Synthesis of Mn(II) and Zn(II) complexes with new macrocyclic Schiff-base ligands containing piperazine moiety: Spectroscopic, structural, cytotoxic and antibacterial properties

Article abstract of DOI:10.1016/j.poly.2017.02.008

The new diamine 2,2′-(piperazine-1,4-diylbis(methylene))dianiline (A¹) was synthesized by reaction of 2-nitrobenzylchloride and piperazine. Its corresponding Mn(II) and Zn(II) macrocyclic Schiff-base complexes were prepared via the metal templa

Full text of DOI:10.1016/j.poly.2017.02.008

Accepted Manuscript
Synthesis of Mn(II) and Zn(II) complexes with new macrocyclic Schiff-base
ligands containing piperazine moiety: spectroscopic, structural, cytotoxic and
antibacterial properties
Hassan Keypour, Masoumeh Mahmoudabadi, Amir Shooshtari, Leila
Hosseinzadeh, Fariba Mohsenzadeh, Robert William Gable
PII:
S0277-5387(17)30118-3
DOI:
http://dx.doi.org/10.1016/j.poly.2017.02.008
POLY 12475
Reference:
Polyhedron
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 December 2016
7 February 2017
8 February 2017
Please cite this article as: H. Keypour, M. Mahmoudabadi, A. Shooshtari, L. Hosseinzadeh, F. Mohsenzadeh, R.W.
Gable, Synthesis of Mn(II) and Zn(II) complexes with new macrocyclic Schiff-base ligands containing piperazine
moiety: spectroscopic, structural, cytotoxic and antibacterial properties, Polyhedron (2017), doi: http://dx.doi.org/
10.1016/j.poly.2017.02.008
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Synthesis of Mn(II) and Zn(II) complexes with new macrocyclic Schiff-base ligands
containing piperazine moiety: spectroscopic, structural, cytotoxic and antibacterial
properties
Hassan Keypour a,*, Masoumeh Mahmoudabadia, Amir Shooshtaria, Leila Hosseinzadehb,
Fariba Mohsenzadehc, Robert William Gable d
aFaculty of Chemistry, Bu-Ali Sina University, Hamedan 65174, Iran
b Pharmaceutical Sciences Research Center, School of Pharmacy Kermanshah, University of
Medical Sciences Kermanshah, Iran
cLaboratory of Plant Cell Biology, Department of Biology, Bu-Ali Sina University, Hamedan
65174, Iran
dSchool of Chemistry, University of Melbourne, Victoria 3010, Australia
* Corresponding authors. Tel.: +98 (81) 38242044; fax: +98 (81) 38272404 (H. Keypour)
E-mail addresses: haskey1@yahoo.com (H. Keypour)

Abstract
The new diamine 2,2'-(piperazine-1,4-diylbis(methylene))dianiline (A1) was synthesized by
reaction of 2-nitrobenzylchloride and piperazine. Its corresponding Mn(II) and Zn(II)
macrocyclic Schiff-base complexes were prepared via the metal templated [1+1]
cyclocondensation of this diamine and 2,6-diacetylpyridine or 2,6- pyridinedicarbaldehyde. The
diamine and all the macrocyclic complexes were characterized by elemental analysis, mass
1
spectrometry and spectroscopic methods such as: FT-IR, H and 13C NMR. Also, the crystal
structures of [MnL1(ClO4)2] (1) and [ZnL1(H2O)2](ClO4)2 (3) complexes were obtained by
single-crystal X-ray crystallography. The cytotoxic properties of all macrocyclic complexes were
studied. The results showed, some of these complexes were more potent than doxorubicin in U87
MG cell line predicating their therapeutic potential in treatment of glioblastoma. Also, the
complexes were tested for in vitro antibacterial properties against some Gram-positive and
Gram-negative bacteria. The complexes had antibacterial properties and in some cases even
more than those of Tobramycin and Tetracycline as standards.
Keywords: Macrocyclic Schiff-base complexes, Piperazine moiety, X-ray structures,
Antibacterial activity, Cytotoxicity.

1. Introduction
Schiff base macrocyclic ligands and their related complexes are an important group of
compounds which have been extensively studied [1-4]. Macrocyclic Schiff-base complexes have
many applications in several areas such as DNA binding and cleavage reagents [5], models of
reaction centers of metalloenzymes [6], magnetic resonance imaging [7-8], sensors [9–11],
catalyst [12–14], antibiotics [15] and can be utilized for metal biosites modeling [16-18]. They
are polydentate systems whose donor atoms are parts of a cyclic hydrocarbon backbone [19].
The most important problem with the synthesis of macrocycles is the cyclization step that
contains pathway in bringing two ends of a chain together. So, the best strategy involves reaction
in the presence of a transition metal ion which increases the yield of the cyclization pathway and
directs the strict course of the reaction towards cyclic products rather than polymeric ones. The
effect has been called the metal-template effect [20-21]. The formation of macrocyclic
complexes depends on the size of the internal cavity, the nature of its donor atoms, the rigidity
and flexibility of the macrocycle, and stereochemical preference of metal ions [22]. Following
previous reports macrocyclic complexes of manganese and zinc have antibacterial and cytotoxic
properties [23-29]. Prompted by these facts and previous reports on synthesis macrocyclic
complexes [30-39], in the present paper, the synthesis and characterization of Mn(II) and Zn(II)
macrocyclic complexes derived from diamine A1 and 2,6-diacetylpyridine or 2,6-
pyridinedicarbaldehyde are discussed. All of the complexes were characterized by elemental
analyses, mass spectrometry and spectroscopy (IR and NMR). The crystal structures of the
macrocyclic complexes 1 and 3 were determined by single-crystal X-ray analyses.

