Synthesis, structure, electrochemical properties, cytotoxic effects and antioxidant activity of 5-amino-8-methyl-4H-benzopyran-4-one and its copper(II) complexes

Polyhedron 31 (2012) 150–158

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Synthesis, structure, electrochemical properties, cytotoxic effects and antioxidant activity of 5-amino-8-methyl-4H-benzopyran-4-one and its copper(II) complexes _ a, Aleksander Kufelnicki b, Magdalena Wozniczka b, Ingo-Peter Lorenz c, Peter Mayer c, Magdalena Grazul Andrzej Józ´wiak d, Malgorzata Czyz e, Elzbieta Budzisz a,⇑ a

Department of Cosmetic Raw Materials Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland Department of Physical and Biocoordination Chemistry, Faculty of Pharmacy, Medical University of Lodz, Muszynskiego 1, 90-151 Lodz, Poland c Department of Chemistry, Ludwig Maximilians University, Butenandtstr. 5-13 (D), D-81377 Munich, Germany d Department of Organic Chemistry, Faculty of Chemistry, University of Lodz, Tamka 12, 91-403 Lodz, Poland e Department of Molecular Biology of Cancer, Medical University of Lodz, Mazowiecka 6/8 Street, 92-215 Lodz, Poland b

a r t i c l e

i n f o

Article history: Received 1 August 2011 Accepted 6 September 2011 Available online 16 September 2011 Keywords: Chromone derivative Synthesis Cytotoxic effect Antioxidant activity Copper(II) complexes

a b s t r a c t The chromone derivative 5-amino-8-methyl-4H-benzopyran-4-one (ligand) (1) has been used to obtain a series of Cu(II) complexes 2–4 as potential anticancer compounds. The molecular structures of ligand 1 and its Cu(II) complex 3 have been determined by X-ray crystallography. The cytotoxicity of all obtained compounds has been evaluated on melanoma A375 cell line. The ability of compounds 1–4 to take part in redox reactions and their antioxidant activity have been studied. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Metal ions play a very important role in many biologically systems [1]. Copper is associated with various biomolecules related to essential physiological activities in human organism. Our effort is focused on these metal–ion complexes because of their promising anticancer and antioxidant agents [2]. For many years numerous researches have been actively investigating copper compounds basing on the hypothesis that endogenous metals may prove less toxic and more potent [3]. It has been established that the properties of copper complexes are largely determined by the nature of ligands and the donor atoms bound to the metal ion. The redox activity of copper(II) ion plays a decisive role in addressing the main effects of this metal. Copper(II) may contribute to the production of free radicals and in regulation and induction of apoptosis [4].

Abbreviations: DMSO, dimethyl sulfoxide; DMF, N,N-dimethylformamide; ABTS, 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide. ⇑ Corresponding author. Tel.: +48 42 677 91 25; fax: +48 42 678 83 98. E-mail address: [email protected] (E. Budzisz). 0277-5387/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2011.09.003

Since Albert Szent-Györgyi discovered a group of flavonoid compounds (chromone derivatives) in 1936, a lot of scientists have tried to explain their folded properties. Chromones are naturally occurring compounds which are able to induce cytotoxic effect in various types of cells. Due to their miscellaneous activities, like for instance anticancer [5], antioxidant, antiproliferative [6], anti HIV, antiinflammatory [7], and many other activities, they are used in several fields like chemistry, biochemistry, genetics, cellular and molecular biology. Since the last decade, metal coordination chemistry has been one of the most efficient strategies in the design of drugs [8]. The main target of chemotherapy is the destruction of tumor cells without any undue influence on proper cells. It is proved that chromone and some of its derivatives are able to form stable complexes with various metal ions which are extractable into organic solvents [9,10]. These complexes are strongly colored, so this feature is widely used in spectrophotometrical determination of metal ions [11]. A lot of research has proved that metal– chromone complexes possess various biological activity in some cases comparable with cisplatin which is an effective anticancer drug [12,13]. Moreover, flavonoids complexes of Cu(II), which are connected with reactions generating free radicals, may in some cases obtain additional anti-oxidant ability [14] e.g., it has been

