Synthesis, characterization and bioactive evaluation of copper(II) 5, 10, 15,20tetrakis[α,α,α,α-2-(2,6-bis(4-methylpiperazine-l- yl-methyl)-4-iminomethyl phenol)phenyl] porphyrin: A picket-fence porphyrin

Article abstract of DOI:10.1016/j.saa.2010.07.005

We are interested in constructing deeper small cavities by adding more bulky substituents at the ortho-phenyl positions of tetrakis(o-aminophenyl) porphyrin. The synthesis of picket-fence porphyrin is initiated by the preparation of the tetrakis(o-nitrophenyl)porphyrin followed by the nitro to amino reduction and subsequent condensation with Schiff base ligand to form imine linkages of porphyrin complexes. The synthesis and characterization of a new series of picket-fence porphyrins and their copper complexes are described. 5,10,15,20-Tetrakis[α,α,o-2-(2,6-bis(4-methyl piperazine-l-yl- methyl)-4-iminomethyl phenol)phenyl] porphyrin can be synthesized from 2,6bis[4-methylpiperazin-l-yl-methyl]-4-formylphenol (L) and 5,10,15,20-tetra[α,α,α,α-o-nitrophenyl]porphyrin. 5,10,15,20-Tetra[α,α-porphyrin was obtained by the reduction of 5,10,15,20-tetra[α,α,α,α-o-nitrophenyl -porphyrin. The spectral, electrochemical, antibacterial, antifungal and cytotoxicity properties of all the picket-fence porphyrin complexes were characterized and studied.

Full text of DOI:10.1016/j.saa.2010.07.005

Spectrochimica Acta Part A 77 (2010) 652–660
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Synthesis, characterization and bioactive evaluation of copper(II) 5,10,15,20-
tetrakis[␣,␣,␣,␣-2-(2,6-bis(4-methylpiperazine-1-yl-methyl)-4-iminomethyl
phenol)phenyl] porphyrin: A picket-fence porphyrin
K. Rajesha, A. Kalilur Rahimana,b, K. Shanmuga Bharathia, S. Sreedarana,c, V. Gangadevid, V. Narayanana,∗
a Department of Inorganic Chemistry, University of Madras, Guindy Campus, Chennai 600 025, Tamil Nadu, India
b PG & Research Department of Chemistry, The New College (Autonomous), Chennai 600 014, India
c Department of Chemistry, Arignar Anna Government Arts College, Vadachennimalai, Attur 636 121, India
d School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, Tamil Nadu, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We are interested in constructing deeper small cavities by adding more bulky substituents at
the ortho-phenyl positions of tetrakis(o-aminophenyl)porphyrin. The synthesis of picket-fence por-
phyrin is initiated by the preparation of the tetrakis(o-nitrophenyl)porphyrin followed by the nitro
to amino reduction and subsequent condensation with Schiff base ligand to form imine linkages
of porphyrin complexes. The synthesis and characterization of a new series of picket-fence por-
phyrins and their copper complexes are described. 5,10,15,20-Tetrakis[␣,␣,␣,␣-2-(2,6-bis(4-methyl
piperazine-1-yl-methyl)-4-iminomethyl phenol)phenyl] porphyrin can be synthesized from 2,6-
bis[4-methylpiperazin-1-yl-methyl]-4-formylphenol (L) and 5,10,15,20-tetra[␣,␣,␣,␣-o-nitropheny1]-
porphyrin. 5,10,15,20-Tetra[␣,␣,␣,␣-o-aminopheny1]-porphyrin was obtained by the reduction of
5,10,15,20-tetra[␣,␣,␣,␣-o-nitropheny1]-porphyrin. The spectral, electrochemical, antibacterial, anti-
fungal and cytotoxicity properties of all the picket-fence porphyrin complexes were characterized and
studied.
Received 7 April 2010
Received in revised form 21 June 2010
Accepted 5 July 2010
Keywords:
Picket-fence porphyrins
N-methyl piperazine
Antibacterial activity
Antifungal activity
Cytotoxicity
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
can be added to the macrocycle to form the desired cavity. In
addition, the metal center in metalloporphyrins is known to cat-
Synthetic metalloporphyrins are employed to investigate the
active-site structure and reactivity of various metalloproteins
[1–7]. The success of these investigations often depends upon
the unique steric properties of the synthetic porphyrin employed.
Steric control at the metal site has been facilitated by the use of
capped, single- and double-strapped, bis-pocket, and picket-fence
type porphyrins. The ortho-substituted tetraphenylporphyrins
have been the subject of considerable recent investigation, par-
ticularly the ortho-amino substituted picket-fence porphyrins [8]
in which long chain or bulky ortho substituents prevent rotation
about the porphyrin-phenyl bond at room temperature. Thus, four
diastereomeric atropisomers, corresponding to the four ways of
distributing the substituents between the two sides of the por-
phyrin can be isolated.
alyze many reactions of commercial interest, such as hydrocarbon
oxidation [9], and dehalogenation reaction. Toward the goal of syn-
thesizing biomimetic catalysts, a number of bis-pocket porphyrins
have been synthesized and evaluated in alkane oxidation reactions
that mimic cytochrome P450 chemistry [10,11].
The exhaustive review [12] provides an excellent coverage of
this field up until the end of 1998, and only selected examples that
are either complementary or introductory to more recent papers
will be presented here. The term picket-fence porphyrin will apply
to any porphyrin bearing functional peripheral groups, even though
these groups are sometimes not bulky enough to be considered as
pickets.
Several picket-fence zinc(II)-porphyrin derivatives have been
studied by Imai and Kyuno [13], and their affinities for pyridine,
piperidine, and quinoline has been determined in toluene and other
non coordinating solvents. This study emphasizes two basic con-
cepts. The first concept is the steric selection of the axial ligand.
This is achieved with the ␣4 atropoisomer for which some obvious
steric repulsion directs the binding on the open face of the por-
phyrin. Consequently, as this side is lacking peripheral substitution,
In fact, metalloporphyrins synthetic strategies are well devel-
oped; almost any kind and combination of peripheral substituents
∗
Corresponding author. Tel.: +91 44 22202793; fax: +91 44 22300488.
E-mail address: vnnara@yahoo.co.in (V. Narayanan).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.07.005

