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Spectrochimica Acta Part A 78 (2011) 80–87

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

alladium(II) and platinum(II) derivatives of benzothiazoline ligands: Synthesis,haracterization, antimicrobial and antispermatogenic activity

rishna Sharma, R.V. Singh, Nighat Fahmi ∗

epartment of Chemistry, University of Rajasthan, Jaipur 302004, India

r t i c l e i n f o

rticle history:eceived 6 April 2010eceived in revised form 3 August 2010ccepted 25 August 2010

eywords:alladium(II) and Platinum(II) complexes

a b s t r a c t

A series of Pd(II) and Pt(II) complexes with two N∩S donor ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and 6-nitro-3-(indolin-2-one)benzothiazoline, have been synthesized by thereaction of metal chlorides (PdCl2 and PtCl2) with ligands in 1:2 molar ratios. All the synthesized com-pounds were characterized by elemental analyses, melting point determinations and a combination ofelectronic, IR, 1H NMR and 13C NMR spectroscopic techniques for structure elucidation. In order to eval-uate the effect of metal ions upon chelation, both the ligands and their complexes have been screened

enzothiazolinespectral studiesntimicrobial and antifertility activity

for their antimicrobial activity against the various pathogenic bacterial and fungal strains. The metalcomplexes have shown to be more antimicrobial against the microbial species as compared to free lig-ands. One of the ligands, 5-chloro-3-(indolin-2-one)benzothiazoline and its corresponding palladium andplatinum complexes have been tested for their antifertility activity in male albino rats. The marked reduc-tion in sperm motility and density resulted in infertility by 62–90%. Significant alterations were foundin biochemical parameters of reproductive organs in treated animals as compared to control group. It is

ffect

concluded that all these e

. Introduction

Interest in coordination chemistry is increasing continuouslyith the preparation of organic ligands containing a variety ofonor groups and it is multiplied manifold when the ligands haveiological importance [1]. The number and diversity of nitrogennd sulfur chelating agents used to prepare new coordination andrganometallic compounds has increased rapidly during the pastew years [2–4]. Sulfur compounds and their metal complexes haventimicrobial activity and showed a high dependence on their sub-titutents [5,6]. Organic compounds containing –NC6H4S– moietyre well known for their significant biological activities. The activityay be due to the presence of multi coordination centers having

he ability to form stable chelates with the essential metal ionshich the organisms need in their metabolism. Transition metal

omplexes of several of these compounds have also been screenedor medicinal properties [7,8]. Various compounds of benzothiazo-ines have been tested for antifertility in male rats and were found

o show significant antifertility activity [9,10].

The coordination chemistry, biological effects and toxicology oflatinum and palladium complexes, such as their requirements inharmacological activities, are areas of increasing research inter-

∗ Corresponding author. Fax: +91 141 2704677.E-mail addresses: rvsjpr@hotmail.com, nighat.fahmi@gmail.com (N. Fahmi).

386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2010.08.076

s may finally impair the fertility of male rats.© 2010 Elsevier B.V. All rights reserved.

est [11]. Pd(II) and Pt(II) both the transition metals shows theiractive nature in various fields. It has been well established thatcertain platinum and palladium complexes are of biological impor-tance due to their anticancer [12], antitumor [13], antiamoebic [14]and catalytic activity [15]. Earlier studies from our laboratory [16]on biological activity of Schiff bases derived from isatins and theirmetal complexes, showed significant enhancement of antibacterialand antifungal activity of the isatin derivatives on complexation. Asurvey of literature revealed that so far no attention has been paidto compare the effects of benzothiazoline ligands with palladiumand platinum derivatives on the reproductive system of male rats.

In view of the diversified chelating behavior of benzothiazo-lines, as well as biological importance of palladium and platinumcomplexes, it has been considered worthwhile to synthesize, char-acterize some new palladium(II) and platinum(II) derivatives ofbenzothiazolines and to investigate their physico-chemical andstructural features as well as the biological activity.

2. Experimental

2.1. Analytical methods and physical measurements

Palladium and platinum salts, PdCl2 and PtCl2 as well as p-chloroaniline and m-nitroaniline were purchased from Lancasterand used as such. 5-Chloroisatin and 6-nitroisatin were preparedin the laboratory by the literature method [17,18]. All the solvents

K. Sharma et al. / Spectrochimica A

Tab

le1

An

alyt

ical

dat

aan

dp

hys

ical

pro

per

ties

ofth

eli

gan

ds

and

thei

rco

mp

lexe

s.

Com

pou

nd

Col

our

Mel

tin

gp

oin

t(◦

C)

Fou

nd

(cal

cd.)