2. Experimental
2.1. Materials
Piperazine, 2-nitrobenzylchloride, 2,6-diacetylpyridine, 2,6-pyridinedicarbaldehyde, metal salts
and all of solvents were purchased from the Merck Company and used without further
purification.
Caution! Perchlorate salts are potentially explosive. Only small amount of material should be
prepared and handled with great care.
2.2. Physical measurements
Infrared spectra were measured using KBr pellets on a BIO-RAD FTS-40A spectrophotometer
(4000-400 cm-1). Mass spectra were recorded on an Agilent technologies (HP) 5973 mass
spectrometer operating at an ionization potential of 70eV. CHN analyses were carried out using a
1
13
Perkin-Elmer, CHNS/O elemental analyzer model 2400 series 2. H and C NMR spectra were
taken in DMSO-d6 on a Bruker Avance 400 MHz spectrometer using Si(CH3)4 as an internal
standard.
2.3. X-ray crystallography
Orange
crystals
of
[MnL1(ClO4)2]
(1)
and
yellow
crystals
of
[ZnL1(H2O)2](ClO4)2(3) suitable for single-crystal X-ray analyses were grown by slow diffusion
of diethyl ether vapor into their acetonitrile solution. A suitable crystal was selected and mounted
on an Oxford Diffraction SuperNova, Dual, Cu at zero, Atlas diffractometer. The crystal was

kept at 130.01(10) K during data collection. With Olex2 [40], the structure was solved with the
SHELXT [41] structure solution program using Direct Methods and refined with the SHELXL
[42] refinement package using Least Squares minimization. Crystallographic data for the
macrocyclic complexes [MnL1(ClO4)2] (1) and [ZnL1(H2O)2](ClO4)2 (3) are listed in Table 1.
<Please insert Table 1>
2.4. Synthesis
2.4.1. Synthesis of 2,2'-(piperazine-1,4-diylbis(methylene))dianiline (A1)
Piperazine (0.43 g, 5 mmol) was dissolved in acetonitrile (30 mL) and heated under refluxing
condition, then K2CO3 (1.80 g, 13 mmol) and 2-nitrobenzylchloride (1.71 g, 10 mmol) were
added. The mixture was refluxed for 48 h, being stirring and then allowed to cool to room
temperature. Solution was dried and water was added and the product was extracted with
chloroform (3×25 mL), the product is a yellow powder. A mixture of this powder (1.65 g, 4.63
mmol), NH4Cl (6.94 g, 130 mmol) and 10 mL of H2O in 100 mL of EtOH was heated to boil and
zinc dust (12.11 g, 185 mmol) in 0.50 g portions at intervals of several minutes was added. The
mixture was then refluxed for 12 h. The resulting solution was cooled to room temperature and
filtered under vacuum. The solution was dried and water was added and the product was
extracted with chloroform (3×25 mL). The white powder was dried and characterized as the pure
compound (Scheme 1). Yield: 0.81 g (55%). Anal. Calc. for C18H24N4 (MW: 296.42): C, 72.94;
H, 8.16; N, 18.90. Found: C, 72.45; H, 8.36; N, 19.19%. EI-MS (m/z): 296 (100.0%). IR (KBr,
cm-1): 3267-3440, 1612 υ (NH2).

<Please insert scheme 1>
2.4.2. Synthesis of complexes
2.4.2.1. General procedure
A solution of M(ClO4)2.6H2O (0.50 mmol) (M: Mn, Zn) and 2,6-pyridinedicarbaldehyde
(complexes:1, 3) or 2,6-diacetylpyridine (complexes: 2, 4) (0.5 mmol) (Scheme 2 and Scheme 3)
was refluxed for 4 hours in absolute methanol (20 mL). After this time, A1 (0.15 g, 0.5 mmol)
dissolved in ethanol was added drop wise to hot solution over a period of 2h. After the solution
was refluxed for 24 h, it was filtered. The powder was dried and characterized. Crystalline
compounds were obtained by slow diffusion of diethyl ether vapor into the acetonitrile solution
of the complexes 1 and 3.
<Please insert scheme 2>
<Please insert scheme 3>
2.4.2.2. [MnL1(ClO4)2](1)
Orange crystals. Yield: 0.20 g (62%). Anal. Calc. for C25H25Cl2MnN5O8 (MW: 649.34): C,
46.24; H, 3.88; N, 10.79. Found:C, 47.65; H, 4.22; N, 10.12%. EI-MS (m/z): 649. IR (KBr, cm-
-
1):1617 υ (C=N), 1585, 1491 [υ (C=C) and υ (C=N)Py], 1121, 619 υ (ClO4 ).
2.4.2.3. [MnL2(ClO4)2](2)

