European Journal of Medicinal Chemistry 72 (2014) 46e51
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Synthesis, characterization, cytotoxicity and antibacterial activity of an anthracenyl-linked bis(pyrazolyl)methane ligand and its zinc(II) complexes Ban-Feng Ruan b, Ying-Zhong Zhu a, Wan-Ding Liu a, Bao-An Song c, **, Yu-Peng Tian a, * a b c
Department of Chemistry, Anhui Province Key Laboratory of Functional Inorganic Material Chemistry, Anhui University, Hefei 230039, PR China School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2013 Received in revised form 16 October 2013 Accepted 23 October 2013 Available online 1 November 2013
Three novel Zn(II) complexes (1e3) with 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) have been prepared and structurally characterized by X-ray crystallography. Among them, 1 is a binuclear Zn(II) complex while 2 and 3 are mononuclear. The in vitro cytotoxic and antibacterial activities of these complexes were also evaluated. The three complexes exhibit cytotoxic specificity and significant antitumor activity. The MIC50 value of complex 1 against Pseudomonas putida reaching 0.011 mg/mL much lower than that the positive control chloromycin (0.182 mg/mL), indicates that complex 1 is a potent antibacterial agent. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: 1,10 -(Anthracen-9-ylmethylene)bis(1Hpyrazole) Zinc(II) complex Crystal structure Cytotoxicity Antibacterial activity
1. Introduction Platinum complexes such as cisplatin and carboplatin are metalbased drugs, which are widely used in cancer chemotherapy. The great success in the clinical treatment of human malignancies has stimulated researches in the area of inorganic antitumor agents. However, their use is extremely hampered by severe toxicity and development of resistance during the therapy [1e3]. In order to overcome these disadvantages, current strategies in the development of novel metallodrugs focused more and more on the use of transition metal complexes containing improved organic ligands [4e6]. Among the nonplatinum complexes for metal based chemotherapy, zinc complexes have been much explored due to the fact that zinc is bioessential elements responsible for numerous bioactivities in living organism [7]. Zinc is the second prominent trace metal in the human body which is critical for numerous cells processes and is major regulatory ion in the metabolism of cells [8]. * Corresponding author. Tel.: þ86 551 65108151. ** Corresponding author. Tel.: þ86 851 3620521. E-mail addresses: [email protected] (B.-A. Song), [email protected] (Y.-P. Tian). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.064
Many features of zinc, such as its abilities in assisting Lewis activation, nucleophile generation, fast ligand exchange, and leaving group stabilization, make ZnII ideal for the catalysis of hydrolytic reactions, including DNA cleavage, which is an important property for use as anticancer drugs. Interaction of dinuclear ZnII complexes with DNA has recently attracted much attention [9,10], owing to their possible applications as new cancer therapeutic agents and their photochemical properties, which make them potential probes of DNA structure and conformation [11]. Zinc complexes with diverse biological activity viz. antibacterial [12], anti-inflammatory [13], for the treatment of Alzheimer disease [14] and antiproliferative, antitumor [15] activity have been reported. Keeping in view of biological and medicinal properties of 9anthracene aldehyde and ZnII, we find it vital to join both moieties in search of designing ZnII complexes that could aggressively work against tumor cells and bacterial species. Herein we have synthesized three novel ZnII complexes with a bis(pyrazolyl) methane ligand, viz. 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole). The crystal structures of the free ligand L2 and its three ZnII complexes [ZnCl2(L2)]2, [ZnBr2L2] and [ZnI2L2] have been determined by X-ray crystallography. The cytotoxicity and antibacterial activity have also been evaluated.
B.-F. Ruan et al. / European Journal of Medicinal Chemistry 72 (2014) 46e51
Scheme 1. Synthesis of L2 and complexes 1e3. Reagent and conditions: a) 9anthracenecarboxaldehyde, anhydrous CoCl2, THF, reflux; b) diethyl ether, ZnX2 (X ¼ Cl, Br, I), rt.
2. Results and discussion 2.1. Synthesis of ligand (L2) and complexes 1e3 The starting material bis(1H-pyrazol-1-yl)methanone (S) was purchased from Aldrich (USA). The ligand namely, 1,10 -(anthracen9-ylmethylene)bis(1H-pyrazole) (L2) was prepared by refluxing a mixture of equimolar quantities of S and 9-anthracene carboxaldehyde in a THF solution (Scheme 1) [16]. All the complexes were synthesized by the reaction of the ethyl acetate solutions of L2 with the corresponding zinc(II) salt (1.00 mmol) (ZnCl2 for 1, ZnBr2 for 2, and ZnI2 for 3). The compounds have been characterized by elemental analysis and IR spectra. Structures of the complexes were further confirmed by X-ray crystallography (CCDC 795201 for L2, 814312 for 1, 806628 for 2, and 800348 for 3). 2.2. Crystal structures of L2 and complexes 1e3 The details of crystallographic data and structure refinement parameters of L2 and complexes 1e3 are summarized in Table 1. Selected bond lengths ( A) and angles are collected in Table 2. The single crystal X-ray analysis reveals that the ligand L2 crystallizes as orthorhombic crystal system with space group
47
P212121. Molecules of L2 consist of two pyrazole rings, oriented in a roughly antiparallel manner with respect to each other, N-bonded to the methine carbon and form the cyclopentadienyl ring (Fig. 1). The 2-(9-anthryl) group is attached to the bis(pyrazolyl)methane unit and rotated by ca. 72.8 out of plane perpendicular to that defined by the NeCeN fragment. The angle between the two pyrazolyl rings is 100.5 . Thus, the planarity of the whole molecule is very poor [17]. As shown in Fig. 1-p, the 1D zigzag chain structure is self-assembled via weak CeH/N (2.720 A) and CeH/p (2.866 and 3.306 A) intermolecular interactions. Complex 1 [ZnCl2(L2)]2, a rare chloro-bridged zinc(II) dimmer, is a discrete neutral centrosymmetric compound (Fig. 2). Each ZnII atom has a distorted square-pyramidal geometry, which coordinates with three chlorine atoms and two nitrogen atoms. The ZneN distances [2.072(2) A and 2.129 (2) A] are quite asymmetric, compared to those found in the only other structurally authenticated Zn(II)Cl2 complex containing a bis(pyrazolyl)methane ligand [ZnCl2{Me2C(Pz)2}][2.058(3) and 2.046(3) A]. The ZneClbriding bond lengths of 2.689(1) A and 2.277(1) A are much more asymmetric than those in [CoCl2(L)2, L ¼ (2-propargyloxyphenyl)bis(pyrazolyl) methane] [18]. Both zinc atoms are bridged by two chlorine atoms (Cl1 and Cl1A), forming a centrosymmetry [Zn(m-Cl)2Zn] ring. The configuration of the six membered ring formed by the bis(pyrazolyl)methane and the Zn centre has a boat configuration with the methine proton in an equatorial position. One of the ligand nitrogen atoms is trans to one of the bridging chlorides, whilst the other nitrogen is trans to the non-bridging Cl-ligand. To be interestingly, the existence of the non-bridging chlorine atoms (Cl2 and Cl2A) lead to the distortion of the pyrazol rings of the ligand, which cause the reduce of the dihedral angle between plane N2(N2A)e Zn(Zn1A)eN4(N4A) and plane Cl1eZneCl1A from theoretical 90 e 54.6 . Thus, this kind of distortion leads to the observed asymmetry in ZneN and ZneCl bridging bond distances. However, the dihedral angles in ZnBr2L2 and ZnI2L2 are 87.3 and 86.9 . Accordingly, the ZneN and ZneX (X ¼ Br and I) bridging bond distances in complexes 2 and 3 are almost the same, respectively. Furthermore, there are CeH/Cl (ca. 2.936 A) and CeH/p (ca. 3.202 A) interactions between the adjacent molecules resulting a 1D chain as shown in Fig. 2-p.
