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Synthesis, characterization and biological properties of a novel copper complex

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The study of copper complex in relation to cancer is important in many ways. A novel copper complex has been synthesized with non toxic ligand, viz. potassium salt of N-(2-hydroxy acetophenone) glycinate (NHAG). The structure of the complex has been
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  Preliminary communication Synthesis, characterization and biological properties of a no v elcopper complex Surajit Majumder 1 , Gouri Sankar Panda 2 , Soumitra Kumar Choudhuri 3, * Department of En v ironmental Carcinogenesis and Toxicology (ECT), Chittaranjan National Cancer Institute (CNCI),37 S.P. Mukherjee Road, Calcutta 700 026, India Recei v ed 20 February 2003; recei v ed in re v ised form 6 August 2003; accepted 6 August 2003 Abstract The study of copper complex in relation to cancer is important in many ways. A no v el copper complex has been synthesized withnon toxic ligand,  v iz. potassium salt of   N  -(2-hydroxy acetophenone) glycinate (NHAG). The structure of the complex has beendetermined by spectroscopic means. Toxicity and antitumor property of the complex has been studied in  v i v o. Though the complexis toxic at higher doses, lower non toxic doses of the complex deplete glutathione (GSH). GSH depleting property of the complexmay be utilized to sensitize drug resistant cells where resistance is due to ele v ated le v el of GSH. #  2003 E´ditions scientifiques et me´dicales Else v ier SAS. All rights reserved. Keywords:  Coordination; Copper; Glutathione;  N  -(2-hydroxy acetophenone) glycinate 1. Introduction Coordination of organic compound with metal i.e.,chelation causes drastic change in the biological prop-erty of the ligand and also the metal moiety [1]. It hasbeen reported that chelation is the cause and cure of many diseases including cancer. The metal copper[Cu(II)] is also in v ol v ed in the causation and cure of cancer [2    / 7]. Copper administration suppresses rathepatoma induced by chemical carcinogen [8]. Cop-per(II) complexes cause tumor cells to redifferentiateinto normal cells [9]. Although physiological role of copper is contro v ersial, it is pro v ed that copper is anessential component of se v eral endogenous antioxidantenzymes [10].Coordination beha v ior of sodium or potassium salt of  N  -(2-hydroxy acetophenone) glycine (NHAG) towardsorganotin moiety has recently been reported [11].Antitumor property of the ligand [NHAG] and someof its organotin complexes has been studied [12]. Thenon-toxic nature, water solubility and strong coordina-tion beha v ior of NHAG deser v e attention [11,12].The present work describes the synthesis of a no v elcopper complex with potassium salt of NHAG; thestructure of the complex has been determined byspectroscopic studies. Antitumor property, toxicity andglutathione (GSH) depletion property in  v i v o of thecomplex has also been reported. 2. Chemistry  2.1. Materials and methods 2.1.1. ChemicalsN  -(2-hydroxy) acetophenone, copper sulphate, gly-cine, DMSO was purchased from Aldrich, NY. Etha-crynic acid (EA), buthionine sulphoxamine (BSO), v erapamil, dithio nitrobenzene (DTNB) were purchasedfrom Sigma Chemical Compamy, St. Louis, MO. Otherchemicals used were of highest purity a v ailable. Abbre v iations:  NHAG,  N  -(2-hydroxy acetophenone) glycine;CuNG, copper  N  -(2-hydroxy acetophenone) glycinate; BSO,buthionine sulphoxamine; EA, ethacrynic acid; DTNB, 5,5 ? -dithiobis (2-nitrobenzoic acid); DMSO, dimethyl sulphoxide; EAC, Ehrlichascites carcinoma.