http://informahealthcare.com/enz ISSN: 1475-6366 (print), 1475-6374 (electronic) J Enzyme Inhib Med Chem, 2014; 29(5): 722–727 ! 2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.845818

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ORIGINAL ARTICLE

Design, synthesis and anticancer activity of oxoaporphine alkaloid derivatives Yong-Biao Wei, Ying-Xin Li, Hui Song, and Xian-Jin Feng Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Guangxi Medical University, Nanning, Guangxi, PR China

Abstract

Keywords

A series of new oxoaporphine derivatives were synthesized and their inhibitory activity of topoisomerase I, cytotoxicity and DNA-binding properties were studied. Oxoaporphine can strongly inhibit topoisomerase I at concentrations of 5–50 mM and the cytotoxicity of the derivatives are more potent than their lead compound. Hypochromism, broadening and red shift in the absorption spectra were observed when these compounds bind to calf thymus DNA (CT DNA). These spectral characteristics were consistent with the intercalative binding of these compounds.

Cytotoxicity, DNA binding, oxoaporphine alkaloids, synthesis, topoisomerase I inhibitor

Introduction Oxoaporphine alkaloids have been isolated from plant species of many genera1. These alkaloids exhibited remarkable biological activities such as potent anticancer2,3, antiviral4, antimicrobial5,6 and lowering blood pressure7 properties. Recent studies showed that Liriodenine8 and Dicentrinone9 (Figure 1) could inhibit topoisomerase II and topoisomerase I, respectively. Although oxoaporphine alkaloids have broad biological activity, the activity is weak to moderate even for the most active compound10. Therefore, these alkaloids are interesting substrates for chemical modification (Figure 1). A great deal of studies indicated that intercalation of planar aromatic molecules into the DNA double helix results in dramatic changes in DNA conformation and can inhibit DNA replication, transcription11 and/or topoisomerase activities. These associative interactions with the DNA molecules can cause dramatic changes in the physiological functions of DNA, which might respond for the cytotoxic behavior of the small molecules. Additionally, previous studies demonstrated that introducing at least one basic side chain into chromophore could increase DNA-binding affinity12. Enlightened by the studies above, a series of oxoaporphine derivatives with different basic side chain were synthesized. Their DNA-binding properties, inhibition of topoisomerase I and antitumor activity were studied likewise.

Experimental Proton (1H) nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300 spectrometer (Rheinstetten, Germany).

Address for correspondence: Yong-Biao Wei, Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Guangxi Medical University, No. 22, Shuangyong Road, Nanning 530021, Guangxi, PR China. E-mail: [email protected]

History Received 3 June 2013 Revised 26 August 2013 Accepted 4 September 2013 Published online 18 June 2014

Chemical shifts are reported in parts per million () relative to tetramethylsilane ( 0.00). UV/visible absorbance spectra were measured on a Shimadzu UV-2450 spectrophotometer (Tokyo, Japan). ESI-MS spectra were obtained using a LCMS-2010A Mass Spectrometer (Tokyo, Japan). Elemental analysis was carried out on an Elementar Vario EL CHNS Elemental Analyzer (Hanau, Germany). All reagents were purchased from either the Aldrich Chemical Co. or the Lancaster Synthesis Co. and used without further purification unless otherwise stated. Dimethyl 7H-dibenzo[de,g]quinolin-7-one -4,5-dicarboxylate (2) A solution of 1 (23.7 g, 0.1 mol) and dimethyl acetylenedicarboxylate (DMAD) (31.2 g, 0.22 mol) in toluene (400 ml) was refluxed for eight days and then set aside overnight, during which yellow crystals separated. This crystalline solid was filtered off and was further purified by recrystallization from chloroform to give dimethyl 7H-dibenzo[de,g]quinolin-7-one-4, 5-dicarboxylate 2 (25%), m.p. 271–273  C. 1H NMR (CDCl3): 4.09 (s, 3H), 4.14 (s, 3H), 7.60 (t, 1H, J ¼ 7.3 Hz), 7.78 (t, 1H, J ¼ 7.3 Hz), 7.93– 8.04 (m, 2H), 8.25 (d, 1H, J ¼ 8.1 Hz), 8.47–8.54 (m, 2H). ESIMS m/z: 348 [M þ H]þ. Anal. Calcd for C20H13NO5: C, 69.16; H, 3.77; N, 4.03. Found: C, 69.32; H, 3.57; N, 4.11. 7-Oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxylic acid (3) To a stirred suspension of 2 (3.5 g, 0.01 mol) in methanol (40 ml) was added a solution of KOH (3 g, 0.05 mol) in water (100 ml). The mixture was heated at reflux for 24 h and left to stand overnight. The methanol was removed under vacuum and the aqueous solution was added HCl until pH ¼ 1–2. The yellow solid was filtered off and was purified by crystallization from nitrobenzene to afford 3 as an amorphous powder (92%).