Table 1 Crystallographic data and structure refinements for L2 and its complexes 1e3.
Formula Formula weight Crystal system Space group Crystal size (mm3) a ( A) b ( A) c ( A) a ( ) b ( ) g ( ) Volume ( A3) Z Dc (mg m3) m (mm1) F (000) q rang ( ) Reflections collected Reflections unique Parameters Goodness-of-fit R1, wR2 [I > 2s(I)] R1, wR2 [all data] Larg. peak/hole (e A) CCDC no
L2
[ZnCl2L2]2
ZnBr2L2
ZnI2L2
C21H16N4 324.38 Orthorhombic P212121 0.30 0.30 0.20 7.544(5) 9.397(5) 23.344(5) 90.000(5) 90.000(5) 90.000(5) 1654.9(15) 4 1.329 0.094 686 2.78e20.07 2907 2214 227 0.839 0.0386, 0.1057 0.0591, 0.1258 0.113/0.137 795201
C42H32Cl4N8Zn2 921.34 Monoclinic P21/c 0.30 0.20 0.20 9.631(5) 11.677(5) 18.013(5) 90.000(5) 100.906(5) 90.000(5) 1989.2(14) 2 1.538 1.517 936 2.303e25.71 3500 2939 254 0.850 0.0312, 0.1015 0.1126, 0.850 0.409/0.362 814312
C21H16Br2N4Zn 549.57 Monoclinic P21/n 0.30 0.30 0.20 8.711(5) 14.007(5) 17.916(5) 90.000(5) 103.843(5) 90.000(5) 2122.5(16) 4 1.720 4.936 1738 2.34e19.74 3697 2053 253 0.949 0.0529, 0.1362 0.1173, 0.1772 0.396/0.551 806628
C21H16I2N4Zn 643.57 Monoclinic P21/n P2(1)/n 8.925(5) 14.269(5) 18.017(5) 90.000(5) 103.854(5) 90.000(5) 2227.7(16) 4 1.919 3.887 2231 1.84e23.10 2975 2457 254 0.856 0.0318, 0.1037 0.0411, 0.1165 0.815/0.711 800348
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Table 2 Selected bond lengths ( A) and angles ( ) for L2 and the complexes 1e3. L2 C14eC15 C15eN3 C15eN1 N3eN4 N1eN2 N4eC20 N3eC19 N1eC16 N2eC17 1 C14eC15 C15eN1 C15eN3 N1eN2 N3eN4 N2eZn1 N4eZn1 Zn1eCl1 Zn1eCl2 2 C14eC15 C15eN1 C15eN3 Zn1eN2 Zn1eN4 Zn1eBr1 Zn1eBr2 3 C14eC15 C15eN1 C15eN3 Zn1eN2 Zn1eN4 Zn1eI1 Zn1eI2
1.515(3) 1.480(3) 1.449(3) 1.344(3) 1.359(3) 1.328(4) 1.346(3) 1.341(3) 1.323(4)
C14eC15eN1 C14eC15eN3 N3eC15eN1 C15eN3eC19 C15eN1eN2 C15eN3eN4 C15eN1eC16 N3eN4eC20 N1eN2eC17
113.0(2) 114.4(2) 108.8(2) 129.2(2) 118.4(2) 118.4(2) 129.5(2) 104.0(2) 103.2(2)
1.514(3) 1.478(3) 1.476(3) 1.366(3) 1.359(3) 2.072(2) 2.129(2) 2.277(1) 2.262(1)
N1eC15eN3 C14eC15eN1 C14eC15eN3 C15eN1eN2 C15eN3eN4 N2eZn1eN4 N2eZn1eCl2 N4eZn1eCl1 Cl1eZn1eCl2
110.8(2) 113.5(2) 111.2(2) 119.9(2) 117.9(2) 84.6(8) 107.5(6) 93.3(6) 126.7(4)
1.512(9) 1.490(9) 1.470(9) 2.067(7) 2.062(5) 2.371(2) 2.315(1)
N1eC15eN3 N2eZn1eN4 N2eZn1eBr2 N4eZn1eBr1 Br1eZn1eBr2
109.6(5) 88.7(3) 115.3(2) 108.8(2) 121.9(6)
1.518(7) 1.470(8) 1.471(7) 2.057(5) 2.057(5) 2.514(9) 2.561(1)
N1eC15eN3 N2eZn1eN4 N2eZn1eI1 N4eZn1eI2 I1eZn1eI2
110.7(4) 88.3(2) 114.8(1) 110.6(1) 120.7(3)
Complex 2 contains only monomeric [ZnBr2L2] units (Fig. 3). Like the complex 1 described above, [ZnBr2L2] contains the bis(pyrazolyl)methane ligand in a boat conformation. The ZnII atom has a distorted tetrahedral geometry, which coordinates with two bromine atoms and two nitrogen atoms. The ZneN distances [2.062(5) A and 2.067(7) A] are almost the same. The ZneBr distances [2.371(2) A and 2.315(1) A] are similar to those reported for [LTZnBr2, LT ¼ bis(3,4,5-trimethylparazol-1-yl)methane] [19]. The :NeZneN is 88.7(3) , which is larger than that of complex 1 [84.6(8) ]. There are CeH/Br (ca. 2.923 A) weak interactions
between the adjacent molecules resulting a 1D chain as shown in Fig. 3-p. Complex 3 also contains only monomeric [ZnI2L2] units (Fig. 4) like complex 2. Like complexes 1 and 2 described above, [ZnI2L2] contains the bis(pyrazolyl)methane ligand in a boat conformation. The ZnII atom also has a distorted tetrahedral geometry, which coordinates with two iodine atoms and two nitrogen atoms. The two ZneN bond lengths are completely the same [2.057 (5) A]. The ZneI distances are 2.514(9) A and 2.561(1) A. The :NeZneN is 88.3(3) , which is also larger than that of complex 1 [84.6(8) ]. There are CeH/I (ca. 3.108 A) weak interactions between the adjacent molecules resulting a 1D chain as shown in Fig. 4-p. 2.3. Cytotoxicity results The free ligand 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) and the metal complexes 1e3 were tested for their antitumor activity in vitro against three human cancer cell lines: A549 (nonsmall cell lung carcinoma), Huh-7 (hepatocarcinoma cell line) and MDA-MB-435 (human breast cancer cell line). The free ligand L2 and the complexes 1e3 DMSO was used for the preparation of the mother solutions, which were diluted with complete medium as soon as possible so that the co-solvent (DMSO) percentage never exceeded 0.1%. 