M. Graz_ ul et al. / Polyhedron 31 (2012) 150–158

proved that a flavonoid–copper (II) complex shows higher antiinflammatory activity than the free flavonoid [15]. In our previous papers we have been investigating the cytotoxic activity of platinum(II) and palladium(II) as well as copper(II) complexes [16–18]. Those studies have shown that copper(II) complexes exhibit activity (sensitivity) especially on melanoma cell line. Numerous successful researches on the synthesis of metal–chromone complexes and their promising clinical results incline us to continue investigations on that kind of complexes. In the present research, we synthesized 5-amino-8-methyl-chromone, analyzed its chemical properties and tested its cytotoxic properties as well as those of its Cu(II) complexes on tumor A375 cell line. 2. Experimental 2.1. Chemical data The melting points were determined using a Buchi Melting Point B540 apparatus and are uncorrected. The IR spectra were recorded on a Pye-Unicam 200G Spectrophotometer in KBr. The 1H NMR spectra were recorded on aVarian Gemini 200 BB, CDCl3, 200 MHz. Satisfactory elemental analyses (±0.4% of the calculated values) were obtained for the new compounds in the Microanalytical Laboratory of the Department of Bioorganic Chemistry (Medical University, Lodz) using a Perkin Elmer PE 2400 CHNS analyser. Cyclic voltammetry measurements were performed on a microAutolab/GPS (general purpose Electrochemical System, Version 4.8, Eco Chemie) computer-controlled electrochemical system. Antioxidant activity was measured on a Varian Cary 50 Bio UV– Vis Spectrophotometer. Caution: Perchlorate salts are potentially explosive and were handled only in small quantities with care. 2.1.1. Preparation of 5-amino-8-methyl-4H-benzopyran-4-one (1) Ligand 1 was obtained from 8-methyl-5-nitro-4H-benzopyran4-one according to the literature procedure given for reduction of nitroacetophenone derivatives [19]. Tin(II) chloride (5.81 g, 30.6 mmol) was added at room temperature to a suspension of 8-methyl-5-nitro-4H-benzopyran-4-one (1.40 g, 7 mmol) in hydrochloric acid (d = 1.19 g/mL, 13.6 mL) which was stirred for 3 h. Then the mixture was stirred for 3 h and after this time it was cooled to 5 °C and adjusted to pH 10 by adding 20% solution of potassium hydroxide. Product 1 was extracted with dichloromethane (4  20 mL) and purified by crystallization from toluene (1.08 g, yield 77%), M.p.: 141–142 °C, (lit. [20] 141–143 °C). FTIR (KBr cm1) (selected bands): m(C–NH2) 3432, 3324; m(C@O) 1648; m(C@C) 1625, 1588, 1569. 1H NMR (CDCl3 d ppm) 2.24 (3H, s, CH3), 6.15 (1H, d, J = 5.9 Hz, 3-H), 6,30 (2H, br. s, NH2), 6.39 (1H, d, J = 8.2 Hz, 6-H), 7.17 (1H, d, J = 8.2 Hz, 7-H), 7.71 (1H, d, J = 5.9 Hz, 2-H). 2.1.2. Synthesis of cis-dichlorido(5-amino-8-methyl-4H-1benzopyran-4-one-j2-N,O) copper(II) (2) 52.5 mg (0.3 mmol) of 5-amino-8-methyl-4H-benzopyran-4one (1) was dissolved in ethyl acetate (12 ml) at room temperature. Next, into a previously prepared solution of 54 mg (0.3 mmol) copper(II) chloride in 1 ml of methanol, the solution of compound 1 was added dropwise with constant stirring whereby the light green solution changed its color to dark green and no precipitate was observed. The solution was stirred for the next 15 min at room temperature and then an orange precipitate was formed. 70 mg of solid 2 was obtained (Yield: 75.4%), FTIR (KBr cm1) (selected bands): m(C–NH2) 3297, 3109; m(C@O) 1635; m(C@C) 1623, 1607, 1537; m(M–N) 436; m(M–Cl) 426. Anal. Calc. for C10H9Cl2CuNO2

