Synthesis of some Cd(II) and Zn(II) complexes of a tetraazamacrocyclic ligand and their antimicrobial activities

Synthetic macrocycles specially the tetraazamacrocycles and their complexes have attracted considerable interests owing to their wide variety of applications. The ligand, 3, 10-C-meso3,5,7,7,10,12,14,14-octamethy1-1,4,8,11tetraazacyclotetradeca-4,11-diene(L 1 ), upon reaction with CdI2, Zn(ClO4)2, and Zn(CH3COO)2 produces corresponding square pyramidal cadmium (II) and zinc (II) complexes of formula [ML 1 X]Yn, (M = Cd or Zn; X = I, CH3COO or H2O; Y= I, ClO4 or CH3COO; n = 1 or 2). Among them, [CdL 1 I]I and [ZnL 1 (H2O)](ClO4)2 undergo substitution reactions with KSCN and NaNO2, respectively to produce octahedral transdiisothiocyanatocadmium(II) complex, trans-[CdL 1 (NCS)2] and square pyramidal mononitratozinc(II) nitrate complex, [ZnL 1 (NO2)](NO2). Antifimgal and antibacterial activities of these complexes against some of phytopathogenic fungi and bacteria have been investigated in this study.


Introduction
Organometalicmacrocycles compounds like tetraazamacrocycles and their complexes have attracted considerable interests due to their wide variety of applications: such as in magnetic resonance imaging (MRI) and radio immunotherapypharmacological, industrial and analytical fields (Bembi et al., 1991;Roy et al., 2011Roy et al., , 2010Roy et al., , 2010aRoy et al., , 2008)).Recent interests have been stimulated by the diagnostic and therapeutic medicinal applications of transition metal complexes of macrocyclic ligands.Moreover concerning biological applications, metal complexes of fourteen membered tetraazamacrocyclic ligands have also been explored for their antifungal and antibacterial (Roy et al. 2009;Shin et al. 2007) activities as well their use as anticancer and antitumor drugs (Kim et al. 2005;Roy et al. 2006).Against this background, a number of copper, nickel and cobalt complexes of 3, 10-C-meso-Me 8tetraazacyclotetradecadiene (L 1 ) (Figure 1) have been synthesized and explored for their anti-fungal and antibacterial activities (Horn et al. 2001;Roy et al. 2011a).Template synthesis of four coordinate copper (II) and nickel (II) complexes has also been achieved (Bembi et al. 1988).However cadmium (II) and zinc (II) complexes of this ligand have not been reported so far.Thus the present investigation describes the synthesis, characterization, antifungal and antibacterial activities of some new cadmium (II) and zinc (II) complexes of 3, 10-C¬meso-Me 8 -tetraazacyclotetradecadiene (L 1 ).

Materials and Methods
All the chemicals and solvents used in the reactions were of AR grade and obtained from commercial sources (Merck, Germany).The solvents were dried using standard literature procedures.Microanalyses of C, H, N and S of complexes have been carried out on a C, H, N, S analyzer at the Inorganic Research Laboratory of the Institut der Anorganische and Angewandte Chemie, Hamburg Universitaet, Germany.The infrared spectra of the metal complexes were taken as KBr discs in the range 4000-400 cm -1 at BCSIR laboratory, Dhaka.Electronic spectra of the samples were recorded on a Schimadzu UV-visible spectrophotometer at BCSIR laboratory Chittagong, Bangladesh.Conductance measurements of the metal complexes were done in CHCl 3 , DMF and DMSO solutions using HANNA instrument with HI 8820N conductivity cell at the University of Chittagong.The 1 H-NMR spectra were recorded in CDC1 3 and DMSO with a 400 MHz Bruker DPX-400 spectrometer using TMS as internal standard at the BCSIR Laboratory, Dhaka, Bangladesh.Magnetic measurements were carried out with a Gouy balance, calibrated against Hg[Co(NCS) 4 ]; susceptibilities were corrected for diamagnetic increments.