Oxoaporphine alkaloid

DOI: 10.3109/14756366.2013.845818

O N

O

O

O N

N

O

O

O H3CO OCH3

Oxoaporphine

Liriodenine

Dicentrinone

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Figure 1. Structure of oxoaporphine, liriodenine and dicentrinone. 1 H-NMR (DMSO-d6, 300 MHz): 7.69 (t, 1H, J ¼ 7.5 Hz), 7.91 (t, 1H, J ¼ 7.5 Hz), 8.09–8.18 (m, 2H), 8.31 (d, 1H, J ¼ 7.7 Hz), 8.61 (d, 1H, J ¼ 8.0 Hz), 8.90 (d, 1H, J ¼ 7.0 Hz); ESI-MS m/z: 318 [M  H]. Anal. Calcd for C18H9NO5: C, 67.72; H, 2.84; N, 4.39. Found: C, 67.61; H, 2.52; N, 4.56.

7H-dibenzo[de,g]quinolin-7-one (4) Finely ground diacid 3 (3.0 g, 0.02 mol) in diphenyl ether (10 ml) was heated to approximately 250  C, and heating was discontinued after a few minutes when the obvious reaction had ceased. The residue was purified by column chromatography (dichloromethane as eluent) to afford compound 4 (60% yield) as yellow needle solid. 1HNMR (DMSO-d6, 300 MHz)  8.9 (d, l H, J ¼ 5.3 Hz), 8.7 (d, l H, J ¼ 7.0 Hz), 8.5 (d, l H, J ¼ 8.0 Hz), 8.3 (m, 2 H), 8.2 (d, l H, J ¼ 8.1 Hz), 7.9 (d, 1 H, J ¼ 7.5 Hz), 7.8 (t, l H, J ¼ 6.9 Hz), 7.6 (t, l H, J ¼ 7.1 Hz); ESI. MS m/z: 232[M þ H]þ Anal. Calcd for C16H9NO: C, 83.10; H, 3.92; N, 6.06. Found: C, 83.21; H, 3.87; N, 6.23. 7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarbonyl dichloride (5) A suspension of 3 (2.07 g, 1 mmol) in benzene (70 ml) with thionyl chloride (3.6 ml, 5 mmol) was heated at 95  C for 5 hours. After cooling, the solvent was removed under reduced pressure, benzene was added in three 25 ml aliquots and removed, and 70 ml of hexane was added. The precipitate was collected by filtration, washed with 20 ml of hexane and dried afford 5 (96% yield). ESI-MS m/z: 356 [M þ H]þ.