151

(M = 309.625 g/mol): C, 38.79; H, 2.93; N, 4.52; Found: C, 38.65; H, 3.18; N, 4.38%; M.p.: 135 °C decomp.; MS-FAB (m/z): 309 [Cu(1)Cl2]+, 274 [Cu(1)Cl+], 238.6 [Cu(1)2+], 176 (100%, 1). 2.1.3. Synthesis of trans-bis{(5-amino-8-methyl-4H-1-benzopyran-4one-j2-N,O)(perchlorato-jO)}copper(II) (3) Ligand 1 (26.25 mg 0.15 mmol) was dissolved in 10 ml of ethyl acetate and copper(II) perchlorate (27.78 mg, 0.075 mmol) in methanol (1 ml) which then was added dropwise with constant stirring to the ligand solution at room temperature. The solution changed its color to yellow-green, and a green solid precipitated. The mixture was stirred for further 10 min, then the solid was filtered off, washed with diethyl ether and dried under reduced pressure and used for further analyses, (Yield: 13 mg, 28.3%), FTIR (KBr cm1) (selected bands): m(C–NH2) 3387, 3218; m(C@O) 1627; m(C@C) 1623, 1571, 1562; m(ClO4) 1134, 1090; m(M–N) 416. Anal. Calc. for C20H18Cl2CuN2O12 (M = 612.804 g/mol): C, 39.28; H, 2.96; N, 4.57; Found: C, 39.68; H, 2.95; N, 4.48%; M.p.: 238.3–240.2 °C; MS-FAB (m/z): 612 [Cu(1)2(ClO4)2], 513 [Cu(1)2ClO4+], 413.8 [Cu(1)2]2+, 238.6 [Cu(1)2+], 176 (100%, 1). 2.1.4. Synthesis of trans-bis{(5-amino-8-methyl-4H-1-benzopyran-4one-j2-N,O)(nitrato-jO)}copper(II) (4) Ligand 1 (87.6 mg 0.5 mmol) was dissolved in 20 ml of ethyl acetate. Copper(II) nitrate (60.4 mg, 0.25 mmol) was dissolved in methanol (3 ml) and was added dropwise with constant stirring to ligand solution at room temperature. The resulting mixture changed its color to dark orange and was stirred for further 10 min. The solid was filtered off, washed with diethyl ether, dried under reduced pressure and used for further analyses, (Yield: 54 mg, 35%). FTIR (KBr cm1) (selected bands): m(C–NH2) 3337, 3251; m(C@O) 1641; m(C@C) 1624, 1587, 1494; m(NO3); 1383; mas(NO3) 1358; ms(NO3) 1232; d(NO3) 838; m(M–N) 422. Anal. Calc. for C20H18CuN4O10 (M = 537.918 g/mol): C, 44.65; H, 3.37; N, 10.42; Found: C, 43.14; H, 3.24; N, 10.04; M.p.: 280.0–280.2 °C; MS-FAB (m/z): 538 [Cu(1)2(NO3)2 +H], 475.1 [Cu(1)2NO3+], 413.8 [Cu(1)22+], 238.0 [Cu(1)2+], 176 (100%, 1). 2.2. X-ray structure analysis X-ray data were collected at 200 K with Mo Ka radiation (k = 0.71073 Å) with a Nonius KappaCCD diffractometer equipped with a rotating anode. The structure was solved with direct methods [21] and refined with SHELXL-97 by full-matrix least-squares on F2 [22]. All non-hydrogen atoms were refined anisotropically. The C-bound hydrogen atoms were placed in ideal geometry riding on their parent atoms. The N-bond hydrogen atoms were freely refined. The crystal data and X-ray details are given in Table 3. 2.3. Potentiometric measurements The protonation constant of the ligand 1 was determined by pH-metric titration of 4 mL samples, at temperature 25 ± 0.1 °C. The total concentration of the ligand in each sample ranged within 7.5  104 and 1.0  103 mol/L. Owing to low solubility in pure water a mixed 5% v/v 1,4-dioxane-water solvent was used. The titrations were carried out with carbonate-free NaOH solution of known concentration (0.1 M, J.T. Baker, titre 0.0997–0.1000). The value of pKw = 13.77 resulted from our acid–base calibrations in the same solvent. The pH was measured with a Molspin Ltd. (Newcastle upon Tyne, England) automatic titration set and combined OSH 10-10 electrode (Metron, Poland). The total volume of the Hamilton microsyringe in the autoburette was 250 lL, the volume increments amounted to 0.0025 mL. The titrations were performed by using MOLSPIN.EXE software after addition of a strong acid (HNO3) in excess to the solution of pure ligand. The electrode

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was calibrated in the log [H+] scale by titration of 0.005 M HNO3 (in 5% dioxane) with 0.1 M NaOH, temperature 25 °C. Then concentration overall protonation constants blh = [LlHh]/[L]l[H]h were calculated by a SUPERQUAD computer program [23]. The speciation diagram was obtained by means of HySS2 (Protonic Software 2000) upon the results of SUPERQUAD refinements [24]. A protonation constant b11 followed from a comprehensive file consisting of five titrations carried out at various concentrations. The value in logarithm 3.77 ± 0.04 was refined at statistical parameters: r = 3.35, x2 = 12.94 (critical: r < 3.00 and x2 < 12.60 at confidence level 0.95). Although the results indicate possible complexation with metals, precipitation observed in the experiments with Cu(II) in 5% dioxane and also other mixed solvents, the complex formation studies in solution could not be carried out. 2.4. Cyclic voltammetry The measurements of ligand 1 and its copper(II) complexes 2–4 were performed on a microAutolab/GPS (general purpose Electrochemical System, Version 4.8, Eco Chemie) computer-controlled electrochemical system. Complexes 2 and 4 were dissolved in the mixture of DMF and water, while complex 3 was dissolved in water. Then Britton-Robinson buffer (pH 7.1 or 6.5, respectively) was added to all samples. All measurements were carried out at room temperature (22.0 ± 0.5 °C). Cyclic voltammograms were obtained under an argon atmosphere. The electrochemical cell employed was a standard three-electrode configuration: platinum (working), Ag/AgCl (reference) and platinum wire (auxiliary) electrodes. The redox couple (Fc+/Fc) of ferrocene (+0.40 V versus NHE) was used as the internal standard. The system was monitored with a personal computer for data acquisition and subsequent analysis. In a typical run the supporting electrolyte and compound solution were added by a micropipette to final concentration 1  103 mol/ L of analyzed compound into a clean, dry cell. After a stirring period of 60 s in order to homogenize the solution, a square cyclic voltammogram was recorded. 2.5. Antioxidant activity Reduction of the ABTS radical cation by antioxidants is the main mechanism of this test. The ABTS radical cation was obtained as a result of reaction of ABTS stock solution (7 mM in water) with 2.45 mM potassium peroxosulfate. For measurements, the ABTS solution was diluted with ethanol to an absorbance of 0.700 ± 0.020 at 754 nm. Stock solutions for the compounds 1–4 were diluted with DMSO. For the photometric assay the ABTS solution and antioxidant solution were mixed for 45 s in ratio 10:1 and measured immediately after one minute at 754 nm in quartz cell with silicon stopper. The next measurements were carried out in defined intervals until 24 h using Scanning Kinetics procedure (UV–Vis Spectrophotometer Cary50 Bio).

