www.elsevier.com/locate/jphotobiol

Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226

Isoquino[4,5-bc]acridines: Design, synthesis and evaluation ofDNA binding, anti-tumor and DNA photo-damaging ability

Peng Yang a, Qing Yang b, Xuhong Qian a,c,*, Lianpeng Tong a, Xiaolian Li a

a State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116012, PR Chinab Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian 116012, PR China

c Shanghai Key Laboratory of Chemical Biology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China

Received 1 December 2005; received in revised form 25 March 2006; accepted 30 March 2006Available online 22 May 2006

Abstract

Several novel isoquino[4,5-bc]acridine derivatives have been designed and synthesized. Their DNA-binding, anti-tumor and DNA-photo-damaging properties were investigated. A4 exhibited the highest anti-tumor activities against both A 549 (human lung cancer cell)and P388 (murine leukemia cells). All these compounds were found to be more cytotoxic against P388 than against A549. Under 365-nmlight irradiation, A3 damaged plasmid DNA pBR322 at <2 lM and cleaved DNA from form I to 100% form II by 50 lM. The mech-anism studies revealed that A3 damaged DNA by electron transfer mechanism and singlet oxygen species.� 2006 Elsevier B.V. All rights reserved.

Keywords: Acridine; DNA binding; DNA photo-cleavage; Anti-tumor activity

1. Introduction

Early in 1913, the antibacterial properties of 3,6-diami-noacridine and 3,6-diamino-10-methylacridinium chloridewere determined [1]. Albert and co-workers subsequentlyreported that 9-aminoacridine was a potential antibacterialagent. The anticancer properties of acridine compoundshave been known since the mid 1960s, since this time theyhave been studied extensively [1,2]. To date, a range of acri-dine derivatives have been reported with a range of chem-ical and physical properties. Their utility in thepharmaceutical industry has also been reported [2]. Stevens[3] have designed and synthesized methylated pentacyclicquinoacridinium salts and found their telomerase-inhibi-tory potency. Dzierzbicka [4] reported that the sugar moi-ety acylated acridine/acridone derivatives exhibited potentin vitro cytotoxic activity against a panel of human cell

1011-1344/$ - see front matter � 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.jphotobiol.2006.03.005

* Corresponding author. Tel.: +86 411 83673466; fax: +86 41183673488.

E-mail addresses: xhqian@dlut.edu.cn, xhqian@ecust.edu.cn (X. Qian).

lines. By cyclization the acridine derivatives with phosgene,Antonini [5] reported the enhanced DNA binding affinitiesof these compounds and their satisfactory results of in vitrocytotoxic properties.

Concerning the biological activities of acridine deriva-tives, the most widespread idea is that their planar ring sys-tems and the basic amino groups contribute to the DNAintercalation [1]. Due to the planar ring systems of naph-thalimide derivatives, in recent years, both their DNAintercalative and anti-tumor abilities have been studiedwidely [6]. Brana’s group made great efforts on studies ofheterocycle-fused naphthalimide derivatives to improvetheir topoisomerase I inhibitory and anti-tumor abilities[7].

The anticancer, antibacterial and DNA photo-damagingproperties of both acridine and naphthalimide DNA inter-calators are their important biological applications [1,6,8].As anti-tumor and antibacterial agents, these compoundsare often properly modified to enhance the DNA bindingaffinity, the inhibitory activity toward DNA regulatoryenzymes such as topoisomerase I/II, and the tumor cellcytotoxicity. When used as DNA photo-cleavers, however,

A1 R=Cl A2 R=SCH2CH3A3 R=HN(CH2CH2)2NCH3A5 R =H

N

N

O

ON

R

NH

N

O

ON

O

A4

Fig. 1. The structures of isoquino[4,5-bc]acridine derivatives A1–A5.

222 P. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226

they are usually linked with photo-reactive groups in orderto make fully use of its DNA affinity [8,9]. So far, however,examples that could take fully advantage of the structuralcharacteristics of both acridine and naphthalimide chro-mophores are less well-reported [8].