5-Fluorouracil and cisplatin served as the control compounds in all assays. The results of the cytotoxicity tested are reported in Table 3. All complexes show IC50 values lower than those of the free ligand L2 and the control compound 5-fluorouracil, but higher than those of another control compound cisplatin. Among the tumor cell lines used here, this series of complexes appear to be more cytotoxic against the A549 tumor cell line with IC50 values range from 7.00 to 8.45 mg/mL. 2.4. Antibacterial activity assays All synthesized complexes were tested for their in vitro antibacterial activity against gram-positive bacterial (Bacillus subtilis and Staphylococcus aureus), gram-negative bacterial (Pseudomonas putida and Escherichia coli) strains using the MTT method (for determination of MIC50). Chloromycin was used as standard drug. The results were given in Table 4. The results of the experiments evidently show that all the three complexes show significant activity against P. putida, a gramnegative strain. The MIC50 values of complexes 1e3 are 0.011, 0.910 and 0.042 mg/mL, respectively. Interestingly, the MIC50 value of complex 1 against P. putida reaching 0.011 mg/mL much lower than that the positive control chloromycin (0.182 mg/mL), indicates that complex 1 is a potent antibacterial agent. As shown in Table 4, complex 1 also exhibits better antibacterial activity than the mononuclear complexes 2 and 3. The increase in antibacterial activity of the binuclear complex 1 may be due to the effect of the metal ion on the normal cell process. Complexation considerably reduces the polarity of the metal ion because of partial sharing of its positive charge with the donor groups. Such complexation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layers of cell membrane [20]. 3. Conclusions
Fig. 1. Molecular structure of L2 using Diamond.
In the present study, three novel Zn(II) complexes with 1,10 (anthracen-9-ylmethylene)bis(1H-pyrazole) have been prepared and their biological activities were preliminary evaluated. Complex 1, a binuclear Zn(II) complex, exhibited strong antibacterial activity
B.-F. Ruan et al. / European Journal of Medicinal Chemistry 72 (2014) 46e51
49
Fig. 2. Molecular structure of complex 1 using Diamond.
against P. putida (MIC50 ¼ 0.011 mg/mL), indicates that it is a potent antibacterial agent. 4. Experimental protocols 4.1. General All reagents and solvents were commercially available highgrade materials. IR spectra were recorded on a Thermo Nicolet FT-IR-870SX spectrophotometer (range 400e4000 cm1) as KBr pellets. The 1H NMR and 13C NMR spectra were recorded on a Bruker DRX 600 model spectrometer in CDCl3 solutions at room temperature with TMS as an internal standard. The ESI-MS spectra were recorded on a Mariner System 5304 Mass spectrometer. Carbon, hydrogen and nitrogen assays were carried out with a CHNeO-Rapid instrument and were within 0.4% of the theoretical values. 4.2. Preparation of 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) To the THF solution (50 mL) of NaH (48.00 mmol, 1.16 g) was added a THF solution (15 mL) of pyrazole (48.00 mmol, 3.27 g) with stirring under the protection of nitrogen. The mixture was stirred for 30 min at 0 C to give a light yellow solution. SOCl2 (24.00 mmol, 2.91 g) was added dropwise into the solution to afford a yellowewhite suspension. After stirring for 40 min, 9Anthracenecarboxaldehyde (4.85 mmol, 1.00 g) and anhydrous CoCl2 (1.46 mmol, 0.19 g) was added and refluxing for 10 h, and finally treated with 40 mL H2O, extracted, dried and separated by column chromatography (petroleum ether/ethyl acetate ¼ 10:1) to afford L2 (0.67 g, 42.58%). X-ray quality single crystals were formed
Fig. 3. Molecular structure of complex 2 using Diamond.
by slow evaporation of the ethyl acetate solution in air for a few days. Light green single crystals. IR (KBr, cm1): 3144(w), 3111(w), 3084(w), 3053(w), 3006(w), 1623(w), 1504(w), 1447(m), 1396(m), 1311(s), 1295(s), 1178(m), 1087(s), 1039(s), 964(m), 911(m), 849(m), 813(s), 771(s), 757(s), 732(s), 609(m); 1H NMR (CDCl3): d ¼ 9.04 (s, 1H), 8.60 (s, 1H), 8.05 (s, 1H) 8.04 (s, 1H) 7.79 (s, 1H) 7.77 (s, 1H) 7.67 (d, J ¼ 1.8, 2H) 7.60 (d, J ¼ 1.8, 4H) 7.28 (d, J ¼ 2.4, 2H) 6.35 (t, J ¼ 2.1, 2H); 13C NMR (CDCl3): d ¼ 143.54, 134.21, 134.13, 133.26, 132.28, 130.27, 127.68, 125.45, 109.45, 107.74; MS(EI):m/z, 324.14; Anal. Calcd for C26H11N4: C, 77.76; H, 4.97; N, 17.27%; Found: C, 77.61; H, 4.99; N, 17.24%. 4.3. Synthesis of the complexes 1e3 An ethyl acetate solution (20 mL) of L2 (1.00 mmol, 0.32 g) was added with stirring to an ethyl acetate solution (20 mL) of the corresponding zinc(II) salt (1.00 mmol), viz. ZnCl2 for 1, ZnBr2 for 2, and ZnI2 for 3. The resulting mixtures were stirred at room temperature for 3 h and then filtered to afford white powder. X-ray quality light green single crystals were formed by slow evaporation of the ethyl acetate solutions (12 mL) in air for a few days, respectively. 4.3.1. [ZnCl2L2]2 (1) IR (KBr, cm1): 3119(w), 3063(w), 1625(w), 1525(w), 1422(s), 1305(s), 1222(m), 1096(m), 1064(s), 912(m), 815(m), 767(s), 732(s), 600(m); 1H NMR (CDCl3): d ¼ 9.47 (s, 1H), 8.86 (s, 1H), 8.39 (s, 1H), 8.20 (d, J ¼ 9, 2H), 8.08 (d, J ¼ 1.8, 2H), 7.60 (t, J ¼ 7.2, 5H), 7.12 (d, J ¼ 2.4, 2H), 6.30 (t, J ¼ 2.7, 2H); 13C NMR (CDCl3): d ¼ 145.53, 137.02, 136.64, 134.23, 132.70, 128.77, 119.94, 109.41; Anal. Calcd for C42H32Cl4N8Zn2: C, 54.75; H, 3.50; N, 12.16%; Found: C, 54.56; H, 3.51; N, 12.21%.
