# Synthesis and Characterization of Biologically Active 10-Membered Tetraazamacrocyclic Complexes of Cr(III), Mn(III), and Fe(III)

Authors: Dharam Pal Singh; Vandna Malik; Ramesh Kumar
Journal: Research Letters in Inorganic Chemistry
 (2009)

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## Abstract

A new series of macrocyclic complexes of type [M(TML)X]X2; where M = Cr(III), Mn(III), or Fe(III); TML is tetradentate macrocyclic ligand and X = Cl−1, NO3−1, CH3COO−1 for Cr(III), Fe(III), and X = CH3COO−1 for Mn(III) has been synthesized by template condensation of succinyldihydrazide and glyoxal. The complexes have been formulated as [M(TML)X]X2 due to 1:2 electrolytic natures of these complexes as shown by conductivity measurements. The complexes have been characterized with the help of elemental analyses, molar conductance, electronic, infrared, far infrared spectral studies and magnetic susceptibilities. On the basis of these studies, a five-coordinate distorted square-pyramidal geometry, in which two nitrogens and two carbonyl oxygen atoms are suitably placed for coordination toward the metal ion, has been proposed for all the complexes. The complexes were tested for their in vitro antibacterial activity. Some of the complexes showed remarkable antibacterial activities against some selected bacterial strains. The minimum inhibitory concentration shown by these complexes was compared with minimum inhibitory concentration shown by some standard antibiotics like linezolid and cefaclor.


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## Body

## 1. Introduction

During  the
past  few decades macrocyclic chemistry has attracted the attention of both
inorganic and bioinorganic chemists. The synthesis of macrocyclic complexes
has been a fascinating area of research and growing at a very fast pace owing
to their resemblance with naturally occurring macrocycles and analytical,
industrial, and medical applications [1–3]. In the present paper a new series of
macrocyclic complexes of Cr(III), Mn(III), and Fe(III) obtained by template
condensation reaction of succinyldihydrazide and glyoxal has been reported. These
complexes were also tested for their in vitro antibacterial activities. Some
complexes showed remarkable antibacterial activities.


## 2. Experimental

All the complexes
were prepared by template method. To a stirring methanolic solution (~50 cm3)
of succinyldihydrazide (10 mmol) was added trivalent chromium, manganese, and
iron salt (10 mmol) dissolved in a minimum quantity of methanol (20 cm3).
The resulting solution was refluxed for 0.5 hour. After that glyoxal (10 mmol)
dissolved in ~20 mL of methanol was added to the refluxing mixture
and refluxed again for 6–8 hours. On overnight cooling, a dark colored precipitate
formed which was filtered, washed with methanol, acetone, and diethyl ether and
dried in vacuo (Yield 45%). The
complexes were found soluble in DMF and DMSO, but were insoluble in common
organic solvents and water. They were found thermally stable up to ~240°C
and then decomposed.


## 3. Pharmacology

### 3.1. In Vitro Antibacterial Activity

Some of the synthesized macrocyclic
complexes were tested for their in vitro antibacterial activity against some bacterial strains using spot-on-lawn on
Muller Hinton Agar by following the reported method [4]. Four test pathogenic
bacterial strains viz Bacillus cereus (MTCC 1272), Salmonella
typhi (MTCC 733), Escherichia
coli (MTCC 739), and Staphylococcus aureus (MTCC 1144) were considered for
determination of Minimum Inhibitory Concentration (MIC) of selected complexes.


### 3.2. Culture Conditions

The test pathogens were subcultured
aerobically using Brain Heart Infusion Agar (HiMedia, Mumbai, India) at 37°C/24 hours. Working cultures were
stored at 4°C in Brain Heart Infusion (BHI) broth (HiMedia,
Mumbai, India), while stock cultures were maintained at −70°C in BHI
broth containing 15% (v/v) glycerol (Qualigens, Mumbai, India). Organisms were
grown overnight in 10 mL BHI broth, centrifuged at 5000 g for 10 minutes, and the
pellet was suspended in 10 mL of phosphate buffer saline (PBS, pH 7.2). Optical
density at 545 nm (OD-545) was adjusted to obtain 108 cfu/mL
followed by plating serial dilution onto plate count agar (HiMedia, Mumbai, India).