2.6.2. Melanoma cell proliferation assay (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay, MTT) An inhibition of A375 cell proliferation by compounds 1–4 was tested by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). Briefly, cells were seeded in 24-well plates and incubated in an atmosphere of 5% CO2 to adhere for 6 h in RPMI 1640 medium containing 10% FBS. Next, they were treated for 2 days with tested compounds 1–4. MTT reagent (thiazolyl blue tetrazolium bromide; Sigma–Aldrich; 0.84 mg/mL in PBS) was added and left in the cultures for 3 h at 37 °C. Next, to dissolve the insoluble blue formazan precipitate produced by reduction, 800 ll solubilization reagent (DMSO in optical grade) was added. The absorbance was determined at 540 nM. For each condition assays were performed in triplicate in three independent experiments and SD was calculated. The mean of the absolute absorbance values given by drug-treated cells was divided by the mean of the absolute absorbance of DMSO treated control sample and expressed as relative number of viable adherent cells. Data show the mean of at least three independent experiments ± SD. IC50 values were calculated by concentration-response curve fitting using a Microsoft Excel–based analytic method.

3. Results and discussion 3.1. Chemistry of 5-amino-8-methyl-4H-benzopyran-4-one and its Cu(II) complexes The route leading to ligand 1 is outlined in Scheme 1. 8-Methyl5-nitro-4H-benzopyran-4-one was obtained from 2-hydroxy-3methylacetophenone [25] according to known procedures [20,26] and was reduced by means of SnCl2 [19] to afford compound 1. Thus obtained 5-amino-8-methyl-4H-benzopyran-4-one (1) was then used as a ligand in the synthesis of complexes with copper(II) ions (Scheme 2). The ligand 1, in the reactions with copper(II) chloride dihydrate in ethyl acetate/methanol (10:1) created only one neutral complex 2 of the formula MLCl2 irrespective of the M:L molar ratio (1:1 or 1:2) in which the reaction proceeded. X-ray determination was not suitable for publication1 but it has indicated the molecular structure 2. In the reaction of 1igand 1 with copper(II) perchlorate hexahydrate or copper(II) nitrate hexahydrate (1:1 or 2:1) in ethyl acetate solution only complexes 3 and 4 in molar ratio of 2:1 were formed (Scheme 2). To sum up, the complexes of ligand 1 with copper(II) were formed in L:Cu(II) molar ratios as follows: 1: copper chloride 1:1, 1: copper perchlorate 2:1 and 1: copper nitrate 2:1. 3.2. Spectroscopic analyses of compound 1 and its Cu(II) complexes 2–4 Structures of all tested compounds 1–4 were confirmed by elemental analysis, MS and IR spectroscopy.

2.6. Cytotoxic activity 2.6.1. Cell culture conditions and drug treatment A375 (human melanoma cancer) adherent cell line with high metastatic potential, derived from a 54 year old female with malignant melanoma (a gift of Prof. Piotr Laidler, Jagiellonian University, Poland) was used in this study. Cells were grown at 37 °C in a humidified atmosphere containing 5% CO2, in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin. For drug exposure experiments, compounds 1–4 were dissolved in DMSO, and diluted with the growth medium RPMI 1640 immediately before use. Equivalent final concentration of DMSO was used in the control cultures.

3.2.1. IR spectra The IR spectrum of the ligand 1 (see Table 1) shows prominent stretching vibrations at 3432 cm1 for mas (NH2) and 3324 cm1 for ms (NH2). This bands shift to lower energies (Dm = 135, 215 cm1 for compound 2; Dm = 45, 106 cm1 for compound 3; Dm = 95, 73 cm1 for compound 4), which shows that the coordination via the amino group has occurred. The most relevant feature of the IR spectra of 2–4 is the shift to lower energy of the m(C@O) band upon coordination of 1 to the metal ion, ranging from Dm = 13 cm1(2) to Dm = 24 cm1(3) and Dm = 7 cm1(4). The absorption observed in 1

Unpublished result

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CH3

CH3

CH3 O

OH

153

CH3 O

HNO3 /H2SO4

O

SnCl2/HCl

CH3 O

O

NO2

NH2

O

O 1

Scheme 1. The synthesis of 5-amino-8-methyl-4H-benzopyran-4-one (1).

Cu Cl

NH2

H3C

1 :1

Cl

O

O CuCl2 x 2H2O

2

OClO3 O

O

H3C

O

Cu(ClO4)2 x6H2O

CH3

Cu H3C

2: 1

NH2

NH2

O

O

O

NH2 OClO3 3 ONO2

Cu(NO3)2 x 6H2O

O

NH2

O

CH 3

Cu

2: 1 H3C

O

O

NH 2 ONO2 4

Scheme 2. The synthesis of copper(II) complexes 2–4 with 5-amino-8-methyl-4H-benzopyran-4-one (1).

