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Palladium(II) carbohydrate complexes of alkyl, aryl and ferrocenyl esters and their cytotoxic activities
Inorganica Chimica Acta 416 (2014) 164–170
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Palladium(II) carbohydrate complexes of alkyl, aryl and ferrocenyl esters and their cytotoxic activities Sadanala Bhavya Deepthi a, Rajiv Trivedi a,⇑, Lingamallu Giribabu a, Pombala Sujitha b, C. Ganesh Kumar b a b
Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka, Hyderabad 500 007, India Chemical Biology Laboratory, Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka, Hyderabad 500 007, India
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 4 March 2014 Accepted 11 March 2014 Available online 26 March 2014 Keywords: Ferrocene Xylofuranose Palladium(II) complexes Electrochemistry Cytotoxicity
a b s t r a c t This article describes the synthesis and cytotoxic activities of palladium(II) carbohydrate complexes of methyl, phenyl and ferrocenyl ester derivatives. The 3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose triazole (2c) was prepared by the Huisgen 1,3-dipolar cycloaddition reaction of 2-ethynyl pyridine and 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (1c). The reaction of this conjugate with [Pd(COD)Cl2] in anhydrous dichloromethane formed the corresponding palladium(II) complex (3c). The ligands and the complexes were characterised by 1H NMR, ESI mass, IR and elemental analysis. The UV–Vis and CV studies were also performed for the metal complexes. The electronic absorption spectra of complexes 3a–c shows the presence of both triazole and ferrocene absorption bands. Electrochemical studies of complexes 3a–c show the presence of a reduction peak at around 0.84 V thereby indicating the conversion of Pd(II) to Pd(0). The in vitro cytotoxic activity was studied against a panel of four different cancer cell lines. It was observed that these compounds exhibited significant cytotoxicity specifically on A549 cancer cell line. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The metallodrug cisplatin has opened up new avenues for exploring the potential biological applications of metal based drugs [1]. However, due to high general toxicity and the development of resistance of the cell lines towards platinum drug, further investigation of cisplatin and its analogues have been hampered [2]. In recent times, metal complexes of palladium, titanium, ruthenium, osmium etc., [3–7] have been studied to investigate their efficacy as anti-cancer agents. There exists a significant similarity between the coordination behaviour of palladium(II) and platinum(II) complexes [8–11]. However, the bioactive properties of the palladium(II) complexes are often less effective due to the tendency of these complexes to hydrolyse rapidly in aqueous solutions, thereby hampering a spectroscopic and electrochemical monitoring [12]. Hence, there is a need to design appropriate ligand system that can perhaps control the rate of hydrolysis of the palladium(II) complexes. For this purpose, several palladium complexes of different nitrogen ligands have been studied for biological applications [13–27]. Among various nitrogen ligand systems, pyridyl 1,2,3-triazoles are well known for their coordination with metals [28–31].
⇑ Corresponding author. Tel.: +91 40 27191667; fax: +91 40 27160921. E-mail addresses: [email protected], [email protected] (R. Trivedi). http://dx.doi.org/10.1016/j.ica.2014.03.018 0020-1693/Ó 2014 Elsevier B.V. All rights reserved.
On the other hand, ferrocene derivatives such as ferrocifen, hydroxyferrocifen, ferrocephanes and ferroquine are well known for their potential anticancer and antimalarial activities respectively [32–39]. In recent years, the synergic effect between ferrocene and other metals such as platinum, palladium, ruthenium and gold have shown moderate to significant cytotoxic activities (Fig. 1) [40–49]. Moreover, it has been observed that several of such bimetallic complexes having ferrocene unit linked via ester and amide moiety show promising cytotoxic activity. Carbohydrates containing metal complexes have been studied extensively for their biological applications [50–57]. In our recent work on palladium complexes of the carbohydrate derived pyridyl triazoles, it was observed that the presence of sugar moiety and the protecting group on the sugar scaffold have significant influence on the biological activity [58]. Consequently, the palladium(II) complexes of 3-OH and 3-O-benzyl-a-D-xylofuranose derivatives displayed reasonable bioactivity. We envisaged that converting the hydroxyl group into an ester group might induce some effect on the bioactivity of the complexes. Hence, in continuation to our recent endeavours on the biological activities of ferrocene– carbohydrate conjugates [59–62], we hereby report on the synthesis, characterisation, electrochemistry and cytotoxic activity of a series of palladium(II) carbohydrate complexes of methyl, phenyl and ferrocenoyl ester derivatives. A comparative electronic and spectroscopic analysis between the organic and organometallic fragments have been discussed.
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473 mg), a solution of ferrocenoyl chloride (approx. 2.5 mmol, 620 mg) in anhydrous dichloromethane (10 mL) was added followed by the drop wise addition of triethylamine (3 mmol, 0.4 mL). The reaction mixture was stirred for 6 h at room temperature, subsequently quenched with water and extracted with dichloromethane. The organic layer was washed with water and dried under vacuum. The compound (1c) was obtained as an orange gel after the column chromatography on neutral alumina using hexane/ethyl acetate (7:3) solvent mixture. Yield: 0.82 g (91%), [a]D25:+21.1 (c 0.1, CHCl3); IR (neat, mmax): 3071, 2981, 2106, 1753, 1712, 1219, 1071 cm 1; 1H NMR (300 MHz, CDCl3): d 1.34 (s, 3H, CH3), 1.56 (s, 3H, CH3), 3.47–3.54 (m, 1H, H-5), 3.59–3.66 (m, 1H, H-5), 4.22 (s, 5H, C5H5), 4.45–4.46 (m, 2H, C5H4), 4.47–4.53 (m, 1H, H-4), 4.59 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.79–4.83 (m, 2H, C5H4), 5.36 (d, 1H, 3JH,H = 3.0 Hz, H-3), 5.98 (d, 1H, 3JH,H = 3.7 Hz, H-1) ppm; 13C (75.5 MHz, CDCl3): d 26.17 (CH3), 26.69 (CH3), 49.15 (C-5), 69.81 (5CCp), 70.0 (2CCp), 70.28 (CCp), 71.82 (2CCp), 78.39 (C-2), 83.43 (C-3), 85.30 (C-4), 104.78 (C-1), 111.86 (CMe2), 170.65 (CO) ppm; ESI-MS (in CH3OH): m/z 428 [M+H]+; Anal. Calc. for C19H21FeN3O5 (427.23): C, 53.41; H, 4.95; N, 9.84. Found: C, 53.29; H, 4.82; N, 9.58%. 3.2. General procedure for the preparation of pyridyl triazole ligands (2a-c)
Fig. 1. Few examples of metal complexes of ferrocene conjugates.
2. Materials and methods Optical rotations were determined on a JASCO J-120 polarimeter. H spectra were recorded for CDCl3 solution on 300 MHz (Avance 300) and 500 MHz (Innova 500) instruments. Chemical shifts for protons were reported by taking tetramethylsilane (TMS) as the internal standard. 13C NMR spectra were recorded at 75.5 MHz on an Avance 300 instrument and the carbon shifts were referenced to the 13C signal of CDCl3 at 77.0 ppm. Coupling constants (J) were expressed in Hz. Infrared spectra were recorded on a Thermo Nicolet Nexus 670 spectrometer using KBr discs. Melting points were determined on a Toshniwal melting point apparatus and are uncorrected. The UV–Vis spectra were recorded on a Varian Cary 500 spectrophotometer over the range 200–550 nm using 1 cm path length cuvettes. The cyclic voltammetry (CV) was performed with a conventional three-electrode configuration consisting of glassy carbon working electrode, platinum wire auxiliary electrode and saturated calomel (SCE) reference electrode. The cyclic voltammograms were recorded on CHI620 model electrochemical analyser, in the presence of 0.1 M tetrabutylammonium perchlorate (TBAP) supporting electrolyte at a scan rate of 0.1 Vs 1. 5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose was prepared according to the literature procedures [59]. Azides 1a and 1b were synthesized according to the literature procedures [62]. The triazole 4 and its metal complex 5 were prepared according to the literature protocol [58] 1
3. Experimental 3.1. Synthesis of 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (1c) To a stirred anhydrous dichloromethane solution (10 mL) of 5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (2.2 mmol,
2-Ethynyl pyridine (1 equiv.) and azido sugars (1a–c) (1 equiv.) were dissolved in a 1:1 mixture of water and tert-butyl alcohol (8 mL). To this solution, sodium ascorbate (0.05 g, 0.2 mmol) and CuSO45H2O (0.02 g, 0.09 mmol) were added. The heterogeneous mixture was stirred at room temperature, till the disappearance of the starting materials. The reaction mixture was quenched with saturated NH4Cl solution and extracted with CH2Cl2 (2 5 mL) and the combined organic layers were dried over anhydrous Na2SO4. The removal of solvent under vacuum resulted in crude mass, which was purified by column chromatography using hexane/ethyl acetate (2:3) solvent mixture. 3.2.1. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(4-(2-pyridyl)-1H1,2,3-triazole-1-yl)-a-D-xylofuranose (2a) On treatment of 3-O-acetyl-5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (1a) (0.13 g, 1 mmol) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2a was obtained as pale yellow solid. Yield: 0.31 g (88%); mp: 108 °C; [a]D24: 36.6 (c 1.0, CHCl3); IR (neat, mmax): 3480, 3136, 2987, 1749, 1602, 1243, 1070 cm 1; 1H NMR (300 MHz, CDCl3): d 1.30 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.14 (s, 3H, CH3), 4.51–4.57 (m, 2H, H-5 and H-2), 4.64–4.71 (m, 1H, H-5), 4.79 (dd, 1H, 3JH,H = 3.8 Hz and 3JH,H = 9.8 Hz, H-4), 5.29 (d, 1H, 3JH,H = 3.8 Hz, H-3), 5.98 (d, 1H, 3JH,H = 3.7 Hz, H-1), 7.23 (dt, 1H, 3JH,H = 2.6 Hz and 3JH,H = 6.0 Hz, Py-5-H), 7.78 (t, 1H, 3JH,H = 6.0 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 6.0 Hz, Py-3-H), 8.28 (s, 1H, triazole), 8.59 (d, 1H, 3JH,H = 6.0 Hz, Py-6-H) ppm; 13C NMR (75.5 MHz, CDCl3): d 20.68 (CH3), 26.05 (CH3), 26.54 (CH3), 48.94 (C-5), 76.28 (C-2), 77.17 (C-3), 83.36 (C-4), 104.78 (C-1), 112.44 (CMe2), 120.21 (C-5 triazole), 122.80 (CPy), 122.99(CPy), 136.82 (CPy), 142.49 (C-4 triazole), 149.30 (CPy), 150.11 (CqPy), 169.54 (CO) ppm; ESI-MS (in CH3OH): m/z 361 [M+H]+; Anal. Calc. for C17H20N4O5 (360.36): C, 56.66; H, 5.59; N, 15.55. Found: C, 56.35; H, 5.04; N, 15.16%. 3.2.2. 3-O-Benzoyl-5-deoxy-1,2-O-isopropylidene-5-(4-(2-pyridyl)1H-1,2,3-triazole-1-yl)-a-D-xylofuranose (2b) On treatment of 5-azido-3-O-benzoyl-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (1b) (0.32 g, 1 mmol) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2b was obtained as pale yellow solid. Yield: 0.39 g (93%); mp: 152 °C; [a]D24: 13.6 (c 1.0, CHCl3); IR (neat, mmax): 3436, 3132, 2980, 1728, 1600, 1265, 1082 cm 1; 1H NMR
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(500 MHz, CDCl3): d 1.32 (s, 3H, CH3), 1.50 (s, 3H, CH3), 4.60–4.64 (m, 1H, H-5), 4.73 (d, 1H, 3JH,H = 2.9 Hz, H-2), 4.80–4.83 (m, 1H, H-5), 4.88 (dd, 1H, 3JH,H = 2.9 Hz and 3JH,H = 9.9 Hz, H-4), 5.58 (d, 1H, 3JH,H = 2.9 Hz, H-3), 6.06 (d, 1H, 3JH,H = 2.9 Hz, H-1), 7.22 (t, 1H, 3JH,H = 5.9 Hz, Py-5-H), 7.46–7.49 (t, 2H, 3JH,H = 7.9 Hz, Bz-3H), 7.61–7.64 (t, 1H, 3JH,H = 7.9 Hz, Bz-4-H), 7.75–7.78 (m, 1H, Py4-H), 8.05 (d, 2H, 3JH,H = 7.9 Hz, Bz-2-H), 8.15 (d, 1H, 3JH,H = 7.9 Hz, Py-3-H), 8.30 (s, 1H, CH of triazole), 8.57 (d, 1H, 3JH,H = 5.9 Hz, Py-6H) ppm; 13C NMR (75.5 MHz, CDCl3): d 26.12 (CH3), 26.50 (CH3), 49.30 (C-5), 76.80 (C-2), 77.69 (C-3), 83.46 (C-4), 104.95 (C-1), 112.59 (CMe2), 120.22 (C-5 triazole), 122.78 (Cpy), 122.96 (Cpy), 128.70 (CPy), 128.63 (2CBz), 129.76 (2CBz), 133.84 (CBz), 136.78 (CqBz), 148.60 (C-4 triazole), 149.33 (Cpy), 150.17 (Cqpy), 165.09 (CO) ppm; ESI-MS (in CH3OH): m/z 423 [M+H]+; Anal. Calc. for C22H22N4O5 (422.43): C, 62.55; H, 5.25; N, 13.26. Found: C, 61.99; H, 5.14; N, 13.01%.