In a continuous attempt to develop highly DNA bindingmolecules, anti-tumor agents and highly active DNAphoto-cleavers [10], we designed and synthesized novel iso-quino[4,5-bc] acridine derivatives A1–A5 (Fig. 1). In thisstudy, naphthalimide active group remained and the quin-oline unit was effectively fused with naphthalimide unit.These compounds were designed to have the obvious elec-tron-deficiency characteristics for the sake of the strongelectron withdrawing effect of two carbonyl groups ofnaphthalimide. Hence, the high DNA intercalative abilitiesof these novel acridine derivatives, their in vitro anti-tumoractivities (in the dark) and their DNA photo-damagingproperties were anticipated.

2. Experiments

2.1. Materials and instruments

All the solvents were of analytic grade. 1H NMR wasmeasured on a Bruker AV-400 spectrometer with chemi-cal shifts reported as parts per million (in DMSO-d6/CDCl3, TMS as an internal standard). Mass spectra weremeasured on a HP 1100 LC–MS spectrometer. Meltingpoints were determined by an X-6 micro-melting pointapparatus and uncorrected. Spectrophotometer. UV/Visabsorption spectra were recorded on Shimadzu UV andfluorescence spectra on Perkin–Elmer LS 50 luminescencespectrophotometer.

2.2. Synthesis

2.2.1. Synthesis of A14-Bromo-1,8-naphthalic anhydride (2.77 g, 10.0 mmol)

and N,N 0-dimethylethylenediamine (1.00 g, 11.4 mmol)

were added to 10 mL ethanol, the reaction mixture stirredat reflux temperature for 3 h, cooled, filtered and thendried, the crude product was obtained as yellow solid(2.95 g, 8.50 mmol, 85% yield). APCI-MS (positive) m/z:348.2 ([M + H]+). (b) The obtained 4-bromo-1,8-naphthal-imide derivative (2 g, 5.76 mmol) and 2-aminobenzoic acid(0.80 g, 6.0 mmol), copper bronze (0.038 g, 0.60 mmol),CuI (0.101 g, 0.53 mmol), were dissolved in 10 mL DMF.The solution was stirred at 100 �C for 24 h, filtered whenit was hot, the filtrate was cooled and poured into the icewater, filtered and dried to get the red product 1. (2.0 g,4.95 mmol, 86% yield) APCI-MS (positive) m/z: 404.3([M + H]+). This product was not purified and was useddirectly in the next step. (c) 1.0 g of 1 was stirred in phos-phorus oxychloride at 110 �C for 12 h, then cooled andpoured into the ice water, N (Et)3 was added when stirredvigorously, filter and dried to give yellow solid A1 (0.88 g,88% yield).

Separated by silica gel chromatography (CHCl3:MeOH = 9:1, v/v) to get pure product A1; m.p. 204.8–205.2 �C. 1H NMR (CDCl3) d (ppm): 2.99 (s, 6H,NCH3), 3.51 (s, br, 2H, NCH2), 4.69–4.72 (t, J = 6.0 Hz,2H, CONCH2), 7.77–7.81 (dd, J1 = 7.6 Hz, J2 = 7.6 Hz,1H), 7.96–8.04 (m, 2H), 8.39–8.41 (d, J = 8.8 Hz, 1H),8.52–8.54 (d, J = 8.4 Hz, 1H), 8.72–8.74 (d, J = 7.6 Hz,1H), 9.52 (s, 1H), 9.77–9.79 (d, J = 8.0 Hz, 1H), ESI-HRMS: Calcd for C23H18ClN3O2 (M + H+): 404.1166,Found: 404.1158. IR (KBr): 2923, 2853, 1702, 1660,1347 cm�1.