* Correspondence and reprints. E-mail address:  soumitra01@ v snl.net (S. Kumar Choudhuri). 1 Junior Research Fellow. 2 Senior Research Fellow. 3 Senior Scientific Officer.European Journal of Medicinal Chemistry 38 (2003) 893    / 898www.else v ier.com/locate/ejmech0223-5234/03/$ - see front matter # 2003 E´ditions scientifiques et me´dicales Else v ier SAS. All rights reserved.doi:10.1016/j.ejmech.2003.08.002   2.2. Synthesis of the ligand  The ligand, potassium  N  -(2-hydroxy acetophenone)glycinate was prepared according to the reportedmethods [11]. In brief, a cold aqueous solution of KOH (1.03 g, in 12 mL) was mixed with cold aqueoussolution of glycine (1.38 g in 12 mL) and held at 15    / 20  8 C in an ice bath with continuous stirring. Anethanolic solution of 2-(hydroxy) acetophenone (2.5 gin 25 mL) was added dropwise. Deep yellow color wasde v eloped and stirring was continued for 1 h followedby 5 h at room temperature. The sol v ent was remo v edby rotary e v aporator. The yellow mass was washed withpet    / ether and precipitated with methanol    / diethyl ethermixture. The crude product was recrystallised frommethanol to yield potassium  N  -(2-hydroxyacetophe-none) glycinate. Yield 75%, m.p. 258    / 260  8 C.  2.3. Synthesis of the copper complex CuNG was synthesized from the ligand, potassium  N  -(2-hydroxy acetophenone) glycinate by its reaction withcopper sulphate; in brief, 0.68 g CuSO 4 , 5H 2 O wasdissol v ed in 5 mL deionised water. 0.785 g potassium  N  -(2-hydroxy acetophenone) glycinate was dissol v ed in 25mL ethanol. The solution of   N  -(2-hydroxy acetophe-none) glycinate (yellow color) was slowly added toCuSO 4  solution (blue color) at room temperature withcontinuous stirring by a magnetic stirrer. The mixturewas further stirred in the magnetic stirrer for 1 h at 45    / 50  8 C. The color of the mixture changed to deep green.The mixture was cooled to room temperature and thegreen precipitate was separated by filtration. Thecompound was dried and recrystallised from DMSO.Yield 40%, m.p.242 8 C. Anal. Calc. C 10 H 15 O 6 NCu: C,39.0; H, 4.87; N, 4.54; Cu, 20.45. Found: C, 40.57; H,5.04; N, 4.65; Cu, 21.24%. 3. Pharmacology 3.1. Biological materials All animals were collected from our animal colony.EAC was maintained as an ascitic tumor in maleSwiss albino mice weighing 18    / 20 g (6    / 8 weeks old). 3.2. Study of in  v i  v o toxicity3.2.1. Effect of CuNG on blood  CuNG (1 mg) was dissol v ed in 1 mL DMSO. Thesolution (0.1 mL) was injected to male Swiss albino mice(i.e. 5mg kg  1 ). Blood was collected from normal andalso from treated mice after different time inter v al of CuNG injection intraperitoneally.Blood was obtained  v ia closed cardiac puncture bymeans of a 22-guage hypodermic needle and a sub-xiphoid approach. Blood from each group (CuNGtreated and untreated) was pooled into separate glasstubes and treated with anticoagulant (heparin).Normal and differential blood count was done fortreated and normal mice. The experiment was repeatedfor four times. 