3.2.3. 5-Deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-5-(4-(2pyridyl)-1H-1,2,3-triazole-1-yl)-a-D-xylofuranose (2c) On treatment of 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (0.43 g, 1 mmol) (1c) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2c was obtained as an orange solid. Yield: 0.47 g (88%); mp: 170 °C; [a]D24: 40.6 (c 0.1, CHCl3); IR (neat, mmax): 3420, 3122, 2990, 1718, 1136, 1039 cm 1; 1H NMR (500 MHz, CDCl3): d 1.33 (s, 3H, CH3), 1.49 (s, 3H, CH3), 4.24 (s, 5H, C5H5), 4.48 (d, 2H, 3JH,H = 1.1 Hz, C5H4), 4.57–4.62 (m, 1H, H-5), 4.67 (d, 1H, 3JH,H = 3.4 Hz, H-2), 4.74–4.79 (m, 1H, H-5), 4.80–4.84 (m, 3H, C5H4 and H-4), 5.46 (d, 3JH,H = 3.2 Hz, 1H, H-3), 6.03 (d, 1H, 3 JH,H = 3.4 Hz, H-1), 7.22 (m, 1H, Py-5-H), 7.77 (t, 1H, 3JH,H = 7.5 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 7.5 Hz, Py-3-H), 8.31 (s, 1H, CH of triazole), 8.58 (d, 1H, 3JH,H = 5.4 Hz, Py-6-H) ppm; 13C (75.5 MHz, CDCl3): d 26.19 (CH3), 26.68 (CH3), 49.30 (C-5), 69.90 (5CCp), 70.15 (2CCp), 70.30 (CCp), 71.92(2CCp), 76.12 (C-2), 77.70 (C-3), 83.53 (C-4), 104.93 (C-1), 112.64 (CMe2), 120.26 (C-5 triazole), 122.94 (CPy), 127.92 (CPy), 136.81 (CPy), 148.69 (C-4 triazole), 149.37 (CPy), 150.20 (CqPy), 170.58 (CO) ppm; ESI-MS (in CH3OH): m/z 531 [M+H]+; Anal. Calc. for C26H26FeN4O5 (482.1): C, 58.88; H, 4.94; N, 10.56. Found: C, 58.38; H, 4.85; N, 10.42%.
3.3. General procedure for the preparation of palladium complexes (3a–c) Under an argon atmosphere, ligands 2a–c (0.17 mmol) and [Pd(COD)Cl2] (0.053 g, 0.17 mmol), were dissolved in dichloromethane (5 mL). The mixture was allowed to stir at room temperature for 2 h and the resulting precipitate was collected by filtration. The solid was washed with diethyl ether and dried in vacuum to get the desired palladium complex.
3.3.1. Synthesis of [Pd(2a)Cl2], (3a) On treatment of acetyl-xylofuranose triazole (0.061 g, 0.17 mmol) (2a) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3a was obtained as pale yellow solid. Yield: 0.071 g (78%); mp: 258 °C (decomp.); IR (neat, mmax): 3461, 3117, 2967, 1750, 1600, 1223, 1036 cm 1; 1H NMR (300 MHz, CDCl3): d 1.29 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.22 (s, 3H, CH3), 4.61 (d, 1H, 3JH,H = 3.5 Hz, H-2), 4.73–4.76 (m, 1H, H-5), 4.83–4.91 (m, 1H, H-5), 5.02 (dd, 3 JH,H = 2.6 Hz and 3JH,H = 9.4, H-4), 5.41 (d, 1H, 3JH,H = 2.6 Hz, H-3), 6.02 (d, 1H, 3JH,H = 3.5 Hz, H-1), 7.21 (t, 1H, 3JH,H = 5.6 Hz, Py-5-H), 8.03–8.06 (m, 2H, Py-4-H and Py-3-H), 8.67 (d, 1H, 3JH,H = 5.6 Hz, Py-6-H), 8.77 (s, 1H, CH of triazole) ppm; ESI-MS (in CH3OH + CH2Cl2): m/z 543 [(M Cl)+H2O+Na]+; Anal. Calc. for C17H20Cl2N4O5Pd (537.69): C, 37.97; H, 3.75; N, 10.42. Found: C, 37.62; H, 3.58; N, 10.27%.
3.3.2. Synthesis of [Pd(2b)Cl2], (3b) On treatment of benzoyl-xylofuranose triazole (0.072 g, 0.17 mmol) (2b) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3b was obtained as pale yellow solid. Yield: 0.71 g (88%); mp: 188 °C; IR (neat, mmax): 3508, 3097, 2986, 1725, 1622, 1265,1099 cm 1; 1H NMR (300 MHz, CDCl3): d 1.31 (s, 3H, CH3), 1.47 (s, 3H, CH3), 4.75 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.85–4.89 (m, 1H, H-5), 5.04–5.09 (m, 2H, H-4 and H-5), 5.63 (d, 1H, 3JH,H = 3.0 Hz, H-3), 6.08 (d, 1H, 3 JH,H = 3.7 Hz, H-1), 7.18 (dt, 1H, 3JH,H = 1.5 Hz and 7.5 Hz, Py-5H), 7.50 (t, 2H, 3JH,H = 7.4 Hz, Bz-3-H), 7.62 (dt, 1H, 3JH,H = 1.5 and 7.5 Hz, Py-4-H), 8.09 (m, 1H, Py-3-H), 8.12 (m, 3H, Bz-2-H and Bz-4-H), 8.65 (d, 1H, 3JH,H = 7.5 Hz, Py-6-H), 8.87 (s, 1H, CH of triazole) ppm; ESI-MS (in CH3OH + CH2Cl2): m/z 605 [(M Cl)+H2O+Na]+; Anal. Calc. for C22H22Cl2N4O5Pd (599.75): C, 44.06; H, 3.70; N, 9.34. Found: C, 43.91; H, 3.62; N, 9.19%. 3.3.3. Synthesis of [Pd(2c)Cl2], (3c) On treatment of ferrocenoyl-xylofuranose triazole (0.090 g, 0.17 mmol) (2c) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3c was obtained as an orange solid. Yield: 0.091 g (76%); mp: 180 °C; IR (neat, mmax): 3490, 3099, 2985, 1717, 1622, 1270, 1127 cm 1; 1H NMR (300 MHz, CDCl3): d 1.30 (s, 3H, CH3), 1.45 (s, 3H, CH3), 4.32 (s, 5H, C5H5), 4.47 (m, 2H, C5H4), 4.66 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.78–4.90 (m, 2H, C5H4), 4.92–4.94 (m, 1H, H-5), 5.05–5.17 (m, 2H, H-4 and H-5), 5.65 (d, 1H, 3JH,H = 3.0 Hz, H-3), 6.07 (d, 1H, 3 JH,H = 3.7 Hz, H-1), 7.16 (t, 1H, 3JH,H = 7.5 Hz, Py-5-H), 8.05 (t, 1H, 3 JH,H = 7.5 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 7.5 Hz, Py-3-H), 8.59 (d, 1H, 3JH,H = 7.5 Hz, Py-6-H), 8.94 (s, 1H, CH of triazole) ppm; ESIMS (in CH3OH+CHCl3): m/z = 711 [(M Cl)+H2O+Na]+; Anal. Calc. for C26H26Cl2FeN4O5Pd (707.67): C, 44.13; H, 3.70; N, 7.92. Found: C, 43.85; H, 3.53.14; N, 7.62%. 3.4. In vitro cytotoxicity testing The cytotoxicity of all the compounds was assessed according to the literature procedures [63]. Cell lines used for testing in vitro cytotoxicity included HeLa derived from human cervical cancer cells (ATCC No. CCL-2), A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast adenocarcinoma cells (ATCC No HTB-22) were obtained from the American Type Culture Collection, Manassas, VA, USA. All the tumour cell lines were maintained in a modified DMEM medium supplemented with 10% fetal bovine serum, along with 1% non-essential amino acids without L-glutamine, 0.2% sodium hydrogen carbonate, 1% sodium pyruvate and 1% antibiotic mixture (10 000 units penicillin and 10 mg streptomycin per mL). The cells were washed and resuspended in the above medium and 100 lL of this suspension was seeded in 96 well-bottom plates. The cells were maintained at 37 °C in a humidified 5% CO2 incubator (Model 2406 Shellab CO2 incubator, Sheldon, Cornelius, OR). After incubation for 24 h, the cells were treated for 2 days with test compounds at concentrations ranging from 0.1 to 100 lM in DMSO (1% final concentration) and were assayed at the end of the second day. Each assay was performed with two internal controls: (1) an IC0 with cells only, (2) an IC100 with media only. After incubation for 48 h, the cells were subjected to the MTT colorimetric assay (5 mg mL 1). The effect of the different test compounds on the viability of the tumour cell lines was measured at 540 nm on a multimode reader (InfiniteÒ M200, Tecan, Switzerland). The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose– response curves. The assay was performed using doxorubicin and cisplatin as standards and 1% DMSO as a vehicle control. To avoid DMSO toxicity, the values obtained for the DMSO control were
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subtracted from those of the test compounds. IC50 values (in lM) are expressed as the average of two independent experiments. 4. Results and discussion 4.1. Synthesis and characterisation The carbohydrate azide ligands (1a–c) were synthesised by the esterification reaction of 5-azido-5-deoxy-1,2-O-isopropylidene-aD-xylofuranose with the corresponding acid chlorides. The azides were then treated with 2-ethynyl pyridine to synthesize the pyridyl triazole ligands using the Huisgen 1,3-dipolar cycloaddition reaction conditions at room temperature. The triazole ligands (2a–c) were reacted with [Pd(COD)Cl2] in dry dichloromethane to afford the corresponding complexes (3a–c) (Scheme 1). The ligands were characterised by 1H, 13C and IR spectroscopy. In the 1H NMR spectra, it was observed that the gem-dimethyl groups of the xylofuranose moiety appeared as two singlets around 1.3 and 1.4 ppm for all the ligands and the triazole proton appeared as a sharp singlet at 8.3 ppm. For the ligands 2a and 2b, the anomeric proton of the carbohydrate moiety appeared as doublet at 5.5–5.9 ppm. For the compound 2a, a sharp singlet for the –CH3 protons was observed at 3.0 ppm. In the case of ferrocenoyl-pyridyl triazole 2c, the protons of the unsubstituted Cp ring were observed at 4.2 ppm and those of the substituted Cp rings were observed at 4.5 and 4.8 ppm. The anomeric proton of the carbohydrate moiety appeared as doublet at around 5.9 ppm. Finally, the complexes were prepared by the reaction of the ligands with [Pd(COD)Cl2] in dry dichloromethane for 2 h at room temperature. The resulting complexes were soluble in dichloromethane, DMF and DMSO but insoluble in methanol. The complexes were characterised by 1H NMR spectroscopy, in which a down field shift of the triazole –CH proton indicated the formation of the complex (see Supporting information). In the 1H NMR spectra of the complexes, the anomeric proton of the xylofuranose appeared as a doublet at 6.0 ppm. The triazole proton was observed as a sharp singlet at 8.7 ppm. All the other signals for the sugar moiety appeared as reported [62]. Repeated attempts for crystallization in appropriate solvents failed to
