ 9.1 Hz, 1H), 7.60
to give orange powder, yield: 20%, mp ¼ 267.2e269.7 C. H NMR
400 MHz, CD OD)
9.61 (d, J ¼ 7.0 Hz, 1H), 8.95 (dd, J ¼ 4.6, 1.5 Hz,
H), 8.64 (dd, J ¼ 7.9, 0.7 Hz, 1H), 7.84 (dd, J ¼ 7.7, 4.7 Hz, 1H), 7.55
(
4
dd, J ¼ 7.8, 4.7 Hz, 1H), 7.47e7.38 (m, 1H), 7.16 (t, J ¼ 6.9 Hz, 1H),
(
1
3
d
13
.52 (t, J ¼ 6.0 Hz, 2H), 3.67 (s, 4H), 2.88 (s, 2H), 2.57 (s, 4H).
C
3
NMR (100 MHz, CDCl ) d
178.2, 173.7, 163.1, 153.7, 149.6, 140.3,
(
2
d, J ¼ 7.5 Hz, 1H), 7.24 (t, J ¼ 7.3 Hz, 1H), 4.52 (t, J ¼ 6.2 Hz, 2H),
13
134.3, 130.5, 128.6, 128.5, 128.2, 127.1, 121.8, 121.2, 117.9, 106.5, 66.9,
.86e2.76 (m, 2H), 2.72 (m, 4H), 2.17e2.03 (m, 2H), 1.86 (m, 4H).
OD) 178.7, 172.6, 164.8, 152.7, 148.5, 134.9,
32.5, 131.4, 128.0, 127.9, 126.9, 126.5, 126.4, 120.7, 117.3, 109.1, 64.1,
C
þ
6
C
1.8, 56.7, 53.8. HRMS (ESI) m/z: 406.1405 [MþH] , calcd for
NMR (100 MHz, CD
3
d
H
20
N
3
O
5
406.1397.
1
5
C
22
þ
3.7, 52.7, 27.0, 22.7. HRMS (ESI) m/z: 438.1212 [MþH] , calcd for
4.8.18. 2-Morpholinoethyl-5,12-dioxo-5,12-dihydroindolizino[2,3-
23 21 3 4
H N O
Cl 438.1215.
g]quinoxaline-11-carboxylate (47)
Using the mixture of dichloromethane and methanol (50:1) as
4
.8.13. 2-(Piperidin-1-yl)ethyl-7-chloro-5,12-dioxo-5,12-
ꢀ
eluent to give red brown powder, yield: 22%, mp ¼ 106.5e108.9 C.
dihydroindolizino[2,3-g]quinoline-6-carboxylate (36)
1
H NMR (400 MHz, CDCl
3
)
d
9.88 (d, J ¼ 7.0 Hz, 1H), 8.94 (d,
Using the mixture of chloroform and methanol (15:1) as eluent
J ¼ 4.7 Hz, 1H), 8.52 (d, J ¼ 9.1 Hz, 1H), 8.47 (d, J ¼ 9.1 Hz, 1H),
.56e7.47 (m, 1H), 7.26 (t, J ¼ 6.9 Hz, 1H), 4.56 (s, 2H), 3.70 (s, 4H),
ꢀ
1
to give orange powder, yield: 40%, mp ¼ 188.4e190.9 C. H NMR
400 MHz, CDCl
9.55 (d, J ¼ 6.9 Hz, 1H), 8.95 (dd, J ¼ 4.6, 1.6 Hz,
H), 8.53 (dd, J ¼ 7.9,1.6 Hz,1H), 7.63 (dd, J ¼ 7.9, 4.7 Hz,1H), 7.32 (d,
J ¼ 7.3 Hz, 1H), 7.03 (t, J ¼ 7.2 Hz, 1H), 4.70 (t, 2H), 2.98 (m, J ¼ 1.7 Hz,
7
2
1
1
(
1
3
)
d
13
.91 (s, 2H), 2.60 (s, 4H). C NMR (100 MHz, CDCl
3
) d 177.2, 171.7,
62.4,148.1, 147.7, 145.7, 145.6,140.7, 129.4,128.6, 128.3,122.7, 121.4,
18.6, 106.9, 66.9, 61.9, 56.7, 53.7. HRMS (ESI) m/z: 407.1366
13
2
H), 2.63 (m, 4H), 1.63 (s, 6H). C NMR (100 MHz, CDCl
3
)
d
178.5,
þ
[
MþH] , calcd for C21
19 4 5
H N O 407.1350.
