Indian Journal of Chemistry Vol. 43A, June 2004, pp. 1233-1238

Synthesis, characterization and nuclease activity of copper(II), nickel(II), cobalt(II) and

iron(II) complexes with oxime-thiosemjcarbazones

K Hussain-Reddy"*, M Surendra Babu", P Suresh Babub

& S Dayanandab

"Department of Chemistry, Sri Kri shnadevaraya University, Anantapur 515 003, India

bDepartment of Biochemistry, Sri Krishnadevaraya University, Anantapur 515003, India

Received 17 January 2003; revised 13 April 2004

A novel ligand viz. l-phenyl- I,2-propanedione-2-oxime thiosemicarbazone (PPDOT) has been synthesized and characterized. Complexes of copper(II), nickel(ll), cobalt(II) and iron(II) with PPDOT have been synthesized and characterized by molar conductance, magnetic moments, electronic, IR and ESR spectroscopy . Electrochemical behaviour of these complexes is investigated by cyclic voltammetric studies. The nuclease activity of these complexes has been carried out on double stranded pBR 322 plasmid DNA by using gel electrophoresis experiments in absence and in the presence of oxidant (H20 2) . Metal complexes of diacetylmonoxime thiosemicarbazone (DAMOT) are also investigated for better compari son

IPC Code: CO I G 51/00; CO I G 3/00; CO I G 53/00; CO 1 G 49/00

Investigations of novel transition metal complexes to probe nucleic acids are the focus of current research l

-5

•

Thiosemicarbazones are biologically active pharmacophores, besides having good complexing ability and their activity enhances on complexation with metal ions6

-9

. Although oximes and their transition metal complexes have been investigated as chemical nucleases 1o

. Metal complexes of ligands containing both OXlmes and thiosemicarbazones are pharmacophores much less investigated. Metal compounds of diacetylmonoxime thiosemjcarbazone (DAMOT) have been characterized but their nuclease activity has not been investigated so for" -13

• Recently we have investigated the nuclease activity of copper complexes of ortho substituted heteroaromatic thiosemicarbazones and semicarbazones 14- 16. In the present report we describe synthesis, characterization and comparative nuclease activity of ligands PPDOT and DAMOT and their Cu(II), Ni(II), Co(ll) and Fe (11) metal complexes.

Experimental All the reagents used in the preparation of ligands

and their metal complexes were of reagent grade (Merck). The solvents used for the synthesis of ligands and metal complexes were distilled before use. All other cherrucals were of AR grade and used without further purification.

Plasmid isolation

The E. coli DH5a strains containing plasmid pBR 322 was grown in Luria broth (LB) medium supplemented with 100 Jlg/ml ampicillin . Cells from 5 ml culture were harvested by centrifuging the culture for IO minutes at 8000 rpm. Plasmid pBR 322 was isolated using Qiagen column following manu facturer' s protocols.

Assay of nuclease activity

The DMF solution containing metal complexes was taken in a clean eppendroff tube and I Jlg of plasmid DNA was added. The contents were incubated for 30 min at 37°C and loaded on 0.8% agarose gel after mixing 5 JlI of loading buffer (0.25% bromophenol blue + 0.25% xylene cyanol + 30% glycerol sterilized distilled water). Electrophoresis was performed at constant voltage till the bromophenol blue reached to the 3/4 of the gel. Further the gel is stained for IO min by immersing it in Ethidium bromide solution (5 Jlg/ml of H20). The gel was then de-stained for 10 min by keeping it in sterile distilled water and plasmid bands were visualized by viewing the gel under transilluminator and photographed 17.

Preparation of PPDOT and DAMOT

In a clean 250-ml round bottom flask , the reaction mixture contammg I-phenyl-I,2-propanedione-2-oxime or diacetalmonoxime (0.03 mol) in 100 ml of 1% HCI-ethanol, thiosemicarbazide (2 .8 g, 0.031 mol ) dissolved in boiling water were taken and refluxed for 3 hrs. On cooling pale yellow compound for 1-phenyl-I,2-propanedione-2-oxime thiosemicarbazone /white product for diacetylmonoxime thiosemicarbazone was formed. It was collected by filtration, washed several times with hot water, small quantities of cold methanol and dried in vacuo. DAMOT was prepared according to literature reports 11 -13.