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produce X-ray quality crystals. Therefore, the crystal structure of these complexes could not be obtained. The absorption spectrum of the ferrocenoyl triazole (2c) was determined by the UV–Vis studies in 0.5 mM dichloromethane solution. The compound exhibited characteristic peaks for the Fe(a1g) ? Fe(e1g) transitions and Fe(e1g) ? Cp(e2g) transitions at around 440 nm and 314 nm respectively [64]. In addition to these transitions, a broad absorption band near 350 nm was observed due to the n ? p⁄ transitions of the carbonyl group (see Supporting information). The formation of palladium complexes was also confirmed by the UV–Visible spectroscopy. The absorption spectroscopy measurements of the complexes were carried out in 0.1 mM dichloromethane solutions over a wavelength region of 250–600 nm (Fig. 2). The absorption spectra of all the complexes consists of absorption bands corresponding to the characteristic p ? p⁄ transitions of the triazole (at around 280 nm) and the metal to ligand charge transfer (MLCT) (at around 294 nm). This was in accordance with the coordination behaviour of the pyridyl triazole ligands reported in the literature [28]. In addition to these transitions, the n ? p⁄ transition of the carbonyl group were also observed for all the metal complexes. In the case of ferrocenoyl complex (3c), the ligand Fe ? Cp transitions overlapped with the MLCT band of the complex. A weak band corresponding to the Fe(a1g) ? Fe(e1g) transition of the ferrocene complexes was observed at 440 nm. 4.2. Electrochemistry The electrochemical characterisation of the triazole ligands and the metal complexes was performed in anhydrous dichloromethane by means of differential pulse voltammetry (dpv). To compare the electrochemical behaviour of the complexes 3a–c, the differential pulse voltammetry experiments were conducted for complex 5. Due to sparingly soluble nature of xylofuranose-palladium complex (5) in dichloromethane, the experiment was performed in DMSO solution. No oxidation peak was observed for the complex (5), but a reduction peak (see Supporting information) for Pd(II)/Pd(0) [28] was observed at around 1.79 V.
Scheme 1. Synthesis of pyridyl triazole ligands and their palladium(II) complexes.
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Absorbance (a.u.)
1.5
3a 3b 3c
1.0
0.5
0.0 250
300
350
400
450
500
550
Wavelength (nm) Fig. 2. UV–Vis spectra of palladium complexes in 0.1 mM dichloromethane solution.
Fig. 4. Differential pulse voltammograms (dpv) for the reduction of palladium complexes in dichloromethane solution at 25 °C.
The ligands 2a and 2b and the corresponding metal complexes 3a and 3b respectively did not show any oxidation process. But, surprisingly, in the case of ferrocenoyl pyridyl triazole (2c), in addition to the Fe(II)/Fe(III) oxidation process (+0.70 V), an oxidation peak due to the triazole functionality was also observed at around +1.16 V (Fig. 3a). This additional peak may be due to the oxidation of the triazole functionality [65]. For the compound 2c, the shift in the oxidation potential of Fe(II)/Fe(III) couple is due to the presence of electron withdrawing ester group on the ferrocene of 2c [66]. In the ferrocenoyl palladium complex (3c), the Fe(II)/Fe(III) oxidation process was observed at +0.682 V and oxidation of the triazole group was observed at +0.972 V (Fig. 3b). All the complexes exhibited irreversible reduction wave, indicating the conversion of Pd(II) to Pd(0) (Fig. 4) [28]. The acetyl complex 3a, exhibited a reduction potential of 0.84 V, while the benzoyl complex (3b) showed irreversible reduction process at around 0.86 V. The ferrocenoyl complex exhibited reduction potential corresponding to Pd(II)/Pd(0) at 0.76 V [28].
For instance, it has been reported by Hanessian and Wang that in the case of L-xylose derived platinum(II) complexes, the presence of free –OH group displayed improved cytotoxicity compared to the methyl ether protected analogue [57]. However, it was found that in the case of D-xylose derived palladium(II) complexes, among 3-OH- and 3-O-benzyl-a-D-xylofuranose derivatives, the 3-O-benzyl protected compounds were found to display better bioactivity [58]. The enhanced cytotoxicity of the 3-O-benzyl protected xylofuranose compounds may be attributed to the lipophilicity of the sugar scaffold [58]. Therefore, taking clue from the observations, it was assumed that an ester protection of the xylofuranose at the 3-O-position could exhibit significant effect on the cytotoxicity of the pyridyl carbohydrate conjugates and their metal complexes. Furthermore, it has also been reported in the literature that, ferrocene linkage to metal complexes via ester moiety displayed significant bioactivity [45]. With these insights, we have prepared three different carbohydrate pyridyl triazole ligands containing ester protection at 3-O-position of the xylofuranose ring and their cytotoxicity results are summarised in Table 1. Within the domain of carbohydrate research, the acetyl as well as the benzoyl moieties are extensively used as protecting groups for the hydroxyl group, hence this report describes their comparison with the ferrocenoyl moiety. From our previous work [58], it was evident that the xylofuranose triazole ligand (4) did not show cytotoxic activity (IC50 value >100 lM) on the tested cell lines, but its metal complex (5) exhibited promising cytotoxicity (IC50 value of <10 lM). To our surprise,
4.3. In vitro cytotoxicity (MTT assay) The compounds were tested for cytotoxicity against different test cell lines up to a concentration of 100 lM. In the metal carbohydrate complexes, apart from the central metal ion, the nature of the sugar scaffold and the protecting groups on the sugar play a key role in determining the biological activities of the complexes.
Fig. 3. Differential pulse voltammograms (dpv) for the oxidation of (a) ligand 2c and (b) complex 3c in dichloromethane solution at 25 °C.
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the funding. S. B. D. and P. S. are grateful to the CSIR, New Delhi, India for the award of research fellowships.
Table 1 Cytotoxicity of pyridyl triazole ligands and complexes. Compound
4b 2a 2b 2c 5b 3a 3b 3c Doxorubicinc Cisplatinc a b c
169
IC50 (lM)a A549
HeLa
MDA-MB-231
MCF-7
>100 36.73 (±0.10) 77.97 ± 0.18) 5.73 (±0.03) 6.4 (±0.03) 19.29 (±0.11) 66.19 (±0.19) 13.18 (±0.07) 0.46 (±0.03) 0.15 (±0.13)
>100 >100 >100 >100 8.3 (±0.01) >100 >100 >100 0.51 (±0.02) 0.23 (±0.10)
>100 >100 >100 >100 9.9 (±0.13) >100 >100 >100 0.91 (±0.06) 0.25 (±0.09)
>100 >100 >100 >100 5.5 (±0.09) >100 >100 >100 1.07 (±0.1) 0.48 (±0.16)
Values are mean of two independent experiments. Ref. [58]. Positive control.
it was observed that all the ligands and metal complexes in the present study exhibited variable cytotoxicity on one specific cell line, namely, the human alveolar adenocarcinoma A549 cell line, while for the remaining cell lines IC50 of more than 100 lM was observed. The acetyl and benzoyl substituted ligands were found to exhibit low cytotoxicity with IC50 values 36.73 lM and 77.97 lM respectively. In the case of ferrocene conjugated pyridyl triazole ligand (2c), the inclusion of an organometallic fragment drastically improved the cytotoxicity (IC50 value of 5.73 lM). This difference in the cytotoxicity of the ligands indicates that ferrocene certainly plays a significant role in influencing the cytotoxicity of the carbohydrate-pyridyl triazole ligands. Moreover, incorporation of Pd(II) on the pyridyl triazole moiety improved the cytotoxicity of acetyl and benzoyl containing carbohydrate metal complexes 3a and 3b (exhibited IC50 values 19.29 and 66.19 lM respectively). However, a low cytotoxicity was observed for ferrocene–palladium complex (IC50 value 13.18 lM) as compared to the free ligand (2c). This decrease in the cytotoxicity of the ferrocene–palladium complex perhaps may be attributed to the steric bulk of the metal complex [47]. These results do indicate the effect of an organometallic unit on the overall biological activity of the ligand, however, much more exhaustive study, by varying the metal as well as the protecting group, is currently under progress. 5. Conclusions The first example for the palladium(II) complex of ferrocene– carbohydrate conjugate has been prepared. The complexes were characterised by UV–Vis spectroscopy, in which the characteristic transitions for the cis-palladium complexes were observed. The electrochemical studies have shown oxidation of the triazole moiety in case of ferrocene–carbohydrate pyridyl triazole ligand (2c), whereas such triazole oxidation was not observed in the case of acetyl and benzoyl derivatives. The complexes exhibited reduction wave corresponding to the Pd(II)/Pd(0) conversion. Interestingly, all the ligands and metal complexes have shown cytotoxicity selectively on A549 cancer cell line. Among all the ligands and metal complexes, only ferrocene containing ligand and metal complex exhibited IC50 values less than 20 lM. The development of carbohydrate metal complexes and their organometallic conjugates are currently under progress in our laboratory. Acknowledgments This work was financially supported by the in-house project of CSIR-IICT (INDIA) MLP-0007. Dr. L.G. thanks the Department of Science and Technology (DST, SR/S1/IC21/2008), New Delhi, for
Appendix A. Supplementary material Supplementary (The Supplementary Information contains the H and 13C NMR spectra of ligands and the 1H NMR spectra of complexes.) data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ica.2014.03.018.
