Synthesis, structures and the biological activity study on the metal complexes of 2-(4-aminophenyl)benzothiazole derivative

Inorganica Chimica Acta 382 (2012) 35–42

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Synthesis, structures and the biological activity study on the metal complexes of 2-(4-aminophenyl)benzothiazole derivative Guo-Wu Lin a, Yue Wang a,⇑, Qiao-Mei Jin a, Tao-Tao Yang a, Jie-Mei Song b, Yi Lu a, Qing-Jie Huang b, Ke Song b, Jun Zhou b, Tao Lu a,c,⇑ a b c

College of Basic Science, China Pharmaceutical University, Nanjing 210009, China School of Pharmacy, China Pharmaceutical University, Nanjing 210009, China State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, China

a r t i c l e

i n f o

Article history: Received 11 November 2010 Received in revised form 12 July 2011 Accepted 1 October 2011 Available online 12 October 2011 Keywords: Benzothiazole Cu(II) complex Crystal structure DNA binding DNA cleavage

a b s t r a c t (2-(4-(Benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5) was synthesized and then its three metal complexes were prepared with structures determined by 1H NMR or X-ray. In all complexes, ZL-5 is deprotonated to generate the neutral complex unit and acts as a tetradentate ligand, providing iminodiacetic N atom and three O atoms to bond with metal ions. Meantime we studied on the interaction of complexes with DNA by ultraviolet spectrum and fluorescent spectrum. Also the cleavage abilities of the complexes on plasmid pBR 322 DNA were studied by gel electrophoresis. We deduce the Co(II) complex 1 with the largest planar structure has the strongest DNA binding ability mainly by the intercalation mode, while the Cu(II) complex 2 with the coordinated water cut the DNA most effectively. And in the oxidative cleavage process of all complexes, the hydroxyl radical is produced which can be captured by DMSO. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Nowadays, health is one of the most important domains we human beings have focused on in our society. However, tumor is the biggest killer of our lives, so there has been steadily increasing research in the field of anticancer therapy over recent years. 2-(4-Aminophenyl)benzothiazole (CJM 126) and its analogues comprise a novel mechanistic class of antitumor agents [1,2]. This nucleus comes from the related structure polyhydroxylated 2-phenylbenzothiazoles, flavone quercetin and the isoflavone genistein, which are tyrosine kinase inhibitors bearing potent antitumor activity [3,4]. It is well known that the compounds with the basic structure of 2-(4-aminophenyl)benzothiazole or its derivatives which have high selectivity and strong activity on tumor cells are a new type of antitumor drugs both in vivo and vitro, especially on women’s breast cancer and ovarian cancer [5,6]. They can also be transformed into prodrugs with a famous example lysyl-amide (NSC 710305) (Fig. 1), which acts as a prodrug to exhibit the ability through hydrolysis to form two parts [7–9] to emit the active pharmaceutical ingredient, and then promote the bioavailability. So if some kind of ligands coordinated with various metal ions to be potent prodrugs, the body absorbs them through all ⇑ Corresponding authors. Fax: +8625 86185163 (Y. Wang), +8625 86185180 (T. Lu). E-mail addresses: [email protected] (Y. Wang), [email protected] (T. Lu). 0020-1693/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ica.2011.10.004

possible means, these prodrugs can be hydrolyzed into two parts which have biological activity, respectively, thus may greatly enhance the antineoplastic activity of the chemotherapeutics and reduce the toxicity as well as resistance of drugs. From this point, we focus on the synthesis the (2-(4-(benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid together with the transition metal complexes. In the recent study, it is reported that various transition metal ions (such as Cu2+, Zn2+, Ni2+ and other ions) can bind to the imidazole group of the histidine residues in the vicinity of the enzyme’s active site pockets. It provides a novel strategy for enhancing the binding affinity of an active site-directed inhibitor by attaching an iminodiacetic acid (IDA)-conjugated Cu2+, which could interact with one of the surface-exposed histidine (His-4) residues in carbonic anhydrase or matrix metalloproteinase inhibitors [10,11]. And a weak inhibitor of carbonic anhydrase (viz., benzenesulfonamide) can be converted into a strong inhibitor by attaching an iminodiacetate IDA–Cu2+ group via a suitable spacer. In such a ‘two-prong’ inhibitor, whereas the benzenesulfonamide group binds at the active site pocket of the enzyme, the IDA–Cu2+ moiety loops around and interacts with one of the surface exposed histidine residues [12]. In our work, the series of transition metal complexes of (2-(4(benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid were studied and the schemes were shown in Fig. 2. The title complex

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F

N

N NH

NH 2

S

intermedium sodium (2-(4-(benzothiazole)phenyl)carbamoylmethyl)iminodiacetate (ZL-4) was synthesized by condensation between equimolar amounts of obtained (ZL-3) with iminodiacetic acid (IDA) in the presence of Na2CO3 in ethanol solution (75%), after acidation of ZL-4, the title ligand (ZL-5) was achieved. At last, reaction with transition metals can produce corresponding metal coordination complexes.

