Synthesis, cytotoxic activities and structure-activity relationships of topoisomerase I inhibitors: Indolizinoquinoline-5,12-dione derivatives

Article abstract of DOI:10.1016/j.bmc.2008.02.036

A series of indolizinoquinoline-5,12-dione derivatives (IQDs) are synthesized and evaluated for their cytotoxic activities toward human lung adenocarcinoma (GLC-82), large-cell lung carcinoma (NCI-H460), promyelocytic leukemia (HL-60) and breast carcinoma (MCF-7) cells by MTT method. Most of the IQDs show significant cytotoxic potency. In addition, the evaluation of structure-activity relationships indicated that the incorporation of electron-withdrawing substituents at the C or D ring will enhance the activities of the target compounds distinctly. The topoisomerase I inhibitory activity is also measured.

Full text of DOI:10.1016/j.bmc.2008.02.036

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4617–4625
Synthesis, cytotoxic activities and structure–activity
relationships of topoisomerase I inhibitors:
Indolizinoquinoline-5,12-dione derivatives
Yu Cheng, Lin-Kun An,* Ning Wu, Xiao-Dong Wang, Xian-Zhang Bu,
Zhi-Shu Huang and Lian-Quan Gu*
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China
Received 4 January 2008; revised 9 February 2008; accepted 11 February 2008
Available online 15 February 2008
Abstract—A series of indolizinoquinoline-5,12-dione derivatives (IQDs) are synthesized and evaluated for their cytotoxic activities
toward human lung adenocarcinoma (GLC-82), large-cell lung carcinoma (NCI-H460), promyelocytic leukemia (HL-60) and breast
carcinoma (MCF-7) cells by MTT method. Most of the IQDs show significant cytotoxic potency. In addition, the evaluation of
structure–activity relationships indicated that the incorporation of electron-withdrawing substituents at the C or D ring will enhance
the activities of the target compounds distinctly. The topoisomerase I inhibitory activity is also measured.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
DNA topoisomerase I (TOP1) controls the processive
relaxation of supercoiled DNA by means of transient
DNA strand breakage and religation,1–4 and that
can be used as a target to screen the anticancer agents.5,6
Camptothecin (CPT) derivatives, the well-known TOP1
inhibitors, are successfully developed as anticancer
drug.6,7 Comptothecin derivatives contain indolizi-
no[1,2-b]quinoline nucleus,8 which is reported to be
pharmacologically active moiety.9 In accordance with
Moore’s theory,10 the molecules contain a planar poly-
cyclic aromatic ring and a conjugated quinone attend
to be the DNA-intercalating TOP inhibitors.11–13 In
view of this reason, we designed and synthesized a series
of indolizinoquinoline-5,12-dione derivatives (IQDs,
Scheme 1) which combined the indolizine with the qui-
nolinedione moiety.12 The cytotoxic activities and the
structure–activity relationships of the prepared com-
pounds were studied. In general, these compounds
exhibited potential cytotoxic activities. DNA-topoiso-
merase I relaxation assay indicated that the possible
mechanism of their action was as a TOP1 inhibitor.
2.1. Chemistry
The indolizinoquinoline-5,12-dione derivatives were
synthesized as shown in Scheme 1. 6,7-Dichloroquino-
line-5,8-dione (1) was obtained by oxidizing 8-hydroxy-
quinoline in concentrated HCl solution with sodium
chlorate or concentrated HNO3.12
The heterocyclic skeleton was formed through a single-
step reaction of substrate 1, pyridine and an active meth-
ylene reagent (AMR).14,15 Two regioisomers (N,N-syn
and N,N-anti isomers) were obtained in the cyclization,
which was different from Yanni’s result.14 The isomers
were isolated in pure form by silicon gel flash column
chromatography and fully characterized by mass spec-
trometry, elementary analysis, and NMR analysis.
The regioisomers, 2a and 2b, were obtained from the
reaction of substrate 1 with ethyl acetoacetate and
pyridine. The NMR spectra, ESI-MS spectrum, elemen-
tary analysis, and melting point were similar to Defant’s
result,15 and confirmed their structure.
The cyclization reaction of 3-substituent pyridine, such
as 3-fluoropyridine, with substrate 1 and ethyl acetoace-
tate gave the regioisomers 7a/b. Mass spectra and
elemental analysis showed that both had the same
Keywords: Indolizinoquinoline-5,12-dione derivatives; Cytotoxic activ-
ity; Structure–activity relationship; Topoisomerase I.
*
Corresponding authors. Tel./fax: +86 20 39332678; e-mail addresses:
lssalk@mail.sysu.edu.cn; cesglq@mail.sysu.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.02.036

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Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
R1
16R4
C
16R4
12
N
O
O
B
O
12
R1
R1
Cl
Cl
(iii),
(i) or (ii)
N
14
+
A
N
9
1
N
1
N
N
D
N
O
or R3CH2NO2
OH
9
AMR =
R2
O
1
O
O
4
4
2-17a: N,N-syn
2-17b: N,N-anti
2a, b: R1 = H, R4 = R2 = CO2Et; 8a, b: R1 = Cl, R4 = R2 = CO2Et;
3a, b: R1 = H, R4 = R2 = COMe; 9a, b: R1 = Br, R4 = R2 = CO2Et;
14a, b: R1 = Cl, R4 = R2 = COMe;
15a, b: R1 = Br, R4 = R2 = COMe;
4a, b: R1 = H, R4 = R2 = CN;
5a, b: R1 = H, R4 = R3 = Me;
6a, b: R1 = H, R4 = R3 = H;
10a, b: R1 = Me, R4 = R2 = CO2Et; 16a, b: R1 = OH, R4 = R2 = COMe;
11a, b: R1 = OH, R4 = R2 = CO2Et; 17a, b: R1 = NH2, R4 = R2 = COMe;
12a, b: R1 = NH2, R4 = R2 = CO2Et;
7a, b: R1 = F, R4 = R2 = CO2Et; 13a, b: R1 = F, R4 = R2 = COMe;
Scheme 1. Synthesis of indolizinoquinoline-5,12-dione derivatives. Reagents and conditions: (i) NaClO3, concd HCl, 50 ꢁC; (ii) concd HNO3, concd
HCl, 90 ꢁC; (iii) EtOH, pyridine derivatives, AMR, refluxing.
formula, C18H11FN2O4. The HMBC spectrum of 7a
indicated that the resonance at d = 179.3 ppm was attrib-
utable to C-14 by long-range hetero-correlation with 12-
H at d = 8.54 ppm. As a consequence, the signal at
d = 172.8 ppm could be assigned to C-7. In the 1H
NMR spectrum, the signal at d = 9.64 ppm (d,
J = 6.8 Hz) could be assigned to H-4. Furthermore,
1H–1H COSY spectrum showed a correlation signal of
H-4 with a hydrogen at d = 7.06–7.16 ppm (m, 2H),
which could be assigned to H-3 and H-2. Therefore,
compound 7a was assigned as the N,N-syn isomer, ethyl
7-fluoro-5,12-dioxo-5,12-dihydroindolizino[2,3-g]quino-
line 6-carboxylate. Similarly, compound 7b could be
assigned as the N,N-anti isomer. These results mean that
the cyclization of 3-substituent pyridine derivatives
instead of pyridine would mainly give the 1-substituent
target compounds.
or 22b, could not be obtained by flash column chroma-
tography techniques and the inseparable mixture was
used to determine the biological activities.
Ethyl 6,11-dioxo-6,11-dihydrobenzo[f]pyrido[1,2-a]in-
dole 12-carboxylate (23) was obtained by cyclization
of pyridine with ethyl acetoacetate and 2,3-dichloro-
1,4-naphtoquinone (21).16,17 Compound 21 was pre-
pared by treating 1,4-naphthoquinone in concentrated
HCl solution with sodium chlorate using Bu4NBr as
phase-transfer catalyst.