1234 INDIAN J CHEM, SEC A, JUNE 2004

The pKa values calculated by Philips-Merritt method are 6.52 (PK,) and 8.55 (PK2). The infrared spectrum of PPDOT shows bands at 3148, 1609 and 1199 cm·1

assigned to v(OH) of oxime, v(C=N) and v(C=S) respectively . The IH-NMR spectrum of PPDOT was recorded in d6-DMSO solvent. It shows signals corresponding to -CH3, -C6H5, -NH2' >NH (hydrazone) and -OH protons at 2 .. 17 (s, 3H), 7.22-7 .56 (m, 5H), 8.74-8.1 7 (2H), 10.71 (s, IH) and 11.75 (s, lH) respectively. Mass spectrum of PPDOT shows molecular ion peak at 237 (mlz) corresponding to its molecular weight. The other important peaks at mlz

178 and 84 correspond to the loss of CH3- C =N-OH

S II and C

6H5NH-C-NH2 molecule respectively. The

peaks at 104 and 43, respectively correspond to the + +

formation of C6H5- C =NH and H3C- C =0.

Synthesis of metal complexes The metal complexes were prepared by mixing hot

ethanolic solution of metal chlorides and PPDOT or DAMOT in the molar ratio of I: 1. In the preparation of iron(ll) complex, an aqueous solution of ferrous ammonium sulphate was used. Metal solution was added to the boiling solution of ligand (2 g, 0.0084 moles) in ethanol and heated under reflux for 3 h. The reaction mixture was cooled and left overnight in refrigerator. Crystalline complexes, which separated out, were collected by filtration, washed with hot distilled water and small quantity of cold methanol.

Molecular weights of the complexes were determined by cryoscopic method using camphor as solvent. Magnetic measurements were carried out in the polycrystalline state on a PAR model ISS vibrating sample magnetometer operating at field strength of 2-10 KG. High purity nickel metal (saturation moment 55 emu/g) was used as a standard.

The molar conductance of the complexes in DMF (10.3 M) soltltion were measured at 28 ± 2°C with Systronic model 303 direct reading conductivity bridge. The electronic spectra of metal complexes were recorded in DMF with a Shimadzu UY-160 A spectrophotometer. The infrared spectra were recorded in the range 4000-180 cm· 1 with Perkin­Elmer 983 G spectrophotometer in KBr discs . ESR spectra were recorded on Yarian E-I 12 X -band spectrophotometer at liquid nitrogen temperature (LNT) and room temperature in both solution (DMF) and solid state. The voltammetric measurements were performed on a BAS CY-27 assembly in conjunction with an X-Y recorder. Measurements were made on the degassed (N2 bubbling for 5 min) solutions in dimethylformamide (10.3 M) containing 0.1 M tetraethylammonium per chlorate as the supporting electrolyte. The three-electrode system consisted of a glassy carbon (working) platinum wire (auxiliary) and Ag/ AgCl (reference) electrode.

Results and discussion The present ligands contain two functional groups

viz. oxime and thiosemicarbazone (Structure I) and their metal complexes are stable at room temperature, non-hygroscopic, insoluble in HbO, but slightly soluble in ethanol and methanol, and readily soluble in DMF and DMSp. The colour, molecular weight and molar conductance data are summarized in

R= C6H5, PPDOT

R= CH" DAMOT

Table I- Physical properties of metal complexes of DAMOT and PPDOT

Complex Colour (Yield %) M.P. (0C) Molecu lar weight Magnetic spin Molar Obs (Cal) fl crr value conductance

[Cu(DAMOT)hCl2 Dark green (80) 187-190 542 (546) 1.53 1.7 56.1 [Ni(DAMOT)12CI 2 Greenish black (54) 220-223 529 (536) 1.85 2.8 29.5 [Co(DAMOT)(H20hhCI2 Reddi sh (30) 235-240' 591 (599) 1.35 3.9 HS 1.7 LS 44.5 [Fe(DAMOT)(H2OhbS04 Black (72) 237-242' 592 (598) 3.54 4.9 HS 0.0 LS 22.3 ICu(PPDOT)hCI2 Black (72) 220-224 650 ± 20(672) 1.38 1.73 LS/HS 67.7 [Ni (PPDOT)1 2C1 2 Brown (68) 240-245* 644 ± 20 (622) 1.39 0.0 LS 2.8 HS 54.5 [CoC PP DOT)( H 20 hhCl2 Black (60) 235-240* 710± 20(734) 1.1 8 1.73 LS 3.87 HS 46.5 [Fe(PPDOT)(H2OhhS04 Reddish (50) 232-235 740 ± 20 (752) 2.06 0.0 LS 4.89 HS 33.5

*Decomposes

NOTES 1235

Table I. The molar conductivity data suggest that the complexes are 1: 1 electrolytes. The magnetic moments (Table 1) of metal complexes are found to be subnormal which may be attributed to the presence of magnetically coupled metal centers in dimeric complexes.