Orange powder. Yield: 0.23 g (69%). Anal. Calc. for C27H29Cl2MnN5O8 (MW: 678.39): C,
47.87; H, 4.32; N, 10.34. Found: C, 48.04; H, 5.02; N, 11.20; %. EI-MS (m/z): 677. IR (KBr,
-
cm-1): 1617 υ (C=N), 1589, 1432 [υ (C=C) and υ (C=N)Py], 1095, 623 υ (ClO4 ).
2.4.2.4. [ZnL1(H2O)2](ClO4)2(3)
Yellow crystals. Yield: 0.21 g (60%). Anal. Calc. for C25H29Cl2ZnN5O10 (MW: 695.84): C,
43.15; H, 4.20; N, 10.06. Found: C, 42.86; H, 4.62; N, 10.20%. EI-MS (m/z): 696. IR (KBr, cm-
-
1): 1637 υ (C=N), 1597, 1493 [υ (C=C) and υ (C=N)Py], 1119, 621 υ (ClO4 ).
2.4.2.5. [ZnL2(H2O)2](ClO4)2(4)
Yellow powder. Yield: 0.22 g (61%). Anal. Calc. for C27H33Cl2ZnN5O10 (MW: 723.89): C,
44.80; H, 4.59; N, 9.67%. Found: C, 45.11; H, 5.02; N, 9.17%. EI-MS (m/z): 724. IR (KBr, cm-
-
1): 1637 υ (C=N), 1583, 1496 [υ (C=C) and υ (C=N)Py], 1145, 627 υ (ClO4 ).
2.4.3. 1H and 13C NMR characterization of compounds
2.4.3.1. Diamine A1
1H NMR (DMSO-d6, ppm) δH = 2.52 (broad, 8Hh), 3.39 (s, 4Hg), 5.31 (s, 4H(NH2)), 6.48 (d, Hb),
13
6.62 (m, Hd), 6.92 (d, He), 7.01(m, Hc). C NMR (DMSO-d6, ppm) δC =52.9 (Cg), 61.3 (Ch),
115.1 (Cb), 116.3 (Cd), 121.3 (Cf), 128.5 (Cc), 130.58 (Ce), 148.2 (Ca).
2.4.3.2. [ZnL1(H2O)2](ClO4)2(3)

1H NMR (DMSO-d6, ppm) δH= 2.10 (s, H(H2O)), 2.34 (broad, 8Hl), 3.40 (s, 4Hk), 7.48 (d, Hf),
7.52 (m, Hh), 7.60 (d, Hi), 7.70(m, Hg), 8.50 (s, Hd), 8.73(m, Ha), 8.75(d, Hb). 13C NMR (DMSO-
d6, ppm) δC =39.4 (Ck), 58.5 (Cl), 120.4 (Cj), 129.7 (Cf), 130.4 (Ch), 131.0 (Cb), 132.0 (Cg), 132.2
(Ci), 143.8 (Ca), 147.0 (Ce), 147.3 (Cc), 158.1 (Cd).
2.4.3.3. [ZnL2(H2O)2](ClO4)2(4)
1H NMR (DMSO-d6, ppm) δH = 2.11(s, H(H2O)), 2.32 (s, H(CH3)), 2.76(broad, 8Hl), 5.23 (s, 4Hk),
6.50 (d, Hf), 6.53 (d, Hi), 6.63 (m, Hg), 6.65 (m, Hh), 6.96(m, Ha), 7.01(d, Hb). 13C NMR
(DMSO-d6, ppm) δC =25.9 (CCH3), 39.4 (Ck), 49.3 (Cl), 115.2 (Cj), 116.3 (Cf), 125.1 (Ch), 128.6
(Cb), 129.7 (Cg), 130.7 (Ci), 139.6 (Ca), 148.2 (Ce), 152.8 (Cc), 199.3 (Cd).
2.5. Cytotoxic assay
H1299 (human nonsmall cell lung carcinoma) cell line was a gift from Prof. G. Storm. The cells
had a homozygous partial deletion of the p53 protein and lacked p53 protein expression. A2780,
human ovary carcinoma cell line, and U87 MG, human glioblastoma carcinoma, were obtained
from Pasteur Institute (Tehran, Iran). In addition, the non-transformed human foreskin fibroblast
cell line Hs27 was used as a comparative control. The cells were cultured in Dulbecco’s
modified Eagle’s medium (DMEM-F12) with 5% (v/v) fetalbovine serum, 100 UmL−1 penicillin,
and 100 mgmL−1 streptomycin. The medium was changed 2-3 days and sub-cultured when the
cell population density reached 70–80% confluence. Cytotoxic effects of the compounds have
been studied in cancer cell lines evaluated by MTT assay. Exponentially growing mammalian
cells (on 96 well plate) were exposed for 24h to different concentrations of compounds. Stock
solutions of the compounds and doxorubicin were prepared in dimethyl sulfoxide (DMSO). The