173.1, 164.2, 153.7, 149.0, 134.8, 133.1, 131.5, 127.5, 127.4, 126.6, 126.5,
1
4
26.4, 120.5, 117.0, 110.2, 63.6, 56.8, 54.6, 25.7, 24.1. HRMS (ESI) m/z:
4
.8.19. 2-Morpholinoethyl-5,12-dioxo-5,12-dihydroindolizino[2,3-
þ
3 4
38.1199 [MþH] , calcd for C23H21ClN O 438.1215.
g]phthalazine-11-carboxylate (48)
Using the mixture of dichloromethane and methanol (100:1) as
ꢀ
1
4
.8.14. 3-(Piperidin-1-yl)propyl-7-chloro-5,12-dioxo-5,12-
eluent to give purple powder, yield: 22%, mp ¼ 165.0e166.5 C. H
dihydroindolizino[2,3-g]quinoline-6-carboxylate (37)
NMR (400 MHz, CDCl
3
)
d
9.90 (d, J ¼ 0.9 Hz, 1H), 9.81 (d, J ¼ 0.9 Hz,
Using the mixture of chloroform and methanol (10:1) as eluent
1H), 9.76 (d, J ¼ 7.0 Hz, 1H), 8.49 (d, J ¼ 9.1 Hz, 1H), 7.56e7.47 (m,
ꢀ
1
to give orange powder, yield: 35%, mp ¼ 183.7e185.2 C. H NMR
400 MHz, CDCl
9.56 (d, J ¼ 6.9 Hz, 1H), 8.93 (dd, J ¼ 4.6, 1.5 Hz,
H), 8.54 (dd, J ¼ 7.9,1.4 Hz,1H), 7.64 (dd, J ¼ 7.9, 4.7 Hz,1H), 7.33 (d,
J ¼ 7.4 Hz, 1H), 7.04 (t, J ¼ 7.2 Hz, 1H), 4.53 (t, J ¼ 5.9 Hz, 2H), 2.98 (t,
1H), 7.28 (m, 1H), 4.57 (m, 2H), 3.73 (m, 4H), 2.89 (s, 2H), 2.64 (s,
13
(
1
3
)
d
3
4H). C NMR (100 MHz, CDCl ) d 179.0, 172.4, 162.2, 146.7, 146.6,
140.3, 137.0, 129.5, 128.3, 127.4, 126.4, 126.1, 121.7, 119.0, 106.8, 66.9,
61.8, 56.8, 53.7. HRMS (ESI) m/z: 407.1701 [MþH] , calcd for
þ
13
J ¼ 7.4 Hz, 2H), 2.88 (m, 4H), 2.83 (m, 6H), 1.84 (m, 2H). C NMR
100 MHz, CDCl 178.9, 172.9, 164.4, 153.5, 148.7, 135.1, 133.2,
31.6, 127.9, 127.6, 127.1, 126.9, 126.5, 120.5, 117.3, 109.7, 64.0, 55.2,
21 19 4 5
C H N O 407.1350.
(
3
) d
1
5
C
4.9. TOP1-mediated relaxation assay
þ
3.7, 24.3, 23.7, 22.6. HRMS (ESI) m/z: 452.1358 [MþH] , calcd for
24
H
23ClN 452.1372.
3
O
4
The compounds were tested for the TOP1 inhibitory activities

206
Q. Yu et al. / European Journal of Medicinal Chemistry 152 (2018) 195e207
using TOP1-mediated relaxation assay [35]. Briefly, reaction
mixture (20 L) with 0.5 g supercoiled pBR322 DNA in Relaxation
Buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, 5 mM MgCl , 15 g/mL
BSA, 40 g/mL DTT) was incubated with 1 unit of calf thymus TOP1
Acknowledgements
m
m
2
m
This work is supported by the National Natural Science Foun-
dation of China (No. 81373257 and 81703668), Guangdong Natural
Science Fund (No. S2013010015609 and 2017A030310396).
m
ꢀ
in the absence or in the presence of compound for 30 min at 37 C.
Then the reaction solution was added with 6 ꢂ loading buffer
(
4
m
L), and was analyzed using a 0.8% agarose gel in TBE buffer at
Appendix A. Supplementary data
4
.6 V/cm for 1.5 h. Gel was stained with 1 ꢂ gel red for 30 min and
subsequently visualized with a UV transilluminator.
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.ejmech.2018.04.040.
4.10. TOP1-mediated nicking assay
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ꢁ4
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5
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