2.2. Cytotoxic activities
Evaluation of the synthetic IQDs for cytotoxic activity
in incline was performed by MTT assay using four dif-
ferent human cancer cell lines, lung adenocarcinoma cell
(GLC-82), large-cell lung carcinoma cell (NCI-H460),
promyelocytic leukemia cell (HL-60) and breast carci-
noma cell (MCF-7). The cultured cell lines were divided
into multi-well microplate and treated with the synthe-
sized compounds. The resulting solutions were detected
and evaluated for their biological activities.
In order to investigate the effects of the existence and the
position of nitrogen atom in this heterocyclic skeleton
on the biological activity, compounds 22a/b and 23
(Scheme 2) were synthesized.
The mixture of regioisomers 22a and 22b were prepared
through cyclization of 6,7-dichloroisoquinoline-5,8-
dione (20), ethyl acetoacetate, and pyridine. The sub-
strate 20 was obtained using isoquinoline as starting
material, through nitration, hydrogenation, and oxida-
tion-chlorination reactions. The pure compound, 22a
The assessments of cytotoxic activities were expressed as
the concentration inhibiting 50% of cancer cell growth
(IC50). The results were summarized in Table 1.
According to Table 1, IQDs showed the significant cyto-
toxic activities with the different substituent groups
against a wide variety of tumor cell lines. The substitu-
ent group at the C ring of heterocyclic skeleton had deci-
sive affect on the IC50 value. Compounds with electron
contributing groups at the C ring, such as 5a, 5b, 6a,
NO2
NH2
(i)
(ii)
N
N
N
and 6b, showed weaker cytotoxic activities (IC50
>
18
19
(iii)
10 lM) against the four human cancer cell lines. When
an electron-withdrawing substituent such as ethoxycar-
bonyl, carbonyl, or nitrile group was incorporated at
the C ring, which would be increased activity.
O
O
O
O
O
O
CO2Et
N
(iv)
(v)
Cl
Cl
X
Y
M
Moreover, an enhancement of cytotoxic activities was
observed by enhancing the substituent’s electron-with-
drawing properties at the D ring. Indeed, the halogen
substituted derivatives showed more potent activity
against the cancer cell lines than the corresponding the
amino or hydroxyl substituted derivatives. Compound
8a with chloro substitution showed a 95-fold activity
20: M = N
22a: X = CH, Y = N
22b: X = N, Y = CH
23: X = Y = CH
21: M = CH
Scheme 2. Synthesis of compounds 22a/b and 23. Reagents and
conditions: (i) H2SO4, KNO3, 0–5 ꢁC; (ii) EtOH, Pd/C, H2; (iii)
NaClO3, concd HCl, 80 ꢁC; (iv) NaClO3, concd HCl, Bu4NBr, ethyl
acetate, 50 ꢁC; (v) pyridine, ethylacetoacetate, ethanol, refluxing.

Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
4619
Table 1. Cytotoxic activities in vitro of IQDs, 22a/b and 23
version of supercoiled plasmid DNA by calf thymus
TOP1 in the presence of selectively synthesized com-
pound 8a. CPT, a well-known TOP1 inhibitor, was used
as a positive control (Fig. 1. B). Compound 8a showed
stronger inhibitory activity than CPT. There were
5.8% and 100% of supercoiled DNA after being treated
with 0.2 lM and 125 lM of 8a, respectively. CPT could
inhibit 81.3% of TOP1’s catalytic activity at 125 lM
concentration.
a
Compound
Cytotoxic activities IC50 (lM)
GLC-82
NCI-H460
HL-60
MCF-7
2a
2b
<0.25
2.40
<0.25
1.90
0.88
14.45
0.86
0.96
6.38
3a
<0.25
1.90
2.50
<0.25
1.40
0.16
7.92
12.70
1.56
3b
14.71
12.67
12.46
>25.00
16.97
14.68
1.37
4a,bb
5a
14.20
21.50
11.50
16.70
<0.25
1.70
12.20
>25.00
15.60
12.30
<0.25
1.20
>25.00
>25.00
>25.00
20.20
7.01
5b
6a
6b
7a
3. Conclusion
7b
8a
5.51
6.52
6.20
7.37
Overall, the screening indicated that the synthesized
IQDs showed significant cytotoxic activities against four
human cancer cell lines. The incorporation of electron-
withdrawing groups at C or D ring would enhance the
activities of the resulting compound. Evaluation of
structure–activity relationships indicated that the exis-
tence of nitrogen atom in the A ring was necessary.
And the position of nitrogen atom made a variable,
moderate effect on the activity. TOP1 relaxation assay
indicated that the topoisomerase I might be a biological
target of IQDs.
<0.25
9.73
2.10
2.20
8b
9a
15.42
9.32
19.90
10.30
20.80
14.50
>25.00
>25.00
>25.00
20.90
>25.00
2.31
<0.25
1.10
1.83
2.00
9b
2.40
11.47
0.22
10a
10b
11a
11b
12a
12b
13a
13b
14a
14b
15a
15b
16a
16b
17a
17b
22a,bb
23
n.d.c
n.d.
9.27
7.20
11.65
>25.00
>25.00
16.43
>25.00
0.17
>25.00
23.30
17.58
19.00
<0.25
6.75
23.80
23.41
23.50
5.26
12.70
6.85
1.39
0.19
9.28
7.83
0.23
1.50
16.10
2.01
4.68
2.20
0.21
16.10
11.70
12.40
17.60
n.d.
4. Experimental
2.15
0.20
1.56
4.1. General experimental
19.50
16.30
12.20
10.30
9.36
24.85
>25.00
>25.00
10.29
2.20
>25.00
>25.00
22.30
15.80
17.43
>25.00
All chemicals and solvents were obtained from commer-
cial suppliers and used without further purification. Calf
thymus topoisomerase I and plasmid pBR322 were pur-
chased from TakaRa Biotechnology (Dalian) Co., Ltd.
Chemical reactions were monitored by thin layer chro-
matography using self-preparative silica gel GF254
plates. Flash column chromatography was performed
with silica gel 200–300 meshes. Melting points were
uncorrected and were determined using a XT-4 appara-
>25.00
>25.00
11.70
>25.00
>25.00
n.d.
a IC50 values, defined as the concentration that inhibited growth by
50%. Every experiment was repeated at least three times.
b The mixture was used to determine the cytotoxic activities.
c n.d. means ‘not determined’.
1
1
tus. H NMR, 13C NMR, H–13C HMQC, and HMBC
spectra were measured on a Varian Mercury-Plus
300 MHz or Bruker UltraShield 400 MHz spectrometer.
Chemical shifts were reported in parts per million (d)
relative to tetramethylsilane (TMS) as internal standard.
For the electrospray (ESI) MS analysis a Finnigan LCQ
Deca XP ion trap mass spectrometer equipped with a
Microsoft Windows NT data system and an ESI inter-
face was used. Elementary analysis was recorded on
an Elementar Vario EL elementary analysis device. IR
absorption was recorded on a Bruker TENSOR 37
spectrophotometer. UV absorption was recorded on a
SHIMADZU UV-2501 PC spectrophotometer.
compared with compound 12a with amino substitution
against GLC-82 cell line.
Study on the structure–activity relationship of heterocy-
clic quinines containing nitrogen indicated that the posi-
tion and number of nitrogen in the A ring was important
for the cytotoxicity.18 Compound 23 showed only a
moderate activity, which probably implied that the exis-
tence of nitrogen in the A ring was necessary. The mix-
ture of isoquinolinedione derivatives (22a and 22b)
showed lower activities than the corresponding pure qui-
nolinedione derivative, 2a or 2b. Comparison of the two
regioisomers, the N,N-syn isomer generally showed
more potential activities than the N,N-anti isomer. The
position of nitrogen atom made a moderate effect on
the activity.