1
References [1] M.A. Fuertes, C. Alonso, J.M. Perez, Chem. Rev. 103 (2003) 645. [2] G. Giaccone, Drugs 59 (2000) 9. [3] G. Jaouen, N. Metzler-Nolte, Medicinal Organometallic Chemistry, Topics in Organometallic Chemistry, 32 ed., Springer, Berlin, 2010. [4] E. Alesio, Bioinorganic Medicinal Chemistry, Wiley–VCH, Weinheim, 2011. [5] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391. [6] A.A. Holder, Annu. Rep. Prog. Chem. Sect. A 107 (2011) 359. [7] C.G. Hartinger, N. Metzler-Nolte, P.J. Dyson, Organometallics 31 (2012) 5677. [8] M.A. Ali, A.H. Mirza, R.J. Butcher, M.T.H. Tarafder, T.B. Keat, T.A.M. Ali, J. Inorg. Biochem. 92 (2001) 141. [9] N. Miklášová, E. Fischer-Fodor, P. Lönnecke, C.I. Tomuleasa, P. Virag, M.P. Schrepler, R. Mikláš, L.S. Dumitrescu, E. Hey-Hawkins, Eur. J. Med. Chem. 49 (2012) 41. [10] L.M.M. Vieira, M.V. de Almeida, M.C.S. Lourenço, F.A.F.M. Bezerra, A.P.S. Fontes, Eur. J. Med. Chem. 36 (2009) 4107. [11] T.V. Serebryanskaya, T. Yung, A.A. Bogdanov, A. Shchebet, S.A. Johnsen, A.S. Lyakhov, L.S. Ivashkevich, Z.A. Ibrahimava, T.S. Garbuzenco, T.S. Kolesnikova, N.I. Melnova, P.N. Gaponik, O.A. Ivashkevich, J. Inorg. Biochem. 120 (2013) 44. [12] S. Abu-Surrah, H.H. Al-Sa’doni, M.Y. Abdalla, Cancer Ther. 6 (2008) 1. [13] T. Rau, R. Alsfasser, A. Zahl, R. van Eldik, Inorg. Chem. 37 (1998) 4223. [14] G. Zhao, H. Lin, P. Yu, H. Sun, S. Zhu, X. Su, Y. Chen, J. Inorg. Biochem. 73 (1999) 145. [15] A.C.F. Caires, Anti Cancer Agents Med. Chem. 7 (2007) 484. [16] P. Chellan, N. Shunmoogam-Gouden, D.T. Hendricks, J. Gut, P.J. Rosenthal, C. Lategan, P.J. Smith, K. Chibale, G.S. Smith, Eur. J. Inorg. Chem. (2010) 3520. [17] S. Padhye, Z. Afrasiabi, E. Sinn, J. Fok, K. Mehta, N. Rath, Inorg. Chem. 44 (2005) 1154. [18] E. Budzisz, M. Miernicka, I.-P. Lorenz, P. Mayer, U. Krajewska, M. Rozalski, Polyhedron 28 (2009) 637. [19] W.M. Motswainyana, M.O. Onani, A.M. Madiehe, M. Saibu, J. Jacobs, L. van Meervelt, Inorg. Chim. Acta 400 (2013) 197. [20] C. Urquiola, M. Vieites, M.H. Torre, M. Cabrera, M.L. Lavaggi, H. Cerecetto, M. González, A. López de Cerain, A. Monge, P. Smircich, B. Garat, D. Gambino, Bioorg. Med. Chem. 17 (2009) 1623. [21] P. Štarha, I. Popa, Z. Trávnícˇek, Inorg. Chim. Acta 363 (2010) 1469. [22] M.E.H. Mazumder, P. Beale, C. Chan, J.Q. Yu, F. Huq, ChemMedChem 7 (2012) 1840. [23] Y. Wang, S. Liu, J. Zhang, S. Li, H. Wei, G. Zhou, X. Qin, Appl. Organomet. Chem. 27 (2013) 373. [24] F. Zamora, V.M. González, J.M. Pérez, J.R. Masaguer, C. Alonso, C. NavarroRanninger, Appl. Organomet. Chem. 11 (1997) 659. [25] E. Budzisz, M. Malecka, B.K. Keppler, V.B. Arion, G. Andrijewski, U. Krajewska, M. Rozalski, Eur. J. Inorg. Chem. (2007) 3728. [26] D. Tsekova, P. Gorolomova, G. Gochev, V. Skumryev, G. Momekov, D. Momekova, G. Gencheva, J. Inorg. Biol. Chem. 124 (2013) 54. [27] S. Rubino, P. Portanova, F. Giammalva, M.A. Girasolo, S. Orecchio, G. Calvaruso, G.C. Stocco, Inorg. Chim. Acta 370 (2011) 207. [28] D. Schweinfurth, R. Pattacini, S. Strobel, B. Sarkar, Dalton Trans. (2009) 9291. [29] K.J. Kilpin, E.J. Gavey, C.J. McAdam, C. Anderson, S.J. Lind, C.C. Keep, K.C. Gordon, J.D. Crowley, Inorg. Chem. 50 (2010) 6334. [30] A. Maisonial, P. Serafin, M. Traïkia, E. Debiton, V. Théry, D.J. Aitken, P. Lemoine, B. Viossat, A. Gautier, Eur. J. Inorg. Chem. (2008) 298. [31] T.L. Mindt, H. Struthers, L. Brans, T. Angelov, C. Schweinsberg, V. Maes, D. Tourwe, R. Schibli, J. Am. Chem. Soc. 128 (2006) 15096. [32] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3. [33] R.H. Fish, G. Jaouen, Organometallics 22 (2003) 2166. [34] C. Ornelas, New J. Chem. 35 (2011) 1973. [35] A. Nguyen, A. Vessières, E.A. Hillard, S. Top, P. Pigeon, G. Jaouen, Chimia 61 (2007) 716. [36] O. Buriez, E.A. Hillard, A. Vessières, D. Hamels, S. Top, G. Jaouen, Y.M. Frapart, D. Mansuy, C. Amatore, Chem. Eur. J. 18 (2012) 6581. [37] D. Hamels, P.M. Dansette, E.A. Hillard, S. Top, A. Vessières, P. Herson, G. Jaouen, D. Mansuy, Angew. Chem., Int. Ed. 48 (2009) 9124. [38] E.A. Hillard, G. Jaouen, Organometallics 30 (2011) 20. [39] C. Biot, G. Glorian, L.A. Maciejewski, J.S. Brocard, J. Med. Chem. 40 (1997) 3715. [40] W. Henderson, S.R. Alley, Inorg. Chim. Acta 322 (2001) 106. [41] A. Rosenfeld, J. Blum, D. Gibson, A. Ramu, Inorg. Chim. Acta 201 (1992) 219. [42] M. Viotte, B. Gautheron, M.M. Kubicki, I.E. Nifant’ev, S.P. Fricker, Metal Based Drugs 2 (1995) 311.
170
S.B. Deepthi et al. / Inorganica Chimica Acta 416 (2014) 164–170
[43] M.C. Gimeno, H. Goitia, A. Laguna, M.E. Luque, M.D. Villacampa, C. Sepúlveda, M.J. Meireles, Inorg. Biochem. 105 (2011) 1373. [44] J. Schulz, A.K. Renfrew, I. Císrová, P.J. Dyson, P. Stpnicˇka, Appl. Organomet. Chem. 24 (2010) 392. [45] M. Auzias, B. Therrien, G. Süss-Fink, P. Štpnicˇka, W.H. Ang, P.J. Dyson, Inorg. Chem. 47 (2008) 578. [46] J. Rajput, J.R. Moss, A.T. Hutton, D.T. Hendricks, C.E. Arendse, C. Imrie, J. Organomet. Chem. 689 (2004) 1553. [47] T.V. Segapelo, S. Lillywhite, E. Nordlander, M. Haukka, J. Darkwa, Polyhedron 36 (2012) 97. [48] W.M. Motswainyana, M.O. Onani, A.M. Madiehe, Polyhedron 41 (2012) 44. [49] D. Talancón, C. López, M. Font-Bardía, T. Calvet, J. Quirante, C. Calvis, R. Messeguer, R. Cortés, M. Cascante, L. Baldomà, J. Badia, J. Inorg. Biochem. 118 (2013) 1. [50] Y. Chen, A. Janczuk, X. Chen, J. Wang, M. Ksebati, P.G. Wang, Carbohydr. Res. 337 (2002) 1043. [51] C.G. Hartinger, A.A. Nazarov, S.M. Ashraf, P.J. Dyson, B.K. Keppler, Curr. Med. Chem. 15 (2008) 2574. [52] I. Brudziñska, Y. Mikata, M. Obata, C. Ohtsuki, S. Yano, Bioorg. Med. Chem. Lett. 14 (2004) 2533. [53] T. Allscher, P. Klüfers, O. Labisch, Carbohydr. Res. 342 (2007) 1419. [54] J. Möker, J. Thiem, Eur. J. Org. Chem. (2009) 4842.
[55] M.E. Cucciolito, R.D. Litto, F.P. Fanizzi, D. Migoni, G. Roviello, F. Ruffo, Inorg. Chim. Acta 363 (2009) 741. [56] Y. Mikata, S. Yano, Curr. Top. Med. Chem. 12 (2012) 145. [57] S. Hanessian, J. Wang, Can. J. Chem. 71 (1993) 886. [58] S.B. Deepthi, R. Trivedi, P. Sujitha, C.G. Kumar, B. Sridhar, S.K. Bhargava, J. Chem. Sci. 124 (2012) 1405. [59] R. Trivedi, S.B. Deepthi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, K.V.S. Ramakrishna, Eur. J. Inorg. Chem. (2012) 2267. [60] R. Trivedi, S.B. Deepthi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, K.V.S. Ramakrishna, Appl. Organomet. Chem. 26 (2012) 369. [61] S.B. Deepthi, R. Trivedi, L. Giribabu, P. Sujitha, C.G. Kumar, Dalton Trans. 42 (2013) 1180. [62] S.B. Deepthi, R. Trivedi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, New J. Chem. 38 (2014) 227. [63] M. Navarra, M. Celano, J. Maiuolo, S. Schenone, M. Botta, A. Angelucci, P. Bramanti, D. Russo, BMC Cancer 10 (2010) 602. [64] Y.L.K. Tan, P. Pigeon, E.A. Hillard, S. Top, M. Plamont, A. Vessières, M.J. McGlinchey, H. Müller-Bunz, G. Jaouen, Dalton Trans. (2009) 10871. [65] B. Happ, D. Escudero, M.D. Hager, C. Friebe, A. Winter, H. Gorls, E. Altuntas, L. González, U.S. Schubert, J. Org. Chem. 75 (2010) 4025. [66] G. Gritzner, J. Kuta, Pure Appl. Chem. 56 (1984) 461.