(%)

Mol

.wt.

fou

nd

(cal

cd.)

HC

NS

Cl

M

Bzt

1H

Bro

wn

102–

105

3.01

(3.1

4)58

.18

(58.

23)

9.12

(9.7

0)10

.99

(11.

10)

––

279.

38(2

88.7

5)B

zt2H

Yel

low

150–

154

2.98

(3.0

3)56

.11

(56.

18)

13.9

2(1

4.03

)10

.53

(10.

71)

––

286.

08(2

99.2

9)[P

d(B

zt1H

) 2]C

l 2B

rick

160–

164

2.36

(2.4

0)44

.50

(44.

55)

7.38

(7.4

2)8.

42(8

.49)

18.2

5(1

8.78

)13

.98

(14.

09)

739.

28(7

54.8

0)[P

d(B

zt1) 2

]B

row

n12

0–12

41.

98(2

.06)

49.2

6(4

9.32

)8.

17(8

.21)

9.36

(9.4

0)10

.08

(10.

39)

15.2

4(1

5.60

)66

7.46

(681

.88)

[Pd

(Bzt

2H

) 2]C

l 2G

rap

hit

e13

2–13

582.

29(2

.33)

43.3

0(4

3.34

)10

.78

(10.

83)

8.20

(8.2

6)9.

02(9

.13)

13.3

2(1

3.73

)75

2.96

(775

.88)

[Pd

(Bzt

2) 2

]B

row

n12

0–12

31.

95(2

.00)

47.7

9(4

7.84

)11

.90

(11.

95)

9.08

(9.1

2)–

14.9

9(1

5.13

)76

1.16

(780

.96)

[Pt(

Bzt

1H

) 2]C

l 2G

reen

140–

143

2.09

(2.1

5)39

.79

(39.

87)

6.38

(6.6

4)7.

43(7

.60)

16.5

8(1

6.81

)2.

.01

(23.

12)

828.

30(8

43.4

9)[P

t(B

zt1) 2

]G

reen

140–

144

1.78

(1.8

3)43

.59

(43.

64)

7.18

(7.2

7)8.

13(8

.32)

9.04

(9.2

0)25

.31

(25.

20)

751.

42(7

70.5

7)[P

t(B

zt2H

) 2]C

l 2G

rap

hit

e14

8–15

02.

01(2

.09)

38.8

2(3

8.89

)9.

68(9

.72)

7.37

(7.4

1)8.

02(8

.20)

22.5

6(2

2.36

)84

4.26

(864

.57)

[Pt(

Bzt

2) 2

]B

row

n14

2–14

51.

70(1

.78)

42.4

1(4

2.48

)10

.45

(10.

61)

8.10

(8.0

2)–

24.3

0(2

4.64

)78

0.58

(791

.67)

cta Part A 78 (2011) 80–87 81

were dried and distilled before use. Molecular weights were deter-mined by the Rast Camphor method. Chlorine was estimated byVolhard’s method. Pd(II) and Pt(II) were estimated gravimetrically.Nitrogen was estimated by the Kjeldahl’s method and sulfur wasestimated by the Messenger’s method. The electronic spectra wererecorded on a Varian–Cary/2390 spectrophotometer at RSIC, I.I.T.,Chennai. Infrared spectra of the ligands and their complexes wererecorded with the help of Nicolet Megna FTIR-550 spectrometeron KBr pellets. 13C NMR and 1H NMR spectra were recorded on aJEOL-AL-300 FT NMR spectrophotometer in DMSO-d6 using TMSas the internal standard. Conductivity measurements were madewith a Systronics model 305 conductivity bridge. The purity of thesynthesized complexes was checked by thin layer chromatography(TLC).

2.2. Preparation of benzothiazolines

The benzothiazolines were prepared by the condensation of2-aminothiophenol with 5-chloro-3-(indolin-2-one) and 6-nitro-3-(indolin-2-one) in 1:1 molar ratio in ethanol, respectively. Thereaction mixture was stirred for 3–4 h and the resulting productwas filtered off, recrystallized from ethanol and dried in vaccum.The analytical results came in good consistence with the proposedformulas as in Table 1 (Fig. 1).

2.3. Preparation of metal complexes

2.3.1. Palladium complexesThe methanolic solution of PdCl2 was mixed with methanolic

solution of benzothiazoline in 1:2 molar ratio. Aqueous ammoniawas added dropwise to the reaction mixture until it was weaklyalkaline (pH ca. 8.0) and this reaction mixture was then heatedunder reflux for about 1 h to synthesize Pd(Bzt)2 type of complexes.