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N4,N5-bis(2-(dimethylamino)ethyl)-7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2b) Compound 5 was treated with N1,N1-dimethylethane-1,2-diamine according to general acylation procedure to give 2b (26%) as yellow solid. 1H-NMR (CDCl3, 300 MHz): 2.4 (s, 12H), 2.7 (t, 2H, J ¼ 5.7), 2.8 (t, 2H, J ¼ 6.0), 3.7 (dd, 2H, J1 ¼ 5.1, J2 ¼ 6.1), 4.0 (dd, 2H, J1 ¼ 5.1, J2 ¼ 6.7), 7.6 (t, 1H, J ¼ 7.6), 7.8 (m, 2H), 8.3 (d, 1H, J ¼ 7.7), 8.5 (d, 1H, J ¼ 8.9), 8.6 (m, 2H), 9.2 (s, 1H), 10.9 (s, 1H) ESI-MS m/z: 460 [M þ H]þ. Anal. Calcd for C26H29N5O3: C, 67.95; H, 6.36; N, 15.24. Found: C, 68.31; H, 6.73; N, 15.92. N4,N5-bis(2-(diethylamino)ethyl)-7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2c) Compound 5 was treated with N1,N1-diethylethane-1,2-diamine according to general acylation procedure to give 2c (38%) as yellow solid. 1H-NMR (CDCl3, 300 MHz): 1.1 (t, 6H, J ¼ 7.1), 1.2 (t, 6H, J ¼ 6.9), 2.6 (q, 4H, J1 ¼ 6.9, J2 ¼ 14.0) 2.7 (q, 4H, J1 ¼ 5.9, J2 ¼ 12.6), 2.8 (t, 4H, J ¼ 6.2), 2.6 (dd, 2H, J1 ¼ 5.7, J2 ¼ 12.0), 3.9 (dd, 2H, J1 ¼ 5.5, J2 ¼ 12.1), 7.6 (t, 1H, J ¼ 7.5), 7.7 (m, 2H), 8.3 (d, 1H, J ¼ 8.1), 8.6 (m, 3H), 9.1 (s, 1H), 10.7 (s, 1H); ESI-MS m/z: 516 [M þ H]þ. Anal. Calcd for C30H37N5O3: C, 69.88; H, 7.23; N, 13.58. Found: C, 70.11; H, 7.77; N, 13.09. 7-oxo-N4,N5-bis(2-(piperidin-1-yl)ethyl)-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2d) Compound 5 was treated with 2-(piperidin-1-yl)ethanamine according to general acylation procedure to give 2d (18%) as yellowish red solid. 1HNMR (CDCl3)  (ppm): 1.6 (m, 4 H), 1.9 (m, 4 H), 2.4 (dd, 12 H, Ji1 ¼ 15.9 Hz, J2 ¼ 8.4 Hz), 3.5 (dd, 4 H, J1 ¼ 12.9 Hz, J2 ¼ 6.4 Hz), 3.7 (s, 2 H), 7.5 (m, 2 H), 7.6 (t, 1 H), 8.1 (d, 1 H, J ¼ 5.2 Hz), 8.2 (dd, 2 H, J ¼ 6.7 Hz), 8.4 (d, 1 H), 9.4 (t, 1 H, J ¼ 5.8 Hz), 10.8 (t, 1 H, J ¼ 4.6 Hz), ESI-MS m/z: 540 [M þ H]þ. Anal. Calcd for C32H37N5O3: C, 71.22; H, 6.91; N, 12.98. Found: C, 72.14; H, 6.53; N, 13.32.

General procedure for the preparation of the amines

7-Oxo-N4,N5-bis(2-(pyrrolidin-1-yl)ethyl)-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2e)

A suspension of the 5 (0.354 g, 0.1 mmol) in 1,4-dioxane (25 ml) was stirred at room temperature, then 0.5 ml triethylamine and 0.3 mmol appropriate amines added thereto. After stirring for 30 minutes at room temperature, the temperature was then raised to 80  C and held at this temperature for about 1 hour. After cooling, the solvent was removed under reduced pressure. The residue was taken up with dichloromethane and the solution was washed with a solution of sodium carbonate, dried over sodium sulfate and concentrated under vacuum. The crude product was purified by column chromatography with chloroform:methanol to afford the title compound.

Compound 5 was treated with 2-(pyrrolidin-1-yl)ethanamine according to general acylation procedure to give 2e (20%) as yellowish red solid. 1HNMR (CDCl3)  (ppm): 1.8 (q, 4 H, J ¼ 6.2 Hz), 1.9 (q, 4 H, J ¼ 6.2 Hz), 2.6 (t, 4 H, J ¼ 5.0 Hz), 2.8 (s, 4 H), 2.9 (dd, 4 H, J1 ¼ 6.5 Hz, J2 ¼ 6.6 Hz), 3.7 (dd, 2 H, J1 ¼ 12.7 Hz, J2 ¼ 6.4 Hz), 4.0 (dd, 2 H, Ji1 ¼ 12.1 Hz, J2 ¼ 6.6 Hz), 7.6 (t, 1 H, J ¼ 7.5 Hz), 7.7 (td, 2 H, J1 ¼ 16.5 Hz, J2 ¼ 7.7 Hz), 8.3 (d, 1 H, J ¼ 8.1 Hz), 8.4 (d, 1 H, J ¼ 8.6 Hz), 8.5 (t, 2 H, J ¼ 7.9 Hz), 9.2 (t, 1 H),10.7 (t, 1 H). ESI-MS m/z: 512 [M þ H]þ. Anal. Calcd for C30H33N5O3: C, 70.43; H, 6.50; N, 13.69. Found: C, 69.85; H, 6.02; N, 13.17.

