Indian Journal of Chemistry Vol. 42A, February 2003, pp. 313-3 I 7

Synthesis, antitumour and antibacterial activity of some Ru(bpY)22+/4-substituted

thiosemicarbazide complexes

UK Mazumder*, Malaya Gupta, A Berat, S Bhattacharya, S Karki, L Manikandan & S Patra

Department of Pharmaceutical Technology , Jadavpur University. Calcutta 700 032

Receil'ed 5 March 2002; revised 4 Ouober 2002

Complexes of N-substituted thiosemicarbazides with Ru(ll)-

2,2' bipyridyl have been prepared and characterized by IR, 1H NMR and UV/vis spectroscopy in addition ttY their elemental analysis. Based on molecular mechanics simulations using HyperChem®, the complexes are designed for anticancer activity agaInst Ehrlisch AsciTes Carcino/lla . Their effects on the hematological profile of the tumour hosts have also been studied. The complexes are found to possess prominent antibacterial activity.

Cisplatin is , at present, the most widely used drug in anticancer therapy but has serious dose-limiting side-effects for example myelotoxicity, nephrotoxicity and neuropathy I. After the discovery of its antineoplastic activity in the late 1960s, many efforts hav~ been made to improve its therapeutic efficacy against a broader tumour panel and reduce host toxicity. In the same period, the screening has been extended also to non-platinum-metal compounds and some promising results have been obtained with complexes of Ru, Rh, Jr and Ti. Among these, the ruthenium complexes have been studied most extensively. Some of these complexes have entered, or are about to enter, clinical trials. The substitution of the central metal atom presents an opportunity for obtaining complexes that are effective against tumours with low side-effects. Some classic ruthenium compounds such as Ru(OMSO) CI 2 1 4 2,

Ru(NH])4Cb', ImH[Ru(lmhCI4]4 and ImH[Ru(Im)(DMSO)CI4] (NAMI-A)5 are well-known antitumour agents. The photochemical and electrochemical behaviour of tris-chelates of ruthenium wilh bipyridine and phenanthroline are well-known6

. Besides, these tris-chelates of ruthenium

* For Correspondence: mazumder_uk@yahoo.co.in tCentral Drugs Laboratory, Calculla 70() 016, India

show intercalative properties with the ON A 7 ill vitro. Based on these findings we designed some tris complexes of Rutheni um (II) of the type

[Ru(bpyhL](CI04)2 (where bpy=2,2'-bipyridine and L= 4 substituted thiosemicarbazide). The synlhesized compounds have been characterized by elemental analysis, magnetic measurement as well as various spectroscopic methods such as UV, IR, 11-1 NMR.

(CIO.),

4-N substituted thiosemicarbazides

[Ru(bpy),(L))(CIO.h

Experimental

[Ru(bpyhJCh.2H 20 8 (0.5 mmol) was dissolved in dry ethanol (50 ml) and this solution was added to the solution of the ligands (L) (0.53 mol), which were synthesized using a reported procedure'! . The reaction mixture was refluxed for 14 h under N:> atmosphere with stirring. After completion of the reaction, a col­our change was observed. The reaction mixture was cooled to room temperature. The complexes were pu­rified by column chromatography . The pure com­plexes were dissolved in a minimum volume of dry ethanol. Excess cold solution of NaCI04 was added to the mixture and the resultant solution cooled at O°C overnight. A microcrystalline precipitate was ob­tained. The crystals were filtered and the solid was washed with vcry little amount of water followed by three 25 ml portion of diethyl ether and clried over CaCl 2 in vacuo. The IR and NMR data for [Ru(bpyh(4-CH j TSC)](C104h: (m.p.>270"C) IR

(KBr pellet): 3000-3300 cm-I (VN II) , 720-750 cnf!

(ves), and 1600-1640 cm· 1 (NH bending).

IH NMR (DMSO-dr,): 0 ppm 7.81 (Nil-NIl:», 9.5-

7.04 (aromatic protons of 2,2'-bipyridinc), 2.47

(- CH:l), 7.2 ( - NH). AJllUX (nm, 10-5 M in mClhanol ): 250, 310 and 430nm. Elemental analysis data are rc­ported in Table I.