4.2. Preparation of compounds 1 and 20
6,7-Dichloroquinoline-5,8-dinone (1) was prepared
according to Shaikh method,12 by treating 8-hydroxy-
quinoline in concentrated HCl solution with sodium
chlorate or concentrated HNO3. The reaction residue
was recrystallized in methanol to obtain the light yellow
solid 1. Yield 18% or 39%, respectively; mp = 221–
223 ꢁC (lit.12 221–223 ꢁC); 1H NMR (300 MHz,
DMSO-d6) d: 9.08 (dd, 1H, J = 4.7, 1.7 Hz), 8.49 (dd,
2.3. TOP1 inhibitory activity
The relative topoisomerase-targeting activities of IQDs
were carried out by TOP1 relaxation assay.19,20 The re-
sult indicated that some of IQDs strongly inhibited the
catalytic activity of TOP1. Figure 1 (A) shows the con-

4620
Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
Figure 1. Inhibition of topoisomerase I relaxation activities of 8a (A) and CPT (B). SC, supercoiled DNA; R, relaxed DNA. The lane 1 was DNA
control without enzyme and drugs. The lane 2 was the control of DNA and enzyme without drugs. The rest lanes contained calf thymus TOP1, DNA
and serially diluted drugs from 0.2 to 125 lM.
1H, J = 7.8, 1.5 Hz), 7.93 (dd, 1H, J = 8.0, 4.7 Hz);18
ESI-MS m/z: 228.3, 230.2 [M+1]+.
1.2 Hz), 4.50 (q, 2H, J = 7.2 Hz), 1.52 (t, 3H,
J = 7.2 Hz); 13C NMR (100 MHz, CDCl3) d: 177.9,
173.2, 162.9, 153.2, 149.2, 139.7, 134.0, 130.2, 128.2,
128.1, 127.8, 126.9, 121.3, 120.6, 117.7, 106.4, 60.8,
13.8; IR mmax (KBr): 1675.8, 1637,4, 1488.3, 1383.4,
1318.6 cmꢀ1; UV/vis kmax (EtOH): 248, 321, 355,
466 nm; ESI-MS m/z: 321.1 [M+1]+, 343.2 [M+Na]+;
C18H12N2O4, calcd: C, 67.50%; H, 3.78%; N, 8.75%;
O, 19.98%; found: C, 67.51%; H, 3.80%; N, 8.74%.
The structure was confirmed through HMQC and
HMBC.15
6,7-Dichloroisoquinoline-5,8-dinone (20) was obtained
by treating 5-aminoisoquinoline (19) in concentrated
HCl solution with sodium chlorate. Mp = 177–179 ꢁC
(lit.15 178–180 ꢁC).
4.3. General preparation of IQDs
The heterocyclic skeleton was cyclized according to a
published method with a slight modification.17,18 Sub-
strate 1 (2.2 mmol) was dissolved in absolute ethanol
(50 mL). The solution was heated to 60 ꢁC. Then, the
AMR (2.6 mmol) and pyridine derivative (6.8 mmol)
were gradually added to the solution. The reaction solu-
tion was refluxed for 16 h, to give a dark-reddish suspen-
sion, and concentrated in vacuum. The residue was
subsequently subjected to flash column chromatography
packed manually with silica gel to give the correspond-
ing target compound.
4.3.3. 6-Acetylindolizino[2,3-g]quinoline-5,12-dione 3a.
Red solid, yield 13%; mp (deg.) = 252 ꢁC, 1H NMR
(300 MHz, CDCl3) d: 9.93 (d, 1H, J = 6.9 Hz), 9.03 (d,
1H, J = 3.6 Hz), 8.53 (d, 1H, J = 7.8 Hz), 8.46 (d, 1H,
J = 9.3 Hz), 7.66 (dd, 1H, J = 7.7, 4.7 Hz), 7.51 (t, 1H,
J = 7.8 Hz), 7.25 (t, 1H, J = 7.5 Hz), 2.88 (s, 3H); 13C
NMR (100 MHz, CDCl3) d: 196.7, 180.9, 173.1, 154.4,
149.7, 139.8, 135.3, 130.6, 129.5, 128.4, 126.9, 126.8,
122.3, 121.8, 118.6, 114.9, 31.9; IR mmax (KBr): 2962.9,
1642.3, 1491.9, 1367.0, 1312.4 cmꢀ1
(EtOH): 245, 327, 361, 480 nm; ESI-MS m/z: 291.3
[M+1]+, [M+Na]+, [2M+Na]+;
313.1 602.9
; UV/vis kmax
4.3.1. Ethyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]quin-
oline 6-carboxylate 2a. Orange solid, yield 31%;
mp = 226–227 ꢁC (lit.15 225–226 ꢁC); 1H NMR
(300 MHz, CDCl3) d: 9.92 (d, 1H, J = 7.2 Hz), 9.01 (d,
1H, J = 3.9 Hz), 8.54 (d, 1H, J = 7.5 Hz), 8.34 (d, 1H,
J = 9.0 Hz), 7.64 (dd, 1H, J = 7.5, 4.5 Hz), 7.49 (t, 1H,
J = 8.0 Hz), 7.24 (t, 1H, J = 6.9 Hz), 4.52 (q, 2H,
J = 7.1 Hz), 1.51 (t, 3H, J = 7.1 Hz); 13C NMR
(100 MHz, CDCl3) d: 179.1, 173.1, 162.9, 154.1, 149.5,
139.9, 135.4, 130.9, 128.6, 128.5, 128.1, 126.9, 122.9,
121.1, 117.9, 106.3, 61.1, 14.3; IR mmax (KBr): 1678.6,
C17H10N2O3, calcd: C, 70.34%; H, 3.47%; N, 9.65%;
O, 16.54%; found: C, 70.31%; H, 3.51%; N, 9.63%.
4.3.4. 11-Acetylindolizino[3,2-g]quinoline-5,12-dione 3b.
Red solid, yield 2.5%; mp (deg.) = 234 ꢁC; 1H NMR
(300 MHz, CDCl3) d: 9.80 (d, 1H, J = 6.8 Hz), 8.99 (d,
1H, J = 3.6 Hz), 8.56 (dd, 1H, J = 7.8, 1.5 Hz), 8.46 (d,
1H, J = 9.0 Hz), 7.67 (dd, 1H, J = 7.7, 4.7 Hz), 7.49
(ddd, 1H, J = 8.6, 6.9, 1.2 Hz), 7.23 (td, 1H, J = 6.9,
1.2 Hz), 2.91 (s, 3H); 13C NMR (100 MHz, CDCl3) d:
196.9, 180.1, 173.7, 153.7, 149.4, 139.9, 134.4, 130.8,
129.4, 128.1, 127.6, 127.5, 122.0, 121.1, 118.5, 115.5,
31.9; IR mmax (KBr): 2962.6, 1681.3, 1634.2, 1580.2,
1485.1, 1312.3 cmꢀ1; UV/vis. kmax (EtOH): 244, 325,
361, 480 nm; ESI-MS m/z: 291.3 [M+1]+, 313.2
[M+Na]+, 603.0 [2M+Na]+; C17H10N2O3, calcd: C,
70.34%; H, 3.47%; N, 9.65%; O, 16.54%; found: C,
70.32%; H, 3.50%; N, 9.63%.
1641.8, 1571.3, 1487.6, 1383.1 cmꢀ1
; UV/vis kmax
(EtOH): 250, 322, 337, 356, 468 nm; ESI-MS m/z:
321.2 [M+1]+, 343.2 [M+Na]+, 663.0 [2M+Na]+;
C18H12N2O4, calcd: C, 67.50%; H, 3.78%; N, 8.75%;
O, 19.98%; found: C, 67.52%; H, 3.77%; N, 8.71%.
