Topoisomerase I-mediated antiproliferative activity of 10-substituted and 12-substituted homocamptothecins

Article abstract of DOI:10.1002/cbdv.201000307

Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue, which showed pronounced inhibitory activity of topoisomerase I. In search of novel hCPT-type anticancer agents, two series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines. The results indicated that the 10-substituted hCPT derivatives had a considerably higher cytotoxic activity than the 12-substituted ones. Among the 10-substituted compounds, 8a, 8b, 9b, and 9i showed an equivalent or even more potent activity than the positive control drug topotecan against the lung cancer cell line A-549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory activity than CPT at a concentration of 100 μM. Copyright

Full text of DOI:10.1002/cbdv.201000307

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Topoisomerase I-Mediated Antiproliferative Activity of 10-Substituted and
12-Substituted Homocamptothecins
by Wei Guo, Wenfeng Liu, Lingjian Zhu, Yongqiang Zhang, Pengfei Cheng, Guoqiang Dong, Chunlin
Zhuang, Jianzhong Yao, Chunquan Sheng*, Zhenyuan Miao*, and Wannian Zhang*
School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, P. R. China
(W. Zhang, phone/fax: 86-21-81871243; e-mail: zhangwnk@hotmail.com;
Z. Miao, phone/fax: 86-21-81871233; e-mail: miaozhenyuan@hotmail.com;
C. Sheng, phone/fax: 86-21-81871233; e-mail: shengcq@hotmail.com)
Homocamptothecin (hCPT) is an E-ring modified camptothecin (CPT) analogue, which showed
pronounced inhibitory activity of topoisomerase I. In search of novel hCPT-type anticancer agents, two
series of hCPT derivatives were synthesized and evaluated in vitro against three human tumor cell lines.
The results indicated that the 10-substituted hCPT derivatives had a considerably higher cytotoxic
activity than the 12-substituted ones. Among the 10-substituted compounds, 8a, 8b, 9b, and 9i showed an
equivalent or even more potent activity than the positive control drug topotecan against the lung cancer
cell line A-549. Moreover, the hCPT analogues 8a and 8b exhibited a higher topoisomerase I inhibitory
activity than CPT at a concentration of 100 mm.
Introduction. – As part of a National Cancer Institute compound screening program,
camptothecin (CPT; Fig. 1) was first characterized in 1966 from Camptotheca
acuminata, a plant with impressive activity against a variety of tumors [1]. However,
an obvious shortcoming was its poor H2O solubility. To overcome this solubility
problem, numerous derivatives had been optimized concerning their H2O solubility, to
facilitate intravenous drug administration. Now, irinotecan (CPT-11) and topotecan
(TPT), as two marketed drugs, are successfully used in clinical practice for colon and
ovarian cancer treatment, respectively.
In an attempt to stabilize the closed lactone of CPT, a seven-membered derivative
was synthesized by Lavergne et al. [2] in 1997, i.e., homocamptothecin (hCPT), a semi-
synthetic CPT analog with a modified E ring, i.e., the natural a-hydroxy-d-lactone E
ring was replaced by a b-hydroxy-e-lactone ring [2]. In this decade, a number of hCPT
derivatives were prepared by total or semi-synthetic strategies, of which two promising
compounds, BN80915 (diflomotecan) and BN80927, are so far in clinical phrase II and I
trials, respectively.
The high cytotoxic activity of 10-substituted CPT derivatives such as SN-38 (active
metabolite of CPT-11), which has been submitted to clinical studies, prompted
researchers to investigate the introduction of other moieties at C(10) of CPTand hCPT.
In previous studies, we synthesized a series of H2O-soluble 10-phosphate ester, 10-
amino acid ester, and 9-(hetarylmethylidene)amino derivatives of hCPT [3–5]. To
discover new compounds with more potent and broad-spectrum antitumor activities,
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Fig. 1. Chemical structures of camptothecin analogues
the design of a series of 10- and 12-alkoxy hCPT derivatives and the study of their
structureꢀactivity relationships are reported here.
Results and Discussion. – Synthesis. Ring A of the 10- and 12-alkoxy hCPTs was
prepared in two steps, which are illustrated in Scheme 1. The isomers 1 and 2 were
prepared from 3-hydroxyacetophenone by nitration (KNO3 and AcOH) [6]. Then,
compounds 1 and 2 were hydrogenated in EtOH with Pd/C as catalyst to give
compounds 3 and 4, respectively (Schemes 2 and 3). Compound 5, which is the building
block that introduces rings C, D, and E into the final hCPT derivatives, was synthesized
according to a reported route of 16 reaction steps, which was optimized in our previous
studies [7–9]. The 10-OH- and 12-OH-substituted hCPTs 6 and 7 were prepared via a
Friedlꢀnder cyclization of ring A with 5 and obtained with a good yield of 85%. When
toluene and p-toluenesulfonic acid (TsOH) were used for the cyclization reaction, a
lower yield of 6 and 7 (40%) was obtained than with DMF and tetramethylsilylchloride

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1541
Scheme 1
a) KNO3, AcOH, 608, overnight; yield 22% for 1, 21% for 2.
Scheme 2
a) H2, Pd/C, EtOH, 308, 3 bar, 3 h; yield 91%. b) Me3SiCl (TMSCl), DMF, 2 h, 1008; yield 85%. c) 1,3-
Dibromopropane or 1,4-dibromobutane, DMF, 608, overnight; yield 61% for 8a, 63% for 8b. d) DMF,
608, N2, 2 h; yield 37–51%.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 3
a) H2, Pd/C, AcOEt, 358, 3 bar, 2 h; yield 90 %. b) TMSCl, DMF, 2 h, 1008; yield 86%. c) 1,3-
Dibromopropane or 1,4-dibromobutane, DMF, 608, overnight; yield 60% for 10a, 64% for 10b. d) DMF,
608, N2, 2 h; yield 50% for 11a, 30% for 11b.