### 3.3. Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration
(MIC) is the lowest concentration of the antimicrobial agent that prevents the
development of viable growth after overnight incubation. Antimicrobial activity
of the compounds was evaluated using spot-on-lawn on Muller Hinton Agar (MHA,
HiMedia, Mumbai, India). Soft agar was prepared by
adding 0.75% agar in Muller Hinton Broth (HiMedia, Mumbai, India).
Soft agar was inoculated with 1% of 108 Cfu/mL of the test pathogen
and 10 mL was overlaid on MHA. From 1000X solution of compound (1 mg/mL of DMSO)
1, 2, 4, 8, 16, 32, 64, and 128X solutions were prepared. Dilutions of standard
antibiotics (Linezolid and Cefaclor) were also prepared in the same manner. 5 μL
of the appropriate dilution was spotted on the soft agar and incubated at 37°C for 24
hours. Zone of inhibition of compounds was considered after subtraction of
inhibition zone of DMSO. Negative control (with no compound) was also observed.


## 3.1. In Vitro Antibacterial Activity

Some of the synthesized macrocyclic
complexes were tested for their in vitro antibacterial activity against some bacterial strains using spot-on-lawn on
Muller Hinton Agar by following the reported method [4]. Four test pathogenic
bacterial strains viz Bacillus cereus (MTCC 1272), Salmonella
typhi (MTCC 733), Escherichia
coli (MTCC 739), and Staphylococcus aureus (MTCC 1144) were considered for
determination of Minimum Inhibitory Concentration (MIC) of selected complexes.


## 3.2. Culture Conditions

The test pathogens were subcultured
aerobically using Brain Heart Infusion Agar (HiMedia, Mumbai, India) at 37°C/24 hours. Working cultures were
stored at 4°C in Brain Heart Infusion (BHI) broth (HiMedia,
Mumbai, India), while stock cultures were maintained at −70°C in BHI
broth containing 15% (v/v) glycerol (Qualigens, Mumbai, India). Organisms were
grown overnight in 10 mL BHI broth, centrifuged at 5000 g for 10 minutes, and the
pellet was suspended in 10 mL of phosphate buffer saline (PBS, pH 7.2). Optical
density at 545 nm (OD-545) was adjusted to obtain 108 cfu/mL
followed by plating serial dilution onto plate count agar (HiMedia, Mumbai, India).


## 3.3. Determination of Minimum Inhibitory Concentration

The minimum inhibitory concentration
(MIC) is the lowest concentration of the antimicrobial agent that prevents the
development of viable growth after overnight incubation. Antimicrobial activity
of the compounds was evaluated using spot-on-lawn on Muller Hinton Agar (MHA,
HiMedia, Mumbai, India). Soft agar was prepared by
adding 0.75% agar in Muller Hinton Broth (HiMedia, Mumbai, India).
Soft agar was inoculated with 1% of 108 Cfu/mL of the test pathogen
and 10 mL was overlaid on MHA. From 1000X solution of compound (1 mg/mL of DMSO)
1, 2, 4, 8, 16, 32, 64, and 128X solutions were prepared. Dilutions of standard
antibiotics (Linezolid and Cefaclor) were also prepared in the same manner. 5 μL
of the appropriate dilution was spotted on the soft agar and incubated at 37°C for 24
hours. Zone of inhibition of compounds was considered after subtraction of
inhibition zone of DMSO. Negative control (with no compound) was also observed.