3.2.2. Effect of CuNG on spleen CuNG (5 mg kg  1 ) in DMSO was injected into maleSwiss mice. 3.3. Preparation of spleen cell suspension Normal and CuNG treated male Swiss mice wereanaesthetized and 70% alcohol was sprayed on abdom-inal region. Spleen was remo v ed aseptically and smallamount of PBS was injected to it; Spleen was rubbedagainst the fine wire mesh of the tissue grinder. The cellsuspension formed is spinned at 1000    / 1500 rpm for 5    / 10 min. The supernatant was discarded and the cellswere washed by spinning in PBS twice at roomtemperature.Cell  v iability was tested by trypan blue and cells werecounted in a phase contrast microscope. The experimentwas repeated for four times. 3.3.1. Effect of CuNG on bone marrow CuNG (5 mg kg  1 ) in DMSO was injected to maleSwiss mice. 3.4. Separation of bone marrow cells Normal and CuNG treated mice were anaesthetizedand the femur bone was cut with the help of a  v ertebratescissor. Bone marrow was flushed with 0.56% KClsolution and centrifuged at 3000 rpm for 15 min at37  8 C.Cells were counted under microscope for treated anduntreated animals. The experiment was repeated forfour times. 3.5. Study of antitumor property of CuNG in  v i  v o Fifty-fi v e male Swiss albino mice 6 weeks of age weredi v ided into six groups; one control group with fi v e miceand fi v e drug treated groups with 10 mice in each group.EAC cells (1  / 10 6 ) were injected i.p. to all mice on day1. On day 2,  v arious doses of the ligand (NHAG)dissol v ed in deionised and autocla v ed water wereinjected i.p. to mice of   v arious groups. No drug wasadministered to the control group. Life monitoring wasrestricted to daily body weight measurement, recordingtime of death. Animals were obser v ed for a period of 60days. Cell yield, ascites  v olume, packed cell  v olume, S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893    /  898 894  mean sur v i v al time (MST) and change in life span of thetreated mice in comparison to control (T/C  v alue) wererecorded. The experiment was repeated for four times. 3.5.1. Effect of CuNG on sur v i  v al of animals Various doses of CuNG dissol v ed in DMSO wereinjected ip to male Swiss mice. Animals were obser v edfor a period of 72 h. The a v erage  v alue of the animalsli v ing (percentage) of three independent experimentswith respect to the doses of CuNG was plotted (Fig. 2).The amount of CuNG required to kill 50% of theanimals (IC 50  v alue) in  v i v o was determined from theplot. 3.6. GSH-depleting properties of CuNG in  v i  v o Cells (1  / 10 6 ) were injected i.p. into male Swiss mice6 weeks of age. On the second day, 1 mg kg  1 CuNGwas injected to mice. On the 12th day animals wereanesthetized, killed and EAC cells were collected. Cells(1  / 10 6 ) were washed in PBS twice. Cells were homo-genized and di v ided into two equal amounts formeasuring GSH and protein.EA, BSO,  v erapamil were also injected in the similarmanner and cells were collected.Normal cells were taken from EAC bearing mice 8weeks of age not undergone any drug treatment.Experiment for each drug treated and control groupwere repeated for four times.GSH was measured by the method of Sedlack andLindsay [13] and protein was measured by Lowry [14]. 4. Results and discussions UV spectrum for the complex and the ligand wastaken in methanol.UV bands for the ligand  l max  (methanol): 271, 350,411.UV bands for the complex  l max  (methanol): 271, 337,380.The change in the UV peak from 350 in the ligand to337 in the complex indicates  p    / p * transition. The shiftof the peak from 411 in the ligand to 380 in the complexalso indicates  p    / p * transition.Important infrared (IR) bands for the ligand appearat: 3410    / 3360, 1689, 1619, 1524, 1466, 1421, 1395, 1318,1269, 1205, 1163, 969, 