Synthesis and antiproliferative activity of indolizinophthalazine-5,12- dione derivatives, DNA topoisomerase IB inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.05.048

A series of novel indolizinophthalazine-5,12-dione derivatives were designed and synthesized by the reaction of 6,7-dichlorophthalazine-5,8-dione with active methylene reagents (AMR) and pyridine derivatives. Some of synthesized compounds exhibited significant in vitro antiproliferative activity at micromolar level toward four human tumor cell lines, including lung adenocarcinoma cell, large-cell lung carcinoma cell, breast carcinoma cell and ardriamycin-resistance breast carcinoma cell. The DNA topoisomerase IB inhibitory assay indicated that DNA topoisomerase IB might be a biological target of the synthesized compounds. A series of novel indolizinophthalazine-5, 12-dione derivatives were synthesized. Their in vitro antiproliferative activity toward four human tumor cell lines and DNA topoisomerase IB inhibitory activity was investigated.

Full text of DOI:10.1016/j.ejmech.2010.05.048

European Journal of Medicinal Chemistry 45 (2010) 3938e3942
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiproliferative activity of indolizinophthalazine-5,12-dione
derivatives, DNA topoisomerase IB inhibitors
*
De-Qing Shen, Zu-Ping Wu, Xi-Wei Wu, Zeng-Yun An, Xiang-Zhang Bu, Lian-Quan Gu ,
*
Zhi-Shu Huang, Lin-Kun An
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, 510275, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 April 2010
Received in revised form
A series of novel indolizinophthalazine-5,12-dione derivatives were designed and synthesized by the
reaction of 6,7-dichlorophthalazine-5,8-dione with active methylene reagents (AMR) and pyridine
derivatives. Some of synthesized compounds exhibited significant in vitro antiproliferative activity at
micromolar level toward four human tumor cell lines, including lung adenocarcinoma cell, large-cell lung
carcinoma cell, breast carcinoma cell and ardriamycin-resistance breast carcinoma cell. The DNA top-
oisomerase IB inhibitory assay indicated that DNA topoisomerase IB might be a biological target of the
synthesized compounds.
2
4 May 2010
Accepted 25 May 2010
Available online 1 June 2010
Keywords:
Indolizinophthalazine-5,12-dione
derivatives
Ó 2010 Published by Elsevier Masson SAS.
Antiproliferative activity
DNA topoisomerase IB
Inhibitor
1. Introduction
could specifically inhibit the activity of TOP1 to cleave single strand
DNA [15]. Preliminary structure-activity relationship (SAR) assess-
DNA topoisomerase IB (TOP1) regulates DNA topological struc-
ture by binding to the duplex DNA, transient DNA single strand
breakage and relegation [1e4], and therefore can be used as
a potent target for anticancer agent design and anticancer drug
screening [5e7]. Comptothecin (CPT) is a well known TOP1 inhib-
itor that can reversibly trap TOP1-DNA cleavage complexes [8], and
two water-soluble comptothecin derivatives, topotecan and irino-
tecan, have been approved by FDA as anticancer drugs [6,9,10].
However, the drugs are substrates of efflux transporters associated
with resistance, which has pushed further discovery of novel TOP1
inhibitors [11e13].
ment indicated that the existence of nitrogen in A ring of IQDs
would be essential for their activity [14]. Furthermore, studies from
other groups also supported that the number and position of
nitrogen atom in heterocyclic quinines had very significant effect
on the bioactivity of the compounds [16e18]. Therefore, this study
would focus on the design, synthesis and biological evaluation of
indolizinophthalazine-5,12-dione derivatives (IPDs), a kind of IQD-
type compounds with two nitrogen atoms in A ring.
2. Results and discussion
In our previous work, we have shown that indolizinoquinoline-
5
,12-dione derivatives (IQDs) acted as potential TOP1 inhibitors,
2.1. Chemistry
and these compounds have showed significant cytotoxic potency
against several cancer cell lines [14]. Further investigation has
indicated that IQDs were a novel type of TOP1 inhibitors which
Synthesis of IPDs was shown in Scheme 1. The key intermediate,
6,7-dichlorophthalazine-5,8-dione (4), was prepared according to
Yoo’s method with modification [19]. Briefly, phthalazine (1) was
nitrated with concentrated HNO
3
, followed by amination and
Abbreviations: IPDs, indolizinophthalazine-5,12-dione derivatives; IQDs, indo-
lizinoquinoline-5,12-dione derivatives; AMR, active methylene reagents; EAA, ethyl
acetylacetate; MAA, methyl acetylacetate; AA, acetylacetone; ECA, ethyl cyanoa-
cetate; NE, nitroethane; CPT, comptothecin; TOP1, DNA topoisomerase IB.
catalytic hydrogenation to afford 5,8-diaminophthalazine (3).