## 4. Results and Discussion

The
analytical data show the formula of macrocyclic complexes as [M(C6H8O2N4)X]X2. The test for anions was positive before
and after decomposing the complexes with concentration of HNO3, indicating
their presence inside as well as outside the coordination sphere. Conductivity
measurements in DMSO indicated them to be electrolytic in nature (140–150 ohm−1 cm2 mol−1) [5]. All compounds gave satisfactory elemental analyses
results as shown in Table 1.Table 1
Analytical data of trivalent chromium, manganese, and iron complexes
derived from succinyldihydrazide and glyoxal. Found
(Calcd.) %.
Serial no.ComplexesMCHNColourMol. Wt.1[Cr(C6H8N4O2)Cl]Cl215.12(15.95)22.39 (22.08)2.43 (2.45)17.19 (17.17)Light  green3262[Cr(C6H8N4O2)(NO3)](NO3)212.78(12.80)17.49 (17.73)1.79 (1.97)24.31 (24.13)Light mustered4063[Cr(C6H8N4O2)(OAc)](OAc)213.15(13.09)36.31 (36.27)4.29 (4.28)14.12 (14.10)Yellowish white3974[Mn(C6H8N4O2)(OAc)](OAc)213.81(13.75)36.10 (36.00)4.19 (4.25)14.01 (14.00)Creamy4005[Fe(C6H8N4O2)Cl]Cl216.84(16.96)21.89 (21.81)2.39 (2.42)16.94 (16.96)Light yellow330.56[Fe(C6H8N4O2)(NO3)](NO3)213.89 (13.65)17.58 (17.56)1.92 (1.95)23.87 (23.90)Creamy4107[Fe(C6H8N4O2)(OAc)](OAc)213.87 (13.96)35.88 (35.91)4.27 (4.23)13.82 (13.95)Light brown401


### 4.1. IR Spectra

In the infrared spectrum of succinyldihydrazide a pair
of band corresponding toν(NH2)
is present at ~3200 cm−1 and ~3250 cm−1, but is absent in the
IR spectra of all the complexes. However, a single broad medium band at
~3350–3400 cm−1 was observed in the spectra of all the complexes which
may be assigned due to ν(NH).
Further no strong absorption band was observed near 1710 cm−1 as
observed in spectrum of glyoxal indicating the absence of >C=O groups
of glyoxal molecule. This confirms the condensation of carbonyl groups of
glyoxal and amino groups of succinyldihydrazide [6]. This fact is further
supported by appearance of a new strong absorption band in the region
~1590–1610 cm−1 in the IR spectra of all complexes which may be
attributed due to ν(C=N)
[7]. These results provide strong evidence for the formation of macrocyclic
frame [8]. The lower value of ν(C=N)
indicates coordination of nitrogens of azomethine to metal [9]. A strong peak
at ~1665 cm−1 in the IR spectrum of succinyldihydrazide is assigned
due to >C=O group of the CONH moiety. This peak
gets shifted to lower frequency (~1625–1640 cm−1) in the spectra of
all the complexes [10] suggesting the coordination of oxygen of amide group
with metal.


### 4.2. Far Infrared Spectra

The far infrared spectra show bands in the region~425–445 cm−1 corresponding to ν(M–N) vibrations in all the complexes. The
bands present at ~300–315 cm−1 are assigned to ν(M–Cl) vibrations. The bands present at
~220–250 cm−1 in all nitrato complexes to ν(M–O) vibrations of nitrato group [11].


### 4.3. Magnetic Measurements and Electronic Spectra

#### 4.3.1. Chromium Complexes

Magnetic moment of chromium complexes
were found in the range of4.20–4.50 B.M. These values of magnetic moment
support the predicted geometry of the complexes [12]. The electronic spectra of
chromium complexes show bands at ~9030–9250, 13020–13350, 17450–18320,
27435–27840, and 34820 cm−1. However, these spectral bands cannot be
interpreted in terms of four or six coordinated environment around the metal
atom. In turn, the spectra are comparable to that of five coordinated Cr(III)
complexes, whose structure has been confirmed with the help of X-ray
measurements [13]. Thus keeping in view, the analytical data and 1 : 2 ionic
nature of these complexes, a five-coordinated square-pyramidal geometry may be
assigned for these complexes. Thus, assuming the symmetry C4V for
these complexes [14], the various spectral bands may be assigned as B14→Ea4, B14→B24, B14→A24, and B14→Eb4. The complexes do not have idealized C4V symmetry but it is being used as approximation in order to try and assign the
electronic absorption bands.