N4,N5-bis(2-morpholinoethyl)-7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2a)

N4,N5-bis(3-morpholinopropyl)-7-oxo-7H-dibenzo[de,g]quinoline-4,5-dicarboxamide (2f)

Compound 5 was treated with 2-(morpholine)ethylamine according to general acylation procedure to give 2a (35%) as yellow solid. 1 H-NMR (CDCl3, 300 MHz): 2.6 (m, 8H), 2.7 (t, 2H, J ¼ 6.1), 2.8 (t, 2H, J ¼ 6.4), 3.6 (q, 2H, J1 ¼ 5.8, J2 ¼ 11.7), 3.7 (t, 4H, J ¼ 3.2), 3.8 (s, 4H), 3.9 (q, 2H, J1 ¼ 4.7, J2 ¼ 10.3), 7.6 (t, 1H, J ¼ 7.4), 7.7 (m, 2H), 8.3 (d, 1H, J ¼ 8.6), 8.4 (d, 2H, J ¼ 8.6), 8.5 (t, 1H, J ¼ 8.6), 9.3 (t, 1H, J ¼ 4.6), 10.8 (t, 1H, J ¼ 4.8) ESI-MS m/z: 544 [M þ H]þ. Anal. Calcd for C30H33N5O5: C, 66.28; H, 6.12; N, 12.88. Found: C, 65.35; H, 6.89; N, 12.07.

Compound 5 was treated with 3-(morpholino)propylamine according to general acylation procedure to give 2f (32%) as yellow solid. 1HNMR(CDCl3): 1.93 (m, 2H), 2.04 (m, 2H), 2.46 (t, 4H), 2.55 (m, 8H), 3.56 (q, 2H, J ¼ 6.7), 3.67 (t, 4H, J ¼ 4.58), 3.78 (t, 4H, J ¼ 4.58), 3.90 (q, 2 H, J ¼ 5.36), 7.58 (dd, 1H, J ¼ 8.0, J ¼ 1.0), 7.7 (m, 2H,), 8.3 (d, 1H, J ¼ 8.0), 8.4 (d, 1H, J ¼ 8.6), 8.5 (m, 2H), 9.1 (t, 1H, J ¼ 6.0), 10.7 (t, 1H, J ¼ 1.0); ESI-MS m/z: 572 [M þ H]þ. Anal. Calcd for C32H37N5O5: C, 67.23; H, 6.52; N, 12.25. Found: C, 68.31; H, 6.92; N, 12.74.

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J Enzyme Inhib Med Chem, 2014; 29(5): 722–727

Topoisomerase I inhibition

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13

A modified relaxation assay was carried out as described . Briefly, inhibitory activity of tested compounds on human DNA Topo I was evaluated by monitoring the enzyme-catalyzed relaxation of a supercoiled DNA substrate. Standard assays were performed in 20 mL of topoisomerase I relaxation buffer (10 mM Tris-HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum albumin, 1 mM spermidine, 5% glycerol). The reaction mixture contained 1 unit of the enzyme, 200 ng of supercoiled pBR322 and various concentrations of tested compound and incubated for 30 min at 37  C. The reactions were terminated by addition of 5 ml of gel loading buffer containing 10% SDS, 100 mM EDTA and 0.125% bromophenol blue. The samples were subjected to electrophoresis in 1% agarose in 45 mM Tris-borate (pH 8.0), 1 mM EDTA at 5 V/cm and then stained with 0.5 mg L1 of ethidium bromide. Spectrometric titration and DNA-binding assay The calf thymus DNA (purchased from Sigma Chemical Co.) was dissolved in double-distilled deionized water with 50 mM NaCl and dialyzed for 48 h against a buffer solution of 5 mM of TrisHCl, at pH 7.2, containing 50 mM NaCl at 4  C. The purity of the DNA was checked by monitoring the value of the ratio of the 260 and 280 nm absorbances. The ratio of 1.9 indicated that DNA was sufficiently free of protein14. The DNA concentrations per nucleotide were determined by absorption spectroscopy using the molar extinction coefficient value of 6600 M1 cm1 at 260 nm15. The solution of DNA was stored at 4  C for a short time and then used. Absorption titration was performed at a fixed concentration of drugs (30 mM) in a sodium phosphate buffer (20 mM sodium phosphate, 150 mM NaCl, pH 6.3). Small aliquots of concentrated CT DNA (3.9 mM) were added into the solution to final concentrations ranged from 0 to 165 mM, and stirred for 5 min before measurement. The parameters, max, red shift, hypochromicity and isosbestic point were found from the absorption spectra. Fluorescence titration was performed at a fixed concentration of drugs (3 mM) in sodium phosphate buffer (10 mM sodium phosphate, 50 mM NaCl, pH 7.0). Small aliquots of concentrated CT DNA (3.0 mM) were added into the solution to final concentrations ranged from 0 to 150 mM, and stirred for 5 min. Fluorescence intensity was measured at Ex 410 nm and Ex/Em 5/ 15 nm. Binding constants were derived from the modified Scatchard equation, r/Cf ¼ Ki(1  Nr)[(1  Nr)/[1(N  1)r]]n1, Scheme 1. Synthesis of 7H-dibenzo[de,g]quinolin-7-one 4. Reagents and conditions: (1) DMAD/toluene/reflux/8 days; (2) KOH/ CH3OH/H2O (85  C); (3) Diphenyl ether (250  C).