Substrate 4 was then obtained through chloro-oxidation of
substrate 3 with NaClO in concentrated hydrochloride acid. IPDs
3
were prepared by a single-step heterocyclic reaction of 6,7-
dichlorophthalazine-5,8-dione (4) with active methylene reagents
(AMR) and pyridine derivatives [20]. The products were purified by
*
Corresponding authors. Tel./fax: þ86 20 39943052.
E-mail addresses: cesglq@mail.sysu.edu.cn (L.-Q. Gu), lssalk@mail.sysu.edu.cn
(
L.-K. An).
0223-5234/$ e see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.05.048

D.-Q. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3938e3942
3939
Scheme 1. Synthesis of indolizinophthalazine-5,12-dinone derivatives.
silica gel flash column chromatography and were fully character-
ized by mass spectra, elemental analysis and NMR spectroscopy.
Using 3-substituted pyridine derivative instead of pyridine as
reagent, the heterocyclic reaction would mainly gave the corre-
sponding 10-substituted product. Exceptionally, the reaction of 4 with
activity. While the two nitrogen atoms existing on
b
-position of A
ring, the electron-withdrawing substituent such as alkoxycarbonyl
group at the C-ring could not increase activity. For example, 5a, 5b
and 5c exhibited moderate or poor antiproliferative activity against
the four cancer cell lines. Similar to indolizinoquinoline-5,12-dione
derivatives and indolizinoquinoxaline-5,12-dione derivatives, the
electron-withdrawing substituent would increase the activity. The
solubility of compound 5g in dimethyl sulfoxide was so poor that its
antiproliferative activity could not be determined.
TOP1 inhibitory activity of IPDs was assayed by measurement of
TOP1-mediated DNA relaxation assay. Comparing with CPT,
synthesized compounds exhibited moderate to more potent
inhibitory activity than CPT. It was worth noting that compound 5d
showed significant antiproliferative activity and stronger TOP1
inhibitory activity than CPT, shown as Fig. 2A. About 23% of TOP1
catalytic activity was inhibited after being treated with 1
and only slight TOP1 catalytic activity was inhibited by 1
(Fig. 2B). After being treated at 125 mM concentration, 5d and CPT
could inhibit 100% and 73% of TOP1 catalytic activity, respectively.
The result indicated that 5d was a stronger TOP1 inhibitor than CPT.
Similar to IPDs, the indolizinoquinoxaline-5,12-dione derivatives,
3-methylpyridine and ethyl acetoacetate gave two major products, 5j
and 6, at the same time. Mass spectra and elemental analysis indicated
1
that 5j and 6 had same formula, C18
of 5j, the doubletsignalat9.53ppm(J¼ 6.8Hz)couldbe assignedtoH-
, and the signals at 7.17 (d, J ¼ 6.8 Hz) and 7.12 (t, J ¼ 6.8 Hz) could be
13 3 4
H N O . In the H NMR spectrum
7
assigned to H-9 and H-8, respectively. Therefore, 5j could be assigned
as ethyl 10-methyl-5,12-dioxo-5,12-dihydroindolizino[2,3-g]phthala-
zine-11-carboxylate, a 10-substituted product. Distinctly, H-7 of
compound 6 appeared at 9.59 ppm as a broaden singlet signal. A
doublet signal in AB type at 8.26 (J ¼ 9.2 Hz) could be assigned to H-10,
and the double doublet signal in AB type at 7.37 (J ¼ 9.2, 1.2 Hz) could
be assigned to H-9. On the basis of spectra, compound 6 could be
assigned as ethyl 8-methyl-5,12-dioxo-5,12-dihydroindolizino[2,3-g]
phthalazine-11-carboxylate, a 8-substituted product shown as Fig. 1.
mM of 5d,
mM of CPT
2.2. Biological activities
in which two nitrogen atoms exist on
inhibitory activity to TOP1 [21]. However, indolizinoquinoxaline-
,12-dione derivatives exhibited lower or potent activity as CPT.
a-position of A ring, exhibited
In vitro biological evaluation of the synthesized IPDs was
5
performed by MTT assay using four different human cancer cell
lines, including lung adenocarcinoma cell (GLC-82), large-cell lung
carcinoma cell (NCI-H460), breast carcinoma cell (MCF-7) and
ardriamycin-resistance breast carcinoma cell (MCF-7/ARD). The
assessment of antiproliferative activity was expressed as
the concentration inhibiting 50% of cancer cell growth (IC50). The
results summarized in Table 1 showed that some of IPDs (5d, 5e, 5h
and 5j) exhibited significant activity toward four human cancer cell
lines in low micromolar range. Similar to indolizinoquinoxaline-
Table 1
In vitro antiproliferative activity of synthesized compounds.