#### 4.3.2. Manganese Complex

The magnetic moment of manganese
complex was found to be 4.85 B.M. The electronic spectrum of manganese complex show
three d-d bands at approximately 12.250, 16.045, and 35.435 cm−1. The
higher energy band at 35465 cm−1 may be assigned due to charge
transfer transitions. The spectrum resembles those reported for five-coordinate square-pyramidal manganese porphyrins [14]. This idea is further supported
by the presence of the broad ligand field band at 20410 cm−1 diagnostic of C4V symmetry and thus the various bands may be assigned
as follows: B15→A15, B15→B25, and B15→E5,
respectively. The band assignment in single electron transition may be made as
dz2→dx2-y2,
dxy→dx2-y2 and dxy,
dyz→dx2-y2,
respectively, in order of increasing energy. However, the complexes do not have
idealized C4V symmetry.


#### 4.3.3. Iron Complexes

The magnetic moments of iron
complexes lay in the range5.82–5.90 B.M. and are in accordance with proposed
geometry of the complexes. The electronic spectra of trivalent iron complexes
show various bands 9825–9975, 15525–15570, 27635–27710 cm−1, and
these bands do not suggest the octahedral or tetrahedral geometry around the
metal atom. The spectral bands are consistent with the range of spectral bands
reported for five coordinate square pyramidal iron (III) complexes [15]. Assuming
C4V symmetry for these complexes, the various bands can be assigned
as dxy→dxz, dyz and dxy→dz2.
Any attempt to make accurate assignment is difficult due to interactions of the
metal-ligand pi-bond systems lifting the degeneracy of the dxz and dyz pair.


## 4.1. IR Spectra

In the infrared spectrum of succinyldihydrazide a pair
of band corresponding toν(NH2)
is present at ~3200 cm−1 and ~3250 cm−1, but is absent in the
IR spectra of all the complexes. However, a single broad medium band at
~3350–3400 cm−1 was observed in the spectra of all the complexes which
may be assigned due to ν(NH).
Further no strong absorption band was observed near 1710 cm−1 as
observed in spectrum of glyoxal indicating the absence of >C=O groups
of glyoxal molecule. This confirms the condensation of carbonyl groups of
glyoxal and amino groups of succinyldihydrazide [6]. This fact is further
supported by appearance of a new strong absorption band in the region
~1590–1610 cm−1 in the IR spectra of all complexes which may be
attributed due to ν(C=N)
[7]. These results provide strong evidence for the formation of macrocyclic
frame [8]. The lower value of ν(C=N)
indicates coordination of nitrogens of azomethine to metal [9]. A strong peak
at ~1665 cm−1 in the IR spectrum of succinyldihydrazide is assigned
due to >C=O group of the CONH moiety. This peak
gets shifted to lower frequency (~1625–1640 cm−1) in the spectra of
all the complexes [10] suggesting the coordination of oxygen of amide group
with metal.


## 4.2. Far Infrared Spectra

The far infrared spectra show bands in the region~425–445 cm−1 corresponding to ν(M–N) vibrations in all the complexes. The
bands present at ~300–315 cm−1 are assigned to ν(M–Cl) vibrations. The bands present at
~220–250 cm−1 in all nitrato complexes to ν(M–O) vibrations of nitrato group [11].


## 4.3. Magnetic Measurements and Electronic Spectra

### 4.3.1. Chromium Complexes

Magnetic moment of chromium complexes
were found in the range of4.20–4.50 B.M. These values of magnetic moment
support the predicted geometry of the complexes [12]. The electronic spectra of
chromium complexes show bands at ~9030–9250, 13020–13350, 17450–18320,
27435–27840, and 34820 cm−1. However, these spectral bands cannot be
interpreted in terms of four or six coordinated environment around the metal
atom. In turn, the spectra are comparable to that of five coordinated Cr(III)
complexes, whose structure has been confirmed with the help of X-ray
measurements [13]. Thus keeping in view, the analytical data and 1 : 2 ionic
nature of these complexes, a five-coordinated square-pyramidal geometry may be
assigned for these complexes. Thus, assuming the symmetry C4V for
these complexes [14], the various spectral bands may be assigned as B14→Ea4, B14→B24, B14→A24, and B14→Eb4. The complexes do not have idealized C4V symmetry but it is being used as approximation in order to try and assign the
electronic absorption bands.