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Palladium(II) carbohydrate complexes of alkyl, aryl and ferrocenyl esters and their cytotoxic activities Sadanala Bhavya Deepthi a, Rajiv Trivedi a,⇑, Lingamallu Giribabu a, Pombala Sujitha b, C. Ganesh Kumar b a b
Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka, Hyderabad 500 007, India Chemical Biology Laboratory, Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Technology (IICT), Uppal Road, Tarnaka, Hyderabad 500 007, India
a r t i c l e
i n f o
Article history: Received 9 January 2014 Received in revised form 4 March 2014 Accepted 11 March 2014 Available online 26 March 2014 Keywords: Ferrocene Xylofuranose Palladium(II) complexes Electrochemistry Cytotoxicity
a b s t r a c t This article describes the synthesis and cytotoxic activities of palladium(II) carbohydrate complexes of methyl, phenyl and ferrocenyl ester derivatives. The 3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose triazole (2c) was prepared by the Huisgen 1,3-dipolar cycloaddition reaction of 2-ethynyl pyridine and 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (1c). The reaction of this conjugate with [Pd(COD)Cl2] in anhydrous dichloromethane formed the corresponding palladium(II) complex (3c). The ligands and the complexes were characterised by 1H NMR, ESI mass, IR and elemental analysis. The UV–Vis and CV studies were also performed for the metal complexes. The electronic absorption spectra of complexes 3a–c shows the presence of both triazole and ferrocene absorption bands. Electrochemical studies of complexes 3a–c show the presence of a reduction peak at around 0.84 V thereby indicating the conversion of Pd(II) to Pd(0). The in vitro cytotoxic activity was studied against a panel of four different cancer cell lines. It was observed that these compounds exhibited significant cytotoxicity specifically on A549 cancer cell line. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction The metallodrug cisplatin has opened up new avenues for exploring the potential biological applications of metal based drugs [1]. However, due to high general toxicity and the development of resistance of the cell lines towards platinum drug, further investigation of cisplatin and its analogues have been hampered [2]. In recent times, metal complexes of palladium, titanium, ruthenium, osmium etc., [3–7] have been studied to investigate their efficacy as anti-cancer agents. There exists a significant similarity between the coordination behaviour of palladium(II) and platinum(II) complexes [8–11]. However, the bioactive properties of the palladium(II) complexes are often less effective due to the tendency of these complexes to hydrolyse rapidly in aqueous solutions, thereby hampering a spectroscopic and electrochemical monitoring [12]. Hence, there is a need to design appropriate ligand system that can perhaps control the rate of hydrolysis of the palladium(II) complexes. For this purpose, several palladium complexes of different nitrogen ligands have been studied for biological applications [13–27]. Among various nitrogen ligand systems, pyridyl 1,2,3-triazoles are well known for their coordination with metals [28–31].
⇑ Corresponding author. Tel.: +91 40 27191667; fax: +91 40 27160921. E-mail addresses: [email protected], [email protected] (R. Trivedi). http://dx.doi.org/10.1016/j.ica.2014.03.018 0020-1693/Ó 2014 Elsevier B.V. All rights reserved.
On the other hand, ferrocene derivatives such as ferrocifen, hydroxyferrocifen, ferrocephanes and ferroquine are well known for their potential anticancer and antimalarial activities respectively [32–39]. In recent years, the synergic effect between ferrocene and other metals such as platinum, palladium, ruthenium and gold have shown moderate to significant cytotoxic activities (Fig. 1) [40–49]. Moreover, it has been observed that several of such bimetallic complexes having ferrocene unit linked via ester and amide moiety show promising cytotoxic activity. Carbohydrates containing metal complexes have been studied extensively for their biological applications [50–57]. In our recent work on palladium complexes of the carbohydrate derived pyridyl triazoles, it was observed that the presence of sugar moiety and the protecting group on the sugar scaffold have significant influence on the biological activity [58]. Consequently, the palladium(II) complexes of 3-OH and 3-O-benzyl-a-D-xylofuranose derivatives displayed reasonable bioactivity. We envisaged that converting the hydroxyl group into an ester group might induce some effect on the bioactivity of the complexes. Hence, in continuation to our recent endeavours on the biological activities of ferrocene– carbohydrate conjugates [59–62], we hereby report on the synthesis, characterisation, electrochemistry and cytotoxic activity of a series of palladium(II) carbohydrate complexes of methyl, phenyl and ferrocenoyl ester derivatives. A comparative electronic and spectroscopic analysis between the organic and organometallic fragments have been discussed.
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165
473 mg), a solution of ferrocenoyl chloride (approx. 2.5 mmol, 620 mg) in anhydrous dichloromethane (10 mL) was added followed by the drop wise addition of triethylamine (3 mmol, 0.4 mL). The reaction mixture was stirred for 6 h at room temperature, subsequently quenched with water and extracted with dichloromethane. The organic layer was washed with water and dried under vacuum. The compound (1c) was obtained as an orange gel after the column chromatography on neutral alumina using hexane/ethyl acetate (7:3) solvent mixture. Yield: 0.82 g (91%), [a]D25:+21.1 (c 0.1, CHCl3); IR (neat, mmax): 3071, 2981, 2106, 1753, 1712, 1219, 1071 cm 1; 1H NMR (300 MHz, CDCl3): d 1.34 (s, 3H, CH3), 1.56 (s, 3H, CH3), 3.47–3.54 (m, 1H, H-5), 3.59–3.66 (m, 1H, H-5), 4.22 (s, 5H, C5H5), 4.45–4.46 (m, 2H, C5H4), 4.47–4.53 (m, 1H, H-4), 4.59 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.79–4.83 (m, 2H, C5H4), 5.36 (d, 1H, 3JH,H = 3.0 Hz, H-3), 5.98 (d, 1H, 3JH,H = 3.7 Hz, H-1) ppm; 13C (75.5 MHz, CDCl3): d 26.17 (CH3), 26.69 (CH3), 49.15 (C-5), 69.81 (5CCp), 70.0 (2CCp), 70.28 (CCp), 71.82 (2CCp), 78.39 (C-2), 83.43 (C-3), 85.30 (C-4), 104.78 (C-1), 111.86 (CMe2), 170.65 (CO) ppm; ESI-MS (in CH3OH): m/z 428 [M+H]+; Anal. Calc. for C19H21FeN3O5 (427.23): C, 53.41; H, 4.95; N, 9.84. Found: C, 53.29; H, 4.82; N, 9.58%. 3.2. General procedure for the preparation of pyridyl triazole ligands (2a-c)
Fig. 1. Few examples of metal complexes of ferrocene conjugates.
2. Materials and methods Optical rotations were determined on a JASCO J-120 polarimeter. H spectra were recorded for CDCl3 solution on 300 MHz (Avance 300) and 500 MHz (Innova 500) instruments. Chemical shifts for protons were reported by taking tetramethylsilane (TMS) as the internal standard. 13C NMR spectra were recorded at 75.5 MHz on an Avance 300 instrument and the carbon shifts were referenced to the 13C signal of CDCl3 at 77.0 ppm. Coupling constants (J) were expressed in Hz. Infrared spectra were recorded on a Thermo Nicolet Nexus 670 spectrometer using KBr discs. Melting points were determined on a Toshniwal melting point apparatus and are uncorrected. The UV–Vis spectra were recorded on a Varian Cary 500 spectrophotometer over the range 200–550 nm using 1 cm path length cuvettes. The cyclic voltammetry (CV) was performed with a conventional three-electrode configuration consisting of glassy carbon working electrode, platinum wire auxiliary electrode and saturated calomel (SCE) reference electrode. The cyclic voltammograms were recorded on CHI620 model electrochemical analyser, in the presence of 0.1 M tetrabutylammonium perchlorate (TBAP) supporting electrolyte at a scan rate of 0.1 Vs 1. 5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose was prepared according to the literature procedures [59]. Azides 1a and 1b were synthesized according to the literature procedures [62]. The triazole 4 and its metal complex 5 were prepared according to the literature protocol [58] 1
3. Experimental 3.1. Synthesis of 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (1c) To a stirred anhydrous dichloromethane solution (10 mL) of 5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (2.2 mmol,
2-Ethynyl pyridine (1 equiv.) and azido sugars (1a–c) (1 equiv.) were dissolved in a 1:1 mixture of water and tert-butyl alcohol (8 mL). To this solution, sodium ascorbate (0.05 g, 0.2 mmol) and CuSO45H2O (0.02 g, 0.09 mmol) were added. The heterogeneous mixture was stirred at room temperature, till the disappearance of the starting materials. The reaction mixture was quenched with saturated NH4Cl solution and extracted with CH2Cl2 (2 5 mL) and the combined organic layers were dried over anhydrous Na2SO4. The removal of solvent under vacuum resulted in crude mass, which was purified by column chromatography using hexane/ethyl acetate (2:3) solvent mixture. 3.2.1. 3-O-Acetyl-5-deoxy-1,2-O-isopropylidene-5-(4-(2-pyridyl)-1H1,2,3-triazole-1-yl)-a-D-xylofuranose (2a) On treatment of 3-O-acetyl-5-azido-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (1a) (0.13 g, 1 mmol) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2a was obtained as pale yellow solid. Yield: 0.31 g (88%); mp: 108 °C; [a]D24: 36.6 (c 1.0, CHCl3); IR (neat, mmax): 3480, 3136, 2987, 1749, 1602, 1243, 1070 cm 1; 1H NMR (300 MHz, CDCl3): d 1.30 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.14 (s, 3H, CH3), 4.51–4.57 (m, 2H, H-5 and H-2), 4.64–4.71 (m, 1H, H-5), 4.79 (dd, 1H, 3JH,H = 3.8 Hz and 3JH,H = 9.8 Hz, H-4), 5.29 (d, 1H, 3JH,H = 3.8 Hz, H-3), 5.98 (d, 1H, 3JH,H = 3.7 Hz, H-1), 7.23 (dt, 1H, 3JH,H = 2.6 Hz and 3JH,H = 6.0 Hz, Py-5-H), 7.78 (t, 1H, 3JH,H = 6.0 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 6.0 Hz, Py-3-H), 8.28 (s, 1H, triazole), 8.59 (d, 1H, 3JH,H = 6.0 Hz, Py-6-H) ppm; 13C NMR (75.5 MHz, CDCl3): d 20.68 (CH3), 26.05 (CH3), 26.54 (CH3), 48.94 (C-5), 76.28 (C-2), 77.17 (C-3), 83.36 (C-4), 104.78 (C-1), 112.44 (CMe2), 120.21 (C-5 triazole), 122.80 (CPy), 122.99(CPy), 136.82 (CPy), 142.49 (C-4 triazole), 149.30 (CPy), 150.11 (CqPy), 169.54 (CO) ppm; ESI-MS (in CH3OH): m/z 361 [M+H]+; Anal. Calc. for C17H20N4O5 (360.36): C, 56.66; H, 5.59; N, 15.55. Found: C, 56.35; H, 5.04; N, 15.16%. 3.2.2. 3-O-Benzoyl-5-deoxy-1,2-O-isopropylidene-5-(4-(2-pyridyl)1H-1,2,3-triazole-1-yl)-a-D-xylofuranose (2b) On treatment of 5-azido-3-O-benzoyl-5-deoxy-1,2-O-isopropylidene-a-D-xylofuranose (1b) (0.32 g, 1 mmol) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2b was obtained as pale yellow solid. Yield: 0.39 g (93%); mp: 152 °C; [a]D24: 13.6 (c 1.0, CHCl3); IR (neat, mmax): 3436, 3132, 2980, 1728, 1600, 1265, 1082 cm 1; 1H NMR
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(500 MHz, CDCl3): d 1.32 (s, 3H, CH3), 1.50 (s, 3H, CH3), 4.60–4.64 (m, 1H, H-5), 4.73 (d, 1H, 3JH,H = 2.9 Hz, H-2), 4.80–4.83 (m, 1H, H-5), 4.88 (dd, 1H, 3JH,H = 2.9 Hz and 3JH,H = 9.9 Hz, H-4), 5.58 (d, 1H, 3JH,H = 2.9 Hz, H-3), 6.06 (d, 1H, 3JH,H = 2.9 Hz, H-1), 7.22 (t, 1H, 3JH,H = 5.9 Hz, Py-5-H), 7.46–7.49 (t, 2H, 3JH,H = 7.9 Hz, Bz-3H), 7.61–7.64 (t, 1H, 3JH,H = 7.9 Hz, Bz-4-H), 7.75–7.78 (m, 1H, Py4-H), 8.05 (d, 2H, 3JH,H = 7.9 Hz, Bz-2-H), 8.15 (d, 1H, 3JH,H = 7.9 Hz, Py-3-H), 8.30 (s, 1H, CH of triazole), 8.57 (d, 1H, 3JH,H = 5.9 Hz, Py-6H) ppm; 13C NMR (75.5 MHz, CDCl3): d 26.12 (CH3), 26.50 (CH3), 49.30 (C-5), 76.80 (C-2), 77.69 (C-3), 83.46 (C-4), 104.95 (C-1), 112.59 (CMe2), 120.22 (C-5 triazole), 122.78 (Cpy), 122.96 (Cpy), 128.70 (CPy), 128.63 (2CBz), 129.76 (2CBz), 133.84 (CBz), 136.78 (CqBz), 148.60 (C-4 triazole), 149.33 (Cpy), 150.17 (Cqpy), 165.09 (CO) ppm; ESI-MS (in CH3OH): m/z 423 [M+H]+; Anal. Calc. for C22H22N4O5 (422.43): C, 62.55; H, 5.25; N, 13.26. Found: C, 61.99; H, 5.14; N, 13.01%.