(TMSCl; 85%). 10-(3-Bromopropoxy)-7-methylhomocamptothecin (8a) and 10-(4-
bromobutoxy)-7-methylhomocamptothecin (8b) were obtained from 1,3-dibromopro-
pane or 1,4-dibromobutane and 10-hydroxy-7-methylhomocamptothecin (6) in DMF.
Compounds 8a and 8b were reacted with different N-containing compounds to afford
the 10-alkoxy-hCPTs 9a–9l. The 12-alkoxy-hCPTs 10a, 10b, 11a, and 11b were
obtained by a similar route (Scheme 3) than the 10-alkoxy hCPTs.
Cytotoxicity. The hCPT derivatives were evaluated for their cytotoxicity against
three cancer cell lines, i.e., A-549 (human lung adenocarcinoma epithelial cell line),
MDA-MB-435 (human breast carcinoma cell line), and HCT-116 (human colon
carcinoma cell line), using the MTT method in vitro. The results are summarized in
Tables 1 and 2. Most of the 10-substitued hCPTs showed antitumor activities against the
three cell lines, especially against the A-549 cell line. Among them, the three
compounds 8a, 8b, and 9b exhibited particularly high inhibitory activities against the A-
549 cell line, with IC50 values lower than 0.6 mm; hence, they were more potent than the
reference drug topotecan against this cell line. But the length of the alkane chain
between the hCPT and the heterocycle seemed not to be related to the activities.
Indeed, compounds 9b and 9d showed higher activities than 9a and 9c, while
compounds 9g, 9i, and 9k showed higher activities than 9h, 9j, and 9l, respectively.
However, the 12-subsituted hCPTs displayed lower activities compared with the 10-

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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Table 1. Cytotoxicity of the 10-Alkoxy-hCPT Derivatives against Three Human Tumor Cell Lines
Compounds
IC50 [mm]a)
A-549
MDA-MB-435
HCT-116
8a
8b
9a
9b
9c
9d
9e
0.58
0.20
22.62
0.35
–
19.67
15.67
–b)
–
76.60
62.88
–
9.82
5.32
–
56.27
–
19.15
–
–
–
9f
–
–
–
9g
9h
9i
9j
9k
9l
3.00
15.03
1.57
9.42
6.16
43.70
2.44
90.28
80.68
–
–
–
27.52
56.00
75.19
–
74.28
–
–
Topotecan
21.79
4.97
a) The IC50 values were determined by using the MTT method. b) –: IC50 >100 mm.
Table 2. Comparison of the Cytotoxicity of the 12-Alkoxy-hCPTs with that of the Most Active 10-Alkoxy-
hCPTs
Compounds
IC50 [mm]a)
A-549
MDA-MB-435
HCT-116
10a
10b
–b)
–
–
–
–
–
11a
11b
88.24
–
–
–
–
–
8a
8b
9b
9i
0.58
0.20
0.35
1.57
2.44
19.67
15.67
–
9.82
5.32
56.27
75.19
4.97
–
Topotecan
21.79
a) The IC50 values were determined by using the MTT method. b) –: IC50 >100 mm.
substituted ones. As shown in Table 2, the 10-substituted derivatives 9i and 9b showed
two to seven times higher activities than topotecan, whereas the 12-subsituted hCPTs
11a and 11b showed very low or no cytotoxic activity against A-549 cells. The previously
published binding mode of hCPT within the active site of topoisomerase I showed that
C(12) of hCTP was located on the minor groove side of the intercalation binding pocket
and that there was less space for a substituent at this position than at the 10-position [5].
The present results showed that the activities of the derivatives with long substituents at
C(12) were drastically decreased, which was in good agreement with the proposed
binding mode [5]. The determined cytotoxic effects of the synthesized hCPT

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
derivatives affirmed that long substituents at C(12) were unfavorable for the activity,
which was similar to CPT derivatives.
Topoisomerase I-Mediated DNA Relaxation. The topoisomerase I-inhibitory
properties of the selected hCPT derivatives 8a, 8b, and 9g were investigated with the
in vitro cleavable-complex assay. Negative supercoiled plasmid pBR322 was incubated
with human topoisomerase I in the absence and presence of 100 mm hCPT derivatives.
A parallel experiment was performed with CPT. The results indicated that compounds
8a and 8b, which were found to be highly cytotoxic agents, had higher topoisomerase I-
inhibitory properties than CPT (Fig. 2), while compound 9g showed the same lower
activity than CPT in the DNA cleavage assay.
Fig. 2. Effect of three homocamptothecin derivatives on topoisomerase I-mediated DNA relaxation. Lane
1: supercoiled DNA pBR322; Lane 2: supercoiled DNA and Topoisomerase I; Lanes 3–6: supercoiled
DNA, Topoisomerase I, and 100 mm of CPT, 8a, 9g, or 8b, respectively.
Conclusions. – In continuation of the work about 10- and 12-alkoxy hCPT
derivatives, 18 new hCPT derivatives were synthesized from our previously published
route [5][7–9]. Preliminary biological studies of these hCPT analogues including the
determination of the inhibitory potential of topoisomerase I-mediated DNA cleavage
reactions and of the in vitro cytotoxicity exhibited that most 10-substituted derivatives
possessed cytotoxic activities. The three compounds 8a, 8b, and 9b showed to be even
more efficient against the human lung adenocarcinoma epithelial cell line A-549 than
topotecan. Moreover, compounds 8a and 8b were found to exhibit a stronger
topoisomerase I-dependent cytotoxic activity than CPT at a concentration of 100 mm.