To obtain the [Pd(BztH)2]Cl2 type of complexes the methanolicsolution of PdCl2 and benzothiazoline in 1:2 molar ratio was stirredon a magnetic stirrer for 2–3 h in presence of few drops of con-centrated HCl. The resulting products were recovered by filtration,washed with methanol and dried in vacuum.

2.3.2. Platinum complexesThe 1:1 water–ethanol solution of PtCl2 was mixed with an

ethanolic solution of benzothiazoline in 1:2 molar ratio. To obtainthe Pt(Bzt)2 type of complexes aqueous ammonia was added drop-wise to the reaction mixture until it was weakly alkaline (pH ca. 8.0).The reaction mixture was then heated under reflux for about 1 h.On cooling, the complexes were separated out which were filteredand washed with ethanol and dried in vacuum.

On the other hand, [Pt(BztH)2]Cl2 type of complexes have beensynthesized by stirring the above reaction mixture (solution ofPtCl2 + benzothiazoline in 1:2 molar ratio) on a magnetic stirrer for2–3 h in presence of few drops of concentrated HCl in place of aque-ous ammonia. The resulting product was recovered by filtration,washed with ethanol and dried in vacuum.

2.4. Biological tests

Antimicrobial activity of the ligands and their corresponding

metal complexes was tested in vitro for the growth inhibitingpotential against various pathogenic fungal and bacterial strains.Fungal strains, Fusarium oxysporum and Alternaria alternata andbacterial strains, P. aeruginosa and E. coli were used. One of theligands, 5-chloro-1H-indol-2,3-dione benzothiazoline and its cor-responding metal complexes have been tested in vivo for theirantifertility activity in male albino rats.

82 K. Sharma et al. / Spectrochimica Acta Part A 78 (2011) 80–87

. X = 5-

22a[orddwmai2aa

2dftaaai1aoticra

%

wc

22trrbs

1cdItGg6

Fig. 1. Synthesis of the ligands

.4.1. In vitro study

.4.1.1. Antibacterial screening. Antibacterial activity was testedgainst P. aeruginosa and E. coli using the paper disc plate method19]. Each of the compounds was dissolved in DMSO and solutionsf the concentrations (500 and 1000 ppm) were prepared sepa-ately. Paper discs of Whatman filter paper (No. 42) of uniformiameter (2 cm) were cut and sterilized in an autoclave. The paperiscs soaked in the desired concentration of the complex solutionsere placed aseptically in the Petri dishes containing nutrient agaredia (agar 20 g + beef extract 3 g + peptone 5 g) seeded with P.

eruginosa and E. coli bacteria separately. The Petri dishes werencubated at 37 ◦C and the inhibition zones were recorded after4 h of incubation. The antibacterial activity of a common standardntibiotic tetracycline was also recorded using the same procedures above at the same concentrations and solvent.

.4.1.2. Antifungal screening. The antifungal activity of the stan-ard fungicide (Flucanazone), ligands and complexes were testedor their effect on the growth of microbial cultures and studied forheir interaction with F. oxysporum and A. alternata using Czapek’sgar medium[20] having the composition, glucose 20 g, starch 20 g,gar-agar 20 g and distilled water 1000 mL.To this medium wasdded requisite amount of the compounds after being dissolvedn dimethylformamide so as to get the certain concentrations (50,00 and 200 ppm). The medium then was poured into Petri platesnd the spores of fungi were placed on the medium with the helpf inoculum’s needle. These Petri plates were wrapped in poly-hene bags containing a few drops of alcohol and were placed in anncubator at 30±2 ◦C. The controls were also run and three repli-ates were used in each case. The linear growth of the fungus wasecorded by measuring the diameter of the fungal colony after 96 hnd the percentage inhibition was calculated by the equation:

I = dC − dT

dC× 100

here dC and dT are the diameters of the fungus colony in theontrol and test plates, respectively.

.4.2. In vivo study

.4.2.1. Antifertility activity. The activity of the synthetic productsowards the biological systems is an important feature of the cur-ent research and the Schiff base metal complexes play a significantole in this direction. In view of such potential interest in theseiologically active compounds, the antifertility activity of someelected compounds has been studied on male albino rates.