Compounds
Antiproliferative activity IC50
(
m
M)a
MCF-7
GLC-82
NCI-H460
MCF-7/ARD
5a
>25.00
>25.00
>25.00
>25.00
5
5
5
5
5
5
5
5
5
5
6
b
c
d
e
f
g
h
i
13.67 ꢀ 1.22
24.74 ꢀ 4.17
1.13 ꢀ 0.23
1.92 ꢀ 0.81
10.77 ꢀ 1.02
9.94 ꢀ 0.97
21.29 ꢀ 3.34
9.32 ꢀ 0.63
1.21 ꢀ 0.42
11.38 ꢀ 0.61
–
23.70 ꢀ 2.26
17.00 ꢀ 1.85
11.22 ꢀ 2.11
0.81 ꢀ 0.48
14.10 ꢀ 0.87
11.73 ꢀ 1.37
–
b
nd
5
,12-dione derivatives [21], the enhancement of number of
1.09 ꢀ 0.44
1.67 ꢀ 0.16
14.70 ꢀ 0.77
–
nitrogen atom at the A ring was not beneficial to antiproliferative
c
–
1.09 ꢀ 0.28
11.59 ꢀ 0.96
10.32 ꢀ 1.79
1.85 ꢀ 0.95
9.87 ꢀ 1.32
>25.00
1.44 ꢀ 0.46
23.5 ꢀ 2.44
1.18 ꢀ 0.37
10.60 ꢀ 1.33
11.11 ꢀ 1.08
>25.00
1.20 ꢀ 0.22
20.8 ꢀ 2.10
1.22 ꢀ 0.84
10.65 ꢀ 0.89
12.22 ꢀ 0.97
nd
1.60 ꢀ 0.87
>25.00
j
k
1.55 ꢀ 0.41
13.23 ꢀ 1.62
18.67 ꢀ 1.78
nd
CPT
a
IC50 values, defined as the concentration that inhibited growth by 50%. Every
experiment was repeated at least three times.
b
“
nd” means “not determined”.
c
Fig. 1. The structure of compound 6.
“–” means “not determined because of poor solubility of the compound”.

3940
D.-Q. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3938e3942
Fig. 2. Inhibition of TOP1 relaxation activity of 5d (A) and CPT (B). SC, supercoiled DNA; R, relaxed DNA. Lane 1 was DNA control without enzyme and drugs. Lane 2 was the control
of DNA and enzyme without drugs. Lanes 3e6 contained enzyme, DNA and serially diluted drugs from 1 to 125 M.
m
Those results indicated that the aza-naphthindolizinedione deriv-
ative might be a kind of compound targeted to TOP1.
4.3. Synthesis of 5,8-diaminophthalazine (3)
According to Kim’s method [19], successful introduction of
amino group to 5-nitrophthalazine gave 5-nitro-8-amino-phtha-
lazine, which (2.6 g, 13.7 mmol) was dissolved in methanol
3
. Conclusion
(
2
150 mL) and hydrogenated under 1 atm of H at room temperature
In summary, a series of novel indolizinoquinoline-5,12-dione
using Pd/C (10%, 0.3 g) as catalyst. The filtrate was concentrated to
about 7 mL after the removal of the catalyst, and the residue was
chilled to give gray solid product 3 (2.1 g), yield 95.8%; H NMR
(
derivatives were designed and synthesized. Some of synthesized
compounds exhibited significant antiproliferative activity toward
four human cancer cell lines or great TOP1 inhibitory activity.
Among them, compound 5d exhibited well consistent anti-
proliferative activity and TOP1 inhibitory activity. This study indi-
cated that the indolizinoquinoline-5,12-dione structure might
serve as a potential pharmacophore for the design of anticancer
chemical entities, and TOP1 might be a biological target.
1
6
300 MHz, DMSO-d ) d: 9.52 (s, 2H), 6.92 (s, 2H), 5.45 (s, 4H); ESI-
þ
MS m/z: 161.1 [Mþ1] .
4.4. Synthesis of 6,7-dichlorophthalazine-5,8-dione (4)
4
. Experimental section
Chlorination-oxidation of substrate 3 was carried out using
NaClO
3
in concentrated HCl according to our previous method to
ꢁ
4.1. General experimental
give yellow crystal 4, yield 54.6%; mp ¼ 223e225 C (lit [19].