Table 1 IR data (m, cm1) for ligand 1 and its complexes 2–4. Compound

m(NH2)

m(C@O)

maromat

1 2 3 4

3432, 3297, 3287, 3337,

1648 1635 1624 1634

1625, 1623, 1623, 1624,

3324 3109 3218 3251

1588, 1607, 1571, 1587,

1569 1537 1562 1494

m(M–N)

mCu–Cl/–ClO4/–NO3-

436 416 422

426 1134, 1090 838, 1232, 1358

Table 2 MS data (m/z) of complexes 2–4. Compound

Molecular ions

Fragment ions

2 3

309 612

4

538

274 [Cu(1)Cl+], 238.6 [Cu(1)2+], 176 [100%,1] 513 [Cu(1)2ClO4+], 413.8 [Cu(1)22+], 238.6 [Cu(1)2+], 176 [100%,1] 475.8 [Cu(1)2NO3+], 413.8 [Cu(1)22+], 176 [100%,1]

the lower area of the spectrum of 2 at about 426 cm1 is assigned to the Cu–Cl stretching vibration [27]. The new bands at 436 to 416 cm1 in the spectra of complexes 2–4 correspond to the copper – nitrogen vibration involving the nitrogen atoms of amine group. The perchlorato complex 3 shows two bands at 1134 and 1090 cm1 corresponding for the splitting of m3 (ClO4) by symmetry reduction and indicating the monodentate perchlorato-O coordination [28]. The nitrato complex 4 has

two bands at 1232 and 1358 cm1 corresponding to ms and mas (NO2) with a separation of Dm = 126 cm1, also pointing towards monodentate coordination [29]. The appearance of a small band at 1383 cm1 for mas (NO3) in the spectrum of complex 4 is the most probably due to the fact that a certain amount of coordinated nitrate transforms to ionic form by pressing KBr pellet [30]. In addition, a weak band at 838 cm1 assignable to vd (NO2) is a further support for the nitrate-O coordination [31].

M. Graz_ ul et al. / Polyhedron 31 (2012) 150–158

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Table 3 Crystallographic data and data collection parameters for ligand 1 and complex 3. 1 Net formula 1

Fig. 1. Molecular structure of ligand 1.

Fig. 2. Molecular structure of complex 3. Thermal ellipsoids drawn at the 50% probability level. Non-labeled non-hydrogen atoms are generated by inversion at the Cu-centre (symmetry operation x, y, z).

3.2.2. Mass spectrometry The main peaks in FAB mass spectra of the Cu(II) complexes are summarized in Table 2. All compounds have been investigated by positive/negative mass spectrometric measurements, giving valuable structural information. For the complexes 2–4 the molecular peaks have been observed at 309, 612 and 538 [M+H]+ in FABMS spectra, respectively. For compounds 3 and 4 strong peaks at m/z 513 and 476, which correspond to the loss of ClO4 or NO3 moieties respectively, have been observed. The FAB mass spectrum of 2 recorded in the positive ion mode contains a peak at m/z 274 due to [Cu(1)Cl+] and at m/z 238.6 due to [Cu(1)+]. In addition, the predominant peak corresponding to the ligand 1 at m/z 176 [100%, 1] was always observed. The data presented above are in good agreement with the results obtained from the elemental analysis and therefore confirm the structure of investigated complexes which have been proposed by X-ray spectroscopy. 3.3. Molecular structures of 1 and 3 3.3.1. Structure discussion of ligand 1 The molecular structure of 5-amino-8-methyl-4H-benzopyran4-one (1) and its copper(II) complex 3 are given in Figs. 1 and 2,

Mr/g mol Crystal size (mm) Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) V (Å3) Z Calculated density (g cm3) l (mm1) Absorption correction Transmission factor range Reflections measured Rint Mean r(I)/I h range Observed reflections x, y (weighting scheme) Hydrogen refinement Reflections in refinement Parameters Restraints R(Fobs) Rw(F2) S Shift/errormax Maximum electron density (e Å3) Minimum electron density (e Å3) CCDC

C10H9NO2 C20H18Cl2CuN2O12 175.184 0.24  0.11  0.08 monoclinic P21/n 10.0318(7) 7.0248(5) 12.0060(8) 90 101.439(7) 90 829.27(10) 4 1.40318(17) 0.099 ‘multi-scan’ 0.96972–1.00000 3335 0.0307 0.0737 3.78–25.35 706 0.0429, 0 mixed 1499 127 1 0.0347 0.0890 0.851 0.001 0.156

3

612.815 0.10  0.07  0.05 monoclinic P21/n 5.5238(2) 20.6639(6) 10.2205(4) 90 101.520(2) 90 1143.10(7) 2 1.78045(11) 1.261 none 9092 0.0540 0.0503 3.59–27.50 1964 0.0460, 0.3300 mixed 2626 178 0 0.0372 0.0978 1.027 0.001 0.461