The structure was confirmed through HMQC and
HMBC.15
4.3.2. Ethyl 5,12-dioxo-5,12-dihydroindolizino[3,2-g]quin-
oline 11-carboxylate 2b. Orange solid, yield 12%; 1H
NMR (300 MHz, CDCl3) d: 9.78 (d, 1H, J = 6.9 Hz),
8.99 (d, 1H, J = 3.6 Hz), 8.55 (d, 1H, J = 7.8 Hz), 8.38
(d, 1H, J = 9.0 Hz), 7.65 (dd, 1H, J = 7.8, 4.5 Hz), 7.47
(ddd, 1H, J = 8.7, 6.9, 0.9 Hz), 7.20 (td, 1H, J = 6.9,
Pure products 4a and 4b could not be obtained by the
flash column chromatographic techniques. 4a (red solid
1
mixture with 4b): H NMR (300 MHz, DMSO-d6) d:
9.77 (d, 1H, J = 7.2 Hz), 9.00 (dd, 1H, J = 4.8, 1.5 Hz),

Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
4621
8.51 (dd, 1H, J = 7.8, 1.5 Hz), 7.90 (d, 1H, J = 9.0 Hz),
7.84 (dd, 1H, J = 7.8, 4.8 Hz), 7.56 (td, 1H, J = 6.9,
1.2 Hz), 7.39 (td, 1H, J = 6.9, 0.9 Hz). 4b (red solid mix-
4.8 Hz), 7.16–7.06 (m, 2H), 4.54 (q, 2H, J = 7.2 Hz),
1.47 (t, 3H, J = 7.2 Hz); 13C NMR (100 MHz, CDCl3)
d: 179.3, 172.8, 163.2, 154:5dðJ1CꢀF ¼ 254:4 HzÞ, 154.4,
153.8, 135.2, 134.7, 130.5, 128:9dðJ2CꢀF ¼ 32:8 HzÞ,
127.4, 127.1, 126.6, 124:8dðJ4CꢀF ¼ 5:2 HzÞ, 122.6,
117:1dðJ3CꢀF ¼ 6:8 HzÞ, 110:5dðJC2 ꢀF ¼ 17:1 HzÞ, 62.3,
14.1; IR mmax (KBr): 1725.2, 1674.9, 1639.1, 1619.1,
1
ture with 4a): H NMR (300 MHz, DMSO-d6) d: 9.51
(d, 1H, J = 7.2 Hz), 8.91 (dd, 1H, J = 4.8, 1.5 Hz), 8.48
(dd, 1H, J = 7.8, 1.5 Hz), 7.91 (d, 1H, J = 9.0 Hz), 7.78
(dd, 1H, J = 7.8, 4.8 Hz), 7.40 (td, 1H, J = 6.9,
1.2 Hz), 7.28 (td, 1H, J = 6.9, 0.9 Hz). ESI-MS m/z(mix-
ture): 274.4 [M+1]+.
1575.9, 1494.0, 1404.5 cmꢀ1
; ESI-MS m/z: 339.0
[M+1]+, 698.9 [2M+Na]+; C18H11FN2O4, calcd: C,
63.91%; H, 3.28%; F, 5.62%; N, 8.28%; O, 18.92%;
found: C, 63.90%; H, 3.31%; F, 5.62%; N, 8.26%. The
structure was confirmed through HMQC and HMBC.
4.3.5. 6-Methylindolizino[2,3-g]quinoline-5,12-dione 5a.
Red solid, yield 15%; mp = 238 ꢁC; 1H NMR
(300 MHz, CDCl3) d: 9.77 (d, 1H, J = 6.6 Hz), 8.99 (d,
1H, J = 3.0 Hz), 8.49 (d, 1H, J = 7.5 Hz), 7.67 (d, 1H,
J = 9.0 Hz), 7.57 (dd, 1H, J = 7.4, 4.8 Hz), 7.27 (t, 1H,
J = 7.4 Hz), 7.11 (t, 1H, J = 6.6 Hz), 2.70 (s, 3H); IR
mmax (KBr): 2925.8, 1731.7, 1659.3, 1633.0, 1574.7,
1477.9, 1318.9 cmꢀ1; UV/vis kmax (EtOH): 253, 340,
380, 504 nm; ESI-MS m/z: 263.3 [M+1]+, 247.0
[2M+Na]+; C16H10N2O2, calcd: C, 73.27%; H, 3.84%;
N, 10.68%; O, 12.20%; found: C, 73.27%; H, 3.85%;
N, 10.66%.
4.3.10. Ethyl 10-fluoro-5,12-dioxo-5,12-dihydroindolizi-
no[3,2-g]quinoline 11-carboxylate 7b. Orange solid, yield
18%; mp = 200–202 ꢁC; 1H NMR (300 MHz, CDCl3) d:
9.49 (dd, 1H, J = 6.3, 1.2 Hz), 9.00 (dd, 1H, J = 4.7,
1.7 Hz), 8.57 (dd, 1H, J = 7.8, 1.8 Hz), 7.68 (dd, 1H,
J = 7.8, 4.5 Hz), 7.14–7.03 (m, 2H), 4.54 (q, 2H,
J = 7.2 Hz), 1.48 (t, 3H, J = 7.2 Hz); 13C NMR
(100 MHz,
CDCl3)
d:
178.3,
173.5,
163.2,
154:6dðJ1CꢀF ¼ 254:9 HzÞ, 153.8, 149.3, 134.7, 131.2,
128:9dðJ2CꢀF ¼ 32:4 HzÞ, 127.4, 127.3, 124:4dðJ4CꢀF
5:3 HzÞ, 121.5, 117:0dðJ3CꢀF ¼ 6:7 HzÞ, 110:3dðJ2CꢀF
¼
¼
4.3.6. 11-Methylindolizino[3,2-g]quinoline-5,12-dione 5b.
Red solid, yield 19%; mp (deg.) = 233 ꢁC; 1H NMR
(300 MHz, CDCl3) d: 9.64 (d, 1H, J = 6.6 Hz), 8.94 (d,
1H, J = 2.6 Hz), 8.58 (d, 1H, J = 7.5 Hz), 7.69 (d, 1H,
J = 9.0 Hz), 7.63 (dd, 1H, J = 7.4, 4.8 Hz), 7.26 (t, 1H,
J = 7.4 Hz), 7.10 (t, 1H, J = 6.6 Hz), 2.74 (s, 3H); IR
mmax (KBr): 2927.5, 1731.1, 1677.4, 1619.7, 1576.8,
1478.3, 1315.7 cmꢀ1; UV/vis kmax (EtOH): 252, 336,
378, 507 nm; ESI-MS m/z: 263.3 [M+1]+; C16H10N2O2,
calcd: C, 73.27%; H, 3.84%; N, 10.68%; O, 12.20%;
found: C, 73.25%; H, 3.82%; N, 10.71%.
17:0 HzÞ, 62.4, 14.0; IR mmax (KBr): 1722.1, 1680.4,
1634.8, 1577.7, 1547.5, 1488.3, 1403.1 cmꢀ1; UV/vis kmax
(EtOH): 244, 319, 463 nm; ESI-MS m/z: 339.0 [M+1]+;
C18H11FN2O4, calcd: C, 63.91%; H, 3.28%; F, 5.62%;
N, 8.28%; O, 18.92%; found: C, 63.87%; H, 3.32%; F,
5.61%; N, 8.27%. The structure was confirmed through
HMQC and HMBC.