ꢁ
1
2
23e225 C); H NMR (300 MHz, DMSO-d
6
)
d
: 9.84 (s, 2H); ESI-MS
ꢂ
CPT was purchased from National Institute For The Control Of
m/z: 227.9 (100%), 229.9 (65%), 231.9 (10%) [M] .
Pharmaceutical And Biological Products. All chemicals and solvents
were obtained from commercial suppliers and used without further
purification. Chemical reactions were monitored by self-prepared
silica gel GF254 plates. Flash column chromatography was per-
formed with silica gel 200e300 meshes. Calf thymus top-
oisomerase I and plasmid pBR322 were purchased from TakaRa
Biotechnology (Dalian) Co., Ltd and used without further purifica-
tion. Melting points were determined using a XT-4 apparatus and
were uncorrected. Nuclear magnetic resonance spectra were
measured on a Varian Mercury-Plus 300 MHz or Bruker UltraShield
4
.5. General synthesis procedure of indolizinophthalazine-5,8-
dione derivatives (5ae5k and 6)
The dichlorosubstituted substrate 4 (503.8 mg, 2.2 mmol) was
dissolved in absolute ethanol (50 mL). The solution was heated to
6
ꢁ
0
C. And then, the pyridine derivative (6.8 mmol) and AMR
(2.6 mmol), such as ethyl acetoacetate, methyl acetoacetate, ethyl
cyanoacetate, acetylacetone, nitromethane or nitroethane, were
added dropwise. The reaction mixture was refluxed for 4 h, and the
dark-reddish suspension was concentrated in vacuo. The residue
was then subjected to flash column chromatography (silica gel) to
give the corresponding target compounds.
4
00 MHz spectrometer. Chemical shifts were reported in parts per
million ( ) relative to tetramethylsilane (TMS) as internal standard.
d
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 interface was used. Elemental analysis was
performed on an Elementar Vario EL elemental analysis device. UV
4.5.1. Synthesis of ethyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]
absorption was recorded on
spectrophotometer.
a
SHIMADZU UV-2501 PC
phthalazine-11-carboxylate (5a)
According to the “General synthesis procedure”, compound 4,
pyridine (0.6 mL, 6.8 mmol) and ethyl acetoacetate (0.3 mL,
2.6 mmol) were used as materials, and a mixture of petroleum
ether and ethyl acetate (11:9) was used as eluent. Red solid 5a
4.2. Synthesis of 5-nitrophthalazine (2)
ꢁ
1
The preparation was carried out according to Shaikh’s method
18]. Briefly, KNO (12.1 g, 119.8 mmol) was added to a solution of
(196.0 mg) was obtained, yield 27.3%; mp ¼ 219e220 C, H NMR
(300 MHz, CDCl
: 9.94 (s, 1H), 9.87 (s, 1H), 9.80 (d, 1H, J ¼ 6.9 Hz),
8.41 (d, 1H, J ¼ 9.0 Hz), 7.57 (dd, 1H, J ¼ 9.0, 6.9 Hz), 7.31 (td, 1H,
[
3
3
) d
phthalazine (5.2 g, 40.0 mmol) in concentrated H
2
SO
4
(98%, 36 mL)
ꢁ
ꢁ
13
in small portion at 0 C. The mixture then was heated to 70e80 C
for 15 h, cooled and diluted with distilled water. The solution was
treated with aqueous NaOH (10 M) to give yellow precipitate.
J ¼ 6.9, 0.9 Hz), 4.52 (q, 2H, J ¼ 7.2 Hz), 1.51 (t, 3H, J ¼ 7.2 Hz);
NMR (100 MHz, CDCl : 178.0, 171.4, 161.5, 145.8, 145.7, 139.3,
128.5, 127.4, 126.4, 125.4, 125.2, 120.6, 120.5, 117.9, 106.2, 60.5, 13.3;
C
3
) d
Recrystallization from ethanol gave yellow crystals 2 (4.5 g), yield
UV/vis
l
max (EtOH,
e
): 206 (22500), 248 (31200), 332 (9800), 500
1
þ
6
8
4.3%; H NMR (300 MHz, MDSO-d
6
)
d
: 10.15 (s, 1H), 9.88 (s, 1H),
(4500) nm; ESI-MS m/z: 322.3 [Mþ1] ; C17
11 3 4
H N O , calcd: C,
.81 (d, 1H, J ¼ 7.8 Hz), 8.60 (d, 1H, J ¼ 8.1 Hz), 8.23(t, 1H, J ¼ 8.1 Hz);
63.55%; H, 3.45%; N, 13.08%; O, 19.92%; found: C, 63.58%; H, 3.47%;
N, 13.07%.