The electronic spectral data of metal complexes are given in Table 2. The spectral data of copper complex is dominated by intense intra-ligand (24390 cm", 18518 cm-') and charge transfer (CT) (33330 cm-', 36231cm-') bands. The presence of a single d-d band may be attributed to the symmetric nature of ligand field. The electronic spectrum of nickel complex exhibits a medium intensity band 18520 cm-' suggesting a square planar geometry for the complex. A charge transfer band 37037 cm-' is also observed in the electronic spectrum of cobalt complex. The electronic spectrum of cobalt complexes recorded in DMF shows two distinct bands (Table 2) attributable to the 4T,g(F) -7 4A2g(F)(V2) and 4T ,g (F) -7 4A 'g (P) (V3) transi tions respectively in an octahedral field. Important ligand field parameters [B (Racah parameter) 708; 10 Dq 7995 and ~35 0.73] are calculated and the values lie in the same range as expected for an octahedral Co(fI) complex. The iron(II) complex exhibits an intense charge transfer

Table 2-Electronic spectral data (em-I) for DAMOT and PPDOT metal complexes

Complex dod L-M

[Cu(DAMOT)hCl2 24390 33330 [Ni(DAMOT)hCh 20410 33110 [Co(DAMOT)(H20)2hCI2 15040 33898

16390 [Fe(DAMOT)(H2OhhS04 14705 27780 [Cu(PPDOT)hCI2 1851 8 36231 [Ni(PPDOT)hCI2 18520 37037 [Co(PPDOT)(H20)~hCI2 14925 37037

16666 [Fe(PPDOT)(H2OhhS04 16666 35714

band at 37037 cm-' assigned to T2g (Fe) -7 11:* (L) transition. The band observed at 16666 cm-' in the electronic spectrum of iron (II) complex is assigned to 5T2g -7 5 B ' g, which is generally observed in 6-coordinate iron(lI) complexes.

The important vibrational bands of metal complexes are included in Table 3. The absence of SH band at 2570 cm-' and presence of NH band at 3233 cm-' in the IR spectrum of ligands suggest that the ligands remain in thione form at least in solid state.

A strong band appearing at 1201 and 1134 cm-' in the spectra of PPDOT and DAMOT is shifted to lower frequency, indicating the involvement of thio­keto sulphur in coordination. A strong band observed at 3414 and 3411 cm-' in the IR spectra of PPDOT and DAMOT disappeared in the spectra of all complexes suggesting deprotonation of oxime OH in the complex formation. The> C = N (imine band) is observed at 1610 and 1600 cm-' in the IR spectra of PPDOT and DAMOT respectively. This band is shifted to lower wave numbers in the spectra of complexes suggesting the participation of imine nitrogen atom in coordination. Broad and strong bands in the IR spectra of Co(ll) and Fe(II) complexes in 3400-3500 cm-' region suggest the presence of coordinated water molecules. The appearances of bands 800-825 cm-' region are assigned to wagging modes of water molecules in iron (II) and cobalt(l]) complexes. Additional bands are observed in Far IR spectra of metal complexes in 500-480 and 365-315 cm-' regions due to v(M-N) and v(M-S) modes respectively. Based on molecular weight determination, magnetic moments, electronic and IR spectra a general structure (Structure II) is assigned for the complexes.

Electron spin resonance spectra of copper complex was recorded in DMF at liquid nitrogen temperature.

Table 3--Selected IR bonds (em-I) with tentative assignments for DAMOT and PPDOT

Complex v (N-H) v (A r-H) v (Ring) v (C=N) v (C=S) v (M-N) v (M-S) v (M-O)

DAMOT 3233 1600 1134 [Cu(DAMOT)hCI2 3027 1551 1072 498 315 [Ni(DAMOT)hCI2 3073 1530 1063 464 347 [Co(DAMOT)(H20hhCI2 3126 1550 1057 477 300 [Fe(DAMOT)(H2OhhS04 3003 1545 1061 460 308 PPDOT 3148 3046 1590-1 369 1610 1201 [Cu(PPDOT)hCl2 3078 1603-1335 1530 1174 484 361 518 [Ni(PPDOT)hCI2 3170 3017 1600-1335 1535 1159 496 318 551 [Co(PPDOT)(H 20 hhCI2 3 142 3052 1623-1372 1549 1166 503 365 529 [Fe(PPDOT)(H2OhI2S04 3060 1627-1316 1512 1158 482 365 532