final concentration of the vehicle in the medium was always 0.5%. Control cells were kept in
medium containing 0.1% DMSO. After 24 h of incubation, the medium was omitted and 0.1 mg
of MTT was added to cells, with plates further incubated for 4 h at 37 °C. The formazan crystals
were solubilized in 0.1 mL of dimethylsulfoxide and the optical density (OD570) was measured
using a microplate reader (Bio Tek Instruments, USA). IC50 values were calculated by plotting
the log10 of percent cell viability versus drug concentrations. In this paper, we studied the
cytotoxic effects of Mn(II) and Zn(II) macrocyclic complexes and compared them to
Doxorubicin.
2.6. Antibacterial assays
The antibacterial effect of four macrocyclic complexes was determined against the three Gram-
positive bacteria: Bacillus subtilis (ATCC 6051), Bacillus thuringiensis (PTCC 1358) and
Staphylococcouss aprophyticus (ATCC 6633), and two Gram negative bacteria: Pectobacterium
Sp., Pseudomonas fluorescens (PTCC 1310). The microorganisms were prepared from Persian
Type Culture Collection, Tehran, Iran. The organisms were sub-cultured in nutrient agar (Oxiod
Ltd.) and nutrient broth for useing in experiments, while diagnostic sensitivity test agar (DST)
(Oxoid Ltd.) was used in antibiotic sensitivity testing. For bioassays, suspensions of
approximately 1.5 ×108 cells per mL in sterile normal saline were prepared [43]. The sensitivity
testing was determined by using the agar-gel diffusion method [44-45]. 20 mcL of each chemical
compound, containing 10 mcg in DMSO, was loaded in each disk. The minimum inhibitory
concentration (MIC) of each compound was also determined using a twofold dilution method
[46]. The isolated bacterial strains were first grown in nutrient broth for 18 h before being used.

The inoculums suspensions were standardized and then were tested against the test compound,
with 20 mcL for each disk in DST medium. The plates were then incubated at 37.0 ± 0.5 °C for
24h after which they were observed for zones of inhibition. These effects were compared with
those of Tobramycin and Tetracycline as standard antibiotics at a concentration of 1 mg/mL [47].
The MIC was also determined by tube dilution techniques in Mueller–Hinton broth (Merck)
according to the procedure [48]. The tests were repeated at least three times for each organism.
3. Results and discussion
The new symmetric diamine A1 was prepared from condensation of 2-nitrobenzylchloride and
piperazine in acetonitrile solution. A1 was characterized by elemental analysis, mass
spectrometry, IR and NMR spectroscopy. The mass spectrum of A1 showed the molecular peak
at m/z = 296, consistent with the molar mass of this compound. Four new Schiff base
macrocyclic complexes, [MnL1(ClO4)2] (1), [MnL2(ClO4)2] (2), [ZnL1(H2O)2](ClO4)2(3) and
[ZnL2(H2O)2](ClO4)2(4) were prepared by template condensation reactions starting from A1 and
2,6-pyridinedicarbaldehyde or 2,6-diacetylpyridine and metal salts (mole ratio1:1:1), in mixture
of methanol and ethanol solvents (scheme. 2). All of complexes were characterized by elemental
analysis, mass spectrometry and IR spectroscopy, and Zinc complexes were characterized by
NMR spectroscopy. Also the crystal structures of macrocyclic complexes (1) and (3) were
determined by single-crystal X-ray crystallography. The infrared spectra of complexes confirmed
the formation of the macrocyclic compounds by the absence of bands of carbonyl and amine
groups of the starting materials. The IR spectra of all complexes exhibited a υ(C=N) vibration in
the range1617–1637 cm-1 and for the perchlorate anions, absorptions were found at