þ
ESI-MS m/z: 176.1 [Mþ1] .

D.-Q. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3938e3942
3941
4.5.2. Synthesis of methyl 5,12-dioxo-5,12-dihydroindolizino[2,3-g]
were used as reagents, and a mixture of petroleum ether and ethyl
phthalazine-11-carboxylate (5b)
According to the “General synthesis procedure”, compound 4,
pyridine (0.6 mL, 6.8 mmol) and methyl acetoacetate (0.3 mL,
acetate (1:1) was used as eluent. Red solid 5f (42.1 mg) was
ꢁ
1
obtained, yield 7.7%; mp > 300 C, H NMR (300 MHz, CDCl ) d: 9.99
3
(s, 1H), 9.88 (s, 1H), 9.72 (dd, 1H, J ¼ 6.9, 0.9 Hz), 7.88 (dd,1H, J ¼ 9.0,
0.9 Hz), 7.50 (dd, 1H, J ¼ 9.0, 6.9 Hz), 7.32 (t, 1H, J ¼ 6.9 Hz), 7.26 (s,
2
.6 mmol) were used as reagents, and a mixture of petroleum ether
and ethyl acetate (1:1) was used as eluent. Red solid 5b (216.8 mg)
1H); UV/vis
l
max (EtOH,
e
): 210 (8600), 256 (9600), 339 (2900), 526
ꢁ
1
þ
was obtained, yield 31.8%; mp > 300 C, H NMR (300 MHz, CDCl
: 9.94 (d, 1H, J ¼ 1.2 Hz), 9.86 (d, 1H, J ¼ 1.2 Hz), 9.80 (d, 1H,
J ¼ 6.9 Hz), 8.41 (d, 1H, J ¼ 9.0 Hz), 7.57 (dd, 1H, J ¼ 9.0, 6.9 Hz), 7.32
3
)
(1300) nm; ESI-MS m/z: 250.1 [Mþ1] ; C14
7 3 2
H N O , calcd: C, 67.47%;
d
H, 2.83%; N,16.86%; O,12.84%; found: C, 68.44%; H, 2.85%; N,16.88%.
13
(
1
1
td, 1H, J ¼ 6.9, 1.2 Hz), 4.06 (s, 3H); C NMR (100 MHz, CDCl
78.0, 171.4, 161.9, 145.8, 145.6, 139.3, 128.6, 127.4, 126.4, 125.4,
25.1, 120.7, 120.5, 118.0, 105.6, 51.3; UV/vis max (EtOH, ): 206
3
)
d:
4.5.7. Synthesis of ethyl 10-fluoro-5,12-dioxo-5,12-dihydroindo-
lizino[2,3-g]phthalazine-11-carboxylate (5g)
l
e
According to the “General synthesis procedure”, compound 4,
3-fluoropyridine (0.6 mL, 6.8 mmol) and ethyl acetoacetate (0.3 mL,
2.6 mmol) were used as reagents, and a mixture of petroleum ether
and ethyl acetate (3:2) was used as eluent. Red solid 5g (156.6 mg)
(
3
2
30200), 248 (34700), 331 (9900), 502 (4800) nm; ESI-MS m/z:
þ
9 3 4
08.1 [Mþ1] ; C16H N O , calcd: C, 62.54%; H, 2.95%; N, 13.68%; O,
0.83%; found: C, 62.52%; H, 2.97%; N, 13.67%.