4.3.11. Ethyl 7-chloro-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 8a. Red solid, yield
16%; mp = 185–190 ꢁC; 1H NMR (300 MHz, CDCl3)
d: 9.58 (d, 1 H, J = 6.9 Hz), 8.98 (d, 1H, J = 4.5 Hz),
8.57 (d, 1H, J = 7.5 Hz), 7.67 (dd, 1H, J = 7.8, 4.5 Hz),
7.36 (d, 1H, J = 7.5 Hz), 7.07 (t, 1H, J = 7.2 Hz), 4.56
(q, 2H, J = 7.2 Hz), 1.49 (t, 3H, J = 7.2 Hz); IR mmax
(KBr): 2924.9, 1733.1,1680.9, 1645.3, 1578.3, 1514.6,
1497.3, 1299.9 cmꢀ1; UV/vis kmax (EtOH): 246, 329,
468 nm; ESI-MS m/z: 355.2, 357.2 [M+1]+;
C18H11ClN2O4, calcd: C, 60.94%; H, 3.13%; Cl, 9.99%;
N, 7.90%; O, 18.04%; found: C, 60.90%; H, 3.15%; Cl,
9.98%; N, 7.87%.
4.3.7. Indolizino[2,3-g]quinoline-5,12-dione 6a. Red solid,
1
yield 13%; H NMR (300 MHz, CDCl3) d: 9.74 (d, 1H,
J = 5.7 Hz), 9.02 (s, br, 1H, J = 4.5 Hz), 8.51 (d, 1H,
J = 6.9 Hz), 7.71 (d, 1H, J = 7.5 Hz), 7.50–7.75 (m,
1H), 7.25–7.32 (m, 2H), 7.10 (s, 1H);15 IR mmax (KBr):
2925.4, 1726.7, 1619.9, 1535.8, 1493.4, 1383.2,
1342.4 cmꢀ1
;
ESI-MS m/z: 249.3 [M+1]+, 518.9
[2M+Na]+; C15H8N2O2, calcd: C, 72.58%; H, 3.25%;
N, 11.28%; O, 12.89%; found: C, 72.60%; H, 3.28%;
N, 11.24%.
4.3.8. Indolizino[3,2-g]quinoline-5,12-dione 6b. Red solid,
yield 8%; 1H NMR (300 MHz, DMSO-d6, TMS) d: 9.49
(d, 1H, J = 6.9 Hz), 8.91 (d, br, 1H, J = 1.8 Hz), 8.48 (d,
1H, J = 7.5 Hz), 7.93 (d, 1H, J = 9.3 Hz), 7.79 (dd, 1H,
J = 7.5, 4.5 Hz), 7.42 (t, 1H, J = 7.5 Hz), 7.30 (t, 1H,
J = 6.9 Hz), 7.19 (s, 1H); IR mmax (KBr): 2961.6,
1736.7, 1678.2, 1620.6, 1577.7, 1488.0, 1391.6,
1337.2 cmꢀ1; UV/vis kmax (EtOH): 251, 326, 363,
481 nm; ESI-MS m/z: 249.2 [M+1]+; C15H8N2O2, calcd:
C, 72.58%; H, 3.25%; N, 11.28%; O, 12.89%; found: C,
72.58%; H, 3.27%; N, 11.29%.
4.3.12. Ethyl 10-chloro-5,12-dioxo-5,12-dihydroindolizi-
no[3,2-g]quinoline 11-carboxylate 8b. Red solid, yield
12%; mp (deg.) = 195 ꢁC; H NMR (300 MHz, CDCl3)
1
d: 9.72 (d, 1H, J = 6.3 Hz), 9.03 (d, br, 1H,
J = 3.3 Hz), 8.52 (d, 1H, J = 7.2 Hz), 7.65 (dd, 1H,
J = 7.8, 4.6 Hz), 7.38 (d, 1H, J = 7.2 Hz), 7.09 (t, 1H,
J = 7.2 Hz), 4.55 (q, 2H, J = 7.2 Hz), 1.47 (t, 3H,
J = 7.2 Hz); IR mmax (KBr): 2924.9, 1731.1, 1670.5,
1639.4, 1618.5, 1498.4, 1457.1 cmꢀ1
; UV/vis kmax
(EtOH): 254, 324, 359, 472 nm; ESI-MS m/z: 355.2,
357.2 [M+1]+; C18H11ClN2O4, calcd: C, 60.94%; H,
3.13%; Cl, 9.99%; N, 7.90%; O, 18.04%; found: C,
60.93%; H, 3.14%; Cl, 9.98%; N, 7.89%.
4.3.9. Ethyl 7-fluoro-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 7a. Orange solid, yield
12%; mp = 197–200 ꢁC;1H NMR (400 MHz, CDCl3) d:
9.64 (d, 1H, J = 6.8 Hz), 9.06 (dd, 1H, J = 4.8, 1.6 Hz),
8.54 (dd, 1H, J = 8.0, 1.6 Hz), 7.67 (dd, 1H, J = 8.0,
4.3.13. Ethyl 7-bromo-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 9a. Orange solid, yield
18%; mp = 210–215 ꢁC; 1H NMR (300 MHz, CDCl3)

4622
Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
d: 9.63 (d, 1H, J = 6.9 Hz), 8.98 (dd, 1H, J = 4.5,
1.5 Hz), 8.55 (dd, 1H, J = 7.8, 1.8 Hz), 7.67 (dd, 1H,
J = 7.8, 4.5 Hz), 7.55 (d, 1H, J = 7.5 Hz), 6.99 (t, 1H,
J = 7.2 Hz), 4.57 (q, 2H, J = 7.2 Hz), 1.50 (t, 3H,
J = 7.2 Hz); IR mmax (KBr): 2925.2, 1727.0, 1679.5,
4.3.18. Ethyl 10-hydroxy-5,12-dioxo-5,12-dihydroindoliz-
ino[3,2-g]quinoline 11-carboxylate 11b. Red solid, yield
13%; mp = 220–223 ꢁC; H NMR (300 MHz, DMSO-
1
d6) d: 11.3 (s, 1H), 9.09 (d, 1H, J = 6.6 Hz), 8.91 (d,
br, 1H, J = 3.9 Hz), 8.47 (dd, 1H, J = 7.8, 0.9 Hz), 7.80
(dd, 1H, J = 7.8, 4.7 Hz), 7.19 (t, 1H, J = 7.2 Hz), 6.73
(d, 1H, J = 7.8 Hz), 4.36 (q, 2H, J = 6.9 Hz), 1.33(t,
3H, J = 6.9 Hz); 13C NMR (100 MHz, DMSO-d6) d:
178.6, 172.0, 164.2, 152.9, 150.0, 148.7, 134.2, 131.6,
129.7, 127.8, 125.3, 119.8, 119.5, 119.0,108.5, 107.8; IR
mmax (KBr): 1681.4, 1681.8, 1479.9, 1385.1 cmꢀ1; ESI-
MS m/z: 335.2 [M-1]ꢀ; C18H12N2O4, calcd: C, 64.29%;
H, 3.60%; N, 8.33%; O, 23.79%; found: C, 64.30%; H,
3.61%; N, 8.30%.
1640.6, 1575.3, 1494.2, 1379.3 cmꢀ1
; UV/vis kmax
(EtOH): 250, 326, 467 nm; ESI-MS m/z: 399.0, 401.0
[M+1]+; C18H11BrN2O4, calcd: C, 54.16%; H, 2.78%;
Br, 20.02%; N, 7.02%; O, 16.03%; found: C, 54.15%;
H, 2.80%; Br, 20.00%; N, 7.01%.
4.3.14. Ethyl 10-bromo-5,12-dioxo-5,12-dihydroindolizi-
no[3,2-g]quinoline 11-carboxylate 9b. Orange solid, yield
16%; 1H NMR (300 MHz, CDCl3) d: 9.79 (d, 1H,
J = 6.6 Hz), 9.02 (d, br, 1H, J = 1.8 Hz), 8.51 (d, 1H,
J = 7.5 Hz), 7.65 (dd, 1H, J = 7.8, 4.5 Hz), 7.57 (d, 1H,
J = 6.9 Hz), 7.02 (t, 1H, J = 6.9 Hz), 4.59 (q, 2H,
J = 7.2 Hz), 1.49 (t, 3H, J = 7.2 Hz); ESI-MS m/z:
399.0, 401.0 [M+1]+, 421.1, 423.1 [M+Na]+;
C18H11BrN2O4, calcd: C, 54.16%; H, 2.78%; Br,
20.02%; N, 7.02%; O, 16.03%; found: C, 54.13%; H,
2.81%; Br, 20.01%; N, 7.02%.