Proven-fertile male albino rats of the Wistar strain, weighing78–200 g (90–100 days old), were used. They were housed in stealages and maintained under standard conditions (12 h light/12 hark; 25±3 ◦C; 35–60% relative humidity). Rat feed (Ashirwad

ndustries Ltd., Chandigarh, India) and water was provided ad libi-um. Animals were divided into six groups of five animals each.roup A served as a vehicle (olive oil) treated control. In this controlroup, only olive oil (0.5 mL/rat/day) was orally administered for0 days. Groups B, C, D, E and F were correspond to ligand (BztH1)

Cl (Bzt1H) and 6-NO2 (Bzt2H).

and metal complexes, [Pt(Bzt1H)2]Cl2, Pt(Bzt1H)2, [Pd(Bzt1H)2]Cl2and Pd(Bzt1)2, respectively. The oral administration of these groupswas given in olive oil at the same doses (10 mg/rat/day) for 60 days.The rats were cohabited with the proestrus females in 1:2 ratio toassess the fertility test by natural mating. The mating exposure testsof various compounds were performed before and on 55th day oftreatment. The presence of sperm cells in the vaginal smears wasaccepted as evidence of copulation. Mated females were separatedand then allowed to complete the term. The number of litters deliv-ered was recorded and used as an index for fertility of the males.Body weights of the experimental rats were monitored throughoutthe study.

All treated male rats were anesthetized on the day 61st withsolvent ether and their testes, epididymis, ventral prostate andseminal vesicles were dissected out and weighed. Final bodyweights of the animals were recorded. Sperm mobility in caudaepididymis and sperm density in testes and cauda epididymis wereassessed. The protein, sialic acid, glycogen, fructose and cholesterolwere estimated in testes, epididymis and accessory sex organs byusing standard laboratory techniques. Results were analysed sta-tistically using student’s t-test.

3. Results and discussion

The metal chloride interacts with the ligands in 1:2 molarratios in the presence of few drops of concentrated HCl to form[M(BztH)2]Cl2 type of complexes as follows:

MCl2 + 2BztH1:2−→HCl

[M(BztH)2]Cl2

However, the replacement of both chloride ions is possible in thepresence of aqueous ammonia. The reaction can be depicted asfollows:

MCl2 + 2BztH1:2−→

NH4OH[M(Bzt)2]+ 2NH4Cl+ 2H2O

where M = Pd(II) and Pt(II) and BztH is the benzothiazolinemolecule.

The reactions proceed easily and all the complexes are coloredsolids. All the complexes are soluble in DMSO, DMF and CHCl3and insoluble in common organic solvents. The molar conductanceof 10−3 M solutions of [M(BztH)2]Cl2 type complexes at the roomtemperature lie in the range of 200–218 ohm−1 mol−1 cm2, indicat-ing that they behave as 1:2 electrolytes. However, the complexesof the type M(Bzt)2 are nonelectrolytes (16–20 ohm−1 mol−1 cm2).The metal complexes are diamagnetic, as expected for square pla-nar d8 complexes [21]. Their magnetic susceptibilities lie in therange 0.3–0.8×10−6 c.g.s. units. The complexes are monomers asrevealed by their molecular weight determinations (Table 1).

3.1. Electronic spectra

The electronic spectra of some representative metal complexeswere recorded in distilled DMSO. In the spectra of these complexes

K. Sharma et al. / Spectrochimica Acta Part A 78 (2011) 80–87 83

Table 2Electronic spectral data (cm−1) of palladium(II) and platinum(II) complexes.

Complexes Transition Spectral bands (cm−1) �1 �2 �3 �1/�2

[Pd(Bzt1H)2]Cl2 1A1g→ 1A2g (�1) 22,222 24,322 3368 1976 1.091A1g→ 1B1g (�2) 24,0381A1g→ 1E1g (�3) 27,624

[Pd(Bzt1)2] 1A1g→ 1A2g (�1) 20,618 22,718 4405 3199 1.161A1g→ 1B1g (�2) 23,9231A1g→ 1E1g (�3) 27,322

[Pt(Bzt2H)2]Cl2 1A1g→ 1A2g (�1) 20,833 22,933 4176 3064 1.141A1g→ 1B1g (�2) 23,8091 1

ttdso1ttbtsTw

3

3

(Teb

3

wbdmdstta2

T1

bm

A1g→ E1g (�3) 27,173[Pt(Bzt2)2] 1A1g→ 1A2g (�1) 20,703

1A1g→ 1B1g (�2) 23,5291A1g→ 1E1g (�3) 27,027

hree d–d spin allowed transitions are observed corresponding tohe transitions from the three lower lying ‘d’ orbital to the empty

x2−y2 orbital. The ground state is 1A1g and excited states corre-ponding to the above transitions are 1A2g, 1B1g and 1E1g in orderf increasing energy. These d–d transition bands in the regions,9,801–20,833, 23,509–24,038 and 27,027–27,624 cm−1 attributedo 1A1g→ 1A2g, 1A1g→ 1B1g and 1A1g→ 1E1g transitions, respec-ively. Three different orbital parameters �1, �2, �3 have alsoeen calculated by first using the correlation, F2 0F2 600 cm−1 forhe Slater–Condon interelectronic repulsion parameters and sub-equently the equations suggested by Gray and Ballhausen [22].he �2/�1 were also calculated (Table 2) and are in close agreementith data reported earlier for the square planar geometry [23].