ꢁ
1
was obtained, yield 21.0%; mp ¼ 160e162 C, H NMR (300 MHz,
CDCl
: 9.97 (s, 1H), 9.86 (s, 1H), 9.50 (d, 1H, J ¼ 6.6 Hz), 7.22e7.13
(m, 2H), 4.54 (q, 2H, J ¼ 7.2 Hz), 1.47 (t, 3H, J ¼ 7.2 Hz); C NMR
4.5.3. Synthesis of 11-acetylindolizino[2,3-g]phthalazine-5,12-
3
) d
13
dione (5c)
According to the “General synthesis procedure”, compound 4,
pyridine (0.6 mL, 6.8 mmol) and acetylacetone (0.3 mL, 2.6 mmol)
were used as reagents, and a mixture of petroleum ether and ethyl
acetate (1:1) was used as eluent. Violet solid 5c (264.0 mg) was
1
(100 MHz, CDCl
3
)
d
: 178.3, 171.1, 161.5, 153.7d ( JCeF ¼ 255.9 Hz),
2
145.9, 145.5, 128.3d ( JCeF ¼ 32.2 Hz), 125.8, 125.0, 124.6, 123.6d
4
3
( JCeF
¼
5.4 Hz), 120.2, 117.2d ( JCeF
¼
6.8 Hz), 110.6d
2
3
( JCeF ¼ 17.2 Hz), 107.5d ( JCeF ¼ 4.4 Hz), 61.6, 13.0; ESI-MS m/z:
ꢁ
1
þ
obtained, yield 40.9%; mp ¼ 226e227 C, H NMR (300 MHz, CDCl
: 9.97 (s, 1H), 9.87 (s, 1H), 9.82 (d, 1H, J ¼ 6.9 Hz), 8.50 (d, 1H,
J ¼ 9.0 Hz), 7.59 (t,1H, J ¼ 7.8 Hz), 7.34 (t,1H, J ¼ 6.9 Hz), 2.88 (s, 3H);
3
)
340.0 [Mþ1] ; C17
3 4
H10FN O , calcd: C, 60.18%; H, 2.97%; F, 5.60%; N,
d
12.39%; O, 18.86%; found: C, 60.15%; H, 2.99%; F, 5.61%; N, 12.43%.
13
C NMR (100 MHz, CDCl
3
)
d
: 195.1, 180.0, 171.3, 145.7, 145.6, 139.0,
4.5.8. Synthesis of ethyl 10-chloro-5,12-dioxo-5,12-dihydroindo-
lizino[2,3-g]phthalazine-11-carboxylate (5h)
129.3, 127.2, 125.5, 125.2, 124.9, 121.2, 119.8, 118.5, 114.4, 30.8; UV/
vis
l
max (EtOH,
e
): 205 (20200), 252 (19700), 335 (6100), 511
According to the “General synthesis procedure”, compound 4,
3-chloropyridine (0.7 mL, 6.8 mmol) and ethyl acetoacetate (0.3 mL,
2.6 mmol) were used as reagents, and a mixture of petroleum ether
and ethyl acetate (3:2) was used as eluent. Red solid 5h (101.0 mg)
ꢂ
(
2600) nm; ESI-MS m/z: 291.0 [M] ; C16
9
H N
3
O
3
, calcd: C, 65.98%; H,
3
.11%; N, 14.43%; O, 16.48%; found: C, 65.97%; H, 3.13%; N, 14.41%.
ꢁ
1
4.5.4. 5,12-Dioxo-5,12-dihydroindolizino[2,3-g]phth-alazine-11-
was obtained, yield 13.0%; mp ¼ 220e222 C, H NMR (300 MHz,
DMSO-d
: 9.92 (s,1H), 9.80 (s,1H), 9.57 (d,1H, J ¼ 6.9 Hz), 7.81 (d,
1H, J ¼ 7.8 Hz), 7.48 (t, 1H, J ¼ 7.2 Hz), 4.50 (q, 2H, J ¼ 7.2 Hz), 1.43 (t,
carbonitrile (5d)
6
) d
According to the “General synthesis procedure”, compound 4,
pyridine (0.6 mL, 6.8 mmol) and ethyl cyanoacetate (0.3 mL,
13
3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl
146.0, 145.3, 132.6, 129.9, 127.8, 126.6, 126.2, 125.6, 124.3, 119.2,
117.1, 109.6, 61.8, 13.0; UV/vis max (EtOH, ): 216 (6700), 253
3
) d: 178.5, 170.8, 162.6,
2
.6 mmol) were used as reagents, and a mixture of dichloro-
methane and methanol (100:1) was used as eluent. Red solid 5d
l
e
ꢁ
1
(
(
232.0 mg) was obtained, yield 38.5%; mp > 300 C, H NMR
400 MHz, DMSO-d
: 9.92 (d, 1H, J ¼ 1.2 Hz), 9.83 (d, 1H,
(15100), 328 (4500), 360 (2800), 499 (2300) nm; ESI-MS m/z: 355.0
ꢂ
6
)
d
(100%), 357.0 (34%) [M] ; C17
H10ClN
3
O
4
, calcd: C, 57.40%; H, 2.83%;
J ¼ 1.2 Hz), 9.64 (d, 1H, J ¼ 7.2 Hz), 8.17 (d, 1H, J ¼ 8.8 Hz), 7.85 (t, 1H,
Cl, 9.97%; N, 11.81%; O, 17.99%; found: C, 57.40%; H, 2.84%; Cl, 9.94%;
N, 11.83%.