4.3.19. Ethyl 7-amino-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 12a. Purple solid, yield
1
24%; mp = 198–200 ꢁC; H NMR (300 MHz, DMSO-
d6) d: 9.16 (d, 1H, J = 6.6 Hz), 8.95 (d, 1H,
J = 3.6 Hz), 8.38 (d, 1H, J = 7.8 Hz), 7.75 (dd, 1H,
J = 7.8, 3.6 Hz), 7.18 (t, 1H, J = 6.9 Hz), 6.70 (d, 1H,
J = 7.5 Hz), 6.04 (s, br, 2H), 4.41 (q, 2H, J = 6.9 Hz),
1.38 (t, 3H, J = 6.9 Hz); IR mmax (KBr): 1718.2,
1673.5, 1621.3, 1573.6, 1494.1, 1392.8 cmꢀ1; UV/vis
kmax (EtOH): 266, 360, 557 nm; ESI-MS m/z: 336.2
[M+1]+; C18H13N3O4, calcd: C, 64.47%; H, 3.91%; N,
12.53%; O, 19.09%; found: C, 64.44%; H, 3.93%; N,
12.51%.
4.3.15. Ethyl 7-methyl-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 10a. Yellow solid, yield
21%; mp (deg.) = 165–170 ꢁC; 1H NMR (300 MHz,
CDCl3) d: 9.73 (d, br, 1H, J = 4.2 Hz), 9.03 (dd, 1H,
J = 4.8, 1.5 Hz), 8.56 (dd, 1H, J = 7.8, 1.5 Hz), 8.23 (d,
1H, J = 9.0 Hz), 7.67 (dd, 1H, J = 7.8, 4.5 Hz), 7.34
(dd, 1H, J = 9.0, 1.5 Hz), 4.50 (q, 2H, J = 6.9 Hz), 2.49
(s, 3H), 1.50 (t, 3H, J = 6.9 Hz); IR mmax (KBr):
4.3.20. Ethyl 10-amino-5,12-dioxo-5,12-dihydroindolizi-
no[3,2-g]quinoline 11-carboxylate 12b. Purple solid, yield
1726.5, 1674.9, 1638.1, 1573.3, 1482.0, 1382.0 cmꢀ1
;
24%; mp = 323 ꢁC; H NMR (300 MHz, DMSO-d6) d:
1
ESI-MS m/z: 335.0 [M+1]+, 357.1 [M+Na]+;
C19H14N2O4, calcd: C, 68.26%; H, 4.22%; N, 8.38%;
O, 19.14%; found: C, 68.25%; H, 4.22%; N, 8.37%.
9.11 (d, 1H, J = 6.6 Hz), 8.92 (dd, 1H, J = 4.5, 1.8 Hz),
8.46 (dd, 1H, J = 7.8, 1.5 Hz), 7.81 (dd, 1H, J = 7.8,
4.8 Hz), 7.19 (dd, 1H, J = 7.5, 6.6 Hz), 6.71 (d, 1H,
J = 7.5 Hz), 4.42 (q, 2H, J = 7.1 Hz), 1.38 (t, 3H,
J = 7.1 Hz); IR mmax (KBr): 1693.1, 1676.3, 1620.3,
1578.8, 1548.4, 1462.2, 1421.6, 1303.2 cmꢀ1; UV/vis kmax
(EtOH): 266, 361, 564 nm; ESI-MS m/z: 336.2 [M+1]+,
358.1 [M+Na]+, 692.9 [2M+Na]+; C18H13N3O4, calcd:
C, 64.47%; H, 3.91%; N, 12.53%; O, 19.09%; found:
C, 64.46%; H, 3.92%; N, 12.52%.
4.3.16. Ethyl 10-methyl-5,12-dioxo-5,12-dihydroindolizi-
no[3,2-g]quinoline 11-carboxylate 10b. Brown solid, yield
1
7%; mp = 160 ꢁC; H NMR (300 MHz, CDCl3) d: 9.52
(d, 1H, J = 6.9 Hz), 8.94 (dd, 1H, J = 4.8, 1.5 Hz), 8.55
(dd, 1H, J = 7.8, 1.8 Hz), 7.65 (dd, 1H, J = 7.8,
4.5 Hz), 7.11 (d, 1H, J = 6.9 Hz), 7.05 (t, 1H,
J = 7.2 Hz), 4.54 (q, 2H, J = 7.2 Hz), 2.54 (s, 3H), 1.48
(t, 3H, J = 7.2 Hz); IR mmax (KBr): 2921.2, 1674.8,
4.3.21. 6-Acetyl-7-fluoro-indolizino[2,3-g]quinoline-5,12-
dione 13a. Yellow solid, yield 18%; mp = 143–145 ꢁC;
1H NMR (300 MHz, CDCl3) d: 9.92 (d, br, 1H,
J = 2.1 Hz), 9.04 (d, 1H, J = 4.5 Hz), 8.54 (d, 1H,
J = 7.8 Hz), 8.49 (d, 1H, J = 9.6 Hz), 7.68 (dd, 1H,
J = 7.5, 4.5 Hz), 7.39 (t, 1H, J = 9.6 Hz), 2.90 (s, 3H);
IR mmax (KBr): 2963.5, 1638.2, 1618.5, 1577.5, 1417.2,
1261.2 cmꢀ1; ESI-MS m/z: 331.0 [M+Na]+; C17H9
FN2O3, calcd: C, 66.24%; H, 2.94%; F, 6.16%; N,
9.09%; O, 15.57%; found: C, 66.24%; H, 2.95%; F,
6.14%; N, 9.06%.
1637.5, 1472.3, 1315.7 cmꢀ1
[M+1]+, [M+Na]+,
357.1
;
ESI-MS m/z: 335.0
690.9
[2M+Na]+;
C19H14N2O4, calcd: C, 68.26%; H, 4.22%; N, 8.38%;
O, 19.14%; found: C, 68.23%; H, 4.23%; N, 8.38%.
4.3.17. Ethyl 7-hydroxy-5,12-dioxo-5,12-dihydroindolizi-
no[2,3-g]quinoline 6-carboxylate 11a. Red solid, yield
1
24%; mp = 221–223 ꢁC; H NMR (300 MHz, DMSO-
d6) d: 9.16 (d, 1H, J = 6.6 Hz), 8.96 (dd, 1H, J = 4.5,
1.5 Hz), 8.38 (dd, 1H, J = 7.8, 1.5 Hz), 7.75 (dd, 1H,
J = 7.8, 4.5 Hz), 7.19 (t, 1H, J = 7.2 Hz), 6.73 (d, 1H,
J = 7.8 Hz), 4.35 (q, 2H, J = 6.9 Hz), 1.33 (t, 3H,
J = 6.9 Hz); 13C NMR (100 MHz, DMSO-d6) d: 179.7,
171.3, 164.2, 153.9, 150.2, 150.0, 134.4, 129.9, 129.5,
127.0, 124.1, 122.0, 121.1, 119.5, 107.9, 107.8, 61.1,
13.9; UV/vis kmax (EtOH): 253, 339, 360, 516 nm; ESI-
MS m/z: 335.2 [M-1]ꢀ; C18H12N2O4, calcd: C, 64.29%;
H, 3.60%; N, 8.33%; O, 23.79%; found: C, 64.29%; H,
3.61%; N, 8.31%.