1236 INDIAN J CHEM, SEC A, JUNE 2004

The spin Hamiltonian orbital reduction and bonding parameters of copper complexes of PPDOT and DAMOT are given in Table 4. The gil and g-L values are computed from the spectrum using tetracyanoethylene (TCNE) free radical as 'g' marker. From the observed values it is clear that gil> g-L > 2.00 which suggest the fact that the unpaired electrons lies

predominantly in the d x2 _ / orbital IS. The gay value

for this complex is greater than 2 indicating covalent property'') . For in-plane n-bonding KII < K-L, while for out-of-plane n-bonding KII > K-L (ref. 15). The observed KII> K.l, relation indicates the presence of out-of-plane n-bonding. The axial symmetry parameter (G) for

where X = water molecule present in Co(ll) and Fe(II) complexes

M = Cu(II), Ni(JI) , Co(ll) and Fe(II)

Y = 1/2 504 in Fe(II); CI in Cu(II), Ni(lI) and Co(ll) complexes

II

these complexes indicates that there is no interaction between copper centers in DMF medium.

The EPR X-band spectra for the binuclear copper complexes are also recorded in solid state at room temperature and liquid nitrogen temperature. At both temperatures the complexes gives a broad signal in the low field region indicating spin-exchange interactions between two copper (II) ions. The two expected spin allowed EPR transitions occur at 2880, 3150 G ; and 3140, 3180 G yielding g iso values 2.163 and 2.087 for copper PPDOT and DAMOT respectively. The absence of hyperfine structure indicates that the interaction would be mainly dipolar in nature.

Table 4--Spin Hamiltanian and orbital reduction parameters of Cu(II) complexes

Parameters [Cu(DAMOT)12C12 [Cu(PPDOT)1 2CI 2

gil 2.2431 2.4175

gJ. 2.0422 2.0731 gay 2.1426 2.2453 G 6.0049 5.86

KII 0.7658 1.0307

KJ. 0.6233 0.85 13 Aall 0.1271 A·J. 0.001395 Aa

av 0.005167 ? 1.0072 a-

P 0.01792 K 0.4288

·Units in cm-I

Table 5-----Cyc1ic voltammelric data of DAMOT and PPDOT complexes

Complex Gain Redox couple CV cathodic CVanodic

t!.Ep EI /2 (V) -iji. EPe Ep.

[Cu(DAMOT)hCl2 0.1 IIII 0. 18 0.49 310 0.335 0.564 [Ni(DAMOT)hCI2 0.1 IV/III -0_93

II Ifll -1 .29 -1 .20 90 -1.245 III1 -1.56 -1.43 130 -1.495

[Co(DAMOT)(H20hhCI2 0.1 II If II -1.12 -0.89 230 -1.005 II/I -1.42 -1.31 110 -1 .365

[Fe(DAMOT)(H2OhhS04 0.1 IIII 0.21 0.39 180 0.33 1.034

[Cu(PPDOT)hCI2 0.1 IIII 0.55 0.42 130 0.485 0.829 [Ni(PPDOT)hCI2 0.1 IV/Ill -0.78 -0.91 140 -0.845

IIIIII -1.32 -1.34 20 -1.33 IlI1 -1.7 -1.60 130 -1.53

[Co(PPDOT)(H20hhCI2 0.1 II 1111 -0.36 -0.58 220 -0.47 -1.06 -0.88 180 -0.97 -1.72 -1.65 70 -1.69

[Fe(PPDOT)(H2OhhS04 0.01 111111 0.40 0.26 140 0 .33 0.569

Recorded in DMF at room temperature with E4 NCI04 as supporting electrolyte; glassy carbon as working electrod:: ; PI wire as auxiliary e lectrode and Ag/AgCl as reference electrode, Scan rate 100 mV S- I.

NOTES 1237

The redox behaviour of complexes has been investigated by cyclic voltammetry at a glassy carbon electrode. Table 5 gives the electrochemical data obtained at glassy carbon electrode in DMF. The cathodic peak current function values were found to be independent of the scan rate. Repeated scans, as well as different scan rates showed that dissociation does not take place in these complexes. The reduction peak of the Fe(JII)/Fe(JI) and Cu(III)/Cu(I1) couple for these complexes is observed at the potential 0.33 0.33, and 0.485 0.564, Y versus Ag/ AgCl for PPDOT and DAMOT respectively, which are similar to the values reported earlier2o

.24

. The non-equivalent current

Nicked (11)---7 Linear (111)---7

Supercoiled (1)---7

intensity of cathodic and anodic peaks (ielia = 0.829, 1.034; 0.569, 0.564 Y at 100 mY S·I) for copper and iron complexes of PPDOT and DMOT respectively indicate a quasi-reversible behaviour25