13
J ¼ 7.8 Hz), 7.63 (t, 1H, J ¼ 6.6 Hz); C NMR (100 MHz, 90%
CDCl CO D) : 175.5, 167.9, 146.6, 146.0, 141.5, 130.9,
þ 10% CF
30.2, 128.6, 128.5, 128.2, 120.2, 119.7, 118.8, 110.9, 83.6; UV/vis
EtOH, ): 205 (10800), 246 (13000), 322 (3800), 493 (1700) nm;
ESI-MS m/z: 275.0 [Mþ1] ; C15
N, 20.43%; O, 11.67%; found: C, 65.73%; H, 2.23%; N, 20.40%.
3
3
2
d
1
(
l
max
4.5.9. Synthesis of ethyl 10-hydroxy-5,12-dioxo-5,12-dihydroin-
dolizino[2,3-g]phthalazine-11-carboxylate (5i)
According to the “General synthesis procedure”, compound 4,
3-hydroxypyridine (646.0 mg, 6.8 mmol) and ethyl acetoacetate
e
þ
H
6
N
4
O
2
, calcd: C, 65.70%; H, 2.21%;
(0.3 mL, 2.6 mmol) were used as reagents, and a mixture of
4
.5.5. Synthesis of 11-methylindolizino[2,3-g]phthalazine-5,12-
dione (5e)
According to the “General synthesis procedure”, compound 4,
petroleum ether and ethyl acetate (3:7) was used as eluent. Violet
ꢁ
1
solid 5i (186.0 mg) was obtained, yield 27.3%; mp ¼ 172e173 C, H
NMR (300 MHz, CDCl : 9.94 (s, 1H), 9.85 (s, 1H), 9.54 (d, 1H,
J ¼ 6.6 Hz), 7.26e7.23 (m, 1H), 7.02 (d, 1H, J ¼ 8.1 Hz), 4.57 (q, 2H,
3
) d
pyridine (0.6 mL, 6.8 mmol) and nitroethane (0.2 mL, 2.6 mmol)
were used as reagents, and a mixture of petroleum ether and ethyl
acetate (3:2) was used as eluent. Violet solid 5e (82.7 mg) was
1
3
J ¼ 7.2 Hz), 1.55 (t, 3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl
3
) d:
177.4, 171.3, 166.5, 150.1, 145.9, 145.6, 132.8, 126.6, 125.5, 125.2,
ꢁ
1
obtained, yield 13.6%; mp ¼ 277e278 C, H NMR (300 MHz, CDCl
: 9.96 (s, 1H), 9.81 (s, 1H), 9.67 (d, 1H, J ¼ 6.9 Hz), 7.75 (d, 1H,
J ¼ 9.0 Hz), 7.38 (d, 1H, J ¼ 7.8 Hz), 7.26e7.20 (m, 1H), 2.72 (s, 3H);
3
)
121.5, 119.8, 119.5, 112.9, 104.9, 62.4, 12.9; UV/vis
(17600), 345 (4600), 553 (900) nm; ESI-MS m/z: 336.1 [M-1] ;
l
max (EtOH,
e
): 252
ꢂ
d
17 11 3 5
C H N O , calcd: C, 60.54%; H, 3.29%; N, 12.46%; O, 23.72%; found:
C, 60.52%; H, 3.32%; N, 12.44%.
1
3
C NMR (100 MHz, CDCl
27.4, 125.5, 125.2, 124.8, 119.0, 118.5, 117.8, 116.5, 8.7; UV/vis
EtOH, ): 214 (14700), 261 (18200), 359 (3600), 547 (1600) nm;
ESI-MS m/z: 263.0 [M] ; C15
3
)
d: 181.1, 168.2, 146.4, 145.3, 137.8, 127.5,
1
(
l
max
e
4.5.10. Synthesis of ethyl 10-methyl-5,12-dioxo-5,12-dihydroindo-
lizino[2,3-g]phthalazine-11-carboxylate (5j) and Ethyl 8-methyl-
5,12-dioxo-5,12-dihydroindolizino[2,3-g]ph-thalazine-11-
carboxylate (6)
ꢂ
9 3 2
H N O , calcd: C, 68.44%; H, 3.45%; N,
15.96%; O, 12.16%; found: C, 68.46%; H, 3.47%; N, 15.95%.