2.2.2. Synthesis of A2A1 (1.0 g, 2.48 mmol) and ethanethiol (0.20 g,

3.23 mmol) was added to 10 mL acetonitrile. The solutionwas refluxed for 10 h, cooled and filtered. Separated by sil-ica gel chromatography (CHCl3: MeOH = 9:1, v/v) to getpure A2 (0.88 g, 83% yield); m.p. 161.0–161.4 �C. 1HNMR (CDCl3) d (ppm): 1.19–1.23 (t, J = 7.2 Hz, 3H,SCH2CH3), 2.51(s, 6H, NCH3), 2.86 (s, br, 2H, NCH2),3.04–3.10 (q, J = 7.2 Hz, 2H, SCH2), 4.44–4.47 (t,J = 7.2 Hz, 2H, CONCH2), 7.75–7.78 (dd, J1 = 8.4 Hz,J2 = 6.8 Hz, 1H), 7.94–8.00 (m, 2H), 8.39–8.41 (d,J = 8.8 Hz, 1H), 8.70–8.72 (d, J = 8.0 Hz, 1H), 8.87–8.89(d, J = 8.8 Hz, 1H), 9.74–9.76 (d, J = 8.0 Hz, 1H), 9.88(s, 1H), ESI-HRMS: Calcd for C25H23N3O2S (M + H+):430.1589. Found: 430.1592. IR (KBr): 3397, 3297, 2962,2852, 1701, 1663, 1346 cm�1.

2.2.3. Synthesis of A3The preparation and purification procedure of A3 was

the similar to that of A2. Separated by silica gel chroma-tography (CHCl3: MeOH = 5:1, v/v) to get pure A3 (86%yield); m.p. 197.6–198.2 �C. 1H NMR (CDCl3) d (ppm):1.62 (s, br, 7H, (CH2CH2)2NCH3), 2.60 (s, 6H,N(CH3)2), 2.93 (s, br, 4H, (CH2CH2)2NCH3), 3.87 (s, br,2H, NCH2), 4.50 (s, br, 2H, CONCH2), 7.62 (s, br, 1H),7.85–7.96 (m, 2H), 8.35–8.37 (d, J = 8.4 Hz, 1H), 8.42–8.44 (d, J = 8.8 Hz, 1H), 8.67–8.69 (d, J = 7.6 Hz, 1H),

P. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226 223

9.45 (s, 1H), 9.70–9.72 (d, J = 8.8 Hz, 1H), ESI-HRMS:Calcd for C23H19N3O2 (M + H+): 468.2400. Found:468.2412. IR (KBr): 2937, 2819, 1698, 1657, 1377 cm�1.

2.2.4. Synthesis of A4Compound 1 (1.0 g, 2.50 mmol) was added to 10 mL of

concentrated sulfuric acid, and the resulting mixture washeated at 90 �C for 3 h, cooled and poured slowly intoice water (100 g) with stirring. NH3H2O was added untila pH of 9 resulted while the temperature remained below5 �C. Separated by silica gel chromatography (CHCl3:MeOH = 9:1, v/v) to get pure A4 (0.57 g, 60% yield);m.p. >300 �C. 1H NMR (d6-DMSO) d (ppm): 2.25 (s,6H, NCH3), 2.55 (s, br, 2H, NCH2), 4.15 (s, br, 2H,CONCH2), 7.39–7.43 (dd, J1 = 6.8 Hz, J2 = 6.8 Hz, 1H),7.81–7.84 (dd, J1 = 7.6 Hz, J2 = 7.2 Hz, 1H), 7.95–8.02(m, 2H), 8.22–8.24 (d, J = 7.6 Hz, 1H), 8.56–8.58 (d,J = 6.8 Hz, 1H), 9.09 (s, 1H), 9.27–9.29 (d, J = 8.8 Hz,1H), ESI-HRMS: Calcd for C23H20N4O2 (M + H+):386.1505. Found: 385.1516. IR (KBr): 3327, 2959, 2817,1699, 1656, 1396 cm�1.

2.2.5. Synthesis of A5(a) 1,1-Carbonyldiimidazole (0.51 g, 3.2 mmol) was

added to a suspension of 1 (0.806 g, 2.0 mmol) in5 mL of dry THF. The mixture was stirred at refluxtemperature for 24 h. It was added dropwise to15 mL THF-H2O (1:1) containing NaBH4 (0.55 g,15 mmol). The resulting solution was stirred for20 min. Then, 5 N HCl was added until a pH of 6and the product precipitated by removing the THFin vacuum, filtered and dried to give a red oil(0.62 g, 50% yield). API-ES (positive) m/z: 390.0(M + H)+. The obtained product was not purifiedand was carried out in the next step.