931, 752, 730.Important IR bands for the complex appear at: 3407    / 2700, 2340, 1630, 1604, 1541, 1465, 1444, 1364,1309,1237, 1160, 1088, 1071, 1035, 965, 941, 861, 785,762, 722, 622, 595, 537, 526, 453.The IR spectrum of the ligand shows broad band at3360    / 3410 cm  1 which may be due to  n OH. The free n OH is generally obser v ed between 3400 and 3500 cm    1 .The obser v ed low  v alue is due to intramolecular H-bonding between H and nitrogen [11]. In the coppercomplex the band shifted to 2700    / 3407 cm  1 . The alkylgroups CH 2 , CH 3  show characteristic deforming bandsat 1466    / 1395 cm  1 and the rocking modes at   / 730cm  1 in the ligand. The rocking mode of the ligandshifts to 762 cm  1 in the complex. The band at 1604cm  1 is assigned due to  n CN in the complex (in theligand the band appears at 1619). The sharp band at1395 cm  1 in the ligand may be assigned due to  n OCOsymmetric that has been shifted to 1364 cm  1 in thecomplex indicating coordination with the Cu-atomthrough the COO  group of the ligand. The band at1269 cm  1 in the ligand may be assigned due to n Ph(CO) which has been shifted to 1237 cm  1 in thecomplex indicating coordination of the phenolic oxygento the Cu-atom. The band at 1689 cm  1 in the ligandmay be assigned due to a  n (OCO) asymmetric  v ibrationand in complex this  v ibration appears as broad band at1630 cm  1 [12].Thus, the IR spectra results pro v ide strong e v idencefor the complexation of the potentially multidentateligand.Proton NMR peak of the ligand in D 2 O appears at7.38    / 7.51 (s, 5H) and 6.59    / 7.6 (s, 3H) for aromaticprotons. CH 2  protons appear at 4.09 (1H, m). CH 3 protons appear at 2.26    / 2.29 (4H, m).Proton NMR peak of CuNG in DMSO- d  6  appear at5.59 (S) for aromatic protons. CH 2  protons appear 3.40(B). CH 3  protons appear at 2.51    / 2.79 (m, 4H). In NMRspectra we obser v e a shift of electron density from theligand to metal moiety. Aromatic protons shifts fromthe range of 7.38    / 7.51 in the ligand to 5.99 in thecomplex. Methelene protons shift from 4.09 in theligand to 3.40 in the complex. The shielding of thearomatic and methylene protons indicate the formationof the complex. Howe v er, the methyl protons in theligand shifted to higher ppm; from ligand 2.26    / 2.29 to2.51    / 2.79 in the complex. Probably, methyl protonswere not pulled from ligand to metal moiety.Mass spectral data is presented in Fig. 1. Theformation of molecular ion peaks indicates that thestructure of the complex is  A  in Fig. 1.Electron paramagnetic resonance (EPR) spectrum of the complex has been recorded in the polycrystallinestate at room temperature. The spectrum shows the v alue of   g    / 2.06 and  g  jj  / 2.20. The  v alue of   g  jj  lowerthan 2.3 indicates a co v alent metal    / ligand en v ironment[15]. The  v alue of   g  jj B / 2.3 in the present Cu-complexgi v es a clear indication of co v alent character of themetal    / ligand bond and delocalisation of the unpairedelectron into the ligand. The trend of   g  jj  /  g    / 2.0023describes the axial symmetry with the unpaired electronresiding in the d x 2   y 2  orbital [16].Magnetic susceptibility study of the present Cu-complex gi v es a magnetic moment  v alue ( m eff  ) of 1.98BM at room temperature.  