This research was supported in part by the Key Project of Science and Technology of Shanghai (No.
09431901700), the Major Special Project for the Creation of New Drugs (No. 2009ZX09301-011), and the
Shanghai Leading Academic Discipline Project (Project No. B906).
Experimental Part
General. Commercial solvents were used without any further purification or pretreatment. Column
chromatography (CC): silica gel 60 G (SiO2; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R.
China). TLC: precoated GF254 silica-gel plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R.
China). 1H- and 13C-NMR Spectra: Bruker 500 NMR spectrometer, at 500 and 125 MHz, resp., in
(D6)DMSO; d in ppm rel. to Me4Si, J in Hz. ESI-MS: API-3000 mass spectrometer; in m/z. Elemental
analysis: MOD-1106 instrument; analses were consistent with the theoretical values within ꢁ0.4%.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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5-Hydroxy-2-nitroacetophenone (¼1-(5-Hydroxy-2-nitrophenyl)ethanone; 1). 3-Hydroxyacetophe-
none (13.6 g, 0.1 mol) was dissolved in AcOH (100 ml) under stirring for 10 min at r.t., and then KNO3
(25.0 g, 0.2 mol) was added slowly over a period of 1 h. After raising the temp. to 608, the mixture was
stirred overnight. Then, the residue was cooled to r.t., poured into H2O (500 ml), and extracted with
AcOEt. The AcOEt extract was washed successively with sat. aq. NaHCO3 and H2O, and concentrated in
vacuo to give an oily residue, which was purified by flash chromatography (FC; SiO2; hexane/AcOEt
2 :1) to afford 1 (4.0 g, 22%). Yellow solid. M.p. 146–1478. 1H-NMR: 2.51 (s, Me); 6.84 (d, J¼2.6,
HꢀC(6)); 6.98 (dd, J¼2.6, 9.1, HꢀC(4)); 8.07 (d, J¼9.1, HꢀC(3)); 11.34 (s, OH). ESI-MS: 180.42 ([Mꢀ
H]ꢀ ).
3-Hydroxy-2-nitroacetophenone (¼1-(3-Hydroxy-2-nitrophenyl)ethanone; 2). Compound 2 was
prepared according to the method described for 1: 3.8 g (21%). Yellow solid. M.p. 144–1468. 1H-NMR:
2.53 (s, Me); 6.85 (d, J¼7.5, HꢀC(4)); 7.24 (d, J¼8.7, HꢀC(6)); 7.60 (t, J¼5.7, HꢀC(5)); 10.53 (s, OH).
ESI-MS: 180.51 ([MꢀH]ꢀ ).
2-Amino-5-hydroxyacetophenone (¼1-(2-Amino-5-hydrophenyl)ethanone; 3). To a soln. of 1 (1.3 g,
7.1 mmol) in EtOH (200 ml) Pd-C (0.1 g) was added as catalyst and hydrogenation followed at 3 bar for
2 h at 308. The resulting suspension was filtered and concentrated under reduced pressure to afford 3
(1.0 g, 91%). Yellow solid. M.p. 162–1658. 1H-NMR: 2.58 (s, Me); 6.52 (t, J¼7.5, HꢀC(3)); 6.89 (dd, J¼
2.7, 8.7, HꢀC(4)); 7.37(d, J¼2.7, HꢀC(6)); 7.27 (s, OH). ESI-MS: 150.41 ([MꢀH]ꢀ ).
10-Hydroxy-7-methylhomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5,10-dihydroxy-12-methyl-
3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 6). The tricyclic ketone 5 (0.3 g,
1.8 mmol) and 1 (0.5 g, 2.0 mmol) were dissolved in DMF at r.t. TMSCl (0.8 ml, 6.5 mmol) was added
drop wise, and then the mixture was refluxing at 1008 for 2 h. The mixture was poured into ice/H2O
(100 ml) and filtered. The crude product was washed with acetone (10 ml) and MeOH (10 ml) to give 6
(0.6 g, 85%). Yellow solid. M.p. >3008 (dec.). 1H-NMR: 0.87 (t, J¼7.5, Me(18)); 1.86 (q, J¼7.6,
CH2(19)); 2.66 (s, Me(7)); 3.47 (q, J¼13.7, CH2(21)); 5.21 (s, CH2(5)); 5.52 (q, J¼15.1, CH2(17)); 5.98 (s,
HOꢀC(20)); 7.30 (d, J¼2.7, HꢀC(9)); 7.32(s, HꢀC(14)); 7.42 (dd, J¼2.7, 9.2, HꢀC(11)); 8.15 (d, J¼9.2,
HꢀC(12)); 10.30 (s, HOꢀC(10)). ESI-MS: 393.10 ([MꢀH]þ ).
10-(3-Bromopropoxy)-7-methylhomocamptothecin (¼10-(3-Bromopropoxy)-5-ethyl-1,4,5,13-tetra-
hydro-5-hydroxy-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 8a). A mixture of 6
(3.0 g, 7.8 mmol), 1,3-dibromopropane (15.9 ml, 156.0 mmol), and anh. K2CO3 (5.4 g, 39.0 mmol) in anh.
DMF (20 ml) was stirred at 608 under N2 overnight. The mixture was filtered, and the residue was poured
into ice H2O (200 ml) and extracted with CH2Cl2. The CH2Cl2 extract was washed successively with H2O
and concentrated in vacuo to give an oily residue, which was purified by FC (SiO2; CH2Cl2/MeOH 100 :5)
1
to afford 7a (2.3 g, 60%). Yellow solid. M.p. >2508. H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.88 (q, J¼7.5,
CH2(19)); 2.35–2.37 (m, CH2(2’)); 2.73 (s, Me(7)); 3.38 (q, J¼13.8, CH2(21)); 3.74 (t, J¼6.0, CH2(3’));
4.32 (t, J¼6.6, CH2(1’)); 5.23 (s, CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.09 (s, HOꢀC(20)); 7.32 (s,
HꢀC(14)); 7.49 (d, J¼2.6, HꢀC(9)); 7.50 (dd, J¼2.7, 9.1, HꢀC(11)); 8.03 (d, J¼9.1, HꢀC(12)).