314 INDIAN J CHEM. SEC A. FEBRUARY 2003

Table I-Analytical data LD,o values for Ru (Il) complexes

Complex

[Ru (hpyh( 4-CH, TSC) I(CI04 h

[Ru(bpYl1( 4-CH,CH2TSC)](CI04h [Ru(bpYh(4-C6 H I ITSC)](Cl04),

IRu(bpyh(4-Ph TSC)](CI04)2

I Ru(bpY)2( 4p-CI Ph TSC)](CI04 )2

I Ru(bpy h( 4p-13rPh TSC) ](CI04 h

IRlI(hpyh(4p-IPh TSC)J(Cl04h [Ru(bpyh(4p-CH, Ph TSC)](CI04)2

[RlI(bpY)2(4p-OCH,Ph TSC)](CI04 h

[Ru(bpY)2(41'-O C2H, Ph TSC) I(CI04h

C

36.23 (36.82)

37.8 (37.76)

41.01 (41.27)

41.11(41.59)

39.25 (39.83)

37.34 (37.76)

35.94 (35.8)

42.X2 (42.37)

41.09 (4 1.53)

42.81 (42.28)

AlltitulIIor activity al/.d hematological effects of the cOlllplexes

In order to test the antitumor potential of the ruthe­nium complexes, EAC a murine cancer cell line was chosen. Male albino Swiss mice weighing 20±2 g were divided into twelve groups A 1 to A 12 (n= 10) and inoculated with 2x 106 EAC cells/mouse. LDsOtO values of the complexes are reported in Table I . The ruthenium complexes were administered at a dose of 2 mg/kg, cisplatin at a dose of 2 mg/kg and normal saline S ml/kg body weight was injected intraperito­neally (i .p.) for nine consecutive days into groups A I to A 12 respectively, 24 h after tumour transplantation. On the tenth day, the animals were sacrificed and the effects of the ruthenium complexes on tumour growth were estimated by evaluating tumour volume and tu­mour cell count. Mean survival time was calculated for groups A I to A 12 (n=6) by a simi lar protocol t t as descri bed above.

For hematological studies 12 , blood was taken from tail vein, 24 h after last dose. Total count of WBC and RBC were done on an automatic blood analyzer.

Assay of antibacterial activity A stock solution of ruthenium complex 2000 )..lg/

ml was made in sterile water containing S% DMF under aseptic conditions and further dilutions were made with the same solvent in a similar manner. All the dilutions and stock were sterili zed by filtration . Solid agar and liquid broth culture media No. I t] were used for all the test organisms except for Pseudomo­lias aeruginosa where the pH was adjusted to 7 .2 . Antimicrobial activity of the complex against differ­ent strains of bacteria was determined by cup-plate

Found (Calcd). % LD,,, ( mg/kg H N body wc ight )

3.33 (3 .2) 13.61 ( 13.67) 3-+

3.31 (3 .42 ) 13.08 ( 13.4) 34

3.68 (3.94) 12 .82 ( 12.48) ' ") .L

3.21 (3.2) 12.51 ( 12.58) 25

2.73 (2.95) 12.12 ( 12.05) n 2.52 (2.79) 11.39 (11.42) 2X

2.88 (2 .65) 10.49 ( 10.82) 27

3.85 (3.40) 12.29 ( 12.36) 26

3.11 (3 .34) 12.08 ( 12.11 ) 25

3.21 (3.52) 12.02 ( 11.90) 2()

method, and the activity was expressed in te rms of diameters of zone of inhibition against diffcrent con­centrations of the drug. Innoculum was prepared by washing a fresh S ml medium slant of test organisms with 5 ml sterile water and further diluting the I ml washing to 10 ml. This suspension (O.IS ml) was added to 15 ml melted medium at a tcmperature of

45-S0°C and plates were prepared. Holes of diameter 6 mm were dug into the agar plates with a sterile borer and filled with different dilutions of the drug.

The plates were incubated for at 35°C for 18 h. The results were compared with that of chloramphenicol, a standard broad spectrum antibiotic.

Results and discussion In order to obtain products of high purity, it was

necessary to use column chromatography . TLC was attempted in order to determine efficient supports and eluent compositions. It was carried out using solvent mixture as CHCb-CH:;OH (80:20) . The solvent mix­ture provided sharp spots of main complex and one or two secondary spots, which had convenient Rr values. Column chromatography was performed with silica as the support with CHCI] -CH]OH as the eluate. It is well -known that thiosemicarbazides in the free state exist in the trans-configuration with respect to the thiocarbonyl sulphur and the terminal nitrogen atom of the thiosemicarbazide moiety. But during complex formation, they became cis to each other l4

. In the IR spectra of the ligands there are two strong bands in the 3000-3300 cm-t region, which corresponds to -NH2 stretching and anti-stretching vibrations , be­sides one or two NH vibration bands in the same re­gion. A strong band near 1600-1640 cm- t corresponds