where r is the molar ratio of bound ligand to DNA, Cf the free ligand concentration, Ki the binding constant and n the binding size in base pairs16,17. Cytotoxicity assay All tumor cell lines, human breast cancer MCF-7 cell, NCI-H460 cell and GLC-82 cell (from ATCC, Tockville, MD) were cultured on RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U ml1 penicillin and 100 g/ml streptomycin in 25 cm2 culture flasks at 37  C in humidified atmosphere with 5% CO2. For the cytotoxicity tests, cells in exponential growth stage were harvested from culture by trypsin digestion and centrifuging at 180 g for 3 min, then resuspended in fresh medium at a cell density of 5  104 cells per ml. The cell suspension was dispensed into a 96-well microplate at 100 ml per well, and incubated in humidified atmosphere with 5% CO2 at 37  C for 24 h, and then treated with the compounds at various concentrations (0, 1, 10, 100 mmol). After 48 h of treatment, 50 ml of 1 mg ml1 MTT solution was added to each well, and further incubated for 4 h. The reaction product was then dissolved with 100 ml of DMSO and the optical density (OD) at 570 nm was recorded. All drug doses were tested in triplicate and the IC50 values were derived by data regression of the OD values versus drug concentrations using a curve-fitting program.

Results and discussion Chemistry Preparation of 2 was carried out by a reported method18. Hydrolysis of 2 by potassium hydroxide gave 3 in 92% yield19. Synthesis of 7 4 was by decarboxylation of 3. Attention to detail was required in the decarboxylation process to obtain good yield (Scheme 1). The solid was then recrystallized in toluene to give a yellow needle solid with an yield of 60%. Oxoaporphine alkaloids are coplanar isoquinoline alkaloids, which possess a 7H-dibenzo[de,g]quinolin-7-one moiety in their structures (Figure 1). The alkaloids can intercalate into the DNA double helix, thus they can inhibit DNA replication, transcription and/or topoisomerase activities11. To improve their DNA-binding ability, the length of the intermediate chain, and consequently its flexibility, was varied in some cases. A series of oxoaporphine derivatives with different basic side chain at 4,5-position of 7H-dibenzo[de,g]quinolin-7-one (2a–f in Figure 2) (general formula Ar-(CONH(CH2)nNR2)2, Ar ¼ 7H-dibenzo[de,g]quinolin-7-one, n¼2 or 3) and 4-position of 7HH3CO

O

O O O

O

N

+

O

reflux

OCH3

N

O

PhMe

O

O

1

NaOH

COOH

92%

N

COOH

2

230∼240 °C 60%

N O

O 3

4

Oxoaporphine alkaloid

DOI: 10.3109/14756366.2013.845818

N

N

O

O

NR2

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Compound

NR2

Compound 3a: n=2; 3e: n=3

N

N(CH3)2

3b: n=2; 3f: n=3

N(CH3)2

2c: n=2;

NEt2

3c: n=2; 3g: n=3

NEt2

2d:n=2

N

2e: n=2

N

2a: n=2; 2f: n=3

N

2b: n=2;

Cl

O

COOH COOH N

CONH(CH2)nNR2

CONH(CH2)nNR2 CONH(CH2)nNR2

Figure 2. Structures of oxoaporphine alkaloid derivatives.