13C-NMR: 8.7; 15.5; 31.6; 32.3; 36.8; 42.9; 50.5; 61.8; 66.4; 73.6; 99.4; 103.9; 122.2; 122.7; 129.3; 129.4;
131.6; 138.8; 144.4; 145.7; 149.8; 156.3; 157.7; 159.5; 172.4. ESI-MS: 511.38 ([MꢀH]ꢀ ). Anal. calc. for
C25H25BrN2O5 (512.09): C 58.49, H 4.91, N 5.46; found: C 58.40, H 4.90, N 5.48.
10-(4-Bromobutoxy)-7-methylhomocamptothecin (¼10-(4-Bromobutoxy)-5-ethyl-1,4,5,13-tetrahy-
dro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 8b). Com-
pound 8b was prepared according to the method described for 8a and obtained as a yellow solid
(2.6 g, 63%). M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.86 (q, J¼14.8, CH2(19)); 1.85–2.04 (m,
CH2(2’)); 2.04–2.35 (m, CH2(3’)); 2.74 (s, Me(7)); 3.38 (q, J¼15.1, CH2(21)); 3.67 (t, J¼6.6, CH2(4’));
4.25 (t, J¼6.3, CH2(1’)); 5.27 (s, CH2(5)); 5.52 (q, J ¼ 15.0, CH2(17)); 6.01 (s, HOꢀC(20)); 7.32 (s,
HꢀC(14)); 7.50 (d, J¼2.4, HꢀC(9)); 7.50 (dd, J¼2.7, 9.1, HꢀC(11)); 8.04 (d, J¼9.0, HꢀC(12)).
13C-NMR: 8.6; 15.5; 27.7; 29.5; 35.2; 36.7; 39.5; 42.8; 50.4; 61.7; 67.6; 73.5; 99.2; 103.7; 122.1; 122.7; 129.3;
131.5; 138.6; 144.3; 145.7; 149.6; 156.3; 157.8; 159.5; 172.3. ESI-MS: 525.22 ([MꢀH]ꢀ ). Anal. calc. for
C26H27BrN2O5 (526.11): C 59.21, H 5.16, N 5.31; found: C 59.13, H 5.17, N 5.29.
7-Methyl-10-(morpholin-3-ylpropoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
12-methyl-10-[3-(morpholin-4-yl)propoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-
dione; 9a). A mixture of 8a (50 mg, 0.1 mmol) and morpholine (0.2 ml) in anh. DMF (10 ml) was stirred

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
at 608 for 2 h under N2 atmosphere. The mixture was poured into ice/H2O (100 ml) and extracted with
CH2Cl2. The CH2Cl2 extract was washed successively with H2O and concentrated in vacuo to give an oily
residue, which was purified by FC (SiO2; CH2Cl2/MeOH 100 :5) to afford 9a (25 mg, 49%). Yellow solid.
M.p. >2508. 1H-NMR: 0.87 (t, J¼7.5, Me(18)); 1.85 (q, J¼7.5, CH2(19)); 2.36 (s, CH2(2’)); 2.74 (s,
Me(7)); 3.07–3.61 (m, CH2(2’’), CH2(3’’), CH2(5’’), CH2(6’’), CH2(21), CH2(3’)); 4.30 (t, J¼1.5,
CH2(1’)); 5.27 (s, CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.07 (s, HOꢀC(20)); 7.33 (s, HꢀC(14)); 7.47 (d, J¼
2.6, HꢀC(9)); 7.51 (dd, J¼2.8, 9.2, HꢀC(11)); 8.05 (d, J¼9.2, HꢀC(12)). ESI-MS: 518.27 ([MꢀH]ꢀ ).
Anal. calc. for C29H33N3O6 (519.24): C 67.04, H 6.40, N 8.09; found: C 67.11, H 6.39, N 8.11.
7-Methyl-10-(morpholin-4-ylbutoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
12-methyl-10-[4-(morpholin-4-yl)butoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 9b). Compound 9b was prepared from 8b and morpholine according to the method described for 9a
and obtained: 20 mg (39%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.82 (q, J¼7.5,
CH2(19)); 1.65–1.85 (m, CH2(2’), CH2(3’)); 2.36–2.38 (m, CH2(2’’), CH2(6’’)); 2.74 (s, Me(7)); 3.06 (q,
J¼13.9, CH2(21)); 3.48–3.58 (m, CH2(4’), CH2(3’’), CH2(5’’)); 4.24 (t, J¼6.3, CH2(1’)); 5.26 (s, CH2(5));
5.50 (q, J ¼ 14.5, CH2(17)); 6.00 (s, HOꢀC(20)); 7.33 (s, HꢀC(14)); 7.46 (d, J¼2.5, HꢀC(9)); 7.75 (dd, J¼
2.6, 9.1, HꢀC(11)); 7.76 (d, J¼9.3, HꢀC(12)). ESI-MS: 532.46 ([MꢀH]ꢀ ). Anal. calc. for C30H35N3O6
(533.25): C 67.52, H 6.61, N 7.87; found: C 67.63, H 6.63, N 7.85.