. The difference fl.Ep = Epe-Epa in all these complexes exceeds the Nernstian requirement of 59/n mY (n = number of electrons involved in oxidation reaction) which suggests the quasi-reversible character of these complexes. All these complexes have large separation (1 00-350 mY) between anodic and cathodic peaks indicating the quasi reversible character. An exception is nickel complex of PPDOT which show a reversible one electron reduction Ni(III)/Ni(1I) couple with fl.Ep

2 J 4 S 6 7 X .) III I I I 2

Nicked (11)--7 Linear (111)--7

Supercoiled (I) --7

Fig. I- Agarose gel (0.8%) show ing the results of e lectrophoresis o f 2 f.ll o f (0. 10 !!g/ml) pBR 322 plasmid ON A; 2 f.ll of 0.1 M Tri s-HC I (PH 8.0) buffer; 2 f.ll ( 100 f.lM) complex in DMF; 12 f.ll of sterili zed water and 2 f.ll of 9.0 mM H20 2 were added, respecti vely, at 25°C (total volume 20 f.ll ). incubation 37°C (30 min): (a}-Lane I: DNA; Lane 2: DNA + H20 2 ; Lane 3: DNA + PPDOT; Lane 4 : DNA + PPDOT + H20 2: lane 5: DNA + [Cu(PPDOT)hCI2: Lane 6: DNA + [Cu(PPDOT)hCI2 + H20 2; Lane 7: DNA + [Ni (PPDOT)hCI2: Lane 8: D A + [Ni(PPDOT)hCI2 + H20 2; Lane 9: DNA+ Co(PPDOT)( H20 hhCI2; Lane 10: DNA + [Co(PPDOT) (H20 h hCI 2 + H20 2: Lane II : DNA + [Fe( PPDOT) (H20 hhS04; Lane l2: DNA + [Fe(PPDOT) (H20 hhSO" + H20 2: (b}-Lane I : DNA: Lane 2: DNA + H20}: Lane 3: DNA + DAMOT: Lane 4: DNA + DAMOT + H20 2: lane 5: DNA + [Cu(DAMOT)hCI2: Lane 6: DNA + [Cu(DAMOT)hCI2 + H20 2; Lane 7: DNA + [Ni (DAMOT)bCI2: Lane 8: DNA + [Ni(DAMOT)hCI 2 + H20 2: Lane 9: DNA + [Co(DAMOT)(H20 h12C I2; Lane 10: DNA + [Co(DAMOT) (H20)2hC12 + H20 2: Lane II: DNA + [Fe(DAMOT) (H20 )2hS04: Lanel2 : DNA + [Fe(DAMOT) (H20 )2hS04 + H20 2·

1238 INDIAN J CHEM, SEC A, JUNE 2004

value equal to 20. The EI/2 values of these complexes are comparable with other complexes with nitrogen and sulphur donor ligands 14. 16 .

The nuclease activity of present ligands and their complexes has been investigated on pBR 322 plasmid DNA by agarose gel electrophoresis in the presence / absence of H20 2. At micro molar concentration, the ligands exhibit no significant activity in absence and in the presence of the oxidant as shown in Fig I. The nuclease activity is greatly enhanced by incorporation of metal ions in the li gands . In absence of oxidants copper and iron compl exes of PPDOT causes discernible DNA cleavage as shown by increase in intensity in form J[

(nicked) and form III (linear) with decrease in intensi ty in from I (super coiled) which is attributed to step-wise conversion of from I to form II and to form Ill. Similar observations were also evident in case of copper and irop complexes of DAMOT and the conversion to linear form was complete. The nuclease act ivity of ni ckel and cobalt complexes with DAMOT is more when compared with PPDOT complexes of nickel and cobalt.

All complexes show much enh anced nuclease activity in the presence of oxidant, which may be due to free radical reacti on (OW) with DNA . The production of hydroxyl radical s due to the reaction between H20 2 and metal complex may be explained as shown below26

.

The OH· radi ca l involves ox idation of deoxyribose moiety fo ll owed by hydrolytic cleavage

'7 of sugar phosphate backbone- .

The hi gher activity of DAMOT complexes is probably due to presence of lipophilic -CH3 group. The lipophilic nature is evaluated by thin layer chro matography for the li gands in 10-3 M alcoholic soluti ons. The stati onary phase was sili ca gel chem ically bonded (Nano-Si NH 2) and mobile phase was a 2:3 mixture of water and methanol. The Rf values [DAMOT - 46; PPDOT- 29 ili mm] indicate more lipophilic nature of DAMOT and its complexes .

Acknowledgements The authors (MSB and KHR) are thankful to the

UGC, New Delhi (Grant No. F 12-118/200 1) fo p'i' financial support.

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