725

O

O

CONH(CH2)nNR2 CONH(CH2)nNR2

Cl SOCI2

O

N

N

O

O

5

O

2a-f

Figure 3. Synthesis of disubstituted derivatives. Reagents and conditions: (1) SOCl2/C6H6; (2) NH2 (CH2)nNR2/Et3N.

dibenzo[de,g]quinolin-7-one (3a–g in Figure 2) (general formula Ar ¼ 7H-dibenzo[de,g]quinolin-7-one, Ar-CONH(CH2)nNR2, n ¼ 2 or 3) were designed and synthesized. 7-Oxo-7H-dibenzo[de,g]quinoline-4,5-dicarbonyl dichloride 5 was obtained in 90% yield by 3 reacted with thionyl chloride20,21. The acid chlorides 5 was then submitted to amidification in 1,4dioxane to obtain amides 2a–2f (Figure 3)22. Based on the favorable biological activity of disubstituted derivatives, monosubstituted derivatives were chosen to avoid the steric repulsion between the two side chains, because the steric repulsion effect in these series of compounds plays an important role. In the monosubstituted compounds, the side chains are amenable to rotation and can easily become coplanar with the oxoaporphine chromophore. Compounds 3a–3f were synthesized using our previous method23. Biology Topoisomerase I inhibition study The effect of 4 on human DNA Topo I was evaluated by the Topo I relaxation assays24. Camptothecin, a well-known Topo I inhibitor, was used as a positive control (Figure 4). The result indicated that 4 could inhibit Topo I activity in dose-dependent manner and completely eliminate Topo I at 50 mM. This is consistent with another study by Woo et al.8,25 who showed that oxoaporphine alkaloids, such as Liriodenine (in Figure 1), could intercalate DNA and inhibit topoisomerase II26. Compared with CPT that fully inhibited Topo I activity at 125 mM (Figure 4), Topo I inhibitory activity of Compound 4 is somewhat two times greater than that of CPT.

Figure 4 Inhibition of Topo I-catalyzed DNA relaxation by 7-oxoaporphine and Camptothecin. Lane 1, pBR322 DNA only. Lane 2, pBR322 DNA and Topo I. Lanes 3–7, pBR322 DNA, Topo I, and various drug concentrations.

DNA-binding properties The DNA-binding properties of compound 4 were evaluated by using UV visible spectral absorbance analysis, which has been used as a convenient tool to detect the interaction between drugs and DNA27. It was observed that the addition of CT DNA to the

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J Enzyme Inhib Med Chem, 2014; 29(5): 722–727

Table 1. Binding constants (Ki) and photometric properties of oxoaporphine derivatives in contact with CT DNA.

Ki 5

1 a

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Compounds ( 10 M ) 4 2a 2b 2c 2d 2e 2f 3a 3b 3c 3e 3f 3g

Red Isosbestic max shift Hypochromicity point b,d b,d b,d (nm) (%) (nm) (nm)

0.21 0.43e 0.67e 0.82e 0.87e 1.02e 0.52e 2.41c 2.26c 3.16c 3.21c 2.45c 4.03c

412 467 462 465 457 466 431 427 425 432 425 430 423

5 16 18 17 19 20 14 4 1 5 5 5 6

6 5 8 8 10 12 7 9 8 16 12 15 20

420 Unclear 506 514 508 511 Unclear 450 Unclear 452 453 452 450

a

Figure 5 Absorption titration of 7-oxoaporphine with CT DNA. Compound 7-oxoaporphine (20 mM) in 10 mM sodium phosphate buffer (pH 7.0) with 150 mM NaCl at increasing CT DNA concentration (arrow: 0–400 mM).

solutions of 4 at the DNA/compound molar ratios varying from 0 to 20 induced a bathochromic shift (5 nm) and hypochromicity (6%) (Figure 5). Hypochromism and the red shift in the absorption spectra indicated a strong electronic interaction between the chromophore of oxoaporphine and the DNA bases28–30, which suggested a close proximity of the oxoaporphine chromophore to the DNA bases through a strong overlap of the states. These spectral changes indicated a strong electronic interaction between the 7H-dibenzo[de,g]quinolin-7-one moiety of oxoaporphine and the DNA bases, which suggested a close proximity of the oxoaporphine chromophore to the DNA bases through a strong overlap of the –* states16. In addition, an isosbestic point was also observed in the spectra. These various spectral changes were consistent with the intercalation of these derivatives into the DNA base stack16. This is confirmed by a previous report in which Liriodenine, an oxoaporphine alkaloid, was found to intercalate into the DNA base pairs (Table 1)9.