.2. IR spectra

.2.1. IR spectra of the ligandsIn the IR spectra of the free benzothiazolines (Bzt1H) and

Bzt2H), the �(NH) stretching band appears at 3265–3380 cm−1.he absence of �(SH) and �(C N) bands is a strong evidence for thexistence of benzothiazoline ring structure rather than the Schiffase structure [24].

.2.2. IR spectra of the metal complexesA comparative study of the IR spectra of the metal complexes

ith that of free ligands shows disappearance of �NH absorptionands in the spectra of the metal complexes which indicates itseprotonation and chelation of nitrogen of the ligands to the centraletal atom. The appearance of new sharp band at 1610–1620 cm−1

ue to �C N in the spectra of the metal complexes very well

upports the fact that the resulting complexes are metal azome-hine derivatives, as the benzothiazoline ring rearranges to givehe Schiff base form [25] which in presence of metal ion finallycts as a monobasic bidentate ligand. The bands due to �(SH) at522–2600 cm−1 are observed in the [M(BztH)2]Cl2 type of com-

able 3H NMR spectral data (ı, ppm) of the ligands and their corresponding complexes.

Compound –NH (isatin ring) (bs) –NH (r

Bzt1H 1190 4.26Bzt2H 1192 4.30[Pd(Bzt1H)2]Cl2 11.91 –[Pd(Bzt1)2] 11.93 –[Pd(Bzt2H)2]Cl2 11.92 –[Pd(Bzt2)2] 11.94 –[Pt(Bzt1H)2]Cl2 11.92 –[Pt(Bzt1)2] 11.94 –[Pt(Bzt2H)2]Cl2 11.93 –[Pt(Bzt2)2] 11.91 –

s, broad signal., multiplet.

22,804 4025 3198 1.13

plexes suggesting the coordination of sulfur to the metal atom inthese complexes. However, no �(M–Cl) band is observed in thespectra of [M(BztH)2]Cl2 type of complexes, suggesting that chlo-ride is ionic in these complexes. The appearance of the bands at350–450 and 300–312 cm−1 due to �(M←N) and �(M–S), respec-tively, further support the coordination of the ligands to the metalions through the azomethine nitrogen and thiolato sulfur atoms.

3.3. 1H NMR spectra

To further confirm the bonding pattern in the metal complexesthe 1H NMR spectra of the free (Bzt1H) and (Bzt2H), and their metalderivatives have been recorded in DMSO-d6 using TMS as internalreference.

3.3.1. 1H NMR spectra of the ligandsThe spectra of free ligands show the –NH proton signals

at ı4.26–4.30 ppm. The free ligands show a complex multi-plet at ı6.50–8.85 for the aromatic protons and a singlet atı11.90–11.94 ppm due to –NH of the isatin ring.

3.3.2. 1H NMR spectra of the complexesThe –NH protons signals disappear in the metal complexes,

indicating the deprotonation of this group on complexation. Inthe [M(BztH)2]Cl2 type of complexes the signals appearing atı4.75–4.80 ppm are due to the SH proton and these are not observedin [M(Bzt)2] type of complexes. The signal due to –NH of the isatin

ring remains unaltered in the complexes indicating that the –NHgroup of the isatin ring is not taking part in the complexation. Thefree ligands show a complex multiplet for the aromatic protonswhich remains more or less at the same position in the spectra ofmetal complexes (Table 3).

ing) (bs) –SH (bs) Aromatic protons (m)

– 6.70–8.74– 6.65–7.204.75 6.74–8.02– 6.72–8.004.78 6.78–8.09– 6.50–8.004.76 6.55–8.85– 6.58–8.264.80 6.60–8.40– 6.90–8.46

84 K. Sharma et al. / Spectrochimica A

F(

3

wiosgmtltna1

lis

4

4

ai

ig. 2. Suggested structures for the metal complexes. M = Pd(II) and Pt(II), X = 6-ClBzt1H) and 6-NO2 (Bzt2H).