H 2N (CH2) 4

S

CJM 126

NSC 710305

O

NH 2

Fig. 1. The structure of 2-(4-aminophenyl)benzothiazole (CJM 126) and 2-(4amino-3-methylphenyl)-5-fluorobenzothiazole (NSC 710305).

2.2.1. 2-(4-Nitrophenyl)benzothiazole (ZL-1) 4-Nitrobenzoic acid (6.7 g, 0.04 mol) was dissolved in polyphosphoricacid (100 mL) at 180 °C. 2-Aminothiophenol (5.0 g, 0.04 mol) was added and the resulting solution was stirred at 180 °C for 5 h. After cooling, the reaction mixture was poured into ice-cold aqueous ammonia (100 mL). The precipitate was collected by filtration and washed with water (50 mL  2). The product was dried and crystallized from acetoacetate to give the 2-(4-nitrophenyl)benzothiazoles (ZL-1) as a white solid (8.3 g, 0.03 mol, yield 81%), m.p.: 160–161 °C; MS m/z 258.0 (M+1).

is mainly consisting of two parts: the 2-(4-aminophenyl)benzothiazole and the iminodiacetic complex, linked by –CO–CH2– group. Also their structures and the binding and cleavage ability with DNA were studied. 2. Experimental 2.1. Apparatus and reagents All solvents and other reagents were all of chemical grade and used as received. Water for solution preparation was distilled water. Calf Thymus DNA (CT DNA) and pBR 322 plasmid DNA were purchased from Sigma. Reaction progress was monitored using analytical thin layer chromatography (TLC) on percolated Merck silica gel Kiesegel 60 F254 plates, and the spots were detected under UV light (254 nm). Melting points were determined with a digital melting point apparatus and are reported uncorrected. 1H NMR spectra was recorded at 300 MHz on a Bruker ARX 300 spectrometers. IR spectra were measured on a Jasco FT/IR-430 spectrophotometer. Mass spectra were recorded on an a Quattro microMS Micromass UK mass spectrometer, and was recorded on an electrospray ionization mass spectrometer as the value m/z. The X-ray measurements were made on a Rigaku RAXIS RAPID diffractometer with a graphite monochromatised Mo Ka radiation (k = 0.71069 Å) using x scan mode. All the UV absorption spectra were recorded on a Shimadzu UV-2100 spectrophotometer after through mixing for 5 min at 25 °C. Electronic absorption spectra and fluorescence spectra were performed with a Shimadzu UVW-2101PC spectrophotometer and a Shimadzu FR-5301 spectrofluorometer at 25 °C, respectively. The gel was photographed on a capturing system gel printer plus TDI. Bands were quantified using the Scion Image for Windows software program based on NHI Image.

2.2.2. 2-(4-Aminophenyl)benzothiazole (ZL-2) 4-Aminobenzoic acid (6.6 g, 0.048 mol) was dissolved in polyphosphoricacid (120 mL) at 200 °C. 2-Aminothiophenol (6.0 g, 0.048 mol) was added and the resulting solution stirred at 200 °C for 5 h. After cooling, the reaction mixture was poured into ice-cold aqueous ammonia (100 mL). The precipitate was collected by filtration and washed with water (50 mL  2). The product was dried and crystallized from acetoacetate to afford the corresponding adduct 2-(4-aminophenyl)benzothiazoles (ZL-2) as a yellow solid (9.9 g, 0.043 mol, yield 90%), m.p.: 155–157 °C; MS m/z 227.3 (M+1).

2.2. Synthesis of the (2-(4-(benzothiazole)phenyl)carbamoylmethyl) iminodiacetic acids (ZL-5) and its complexes 1–3

2.2.3. 2-(4-Chloroacetamidophenyl)benzothiazole (ZL-3) In a 500 mL single-necked, round-bottomed flask equipped with a magnetic stirrer, the following were placed: 2-(4-aminophenyl)benzothiazoles (ZL-2) (8.0 g, 0.035 mol), triethylamine (14.8 mL, 0.106 mol) and anhydrous acetone (200 mL), then the chloroacetyl chloride (8.5 mL, 0.106 mol) in anhydrous acetone (10 mL) was added drop wise to the stirred mixture. After the mixture was boiled for 4 h, the precipitate was collected and washed with water (50 mL  2) to give 2-(4-chloroacetamidophenyl)benzothiazoles (ZL-3) as pale-yellow powder (9.2 g, 0.03 mol, yield 86%). m.p.: 214–215 °C; MS m/z 303.5 (M+1), dH 1H NMR (DMSOd6) 10.80 (1H, s, –CO–NH–), 8.20–7.44 (8H, m, Ar–H  8), 4.33 (2H, s, –CH2Cl).