Fig. 4. Molecular structure of complex 3 using Diamond.
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B.-F. Ruan et al. / European Journal of Medicinal Chemistry 72 (2014) 46e51
4.5. Cytotoxicity tests in cancer cell lines
Table 3 Antitumor activity (IC50, mg/mL) of L2 and complexes 1e3. IC50 SD (mg/mL)a
Comp. no
A549 L2 1 2 3 5-F Cisplatin
Huh-7
9.77 7.51 8.45 7.00 24.87 4.50
2.94 1.36 1.76 1.16 1.91 0.45
27.97 13.67 14.97 16.63 20.03 2.82
MDA-MB-435
4.05 2.25 4.46 5.01 1.25 0.71
23.77 16.53 14.17 15.67 25.60 3.42
1.66 1.20 3.76 4.85 1.02 0.23
a Values are the average of three independent experiments run in triplicate. Variation was generally 5e10%.
4.3.2. [ZnBr2L2] (2) IR (KBr, cm1): 3151(m), 3055(w), 1624(w), 1526(w), 1417(s), 1369(w), 1298(s), 1211(m),1184(m), 1095(m), 1064(s), 984(m), 912(m), 875(m), 812(s), 785(s), 765(s), 733(s), 595(m); 1H NMR (CDCl3): d ¼ 9.51 (s, 1H), 8.86 (s, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 8.09 (d, J ¼ 1.8, 2H), 7.78 (s, 1H), 7.67 (s, 1H), 7.60 (s, 2H), 7.49 (d, J ¼ 7.2, 1H), 7.10 (d, J ¼ 2.4, 2H) 6.30 (t, J ¼ 2.4, 2H); 13C NMR (CDCl3): d ¼ 145.50, 137.13, 136.66, 134.43, 133.94, 132.5, 130.67, 128.6, 119.94, 109.37; Anal. Calcd for C21H16Br2N4Zn: C, 45.89; H, 2.93; N, 10.19%; Found: C, 45.97; H, 2.92; N, 10.14%.
The antitumor activity of the prepared compounds against A549, Huh-7 and MDA-MB-435 cell lines were evaluated as described elsewhere with some modifications [22]. Target tumor cell lines were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 2 104 cells mL1 with the complete medium, 100 mL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was permitted at 37 C, 5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Tested samples at pre-set concentrations were added to 6 wells with 5-Fu and cisplatin coassayed as positive reference. After 48 h exposure period, 40 mL of PBS containing 2.5 mg mL1 of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))) was added to each well. 4 h later, 100 mL extraction solutions (10% SDSe 5% isobutyl alcohole0.01 M HCl) were added. After an overnight incubation at 37 C, the optical density was measured at a wavelength of 570 nm on an ELISA microplate reader. In all experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times. The results were summarized in Table 3. 4.6. Antibacterial assay
2
4.3.3. [ZnI2L ] (3) IR (KBr, cm1): 3149(m), 1623(w), 1515(w), 1419(s), 1298(s), 1211(m), 1185(w), 1094(m), 1064(s), 985(w), 875(w), 811(m), 761(s), 732(s), 600(m); 1H NMR (CDCl3): d ¼ 9.52 (s, 1H), 8.88 (s, 1H), 8.54 (s, 1H), 8.21 (s, 1H), 8.10 (s, 1H) 7.61 (t, J ¼ 7.2, 5H), 7.09 (d, J ¼ 1.8, 2H), 6.30 (t, J ¼ 2.4, 2H); 13C NMR (CDCl3): d ¼ 145.67, 137.35, 136.73, 134.27, 132.52, 130.72, 128.42, 124.85,119.29, 109.29; Anal. Calcd for C21H16I2N4Zn: C, 39.19; H, 2.51; N, 8.71%; Found: C, 39.32; H, 2.51; N, 8.67%. 4.4. Crystallographic structure determination Single crystal X-ray diffraction measurements for L2 and the complexes 1e3 were carried out on a Siemens Smart 1000 CCD diffractometer equipped with a graphite crystal monochromator situated in the incident beam for data collection at room temperature. The determination of unit cell parameters and data collections were performed with Mo-Ka radiation (l ¼ 0.71073 A). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods with SHELXL-97 [21]. All the non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was performed by fullmatrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically and riding on the concerned atoms. The crystal data and structure refinement were listed in Table 1. The selected bond lengths ( A) and angles ( ) were listed in Table 2.
Table 4 Antibacterial activity (MIC50, mg/mL) of L2 and complexes 1e3. Comp. no
B. subtilis L2 1 2 3 Chloromycin a
3.258 5.783 9.442 32.683 1.255
2.132 5.777 1.032 0.581 0.499
Each value is the mean S.D. (n ¼ 3).
0.329 0.762 0.014 0.236 0.302
P. putida 1.543 0.011 0.910 0.042 0.182
This work was supported by a grant for the National Natural Science Foundation of China (21071001, 21271004, 51372003 and 21271003), the Natural Science Foundation of Anhui Province (1208085MB22, 1308085MB24), Ministry of Education Funded Projects Focus on returned overseas scholar, Program for New Century Excellent Talents in University (China) and Doctoral Program Foundation of Ministry of Education of China (20113401110004).
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.10.064.
Gram-negative S. aureus
0.513 0.762 0.975 1.314 0.099
Acknowledgment
Appendix A. Supplementary data
MIC50 SD (mg/mL)a Gram-positive
The antibacterial activities of the synthesized compounds was tested against two Gram-positive bacterial strains (B. subtilis ATCC6633, S. aureus ATCC25923) and two Gram-negative bacterial strains (P. putida ATCC49128, E. coli ATCC35218), using method recommended by Clinical Laboratory Standards Institute (CLSI) [23]. The MIC50s of the test compounds were determined by a colorimetric method using the dye MTT. A stock solution of the synthesized compound (1000 mg/mL) in DMSO was prepared and graded quantities of the test compounds were incorporated in specified quantity of sterilized liquid medium (50% (v/v) of DMSO in PBS). A specified quantity of the medium containing the test compound was poured into 96-well plates. Suspension of the microorganism was prepared to contain approximate 105 cfu mL1 and applied to 96-well plates with serially diluted compounds to be tested and incubated at 37 C for 24 h. The optical density (OD) was measured with a microplate reader at 550 nm. The results were summarized in Table 4.