approximately 1100 and 620 cm-1. The cytotoxic and antibacterial activities of the complexes
were studied.
3.1. X-ray structures
Orange crystals of [MnL1(ClO4)2] (1) and yellow crystals of [ZnL1(H2O)2](ClO4)2(3) suitable for
single-crystal X-ray analyses were grown by slow diffusion of diethyl ether vapor into
acetonitrile solution. For complex(1), the Mn atom was in a distorted pentagonal bipyramidal
environment, surrounded by five nitrogen atoms from the ligand, with two monodentate
perchlorate anions coordinating to Mn in the axial positions. The pyridyl nitrogen (N2) and the
imine nitrogen atoms (N1 and N3) are co-planar with Mn atom, with the tertiary amine
nitrogens, N4 and N5, lying above and below this plane (N4: 0.211(3) Å and N5 -0.849(3) Å)
respectively. The rms deviation of the five nitrogen atoms from being planar is 0.312 Å, with Mn
lying 0.0771(8) Å out of the least-squares plane. All Mn-O and Mn-N distances were between
2.201 and 2.425Å. The 10 atoms of the pyridine and imine nitrogen system were coplanar to
within 0.0803(13) Å, while the dihedral angles between this plane and the imine aromatic rings
were 27.59(7)° and 37.89°(5), respectively. Weak C-H...Cl and C-H...O interactions (Table 3)
link the molecules into a 2D network lying in the ac plane.
For complex 3, the coordination environment of the Zn atom, and the conformation of the ligand,
are similar to the one found for Mn complex. Zn atom is in a distorted pentagonal bipyramidal
environment, surrounded by five nitrogen atoms from the ligand, with two water molecules
coordinating to Zn in the axial positions. The pyridine nitrogen (N2) and the imine nitrogen
atoms, (N1 and N3) are co-planar with the Zn atom, with the tertiary amine nitrogens, N4 and

N5, lying above and below the plane (N4: 0.292(3) Å and N5 -0.754(3) Å). The rms deviation of
the five nitrogen atoms from being planar is 0.311 Å, with the Zn lying 0.0350(8) Å out of the
least-squares plane. All Zn-O and Zn-N distances are between 2.141 & 2.486 Å. The 10 atoms of
the pyridine and imine nitrogen system are coplanar to within 0.0742(14) Å, while the dihedral
angles between this plane and the imine aromatic rings are 25.82(7)° and 40.92°(5), respectively.
Both perchlorate anions are non-coordinating, however both are involved in hydrogen bonds
with the coordinating water molecules linking the moieties into a 2D network parallel to the
plane (Table 4). Additional weak C-H...O interactions, link the ions into a 3D network. The
ORTEP views of complexes 1 and 3 are shown in Figs. 1 and 2, respectively. Crystallographic
data and structure refinement parameters are given in Table 1 and selected bond distances and
angles are given in Table 2.
Seven coordination, pentagonal bypyramidal, is unusual for first row transition elements, where
octahedral coordination tends to predominate. A search of the Cambridge Structural Database
[49] found 114 seven coordinate Mn(II) complexes, compared to 986 six coordinate complexes,
while seven-coordinate Zn(II) complexes were even rarer, with only 31 entries, compared to
1509 six coordinate complexes. The present complexes 1 and 3 can be compared to the seven
coordinates Mn(II) and Zn(II) complexes of 2,6-diacetylpyridine bis(thiosemicarbazone)
([Mn(daptsc)(H20)2](ClO4)2 (A) and [Zn(H2daptsc)(H20)2](N03)2 (B)) [50- 51]; in both cases the
ligand adopts a pentagonal planar arrangement of the five donor atoms, with two water
molecules coordinating in the apical positions. The maximum deviations from the least squares
plane of the five donor atoms involve the imine N atoms, 0.14 and -0.14 Å for the complex A,
and 0.166 and -0.205 Å for the complex B, indicating that the 2,6-diacetylpyridine
bis(thiosemicarbazone) ligand is coordinating to the metal in a more planar arrangement than the

ligand, L1, used here for 1 and 3. The Mn-O bond length of 2.229(3) Å and the Mn-N bond
lengths of 2.268(3) and 2.364(2) Å, are similar to those found here for 1, while the Zn-O bond
lengths of 2.128(9) and 2.13(1) Å, and the Zn-N bond lengths of 2.213(7) -2.348(9) Å, are
similar to those found here for 3. Slight differences in the geometry around the metal atom are
presumably due to the 2,6-diacetylpyridine bis(thiosemicarbazone) being an open-ended
macrocycle, with N3S2 donor atoms, while L1 is a fully enclosed macrocycle, with five nitrogen
donor atoms. The views of complexes A and B are shown in Fig. 3.
The structures of two complexes with similar ligands are known ([MnL(CH3CN)] (C) and
[CdL(CH3OH)] (D)) [52-53], where the amine nitrogens (corresponding to N4 and N5 here) are
secondary amines, with the two amine nitrogen atoms being linked by only one ethyl group,
rather than two ethyl groups as for the present ligands. The differences between the ligands result
in a major change in the conformation of the ligand and in the coordination environment of the
metal ions. Whereas the metal in the present complexes are in a seven coordinate, distorted
pentagonal planar environment, the metals in the structures [MnL(CH3CN)] (C) and
[CdL(CH3OH)] (D) are six coordinate, in a coordination environment that is between distorted
pentagonal pyramidal and distorted octahedral. The rms deviations of the five nitrogens from the
least squares planes for [MnL(CH3CN)] (C) and [CdL(CH3OH)] (D) are 0.653 Å and 0.677 Å,
respectively, with Mn atom lying 0.265 Å and the Cd atom lying 0.213 Å out of the respective
least squares planes. 10 atoms of the pyridine and imine nitrogen system are coplanar within
0.114 Å, for A, and 0.165 Å for C, while the dihedral angles between this plane and the imine
aromatic rings are 46.42° and 55.70° for A, and 45.95° and 51.43°, for C, respectively. The
present ligand, with tertiary amines, results in the conformation of the ligand being more planar
than the ligand with secondary amines. The five nitrogen atoms are closer to being planar,