0.125

0.431

795611

795612

selected bond lengths and angles are listed in Table 6 and 7, hydrogen bonds are listed in Table 8. The crystallographic data of both are summarized in Table 3. Ligand 1 is exactly planar, even with all substituents O1, N and C10 showing only a deviation of 2° from planarity. Whereas all C–C bond lengths within the phenyl ring lie between 1.374(3) Å (C2–C3) and 1.428(2) Å (C1–C6) with typical aromatic values, the quinoid bond C8–C9 = 1.333(3) Å is the shortest one. The exocyclic bond C7–O1 = 1.243(2) Å is typical for a C@O double bond, the cyclic ones, however, C5–O2 = 1.382(2) Å and C9–O2 (1.348(2) Å) are rather longer showing only single bond character. In between these two values the C1–N bond = 1.367(3) Å is found. All the bond angles of both rings lie more or less about 120° with the only exceptions C4–C5–O2 (114.78(16)°) and C8–C9–O2 (124.18(18)°). In the crystal of 1 two hydrogen bonds were found between the exocyclic oxygen O1 as hydrogen acceptor in hydrogen bonds of the type N–H  O1, both having the ketooxygen as acceptor. One of the hydrogen bonds is intramolecular, the other one is directed to an adjacent molecule leading to zig– zag chains along [0 1 0]. 3.3.2. Structure discussion of complex 3 The centrosymmetric complex 3 shows a distorted octahedron with both chromone ligands chelating by N, O atoms in the equatorial plane and equal chelating atoms in trans-position to each other. Both perchlorato-O-ligands are bound in the axial position. Whereas the equatorial bond lengths Cu–O1 (1.933(2) Å) and Cu–N (1.968(2) Å) are rather similar, that of Cu–O3 (2.433(2) Å) is very long caused by the Jahn-Teller distortion. The perchlorato ligand is almost an ideal tetrahedron (O3–Cl–On 109.14° and

M. Graz_ ul et al. / Polyhedron 31 (2012) 150–158

On–Cl–Om 110.12°). The four bonds Cl–On (n = 3–6) lie in the range 1.422(2)–1.456(2) Å, whereby the longest is formed by coordinating O3. The bite angle (N–Cu–O1) of the planar chelating ligand is 92.54(8)°. The complex is not perfectly planar, since an angle of 12.51(8)° is formed by the least-square planes of Cu and the coordinating chromone atoms on the one hand and the ten-membered bicyclic core of chromone on the other hand. In the crystal of 3 two hydrogen bonds were found between the amino N and oxygen atoms O3 and O6 of the perchlorato-ligand. A third but weak one is formed by the ring carbon C9. Thus, the molecules are linked to infinite strands along [1 0 0] by hydrogen bonds between the amino group and the copper-bound oxygen of the perchlorate moiety. 3.4. Protonation constants of 5-amino-8-methyl-4H-benzopyran-4one (1) Similarly as for L = 2-ethanimidoyl-2-methoxy-2H-1,2-benzoxaphosphinin-4-ol-2-oxide (log b11 = 3.70 ± 0.01) [32], the refinements for compound 1 indicate only one functional group. Compound 1 behaves as a monoprotonated molecule within pH 2.5–5.0 (Fig. 3). Since protonation of a carbonillic group needs very acidic conditions, the results are indicating that of the two potential donors, 4-O and 3-N atom, only the amino group is the protonated function. The presently measured protonation constant log b11 = 3.77 ± 0.04 is also of the same order as for the ligand under comparison, which shows the essential role of O1 in one of the two six-membered rings and a negligible influence of both the phosphonate group at C2 and methyl group in C8. It is also worthy

155

to mention that titration above neutral pH resulted in distortions caused by further precipitation. 3.5. Cyclic voltammetry Because of many suggestions that cytotoxic activity of Cu(II) complexes could be related to their abilities to take part in redox reactions [33,34] we decided to test the electrochemical properties of all obtained complexes 2–4 and ligand 1 as well. Complexes were electrochemically measured electrochemically at platinum (working), Ag/AgCl (reference) and platinum wire (auxiliary) electrodes in H2O/DMF solution using cyclic voltammetry. A representative voltammogram has been depicted in Figs. 4a and 4b. The potential data are given in Table 4. The measurements were performed in pH 6.5 (pH corresponds to pH which was measured in cytosol of cancer cells) and pH 7.1 (which corresponds to pH which was measured in cytosol of normal cells)[35,36]. Complexes 2 and 4 were dissolved in the mixture of DMF and water, while complex 3 was dissolved in water. Then Britton-Robinson buffer (pH 7.1 or 6.5, respectively) was added to all samples. Table 4 shows the cyclic voltammograms for 1  103 mol/L of copper(II) complexes 2–4 obtained by scanning the potential between 1 and +1 V versus Ag/AgCl at a scan rate of 0.10 V/s. The cyclic voltammetric analysis indicated that the peak-to-peak separation DEp for all of tested complexes is in the range 161–249 mV. The results for complex 2 are presented in Fig. 4. All complexes are involved in Cu(I)–Cu(II) oxidation process. It can be seen in Table 4 that the pH of environment has no influence on the reversibility of redox processes of compounds 2–4 which are quasi-reversible in both used buffers (pH values 6.5 and 7.1). Our potentiometric measurements clearly showed that ligand is completely deprotonated in the used conditions (see Section 3.4). Free ligand 1 does not indicate any electrochemical activity in the working potential region in any pH. 3.6. Antioxidant activity

Fig. 3. Species distribution of L = 5-amine-8-methyl-chromone in 5% dioxane. LH denotes the monoprotonated form of L (free-ligand). CL = 1.0  103 mol/L.