m eff   / 1.98 BM indicates one S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893    /  898  895  electron paramagnetism. This  v alue is higher than thespin-only  v alue of 1.73 BM for one unpaired electron.The higher  v alue of the magnetic moment indicates thatCuNG is monomeric in nature and there is no metal    / metal interaction along the axial position in the complex[17].Study of hematological toxicity in  v i v o is presented inTable 1.The ligand is nontoxic up to 100 mg kg  1 . There is nochange in hematological parameters at a dose of 50 and100 mg kg  1 of the ligand. The effect was studied at v arious time inter v als like 1, 10 and 25 days of theligand. CuNG is toxic at a dose of 25 mg kg  1 . Lowerdoses like 6 or 10 mg kg  1 do not show any toxic effect.Slight toxicity is shown at 12 mg kg  1 . The resultspresented in Table 1 are based on four independentexperiments.The effect of the complex on spleen and bone marrowis presented in Table 2. The ligand is nontoxic up to 100mg kg  1 . There is no change in spleen or bone marrowcells at a dose of 50 and 100 mg kg  1 of the ligand.CuNG is toxic at a dose of 25 mg kg  1 . Lower doseslike 6 or 10 mg kg  1 do not show any toxic effect; 12 mgkg  1 shows slight toxicity.Toxicity of CuNG in  v i v o is presented in Fig. 2.CuNG at a dose of 35 mg kg  1 kills 50% of the animals Fig. 1. Mass fragmentation of CuNG.Table 1Effect of CuNG on hematological parameters of male albino Swiss miceDrug Dose (mg kg  1 ) Haemoglobin (Hb) (gm/dl) WBC (  / 10 3 ) RBC (  / 10 6 ) Lymphocyte (%) Neutrophil (%)1D 10D 25D 10D 10D 10D 10D    /    /  11.77 9 / 0.31 11.89 9 / 0.35 11.91 9 / 0.41 2.9 9 / 0.08 5.6 9 / 0.21 59.2 9 / 0.83 36.07 9 / 0.33Ligand 50 11.57 9 / 0.49 11.62 9 / 0.31 11.81 9 / 0.37 2.93 9 / 0.12 5.87 9 / 0.25 52.03 9 / 053 42.07 9 / 1.41Ligand 100 11.83 9 / 0.29 11.59 9 / 0.14 11.91 9 / 0.41 2.84 9 / 0.21 5.77 9 / 0.13 51.67 9 / 1.83 40.07 9 / 3.33CuNG 6 11.9 9 / 0.96 11.38 9 / 0.28 11.93 9 / 0.31 2.89 9 / 0.17 5.6 9 / 0.08 59.0 9 / 2.83 41.07 9 / 0.8CuNG 10 11.71 9 / 0.32 11.28 9 / 0.13 11.6 9 / 0.51 2.79 9 / 0.13 5.5 9 / 0.11 58.6 9 / 2.1 40.10 9 / 0.72CuNG 12 11.9 9 / 0.96 10.92 9 / 0.31 10.73 9 / 0.47 2.36 9 / 0.25 3.9 9 / 0.21 57.2 9 / 2.83 36.87 9 / 1.66CuNG 25 8.3 9 / 0.22 8.43 9 / 0.35 8.41 9 / 0.31 1.25 9 / 0.04 2.6 9 / 0.08 51.53 9 / 1.01 28.02 9 / 1.67The data are means 9 / S.D. of four independent experiments. CuNG at a dose of 10 mg kg  1 has no toxic effect on hematological parameters (  p B / 0.001) when compared with untreated control.Table 2Effect of CuNG on spleen and bone marrow of male Swiss albino miceDrug Dose (mgkg  1 )Spleen cell (  / 10 3 )Bone marrow cell (  / 10 3 )10D 10D    /    /  4.7 9 / 0.82 1.27 9 / 0.06Logan 50 4.65 9 / 1.82 1.2 9 / 0.82Ligand 100 4.45 9 / 0.41 1.3 9 / 0.82CuNG 6 4.65 9 / 0.45 1.01 9 / 0.13CuNG 10 4.61 9 / 0.31 0.97 9 / 0.08CuNG 12 3.95 9 / 1.15 0.7 9 / 0.08CuNG 25 3.17 9 / 0.03 0.65 9 / 0.04The data are means 9 / S.D. of four independent experiments. CuNGat a dose of 10 mg kg  1 is significantly non toxic to spleen and bonemarrow when compared with untreated control (  p B / 0.001). S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893    /  898 896  (IC 50  v alue). The graph is based on the a v erage  v alue of three independent experiments.The antitumor property of CuNG is presented inTable 3. Neither the ligand nor the complex showed anyantitumor property in  v i v o as reflected in T/C  v alue. Theligand is non toxic at 120 mg kg  1 . CuNG is quite toxicat a dose of 35 mg kg  1 .Depletion of GSH by CuNG is shown in Table 4.CuNG is stronger GSH depleting agent than therecommended drug EA [18] at a dose of 10 mg kg  1 .Most of the GSH