K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
653
only non selective binding is observed, and the small variations may
be attributed to distortions in the porphyrin skeleton. The second
concept is that interactions as weak as CH–␲ interactions can con-
tribute to enhance the affinity of the receptor for an aromatic axial
ligand in the cases of trans-␣2 atropoisomers. The most common
approach, first used by Groves and Meyers [14], which involves
attaching chiral units to preformed porphyrins such as amino- or
hydroxy-substituted TPP derivatives.
Picket-fence porphyrin Fe(II) and Co(II) complexes, developed
by Collman et al. is an excellent model of natural hemoproteins,
and their static and dynamic properties have been studied exten-
sively by using a variety of physicochemical methods [15,16]. These
compounds have four atropisomers by the restricted rotation of the
phenyl rings, and some interesting points have appeared for the
differences in chemical properties among these isomers.
Porphyrin-based compounds exert antibacterial action against
the sexually transmitted pathogens Neisseria gonorrhoeae and
Haemophilus ducreyi [17]. A survey of the literature showed that
the metalloporphyrins are relatively unstudied for their potential
antimicrobial and anticancer activity for exploitation in medicine
and industry. Only a handful of these metalloporphyrins have been
completely studied for their biological activity.
Fig. 1. Synthesis of 2,6-bis(4-methylpiperazine-1-yl-methyl)-4-formylphenol (L).
2.2. Synthesis of 2,6-bis(4-methylpiperazine-1-yl-methyl)-4-
formylphenol (L)
The Mannich reaction [18] was carried out by taking p-
hydroxybenzaldehyde (4.27 g, 35 mmol) in methanol (150 ml) and
para-formaldehyde (2.21 g, 70 mmol) was added slowly with stir-
ring and then it was heated to reflux for 1 h. To this solution
N-methylpiperazine (7.76 ml, 70 mmol) was added and heated to
reflux for 24 h. The solvent was evaporated to half the volume and
the resulting solution was neutralized with 5% Na2CO3 solution and
extracted with chloroform. The chloroform extract was dried with
anhydrous Na2SO4, filtered and evaporated to dryness, dried with
diethyl ether. Yield. 8.1 g (Fig. 1). Molecular formula: C19H30N4O2,
Molecular weight: 346 Calculated (%): C, 65.87; H, 8.73; N, 16.17;
Found (%): C, 65.94; H, 8.62; N, 16.23; Mass (EI) m/z: 346 (M+);
Selected IR data (KBr disc, ꢀ/cm−1): 1682 ꢀ(C O), 3446 ꢀ(OH), 1458
ꢀ(–N–CH3); 1H NMR (ı ppm in CDCl3): ∼2.22 (s, 6H, N–CH3), ∼2.45
(br s, 16H, methylene protons), ∼3.64 (s, 4H, benzylic protons), 7.7
(s, 1H, phenolic OH) ∼7.3 (s, 2H, aromatic protons), ∼9.8 (s, 1H,
aldehyde proton).
The present study focused on screening the picket-fence por-
phyrin complexes for their potential in antibacterial, antifungal and
anticancer activity against human pathogenic bacteria, fungi and
some human cancer cell lines.
2. Experimental
2.1. Materials and methods
o-Nitrobenzaldehyde (AR), p-hydroxybenzaldehyde, para-
formaldehyde (AR), nitrobenzene (AR), tin(II) chloride (AR),
N-methylpiperazine (AR) and dichloromethane were purchased
from Qualigens chemicals. Solvents were purified according to
standard procedure and stored over molecular sieves.