Synthesis of cadmium(II) complexes 2.2.1. Synthesis of [CdL 1 I]I
The free ligand L 1 (0.308 g, 1.0 mmol) and cadmium (II) iodide (0.366g, 1.0 mmol) were dissolved separately in hot methanol (40 mL) and mixed.The reaction mixture was heated for 30 min on a steam bath to ensure the completion of the reaction and then allowed to cool.After one day the white product [CdL 1 I]I was filtered off, washed with methanol followed by diethyl ether and dried in desiccators over silica gel.Decomposition point: 144°C.Yield 65% (found: C, 32.08; 14, 5.39; N, 8.33%; C I8 H 36 N 4 CdI 2 requires C, 32.04; H, 5.38; N, 8.30%).

Synthesis of axial ligand substitution product, [CdL 1 (SCN) 2 from [CdL 1 I]I
A suspension of [CdL 1 I]I (0.674 g, 1.0 mmol) in hot methanol (40 mL) was added to a solution of KSCN (0.194 g, 2.0 mmol) in hot methanol (40 mL) and the mixture was heated for 30 min on a steam bath and then allowed to cool.After one hour the white product [CdL I (SCN) 2 ] was filtered off, washed with methanol followed by diethyl ether and dried in a desiccator over silica gel.A solution of ligand L 1 ( 0.308 g, 1.0 mmol) in hot methanol (40 mL) was mixed with a solution of zinc (II) perchlorate hexahydrate (0.372g, 1.0 mmol) in hot methanol (40 mL).The resulting solution was refluxed for 30 min on a steam bath and then allowed to cool.After one day the white product [ZnL 1 (H 2 O)](ClO 4 ) 2 was filtered off, washed with methanol followed by diethyl ether and dried in a desiccator over silica gel.Decomposition point: 164°C.Yield 60% (Found: C, 36.86;H, 6.32; N, 9.39%; C 18 H 38 N 4 ZnC1 2 O 9 rerquires C, 36.59;H, 6.48; N, 9.48%).

Synthesis of [ZaL l (CH 3 COO)](ClO 4 )
To a filtered solution of zinc (II) acetate dihydrate (0.219 g, 1.0 mmol) in hot methanol (40 mL), a suspension of ligand salt L 1 .2HClO4 (0.509 g, 1.0 mmol) in the same solvent (40 mL) was added.A white product separated out after heating for 30 min.The reaction mixture was heated for further 30 minutes on a steam bath while the volume was reduced to 40 cm 3 and then allowed to cool.After one day the white product [ZaL l (CH 3 COO)](ClO 4 ) was filtered off, washed with methanol followed by diethyl ether and dried in a desiccator over silica gel.Melting point: above 220°C.Yield 68% (found:, C, 45.28; H, 7.41; N, 10.49%) C 20 H 39 N 4 ZnClO 6 , requires C, 45.12; H, 7.38; N, 10.52%).

Antifitngal activities
The test organisms are phytopathogenic.For this reason, all steps of the work were done with high precaution and aseptic condition.PDA was used as a growth medium for the test.DMSO and chloroform was used as a solvent initially to prepare solution of the ligand and complexes.Such solutions were then mixed with the sterilized PDA to maintain desired concentration (0.01%) of the compounds and the mixture (20 cm 3 ) was poured in each Petri dish.Linear growth of the fungus was measured in mm after five days of incubation at (25 ± 2) °C.

Antibacterial activities
Antibacterial activities of these complexes against selected bacteria were assessed by the disc diffusion method.Paper disc of 6 mm in diameter and Petri plate of 70 mm in diameter were used throughout the experiment.Pour plates were made with sterilized melted NA (45 °C) and after solidification of pour plates, the test organisms (suspension) were spread uniformly over the pour plate with sterilized glass rod separately.The paper discs after soaking with test chemicals (1% in CHC1 3 ) were placed at the center of the inoculated pour plate.A control plate was also maintained in each case with CHC1 3 .The plates firstly were kept for 4 hours at low temperature (4 °C) and the test chemicals diffused from disc to the surrounding medium by this time.The plates were then incubated at (35±2) °C for growth of test organisms and were observed at 24-hour intervals.The activity is expressed in terms of diameter of zone of inhibition in mm.