(b) The active MnO2 (3.5 g) was added to the solution ofthe obtained orange solid (1.0 g, 2.57 mmol) in 50 mLof chloroform, and the mixture was stirred at roomtemperature for 3 days. The MnO2 was filtered, andthe filter cake was washed with hot chloroform. Thefiltrate was evaporated in vacuum to give a red oilof 0.82 g (82% yield). API-ES (positive) m/z: 388.1(M + H)+. The obtained product was not purifiedand was carried out in the next step.

(c) The obtained compound was dissolved in trifluoro-acetic acid. The solution was stirred at room temper-ature for 24 h under nitrogen. Then, it was pouredinto ice water with 5% NH3H2O, filtered and driedto give yellow solid. Separated by silica gel chroma-tography (CHCl3: MeOH = 9:1, v/v) to get pure A5

(0.31 g, 40% yield); m.p. 191.0–193.2 �C. 1H NMR(CDCl3) d (ppm): 2.46 (s, 6H, NCH3), 2.81 (s, br, 2H, NCH2), 4.41–4.44 (t, J = 6.4 Hz, 2H, CONCH2),7.68–7.71 (dd, J1 = 7.2 Hz, J2 = 7.6 Hz, 1H), 7.94–8.02 (m, 2H), 8.13–8.15 (d, J = 8.8 Hz, 1H), 8.40–8.42 (d, J = 8.8 Hz, 1H), 8.71–8.73 (d, J = 8.0 Hz,

1H), 8.97 (s, 1H), 9.04 (s, 1H), 9.75–9.77 (d,J = 8.4 Hz, 1H), ESI-HRMS: Calcd forC23H19N3O2 (M + H+): 370.1556. Found: 370.1565.IR (KBr): 2926, 2854, 1735, 1632, 1384 cm�1.

2.3. Spectroscopic measurements and DNA binding studies

A3 were dissolved in absolute ethanol to give 10�5 Msolutions and rhodamine B in ethanol was used as quantumyield standard.

DNA binding studies was carried out in the buffer ofTris (tris (hydroxymethyl) aminomethane)–HCl (30 mM,pH 7.0). 0.1 mL A3 DMSO solution (10�3–10�4 M) wasdiluted with buffer to 10 mL. Fluorescence wavelengthand intensity area of samples were measured.

2.4. Photo-cleavage of supercoiled pBR322DNA

Irradiation was performed with lamp (2.3 mW/cm2, Co(NO3)2, 365 nm), placed at 20 cm from samples. A filterwas used to filter off short wavelength light preventingDNA from photo-cleavage. The irradiated samples con-tained pBR322 DNA (0.5 lg) dissolved in Tris–HCl buffer(20 mM, pH 7.5) and the examined compounds. Super-coiled DNA runs at position I, nicked DNA at positionII and linear DNA at position III. The samples were ana-lysed by gel electrophoresis in 1% agarose gel and wasstained with ethidium bromide. Experiments were repeatedin triplicate.

2.5. Molecular modeling methods

AM1 semi-empirical method was used to carry out thecalculation of energies of A1–A5 at ground state, excitedsinglet and triplet state and the n = 1r was selected in allcases.

3. Results and discussion

3.1. Synthesis and spectra

The synthesis of A1–A5 was outlined in Scheme 1. Thestarting material, 4-bromo-1,8-naphthalic anhydride,reacted with N,N 0-dimethylethylenediamine in ethanol for3 h. And then, the obtained naphthalimide derivative wascoupled with 2-aminobenzoic acid in DMF, catalyzed byCuI and Cu [11]. Compound A5 was synthesized basedon the reported procedure [12]. The yield of ring closurewas satisfactory due to the electron-rich property of 3-siteof 1,8-naphthalic anhydride [12,13]. Compounds A2 andA3 were synthesized by treatment of A1 with different sub-stituents. A4 was obtained by dehydration reaction inH2SO4. All of their structures were confirmed by IR, 1HNMR and HRMS. The UV–Vis and fluorescent spectrafor these compounds were measured and the data wereshown in Table 1.