Synthesis of the (2-(4-(benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acids (ZL-5) was outlined in Scheme 1. For the first, the 2-(4-nitrophenyl)benzothiazole (ZL-1) and 2-(4-aminophenyl)benzothiazole (ZL-2) were obtained by the condensation in polyphosphoric acid (PPA) at 180–200 °C, (ZL-2) also can get form (ZL-1) by reduction of nitro; Secondly, reaction of amines with chloroacetyl chloride in refluxing dry acetone gave the 2-(4-chloroacetamidophenyl)benzothiazoles (ZL-3). Then, the key

2.2.4. (2-(4-(Benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5) In a 150 mL single-necked, round-bottomed flask equipped with a magnetic stirrer, the following were placed: 2-(4-chloroacetamidophenyl)benzothiazoles (ZL-3) (5.0 g 0.017 mol), iminodiacetic acid (IDA) (2.2 g 0.018 mol), sodium carbonate (3.0 g 0.028 mol) and 75% ethanol (200 mL). The stirred mixture was heated under reflux for 10 h, then cooled to room temperature to

H 3C N

H 3C

N

N

H3 C

H

OH 2

O

N

N H

O C

O N

O Cu

Cu

Co S

H3 C

H N

O

O

O

.5H 2O

N

O

O

S N H

C

N

O

O

O

Fig. 2. The chemical structure of complex 1–3.

S N

N H

O C

2H2 O

O N

O

O

O

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NH2 + O 2N

COOH

a

N NO 2

S

SH

ZL-1 b

NH 2 + H 2N

COOH

c

N

d

NH 2 S

SH

N NHCOCH 2Cl S

ZL-2 e

CH 2COONa

N NHCOCH 2 N S

CH 2COONa

ZL-4 g

ZL-3 f

CH 2COOH

N S

NHCOCH 2 N ZL-5

CH 2COOH

Complexes 1-3

Scheme 1. Reagents and conditions: (a) polyphosphoric acid, 180 °C, 5 h; (b) Fe powder, NH4Cl, 75% C2H5OH, reflux 2 h; (c) polyphosphoric acid, 200 °C, 5 h; (d) chloroacetyl chloride; acetone, reflux 4 h, then 10% Na2CO3; (e) iminodiacetic acid (IDA), Na2CO3, 75% C2H5OH, reflux 2 h; (f) 4% HCl; (g) CuCl22H2O or CoCl22H2O, DMF, several months.

give the key intermediate (ZL-4). The reaction mixture was precipitated at pH 2–3 with 5% HCl, and collected by filtration in vacuum, washed with water and methanol, dried and crystallized from absolute methanol to afford the corresponding adduct (2-(4-(benzothiazole)phenyl)carbamoylmethyl)iminodiacetic acid (ZL-5) (6.1 g, 0.015 mol, yield 88.3%)as a white precipitate. m.p.: 223– 226 °C; MS m/z 400.4 (M+1); dH 1H NMR (DMSO-d6) d 12.61 (2H, s, –COOH  2), d 10.62 (1H, s, –CO–NH–), 8.20–7.40 (m, 8H, Ar– H), 4.33 (2H, s, –CH2Cl), d 3.54 (4H, s, –CH2–  2). 2.2.5. Metal coordination compounds Orange platelet crystals of ZL-5-Co-Phen (complex 1) were obtained by slow evaporation of a DMF solution of (ZL-5), 1, 10-phenanthroline and CoCl2.6H2O (molar ratio 1:1:1) at room temperature for several weeks. Blue prism crystals of ZL-5-Cu-1 (complex 2) were obtained by slow evaporation of a DMF solution of (ZL-5) and CuCl2. 2H2O (molar ratio 1:1) at room temperature for several weeks. Blue needle crystals of ZL-5-Cu-2 (complex 3) were obtained by slow evaporation of a DMF solution of (ZL-5) and CuCl22H2O (molar ratio1:1) at pH 3–4 at room temperature for several weeks. 2.3. Study with UV spectroscopy The absorption spectra of the interaction of complexes with CT DNA have been recorded for a constant complex concentration with increasing amount of CT DNA. Complexes were dissolved in a DMF-methanol mixture (4:1 v/v).

buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) and complex solution with ascorbate at a 10-fold molar excess relative to the complex to yield a total volume of 18 lL [15]. The complex with different concentration was dissolved in DMF and MeOH with volume ratio 4:1. After mixing, the sample was incubated at 37 °C for 90 min. The reaction was quenched by the addition of sodium ethylene diamine tetraacetic acid (EDTA-2 Na) and 5 lL of loading buffer (0.25% bromphenol blue, 50% glycerol). Then the solution was subjected to electrophoresis. Electrophoresis was achieved at 80 V for 70 min in apt TAE buffer (40 mM Tris acetate/ 1 mM EDTA). The gel was photographed on a capturing system gel printer plus TDI. Bands were quantified using the Scion Image for Windows software based on NHI Image. 2.6. Research on the mechanism of cleavage of pBR 322 Reactions were carried out by the method mentioned above. Also, to study the cleavage mechanism, DMSO, KI and NaN3 was added respectively as comparison. After mixing, the sample was incubated at 37 °C for 90 min. The reactions were quenched at appropriate time by the addition of sodium ethylene diamine tetraacetic acid (EDTA-2 Na) and 5 lL of loading buffer (0.25% bromphenol blue, 50% glycerol). Then the solution was subjected to electrophoresis on 0.8% agarose gel in TAE buffer (40 mM Tris acetate/1 mM EDTA) at 80 V and visualized by ethidium bromide staining. 3. Results and discussion

2.4. Study with fluorescence spectroscopy

3.1. X-ray crystal analysis

Ethidium bromide (EB) emits intense fluorescence light in the presence of DNA, due to strong intercalation between the adjacent DNA base pairs. The competitive binding of the complexes to DNA reduces the emission intensity of EB with DNA [13,14]. In the binding study, the fluorescence spectra (kex = 520 nm, kem = 590 nm) were recorded at room temperature and complexes solution with original concentration of 2.0  104 M were added to a volume of 2 mL of the EB–DNA solution to get the final concentration of DNA and EB being 4.0  105 M and 4.0  106 M, respectively. Five minutes later after adding the complexes, the fluorescence intensity of the mixture was recorded.

The X-ray measurements were made on a Rigaku RAXIS RAPID diffractometer with a graphite monochromatised Mo Ka radiation (k = 0.71069 Å) using x scan mode. A summary of the crystallographic data and structure refinements is given in Table 1. The data were corrected for Lorentz and polarization effects. The structure was solved by direct methods [16] using the crystal structure [17] software package. The refinement was performed using SHELXL-97 [18]. All H atoms were located from difference Fourier maps and treated as riding, with C–H distance of 0.93, phenol N–H distance of 0.86 and Uiso(H) values equal to 1.2 Ueq(C) and Uiso(water H) values equal to 1.5 Ueq(C) (Ueq is the equivalent isotropic displacement parameter for the pivot atom). P The function of w (Fo2–Fc2)2 was minimized by using the weight scheme of w = 1/[r2(Fo2) + (aP)2 + bP], where P = (Fo2 + P P P 2Fc2)/3. Final R [= (|Fo||Fcj)/ |Fo|], Rw [=( w(|Fo|  |Fc|)2/ P P 2 1/2 w|Fo| ) ] and S (goodness of fit) [=( w(|Fo|  |Fc|)2/(MN)1/2),

2.5. Research on the cleavage of pBR 322 by complexes Reactions were carried out in a total volume of 18 lL by mixing 1 lL of supercoiled (SC) pBR 322 plasmid DNA (0.25 lg/lL), a

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Table 1 Crystal data and structure refinement for complexes 1–3. Complex

1

2

3

Formula Formula weight Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å) Z Crystal size (mm) Shape Color Dcalc (g cm3) Reflections collected Unique reflections Absorption coefficient (mm1) F(0 0 0) R (F2 > 2r(F2)) wR (F2) Goodness of fit (GOF) n F2 Number of variables

C31H33CoN5O10S 726.63 triclinic  P1

C22H24CuN4O7S 552.05 monoclinic P21/c 25.160(5) 7.1510(14) 13.609(3) 90.00 93.43(3) 90.00 2444.1(8) 4 0.4  0.2  0.1 prism blue 1.500 4771 2980 1.029 1140 0.0800 0.1919 1.028 281

C22H24CuN4O7.50S 560.05 orthorhombic Pbca 22.359(5) 6.7830(14) 39.112(8) 90.00 90.00 90.00 5932(2) 8 0.2  0.2  0.1 needle blue 1.254 5294 2100 0.850 2312 0.0826 0.1913 1.011 301

8.9990(7) 12.7766(7) 14.0652(8) 92.661(2) 102.770(2) 97.389(2) 1559.39(17) 2 0.5  0.5  0.2 platelet orange 1.548 7094 6151 0.662 646.00 0.0409 0.1176 1.118 434