Cadmium (II) complexes 3.1.1. Monoiodocadmium (II) iodide complex
Reaction of methanolic solution of free ligand L 1 with cadmium (II) iodide produced a white complex, which on the basis of analytical data was formulated as [CdL 1 I]I.The infrared spectrum of this complex showed (Table1) V NH band at around 3217 cm -1 and other characteristic bands for  C=N , c-c,  C-H ,  CH3 , and  Cd-N at 1655 cm -1 , 1161 cm -1 , 2966 cm -1 , 1369 cm -1 and 513 cm -1 respectively.Since the spectrum was not run below 400 cm -1 , so the expected Cd-I band around 260 cm -1 could not be recorded.The molar conductivity values (Table 3) of 70 ohm -1 cm 2 mol -1 in DMF and 62 ohm -1 cm 2 mol -1 in DMSO solution indicate that the complex is 1:1 electrolyte in nature.This evidence supports a square pyramidal geometry (Figure 2) for this complex.
Table 3. Conductivity data for the complexes.

Trans-diisothiocyanato cadmium (II) complex
The above mentioned complex [CdL 1 I)I had undergone substitution and addition reactions simultaneously with KSCN, producing a white product of formula, [CdL 1 (SCN) 2 ].The infrared spectrum of this complex shows  NH band at 3217 cm -1 ,  CN band at 1654 cm -1 and other characteristic bands for  C-C ,  C-H ,  CH3 , and  Cd-N at 1161 cm - 1 , 2966 cm -1 , 1369 cm -1 and 513 cm -1 , respectively .The spectrum further showed  CN band at 2015 cm -1 ,  CS at 840 cm -1 and  NCS band at 480 cm -1 , indicating coordination by the -NCS moiety.
The 'H-NMR spectrum of this complex showed three sharp singlets at 1.27 ppm, 1.37 ppm and 1.90 ppm each corresponding to 611.The first two singlets at 1.27 ppm and 1.37 ppm were assigned to the protons of gemdimethyl groups on C7 and CI4 having equatorial and axial orientations and the third downfield singlet at 1.90 ppm was due to the protons of methyl groups on sp 2 carbons.The spectrum also showed singlet at 1.05 ppm corresponding to 6H.The position of this upfield doublet was assigned to the equatorial chiral methyl protons.
The other multiplets at 1.85 ppm, 2.40 ppm and 2.77 ppm, 3.01 ppm and 3.78 ppm were attributed to the methylene, methine and NH protons.This observation indicated that though axial substitution as well as axial addition had taken place on square pyramidal [CdL 1 I]I to form octahedral trans-[CdL 1 (NCS) 2 ], the stereochemistry of the ligand of the newly formed complex remained the same.
The conductance value of 0 ohm -1 em 2 mo1 -1 for this complex in CHC1 3 strongly supports the nonelectrolytic nature of this complex, indicating coordination by the thiocyanate ions.Thus an octahedral structure (Figure 3) can be assigned for [CdL 1 (NCS) 2 ].

Zinc (II) complexes 3.2.1. Aquazinc (II) diperchlorate complex
Interaction of L 1 with zinc (II) perchlorate hexahydrate in methanolic solution produced white solid product, which on the basis of analytical data coupled with 1 H-NMR results was formulated as [ZnL I (H 2 O)](C1O 4 ) 2 , The IR spectrum of [ZnL 1 (H 2 O)](C1O 4 ) 2 showed  NH band at 3227 cm -1 ,  C=N band at 1660 cm -1 ' and other characteristic  C-H ,  C-C and  Zn-N bands at the expected region (Table 1).The spectrum further showed bands at 1116 cm -1 and 624 cm -1 which supported the presence of C10 4 -ion.Presence of  OH bands at 3473 cm -1 and  H2O band at 1660 cm -1 overlapped with  C=N band were attributed to the presence of coordinated H 2 O molecule.The 1 H-NMR spectrum (Table 2) of this complex displayed three sharp singlets at 1.26 ppm, 1.36 ppm and 1.89 ppm each corresponding to 611.The first two singlets at 1.26 ppm and 1.36 ppm were assigned to the gemdimethyl protons on C1 and C14 having equatorial and axial orientations and the third downfield singlet at 1.89 ppm was accounted for protons of methyl groups on sp 2 carbons.The spectrum also showed a singlet at 1.05 ppm corresponding to 6H of chiral methyls.The position of this upfield doublet was assigned to the equatorial chiral methyl protons.The other multiplets at 2.43 ppm and 2.99 ppm, 3.23 ppm and 3.77 ppm were due to the methylene, methine and NH protons.The molar conductivity values (Table 3) of 193 ohm -1 cm 2 mol -1 in DMSO and that of 158 ohm -1 cm 2 mol -1 in DMF solution of this complex were in agreement with a 1:2 electrolytic square pyramidal structure (Figure 4) for [ZnL 1 (H 2 O)](C1O 4 ) 2 .