allowing a more planar coordination of the ligand to the metal, and less steric hindrance,
permitting a seven coordinate geometry around the metal. Space fill models (Mercury) for A and
B show that the conformation of the ligand does not permit 7-coordination due to steric
hindrance, while for the present complexes, the conformation of the ligand easily allows seven
coordination. The macrocycle in C and D, with secondary amines, is flexible enough to allow the
metal to achieve a 6-coordination environment, with an additional donor molecule, even if the
coordination environment is highly distorted. The present ligand, with tertiary amines, is less
flexible, which results in the five nitrogen donor atoms being in a, somewhat distorted,
pentagonal planar arrangement, thus permitting 7-coordination, with 2 additional donor atoms.
The views of complexes C and D are shown in Fig. 4. The Space Fill diagrams for the complexes
1, 3, C and D are shown in Fig. 5.
<Please insert Fig. 1>
<Please insert Fig. 2>
<Please insert Fig. 3>
<Please insert Fig. 4>
<Please insert Fig. 5>
<Please insert Table 2>
<Please insert Table 3>
<Please insert Table 4>
3.2. Cytotoxic activity

Cytotoxic activity of the four macrocyclic complexes was evaluated against three cell lines of
human cancer including H1299, A2780 and U37 MG and a normal cell line Hs27 using MTT
method, and the IC50 values for each compound were determined. As shown in Figures 6,
complex 3 efficiently inhibited all cell growth tested in a dose dependent manner. The IC50 of
complex 3 was 40 ±3.28 mcM, 25 ± 4.02 mcM, and 20±1.52 mcM in H1299, A2780 and U87
MG cells, respectively. Also, all complexes had a potent cytotoxic effect on U87 MG human
glioblastoma carcinoma cells. The IC50 values for doxorubicin were 4.2±0.89, 27.3±3.9,
23.3±1.7 in A2780, H1299 and U87 MG cell lines, respectively. According to results, complex 3
was more potent than doxorubicin in U87 MG cell line predicting their therapeutic potential in
treatment of glioblastoma. In contrast to what was observed for the cancer cell lines, no
cytotoxicity was observed in the non-transformed Hs27 cell line at concentration up to 75 mcM
(Fig 6). Therefore, the apparent absence of cytotoxicity of all compounds to the non-transformed
Hs27 cell line in vitro is interesting, but more studies are required on other non-transformed cell
lines.
<Please insert Fig. 6>
3.3. Antibacterial properties
Antibacterial properties of four macrocyclic complexes were studied against a number of Gram-
positive and Gram-negative bacterial strains (Table 5). All of these complexes inhibited the
growth of bacterial strains, producing a zone of inhibition of diameter 7.0–32.0 mm. The most
effective compounds were 1 and 2 against B. Thuringiensis and Pectobacterium SP. In some
cases, these compounds were even more effective than the standard antibiotics Tobramycin and
Tetracycline. Since the comparison of the size of inhibition zones is generally not trustworthy,

the MIC values of the compounds were also determined. The results indicated that the MIC
values against the tested organisms varied between 4 against Pectobacterium Sp. to 12 against B.
thuringiensis lgmL-1. The standard antibiotic tobramycin showed MIC values 4 lg mL-1 and
Tetracycline 8 lgmL-1. Hence, these compounds have stronger properties than the standard
antibiotics against some bacterial strains.
<Please insert Table 5>
4. Conclusion
This paper involved the synthesis and spectroscopic characterization of new symmetric diamine
A1 that was prepared from condensation of 2-nitrobenzylchloride and piperazine in acetonitrile
solution. Four macrocyclic Schiff-base complexes of Mn(II) and Zn(II) were prepared via the
metal template [1+1] cyclocondensation of 2,6-pyridinedicarbaldehydeor 2,6-diacetylpyridine
with a new diamine A1 All of products were characterized by using different physical
.
techniques. Molecular structures of complexes 1 and 3 were revealed by single crystal X-ray
analysis. The crystal structure of 1 and 3 shows distorted pentagonal bipyramidal geometry. All
products were investigated for Cytotoxic and antibacterial properties. The results of cytotoxic
activity studies indicated complex 3 was more potent than doxorubicin as standard in U87 MG
cell line predicating their therapeutic potential in treatment of glioblastoma. The results of
antibacterial studies indicate that complexes 1 and 2 against B. thuringiensis, S. saprophyticus
and Pectobacterium SP. exhibit more activity than Tobramycin and Tetracycline as standards.
Acknowledgements