3.2.3. 5-Deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-5-(4-(2pyridyl)-1H-1,2,3-triazole-1-yl)-a-D-xylofuranose (2c) On treatment of 5-azido-5-deoxy-3-O-ferrocenoyl-1,2-O-isopropylidene-a-D-xylofuranose (0.43 g, 1 mmol) (1c) with 2-ethynyl pyridine (0.10 g, 1 mmol), 2c was obtained as an orange solid. Yield: 0.47 g (88%); mp: 170 °C; [a]D24: 40.6 (c 0.1, CHCl3); IR (neat, mmax): 3420, 3122, 2990, 1718, 1136, 1039 cm 1; 1H NMR (500 MHz, CDCl3): d 1.33 (s, 3H, CH3), 1.49 (s, 3H, CH3), 4.24 (s, 5H, C5H5), 4.48 (d, 2H, 3JH,H = 1.1 Hz, C5H4), 4.57–4.62 (m, 1H, H-5), 4.67 (d, 1H, 3JH,H = 3.4 Hz, H-2), 4.74–4.79 (m, 1H, H-5), 4.80–4.84 (m, 3H, C5H4 and H-4), 5.46 (d, 3JH,H = 3.2 Hz, 1H, H-3), 6.03 (d, 1H, 3 JH,H = 3.4 Hz, H-1), 7.22 (m, 1H, Py-5-H), 7.77 (t, 1H, 3JH,H = 7.5 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 7.5 Hz, Py-3-H), 8.31 (s, 1H, CH of triazole), 8.58 (d, 1H, 3JH,H = 5.4 Hz, Py-6-H) ppm; 13C (75.5 MHz, CDCl3): d 26.19 (CH3), 26.68 (CH3), 49.30 (C-5), 69.90 (5CCp), 70.15 (2CCp), 70.30 (CCp), 71.92(2CCp), 76.12 (C-2), 77.70 (C-3), 83.53 (C-4), 104.93 (C-1), 112.64 (CMe2), 120.26 (C-5 triazole), 122.94 (CPy), 127.92 (CPy), 136.81 (CPy), 148.69 (C-4 triazole), 149.37 (CPy), 150.20 (CqPy), 170.58 (CO) ppm; ESI-MS (in CH3OH): m/z 531 [M+H]+; Anal. Calc. for C26H26FeN4O5 (482.1): C, 58.88; H, 4.94; N, 10.56. Found: C, 58.38; H, 4.85; N, 10.42%.
3.3. General procedure for the preparation of palladium complexes (3a–c) Under an argon atmosphere, ligands 2a–c (0.17 mmol) and [Pd(COD)Cl2] (0.053 g, 0.17 mmol), were dissolved in dichloromethane (5 mL). The mixture was allowed to stir at room temperature for 2 h and the resulting precipitate was collected by filtration. The solid was washed with diethyl ether and dried in vacuum to get the desired palladium complex.
3.3.1. Synthesis of [Pd(2a)Cl2], (3a) On treatment of acetyl-xylofuranose triazole (0.061 g, 0.17 mmol) (2a) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3a was obtained as pale yellow solid. Yield: 0.071 g (78%); mp: 258 °C (decomp.); IR (neat, mmax): 3461, 3117, 2967, 1750, 1600, 1223, 1036 cm 1; 1H NMR (300 MHz, CDCl3): d 1.29 (s, 3H, CH3), 1.44 (s, 3H, CH3), 2.22 (s, 3H, CH3), 4.61 (d, 1H, 3JH,H = 3.5 Hz, H-2), 4.73–4.76 (m, 1H, H-5), 4.83–4.91 (m, 1H, H-5), 5.02 (dd, 3 JH,H = 2.6 Hz and 3JH,H = 9.4, H-4), 5.41 (d, 1H, 3JH,H = 2.6 Hz, H-3), 6.02 (d, 1H, 3JH,H = 3.5 Hz, H-1), 7.21 (t, 1H, 3JH,H = 5.6 Hz, Py-5-H), 8.03–8.06 (m, 2H, Py-4-H and Py-3-H), 8.67 (d, 1H, 3JH,H = 5.6 Hz, Py-6-H), 8.77 (s, 1H, CH of triazole) ppm; ESI-MS (in CH3OH + CH2Cl2): m/z 543 [(M Cl)+H2O+Na]+; Anal. Calc. for C17H20Cl2N4O5Pd (537.69): C, 37.97; H, 3.75; N, 10.42. Found: C, 37.62; H, 3.58; N, 10.27%.
3.3.2. Synthesis of [Pd(2b)Cl2], (3b) On treatment of benzoyl-xylofuranose triazole (0.072 g, 0.17 mmol) (2b) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3b was obtained as pale yellow solid. Yield: 0.71 g (88%); mp: 188 °C; IR (neat, mmax): 3508, 3097, 2986, 1725, 1622, 1265,1099 cm 1; 1H NMR (300 MHz, CDCl3): d 1.31 (s, 3H, CH3), 1.47 (s, 3H, CH3), 4.75 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.85–4.89 (m, 1H, H-5), 5.04–5.09 (m, 2H, H-4 and H-5), 5.63 (d, 1H, 3JH,H = 3.0 Hz, H-3), 6.08 (d, 1H, 3 JH,H = 3.7 Hz, H-1), 7.18 (dt, 1H, 3JH,H = 1.5 Hz and 7.5 Hz, Py-5H), 7.50 (t, 2H, 3JH,H = 7.4 Hz, Bz-3-H), 7.62 (dt, 1H, 3JH,H = 1.5 and 7.5 Hz, Py-4-H), 8.09 (m, 1H, Py-3-H), 8.12 (m, 3H, Bz-2-H and Bz-4-H), 8.65 (d, 1H, 3JH,H = 7.5 Hz, Py-6-H), 8.87 (s, 1H, CH of triazole) ppm; ESI-MS (in CH3OH + CH2Cl2): m/z 605 [(M Cl)+H2O+Na]+; Anal. Calc. for C22H22Cl2N4O5Pd (599.75): C, 44.06; H, 3.70; N, 9.34. Found: C, 43.91; H, 3.62; N, 9.19%. 3.3.3. Synthesis of [Pd(2c)Cl2], (3c) On treatment of ferrocenoyl-xylofuranose triazole (0.090 g, 0.17 mmol) (2c) with [Pd(COD)Cl2] (0.05 g, 0.17 mmol), 3c was obtained as an orange solid. Yield: 0.091 g (76%); mp: 180 °C; IR (neat, mmax): 3490, 3099, 2985, 1717, 1622, 1270, 1127 cm 1; 1H NMR (300 MHz, CDCl3): d 1.30 (s, 3H, CH3), 1.45 (s, 3H, CH3), 4.32 (s, 5H, C5H5), 4.47 (m, 2H, C5H4), 4.66 (d, 1H, 3JH,H = 3.7 Hz, H-2), 4.78–4.90 (m, 2H, C5H4), 4.92–4.94 (m, 1H, H-5), 5.05–5.17 (m, 2H, H-4 and H-5), 5.65 (d, 1H, 3JH,H = 3.0 Hz, H-3), 6.07 (d, 1H, 3 JH,H = 3.7 Hz, H-1), 7.16 (t, 1H, 3JH,H = 7.5 Hz, Py-5-H), 8.05 (t, 1H, 3 JH,H = 7.5 Hz, Py-4-H), 8.17 (d, 1H, 3JH,H = 7.5 Hz, Py-3-H), 8.59 (d, 1H, 3JH,H = 7.5 Hz, Py-6-H), 8.94 (s, 1H, CH of triazole) ppm; ESIMS (in CH3OH+CHCl3): m/z = 711 [(M Cl)+H2O+Na]+; Anal. Calc. for C26H26Cl2FeN4O5Pd (707.67): C, 44.13; H, 3.70; N, 7.92. Found: C, 43.85; H, 3.53.14; N, 7.62%. 3.4. In vitro cytotoxicity testing The cytotoxicity of all the compounds was assessed according to the literature procedures [63]. Cell lines used for testing in vitro cytotoxicity included HeLa derived from human cervical cancer cells (ATCC No. CCL-2), A549 derived from human alveolar adenocarcinoma epithelial cells (ATCC No. CCL-185), MDA-MB-231 derived from human breast adenocarcinoma cells (ATCC No. HTB-26) and MCF7 derived from human breast adenocarcinoma cells (ATCC No HTB-22) were obtained from the American Type Culture Collection, Manassas, VA, USA. All the tumour cell lines were maintained in a modified DMEM medium supplemented with 10% fetal bovine serum, along with 1% non-essential amino acids without L-glutamine, 0.2% sodium hydrogen carbonate, 1% sodium pyruvate and 1% antibiotic mixture (10 000 units penicillin and 10 mg streptomycin per mL). The cells were washed and resuspended in the above medium and 100 lL of this suspension was seeded in 96 well-bottom plates. The cells were maintained at 37 °C in a humidified 5% CO2 incubator (Model 2406 Shellab CO2 incubator, Sheldon, Cornelius, OR). After incubation for 24 h, the cells were treated for 2 days with test compounds at concentrations ranging from 0.1 to 100 lM in DMSO (1% final concentration) and were assayed at the end of the second day. Each assay was performed with two internal controls: (1) an IC0 with cells only, (2) an IC100 with media only. After incubation for 48 h, the cells were subjected to the MTT colorimetric assay (5 mg mL 1). The effect of the different test compounds on the viability of the tumour cell lines was measured at 540 nm on a multimode reader (InfiniteÒ M200, Tecan, Switzerland). The IC50 values (50% inhibitory concentration) were calculated from the plotted absorbance data for the dose– response curves. The assay was performed using doxorubicin and cisplatin as standards and 1% DMSO as a vehicle control. To avoid DMSO toxicity, the values obtained for the DMSO control were
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subtracted from those of the test compounds. IC50 values (in lM) are expressed as the average of two independent experiments. 4. Results and discussion 4.1. Synthesis and characterisation The carbohydrate azide ligands (1a–c) were synthesised by the esterification reaction of 5-azido-5-deoxy-1,2-O-isopropylidene-aD-xylofuranose with the corresponding acid chlorides. The azides were then treated with 2-ethynyl pyridine to synthesize the pyridyl triazole ligands using the Huisgen 1,3-dipolar cycloaddition reaction conditions at room temperature. The triazole ligands (2a–c) were reacted with [Pd(COD)Cl2] in dry dichloromethane to afford the corresponding complexes (3a–c) (Scheme 1). The ligands were characterised by 1H, 13C and IR spectroscopy. In the 1H NMR spectra, it was observed that the gem-dimethyl groups of the xylofuranose moiety appeared as two singlets around 1.3 and 1.4 ppm for all the ligands and the triazole proton appeared as a sharp singlet at 8.3 ppm. For the ligands 2a and 2b, the anomeric proton of the carbohydrate moiety appeared as doublet at 5.5–5.9 ppm. For the compound 2a, a sharp singlet for the –CH3 protons was observed at 3.0 ppm. In the case of ferrocenoyl-pyridyl triazole 2c, the protons of the unsubstituted Cp ring were observed at 4.2 ppm and those of the substituted Cp rings were observed at 4.5 and 4.8 ppm. The anomeric proton of the carbohydrate moiety appeared as doublet at around 5.9 ppm. Finally, the complexes were prepared by the reaction of the ligands with [Pd(COD)Cl2] in dry dichloromethane for 2 h at room temperature. The resulting complexes were soluble in dichloromethane, DMF and DMSO but insoluble in methanol. The complexes were characterised by 1H NMR spectroscopy, in which a down field shift of the triazole –CH proton indicated the formation of the complex (see Supporting information). In the 1H NMR spectra of the complexes, the anomeric proton of the xylofuranose appeared as a doublet at 6.0 ppm. The triazole proton was observed as a sharp singlet at 8.7 ppm. All the other signals for the sugar moiety appeared as reported [62]. Repeated attempts for crystallization in appropriate solvents failed to