### 4.3.2. Manganese Complex

The magnetic moment of manganese
complex was found to be 4.85 B.M. The electronic spectrum of manganese complex show
three d-d bands at approximately 12.250, 16.045, and 35.435 cm−1. The
higher energy band at 35465 cm−1 may be assigned due to charge
transfer transitions. The spectrum resembles those reported for five-coordinate square-pyramidal manganese porphyrins [14]. This idea is further supported
by the presence of the broad ligand field band at 20410 cm−1 diagnostic of C4V symmetry and thus the various bands may be assigned
as follows: B15→A15, B15→B25, and B15→E5,
respectively. The band assignment in single electron transition may be made as
dz2→dx2-y2,
dxy→dx2-y2 and dxy,
dyz→dx2-y2,
respectively, in order of increasing energy. However, the complexes do not have
idealized C4V symmetry.


### 4.3.3. Iron Complexes

The magnetic moments of iron
complexes lay in the range5.82–5.90 B.M. and are in accordance with proposed
geometry of the complexes. The electronic spectra of trivalent iron complexes
show various bands 9825–9975, 15525–15570, 27635–27710 cm−1, and
these bands do not suggest the octahedral or tetrahedral geometry around the
metal atom. The spectral bands are consistent with the range of spectral bands
reported for five coordinate square pyramidal iron (III) complexes [15]. Assuming
C4V symmetry for these complexes, the various bands can be assigned
as dxy→dxz, dyz and dxy→dz2.
Any attempt to make accurate assignment is difficult due to interactions of the
metal-ligand pi-bond systems lifting the degeneracy of the dxz and dyz pair.


## 4.3.1. Chromium Complexes

Magnetic moment of chromium complexes
were found in the range of4.20–4.50 B.M. These values of magnetic moment
support the predicted geometry of the complexes [12]. The electronic spectra of
chromium complexes show bands at ~9030–9250, 13020–13350, 17450–18320,
27435–27840, and 34820 cm−1. However, these spectral bands cannot be
interpreted in terms of four or six coordinated environment around the metal
atom. In turn, the spectra are comparable to that of five coordinated Cr(III)
complexes, whose structure has been confirmed with the help of X-ray
measurements [13]. Thus keeping in view, the analytical data and 1 : 2 ionic
nature of these complexes, a five-coordinated square-pyramidal geometry may be
assigned for these complexes. Thus, assuming the symmetry C4V for
these complexes [14], the various spectral bands may be assigned as B14→Ea4, B14→B24, B14→A24, and B14→Eb4. The complexes do not have idealized C4V symmetry but it is being used as approximation in order to try and assign the
electronic absorption bands.


## 4.3.2. Manganese Complex

The magnetic moment of manganese
complex was found to be 4.85 B.M. The electronic spectrum of manganese complex show
three d-d bands at approximately 12.250, 16.045, and 35.435 cm−1. The
higher energy band at 35465 cm−1 may be assigned due to charge
transfer transitions. The spectrum resembles those reported for five-coordinate square-pyramidal manganese porphyrins [14]. This idea is further supported
by the presence of the broad ligand field band at 20410 cm−1 diagnostic of C4V symmetry and thus the various bands may be assigned
as follows: B15→A15, B15→B25, and B15→E5,
respectively. The band assignment in single electron transition may be made as
dz2→dx2-y2,
dxy→dx2-y2 and dxy,
dyz→dx2-y2,
respectively, in order of increasing energy. However, the complexes do not have
idealized C4V symmetry.


## 4.3.3. Iron Complexes

The magnetic moments of iron
complexes lay in the range5.82–5.90 B.M. and are in accordance with proposed
geometry of the complexes. The electronic spectra of trivalent iron complexes
show various bands 9825–9975, 15525–15570, 27635–27710 cm−1, and
these bands do not suggest the octahedral or tetrahedral geometry around the
metal atom. The spectral bands are consistent with the range of spectral bands
reported for five coordinate square pyramidal iron (III) complexes [15]. Assuming
C4V symmetry for these complexes, the various bands can be assigned
as dxy→dxz, dyz and dxy→dz2.
Any attempt to make accurate assignment is difficult due to interactions of the
metal-ligand pi-bond systems lifting the degeneracy of the dxz and dyz pair.