Table 2 Bond distances (Å) and angles (°) for compounds. Complex

Bond lengths

Bond angles

1

Co–O2 Co–O4 Co–O5 Co–N3 Co–N4 Co–N5

2.073(2) 2.058(2) 2.094(14) 2.197(16) 2.101(2) 2.168(16)

O2–Co–O4 O2–Co–O5 O2–Co–N3 O2–Co–N4 O2–Co–N5 O4–Co–O5 O4–Co–N3 O4–Co–N4

154.49(5) 94.28(5) 79.76(5) 104.19(5) 85.51(5) 95.28(5) 78.63(5) 98.96(5)

O4–Co–N5 O5–Co–N3 O5–Co–N4 O5–Co–N5 N3–Co–N4 N3–Co–N5 N4–Co–N5

88.92(5) 80.30(5) 92.51(6) 170.28(6) 172.11(6) 109.17(6) 78.15(6)

2

Cu–O4 Cu–O6 Cu–O2 Cu–N1 Cu–O7 Cu–O5

1.949(5) 1.952(5) 1.962(4) 2.020(5) 2.361(5) 2.398(4)

O4–Cu–O6 O4–Cu–O2 O6–Cu–O2 O4–Cu–N1 O6–Cu–N1 O2–Cu–N1 O4–Cu–O7 O6–Cu–O7

95.9(2) 167.25(18) 96.2(2) 85.48(19) 169.20(19) 83.38(18) 86.04(18) 96.56(19)

O2–Cu–O7 N1–Cu–O7 O4–Cu–O5 O6–Cu–O5 O2–Cu–O5 N1–Cu–O5 O7–Cu–O5

88.62(17) 94.22(18) 86.52(19) 88.79(18) 97.71(18) 80.58(16) 171.24(17)

3

Cu–O2 Cu–O5 Cu–O3 Cu–N2 Cu–O1

1.927(5) 1.934(5) 1.960(5) 2.029(6) 2.308(5)

O2–Cu–O5 O2–Cu–O3 O5–Cu–O3 O2–Cu–N2

96.5(2) 94.4(2) 166.1(2) 178.0(2)

O3–Cu–N2 O2–Cu–O1 O5–Cu–O1 O3–Cu–O1

86.9(2) 99.8(2) 96.9(2) 89.88(19)

where M = no. of reflections and N = no. of variables used for the refinement] are given in Table 1. Anisotropic displacement coefficients were refined for all non-hydrogen atoms. Selected bond distances and angles are listed in Table 2. 3.2. Structure studies 3.2.1. The structure of complex 1 (ZL-5-Co-phen) Complex 1 (ZL-5-Co-phen) contains an [Co(ZL-5)(phen)] complex moiety (phen is 1,10-phenanthroline) and five water molecules in the asymmetric unit. The detailed structure description was published in our previous work [19]. 3.2.2. The structure of complex 2 (ZL-5-Cu-1) Complex 2 (ZL-5-Cu-1) contains an neutral [Cu(ZL5)(DMF)(H2O)] moiety (DMF is dimethylformamide) in the

asymmetric unit (Fig. 3). In complex 2 the Cu2+ ion is bonded to the amide O atom of DMF ligand and the O atoms of water molecules, one iminodiacetic N atom and three O atoms from ZL-5 ligand. Here, each ZL-5 acts as a tetradentate ligand to generate a slightly distorted octahedral coordination geometry, with the amide O6 atom of DMF, the iminodiacetic N1 atom of ZL-5 ligand and two carboxylate O2 and O4 atom from iminodiacetate occupying the equatorial plane, leaving two apex positions for carboxyl O5 atom from ZL-5 ligand and the activating water O7 atom occupying the axial direction. The bond length of Cu–O5 and Cu–O7 are 2.398(4) Å and 2.361(5) Å, respectively in axial direction, significantly longer than its equatorial location on the other bond length (from 1.949(5) to 2.020(5) Å), which can be explained by John–Teller effect. The whole structure is stabilized by the intermolecular hydrogen bond, together with the p–p interaction between the adjacent (2phenyl)benzothiazole planes.

G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42

39

Fig. 4. ORTEP drawing of complex 3.

(2-phenyl)benzothiazole fracture extending and forming p–p interaction to make the whole structure more stable. 3.3. Study with UV spectroscopy

Fig. 3. ORTEP drawing of complex 2.