7-Methyl-10-(pyrrolidin-3-ylpropoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
12-methyl-10-[3-(pyrrolidin-1-yl)propoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-
dione; 9c). Compound 9c was prepared from 8a and pyrrolidine according to the method described for
9a: 20 mg (41%). Yellow solid. M.p. >2508. 1H-NMR: 0.79 (t, J¼7.3, Me(18)); 1.83–1.84 (m, CH2(19));
1.86–1.89 (m, CH2(3’’), CH2(4’’)); 1.90–2.20 (m, CH2(2’)); 2.74 (s, Me(7)); 3.15–3.17 (m, CH2(1’’),
CH2(4’’)); 3.19 (q, J¼13.7, CH2(21)); 3.50–3.54 (m, CH2(3’)); 4.30–4.32 (m, CH2(1’)); 4.65 (s, CH2(5));
4.80 (q, J ¼ 15.0, CH2(17)); 6.49 (s, HOꢀC(20)); 7.31 (s, HꢀC(14)); 7.50–7.51 (m, HꢀC(9), HꢀC(11));
8.07 (d, J¼8.3, HꢀC(12)). ESI-MS: 503.24 ([MꢀH]ꢀ ). Anal. calc. for C29H33N3O5 (503.24): C 69.17, H
6.60, N 8.34; found: C 69.03, H 6.58, N 8.36.
7-Methyl-10-(pyrrolidin-4-ylbutoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
12-methyl-10-[4-(pyrrolidin-1-yl)butoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 9d). Compound 9d was prepared from 8b and pyrrolidine according to the method described for 9a:
1
25 mg (51%). Yellow solid. M.p. >2508. H-NMR: 0.80 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.4, CH2(19));
1.93–1.98 (m, CH2(2’), CH2(3’), CH2(3’’), CH2(4’’)); 2.74 (s, Me(7)); 3.20–3.22 (m, CH2(2’’), CH2(5’’),
CH2(21)); 3.33–3.50 (m, CH2(1’), CH2(4’)); 4.24 (s, CH2(5)); 4.86 (q, J ¼ 15.1, CH2(17)); 6.50 (s,
HOꢀC(20)); 7.31 (s, HꢀC(14)); 7.32 (d, J¼2.2, HꢀC(9)); 7.63 (dd, J¼2.4, 9.3, HꢀC(11)); 7.76 (d, J¼3.2,
HꢀC(12)). ESI-MS: 516.27 ([MꢀH]ꢀ ). Anal. calc. for C30H35N3O5 (517.26): C 69.61, H 6.82, N 8.12;
found: C 69.49, H 6.81, N 8.13.
7-Methyl-10-[3-(methylpiperazin-4-yl)propoxy]homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-
5-hydroxy-12-methyl-10-[3-(4-methylpiperazin-1-yl)propoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-
b]quinoline-3,15-dione; 9e). Compound 9e was prepared from 8a and 4-methylpiperazine according to
the method described for 9a: 19 mg (37%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J ¼ 7.4, Me(18));
1.87 (q, J¼7.4, CH2(19)); 1.88–2.23 (m, CH2(2’)); 2.50 (s, Me(4’’)); 2.64 (s, Me(7)); 3.03–3.08 (m,
CH2(2’’), CH2(3’’), CH2(5’’), CH2(6’’)); 3.34 (q, J¼13.8, CH2(21)); 4.25 (t, J¼6.2, CH2(3’)); 4.27 (t, J¼
6.3, CH2(1’)); 5.27 (s, CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.00 (s, HOꢀC(20)); 7.32 (s, HꢀC(14)); 7.47 (d,
J¼2.6, HꢀC(9)); 7.51 (dd, J¼2.6, 9.1, HꢀC(11)); 8.05 (d, J¼9.2, HꢀC(12)). ESI-MS: 531.68 ([MꢀH]ꢀ ).
Anal. calc. for C30H36N4O5 (532.27): C 67.65, H 6.81, N 10.52; found: C 67.77, H 6.82, N 10.50.
7-Methyl-10-[4-(methylpiperazin-4-yl)butoxy]homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-
hydroxy-12-methyl-10-[4-(4-methylpiperazin-1-yl)butoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-
b]quinoline-3,15-dione; 9f). Compound 9f was prepared from 8b and 4-methylpiperazine according to
the method described for 9a: 20 mg (39%). Yellow solid. M.p. >2508. 1H-NMR: 0.86 (t, J¼7.4, Me(18));
1.87 (q, J¼7.5, CH2(19)); 1.81–1.86 (m, CH2(2’), CH2(3’)); 2.30 (s, Me(4’’)); 2.50–2.72 (m, CH2(2’’),
CH2(3’’), CH2(5’’), CH2(6’’)); 2.73 (s, Me(7)); 3.45–3.48 (m, CH2(4’)); 4.20–4.21 (m, CH2(1’)); 3.37 (q,
J¼13.5, CH2(21)); 5.26 (s, CH2(5)); 5.53 (q, J ¼ 15.0, CH2(17)); 6.01 (s, HOꢀC(20)); 7.32 (s, HꢀC(14));
7.46–7.50 (m, HꢀC(11)); 8.04 (d, J¼9.1, HꢀC(12)). ESI-MS: 545.54 ([MꢀH]ꢀ ). Anal. calc. for
C31H38N4O5 (546.28): C 68.11, H 7.01, N 10.25; found: C 68.22, H 7.03, N 10.23.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1547
7-Methyl-10-(piperidin-3-ylpropoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
12-methyl-10-[3-(piperidin-1-yl)propoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 9g). Compound 9g was prepared from 8a and piperidine according to the method described for 9a:
26 mg (51%). Yellow solid. M.p. >2508. 1H-NMR: 0.86 (t, J¼7.4, Me(18)); 2.07–2.43 (m, CH2(3’’),
CH2(4’’), CH2(5’’), CH2(19)); 2.43 (s, CH2(2’)); 2.74 (s, Me(7)); 3.29–3.60 (m, CH2(2’’), CH2(6’’),
CH2(21)); 3.80 (s, CH2(3’)); 4.28 (s, CH2(1’)); 5.26 (s, CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.01 (s,
HOꢀC(20)); 7.33 (s, HꢀC(14)); 7.37–7.45 (m, HꢀC(9), HꢀC(11)); 8.05 (d, J¼9.0, HꢀC(12)). 13C-NMR:
8.6; 15.7; 25.9; 36.8; 41.4; 42.9; 43.4; 50.4; 53.6; 61.8; 63.8; 66.3; 67.4; 73.5; 99.7; 110.7; 116.1; 122.5; 128.1;
129.2; 129.4; 140.0; 140.1; 145.7; 150.5; 155.4; 156.2; 159.5; 172.3. ESI-MS: 516.78 ([MꢀH]ꢀ ). Anal. calc.