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produce X-ray quality crystals. Therefore, the crystal structure of these complexes could not be obtained. The absorption spectrum of the ferrocenoyl triazole (2c) was determined by the UV–Vis studies in 0.5 mM dichloromethane solution. The compound exhibited characteristic peaks for the Fe(a1g) ? Fe(e1g) transitions and Fe(e1g) ? Cp(e2g) transitions at around 440 nm and 314 nm respectively [64]. In addition to these transitions, a broad absorption band near 350 nm was observed due to the n ? p⁄ transitions of the carbonyl group (see Supporting information). The formation of palladium complexes was also confirmed by the UV–Visible spectroscopy. The absorption spectroscopy measurements of the complexes were carried out in 0.1 mM dichloromethane solutions over a wavelength region of 250–600 nm (Fig. 2). The absorption spectra of all the complexes consists of absorption bands corresponding to the characteristic p ? p⁄ transitions of the triazole (at around 280 nm) and the metal to ligand charge transfer (MLCT) (at around 294 nm). This was in accordance with the coordination behaviour of the pyridyl triazole ligands reported in the literature [28]. In addition to these transitions, the n ? p⁄ transition of the carbonyl group were also observed for all the metal complexes. In the case of ferrocenoyl complex (3c), the ligand Fe ? Cp transitions overlapped with the MLCT band of the complex. A weak band corresponding to the Fe(a1g) ? Fe(e1g) transition of the ferrocene complexes was observed at 440 nm. 4.2. Electrochemistry The electrochemical characterisation of the triazole ligands and the metal complexes was performed in anhydrous dichloromethane by means of differential pulse voltammetry (dpv). To compare the electrochemical behaviour of the complexes 3a–c, the differential pulse voltammetry experiments were conducted for complex 5. Due to sparingly soluble nature of xylofuranose-palladium complex (5) in dichloromethane, the experiment was performed in DMSO solution. No oxidation peak was observed for the complex (5), but a reduction peak (see Supporting information) for Pd(II)/Pd(0) [28] was observed at around 1.79 V.
Scheme 1. Synthesis of pyridyl triazole ligands and their palladium(II) complexes.
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Absorbance (a.u.)
1.5
3a 3b 3c
1.0
0.5
0.0 250
300
350
400
450
500
550
Wavelength (nm) Fig. 2. UV–Vis spectra of palladium complexes in 0.1 mM dichloromethane solution.
Fig. 4. Differential pulse voltammograms (dpv) for the reduction of palladium complexes in dichloromethane solution at 25 °C.
The ligands 2a and 2b and the corresponding metal complexes 3a and 3b respectively did not show any oxidation process. But, surprisingly, in the case of ferrocenoyl pyridyl triazole (2c), in addition to the Fe(II)/Fe(III) oxidation process (+0.70 V), an oxidation peak due to the triazole functionality was also observed at around +1.16 V (Fig. 3a). This additional peak may be due to the oxidation of the triazole functionality [65]. For the compound 2c, the shift in the oxidation potential of Fe(II)/Fe(III) couple is due to the presence of electron withdrawing ester group on the ferrocene of 2c [66]. In the ferrocenoyl palladium complex (3c), the Fe(II)/Fe(III) oxidation process was observed at +0.682 V and oxidation of the triazole group was observed at +0.972 V (Fig. 3b). All the complexes exhibited irreversible reduction wave, indicating the conversion of Pd(II) to Pd(0) (Fig. 4) [28]. The acetyl complex 3a, exhibited a reduction potential of 0.84 V, while the benzoyl complex (3b) showed irreversible reduction process at around 0.86 V. The ferrocenoyl complex exhibited reduction potential corresponding to Pd(II)/Pd(0) at 0.76 V [28].
For instance, it has been reported by Hanessian and Wang that in the case of L-xylose derived platinum(II) complexes, the presence of free –OH group displayed improved cytotoxicity compared to the methyl ether protected analogue [57]. However, it was found that in the case of D-xylose derived palladium(II) complexes, among 3-OH- and 3-O-benzyl-a-D-xylofuranose derivatives, the 3-O-benzyl protected compounds were found to display better bioactivity [58]. The enhanced cytotoxicity of the 3-O-benzyl protected xylofuranose compounds may be attributed to the lipophilicity of the sugar scaffold [58]. Therefore, taking clue from the observations, it was assumed that an ester protection of the xylofuranose at the 3-O-position could exhibit significant effect on the cytotoxicity of the pyridyl carbohydrate conjugates and their metal complexes. Furthermore, it has also been reported in the literature that, ferrocene linkage to metal complexes via ester moiety displayed significant bioactivity [45]. With these insights, we have prepared three different carbohydrate pyridyl triazole ligands containing ester protection at 3-O-position of the xylofuranose ring and their cytotoxicity results are summarised in Table 1. Within the domain of carbohydrate research, the acetyl as well as the benzoyl moieties are extensively used as protecting groups for the hydroxyl group, hence this report describes their comparison with the ferrocenoyl moiety. From our previous work [58], it was evident that the xylofuranose triazole ligand (4) did not show cytotoxic activity (IC50 value >100 lM) on the tested cell lines, but its metal complex (5) exhibited promising cytotoxicity (IC50 value of <10 lM). To our surprise,
4.3. In vitro cytotoxicity (MTT assay) The compounds were tested for cytotoxicity against different test cell lines up to a concentration of 100 lM. In the metal carbohydrate complexes, apart from the central metal ion, the nature of the sugar scaffold and the protecting groups on the sugar play a key role in determining the biological activities of the complexes.
Fig. 3. Differential pulse voltammograms (dpv) for the oxidation of (a) ligand 2c and (b) complex 3c in dichloromethane solution at 25 °C.
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the funding. S. B. D. and P. S. are grateful to the CSIR, New Delhi, India for the award of research fellowships.
Table 1 Cytotoxicity of pyridyl triazole ligands and complexes. Compound
4b 2a 2b 2c 5b 3a 3b 3c Doxorubicinc Cisplatinc a b c
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IC50 (lM)a A549
HeLa
MDA-MB-231
MCF-7
>100 36.73 (±0.10) 77.97 ± 0.18) 5.73 (±0.03) 6.4 (±0.03) 19.29 (±0.11) 66.19 (±0.19) 13.18 (±0.07) 0.46 (±0.03) 0.15 (±0.13)
>100 >100 >100 >100 8.3 (±0.01) >100 >100 >100 0.51 (±0.02) 0.23 (±0.10)
>100 >100 >100 >100 9.9 (±0.13) >100 >100 >100 0.91 (±0.06) 0.25 (±0.09)
>100 >100 >100 >100 5.5 (±0.09) >100 >100 >100 1.07 (±0.1) 0.48 (±0.16)
Values are mean of two independent experiments. Ref. [58]. Positive control.
it was observed that all the ligands and metal complexes in the present study exhibited variable cytotoxicity on one specific cell line, namely, the human alveolar adenocarcinoma A549 cell line, while for the remaining cell lines IC50 of more than 100 lM was observed. The acetyl and benzoyl substituted ligands were found to exhibit low cytotoxicity with IC50 values 36.73 lM and 77.97 lM respectively. In the case of ferrocene conjugated pyridyl triazole ligand (2c), the inclusion of an organometallic fragment drastically improved the cytotoxicity (IC50 value of 5.73 lM). This difference in the cytotoxicity of the ligands indicates that ferrocene certainly plays a significant role in influencing the cytotoxicity of the carbohydrate-pyridyl triazole ligands. Moreover, incorporation of Pd(II) on the pyridyl triazole moiety improved the cytotoxicity of acetyl and benzoyl containing carbohydrate metal complexes 3a and 3b (exhibited IC50 values 19.29 and 66.19 lM respectively). However, a low cytotoxicity was observed for ferrocene–palladium complex (IC50 value 13.18 lM) as compared to the free ligand (2c). This decrease in the cytotoxicity of the ferrocene–palladium complex perhaps may be attributed to the steric bulk of the metal complex [47]. These results do indicate the effect of an organometallic unit on the overall biological activity of the ligand, however, much more exhaustive study, by varying the metal as well as the protecting group, is currently under progress. 5. Conclusions The first example for the palladium(II) complex of ferrocene– carbohydrate conjugate has been prepared. The complexes were characterised by UV–Vis spectroscopy, in which the characteristic transitions for the cis-palladium complexes were observed. The electrochemical studies have shown oxidation of the triazole moiety in case of ferrocene–carbohydrate pyridyl triazole ligand (2c), whereas such triazole oxidation was not observed in the case of acetyl and benzoyl derivatives. The complexes exhibited reduction wave corresponding to the Pd(II)/Pd(0) conversion. Interestingly, all the ligands and metal complexes have shown cytotoxicity selectively on A549 cancer cell line. Among all the ligands and metal complexes, only ferrocene containing ligand and metal complex exhibited IC50 values less than 20 lM. The development of carbohydrate metal complexes and their organometallic conjugates are currently under progress in our laboratory. Acknowledgments This work was financially supported by the in-house project of CSIR-IICT (INDIA) MLP-0007. Dr. L.G. thanks the Department of Science and Technology (DST, SR/S1/IC21/2008), New Delhi, for
Appendix A. Supplementary material Supplementary (The Supplementary Information contains the H and 13C NMR spectra of ligands and the 1H NMR spectra of complexes.) data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ica.2014.03.018.