3.2.3. The structure of complex 3 (ZL-5-Cu-2) Complex 3 (ZL-5-Cu-2) contains an [Cu(ZL-5)(DMF)] unit and two free water molecules (Fig. 4). Each Cu2+ ion is bonded to the amide O atom of DMF ligand and one iminodiacetic N atom of ZL-5 ligand, and three O atoms from ZL-5 ligand. Here, ZL-5 also acts as a tetradentate ligand through two iminodiacetic O, one iminodiacetic N, and one amide O atom, while each DMF ligand act as monodentate ligand through N atoms. It generated a slightly distorted square pyramid geometry, with the amide O2 atom of DMF, the iminodiacetic N2 atom, O3 atom and O5 atom from ZL5 ligand occupying the equatorial plane, while carboxylate O1 atoms of ZL-5 ligand occupying the axial plane. By comparison, in all three complexes, ZL-5 is deprotonated to generate the neutral complex unit. The part of the phenyl benzothiazole is planar, indicating by the mean dihedral angle of phenyl and benzothiazole 3.394 Å, with the mean deviation from the plane by 0.0229 Å. The part of the iminodiacetic acid together with –CO– CH2– is twisted to provide four coordination atoms, thus forming three twisted five-member chelated rings, and leaving the basic

Solutions of CT DNA in 50 mM NaCl/5 mM Tris–HCl (pH 7.4) gave a ratio of UV absorbance at 260 and 289 nm of 1.8–1.9:1, indicating that the DNA was sufficiently free of protein [20]. The concentration of CT DNA was determined spectrophotometrically by employing an extinction coefficient of 6600 M1 cm1 at 260 nm [21]. The absorption spectra of complex 1 with increasing amount of CT DNA at a constant concentration of the complex were given in Fig. 5. As the DNA concentration is increased, the main band of this complex at 331 nm exhibit hypochromism with 3 nm red shift, which suggest the complex 1 can strongly bind to DNA by intercalating interaction. Because of structural similarity, a similar result was found for complex 2 and 3. As the DNA concentration increasing, the maximum absorption bands of these two complexes at 331 nm exhibit hyperchromism with 3 nm blue shift. The hyperchromism effects observed suggest that the contacts between the molecule of the complex and groove of DNA are very important. From the absorption of three complexes, we can deduce the maximum absorption at 331 nm may attributed to the p–p⁄ transition by the ligand ZL-5. In all cases, as the result of complex formation, the strong band at 331 nm as well as the other weak bands at near 347 nm remarkably changed in their intensity with a little red-shift or blue-shift with equal absorption point, indicating the formation of new DNA-complexes compounds, but three complexes had a different binding mode due to their different structures. 3.4. Study with fluorescence spectroscopy Fluorescent emission of EB bound to DNA in the presence of complex 1 is shown in Fig. 6. With the concentration of DNA

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Fig. 5. UV–Vis absorption spectra of complex 1–3 (from left to right) with increasing amount of CT DNA. The ratio of CDNA/Ccomplex in each curve was increasing along arrow direction.

lane

1

2

3

4

Form II Form III Form I Fig. 8a. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA (0.25 lg/lL) by complex 1 in the presence of 10-fold excess of ascorbate for 90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane 2, complex 1 (5  106 M); lane 3, complex 1 (5  105 M); lane 4, complex 1 (5  104 M).

Fig. 6. Fluorescence emission spectra (excited at 520 nm) in the EB-CT DNA system with increasing amounts of 2.0  104 M of complex 1.

increasing. In three cases, the emission band of the DNA–EB system decreased in intensity with the increase of the concentration of the complexes results from all complexes replacing EB from the DNA– EB system. Such a characteristic change is often observed in the intercalative DNA interaction. In comparison, it is apparent from the plot shown Fig. 7 that EB bound DNA was efficiently quenched by complex 1, in which displacement process of complex occurred. Deducing from the slope of the quenching plot with the complex concentration, the DNA binding propensity can be reflected as the relative order: complex 1 > complex 2 > complex 3. This observation suggests that the complex competes with the intercalative EB binding to DNA in a different degree which is coinciding with their solid structure in which complex 1 has a largest plane coordinated to ancillary phen ligand. Thus the degree of intercalation interaction was further testified by the fluorescence quenching propensity besides the absorption spectrum results. 3.5. pBR 322 DNA cleavage promoted by three complexes

Fig. 7. The emission intensity of CT DNA-bonded EB(DNA/EB = 10/1) when adding different concentrations of three complexes in 50 mM Tris–HCl buffer (pH 7.4) at room temperature on addition of complex 1 (d), complex 2 (s), complex 3 (.).

increased, the fluorescence intensity was largely decreased. The fluorescence intensities were plotted against the concentration of three complexes to get slopes in Fig. 7. The emission intensity of the DNA–EB system (Ex = 520 nm) decreased apparently with the concentration of each complex