for C30H35N3O5 (517.26): C 69.61, H 6.82, N 8.12; found: C 69.52, H 6.84, N 8.10.
7-Methyl-10-(piperidin-4-ylbutoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
methyl-10-[4-(piperidin-1-yl)butoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
9h). Compound 9h was prepared from 8b and piperidine according to the method described for 9a: 22 mg
(44%). Yellow solid. M.p. >2508. 1H-NMR: 0.80 (t, J¼7.4, Me(18)); 1.23(q, J¼7.5, CH2(19)); 1.55–1.64
(m, CH2(2’), CH2(3’)); 1.81–1.87 (m, CH2(3’’), CH2(4’’), CH2(5’’)); 2.74 (s, Me(7)); 2.87–3.03 (m,
CH2(2’’), CH2(6’’)); 3.46–3.48 (m, CH2(4’), CH2(21)); 4.26–4.28 (m, CH2(1’)); 5.27 (s, CH2(5)); 5.55 (q,
J ¼ 14.8, CH2(17)); 6.00 (s, HOꢀC(20)); 7.28 (s, HꢀC(14)); 7.48–7.52 (m, HꢀC(9), HꢀC(11)); 8.07 (d, J¼
9.2, HꢀC(12)). ESI-MS: 530.52 ([MꢀH]ꢀ ). Anal. calc. for C31H37N3O5 (531.27): C 70.03, H 7.01, N 7.90;
found: C 70.14, H 7.04, N 7.89.
10-[3-(Diethylamino)propoxy]-7-methylhomocamptothecin (¼10-[3-(Diethylamino)propoxy]-5-
ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-
3,15-dione; 9i). Compound 9i was prepared from 8a and Et2NH according to the method described for
9a: 22 mg (45%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.16–1.18 (m, 2
MeCH2N); 1.86 (q, J¼7.4, CH2(19)); 2.15–2.18 (m, CH2(2’)); 2.73 (s, Me(7)); 3.04–3.07 (m,
2 MeCH2N); 3.38 (q, J¼13.7, CH2(21)); 3.46–3.49 (m, CH2(3’)); 4.29–4.31 (m, CH2(1’)); 5.28 (s,
CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.00 (s, HOꢀC(20)); 7.32 (s, HꢀC(14)); 7.47 (d, J¼2.5, HꢀC(9));
7.51 (dd, J¼2.7, 9.2, HꢀC(11)); 7.76 (d, J¼9.2, HꢀC(12)). ESI-MS: 503.52 ([MꢀH]ꢀ ). Anal. calc. for
C29H35N3O5 (505.26): C 68.89, H 6.98, N 8.31; found: C 68.99, H 6.98, N 8.32.
10-[4-(Diethylamino)butoxy]-7-methylhomocamptothecin (¼10-[4-(Diethylamino)butoxy]-5-ethyl-
1,4,5,13-tetrahydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 9j). Compound 9j was prepared from 8b and Et2NH according to the method described for 9a:
1
25 mg (51%). Yellow solid. M.p. >2508. H-NMR: 0.80 (t, J¼7.4, Me(18)); 1.23 (q, J¼7.5, CH2(19));
1.55–1.64 (m, CH2(2’), CH2(3’)); 1.81–1.87 (m, 2 MeCH2N); 2.74 (s, Me(7)); 2.87–3.03 (m, 2 MeCH2N);
3.33–3.48 (m, CH2(4’), CH2(21)); 4.24–4.26 (m, CH2(1’)); 5.27(s, CH2(5)); 5.55(q, J ¼ 14.8, CH2(17));
6.00 (s, HOꢀC(20)); 7.28 (s, HꢀC(14)); 7.48–7.53 (m, HꢀC(9), HꢀC(11)); 8.07 (d, J¼9.2, HꢀC(12)). ESI-
MS: 520.95 ([MþH]þ ). Anal. calc. for C30H37N3O5 (519.27): C 69.34, H 7.18, N 8.09; found: C 69.44, H
7.19, N 8.05.
10-(Imidazol-3-ylpropoxy)-7-methylhomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-
10-[3-(1H-imidazol-1-yl)propoxy]-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-
dione; 9k). Compound 9k was prepared from 8a and 1H-imidazole according to the method described for
9a: 25 mg (45%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.86, (q, J¼6.4, CH2(19));
2.28 (m, CH2(2’)); 2.71 (s, Me(7)); 3.38 (q, J¼13.9, CH2(21)); 4.17 (t, J¼6.0, CH2(3’)); 4.22 (t, J¼6.9,
CH2(1’)); 5.22 (s, CH2(5)); 5.52 (q, J ¼ 14.9, CH2(17)); 6.00 (s, HOꢀC(20)); 6.94 (s, HꢀC(4’’)); 7.30 (s,
HꢀC(5’’)); 7.33 (s, HꢀC(14)); 7.44 (s, HꢀC(9)); 7.50 (dd, J¼2.6, 9.0, HꢀC(11)); 7.73 (s, HꢀC(2’’)); 8.04 (d,
J¼9.0, HꢀC(12)). ESI-MS: 499.49 ([MꢀH]ꢀ ). Anal. calc. for C28H28N4O5 (500.21): C 67.19, H 5.64, N
11.19; found: C 67.07, H 5.63, N 11.18.