depleting agents reported in literatureare effecti v e in  v itro and are toxic at required doseswhen applied in  v i v o [18    / 20]. Though CuNG is toxic athigher doses, low non-toxic doses like 2    / 5 mg kg  1 maybe effecti v e to deplete GSH. 5. Conclusions CuNG is toxic at higher doses and depletes GSH e v enin nontoxic doses. It has been reported that a number of drug resistant cells ha v e higher le v el of GSH comparedto drug sensiti v e cells and modulation of cellular GSHhomeostasis sensitizes drug resistant cancer cells to awide range of chemotherapeutic drugs [21]. The nontoxic dose of CuNG may be utilized to deplete GSH in anumber of drug resistant cell lines and thereby o v ercomedrug resistance where resistant is due to higher le v el of GSH. The study of the effect of CuNG on  v arioussensiti v e and drug resistant cells are in progress. 6. Experimental protocols 6.1. Chemistry UV    / v is spectra was recorded in Shimadzu UV 160 Aand in Varian Cary 100 Scan in the range of 800    / 200m m .IR spectra were recorded in Perkin    / Elmer RX 1 FTspectrophotometer in KBR discs in the range 4500    / 500cm  1 .Proton NMR spectra was recorded in DMSO- d  6  on aBruker ACF 300 spectrometer at 300.13 MHz referenceto Me 4 Si (0.0 ppm).Mass spectrum was recorded in an AEI MS-30 massspectrometer.C, H, N was measured by Perkin    / Elmer 2400 SeriesII CHN analyzer.Copper was measured by atomic absorption spectro-photometer Varian Spectra 200 FS, hollow cathodelamp, flame type: air acetylene; replicate 3; wa v elength324.8 nm.EPR spectrum (Fig. 3) was recorded in Varian E 109Cat room temperature at field set: 3200G; scan range 4  / 1 K; recei v er gain 4.0  / 10 3 ; scan time 4 min; modula-tion 1.25  / 10; time constant 0.250; frequency 9.1 GHz,power 30 dB; temp. 295 K; state: microcrystalline.Magnetic moment was measured in EG & G  v ibratingsample magnetometer (model 155). 6.2. Biological e v aluation GSH was measured following the method of Sedlackand Lindsay [13]. Briefly, to 2  / 10 5 cells in 0.2 mL PBS,4.8 mL EDTA (0.2 M) was added and kept on ice bathfor 10 min. Then 4 mL deionised water and 1 mL of 5%trichloroacetic acid (TCA) were added. The mixture wasagain kept on ice for 10    / 15 min and then centrifuged at3000 rpm for 15 min. Two milliliters of supernatant was Fig. 2. In  v i v o toxicity of CuNG. The plot is drawn based upon thea v erage  v alue of three independent experiments of animals li v ing(percentage) with respect to dose of CuNG. IC 50  v alue of CuNG is 35mg kg  1 .Table 3Effect of CuNG on EAC bearing male Swiss mice (10 days after CuNG injection)Parameters Control Ligand CuNG (mg kg  1 )50 100 6 12 35Cells in whole ascites fluid (10 6 ) 170 9 / 10.2 173 9 / 1.7 171 9 / 2.49 174.4 9 / 1.63 154 9 / 3.27 138 9 / 1.67Total  v olume (mL) (tumor cells  / ascites fluid) 7.5 9 / 1.48 7.13 9 / 0.2 7.77 9 / 0.13 7.27 9 / 0.13 5.4 9 / 0.52 5.37 9 / 0.17Total packed cell  v olume (mL) 5.3 9 / 0.43 5.4 9 / 0.16 5.07 9 / 0.13 4.93 9 / 0.19 4 9 / 0.16 3.4 9 / 0.16Mean sur v i v al time (MST) (days) 33.5 9 / 2.73 36 9 / 1.63 34 9 / 1.63 30 9 / 1.63 23 9 / 1.26 17 9 / 0.82T/C (%) 100 107.46 107.46 89.55 69.64 50.75The data are means 9 / S.D. of four independent experiments. CuNG at non toxic dose (10 mg kg  1 ) has no antitumor property when comparedwith untreated control (  p B / 0.005). S. Majumder et al. / European Journal of Medicinal Chemistry 38 (2003) 893    /  898  897
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