NOTES 315

to -NH2 bending vibration and a band at 720-750 cm-I

corresponds to C=S stretching vibration. On com­plexation both the -NH2 bending and C=S stretching vibrations are lowered by 20-30 cm'l. Theseobserva­tions clearly indicate the participation of the NH2 group and thiocarbonyl sulphur in coordination to the metal ion . Representative IH NMR data, were ob­tained for all of the ten complexes. For [Ru(bpyh(CH,NHCSNHNH2)](CI04h, a complex series of resonance extending from 8 9.5 to 7.04 (area 16) was assigned to the aromatic protons on 2,2'­bipyridine. The molecule possesses C2 element of symmetry so that the eight bipyridine protons are ex­pected to be unique. A first order coupling scheme predicts four doublets and four triplets. Such splitting pattern have been observed for cis-(bpyhRuCI2 and cis-(dmbphRu(NH3h (ref. 15) (where dmbp is 5,5'­dimethyl-2,2'-bipyridine). In the aliphatic region one methyl resonance peak situated at 8 2.47 ppm (singlet, area 3) corresponds to the protons of the methyl group of 4-methylthiosemicarbazide. The relative ratio of aromatic protons to the methyl proton was 16 to 3 as expected for the proposed structure. The complex [Ru(bpyh(4-(p-methylphenyl) thiosemicarbazide)] (C104h showed a complicated pattern of resonance from 8 7.02 to 9.67 ppm. The observed pattern can be assigned as a superimposition of the aromatic protons on the bipyridine with those of phenyl group of 4-(p­methyl phenyl) thiosemicarbazide. The para methyl

group exhibited a sharp singlet. The relative ratio of aromatic protons to the methyl protons was 28 to 9 as expected for the proposed structure. These complexes show broad and intense visible bands between 350 to 450 nm due to metal to ligand charge transfer transi­tion I4.15

. In the UV region the bands at 290 nm and 3 \0 nm are assigned to bipyridine ligand. 1[-1[* charge transfer transitions l4. The same transition is found in free 2,2' -bipyridine at 280 nm so that coordination of the ligand results in a red shift in the transition en­ergy. There are also two shoulders at 390 and 500 nm which are tentatively attributed to a metal to ligand charge transfer transitions involving bipyridine li­gand.

The results of the antitumor screening studies clearly demonstrate the tumour inhibitory activity of the Ru complexes against the transplantable murine tumour cell line (Table 2). The mechanism by which these compounds mediate its antitumor effect is still to be elucidated. In the EAC bearing mice, cells are present in the peritoneal cavity and the compounds were administered directly into the peritoneum. Thus, tumour inhibition might be due to direct cytotoxic effect of the compounds on the tumour cells. The ef­fect of these compounds on DNA synthesis is yet un­known but certain structurally-related tris chelates of ruthenium are reported to have DNA binding property ill vitro7. Likewise the action of the synthesized com­pounds could also be mediated via its effect, if any,

Table 2-EfTect of Ru (II) complexes on tumour growth in EAC bearing mi ce

Complex Tumor volume Viable/non-vi<Jble MST (m l) cell count r<Jtio (clays)

Tumor control 5.2±0.01 24.01 21±O.RI

[Ru(bpy h( 4-CH, TSC)](CI04h 4.5±0.O4 1.52 23±O.47

[R u(bpy he 4-CH1CH 2 TSC)](CI04h 4.3±0.02 135 25±0.62

IRlI(bpyh(4-Cr,H J JTSC)](CI04h 3.9±0.08* 2.09 23±0.56

[RlI(bpyh(4-Ph TSC)](CI04)2 2.0±0.03* 0.61 32±0.24*

[Ru(bpyh(4p-CIPh TSCl](CI04l2 2.7±0.02* 0.52 24±0.26

[RlI(bpyh(4p-BrPh TSC)J(CI04l2 2.8±0.03 * 0.54 26±0.36

[RlI(bpyh(4p-IPh TSC))(C I0 4h 2.5±0.06* 0.48 28±O.S4*

[RlI(bpY)2(4p-CH, Ph TSC)](CI04h 2.6±0.02* 0.56 2R±O.35 *

[RlI(bpyh(4p-OCH, Ph TSC)](CI04) 2 2.5±0.04* 0.42 27±0.33*

[Ru(bpyh(4p-O C2H, Ph TSCl](CI04h 2.2±O.01 * 0.41 26±0.32

Cisplatin 0.2±0.002* 0.01 22±0.14

Values are mean ±SEM *P < 0.01 when compared to the control.