10-(Imidazol-4-ylbutoxy)-7-methylhomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-10-
[4-(1H-imidazol-1-yl)butoxy]-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-di-
one; 9l). Compound 9l was prepared from 8b and 1H-imidazole according to the method described for
9a: 25 mg (39%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.80–1.86 (m, CH2(19),
CH2(3’)); 1.90–1.95 (m, CH2(2’),); 2.76 (s, Me(7)); 3.10 (q, J¼13.7, CH2(21),; 4.19–4.28 (m, CH2(1’),
CH2(4’)); 5.28 (s, CH2(5)); 5.50 (q, J ¼ 15.0, CH2(17)); 6.00 (s, HOꢀC(20)); 6.93 (s, HꢀC(4’’)); 7.07 (s,
HꢀC(5’’)); 7.32 (s, HꢀC(14)); 7.45 (d, J¼2.3, HꢀC(9)); 7.50 (dd, J¼2.6, 9.0, HꢀC(11)); 7.73 (s, HꢀC(12));

1548
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
8.05 (d, J¼9.1, HꢀC(2’’)). ESI-MS: 513.67 ([MꢀH]ꢀ ). Anal. calc. for C29H30N4O5 (514.22): C 67.69, H
5.88, N 10.89; found: C 67.76, H 5.89, N 10.87.
2-Amino-3-hydroxyacetophenone (¼1-(2-Amino-3-hydroxyphenyl)ethanone; 4). The title com-
pound was prepared according to the method described for 3: 1.0 g (90%). Yellow solid. M.p. 179–1818.
1H-NMR: 2.50 (s, Me); 6.40 (t, J¼7.9, HꢀC(5)); 6.69 (s, NH2); 6.80 (dd, J¼7.8, 1.1, HꢀC(4)); 7.25 (d, J¼
8.1, HꢀC(6)); 9.66 (s, OH). ESI-MS: 150.30 ([MꢀH]ꢀ ).
12-Hydroxy-7-methylhomocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5,8-dihydroxy-12-methyl-
3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 7). Compound 7 was prepared from 4
and 5 according to the method described for 6: 0.6 g (86%). Yellow solid. M.p. >3008 (dec). 1H-NMR:
0.87 (t, J¼7.2, Me(18)); 1.87 (q, J¼8.7, CH2(19)); 2.73 (s, Me(7)); 3.47 (q, J¼14.0, CH2(21)); 5.28 (s,
CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.04 (s, HOꢀC(20)); 7.18 (d, J ¼ 7.3, HꢀC(11)); 7.54 (m, HꢀC(10),
HꢀC(14)); 7.63 (d, J¼8.3, HꢀC(9)); 9.95 (s, HOꢀC(12)). ESI-MS: 391.83 ([MꢀH]ꢀ ).
12-(3-Bromopropoxy)-7-methylhomocamptothecin (¼ 8-(3-Bromopropoxy)-5-ethyl-1,4,5,13-tetra-
hydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 10a).
Compound 10a was prepared from 7 and 1,3-dibromopropane according to the method described for
1
8a: 60%. Yellow solid. M.p. >2508. H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.86 (q, J¼8.7, CH2(19)); 2.43–
2.50 (m, CH2(2’)); 2.75 (s, Me(7)); 3.38 (q, J¼13.7, CH2(21)); 3.83 (t, J¼6.5, CH2(3’)); 4.36–4.40 (m,
CH2(1’)); 5.28 (s, CH2(5)); 5.52 (q, J ¼ 15.0, CH2(17)); 6.09 (s, HOꢀC(20)); 7.33 (s, HꢀC(14)); 7.37 (d, J¼
7.2, HꢀC(11)); 7.63 (t, J¼8.1, HꢀC(10)); 7.80 (d, J¼8.4, HꢀC(9)). ESI-MS: 511.53 ([MꢀH]ꢀ ). Anal.
calc. for C25H25BrN2O5 (512.09): C 58.49, H 4.91, N 5.46; found: C 58.37, H 4.90, N 5.47.
12-(4-Bromobutoxy)-7-methylhomocamptothecin (¼ 8-(4-Bromopropoxy)-5-ethyl-1,4,5,13-tetrahy-
dro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione; 10b). Com-
pound 10b was prepared from 7 and 1,4-dibromobutane according to the method described for 8a: 64%.
1
Yellow solid. M.p. >2508. H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.87 (q, J¼7.2, CH2(19)); 2.10–2.11 (m,
CH2(3’)); 2.35–2.37 (m, CH2(2’)); 2.75 (s, Me(7)); 3.38 (q, J¼13.8, CH2(21)); 3.78–3.82 (m, CH2(4’));
4.28–4.31 (m, CH2(1’)); 5.28 (s, CH2(5)); 5.52 (q, J ¼ 15.1, CH2(17)); 6.04 (s, HOꢀC(20)); 7.31 (d, J¼8.0,
HꢀC(11)); 7.34 (s, HꢀC(14));7.63 (t, J¼8.1, HꢀC(10)); 7.77 (d, J¼8.5, HꢀC(9)). 13C-NMR: 8.7; 15.7;
27.6; 30.2; 35.4; 36.7; 42.9; 50.5; 61.7; 68.6; 73.5; 99.8; 110.5; 116.1; 122.5; 128.1; 129.2; 129.5; 140.0; 140.1;
145.8; 150.6; 155.4; 156.1; 159.5; 172.3. ESI-MS: 527.85 ([MþH]þ ). Anal. calc. for C26H27BrN2O5
(526.11): C 59.21, H 5.16, N 5.31; found: C 58.33, H 5.18, N 5.29.