We are grateful to the Faculty of Chemistry of Bu-Ali Sina University, for financial supports.
Appendix A. Supplementary data
CCDC 1491879 and 1491880 contain the supplementary crystallographic data for [MnL1(ClO4)2]
and [ZnL1(H2O)2](ClO4)2 compounds, respectively. These data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033; or e303 mail:
deposit@ccdc.cam.ac.uk
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Table 1.
Crystal data and structure refinement for macrocyclic complexes [MnL1(ClO4)2] (1) and [ZnL1(H2O)2](ClO4)2 (3).
Identification code
Empirical formula
Formula weight
T (K)
[MnL1(ClO4)2] (1)
C25H25Cl2MnN5O8
649.34
[ZnL1(H2O)2](ClO4)2 (3)
C25H29Cl2N5O10Zn
695.80
130.01(10)
triclinic
130.00(10)
Crystal system
Space group
Unit cell dimensions
a (Å)
monoclinic
P¯1
P21/c
9.4526(5)
10.9939(7)
14.3110(9)
83.879(5)
72.528(5)
66.272(6)
1298.52(15)
2
14.0988(6)
10.7745(4)
18.3415(6)
90
b (Å)
c (Å)
α (°)
β (°)
92.392(4)
90
γ (°)
Volume (Å3)
2783.79(17)
4
Z
Dcalc (mg/m3)
Absorption coefficient
(mm-1)
1.661
1.660
0.776
1.142
F(000)
666.0
1432.0
Crystal size (mm3)
0.296 × 0.221 × 0.141
MoKα (λ = 0.71073)
6.57 to 60.842
-11 ≤ h ≤ 13
0.277 × 0.219 × 0.172
MoKα (λ = 0.71073)
6.596 to 60.99
-19 ≤ h ≤ 14
Radiation
2Θ range for data collection (°)
Index ranges
-13 ≤ k ≤ 15
-15 ≤ k ≤ 14
-18 ≤ l ≤ 18
-25 ≤ l ≤ 25
Reflections collected
13427
29112
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
6761 [Rint = 0.0306, Rsigma = 0.0478]
6761/0/370
7705 [Rint = 0.0340, Rsigma = 0.0339]
7705/0/404
1.051
1.046
Final R indexes [I>=2σ (I)]
R1 = 0.0412
R1 = 0.0399
wR2 = 0.0963
R1 = 0.0522
wR2 = 0.0928
R1 = 0.0506
Final R indexes [all data]
wR2 = 0.1070
0.44 and -0.68
wR2 = 0.1013
Largest differences in peak
and hole ( e Å-3)
0.92 and -0.81

Table 2.
Bond Lengths and Bond Angles for complexes [MnL1(ClO4)2] (1)and [ZnL1(H2O)2](ClO4)2 (3)
[MnL1(ClO4)2] (1)
Length (Å)
[ZnL1(H2O)2](ClO4)2 (3)
Length (Å)
Bond
Bond
Mn1- O1
Mn1- O5
Mn1- N1
Mn1- N2
Mn1- N3
Mn1- N4
Mn1- N5
Bond
2.2871(15)
2.2488(15)
2.3920(17)
2.2007(16)
2.4247(17)
2.2786(16)
2.3199(16)
Angle (˚)
Zn1- O1
Zn1- O2
Zn1- N1
Zn1- N2
Zn1- N3
Zn1- N4
Zn1- N5
Bond
2.1684(16)
2.1407(17)
2.3637(17)
2.1640(17)
2.4859(18)
2.2234(17)
2.2397(18)
Angle (˚)
O1- Mn1- N1
O1- Mn1- N3
O1- Mn1- N5
O5- Mn1-O1
O5- Mn1-N1
O5- Mn1-N3
O5- Mn1-N4
O5- Mn1-N5
N1- Mn1-N3
N2- Mn1-O1
N2- Mn1-O5
N2- Mn1-N1
N2- Mn1-N3
N2- Mn1-N4
N2- Mn1-N5
N4- Mn1-O1
N4- Mn1-N1
N4- Mn1-N3
N4- Mn1-N5
N5- Mn1-N1
N5- Mn1-N3
92.11(6)
83.26(6)
78.62(6)
174.69(6)
89.61(6)
92.31(6)
80.39(6)
106.65(6)
142.23(6)
85.21(6)
90.58(6)
71.31(6)
70.96(6)
148.67(6)
145.42(6)
101.59(6)
137.84(6)
79.44(6)
O1- Zn1- N1
O1- Zn1- N3
O1- Zn1- N4
O1- Zn1- N5
O2- Zn1- O1
O2- Zn1- N1
O2- Zn1- N2
O2- Zn1- N3
O2- Zn1- N4
O2- Zn1- N5
N1- Zn1- N3
N2- Zn1- O1
N2- Zn1- N1
N2- Zn1- N3
N2- Zn1- N4
N2- Zn1- N5
N4- Zn1- N1
N4- Zn1- N3
N4- Zn1- N5
N5- Zn1- N1
N5- Zn1- N3
92.97(6)
81.96(6)
105.98(6)
83.84(6)
169.15(7)
80.84(7)
88.13(7)
97.51(7)
84.37(7)
103.56(7)
142.56(6)
81.45(6)
71.86(6)
70.70(6)
145.92(7)
146.58(7)
138.74(6)
77.40(6)
67.25(7)
79.16(6)
65.39(6)
78.90(6)
135.70(6)
136.27(6)