a , b

c , d

h

e ,f ,g

A 4

A 5

A 1 ~ A 3

1

O OO

B r

N OO

H N

C O O H

N

Scheme 1. Syntheses of Isoquino[4,5-bc] acridines derivatives. (a) N,N 0-dimethylethylenediamine, ethanol, reflux, 3 h, 85% yield; (b) 2-aminobenzoic acid,Cu/CuI, DMF, 100 �C, 24 h, 86% yield; (c) phosphorus oxychloride, 110 �C, 12 h, 88% yield; (d) ethanethiol/N-methylpiperazine, acetonitrile, reflux, 10 h,83%/86% yield; (e) CDI, NaBH4, THF, reflux, 24 h, 50% yield; (f) MnO2, chloroform, room temperature, 3 days, 82% yield; (g) TFA, room temperature,24 h, 40% yield; (h) H2SO4, 90 �C, 3 h, 60% yield.

Table 1Spectra dataa,b and cytotoxicities (A-549c, P388d) of compounds

Compound UV kmax/nm (log e) FL kmax/nm (U) Stokes’ shift/nm Cytotoxicity (IC50, lM)

A549 P388

A1 377 (3.65) 447 (0.03) 70 13.18 0.409A2 425 (3.71) 520 (0.10) 95 37.40 0.218A3 450 (3.56) 557 (0.02) 107 20.81 1.170A4 418 (5.36) 495 (0.52) 77 0.133 0.016A5 378 (3.75) 457 (0.10) 79 7.890 0.198

a In absolute ethanol.b With rhodamine B in ethanol as quantum yield standard (U = 0.97).c Cytotoxicity (CTX) against human lung cancer cell (A549) was measured by sulforhodamine B dye-staining method [15].d CTX against murine leukemia cells (P388) was measured by microculture tetrazolium-formazan method [16].

Fig. 2. Fluorescence spectra before and after interaction of compound A3

and CT-DNA. Curves F and F-CT corresponded to compound A3 beforeand after being mixed with DNA. Numbers 1–4 indicated the concentra-tion of B2, 5, 10, 20, 40 lM, respectively. DNA applied was 50 lM (bp).

224 P. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226

From Table 1, we could see that with a strong electron-donating group, N(CH2CH2)2NCH3, A3 showed the lon-gest absorption and fluorescence wavelength, which madeit have potential value as photonuclease for safe applica-tion. In addition, the fluorescence quantum yield of A3

was relatively low and its stokes shift (107 nm) was thelargest one among these compounds. We proposed thatthis unusually large stokes shift was presumably due tothe twisted intramolecular charge transfer (TICT) statebetween N(CH2CH2)2NCH3 group and acridinefluorophore.

The Scatchard binding constant between calf thymusDNA (CT-DNA) with A3, used as an example, was moni-tored by fluorescence spectroscopy technique (in 30 mMTris–HCl buffer, pH 7.0) [14]. As shown in Fig. 2, the emis-sion intensity of the free dye was quenched upon the addi-tion of CT-DNA. Its Scatchard binding constant wasdetermined to be 3.55 · 105 M�1, indicating the efficientDNA binding affinity.

3.2. Anti-tumor activity

Sulforhodamine B (SRB) assay against A549 (humanlung cancer cell) and MTT tetrazolium dye assay againstP388 (murine leukemia cell) were used to evaluate theanti-tumor activities of these compounds [15,16]. IC50 rep-resents the drug concentration (micro-molar) required to

inhibit cell growth by 50%. Their cytotoxicities in the dark(as IC50 values) were listed in Table 1.

It could be seen from Table 1 that the compound (A4)with electron-withdrawing carbonyl groups at 8-positionexhibited the highest cytotoxicity against both A549(0.133 lM) and P388 (0.016 lM). For A549, the order ofanti-tumor activities of these compounds was revealed asA4 > A5 > A1 > A3 > A2. As far as P388 was concerned,the order was A4 > A5 > A2 > A1 > A3. It was clear thatboth A2 (13.18 lM) and A3 (37.40 lM) with the electron-donating group at 8-position exhibited the relatively lower

P. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226 225

activities against A549 and the compound A5 without anysubstituent was of moderate cytotoxicity against both A549(7.89 lM) and P388 (0.198 lM).