7-Methyl-12-(4-piperidylbutoxy)homocamptothecin (¼ 5-Ethyl-1,4,5,13-tetrahydro-5-hydroxy-12-
methyl-8-[4-(piperidin-1-yl)butoxy]-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-dione;
11a). Compound 11a was prepared from 10b and piperidine according to the method described for 9a:
25 mg (50%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.63–1.66 (m, CH2(3’’),
CH2(4’’), CH2(5’’), CH2(19)); 1.84–1.99 (m, CH2(2’), CH2(3’)); 2.75 (s, Me(7)); 2.89–3.39 (m, CH2(2’’),
CH2(6’’)); 3.10 (q, J¼14.0, CH2(21)); 3.49–3.51 (m, CH2(4’)); 4.30 (m, CH2(1’)); 5.23 (s, CH2(5)); 5.55 (q,
J ¼ 15.1, CH2(17)); 6.10 (s, HOꢀC(20)); 7.32 (s, HꢀC(14)); 7.33 (d, J¼8.3, HꢀC(11)); 7.63 (t, J¼8.1,
HꢀC(10)); 7.77 (d, J¼8.3, HꢀC(9)). ESI-MS: 530.86 ([MꢀH]ꢀ ). Anal. calc. for C31H37N3O5 (531.27): C
70.03, H 7.01, N 7.90; found: C 70.16, H 7.03, N 7.88.
12-[3-(Diethylamino)propoxy]-7-methylhomocamptothecin (¼ 8-(3-Diethylamino)propoxy)-5-eth-
yl-1,4,5,13-tetrahydro-5-hydroxy-12-methyl-3H,15H-oxepino[3’,4’:6,7]indolizino[1,2-b]quinoline-3,15-
dione; 11b). Compound 11b was prepared from 10a and according to the method described for 9a: 22 mg
(30%). Yellow solid. M.p. >2508. 1H-NMR: 0.87 (t, J¼7.4, Me(18)); 1.18–1.30 (m, 2 MeCH2N); 1.86 (q,
J¼7.4, CH2(19)); 2.47–2.49 (m, CH2(2’)); 2.75 (s, Me(7)); 3.08–3.15 (m, 2 MeCH2N); 3.38 (q, J¼13.8,
CH2(21)); 4.29–4.34 (m, CH2(1’), CH2(3’)); 5.27 (s, CH2(5)); 5.52 (q, J ¼ 15.3, CH2(17)); 6.05 (s,
HOꢀC(20)); 7.29 (s, HꢀC(14)); 7.33 (d, J¼8.0, HꢀC(11)); 7.63 (t, J¼8.0, HꢀC(10)); 7.76 (d, J¼8.3,
HꢀC(9)). ESI-MS: 503.52 ([MꢀH]ꢀ ). Anal. calc. for C29H35N3O5 (505.26): C 68.89, H 6.98, N 8.31;
found: C 68.97, H 6.99, N 8.29.
Cytotoxicity Assay. The prepared compounds were tested for their in vitro cytotoxic activities against
three cell lines, i.e., A-549 (human lung adenocarcinoma epithelial cell line), MDA-MB-435 (human
breast carcinoma cell line), and HCT-116 (human colon carcinoma cell line), using a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)-based assay. The cells were plated
in 96-well plates at 1200 cells/well and incubated at 378 for 24 h. Then, the test compounds were added at

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
1549
different concentrations into triplicate wells, and 0.1% DMSO was used for the control. After 3 d of
incubation at 378, 20 ml of MTT soln. (5 mg/ml) was added to each well, and after shaking for 1 min the
plate was incubated for further 4 h. The formazan crystals were dissolved with 100 ml of DMSO. The
absorbance (OD) at 570 nm was quantified with a microplate reader (WellscanMK-2, Labsystems Co.,
Ltd., Finland). Wells containing no test compounds were used as blanks. The survival of the cells was
expressed as percentage relative to untreated control cells.
Inhibition of Topoisomerase I-Mediated DNA Relaxation. Camptothecin (CPT) was obtained from
Tianzunzezhong, P. R. China, and calf thymus topoisomerase I (Topo I), reaction buffer, bovine serum
albumin (BSA), loading buffer, and supercoilded DNA pBR322 were purchased from TaKaRa
Biotechnology Co., Ltd., P. R. China. To 16 ml dist. H2O, 2 ml reaction buffer containing 0.25 mg
supercoiled DNA and 0.5 U Topo I, 2 ml 0.1% BSA, and 0.02 ml test compound soln. in 1.5-ml sample tube
(final concentration 100 mm) were added. The mixture was incubated at 378 for 15 min. The reactions
were stopped by adding SDS (0.5% final concentration), and 3.5 ml of 6ꢂloading buffer (0.1 mm EDTA,
7% glycerol, 0.01% xylene cyanol FF, and 0.01% bromopenol blue) was added to the mixtures, which
were electrophoresed in 0.8% agarose gel in TAE buffer (0.4m TRIS, 0.01m EDTA, and 0.2m AcOH,
pH 8.5) for 40 min at 120 V. The gels were stained with ethidium bromide at r.t. and photographed with
an UV transilluminator (Tannon 2500, Tianneng Co., Ltd., P. R. China).
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Received November 4, 2010