.4. 13C NMR spectra

The comparison of the 13C NMR spectrum of benzothiazolinesith their corresponding metal complexes reveals some useful

nformation about the mode of bonding as well as the geometryf compounds. The signal observed at ı162.56–165.42 ppm in thepectrum of free benzothiazolines has been assigned to >C(R)N–roup. A downfield shift of this carbon signal in the spectrum ofetal complexes which appears at ı172.30–174.24 ppm confirms

he formation of >C N– group by rearrangement of benzothiazo-ine ring and subsequent formation of Schiff base derivative withhe formation of M←N and M–S bonds. The aromatic carbon sig-als are observed in the range ı120.31–149.97 ppm, which remainlmost same in the spectra of metal complexes. Thus, the 1H and3C NMR spectra, confirms the monobasic bidentate nature of theigands, which has already been suggested by the IR spectral stud-es, discussed above. On the basis of the above discussion, followingtructures have been suggested for the metal complexes (Fig. 2).

. Bioassay

.1. Antimicrobial activity

The antimicrobial screening data show that both the ligandsnd their complexes individually exhibited varying degrees ofnhibitory effects on the growth of the tested bacterial and fun-

cta Part A 78 (2011) 80–87

gal species. The antibacterial (Fig. 3) and antifungal (Fig. 4) resultsevidently show that the activity of the ligands became more pro-nounced when coordinated to the metal. The increased activity ofthe metal chelates can be explained on the basis of chelation the-ory [26], according to which the polarities of the ligand and thecentral metal atom are reduced through charge equilibration overthe whole chelate ring. This increases the lipophilic character ofthe metal chelate and favours its permeation through the lipoidlayer of the bacterial membranes. The variation in the effective-ness of different compounds against different organisms dependseither on the impermeability of the cells of the microbes or thedifference in ribosomes of microbial cells [27]. It has also been pro-posed that concentration plays a vital role in increasing the degreeof inhibition; as the concentration increases, the activity increases.

4.2. Antifertility activity

4.2.1. Body and organ weightsAdministration of ligand(Bzt1H) and its metal complexes

[Pt(Bzt1H)2]Cl2, Pt(Bzt1H)2 [Pd(Bzt1H)2]Cl2 and Pd(Bzt1)2 did notaffect the body weights of treated animals. During the study, all thetreated animals showed the normal behavior and they were healthyin appearance. However, a significant (P≤0.01– to P≤0.001) reduc-tion was observed in the weights of testes, epididymides andaccessory sex organs (seminal vesicles and ventral prostate) in ratstreated with these compounds than those of the control group(Table 4). Reduction in weights may reflect a declined amount andsynthesis of androgen within these organs [28].

4.2.2. Sperm motility and sperm densityA significant (P≤0.01 to P≤0.001) decline in sperm density

in testes and epididymis were noticed in rats treated with lig-and and its metal complexes. Sperm motility in cauda epididymiswas also decreased significantly in experimental animals (Table 5).Decrease in sperm motility and density could compromise thefertility [29]. These depletions suggest alterations in sperm mat-uration and sperm production [30]. Sperm count is considered tobe one of the important factors that affect fertility. Low sperm con-centration is associated with low fertility. This may be related todecrease testicular size, which may be caused by androgen depri-vation. Sperm must be motile to penetrate through the survivalmucus and to migrate through the female genital tract to the site offertilization. Thus, sperm motility is one of the most important pre-dictors of sperm fertilizing ability. In this investigation, the motilityof spermatozoa collected from cauda epididymis was hampered incase of the ligand and its metal complexes as compared to controlgroup. Sperm motility may be affected by inhibition of adenosinetriphosphate (ATP) by uncoupling of oxidative phosphorylation andthus renders the spermatozoa immotile. Suppressed sperm motilityand density may cause of 62–90% infertility.

4.3. Biochemical changes

4.3.1. Protein and sialic acidProtein and sialic acid contents of testes, epididymis, ventral

prostate and seminal vesicle were reduced after the treatment withthe ligand and its metal complexes (Tables 6 and 7).

4.3.2. Cholesterol, glycogen and fructoseReduction in testicular glycogen and fructose content of semi-

nal vesicle was noticed in animals treated with ligand and its metalcomplexes whereas testicular cholesterol was increased (Table 8).Thus the treatment of compounds B, C, D, E and F brought aboutthe alteration in biochemical parameters. The reduction of pro-tein contents in reproductive organs may reflect the alteration in

K. Sharma et al. / Spectrochimica Acta Part A 78 (2011) 80–87 85

Fig. 3. Antibacterial screening of the ligands and their metal complexes.

Fig. 4. Antifungal screening of the ligands and their metal complexes.