It could also be seen that all these compounds werefound to be more cytotoxic against P388 than againstA549. A2 exhibited the most excellent selective anti-tumoractivity and was nearly 172-fold more active towards P388(IC50, 0.218 lM) than against A549 (IC50, 37.40 lM).Compared to the others, A4 resulted in the slightestincreased ratio of anti-tumor activity, which was approxi-mately 8-fold more active. For A1, A3 and A5, theincreased ratio was 32-, 18- and 40-fold, respectively.

3.3. DNA photo-damaging ability

DNA photo-cleaving activities of A1–A5 were evaluatedin 20 mM Tris–HCl (pH 7.5) buffered aqueous solutions inthe presence of supercoiled pBR322 plasmid DNA. Thephotosensitized DNA cleavage products were analysed by1% agarose gel electrophoresis. In the dark, they did notpromote DNA strand breaks. When they were irradiatedunder 365 nm light, the DNA strand break was detectedin the relaxed, open circular form. Fig. 3(a) revealed thatA3 induced the most efficient cleavage. During irradiation,supercoiled plasmid DNA pBR322 started to be damagedby 2 lM of A3 and was cleaved from form I to 100% form

Fig. 3. Photo-cleavage of supercoiled pBR322 DNA in the buffer of Tris–HCl (20 mM, pH 7.5) containing 20% acetonitrile under 365 nm lightirradiation (a) DNA photo-cleavage by compounds (100 lM) for 2 h.Lanes 1–5, A5, A4, A3, A2, A1 and DNA, respectively, lane 6, DNA alone(hm), lane 7, DNA alone (no hm). (b) DNA photo-cleavage by A3 atvarious concentrations for 2 h. Lane 1, DNA alone (no hm); lane 2, DNAalone (hm); lanes 3–6, A3 at concentration of 50, 25, 5, 2 lM, respectively.(c) Effects of additives on DNA photo-cleavage by A3 (25 lM) for 2 h.Lanes 1–5, DNA and A3 in the presence of DMSO (1.4 M), DTT(30 mM), SOD (100 lg/mL), ethanol (1.7 M), NaN3 (100 mM), lanes 6,DNA and A3, lane 7, DNA alone (hm), lane 8, DNA alone (no hm). (d)Effect of D2O on DNA photo-cleavage by A3 (25 lM) for 2 h. Lane 1,DNA and A3 in 70% D2O, lanes 2, DNA and A3 in H2O, lane 3, DNAalone (hm), lane 4, DNA alone (no hm). (e) Inhibitory effect of NaN3 on theformation of Form II DNA photo-induced by A3 (25 lM) for 2 h. Lanes1–5, DNA and A3 in the presence of NaN3 at concentrations of 200, 100,75, 50 and 25 mM, respectively, lane 6, DNA alone (hm), lane 7, DNAalone (no hm). (f) Inhibitory effect of NaN3 by compounds (50 lM) for 1 h.Lanes 1, 3 and 5, DNA and A4, A3, A2 in the presence of NaN3

(100 mM); lanes 2, 4 and 6, DNA and A4, A3, A2; lane 7, DNA alone (hm);lane 8, DNA alone (no hm).

II by 50 lM of A3 (Fig. 3(b)). The order of the DNAphoto-damaging efficiency was A3 > A2 > A4 � A1 � A5.

A mechanistic experiment was performed by the addi-tion of sodium azide NaN3 (singlet oxygen quencher),dithiothreitol (DTT, superoxide radical scavenger), ethanol(radical scavenger) and superoxide dismutase (SOD, super-oxide radical killer), respectively (Fig. 3(c)). It was shownin Fig. 3(c) that DTT and SOD could obviously inhibitthe DNA cleaving activity. As we know, both DTT andSOD could retard the superoxide anion radicals and thelatter was formed through electrons transferring fromnucleobase to the intercalator. Therefore, we supposed thatthere existed the electron transfer mechanism in this DNAphoto-damaging process.