3 mM drug in 10 mM NaH2PO4-Na2HPO4 (pH 7.0) buffer containing 50 mM NaCl at room temperature. b 20 mM drug in 10 mM NaH2PO4-Na2HPO4 (pH 7.0) buffer containing 150 mM NaCl at room temperature. c Obtained from spectrofluorimetric titration at ex ¼ 430 nm and em ¼ 450 nm. d Obtained at max. e Obtained from spectrofluorimetric titration at ex ¼ 464 nm and em ¼ 470 nm. Table 2. IC50 cytotoxicity values (mM) of oxoaporphine derivatives against tumor cells. Compound 4 2a 2b 2c 2d 2e 2f 3a 3b 3c 3e 3f 3g

MCF-7

NCI-H460

GLC-82

15.0 9.21 9.01 6.75 5.11 3.58 8.28 2.63 2.96 1.94 1.86 9.22 1.16

45.4 31.37 17.00 20.15 11.06 10.21 18.33 6.39 8.43 6.79 2.45 8.08 4.12

35.4 23.51 26.28 25.01 21.22 10.18 20.91 36.93 9.90 28.30 43.01 35.01 5.42

Cytotoxicity profiles Table 2 shows the IC50 values of compounds against three types of tumor cells (human breast cancer MCF-7 cell line, NCI-H460 cell line and GLC-82 cell line) in culture. According to the Table 2, it was clear that all of monosubstituted ligands exhibited significant higher cytotoxicity than that of disubstituted derivatives. An explanation for the lower activity of disubstituted derivatives is that, steric repulsion between the two side chains makes it not easily become coplanar between basic side chain and the oxoaporphine chromophore and their larger molecular weights make them more difficult to diffuse through the plasma membrane. While there is almost no steric repulsion in monosubstituted derivatives, therefore, the monosubstituted derivatives can easily intercalate into the DNA molecules, thus increase the DNA-binding affinity. The binding constants and cytotoxicity values are detailed in Tables 1 and 2, respectively. 7H-dibenzo[de,g]quinolin-7-one 4 has a weak biological activity. The disubstituted derivatives showed more potency than their lead compound in cytotoxicity assay. These results indicate that the amino side chains introduced into the lead compound appeared to be the major contributors to the DNA binding and cytotoxicity enhancement. All the monosubstituted ligand compounds had higher DNA-binding

constant and cell cytotoxicity than those of disubstituted derivatives (see Tables 1 and 2). We proposed that different DNA binding constant of monosubstituted derivatives and disubstituted derivatives are due to the conformational rotational difference of the side chains. It may because in disubstituted derivatives, there are steric repulsion between the two side chains, thus there is high-energy barrier for the two side chains to become coplanar with the oxoaporphine chromophore. Due to this constrain, disubstituted derivatives cannot easily intercalate into the DNA. In that sense, ortho-substitution groups that cannot become coplanar with the oxoaporphine chromophore are unfavorable for designing DNA-intercalators as antitumor drugs.

Conclusion A series of oxoaporphine alkaloid derivatives were synthesized and evaluated for their anticancer activities. All derivatives had a higher DNA-binding constant and more potent cytotoxicity activity than their lead compound. Moreover, the UV spectroscopic studies indicated that the compounds bind to DNA in an intercalative mode. The DNA-binding affinity is in well

DOI: 10.3109/14756366.2013.845818

agreement with the IC50 values. The present results indicate that introduction of the amino side chains can dramatically increase the anticancer activities of oxoaporphine alkaloids.

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This project was supported by Natural Science Foundation of Guangxi Province of China (2013GXNSFBA019158) and the Doctor Foundation of Guangxi Medical University (GXMUYSF201207).

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Design, synthesis and anticancer activity of oxoaporphine alkaloid derivatives.

A series of new oxoaporphine derivatives were synthesized and their inhibitory activity of topoisomerase I, cytotoxicity and DNA-binding properties we...
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