The antioxidant activity of compounds 1–4 has been determined by using assays measuring the abilities of tested compounds to reduction of ABTS radical cation. Table 5 shows the antioxidant behavior of the compounds 1–4 expressed as values of first-order rate constants and corresponding half-life periods. Influence of incubation time on the suppression of the absorbance of ABTS radical cation has been tested (Fig. 5). It is clearly visible that the rate of absorbance changes decreases in time.

0.750x10-5 0.500x10-5

i/A

0.250x10-5 0 -0.250x10-5 -0.500x10-5 -0.750x10-5 -1.250 -1.000 -0.750 -0.500 -0.250

0

0.250 0.500 0.750 1.000 1.250

E/V Fig. 4a. Cyclic voltammogram of complex 2 at pH 6.5.

M. Graz_ ul et al. / Polyhedron 31 (2012) 150–158

156

0.750x10-5 0.500x10-5

i/A

0.250x10-5 0 -0.250x10-5 -0.500x10-5 -0.750x10-5 -1.250 -1.000 -0.750 -0.500 -0.250

0

0.250 0.500 0.750 1.000 1.250

E/V Fig. 4b. Cyclic voltammogram of complex 2 at pH 7.1.

Table 4 Cyclic voltammetric data* obtained for complexes of 5-amino-8-methyl-4H-benzopyran-4-one with Cu(II) ions.

*

pH

Compound

Concentration (mol/L)

Epc (V)

Epa (V)

DEp (mV)

6.5 6.5 6.5

2 3 4

1  103 1  103 1  103

0.180 0.233 0.282

0.019 0.023 0.033

161 210 249

7.1 7.1 7.1

2 3 4

1  103 1  103 1  103

0.224 0.219 0.282

0.048 0.043 0.043

176 176 239

Table 5 The values of first-order rate constants and corresponding half-life periods of compounds 1–4 at different concentrations.

1

2

3

4

Cu–O1 1.933(2) Cl–O3 1.456(2) Cl–O6 1.434(2) O1–Cu–O3 91.40(7) O3–Cu–N 88.06(9) Cu–O3–Cl 130.2(1)

Cu–O3 2.453(2) Cl–O4 1.422(2) O1–C7 1.260(3) O1–Cu–N 92.55(8) O3–Cu–O3a 180.00 C1–C6–C7 125.6(2)

Cu–N 1.968(2) Cl–O5 1.423(2) N–C1 1.451(3) O1–Cu–O1a 180.00 N–Cu–Na 180.00

Table 8 Hydrogen bond details. Bond lengths (in Å) and angles (in °). Symmetry codes: i = 0.5  x, y  0.5, 0.5  z; ii = 1  x, y, z; iii = x, y, z; iv = x, y, 1  z.

DEp = Epa  Epc, where Epa and Epc are anodic and cathodic potentials.

Compound

Table 7 Selected bond lengths (in Å) and angles (in °) for 3. Symmetry code a = x, y, z.

Concentration (lM)

k (min1) ±103

t(0.5) (min)

R2

1.5 5 10 15

2.611 ± 0.098 5.107 ± 0.261 7.565 ± 0.468 5.727 ± 0.398

265.5 ± 10.0 135.7 ± 6.9 91.6 ± 5.7 121.0 ± 8.4

0.9820 0.9670 0.9527 0.9409

1.5 5 10 15

4.119 ± 0.106 4.668 ± 0.157 5.755 ± 0.122 4.314 ± 0.168

168.3 ± 4.3 148.5 ± 5.0 120.4 ± 2.6 160.7 ± 6.3

0.9914 0.9855 0.9942 0.9806

1.5 5 10 15

1.230 ± 0.034 4.403 ± 0.164 5.653 ± 0.310 5.264 ± 0.226

563.5 ± 15.6 157.4 ± 5.9 122.6 ± 6.7 131.7 ± 5.7

0.9900 0.9824 0.9623 0.9767

1.5 5 10 15

2.637 ± 0.082 5.152 ± 0.210 4.574 ± 0.216 4.546 ± 0.274

262.9 ± 8.2 134.5 ± 5.5 151.5 ± 7.2 152.5 ± 9.2

0.9875 0.9788 0.9719 0.9548

Compound

D

H

A

D–H

H  A

D–A

D–H–A

1 1 3 3 3

N N N N C9

H11 N12 H1 H2 H9

O1i O1 O3ii O6iii O6iv

0.94(2) 0.910(10) 0.80(3) 0.88(3) 0.95(3)

2.03(2) 1.887(17) 2.17(3) 2.23(3) 2.45(3)

2.929(2) 2.676(3) 2.969(3) 3.076(3) 3.374(3)

160.1(18) 144(2) 173(3) 162(2) 164(3)