E. coli 0.188 0.002 0.041 0.037 0.040
4.799 6.991 8.918 9.259 3.041
0.681 0.845 0.950 0.967 0.479
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Short communication
Synthesis, characterization, cytotoxicity and antibacterial activity of an anthracenyl-linked bis(pyrazolyl)methane ligand and its zinc(II) complexes Ban-Feng Ruan b, Ying-Zhong Zhu a, Wan-Ding Liu a, Bao-An Song c, **, Yu-Peng Tian a, * a b c
Department of Chemistry, Anhui Province Key Laboratory of Functional Inorganic Material Chemistry, Anhui University, Hefei 230039, PR China School of Medical Engineering, Hefei University of Technology, Hefei 230009, PR China Key Laboratory of Green Pesticide and Agriculture Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2013 Received in revised form 16 October 2013 Accepted 23 October 2013 Available online 1 November 2013
Three novel Zn(II) complexes (1e3) with 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) have been prepared and structurally characterized by X-ray crystallography. Among them, 1 is a binuclear Zn(II) complex while 2 and 3 are mononuclear. The in vitro cytotoxic and antibacterial activities of these complexes were also evaluated. The three complexes exhibit cytotoxic specificity and significant antitumor activity. The MIC50 value of complex 1 against Pseudomonas putida reaching 0.011 mg/mL much lower than that the positive control chloromycin (0.182 mg/mL), indicates that complex 1 is a potent antibacterial agent. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: 1,10 -(Anthracen-9-ylmethylene)bis(1Hpyrazole) Zinc(II) complex Crystal structure Cytotoxicity Antibacterial activity
1. Introduction Platinum complexes such as cisplatin and carboplatin are metalbased drugs, which are widely used in cancer chemotherapy. The great success in the clinical treatment of human malignancies has stimulated researches in the area of inorganic antitumor agents. However, their use is extremely hampered by severe toxicity and development of resistance during the therapy [1e3]. In order to overcome these disadvantages, current strategies in the development of novel metallodrugs focused more and more on the use of transition metal complexes containing improved organic ligands [4e6]. Among the nonplatinum complexes for metal based chemotherapy, zinc complexes have been much explored due to the fact that zinc is bioessential elements responsible for numerous bioactivities in living organism [7]. Zinc is the second prominent trace metal in the human body which is critical for numerous cells processes and is major regulatory ion in the metabolism of cells [8]. * Corresponding author. Tel.: þ86 551 65108151. ** Corresponding author. Tel.: þ86 851 3620521. E-mail addresses: [email protected] (B.-A. Song), [email protected] (Y.-P. Tian). 0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.10.064
Many features of zinc, such as its abilities in assisting Lewis activation, nucleophile generation, fast ligand exchange, and leaving group stabilization, make ZnII ideal for the catalysis of hydrolytic reactions, including DNA cleavage, which is an important property for use as anticancer drugs. Interaction of dinuclear ZnII complexes with DNA has recently attracted much attention [9,10], owing to their possible applications as new cancer therapeutic agents and their photochemical properties, which make them potential probes of DNA structure and conformation [11]. Zinc complexes with diverse biological activity viz. antibacterial [12], anti-inflammatory [13], for the treatment of Alzheimer disease [14] and antiproliferative, antitumor [15] activity have been reported. Keeping in view of biological and medicinal properties of 9anthracene aldehyde and ZnII, we find it vital to join both moieties in search of designing ZnII complexes that could aggressively work against tumor cells and bacterial species. Herein we have synthesized three novel ZnII complexes with a bis(pyrazolyl) methane ligand, viz. 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole). The crystal structures of the free ligand L2 and its three ZnII complexes [ZnCl2(L2)]2, [ZnBr2L2] and [ZnI2L2] have been determined by X-ray crystallography. The cytotoxicity and antibacterial activity have also been evaluated.
B.-F. Ruan et al. / European Journal of Medicinal Chemistry 72 (2014) 46e51
Scheme 1. Synthesis of L2 and complexes 1e3. Reagent and conditions: a) 9anthracenecarboxaldehyde, anhydrous CoCl2, THF, reflux; b) diethyl ether, ZnX2 (X ¼ Cl, Br, I), rt.
2. Results and discussion 2.1. Synthesis of ligand (L2) and complexes 1e3 The starting material bis(1H-pyrazol-1-yl)methanone (S) was purchased from Aldrich (USA). The ligand namely, 1,10 -(anthracen9-ylmethylene)bis(1H-pyrazole) (L2) was prepared by refluxing a mixture of equimolar quantities of S and 9-anthracene carboxaldehyde in a THF solution (Scheme 1) [16]. All the complexes were synthesized by the reaction of the ethyl acetate solutions of L2 with the corresponding zinc(II) salt (1.00 mmol) (ZnCl2 for 1, ZnBr2 for 2, and ZnI2 for 3). The compounds have been characterized by elemental analysis and IR spectra. Structures of the complexes were further confirmed by X-ray crystallography (CCDC 795201 for L2, 814312 for 1, 806628 for 2, and 800348 for 3). 2.2. Crystal structures of L2 and complexes 1e3 The details of crystallographic data and structure refinement parameters of L2 and complexes 1e3 are summarized in Table 1. Selected bond lengths ( A) and angles are collected in Table 2. The single crystal X-ray analysis reveals that the ligand L2 crystallizes as orthorhombic crystal system with space group
47
P212121. Molecules of L2 consist of two pyrazole rings, oriented in a roughly antiparallel manner with respect to each other, N-bonded to the methine carbon and form the cyclopentadienyl ring (Fig. 1). The 2-(9-anthryl) group is attached to the bis(pyrazolyl)methane unit and rotated by ca. 72.8 out of plane perpendicular to that defined by the NeCeN fragment. The angle between the two pyrazolyl rings is 100.5 . Thus, the planarity of the whole molecule is very poor [17]. As shown in Fig. 1-p, the 1D zigzag chain structure is self-assembled via weak CeH/N (2.720 A) and CeH/p (2.866 and 3.306 A) intermolecular interactions. Complex 1 [ZnCl2(L2)]2, a rare chloro-bridged zinc(II) dimmer, is a discrete neutral centrosymmetric compound (Fig. 2). Each ZnII atom has a distorted square-pyramidal geometry, which coordinates with three chlorine atoms and two nitrogen atoms. The ZneN distances [2.072(2) A and 2.129 (2) A] are quite asymmetric, compared to those found in the only other structurally authenticated Zn(II)Cl2 complex containing a bis(pyrazolyl)methane ligand [ZnCl2{Me2C(Pz)2}][2.058(3) and 2.046(3) A]. The ZneClbriding bond lengths of 2.689(1) A and 2.277(1) A are much more asymmetric than those in [CoCl2(L)2, L ¼ (2-propargyloxyphenyl)bis(pyrazolyl) methane] [18]. Both zinc atoms are bridged by two chlorine atoms (Cl1 and Cl1A), forming a centrosymmetry [Zn(m-Cl)2Zn] ring. The configuration of the six membered ring formed by the bis(pyrazolyl)methane and the Zn centre has a boat configuration with the methine proton in an equatorial position. One of the ligand nitrogen atoms is trans to one of the bridging chlorides, whilst the other nitrogen is trans to the non-bridging Cl-ligand. To be interestingly, the existence of the non-bridging chlorine atoms (Cl2 and Cl2A) lead to the distortion of the pyrazol rings of the ligand, which cause the reduce of the dihedral angle between plane N2(N2A)e Zn(Zn1A)eN4(N4A) and plane Cl1eZneCl1A from theoretical 90 e 54.6 . Thus, this kind of distortion leads to the observed asymmetry in ZneN and ZneCl bridging bond distances. However, the dihedral angles in ZnBr2L2 and ZnI2L2 are 87.3 and 86.9 . Accordingly, the ZneN and ZneX (X ¼ Br and I) bridging bond distances in complexes 2 and 3 are almost the same, respectively. Furthermore, there are CeH/Cl (ca. 2.936 A) and CeH/p (ca. 3.202 A) interactions between the adjacent molecules resulting a 1D chain as shown in Fig. 2-p.