4.3.22. 11-Acetyl-10-fluoro-indolizino[3,2-g]quinoline-5,12-
dione 13b. Yellow solid, yield 11%; mp = 141–143 ꢁC; 1H
NMR (300 MHz, CDCl3) d: 9.64 (d, 1H, J = 6.6 Hz),
9.06 (d, 1H, J = 4.2 Hz), 8.53 (d, 1H, J = 7.5 Hz), 7.67
(dd, 1H, J = 7.8, 4.5 Hz), 7.17–7.05 (m, 2H), 2.81 (s,
3H); IR mmax (KBr): 2963.7, 1637.3, 1617.3, 1558.5,
1418.4, 1261.4 cmꢀ1; ESI-MS m/z: 308.0 [M+1]+, 331.0
[M+Na]+; C17H9FN2O3, calcd: C, 66.24%; H, 2.94%;

Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
4623
F, 6.16%; N, 9.09%; O, 15.57%; found: C, 66.28%; H,
2.96%; F, 6.13%; N, 9.07%.
9.71 (d, 1H, J = 6.6 Hz), 9.04 (dd, 1H, J = 4.5, 1.5 Hz),
8.51 (dd, 1H, J = 7.8, 1.5 Hz), 7.67 (dd, 1H, J = 7.8,
4.5 Hz), 7.23 (dd, 1H, J = 7.5, 6.6 Hz), 7.01 (d, 1H,
J = 7.5 Hz), 2.96 (s, 3H); IR mmax (KBr): 3415.2,
1963.6, 1637.7, 1618.3, 1576.5, 1541.7, 1456.8,
1261.8 cmꢀ1; ESI-MS m/z: 307.1 [M+1]+; C17H10N2O4,
calcd: C, 66.67%; H, 3.29%; N, 9.15%; O, 20.90%;
found: C, 66.66%; H, 3.31%; N, 9.13%.
4.3.23. 6-Acetyl-7-chloro-indolizino[2,3-g]quinoline-5,12-
dione 14a. Red solid, yield 9%; mp (deg.) = 128–130 ꢁC;
1H NMR (300 MHz, CDCl3) d: 9.88 (d, br, 1H,
J = 1.8 Hz), 9.04 (d, br, 1H, J = 3.9 Hz), 8.54 (d, 1H,
J = 7.8 Hz), 8.42 (d, 1H, J = 9.6 Hz), 7.69 (dd, 1H,
J = 7.5, 4.5 Hz), 7.39 (dd, 1H, J = 9.6, 1.8 Hz), 2.88 (s,
3H); IR mmax (KBr): 2963.6, 1637.8, 1618.7, 1559.2,
4.3.29. 6-Acetyl-7-amino-indolizino[2,3-g]quinoline-5,12-
dione 17a. Purple solid, yield 11%; mp = 155–158 ꢁC;
1H NMR (300 MHz, CDCl3) d: 9.48 (d, 1H,
J = 6.6 Hz), 9.01 (d, 1H, J = 4.5 Hz), 8.50 (d, 1H,
J = 7.8 Hz), 7.62 (dd, 1H, J = 7.8, 4.5 Hz), 7.06 (t, 1H,
J = 6.9 Hz), 6.59 (d, 1H, J = 7.5 Hz), 2.86 (s, 3H); IR
mmax (KBr): 3419.3, 1733.4, 1716.3, 1668.9, 1617.9,
1417.9, 1261.5 cmꢀ1
;
ESI-MS m/z: 324.0, 326.0
[M+1]+, 347.0, 348.2 [M+Na]+; C17H9ClN2O3, calcd:
C, 62.88%; H, 2.79%; Cl, 10.92%; N, 8.63%; O,
14.78%; found: C, 62.87%; H, 2.80%; Cl, 10.90; N,
8.62%.
4.3.24. 11-Acetyl-10-chloro-indolizino[3,2-g]quinoline-5,12-
1542.3, 1489.0, 1396.7 cmꢀ1
; ESI-MS m/z: 306.0
1
dione 14b. Red solid, yield 15%; mp = 135–138 ꢁC; H
[M+1]+, 328.0 [M+Na]+; C17H11N3O3, calcd: C,
66.88%; H, 3.63%; N, 13.76%; O, 15.72%; found: C,
66.86%; H, 3.65%; N, 13.77%.
NMR (300 MHz, CDCl3) d: 9.60 (d, 1H, J = 6.9 Hz),
8.99 (d, 1H, J = 3.6 Hz), 8.57 (d, 1H, J = 7.5 Hz), 7.68
(dd, 1H, J = 7.5, 4.5 Hz), 7.34 (d, 1H, J = 7.2 Hz), 7.07
(t, 1H, J = 7.2 Hz), 2.82 (s, 3H); IR mmax (KBr):
4.3.30. 11-Acetyl-10-amino-indolizino[3,2-g]quinoline-5,12-
dione 17b. Purple solid, yield 18%; mp = 138–140 ꢁC; 1H
NMR (300 MHz, CDCl3) d: 9.37 (d, 1H, J = 6.3 Hz),
8.97 (d, 1H, J = 3.9 Hz), 8.58 (d, 1H, J = 8.1 Hz), 7.67
(dd, 1H, J = 8.1, 4.8 Hz), 7.05 (t, 1H, J = 7.5 Hz), 6.58
(d, 1H, J = 7.8 Hz), 2.89 (s, 3H); IR mmax (KBr):
1637.4, 1618.9, 1558.3, 1541.8, 1418.6, 1260.1 cmꢀ1
;
ESI-MS m/z: 324.0, 326.0 [M+1]+; C17H9ClN2O3, calcd:
C, 62.88%; H, 2.79%; Cl, 10.92%; N, 8.63%; O, 14.78%;
found: C, 62.88%; H, 2.80%; Cl, 10.89; N, 8.61%.
4.3.25. 6-Acetyl-7-bromo-indolizino[2,3-g]quinoline-5,12-
3414.7, 1716.3, 1637.1, 1618.2, 1541.9, 1457.1 cmꢀ1
;
1
dione 15a. Red solid, yield 12%; mp = 165–168 ꢁC; H
ESI-MS m/z: 328.0 [M+Na]+; C17H11N3O3, calcd: C,
66.88%; H, 3.63%; N, 13.76%; O, 15.72%; found: C,
66.88%; H, 3.66%; N, 13.75%.
NMR (300 MHz, CDCl3) d: 9.65 (d, 1H, J = 6.9 Hz),
9.03 (d, 1H, J = 3.9 Hz), 8.56 (d, 1H, J = 7.8 Hz), 7.65
(d, 1H, J = 7.5 Hz), 7.53 (dd, 1H, J = 7.5, 4.5 Hz), 6.99
(t, 1H, J = 7.2 Hz), 2.87 (s, 3H); IR mmax (KBr): 1702.7,
1684.8, 1636.2, 1557.9, 1508.3, 1489.2, 1399.3,
1250.9 cmꢀ1; ESI-MS m/z: [M+1]+; C17H9BrN2O3, calcd:
C, 55.31%; H, 2.46%; Br, 21.64%; N, 7.59%; O, 13.00%;
found: C, 55.31%; H, 2.47%; Br, 21.63%; N, 7.57%.
4.4. Preparation of 5-aminoisoquinoline 19
Isoquinoline (3.0 g, 0.023 mol) was dissolved in concen-
trated H2SO4 (15 mL), a considerable amount of heat
being evolved, and the solution was then cooled to
0 ꢁC and stirred mechanically while KNO3 (2.4 g,
0.024 mol) in H2SO4 (170 mL) was added dropwise over
2 h. After being kept below 50 ꢁC for 4 h, the reaction
was poured into ice and neutralized with cold aqueous
ammonia. Filtration and recrystallization from ethanol
gave the yellow solid 5-nitroisoquinoline 18: 1.9 g, yield
48%; mp = 108–111 ꢁC (lit.21 108–110 ꢁC).