Therefore obtained results were presented as a logarithmic function: ln(A  A1) = f(t), where the infinite value A1 is sufficiently estimated by the absorbance value after 24 h (Fig. 5). This type of dependence corresponds to first-order reaction with rate constant k. The dependence obtained is essentially rectilinear with determination coefficients within 0.94–0.99 (Table 5). The half-life periods of ABTS radical cation scavenging by tested compounds were relatively shorter in concentrations higher than 5 lM. However it is impossible to see any increase of rate of this process for complexes 2–4 in comparison to ligand 1. Therefore significant antioxidant activity may be attributed to ligand 1, while its complexes do not indicate any increase in activity. 3.7. Cytotoxic activity

Table 6 Selected bond lengths (in Å) and angles (in °) for 1. C1–C2 1.383(3) C4–C5 1.384(3) C7–C8 1.436(3) O2–C9 1.348(2) C4–C10 1.506(3) C1–C2–C3 120.80(18) C3–C4–C5 115.40(18) C6–C7–C8 115.28(17) C8–C9–O2 124.18(18)

C2–C3 1.374(3) C5–C6 1.397(2) C8–C9 1.333(3) O1–C7 1.243(3)

C3–C4 1.382(3) C6–C7 1.455(3) O2–C5 1.382(2) C1–N 1.367(3)

C1–C6–C7 125.6(2) C4–C5–C6 124.49(16) C7–C8–C9 120.91(19)

C2–C3–C4 123.33(19) C5–C6–C7 119.96(15) C5–O2–C9 118.92(15)

The ligand 1 and its complexes 2–4 were tested for their cytotoxic activity against human metastatic melanoma A375 cell line. MTT assay was used to assess the influence of the drugs on the metabolic activity of adherent cells in relation to untreated (control) cells. Concentration–response analyses performed in the range of concentration from 1 to 200 lM are presented in Fig. 6 In general, A375 cells showed the highest sensitivity to compound 3, while they were more resistant to compounds 2 and 4. The activity of compound 3 however was, about 6 times lower when compared with that of cisplatin used against A375 cell line [37,38].

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157

Fig. 5. Absorbance suppression of the ABTS radical cation in time. Concentration values in micromoles were expressed as markers.

IC50 values obtained for melanoma A375 cells treated with 2, 3, and 4 for 2 days were about 153, 114 and 164 lM, respectively. Ligand 1 had very low cytotoxic activity against A375 cell line, it reduced cell proliferation to 82.8% compared to the control cells when used at the concentration of 200 lM. Therefore, it is reasonable to assume that cytotoxic activity of tested compounds is caused more by the complexes activity than by the ligand alone. In addition, we could speculate that the cytotoxic activity was not cause by changes in redox status of the cells for at least two reasons. First of all, ligand 1 exerted moderate antioxidant activity but not anticancer activity, and complexes did not show enhanced antioxidant activity but increased cytotoxic activity. This explanation is supported by the observation that melanocytes and their malignant

Cell proliferation (% of control)

100 80 60 40

1 2

20

counterparts, constantly exposed to oxidative stress induced by UV radiation and quinone toxicity from melanin synthesis, have to be more efficient in scavenging ROS than other cell types. This means that changes in redox status are very efficiently neutralized in those cells, and therefore they are usually not cytotoxic [39]. Further experiments are necessary to elucidate the mechanism of cytotoxic action of tested compounds.

4. Conclusions Ligand 5-amino-8-methyl-4H-benzopyran-4-one (1) and its three new copper(II) complexes 2–4 were synthesized and were characterized by IR, mass spectroscopy and elemental analysis. Ligand 1 possesses only one functional group of the two potential donors, 4-O and 5-N atoms. Compounds 1–4 are involved in redox reactions and the reversibility of that processes is not dependent on the pH of environment. Antioxidant activity of tested compound 2–4 is related solely to ligand 1. All obtained compounds were tested for in vitro antitumor activities against human melanoma cell line A375. All of these results demonstrated that this new synthesized complexes 2–4 have better anti-proliferative activities in comparison with ligand 1. The highest anticancer activity was observed in cultures treated with copper(II) complex containing perchlorate (complex 3). Its activity in A375 cell cultures was, however, 6 times lower than that observed in cultures treated with cisplatin.

3 4

Acknowledgments

0 10

100

Concentration [µM]

Fig. 6. The inhibitory effects of ligand 1 and its Cu(II) complexes 2–4 on human melanoma A375 cell proliferation. Metabolic activity of adherent cells in cell cultures treated with tested complexes are expressed as percentage of that observed in the control culture. The data are mean ± SD of three independent experiments done in triplicates.

The authors would like to thank to Dr. Agata Szuławska-Mroczek for culturing melanoma cells and to Dr. Sławomira Skrzypek for help in cyclic voltammetric analysis. This work was supported by grants from the Medical University of Lodz No. 503/3-066-02/ 503-01 (to E. Budzisz) and No. 503/3-014-02/503-01 (to A. Kufelnicki).

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Appendix Appendix. A. Supplementary data CCDC 795611, 795612 contains the supplementary crystallographic data for 1 and 3. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336 033; or e-mail: [email protected].

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