Table 1 Crystallographic data and structure refinements for L2 and its complexes 1e3.
Formula Formula weight Crystal system Space group Crystal size (mm3) a ( A) b ( A) c ( A) a ( ) b ( ) g ( ) Volume ( A3) Z Dc (mg m3) m (mm1) F (000) q rang ( ) Reflections collected Reflections unique Parameters Goodness-of-fit R1, wR2 [I > 2s(I)] R1, wR2 [all data] Larg. peak/hole (e A) CCDC no
L2
[ZnCl2L2]2
ZnBr2L2
ZnI2L2
C21H16N4 324.38 Orthorhombic P212121 0.30 0.30 0.20 7.544(5) 9.397(5) 23.344(5) 90.000(5) 90.000(5) 90.000(5) 1654.9(15) 4 1.329 0.094 686 2.78e20.07 2907 2214 227 0.839 0.0386, 0.1057 0.0591, 0.1258 0.113/0.137 795201
C42H32Cl4N8Zn2 921.34 Monoclinic P21/c 0.30 0.20 0.20 9.631(5) 11.677(5) 18.013(5) 90.000(5) 100.906(5) 90.000(5) 1989.2(14) 2 1.538 1.517 936 2.303e25.71 3500 2939 254 0.850 0.0312, 0.1015 0.1126, 0.850 0.409/0.362 814312
C21H16Br2N4Zn 549.57 Monoclinic P21/n 0.30 0.30 0.20 8.711(5) 14.007(5) 17.916(5) 90.000(5) 103.843(5) 90.000(5) 2122.5(16) 4 1.720 4.936 1738 2.34e19.74 3697 2053 253 0.949 0.0529, 0.1362 0.1173, 0.1772 0.396/0.551 806628
C21H16I2N4Zn 643.57 Monoclinic P21/n P2(1)/n 8.925(5) 14.269(5) 18.017(5) 90.000(5) 103.854(5) 90.000(5) 2227.7(16) 4 1.919 3.887 2231 1.84e23.10 2975 2457 254 0.856 0.0318, 0.1037 0.0411, 0.1165 0.815/0.711 800348
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Table 2 Selected bond lengths ( A) and angles ( ) for L2 and the complexes 1e3. L2 C14eC15 C15eN3 C15eN1 N3eN4 N1eN2 N4eC20 N3eC19 N1eC16 N2eC17 1 C14eC15 C15eN1 C15eN3 N1eN2 N3eN4 N2eZn1 N4eZn1 Zn1eCl1 Zn1eCl2 2 C14eC15 C15eN1 C15eN3 Zn1eN2 Zn1eN4 Zn1eBr1 Zn1eBr2 3 C14eC15 C15eN1 C15eN3 Zn1eN2 Zn1eN4 Zn1eI1 Zn1eI2
1.515(3) 1.480(3) 1.449(3) 1.344(3) 1.359(3) 1.328(4) 1.346(3) 1.341(3) 1.323(4)
C14eC15eN1 C14eC15eN3 N3eC15eN1 C15eN3eC19 C15eN1eN2 C15eN3eN4 C15eN1eC16 N3eN4eC20 N1eN2eC17
113.0(2) 114.4(2) 108.8(2) 129.2(2) 118.4(2) 118.4(2) 129.5(2) 104.0(2) 103.2(2)
1.514(3) 1.478(3) 1.476(3) 1.366(3) 1.359(3) 2.072(2) 2.129(2) 2.277(1) 2.262(1)
N1eC15eN3 C14eC15eN1 C14eC15eN3 C15eN1eN2 C15eN3eN4 N2eZn1eN4 N2eZn1eCl2 N4eZn1eCl1 Cl1eZn1eCl2
110.8(2) 113.5(2) 111.2(2) 119.9(2) 117.9(2) 84.6(8) 107.5(6) 93.3(6) 126.7(4)
1.512(9) 1.490(9) 1.470(9) 2.067(7) 2.062(5) 2.371(2) 2.315(1)
N1eC15eN3 N2eZn1eN4 N2eZn1eBr2 N4eZn1eBr1 Br1eZn1eBr2
109.6(5) 88.7(3) 115.3(2) 108.8(2) 121.9(6)
1.518(7) 1.470(8) 1.471(7) 2.057(5) 2.057(5) 2.514(9) 2.561(1)
N1eC15eN3 N2eZn1eN4 N2eZn1eI1 N4eZn1eI2 I1eZn1eI2
110.7(4) 88.3(2) 114.8(1) 110.6(1) 120.7(3)
Complex 2 contains only monomeric [ZnBr2L2] units (Fig. 3). Like the complex 1 described above, [ZnBr2L2] contains the bis(pyrazolyl)methane ligand in a boat conformation. The ZnII atom has a distorted tetrahedral geometry, which coordinates with two bromine atoms and two nitrogen atoms. The ZneN distances [2.062(5) A and 2.067(7) A] are almost the same. The ZneBr distances [2.371(2) A and 2.315(1) A] are similar to those reported for [LTZnBr2, LT ¼ bis(3,4,5-trimethylparazol-1-yl)methane] [19]. The :NeZneN is 88.7(3) , which is larger than that of complex 1 [84.6(8) ]. There are CeH/Br (ca. 2.923 A) weak interactions
between the adjacent molecules resulting a 1D chain as shown in Fig. 3-p. Complex 3 also contains only monomeric [ZnI2L2] units (Fig. 4) like complex 2. Like complexes 1 and 2 described above, [ZnI2L2] contains the bis(pyrazolyl)methane ligand in a boat conformation. The ZnII atom also has a distorted tetrahedral geometry, which coordinates with two iodine atoms and two nitrogen atoms. The two ZneN bond lengths are completely the same [2.057 (5) A]. The ZneI distances are 2.514(9) A and 2.561(1) A. The :NeZneN is 88.3(3) , which is also larger than that of complex 1 [84.6(8) ]. There are CeH/I (ca. 3.108 A) weak interactions between the adjacent molecules resulting a 1D chain as shown in Fig. 4-p. 2.3. Cytotoxicity results The free ligand 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) and the metal complexes 1e3 were tested for their antitumor activity in vitro against three human cancer cell lines: A549 (nonsmall cell lung carcinoma), Huh-7 (hepatocarcinoma cell line) and MDA-MB-435 (human breast cancer cell line). The free ligand L2 and the complexes 1e3 DMSO was used for the preparation of the mother solutions, which were diluted with complete medium as soon as possible so that the co-solvent (DMSO) percentage never exceeded 0.1%. 5-Fluorouracil and cisplatin served as the control compounds in all assays. The results of the cytotoxicity tested are reported in Table 3. All complexes show IC50 values lower than those of the free ligand L2 and the control compound 5-fluorouracil, but higher than those of another control compound cisplatin. Among the tumor cell lines used here, this series of complexes appear to be more cytotoxic against the A549 tumor cell line with IC50 values range from 7.00 to 8.45 mg/mL. 2.4. Antibacterial activity assays All synthesized complexes were tested for their in vitro antibacterial activity against gram-positive bacterial (Bacillus subtilis and Staphylococcus aureus), gram-negative bacterial (Pseudomonas putida and Escherichia coli) strains using the MTT method (for determination of MIC50). Chloromycin was used as standard drug. The results were given in Table 4. The results of the experiments evidently show that all the three complexes show significant activity against P. putida, a gramnegative strain. The MIC50 values of complexes 1e3 are 0.011, 0.910 and 0.042 mg/mL, respectively. Interestingly, the MIC50 value of complex 1 against P. putida reaching 0.011 mg/mL much lower than that the positive control chloromycin (0.182 mg/mL), indicates that complex 1 is a potent antibacterial agent. As shown in Table 4, complex 1 also exhibits better antibacterial activity than the mononuclear complexes 2 and 3. The increase in antibacterial activity of the binuclear complex 1 may be due to the effect of the metal ion on the normal cell process. Complexation considerably reduces the polarity of the metal ion because of partial sharing of its positive charge with the donor groups. Such complexation could enhance the lipophilic character of the central metal atom, which subsequently favors its permeation through the lipid layers of cell membrane [20]. 3. Conclusions
Fig. 1. Molecular structure of L2 using Diamond.