3.5.1. Cleavage ability of pBR 322 by the complexes at different concentration The metal complexes cause random cuts to one of the DNA strands. As a consequence, the supercoiled form opens to form an open circular after one nick and subsequently to a linear form if two nicks on complementary strands are within a short distance. Finally, the DNA degraded into small pieces of different size which cannot be detected in our assay. The cleavage products were subjected to gel electrophoretic separation and the gels analyzed after ethidium bromide staining [22–24]. The oxidative DNA cleavage activity of the complexes was studied by gel electrophoresis using SC pBR 322 DNA in Tris–HCl buffer (pH 7.4). Control experiment done under the same conditions does not show any apparent cleavage activity. The conversion of the supercoiled pBR 322 DNA (SC DNA, Form I) to the nicked DNA (NC DNA, Form II) and linear DNA (Form III) becomes more efficient when increasing the concentration of the complexes. With

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lane

1

2

3

4

5

Form II Form III Form I Fig. 8b. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA(0.25 lg/lL) by complex 2 in the presence of 10-fold excess of ascorbate for 90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane 2, complex 2 (2.5  107 M); lane 3, complex 2 (2.5  106 M); lane 4, complex 2 (2.5  105 M); lane 5, complex 2 (2.5  104 M).

lane

1

2

3

4

5

Form II Form III Form I Fig. 8c. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA(0.25 lg/lL) by complex 3 in the presence of 10-fold excess of ascorbate for 90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane 2, complex 3 (5  107 M); lane 3, complex 3 (5  106 M); lane 4, complex 3 (5  105 M); lane 5, complex 3 (5  104 M).

Table 3 Oxidation cleavage data of SC pBR 322 by complex 1. Lane

Concentration

Supercoiled (Form I) (%)

1 2 3 4

DNA control 5  106 M 5  105 M 5  104 M

100 85.89 8.48

Nicked (Form II) (%)

Linear (Form III) (%)

14.11 91.52 57.96

42.04

Fig. 9. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA(0.25 lg/lL) by ligand ZL-5 in the presence of 10-fold excess of ascorbate for 90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane 2, ZL-5 (5  106 M); lane 3, ZL-5 (5  105 M); lane 4, ZL-5 (5  104 M), lane 5, ZL-5 (2.5  104 M).

Table 6 Oxidation cleavage data of SC pBR 322 by ligand ZL-5. Lane

Concentration

Supercoiled (%)

1 2 3 4 5

DNA control 5  106 5  105 5  104 2.5  104

100 100 100 100 100

Lane

Compounds

1 2 3 4 5

DNA control DNA + complex DNA + complex DNA + complex DNA + complex NaN3

Concentration

Supercoiled (%)

DNA control 2.5  107 M 2.5  106 M 2.5  105 M 2.5  104 M

100 45.79 37.52

Nicked (%)

Linear (%)

Concentration

Supercoiled (Form I) (%)

1 2 3 4 5

DNA control 5  107 M 5  106 M 5  105 M 5  104 M

100 93.84 90.20 3.84

42.96 49.88 92.39

11.25 12.60 7.61

Nicked (Form II) (%) 6.16 9.80 57.83 45.76

1

2

Nicked (%)

100 44.80 59.65 47.35 46.55

55.20 40.35 52.65 53.45

3

4

Linear

5

Form I Fig. 10a. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA (0.25 lg/lL) by complex 1 in the presence of 10-fold excess of ascorbate for 90 min in a buffer solution (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1, DNA control; lane 2, DNA + complex 1 (2.5  105 M); lane 3, DNA + complex 1 (2.5  105 M) + 2 lL DMSO; lane 4, DNA + complex 1 (2.5  105 M) + 10 mM KI; lane 5, DNA + complex 1 (2.5  105 M) + 10 mM NaN3.

Table 5 Oxidation cleavage data of SC pBR 322 by complex 3. Lane

1 1 + 2 lL DMSO 1 + 10 mM KI 1 + 10 mM

Supercoiled (%)

Form II

Table 4 Oxidation cleavage data of SC pBR 322 by complex 2.

1 2 3 4 5

Linear

Table 7 Oxidation cleavage data of SC pBR 322 by complex 1.

lane

Lane

Nicked

lane Linear (Form III) (%)

1

2

3

4

5

Form II Form III

38.33 54.24

the increasing concentration of the complex, the DNA cleavage propensity is apparently enhanced, as is shown in Figs. 8a–c. Especially the best cleavage result is obtained on the concentration of 5  105 (complex 1), 2.5  107 M (complex 2) and 5  105 M (complex 3), when degradation to the linear form was observed. With more high concentration, the linear form of complex 2 was almost disappearing to footprint for it is furthermore degraded to