Table 3.
Hydrogen bonds for complex (1).
_____________________________________________________________
D H A
d(D-H) (Å) d(H-A) (Å) d(D-A) (Å) D-H-A (°)
_____________________________________________________________
C3 H3 Cl21
C3 H3 O71
0.95
0.95
0.95
0.99
2.95
2.62
2.50
2.58
3.558(2)
3.488(3)
3.319(3)
3.451(3)
123.0
151.8
144.2
147.0
C5 H5 O32
C14 H14A O23
_____________________________________________________________
3
11-X, 1-Y, 2-Z; 22-X, 1-Y, 1-Z; -1+X, +Y, +Z

Table 4.
Hydrogen bonds for complex (3).
_____________________________________________________________
D H A
d(D-H) (Å) d(H-A) (Å) d(D-A) (Å) D-H-A (°)
_____________________________________________________________
O1 H1A O71
O1 H1B O7
0.77(3)
0.82(4)
0.74(4)
0.72(4)
0.99
2.06(3)
1.99(4)
2.17(4)
2.07(4)
2.65
2.808(2)
2.775(2)
2.882(3)
2.787(3)
3.592(3)
3.334(3)
3.346(3)
3.546(3)
164(3)
160(3)
164(4)
174(4)
159.5
126.4
164.5
151.3
O2 H2A O42
O2 H2B O3
C14 H14B O3
C14 H14B O4
C15 H15A O63
C15 H15B O8
0.99
2.65
0.99
2.38
0.99
2.65
_____________________________________________________________
1-X, 1-Y, 1-Z; 21-X, 1/2+Y, 3/2-Z; 31-X, -1/2+Y, 3/2-Z

Table 5.
Antibacterial activity of macrocyclic complexes that was expressed as diameter of inhabitation zone (mm) and
minimum inhibitory concentration (MIC)
Zone of inhabitation (mm)
standard
Main compounds
Microorganisms
Tobramycin
(10 mg mL-1)
4
Tetracyclin
(10 mg mL-1)
8
(1)
4
(2)
4
(3)
12
(4)
12
MIC (mcg/ml)
Gram (+)
B. Thuringiensis
S. Saprophyticus
B. Subtilis
6
22
13
8
15
15
30
20
0
32
21
0
8
0
15
7
0
8
Gram (-)
P. Fluorescens
Pectobacterium
SP
5
4
0
6
0
0
0
7
0
7
28
29

Figure caption:
Scheme 1. The process for the synthesis of diamine (A1).
Scheme 2. The process for the Synthesis of macrocyclic complexes [MnL1(ClO4)2] (1),
[MnL2(ClO4)2] (2), [ZnL1(H2O)2](ClO4)2 (3) and [ZnL2(H2O)2](ClO4)2 (4)
Scheme 3. Schematic structures of macrocyclic complexes (1), (2), (3) and (4)
Fig. 1. ORTEP representation of [MnL1(ClO4)2] (1). Displacement ellipsoids are drawn at the 50
% probability level. The H atomsare omitted for clarity.
Fig. 2. ORTEP representation of [ZnL1(H2O)2](ClO4)2 (3). Displacement ellipsoids are drawn at
-
the 50 % probability level. The H atoms and counter ions (ClO4 ) are omitted for clarity
Fig. 3. ORTEP representation of [Mn(daptsc)(H20)2](ClO4)2 (A) and[Zn(H2daptsc)(H20)2](N03)2
(B) [50-51].
Fig. 4. The structures of [MnL(CH3CN)] (C) and [CdL(CH3OH)] (D) [52-53].
Fig. 5. Space Fill diagrams for the complexes 1, 3, [MnL(CH3CN)] (A) and [CdL(CH3OH)] (B).
Fig 6. Cell viability of H1299, A2780, U87 MG and Hs27 cell lines after 24 hr treatment with
different concentrations of 1, 2, 3, 4 Data are expressed as Mean ± S.E.M three experiments.

Scheme 1. The process for the Synthesis of diamine (A1).

Scheme 2. The process for the Synthesis of macrocyclic complexes [MnL1(ClO4)2] (1), [MnL2(ClO4)2],
[ZnL1(H2O)2](ClO4)2 (3) and [ZnL2(H2O)2](ClO4)2 (4)