## 5. Biological Assay

The minimum
inhibitory concentration (MIC) shown by the complexes against these bacterial
strains was compared with MIC shown by standard antibioticsLinezolid and Cefaclor
(Table 2).
Complex 1 showed an MIC of 8 μg/mL
against bacterial strain Escherichia
coli (MTCC 739), which is equal to MIC shown by standard
antibiotic Cefaclor against the same
bacterial strain. Complex 3 registered an MIC of 8 μg/mL, against bacterial strain Bacillus cereus (MTCC 1272), which is equal to MIC shown by standard
antibiotic Cefaclor against the same
bacterial strain. Further complexes 3 and 7 showed a minimum inhibitory
concentration of 32 μg/mL against
bacterial strain Salmonella typhi (MTCC 733), which is equal to MIC shown by standard
antibiotic Linezolid against the same
bacterial strain. The MIC of complex 4 against Escherichia coli (MTCC 739) was found to be 16 μg/ml,
which is equal to the MIC shown by standard antibiotic Linezolid against the same bacterial strain. Complex 6 registered
an MIC of 4 μg/mL against bacterial
strain Staphylococcus aureus (MTCC 1144) which is
equal to MIC shown by standard antibiotic Linezolid against the same bacterial strain. Among the series under
test for determination of MIC, complexes 1 and 3 were found most potent as
compared to other complexes. However, complexes 2 and 5 showed poor antibacterial
activity or no activity against all bacterial strains among the whole series. (Table 2).Table 2
Minimum Inhibitory Concentration (MIC) shown by complexes against test bacteria by
using agar dilution assay. (—) No activity, a:Bacillus cereus (MTCC 1272); b: Staphylococcus aureus (MTCC 1144); c: Escherichia coli (MTCC 739); d: Salmonella typhi (MTCC 733); Cefaclor and Linezolid are standard
antibiotics.
Serial no.ComplexesMIC (μg/mL)abcd(1)[Cr(C6H8N4O2)Cl]Cl23232864(2)[Cr(C6H8N4O2)(NO3)](NO3)2——64>128(3)[Cr(C6H8N4O2)(OAc)](OAc)28646432(4)[Mn(C6H8N4O2)(OAc)](OAc)264>1281664(5)[Fe(C6H8N4O2)Cl]Cl2>1286464>128(6)[Fe(C6H8N4O2)(NO3)](NO3)232464>128(7)[Fe(C6H8N4O2)(OAc)](OAc)2>12864>12832Cefaclor82816Linezolid441632


## 6. Conclusions

### 6.1. Chemistry

Based
on elemental analyses, conductivity and magnetic measurements, electronic IR,
and far IR spectral studies, the structure as shown in Figure1 may be proposed for these complexes.Figure 1


### 6.2. Biological Assay

It has
been suggested that chelation/coordination reduces the polarity of the metal ion
mainly because of partial sharing of its positive charge with donor group
within the whole chelate ring system [16]. This process of chelation thus
increases the lipophilic nature of the central metal atom, which in turn,
favors its permeation through the lipoid layer of the membrane thus causing
the metal complex to cross the bacterial membrane more effectively thus
increasing the activity  of the complexes.


## 6.1. Chemistry

Based
on elemental analyses, conductivity and magnetic measurements, electronic IR,
and far IR spectral studies, the structure as shown in Figure1 may be proposed for these complexes.Figure 1


## 6.2. Biological Assay

It has
been suggested that chelation/coordination reduces the polarity of the metal ion
mainly because of partial sharing of its positive charge with donor group
within the whole chelate ring system [16]. This process of chelation thus
increases the lipophilic nature of the central metal atom, which in turn,
favors its permeation through the lipoid layer of the membrane thus causing
the metal complex to cross the bacterial membrane more effectively thus
increasing the activity  of the complexes.



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*Source: 824561-2009-04-26.xml*