Interestingly, Fig. 3(c) (lanes 5) showed clearly thatNaN3 inhibited the DNA photo-cleavage. It is well knownthat NaN3 is an excellent singlet oxygen quencher, andD2O makes the lifetime of singlet oxygen longer [17,18].To further confirm whether singlet oxygen was involvedor not, the trapping experiment was carried out usingsodium azide NaN3 and deuterium oxide D2O. By replac-ing the reaction media of H2O with 70% D2O (Fig. 3(d)),the DNA cleaving efficiency was actually enhanced. Itwas found in Fig. 3(e) that the DNA damage was signifi-cantly suppressed with the increased concentration ofNaN3 and was almost completely inhibited in the presenceof 100 mM NaN3. Furthermore, the difference of singletoxygen generation efficiencies between A2, A3 and A4 wereexamined by adding NaN3 of 100 mM. It could be seenfrom Fig. 3(f) that NaN3 could inhibit DNA photo-cleav-age of these tested compounds to different extents and mostobviously, it retarded the DNA cleave by A3. This indi-cated that A3 could produce singlet oxygen more efficientlythan the others during irradiation.

It is known that the singlet oxygen is a highly reactivespecies that preferentially oxidize guanine (G) and it is gen-erated because the photo-excited compounds convert thetriplet state energy to triplet oxygen [8]. Generally speak-ing, the higher is the triplet state energy of an exited com-pound, the more possibly it produces the singlet oxygen.With AM1 semi-empirical quantum calculations (Hyper-chem 7.0), in this study, we calculated the energies ofA1–A5 at ground state, excited singlet and triplet state(Table 2). It could be seen that A3 was of the higher excitedtriplet state energy than the other compounds, which mayaccount for its highest efficiency of singlet oxygen genera-tion, as shown in Fig. 3(f).

Table 2The calculated energies of A1–A5 at ground state, excited singlet andtriplet state

Compound Ground state/eV Excited singlet/eV Excited triplet/eV

A1 �9.050481 �2.006789 �4.162990A2 �8.670872 �1.915859 �4.043906A3 �8.306059 �1.476122 �3.582076A4 �8.997966 �1.689520 �3.822735A5 �9.036777 �1.826130 �3.992400

226 P. Yang et al. / Journal of Photochemistry and Photobiology B: Biology 84 (2006) 221–226

From the above mentioned, we were confirmed that dur-ing the DNA photo-cleavage process of A3, the higher gen-eration efficiency of singlet oxygen were obtained for thesake of its greater excited triplet state energy and at thesame time, electrons transfer efficiency from nucleobasesto DNA intercalators could be enhanced by the strong elec-tron-donating group at its 8-position. Therefore, it is notdifficult to understand the highest photo-damaging activityof A3.

4. Conclusions

In summary, the present work demonstrates the designand synthesis of novel DNA intercalators isoquino[4,5-bc]acridines derivatives, A1–A5. For both P388 andA549, A4 exhibited the highest cytotoxicity and IC50 valueswere 0.016 lM and 0.133 lM, respectively. All these com-pounds were found to be more cytotoxic against P388 thanagainst A549. A2 was nearly 172-fold more active towardsP388 than against A549. Under 365 nm light irradiation,A3 exhibited the greatest DNA cleavage activity due tothe highest efficiency of both electron transfer and singletoxygen generation and it could damage circular super-coiled pBR322 DNA at <2 lM. During irradiation, thisfamily of novel DNA intercalators was shown to damageDNA by electron transfer mechanism and 1O2 reactivespecies.

Acknowledgements

Financial support by the National Key Project for BasicResearch (2003CB114400) and under the auspices of Na-tional Natural Science Foundation of China is greatlyappreciated.

References

[1] A. Albert, The Acridines, second ed., Edward Arnold Publishers Ltd.,London, 1966.

[2] M. Demeunynck, F. Charmantray, A. Martelli, Interest of acridinederivatives in the anticancer chemotherapy, Curr. Pharm. Des. 7(2001) 1703–1724.

[3] R.A. Heald, C. Modi, J.C. Cookson, I. Hutchinson, C.A.Laughton, S.M. Gowan, L.R. Kelland, M.F.G. Stevens, Antitu-mor polycyclic acridines: synthesis and telomerase-inhibitoryactivity of methylated pentacyclic acridinium salts, J. Med. Chem.45 (2002) 590–597.