4.3.26. 11-Acetyl-10-bromo-indolizino[3,2-g]quinoline-5,12-
1
dione 15b. Red solid, yield 17%; mp = 180–182 ꢁC; H
NMR (300 MHz, DMSO-d6) d: 9.67 (d, 1H,
J = 6.9 Hz), 8.91 (d, 1H, J = 4.5 Hz), 8.56 (d, 1H,
J = 8.1 Hz), 7.86 (dd, 1H, J = 7.5, 4.5 Hz), 7.71 (d, 1H,
J = 7.5 Hz), 7.13 (t, 1H, J = 7.2 Hz), 2.80 (s, 3H); IR
mmax (KBr): 2963.2, 1637.3, 1611.6, 1558.2, 1541.8,
1418.4,
1261.3 cmꢀ1
;
ESI-MS
m/z:
[M+1]+;
5-Nitroisoquinoline 18 (2.5 g, 0.014 mol) and palladium
charcoal (10%, 0.13 g) in EtOH (35 mL) was stirred at
room temperature for 4 h in hydrogen atmosphere.
The catalyst was removed by filtration. The filtrate
was evaporated under vacuum, and the residue was
purified by silica gel chromatography to give 19: 2.0 g,
yield 96.6%; mp = 128–129 ꢁC (lit.22 128–129 ꢁC).
C17H9BrN2O3, calcd: C, 55.31%; H, 2.46%; Br,
21.64%; N, 7.59%; O, 13.00%; found: C, 55.30%; H,
2.50%; Br, 21.61%; N, 7.56%.
4.3.27. 6-Acetyl-7-hydroxy-indolizino[2,3-g]quinoline-5,12-
dione 16a. Purple solid, yield 5%; H NMR (300 MHz,
1
CDCl3) d: 12.42 (s, 1H), 9.58 (d, 1H, J = 6.6 Hz), 9.09
(d, br, 1H, J = 2.4 Hz), 8.56 (dd, 1H, J = 7.8, 1.8 Hz),
8.12 (d, br, 1H, J = 5.1 Hz), 7.94 (dd, br, 1H, J = 8.7,
1.2 Hz), 7.72 (dd, 1H, J = 8.7, 5.7 Hz), 2.98 (s, 3H);
ESI-MS m/z: 307.1 [M+1]+; C17H10N2O4, calcd: C,
66.67%; H, 3.29%; N, 9.15%; O, 20.90%; found: C,
66.65%; H, 3.32%; N, 9.14%.
4.5. Preparation of 2,3-dichloro-1,4-naphtoquinone 21
1,4-Naphthoquinone (5.0 g, 0.030 mol) was dissolved in
ethyl acetate (150 mL) at 50 ꢁC. The solution was added
with concentrated HCl (150 mL) and Bu4NBr (0.5 g),
and then stirred. Sodium chlorate (10.6 g, 0.10 mol)
was added to the solution over 15 min, and stirred for
20 h. The precipitate was removed by filtration, and
the filtrate was washed with water. The organic layer
was concentrated under vacuum. The residue was crys-
4.3.28. 11-Acetyl-10-hydroxy-indolizino[3,2-g]quinoline-
5,12-dione 16b. Purple solid, yield 28%; mp = 200–
1
203 ꢁC; H NMR (300 MHz, CDCl3) d: 12.33 (s, 1H),

4624
Y. Cheng et al. / Bioorg. Med. Chem. 16 (2008) 4617–4625
tallized in BuOH (20 mL) to give the yellow solid 21:
5.3 g, yield 73%; mp = 194–195 ꢁC (lit.23 195 ꢁC).
DMSO (100 lL solvent including 10% SDS, 5% isopro-
panol and 0.01 mol/L HCl as for HL-60) for optical den-
sity reading at 492 nm.
4.6. Preparation of regioisomers 22a and 22b
4.10. TOP1 inhibitory assay
According to the general cyclization procedure (Step
4.3), using 6,7-dichloro isoquinoline-5,8-dione as mate-
rial instead of substrate 1, the reaction gave inspera-
tive regioisomers.15 22a (red solid mixture with 22b):
1H NMR (300 MHz, CDCl3) d: 9.71 (d, 1H,
J = 7.2 Hz), 9.34 (s, 1H), 8.91 (d, 1H, J = 5.1 Hz),
8.23 (d, 1H, J = 9.0 Hz), 7.89 (d, 1H, J = 5.1 Hz),
7.38 (t, 1H, J = 8.1 Hz), 7.12 (t, 1H, J = 6.6 Hz),
4.51 (q, 2H, J = 7.2 Hz), 1.50 (t, 3H, J = 7.2 Hz).
22b (red solid mixture with 22a): 1H NMR
(300 MHz, CDCl3) d: 9.68 (d, 1H, J = 7.2 Hz), 9.31
(s, 1H), 8.91 (d, 1H, J = 5.1 Hz), 8.23 (d, 1H,
J = 9.0 Hz), 7.91 (d, 1H, J = 4.8 Hz), 7.40 (t, 1H,
J = 7.5 Hz), 7.14 (t, 1H, J = 6.0 Hz), 4.51 (q, 2H,
J = 7.2 Hz), 1.50 (t, 3H, J = 7.2 Hz). ESI-MS m/z
(mixture): 321.0 [M+1]+.
The topoisomerase I relaxation assay was performed as
mentioned in the reported method.19,20 Briefly, relaxa-
tion of 0.5 lg plasmid pBR322 was performed in
20 lL of topoisomerase I relaxing buffer (20 mM Tris
pH 7.5, 0.1 mM EDTA, 10 mM MgCl2, 50 lg/mL acet-
ylated BSA, 100 mM KCl) in the presence or the ab-
sence of the test drugs previously dissolved in DMSO
solution. Reactions were started by the addition of
DNA, control groups were either DNA alone or
DNA treated with TOP1. The drugs and DNA in the
presence of TOP1 was incubated for 30 min at 37 ꢁC.
The reaction was terminated by the addition of 4 lL
6· loading buffer. Products were then run in a 1% aga-
rose gel in 45 mM Tris–borate (pH 8.0), 1 mM EDTA
at 5 V/cm. Gels were stained with ethidium bromide
and visualized with a UV transilluminator. Images
were acquired and quantified through AlphaEaseFC
software.
4.7. Preparation of Ethyl 6,11-dioxo-6,11-dihydro-
benzo[f]-pyrido[1,2-a]indole 12-carboxylate 23
According to the general cyclization procedure (Step
4.3), using 2,3-dichloro-1,4-naphtoquinone as material
instead of substrate 1, the reaction gave pure 23. Or-
ange-brown solid, yield 27%; 1H NMR (300 MHz,
CDCl3) d: 9.82 (d, 1H, J = 7.2 Hz), 8.29 (d, 1H,
J = 9.0 Hz), 8.21–8.18 (m, 2H), 7.70 (d, 1H,
J = 5.7 Hz), 7.69 (d, 1H, J = 7.2 Hz), 7.41 (t, 1H,
J = 8.1 Hz), 7.14 (td, 1H, J = 7.2, 0.9 Hz), 4.51 (q, 2H,
J = 7.2 Hz), 1.50 (t, 3H, J = 7.2 Hz);24 IR mmax (KBr):
1673.3, 1638.2, 1591.8, 1509.8, 1321.3 cmꢀ1; ESI-MS
m/z: 320.1 [M+1]+, 342.2 [M+Na]+, 660.9 [2M+Na]+.
Acknowledgments
This work was supported by the NSFC/RGC Joint Re-
search Scheme (Grants 20710006), Natural Science
Foundation of China (20772159) and Guangdong Nat-
ural Science Foundation (4300235).
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