In the present study, three novel Zn(II) complexes with 1,10 (anthracen-9-ylmethylene)bis(1H-pyrazole) have been prepared and their biological activities were preliminary evaluated. Complex 1, a binuclear Zn(II) complex, exhibited strong antibacterial activity
B.-F. Ruan et al. / European Journal of Medicinal Chemistry 72 (2014) 46e51
49
Fig. 2. Molecular structure of complex 1 using Diamond.
against P. putida (MIC50 ¼ 0.011 mg/mL), indicates that it is a potent antibacterial agent. 4. Experimental protocols 4.1. General All reagents and solvents were commercially available highgrade materials. IR spectra were recorded on a Thermo Nicolet FT-IR-870SX spectrophotometer (range 400e4000 cm1) as KBr pellets. The 1H NMR and 13C NMR spectra were recorded on a Bruker DRX 600 model spectrometer in CDCl3 solutions at room temperature with TMS as an internal standard. The ESI-MS spectra were recorded on a Mariner System 5304 Mass spectrometer. Carbon, hydrogen and nitrogen assays were carried out with a CHNeO-Rapid instrument and were within 0.4% of the theoretical values. 4.2. Preparation of 1,10 -(anthracen-9-ylmethylene)bis(1H-pyrazole) (L2) To the THF solution (50 mL) of NaH (48.00 mmol, 1.16 g) was added a THF solution (15 mL) of pyrazole (48.00 mmol, 3.27 g) with stirring under the protection of nitrogen. The mixture was stirred for 30 min at 0 C to give a light yellow solution. SOCl2 (24.00 mmol, 2.91 g) was added dropwise into the solution to afford a yellowewhite suspension. After stirring for 40 min, 9Anthracenecarboxaldehyde (4.85 mmol, 1.00 g) and anhydrous CoCl2 (1.46 mmol, 0.19 g) was added and refluxing for 10 h, and finally treated with 40 mL H2O, extracted, dried and separated by column chromatography (petroleum ether/ethyl acetate ¼ 10:1) to afford L2 (0.67 g, 42.58%). X-ray quality single crystals were formed
Fig. 3. Molecular structure of complex 2 using Diamond.
by slow evaporation of the ethyl acetate solution in air for a few days. Light green single crystals. IR (KBr, cm1): 3144(w), 3111(w), 3084(w), 3053(w), 3006(w), 1623(w), 1504(w), 1447(m), 1396(m), 1311(s), 1295(s), 1178(m), 1087(s), 1039(s), 964(m), 911(m), 849(m), 813(s), 771(s), 757(s), 732(s), 609(m); 1H NMR (CDCl3): d ¼ 9.04 (s, 1H), 8.60 (s, 1H), 8.05 (s, 1H) 8.04 (s, 1H) 7.79 (s, 1H) 7.77 (s, 1H) 7.67 (d, J ¼ 1.8, 2H) 7.60 (d, J ¼ 1.8, 4H) 7.28 (d, J ¼ 2.4, 2H) 6.35 (t, J ¼ 2.1, 2H); 13C NMR (CDCl3): d ¼ 143.54, 134.21, 134.13, 133.26, 132.28, 130.27, 127.68, 125.45, 109.45, 107.74; MS(EI):m/z, 324.14; Anal. Calcd for C26H11N4: C, 77.76; H, 4.97; N, 17.27%; Found: C, 77.61; H, 4.99; N, 17.24%. 4.3. Synthesis of the complexes 1e3 An ethyl acetate solution (20 mL) of L2 (1.00 mmol, 0.32 g) was added with stirring to an ethyl acetate solution (20 mL) of the corresponding zinc(II) salt (1.00 mmol), viz. ZnCl2 for 1, ZnBr2 for 2, and ZnI2 for 3. The resulting mixtures were stirred at room temperature for 3 h and then filtered to afford white powder. X-ray quality light green single crystals were formed by slow evaporation of the ethyl acetate solutions (12 mL) in air for a few days, respectively. 4.3.1. [ZnCl2L2]2 (1) IR (KBr, cm1): 3119(w), 3063(w), 1625(w), 1525(w), 1422(s), 1305(s), 1222(m), 1096(m), 1064(s), 912(m), 815(m), 767(s), 732(s), 600(m); 1H NMR (CDCl3): d ¼ 9.47 (s, 1H), 8.86 (s, 1H), 8.39 (s, 1H), 8.20 (d, J ¼ 9, 2H), 8.08 (d, J ¼ 1.8, 2H), 7.60 (t, J ¼ 7.2, 5H), 7.12 (d, J ¼ 2.4, 2H), 6.30 (t, J ¼ 2.7, 2H); 13C NMR (CDCl3): d ¼ 145.53, 137.02, 136.64, 134.23, 132.70, 128.77, 119.94, 109.41; Anal. Calcd for C42H32Cl4N8Zn2: C, 54.75; H, 3.50; N, 12.16%; Found: C, 54.56; H, 3.51; N, 12.21%.
Fig. 4. Molecular structure of complex 3 using Diamond.