Mononitrozinc(II) nitrite complex
The complex, [ZnL 1 (H 2 O)](C1O 4 ) 2, had undergone substitution reaction with NaNO 2 in methanolic solution yielding a white product of molecular formula [ZnL 1 (NO 2 )](NO 2 ).The infrared spectrum (Table 1) of this complex showed  NH ,  C-H ,  CH3 ,  C-C and  Zn-N stretching bands in the expected region.Moreover the complex displayed the  asym (NO 2 ) and  sym (NO 2 ) bands at 1458 cm -1 and 1332 cm -1 respectively.The spectrum further displayed band at 835 cm -1 due to wagging mode of N-bonded nitro complex which was absent in 0-bonded nitrito complexes.15Presence of  Zn-N band at 430 cm -1 and other bands in the proper region strongly supported that the complex was N-bonded nitro complex.Absence of  ClO4 and V OH bands and presence of  NO2 bands demonstrated that ClO 4 and H 2 O were completely replaced by NO 2 groups.The molar conductivity values (Table 3) of 85 ohm -1 cm 2 mol -1 in DMSO solutions and 105 ohm -1 cm 2 mol -1 in DMF of this complex were in agreement with 1:1 electrolytic square pyramidal structure ( Figure 5) of the complex.

Monoacetatozinc(II) perchlorate complex
Interaction of L I .2HC10 4 with zinc (II) acetate dihydrate in methanolic solution produced a white solid product.Based on all experimental and analytical data the product was formulated as [ZnL I (CH 3 COO)](C1O 4 ).The infrared spectrum (Table 1) of this complex showed V NH band at 3190cm -1 ,  C=N band at 1661 cm -1 and other characteristic  C-H ,  CH3 ,  C-C and  Zn-N bands at the expected region.The spectrum further showed bands at 1129 cm -1 and 624 cm -1 , which supported the presence of C1O 4 -ion.Appearance of a band at around 1602 cm -1 indicated the presence of coordinated CH 3 COO -group.
Molar conductivity values (Table 3) of 68 ohm -1 cm 2 mol -1 in DMSO and 125 ohm -1 cm 2 mol -1 in DMF indicated that the complex behaved as 1:1 electrolyte in solution.This behavior supported the square pyramidal structure (Figure 6) of the complex.

Antimicrobial activities
The test organisms are phytopathogenic.For this reason, all steps of the work were done with high precaution and aseptic condition.And the percentage inhibition of mycelia growth of the test fungus/bacteria was calculated by using following equation:

Percentage of inhibition = (CT/C) × 100
Here, C= Diameter of the fungal/ bacterial colony in the control T = Diameter of the fungal/ bacterial colony in the treated