Table 4Effects of the ligands and their complexes on body and organ weights of male rats.

Group Treatment Body weight (g) Organ weight (mg/100 g b wt.)

Initial Final Testes Epididymis Seminal vesicle Ventral prostrate

A Control 182.18 ± 16 203.84 ± 19 1226 ± 72 472.34 ± 56 473.74 ± 37 261.55 ± 19B Bzt1H 188.42 ± 15 210.15 ± 20* 873.37 ± 32** 283.36 ± 30*** 279.81 ± 26** 194.32 ± 15***

C [Pt(Bzt1H)2]Cl2 178.12 ± 18 204.52 ± 10* 655.02 ± 39** 217.47 ± 15*** 188.36 ± 24** 141.04 ± 9**

D [Pt(Bzt1H)2] 183.84 ± 21 199.40 ± 17* 693.77 ± 49*** 222.40 ± 10*** 207.65 ± 18*** 132.30 ± 13**

E [Pd(Bzt1H)2]Cl2 194.16 ± 12 221.63 ± 25* 699.36 ± 41*** 200.03 ± 9*** 174.37 ± 20** 127.07 ± 14**

F [Pd(Bzt1)2] 200.38 ± 19 223.15 ± 18* 705.13 ± 46*** 209.36 ± 11*** 185.12 ± 10** 120.93 ± 16**

Values means± SE of six determinations.Groups B compared with Group A.Groups C, D, E and F compared with Group B.

* N.S. or Nonsignificant.** P < 0.001

*** P = 0.01.

86 K. Sharma et al. / Spectrochimica Acta Part A 78 (2011) 80–87

Table 5Effects of the ligands and their complexes on sperm dynamics of male rats.

Group Treatment Sperm motility (%) cauda epididymis Sperm density (million/mL) Fertility (%)

Testes Epididymis

A Control 73.86 ± 2.28 3.52 ± 0.24 59 ± 5.1 95 (positive)B Bzt1H 66.14 ± 2.00*** 2.22 ± 0.42*** 44 ± 3.7*** 62 (negative)C [Pd(Bzt1H)2]Cl2 58.73 ± 2.89*** 1.08 ± 0.38*** 32 ± 1.8** 78 (negative)D [Pd(Bzt1)2] 52.51 ± 3.11** 0.867 ± 0.40*** 39 ± 2.7*** 84 (negative)E [Pt(Bzt1H)2]Cl2 55.62 ± 2.79*** 0.995 ± 0.34*** 35 ± 1.6*** 90 (negative)F [Pt(Bzt1H)2] 48.77 ± 3.91** 1.00 ± 0.30*** 31 ± 2.2*** 73 (negative)

Values means± SE of six determinations.Groups B compared with Group A.Groups C, D, E and F compared with Group B.*N.S. or Nonsignificant.

** P < 0.001*** P = 0.01.

Table 6Changes in sialic acid contents of genital organs of male rats after treatment with ligand and their corresponding metal complexes.

Group Treatment Sialic acid (mg/g)

Testes Epididymis Seminal vesicle Ventral prostate

A Control 8.67 ± 0.19 7.42 ± 0.24 7.86 ± 0.27 8.13 ± 0.17B Bzt1H 7.13 ± 0.29** 5.90 ± 0.20** 6.65 ± 0.13** 6.63 ± 0.31**

C [Pd(Bzt1H)2]Cl2 6.24 ± 0.11*** 4.98 ± 0.29*** 5.95 ± 0.19*** 4.98 ± 0.18**

D [Pd(Bzt1)2] 5.95 ± 0.24** 5.10 ± 0.23*** 5.42 ± 0.24** 4.72 ± 0.30**

E [Pt(Bzt1H)2]Cl2 6.09 ± 0.17*** 4.77 ± 0.18** 4.96 ± 0.30** 5.55 ± 0.15***

F [Pt(Bzt1H)2] 6.12 ± 0.22*** 4.82 ± 0.25** 5.31 ± 0.27** 5.19 ± 0.13**

Values means± SE of six determinations.Groups B compared with Group A.Groups C, D, E and F compared with Group B.*N.S. or Nonsignificant.

** P < 0.001*** P = 0.01.

Table 7Changes in protein contents of genital organs of male rats after treatment with ligand and their corresponding metal complexes.