[4] K. Dzierzbicka, A.M. Kolodziejczyk, Synthesis and antitumoractivity of conjugates of muramyldipeptide or normuramyldipeptidewith hydroxyacridine/acridone derivatives, J. Med. Chem. 46 (2003)183–189.

[5] I. Antonini, P. Polucci, A. Magnano, S. Sparapani, S. Martelli,Rational design, synthesis, and biological evaluation of bis(pyrim-ido[5,6,1-de]acridines) and bis(pyrazolo[3,4,5-kl]acridine-5-carboxa-mides) as new anticancer agents, J. Med. Chem. 47 (2004) 5244–5250.

[6] P.F. Bousquet, M.F. Brana, D. Conlon, K. Fitzgerald, M.D. Perron,C. Cocchiaro, R. Miller, M. Moran, J. George, X.D. Qian, G.Keilhauer, C.A. Romerdahl, Preclinical evaluation of LU 79553: anovel bis-naphthalimide with potent antitumor activity, Cancer Res.55 (1995) 1176–1189.

[7] M.F. Brana, M. Cacho, A. Gradillas, B. Pascual-Teresa, A. Ramos,Intercalators as anticancer drugs, Curr. Pharm. Des. 7 (2001) 1745–1780.

[8] B. Amitage, Photocleavage of nucleic acids, Chem. Rev. 98 (1998)1171–1200.

[9] J. Joseph, N.V. Eldho, D. Ramaiah, Design of photoactivated DNAoxidizing agents: synthesis and study of photophysical properties andDNA interactions of novel viologen-linked acridines, Chem. Eur. J. 9(2003) 5926–5935.

[10] Q. Yang, X.H. Qian, J.Q. Xu, Y.S. Sun, Y.G. Li, Thio-heterocylicnaphthalimides with aminoalkyl side chains: novel alternative toolsfor photodegradation of genomic DNA without impairment onbioactivities of proteins, Bioorg. Med. Chem. 13 (2005) 1615–1622.

[11] O. Baudoin, F.M.P. Teulade, J.P. Vigneron, J.M. Lehn, Cyclobisin-tercal and macrocycles: synthesis and physicochemical properties ofmacrocyclic polyamines containing two crescent-shaped dib-enzophenanthroline subunits, J. Org. Chem. 62 (1997) 5458–5470.

[12] S. Feng, C.A. Panetta, D.E. Graves, An unusual oxidation of abenzylic methylene group by thionyl chloride: a synthesis of 1,3-dihydro-2-[2-(dimethylamino)ethyl]-1,3-dioxopyrrolo[3,4-c]acridinederivatives, J. Org. Chem. 66 (2001) 612–616.

[13] R.M. AchesonThe Chemistry of Heterocyclic Compounds, vol. 9,Wiley Interscience, New York, 1973, pp. 563–593.

[14] M. Gupta, R. Ali, Fluorescence studies on the interaction offurocoumarins with DNA in the dark, J. Biochem. 95 (1984) 1253–1257.

[15] P. Skehan, R. Storeny, D. Scudiero, A. Monks, J. McMahon, D.Vistica, J.T. Warren, H. Bokesc, S. Kenney, M.R. Boyd, Newcolorimetric cytotoxicity assay for anticancer-drug screening, J. Natl.Cancer Inst. 82 (1990) 1107–1112.

[16] M. Kuroda, Y. Mimaki, Y. Sashida, T. Hirano, K. Oka, A. Dobashi,H. Li, N. Harada, Novel cholestane glycosides from the bulbs ofornithogalum saundersiae and their cytostatic activity on leukeminHL-60 and MOLT-4 cells, Tetrahedron 53 (1997) 11549–11562.

[17] T.P.A. Devasagayam, S. Steenken, M.S.W. Obendorf, W.A. Schulz,H. Sies, Formation of 8-hydroxy(deoxy)guanosine and generation ofstrand breaks at guanine residues in DNA by singlet oxygen,Biochemistry 30 (1991) 6283–6289.

[18] A.U. Khan, P. DiMascio, M.H.G. Medeiros, T. Wilson, Spermi-dine protection of plasmid DNA against single-strand breaksinduced by singlet oxygen, Proc. Natl. Acad. Sci. USA 89 (1992)11428–11430.