1
References [1] M.A. Fuertes, C. Alonso, J.M. Perez, Chem. Rev. 103 (2003) 645. [2] G. Giaccone, Drugs 59 (2000) 9. [3] G. Jaouen, N. Metzler-Nolte, Medicinal Organometallic Chemistry, Topics in Organometallic Chemistry, 32 ed., Springer, Berlin, 2010. [4] E. Alesio, Bioinorganic Medicinal Chemistry, Wiley–VCH, Weinheim, 2011. [5] C.G. Hartinger, P.J. Dyson, Chem. Soc. Rev. 38 (2009) 391. [6] A.A. Holder, Annu. Rep. Prog. Chem. Sect. A 107 (2011) 359. [7] C.G. Hartinger, N. Metzler-Nolte, P.J. Dyson, Organometallics 31 (2012) 5677. [8] M.A. Ali, A.H. Mirza, R.J. Butcher, M.T.H. Tarafder, T.B. Keat, T.A.M. Ali, J. Inorg. Biochem. 92 (2001) 141. [9] N. Miklášová, E. Fischer-Fodor, P. Lönnecke, C.I. Tomuleasa, P. Virag, M.P. Schrepler, R. Mikláš, L.S. Dumitrescu, E. Hey-Hawkins, Eur. J. Med. Chem. 49 (2012) 41. [10] L.M.M. Vieira, M.V. de Almeida, M.C.S. Lourenço, F.A.F.M. Bezerra, A.P.S. Fontes, Eur. J. Med. Chem. 36 (2009) 4107. [11] T.V. Serebryanskaya, T. Yung, A.A. Bogdanov, A. Shchebet, S.A. Johnsen, A.S. Lyakhov, L.S. Ivashkevich, Z.A. Ibrahimava, T.S. Garbuzenco, T.S. Kolesnikova, N.I. Melnova, P.N. Gaponik, O.A. Ivashkevich, J. Inorg. Biochem. 120 (2013) 44. [12] S. Abu-Surrah, H.H. Al-Sa’doni, M.Y. Abdalla, Cancer Ther. 6 (2008) 1. [13] T. Rau, R. Alsfasser, A. Zahl, R. van Eldik, Inorg. Chem. 37 (1998) 4223. [14] G. Zhao, H. Lin, P. Yu, H. Sun, S. Zhu, X. Su, Y. Chen, J. Inorg. Biochem. 73 (1999) 145. [15] A.C.F. Caires, Anti Cancer Agents Med. Chem. 7 (2007) 484. [16] P. Chellan, N. Shunmoogam-Gouden, D.T. Hendricks, J. Gut, P.J. Rosenthal, C. Lategan, P.J. Smith, K. Chibale, G.S. Smith, Eur. J. Inorg. Chem. (2010) 3520. [17] S. Padhye, Z. Afrasiabi, E. Sinn, J. Fok, K. Mehta, N. Rath, Inorg. Chem. 44 (2005) 1154. [18] E. Budzisz, M. Miernicka, I.-P. Lorenz, P. Mayer, U. Krajewska, M. Rozalski, Polyhedron 28 (2009) 637. [19] W.M. Motswainyana, M.O. Onani, A.M. Madiehe, M. Saibu, J. Jacobs, L. van Meervelt, Inorg. Chim. Acta 400 (2013) 197. [20] C. Urquiola, M. Vieites, M.H. Torre, M. Cabrera, M.L. Lavaggi, H. Cerecetto, M. González, A. López de Cerain, A. Monge, P. Smircich, B. Garat, D. Gambino, Bioorg. Med. Chem. 17 (2009) 1623. [21] P. Štarha, I. Popa, Z. Trávnícˇek, Inorg. Chim. Acta 363 (2010) 1469. [22] M.E.H. Mazumder, P. Beale, C. Chan, J.Q. Yu, F. Huq, ChemMedChem 7 (2012) 1840. [23] Y. Wang, S. Liu, J. Zhang, S. Li, H. Wei, G. Zhou, X. Qin, Appl. Organomet. Chem. 27 (2013) 373. [24] F. Zamora, V.M. González, J.M. Pérez, J.R. Masaguer, C. Alonso, C. NavarroRanninger, Appl. Organomet. Chem. 11 (1997) 659. [25] E. Budzisz, M. Malecka, B.K. Keppler, V.B. Arion, G. Andrijewski, U. Krajewska, M. Rozalski, Eur. J. Inorg. Chem. (2007) 3728. [26] D. Tsekova, P. Gorolomova, G. Gochev, V. Skumryev, G. Momekov, D. Momekova, G. Gencheva, J. Inorg. Biol. Chem. 124 (2013) 54. [27] S. Rubino, P. Portanova, F. Giammalva, M.A. Girasolo, S. Orecchio, G. Calvaruso, G.C. Stocco, Inorg. Chim. Acta 370 (2011) 207. [28] D. Schweinfurth, R. Pattacini, S. Strobel, B. Sarkar, Dalton Trans. (2009) 9291. [29] K.J. Kilpin, E.J. Gavey, C.J. McAdam, C. Anderson, S.J. Lind, C.C. Keep, K.C. Gordon, J.D. Crowley, Inorg. Chem. 50 (2010) 6334. [30] A. Maisonial, P. Serafin, M. Traïkia, E. Debiton, V. Théry, D.J. Aitken, P. Lemoine, B. Viossat, A. Gautier, Eur. J. Inorg. Chem. (2008) 298. [31] T.L. Mindt, H. Struthers, L. Brans, T. Angelov, C. Schweinsberg, V. Maes, D. Tourwe, R. Schibli, J. Am. Chem. Soc. 128 (2006) 15096. [32] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem. 54 (2011) 3. [33] R.H. Fish, G. Jaouen, Organometallics 22 (2003) 2166. [34] C. Ornelas, New J. Chem. 35 (2011) 1973. [35] A. Nguyen, A. Vessières, E.A. Hillard, S. Top, P. Pigeon, G. Jaouen, Chimia 61 (2007) 716. [36] O. Buriez, E.A. Hillard, A. Vessières, D. Hamels, S. Top, G. Jaouen, Y.M. Frapart, D. Mansuy, C. Amatore, Chem. Eur. J. 18 (2012) 6581. [37] D. Hamels, P.M. Dansette, E.A. Hillard, S. Top, A. Vessières, P. Herson, G. Jaouen, D. Mansuy, Angew. Chem., Int. Ed. 48 (2009) 9124. [38] E.A. Hillard, G. Jaouen, Organometallics 30 (2011) 20. [39] C. Biot, G. Glorian, L.A. Maciejewski, J.S. Brocard, J. Med. Chem. 40 (1997) 3715. [40] W. Henderson, S.R. Alley, Inorg. Chim. Acta 322 (2001) 106. [41] A. Rosenfeld, J. Blum, D. Gibson, A. Ramu, Inorg. Chim. Acta 201 (1992) 219. [42] M. Viotte, B. Gautheron, M.M. Kubicki, I.E. Nifant’ev, S.P. Fricker, Metal Based Drugs 2 (1995) 311.
170
S.B. Deepthi et al. / Inorganica Chimica Acta 416 (2014) 164–170
[43] M.C. Gimeno, H. Goitia, A. Laguna, M.E. Luque, M.D. Villacampa, C. Sepúlveda, M.J. Meireles, Inorg. Biochem. 105 (2011) 1373. [44] J. Schulz, A.K. Renfrew, I. Císrová, P.J. Dyson, P. Stpnicˇka, Appl. Organomet. Chem. 24 (2010) 392. [45] M. Auzias, B. Therrien, G. Süss-Fink, P. Štpnicˇka, W.H. Ang, P.J. Dyson, Inorg. Chem. 47 (2008) 578. [46] J. Rajput, J.R. Moss, A.T. Hutton, D.T. Hendricks, C.E. Arendse, C. Imrie, J. Organomet. Chem. 689 (2004) 1553. [47] T.V. Segapelo, S. Lillywhite, E. Nordlander, M. Haukka, J. Darkwa, Polyhedron 36 (2012) 97. [48] W.M. Motswainyana, M.O. Onani, A.M. Madiehe, Polyhedron 41 (2012) 44. [49] D. Talancón, C. López, M. Font-Bardía, T. Calvet, J. Quirante, C. Calvis, R. Messeguer, R. Cortés, M. Cascante, L. Baldomà, J. Badia, J. Inorg. Biochem. 118 (2013) 1. [50] Y. Chen, A. Janczuk, X. Chen, J. Wang, M. Ksebati, P.G. Wang, Carbohydr. Res. 337 (2002) 1043. [51] C.G. Hartinger, A.A. Nazarov, S.M. Ashraf, P.J. Dyson, B.K. Keppler, Curr. Med. Chem. 15 (2008) 2574. [52] I. Brudziñska, Y. Mikata, M. Obata, C. Ohtsuki, S. Yano, Bioorg. Med. Chem. Lett. 14 (2004) 2533. [53] T. Allscher, P. Klüfers, O. Labisch, Carbohydr. Res. 342 (2007) 1419. [54] J. Möker, J. Thiem, Eur. J. Org. Chem. (2009) 4842.
[55] M.E. Cucciolito, R.D. Litto, F.P. Fanizzi, D. Migoni, G. Roviello, F. Ruffo, Inorg. Chim. Acta 363 (2009) 741. [56] Y. Mikata, S. Yano, Curr. Top. Med. Chem. 12 (2012) 145. [57] S. Hanessian, J. Wang, Can. J. Chem. 71 (1993) 886. [58] S.B. Deepthi, R. Trivedi, P. Sujitha, C.G. Kumar, B. Sridhar, S.K. Bhargava, J. Chem. Sci. 124 (2012) 1405. [59] R. Trivedi, S.B. Deepthi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, K.V.S. Ramakrishna, Eur. J. Inorg. Chem. (2012) 2267. [60] R. Trivedi, S.B. Deepthi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, K.V.S. Ramakrishna, Appl. Organomet. Chem. 26 (2012) 369. [61] S.B. Deepthi, R. Trivedi, L. Giribabu, P. Sujitha, C.G. Kumar, Dalton Trans. 42 (2013) 1180. [62] S.B. Deepthi, R. Trivedi, L. Giribabu, B. Sridhar, P. Sujitha, C.G. Kumar, New J. Chem. 38 (2014) 227. [63] M. Navarra, M. Celano, J. Maiuolo, S. Schenone, M. Botta, A. Angelucci, P. Bramanti, D. Russo, BMC Cancer 10 (2010) 602. [64] Y.L.K. Tan, P. Pigeon, E.A. Hillard, S. Top, M. Plamont, A. Vessières, M.J. McGlinchey, H. Müller-Bunz, G. Jaouen, Dalton Trans. (2009) 10871. [65] B. Happ, D. Escudero, M.D. Hager, C. Friebe, A. Winter, H. Gorls, E. Altuntas, L. González, U.S. Schubert, J. Org. Chem. 75 (2010) 4025. [66] G. Gritzner, J. Kuta, Pure Appl. Chem. 56 (1984) 461.