Group Treatment Protein (mg/g)

Testes Epididymis Seminal vesicle Ventral prostate

A Control 198.29 ± 9.28 217.41 ± 6.99 220.35 ± 7.14 237.60 ± 10.64B Bzt1H 153.38 ± 6.22** 186.25 ± 8.63** 182.20 ± 6.96** 191.84 ± 9.58**

C [Pd(Bzt1H)2]Cl2 122.96 ± 6.12** 147.94 ± 5.95** 155.01 ± 6.11** 149.93 ± 4.72**

D [Pd(Bzt1)2] 117.12 ± 5.27** 139.44 ± 5.94** 149.51 ± 5.59** 154.40 ± 7.70**

E [Pt(Bzt1H)2]Cl2 120.04 ± 5.99** 132.79 ± 6.89** 152.26 ± 6.69** 152.16 ± 5.86**

F [Pt(Bzt1H)2] 114.56 ± 6.31** 141.62 ± 7.92** 167.27 ± 5.71*** 150.33 ± 6.31**

Values means± SE of six determinations.Groups B compared with Group A.Groups C, D, E and F compared with Group B.*

TC

VGG*

N.S. or Nonsignificant.** P < 0.001

*** P = 0.01.

able 8hanges in cholesterol, glycogen and fructose levels in rats after treatment with ligand an

Group Treatment Seminal vesic

Fructose

A Control 4.71 ± 0.16B Bzt1H 3.75 ± 0.21**

C [Pd(Bzt1H)2]Cl2 2.88 ± 0.20**

D [Pd(Bzt1)2] 2.75 ± 0.19**

E [Pt(Bzt1H)2]Cl2 2.53 ± 0.23**

F [Pt(Bzt1H)2] 2.79 ± 0.15**

alues means± SE of six determinations.roups B compared with Group A.roups C, D, E and F compared with Group B.

N.S. or Nonsignificant.** P < 0.001

*** P = 0.01.

d its corresponding metal complexes.

le Testicular amount (mg/g)

Cholesterol Glycogen

8.13 ± 0.11 5.14 ± 0.198.96 ± 0.29*** 4.12 ± 0.13**

* 10.18 ± 0.22** 3.60 ± 0.20***

10.36 ± 0.24*** 3.24 ± 0.12**

9.95 ± 0.20** 3.39 ± 0.14**

10.49 ± 0.31** 3.17 ± 0.18**

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[[[[34] K.M. Avari, D.A. Bhiwgada, Indian J. Exp. Biol. 30 (1992) 1118.[35] M.K. Saini, M. Swami, N. Fahmi, Kusum Jain, R.V. Singh, J. Coord. Chem. 62 (2009)

3986.[36] P.K. Sharma, H. Rehwani, A.K. Rai, R.S. Gupta, Y.P. Singh, Bioinorg. Chem. Appl.

2006 (2006) 16895, doi:10.1155/BCA/2006/16895.[37] T. Pandey, R.V. Singh, Main Group Met. Chem. 23 (2000) 345.

K. Sharma et al. / Spectrochi

esticular function [31]. The structural integrity of acrosomal mem-rane is dependent upon sialic acid and due to alteration in itsontent, the motility and fertilizing capacity of sperm may also beffected [32]. Testicular glycogen was found to be decreased at aignificant level, it may be correlated to diminished postmeioticerm cells (secondary spermatocytes and spermatids) which arehe site of glucose metabolism. The fructose content of the sem-nal vesicle was decreased significantly. It may be suggested thathese compounds hamper the glycolytic metabolism of sperma-ozoa resulting in abnormal sperm function [33]. The significantP < 0.001) elevation in concentration of testicular cholesterol mayndirectly indicate the reduced level of circulating testosteronend thus impairment of spermatogenesis takes place [34]. Theseesults may also be correlated with the well-known fact that sulfur-ontaining compounds produce infertility in male rats [35]. Thus, itan be postulated that coordination through sulfur atoms induceshe sterilizing activity in the biological systems.

. Conclusion

We have synthesized biologically relevant ligands and theird(II) and Pt(II) metal ion complexes. Based on various physico-hemical investigations a square planar environment around theetal ions has been proposed. The complexes showed higher

ntimicrobial and antifertility activities as compared to the parentigands. A large number of coordination complexes of nitro-en/sulfur donor ligands have been extensively screened forheir biological activity. A comparison has been made betweenntimicrobial and antispermatogenic activities of the investigatedomplexes with the similar type of complexes studied by otheresearchers [36,37]. The results indicated that the palladium andlatinum complexes exhibited similar antimicrobial and antisper-atogenic activities.

cknowledgements

The authors, Prof. R.V. Singh, Krishna Sharma are thankful toSIR and Nighat Fahmi is thankful to UGC (Grant No. 36-1/2008RAJ) (SR)), New Delhi, for financial assistance.

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