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4.5. Cytotoxicity tests in cancer cell lines
Table 3 Antitumor activity (IC50, mg/mL) of L2 and complexes 1e3. IC50 SD (mg/mL)a
Comp. no
A549 L2 1 2 3 5-F Cisplatin
Huh-7
9.77 7.51 8.45 7.00 24.87 4.50
2.94 1.36 1.76 1.16 1.91 0.45
27.97 13.67 14.97 16.63 20.03 2.82
MDA-MB-435
4.05 2.25 4.46 5.01 1.25 0.71
23.77 16.53 14.17 15.67 25.60 3.42
1.66 1.20 3.76 4.85 1.02 0.23
a Values are the average of three independent experiments run in triplicate. Variation was generally 5e10%.
4.3.2. [ZnBr2L2] (2) IR (KBr, cm1): 3151(m), 3055(w), 1624(w), 1526(w), 1417(s), 1369(w), 1298(s), 1211(m),1184(m), 1095(m), 1064(s), 984(m), 912(m), 875(m), 812(s), 785(s), 765(s), 733(s), 595(m); 1H NMR (CDCl3): d ¼ 9.51 (s, 1H), 8.86 (s, 1H), 8.50 (s, 1H), 8.20 (s, 1H), 8.09 (d, J ¼ 1.8, 2H), 7.78 (s, 1H), 7.67 (s, 1H), 7.60 (s, 2H), 7.49 (d, J ¼ 7.2, 1H), 7.10 (d, J ¼ 2.4, 2H) 6.30 (t, J ¼ 2.4, 2H); 13C NMR (CDCl3): d ¼ 145.50, 137.13, 136.66, 134.43, 133.94, 132.5, 130.67, 128.6, 119.94, 109.37; Anal. Calcd for C21H16Br2N4Zn: C, 45.89; H, 2.93; N, 10.19%; Found: C, 45.97; H, 2.92; N, 10.14%.
The antitumor activity of the prepared compounds against A549, Huh-7 and MDA-MB-435 cell lines were evaluated as described elsewhere with some modifications [22]. Target tumor cell lines were grown to log phase in RPMI 1640 medium supplemented with 10% fetal bovine serum. After diluting to 2 104 cells mL1 with the complete medium, 100 mL of the obtained cell suspension was added to each well of 96-well culture plates. The subsequent incubation was permitted at 37 C, 5% CO2 atmosphere for 24 h before the cytotoxicity assessments. Tested samples at pre-set concentrations were added to 6 wells with 5-Fu and cisplatin coassayed as positive reference. After 48 h exposure period, 40 mL of PBS containing 2.5 mg mL1 of MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide))) was added to each well. 4 h later, 100 mL extraction solutions (10% SDSe 5% isobutyl alcohole0.01 M HCl) were added. After an overnight incubation at 37 C, the optical density was measured at a wavelength of 570 nm on an ELISA microplate reader. In all experiments three replicate wells were used for each drug concentration. Each assay was carried out at least three times. The results were summarized in Table 3. 4.6. Antibacterial assay
2
4.3.3. [ZnI2L ] (3) IR (KBr, cm1): 3149(m), 1623(w), 1515(w), 1419(s), 1298(s), 1211(m), 1185(w), 1094(m), 1064(s), 985(w), 875(w), 811(m), 761(s), 732(s), 600(m); 1H NMR (CDCl3): d ¼ 9.52 (s, 1H), 8.88 (s, 1H), 8.54 (s, 1H), 8.21 (s, 1H), 8.10 (s, 1H) 7.61 (t, J ¼ 7.2, 5H), 7.09 (d, J ¼ 1.8, 2H), 6.30 (t, J ¼ 2.4, 2H); 13C NMR (CDCl3): d ¼ 145.67, 137.35, 136.73, 134.27, 132.52, 130.72, 128.42, 124.85,119.29, 109.29; Anal. Calcd for C21H16I2N4Zn: C, 39.19; H, 2.51; N, 8.71%; Found: C, 39.32; H, 2.51; N, 8.67%. 4.4. Crystallographic structure determination Single crystal X-ray diffraction measurements for L2 and the complexes 1e3 were carried out on a Siemens Smart 1000 CCD diffractometer equipped with a graphite crystal monochromator situated in the incident beam for data collection at room temperature. The determination of unit cell parameters and data collections were performed with Mo-Ka radiation (l ¼ 0.71073 A). Unit cell dimensions were obtained with least-squares refinements, and all structures were solved by direct methods with SHELXL-97 [21]. All the non-hydrogen atoms were located in successive difference Fourier syntheses. The final refinement was performed by fullmatrix least-squares methods with anisotropic thermal parameters for non-hydrogen atoms on F2. The hydrogen atoms were added theoretically and riding on the concerned atoms. The crystal data and structure refinement were listed in Table 1. The selected bond lengths ( A) and angles ( ) were listed in Table 2.
Table 4 Antibacterial activity (MIC50, mg/mL) of L2 and complexes 1e3. Comp. no
B. subtilis L2 1 2 3 Chloromycin a
3.258 5.783 9.442 32.683 1.255
2.132 5.777 1.032 0.581 0.499
Each value is the mean S.D. (n ¼ 3).
0.329 0.762 0.014 0.236 0.302
P. putida 1.543 0.011 0.910 0.042 0.182
This work was supported by a grant for the National Natural Science Foundation of China (21071001, 21271004, 51372003 and 21271003), the Natural Science Foundation of Anhui Province (1208085MB22, 1308085MB24), Ministry of Education Funded Projects Focus on returned overseas scholar, Program for New Century Excellent Talents in University (China) and Doctoral Program Foundation of Ministry of Education of China (20113401110004).
Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.10.064.
Gram-negative S. aureus
0.513 0.762 0.975 1.314 0.099
Acknowledgment
Appendix A. Supplementary data
MIC50 SD (mg/mL)a Gram-positive
The antibacterial activities of the synthesized compounds was tested against two Gram-positive bacterial strains (B. subtilis ATCC6633, S. aureus ATCC25923) and two Gram-negative bacterial strains (P. putida ATCC49128, E. coli ATCC35218), using method recommended by Clinical Laboratory Standards Institute (CLSI) [23]. The MIC50s of the test compounds were determined by a colorimetric method using the dye MTT. A stock solution of the synthesized compound (1000 mg/mL) in DMSO was prepared and graded quantities of the test compounds were incorporated in specified quantity of sterilized liquid medium (50% (v/v) of DMSO in PBS). A specified quantity of the medium containing the test compound was poured into 96-well plates. Suspension of the microorganism was prepared to contain approximate 105 cfu mL1 and applied to 96-well plates with serially diluted compounds to be tested and incubated at 37 C for 24 h. The optical density (OD) was measured with a microplate reader at 550 nm. The results were summarized in Table 4.
E. coli 0.188 0.002 0.041 0.037 0.040
4.799 6.991 8.918 9.259 3.041
0.681 0.845 0.950 0.967 0.479
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