316 INDIAN J CHEM, SEC A, FEBRUARY 2003

Table 3-Effecl of Ru complexes on the hematological profile of the tumour hosts

Compound Hemoglobin RI3C count W/3C count (mg/dl) (cells x 10K

) (cells x I(h

Norillal control 12.2±D.04 7.2±0.02 6.I±O.OI

TUlllor Control R.I ±O.OS 4.I±O.03 19.2±O.O2

I Ru(bpYH4-CH ,TSC)](C10~h 10.0±0.03 S.O±O.03 17.4±0.04

/ Ru(bpy h( 4-CH,CH~ TSC) I(C104 h 9.4±0.OS 4.4±0.OS 17.2±O.OS

I Ru(bpyh(4-C(,f-I"TSC) I(CI04h 9.S±O.02 4.S±O.06 I x.ti±(J.m

I Ru(bpy),(4-l'h TSC)l(CI04)~ 11.1±0.O2 n.O±O.04 9.2±O.OI

IRu(bpyh(4p-CIPh TSC)](CI04 ), IO.2±0.04 n. I±004 10.6±0.O3

/ Ru(bpy),(4p-f3rPh TSC)](CI04 )2 11.2±0.04 S.7±O.07 11.I±O.02

I Ru(bpYh(4p-lph TSC) ](CI04h 11.3±O.(J6 6.3±O.OS 11.2±O.OS

I Ru(bpy),(4p-CH,Ph T5C)](CI04h 10.7±0.O2 S.9±0.02 12.3±O.O2

I Ru(bpyh(4p-OCH,Ph TSC)](CI04h II .O±O.04 6.0±O.O6 13.4±O.03

I Ru(bpYh(4p-0 C2H,Ph TSC) )(C104h 11.2±O.O3 S.6±0.OS 12.2±O.04

Cisplati n 7.1±0.OS 4.I±O.04 S.2±O.05

"Valucs are mean ± SEM

T able 4-Antimicrobial activity of the Ru complexes at 200 Ilg/ ml *

Compound KleiJ.I'i/!ae p"ell- Pset/(IIIIOIUlS Shigella Staphy lococci/s E.I'cherecliia coli /ilO/l eae ouregcJJo.\'(I SOllllei aurells

I Ru(bpyh(4-ClI ,T5C) I(C104h 12 12 II 12 12

/ Ru(bpy)~( 4-CH,CH2 T5C) I(C104 h II II 10 14 12

I Ru(bpYh(4-Cr.H "TSC)](CI04h II 12 IS 14 13

/ Ru(bpYh(4-Ph TSC)](CI04h 13 10 10 10 II

IRu(bpyh(4p-CIPh TSC)](CI04), 13 10 10 9 II

I R u(bPY h( 4p- B rPh T5C) I(CI04h II II 12 13 12

/Ru (bpy);(4p-IPh TSC)](CI04h II 10 13 13 12

/ Ru(bpyh(4p-CII , l'h TSC)I(CI04h 10 II II 12 10

I Ru(bpy )e( 4p-OCH, Ph TSC) I(CI04), II 12 10 13 II

/Ru(bpyh(4p-O C,H,Ph T5C)I(CIO,), 10 II II 13 10

Ch Imamphenicol 16 IR 20 20 16

* Valucs arc zone o f inhibition [mill , including thc diameter of the bore (6 mm)]

011 the DNA . Myelosuppression is a frequent and major complication of cancer chemotherapy. Com­pared to the pretreatment values in EAC, treatment with Ru complexes and subsequent tumour inhibition resulted in appreciable improvements in hemoglobin content, RBC and WBC counts (Table 3). These ob­servations assume great significance as anemia is a common complication in cancer l6 and the situation aggravates further during chemotherapy since a ma-

jority of antineoplastic agents exert suppressive ef­fects on erythropoiesis 17 and thereby limiting the use of these drugs. The improvements in hematological profile of the tumour bearing mice following the treatment with ruthenium compounds could be secon­dary to tumour regression or due to the action of the compounds itself. [n any case, the results of the pres­ent study are encouraging as these compounds exhibit significant reduction in the tumour burden and caused

NOTES 317

prolongation of lifespan of the hosts . Improvements, rather than aggravation, of tumour associated hema­tological complications such as anemia and bone mar­row suppression was noticed .

The complexes were found to possess antibacterial activity against both Gram +ve and Gram -ve stains of bacteria (Table 4). The mode of action may be dif­ferent from that of its antitumor activity. It was found however that the cycloalkyl and the alkyl substituents showed greater activity than the phenyl and para sub­stituted phenyl substituents whereas, the reverse was true for the antitumor activity. Among the para sub­stituted 4-phenyl thiosemicarbazide containing com­plexes the ethoxy and halogen derivatives were more active. The size of the halogen atom may also play an important part as the iodo and the bromo derivatives showed more activity than the chloro derivative. molecular mechanics simulations suggest that among the phenylthiosemicarbazide complexes the N4 atom is absolutely planar in geometry with the rest of the ligand molecule as would be expected in the free li­gand. This might have occurred due to repulsion of the phenyl ring hydrogen atoms with those of the bi­pyridine moiety. Further, the alkyl and cycloalkyl substituents provide for a more branched and or puck­ered stereochemistry, which may be vital for antimi­crobial activity. Overall, the complexes of this type were found to be potential bioactive material against tumours and pathogenic bacteria. Further work needs to be done to ascertain their mode of action and opti­mize the leads for synthesis.

Acknowledgement The authors are thankful to the University Grants

Commission and the All India Council for Technical Education for the funds utilized in this project.

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