Form I

Fig. 10b. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA (0.25 lg/lL) by complex 2 in the presence of 10-fold excess of ascorbate in a buffer solution for 90 min (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1: DNA control; lane 2: DNA + complex 2 (2.5  105 M); lane 3: DNA + complex 2 (2.5  105 M) + 2 lL DMSO; lane 4: DNA + complex 2 (2.5  105 M) + 10 mM KI; lane 5: DNA + complex 2 (2.5  105 M) + 10 mM NaN3.

small pieces, while the Form II and form III in complex 1 and 3 are still coexisting. The oxidation cleavage data of SC pBR 322 by all complexes were listed in Table 3–5. By comparison, complex

42

G.-W. Lin et al. / Inorganica Chimica Acta 382 (2012) 35–42

Table 8 Oxidation cleavage data of SC pBR 322 by complex 2. Lane

Compounds

1 2 3 4 5

DNA + control DNA + complex DNA + complex DNA + complex DNA + complex

lane

Supercoiled

Nicked

Linear

53.42 57.31 50.41 61.14

46.58 29.75 49.59 38.86

100 2 2 + 2 lL DMSO 2 + 10 mM KI 2 + 10 mM NaN3

1

12.94

2

3

4

5

Form II Form I

Fig. 10c. Agarose gel electrophoresis patterns for the cleavage of pBR 322 plasmid DNA (0.25 lg/lL) by complex 3 in the presence of 10-fold excess of ascorbate in a buffer solution for 90 min (containing 50 mM Tris–HCl and 50 mM NaCl, pH 7.4) at 37 °C. Key: lane 1: DNA control; lane 2: DNA + complex 3 (2.5  105 M); lane 3: DNA + complex 3 (2.5  105 M) + 2 lL DMSO; lane 4: DNA + complex 3 (2.5  105 M) + 10 mM KI; lane 5: DNA + complex 3 (2.5  105 M) + 10 mM NaN3.

Table 9 Oxidation cleavage data of SC pBR 322 by complex 3. Lane

Compounds

1 2 3 4 5

DNA + control DNA + complex DNA + complex DNA + complex DNA + complex NaN3

with the complexes alone. But it is apparent that DMSO plays as an effective free catcher to capture the hydroxyl radical during the oxidative cleavage process.

3 3 + 2 lL DMSO 3 + 10 mM KI 3 + 10 mM

Supercoiled (%)

Nicked (%)

100 77.88 91.40 60.68 74.46

22.12 8.60 39.32 25.54

Linear

4. Conclusion Herein, we built a new way to make the bioactive carriers (2-(4aminophenyl)benzothiazole) linked to the small metal complexes for the research of the complexes bearing possible biological activity. On this base, we study on the interaction of coordination compounds and DNA systematically through two methods: gel electrophoresis and optical spectrum which includes ultraviolet spectrum and fluorescent spectrum. By fluorescence titration method, we deduce the complex 1 with the largest planar structure has the strongest DNA binding ability mainly by the intercalation mode by its largest planar structure. And the electrophoresis result indicates the ability of the complex 2 cutting the DNA most effectively for the coordinated water. In the nuclear oxidative cleavage process of all complexes, the hydroxyl radical is produced which can be captured by DMSO. The study above will be helpful on the complexes design and the chemical drug development. Appendix A. Supplementary material CCDC 670230, 669578, 741991 contain the supplementary crystallographic data for complex 1–3. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. References

2 shows the highest cleavage ability for its coordinated water may produce the OH radical in the oxidizing process which will accelerate the cleavage reaction. By comparison with the reported other copper complexes [25–27], complex 2 shows the most effective DNA cleavage probability. We also studied the interaction between the ligand alone and DNA by electrophoresis method. The result got in the same system and the same concentration (Fig. 9 and Table 6) showed that the ligand alone does not cause the nuclear oxidative cleavage process of plasmid PBR 322. 3.5.2. Mechanism of pBR 322 DNA cleavage To verify the reactive oxygen species are responsible for the cleavage, reactions were carried out in the presence of typical scavengers for single oxygen (NaN3), for superoxide (KI), and for hydroxyl radical (DMSO). As the data of complex 1 showed in Fig. 10a and Table 7, the lane 3 adding DMSO is different from others. The Form I is more than that of the lane 2, lane 4, lane 5. So the DMSO plays as an important inhibitor. The data of complex 2 shown in Fig. 10b and Table 8 and those of complex 3 shown in Fig. 10c and Table 9, the lane adding DMSO still have more Form I than the others, verifying that the DMSO plays as an inhibitor in complexes. However, for the Form II and Form I the lane 2, lane 4, lane 5 stay almost in the same level. So, both KI and NaN3 do not have the evident effect on inhibiting the metal complex cutting DNA. That means the cleavage isn’t influenced by N3, neither is it influenced by normal ions, such as iodine ion for the lane adding NaN3 or KI does not present essentially differences from the lane

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