Fungitoxicity study
Antifungal activities of these complexes are summarized in Table 4. Screenings were conducted against selective phytopathogenic fungi, Macrophomina phaseolina, Alternaria alternate and Colletotrichum corcolei.These fungi are phytopathogens of important crop plants such as jute, chilli, brinjal etc.Control of such pathogens by non-hazardous fungicides has been a major concern, especially as fungi gradually develop resistance to known fungicides.It is evident from the results presented in Table 4 that the macrocycle and its complexes showed some antifungal activity.Generally, previous studies have shown that the activity of the macrocyclic ligands decrease upon coordination (Bembi et al. 1991;Roy et al. 2010Roy et al. , 2009Roy et al. , 2008;;Shin et al. 2007) but in the present study, these complexes behaved differently.-means not done A comparison of the activities of the present ligand and complexes showed that the present compounds showed varied amount of effects on the inhibition of mycelial growth and most of the complexes exhibited greater anti-fungal activities than its corresponding ligand specially against Alternariaalternata.It was also observed that different complexes, e.g., acetate, thiocyanate, aqua and nitro complexes of same macrocyclic ligand had different effects on these organisms.These observations suggested that the nature of metal ion and axial ligand / counter ion played a significant role in the inhibition of micelial growth.

Antibacterial study
Anti-bacterial activities of the macrocycles and their complexes have so far been studied for few cases only (Bembi et al. 1991;Roy et al. 2010a;Shin et al. 2007;Hazari et al. 2006).Antibacterial activities of these complexes are summarized in Table 5.The results showed that though some of the complexes show different antibacterial activities to a measurable extent but the ligand and some complexes did not show any such activity as compared to our previous study (Roy et al. 2009;Shin et al. 2007).
Table 5. Antibacterial activities of the ligand and its metal complexes.

Conclusions
Inhibition power of the some newly prepared complexes on a particular bacterial growth was measured in these researches.Some of the complexes are found to exhibit higher antibacterial activities than their corresponding ligand.However for a clear understanding of the functions responsible for antibacterial activities of macrocycles, and their complexes, more studies are needed to be performed with a series of analogous ligands and their complexes against a series of bacteria.

Figure 3 .
Figure 3. Structure of [CdL 1 (NCS) 2 ].3.2.Zinc (II) complexes 3.2.1.Aquazinc (II) diperchlorate complexInteraction of L 1 with zinc (II) perchlorate hexahydrate in methanolic solution produced white solid product, which on the basis of analytical data coupled with 1 H-NMR results was formulated as [ZnL I (H 2 O)](C1O 4 ) 2 , The IR spectrum of [ZnL 1 (H 2 O)](C1O 4 ) 2 showed  NH band at 3227 cm -1 ,  C=N band at 1660 cm -1 ' and other characteristic  C-H ,  C-C and  Zn-N bands at the expected region (Table1).The spectrum further showed bands at 1116 cm -1 and 624 cm -1 which supported the presence of C10 4 -ion.Presence of  OH bands at 3473 cm -1 and  H2O band at 1660 cm -1 overlapped with  C=N band were attributed to the presence of coordinated H 2 O molecule.The 1 H-NMR spectrum (Table2) of this complex displayed three sharp singlets at 1.26 ppm, 1.36 ppm and 1.89 ppm each corresponding to 611.The first two singlets at 1.26 ppm and 1.36 ppm were assigned to the gemdimethyl protons on C1 and C14 having equatorial and axial orientations and the third downfield singlet at 1.89

Figure 7 .
Figure 7. Picture showing inhibition zone against E. coli by the synthesized complexes.

Table 1 . Selected IR bands of the complexes.
The 1 H-NMR spectrum of this complex displayed (Table2) four sharp singlets at 1.26 ppm, 1.36 ppm, 1.90 ppm and 2.09 ppm corresponding to 6H, 6H, 6H and 4H, respectively.The singlets at 1.26 ppm and 1.36 ppm were assigned to the gem-dimethyl groups having equatorial and axial orientation.The third singlet at 1.90 ppm was due to 6H equatorially oriented methyl protons on sp 2 carbons at C5 and C12 positions.The fourth singlet at 2.09 ppm corresponding to 4H was attributed to CH, protons at C6 and C13.However appearance of only upfield doublet at 1.05 ppm was attributed to the equatorially oriented chiral methyl protons on C3 and C10 positions.The other multiplets at 2.75 ppm, 3.20 ppm and 3.95 ppm were presumably due to methylene, methine and NH protons.