4.5.6. Synthesis of indolizino[2,3-g]phthalazine-5,12-dione (5f)
According to the “General synthesis procedure”, compound 4,
According to the “General synthesis procedure”, compound 4,
pyridine (0.6 mL, 6.8 mmol) and nitromethane (0.2 mL, 2.6 mmol)
3-methylpyridine (632.0 mg, 6.8 mmol) and ethyl acetoacetate

3942
D.-Q. Shen et al. / European Journal of Medicinal Chemistry 45 (2010) 3938e3942
(
0.3 mL, 2.6 mmol) were used as reagents, and a mixture of
4.8. TOP1 inhibitory assay
dichloromethane and methanol (100:1) was used as eluent. Two
main products, 5j (138.9 mg) and 6 (88.4 mg), were obtained at the
same time.
The TOP1 relaxation assay was performed as mentioned in the
reported method with slight modification [24,25]. Briefly, relaxa-
ꢁ
1
Product 5j: violet solid, yield 18.8%; mp ¼ 269e270 C, H NMR
400 MHz, CDCl
: 9.92 (s, 1H), 9.77 (s, 1H), 9.53 (d, 1H, J ¼ 6.8 Hz),
.17 (d, 1H, J ¼ 6.8 Hz), 7.12 (t, 1H, J ¼ 6.8 Hz), 4.50 (q, 2H, J ¼ 7.2 Hz),
tion of 0.25
relaxing buffer (20 mM Tris pH 7.5, 0.1 mM EDTA, 10 mM MgCl
50 g/mL acetylated BSA, 100 mM KCl) in the presence or the
absence of the test drugs (125 M) previously dissolved in DMSO
mg plasmid pBR322 was performed in 20 mL of TOP1
(
7
2
3
)
d
2
,
m
13
.51 (s, 3H), 1.42 (t, 3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl
78.9, 170.0, 163.9, 146.2, 145.3, 135.6, 130.2, 127.6, 126.5, 125.2,
25.0, 124.6, 118.6, 117.7, 109.2, 61.6, 17.8, 13.0; UV/vis max (EtOH, ):
3
)
d:
m
1
solution. Reactions were started by the addition of 0.5 U TOP1, and
control groups were either DNA alone or DNA treated with TOP1.
1
l
e
2
3
15 (19300), 248 (24200), 338 (6200), 520 (3000) nm; ESI-MS m/z:
The drugs and DNA in the presence of TOP1 was incubated for
þ
ꢁ
36.1 [Mþ1] ; C18
H
13
N
3
O
4
, calcd: C, 64.47%; H, 3.91%; N, 12.53%; O,
30 min at 37 C. The reaction was terminated by the addition of 4
mL
19.09%; found: C, 64.49%; H, 3.92%; N, 12.50%.
6 X loading buffer. Products were then run in a 1% agarose gel in
45 mM Triseborate (pH 8.0), 1 mM EDTA at 5 V/cm [26]. Gels were
stained with ethidium bromide and visualized with a UV trans-
illuminator. Images were acquired and quantified through
AlphaEaseFC software.
ꢁ
1
Product 6: red powder, yield 12.0%, mp ¼ 256e257 C, H NMR
400 MHz, CDCl : 9.90 (s, 1H), 9.82 (s, 1H), 9.59 (s, br, 1H), 8.26 (d,
H, J ¼ 9.2 Hz), 7.37 (dd, 1H, J ¼ 9.2, 1.2 Hz), 4.45 (q, 2H, J ¼ 7.2 Hz),
(
1
2
3
) d
13
.44 (s, 3H), 1.44 (t, 3H, J ¼ 7.2 Hz); C NMR (100 MHz, CDCl
3
) d:
178.0,171.2,161.6,145.9,145.7,138.0, 131.6, 128.8, 126.1,125.6, 125.5,
þ
1
25.3, 120.2, 119.7, 106.2, 60.4, 17.7, 13.3; ESI-MS m/z: 336.1 [Mþ1] ;
, calcd: C, 64.47%; H, 3.91%; N, 12.53%; O, 19.09%; found:
C, 64.48%; H, 3.92%; N, 12.50%.
Acknowledgments
18 13 3 4
C H N O
This work was supported by the Natural Science Foundation of
China (No. 30801425), National
S & T Major Project (No.
4
.5.11. Synthesis of 11-acetyl-10-fluoroindolizino[2,3-g]phthalaz-
2009ZX09103-042) and Health Department of Guangdong Prov-
ine-5,12-dione (5k)
ince (No. B2009048).
According to the “General synthesis procedure”, compound 4,
3
2
-fluoropyridine (0.6 mL, 6.8 mmol) and acetylacetone (0.3 mL,
.6 mmol) were used as reagents, and a mixture of petroleum ether
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