2.3. Synthesis of 5,10,15,20-Tetrakis[˛,˛,˛,˛-2-(2,6-bis(4-methyl
piperazine-1-yl-methyl)-4-iminomethyl phenol)phenyl]
porphyrin [(˛4-TNH2PP)L]
IR spectra of the compounds were recorded on Schimadzu
FT-IR 8300 series instrument using potassium bromide pellets.
The IR spectra were recorded in the region 400–4000 cm−1. The
UV–vis spectra were recorded on a Hitachi 320 double beam
spectrophotometer. The concentration of the samples used for
these measurements ranged from ∼2 × 10−6 M (Soret band) to
∼5 × 10−4 M (Q bands). The emission spectra were recorded on
a Perkin-Elmer LS-5B fluorescence spectrophotometer. Fluores-
cence lifetime measurements were carried out in a Picosecond
laser excited, time-correlated single-photon counting spectropho-
tometer. The excitation source is a tunable Ti-sapphire (TSUNAMI,
SPECTRA PHYSICS, USA). The photons were counted by a MCP-
PMT (Hamamatso-4878) and the data were analyzed by IBM
software. The C, H, N contents of the porphyrins and their com-
plexes were carried out using a Carl Erba Elemental analyzer Model
1106. The FAB-MS spectra were recorded on a Jeol SX 102/DA-
6000 mass spectrometer using p-nitrobenzyl alcohol (NBA) matrix
and EI-MS spectra on Jeol DX-303 mass spectrometer. Electro-
chemical measurements were performed at room temperature.
The cyclic voltammograms of 10−3 M solution of complexes in
dichloromethane were obtained on a CHI 600A electrochemical
analyzer. The measurements were carried out under oxygen free
conditions using a three-electrode cell in which a glassy carbon
electrode was the working electrode, a saturated Ag/AgCl electrode
was the reference electrode and a platinum wire was used as the
auxiliary electrode. Tetra(n-butyl)ammonium perchlorate (TBAP)
was used as the supporting electrolyte. Antifungal and antibacte-
rial activities of the complexes were tested by the cup plate method
using nutrient agar.
5,10,15,20-Tetra(␣,␣,␣,␣-2-aminopheny1)-porphyrin [19] (␣4-
TNH2PPH2) (1.2 g, 1.7 mmol) and 2,6-bis(4-methylpiperazin-1-yl-
methyl)-4-formylphenol (L) (2.46 g, 7.1 mmol) were dissolved in
methanol. The solution was stirred for 2 h and then heated under
reflux for 8 h. The resulting solution was evaporated to get the
crude product. This was then dissolved in dichloromethane and
chromatographed over silica gel. The title compound was isolated
by removal of the solvent from the first fraction and dried in
vacuum. Yield 0.78 g (Fig. 2). Analytical data for C120H146N24O4;
Formula weight: 1988.18; Calculated (%): C, 72.48; H, 7.39; N,
16.89. Found (%): C, 72.39; H, 7.31; N, 16.80. UV–vis (CH2Cl2, nm)
419 (4.92) (Soret band), 514 (3.88), 552 (3.28), 591 (3.10), 647
(2.98) (Q-band), Fluorescence (CH2Cl2, nm) 660,720; 1H NMR (in
ppm relative to TMS, CDCl3): 8.92 (s, 8H, ␤-pyrrole), 7.98 (d, 8H,
3,6-(4-aminophenyl)), 7.02 (d, 8H, 4,5-(4-aminophenyl)), 8.85 (s,
4H, imine), ∼2.20 (s, 24H, N–CH3), ∼2.44 (br s, 64H, methylene
protons), ∼3.65 (s, 16H, benzylic protons), ∼7.2 (s, 8H, aromatic
protons), ∼9.8 (s, 4H, phenolic OH), −2.75 (s, 2H, pyrrole NH).
Selected IR (KBr, ꢀ/cm−1) 3319 (ꢀpyrrole NH), 1622 ꢀ(CH N–), 1462
ꢀ(–N–CH3).
2.4. Synthesis of copper(II) 5,10,15,20-tetrakis[˛,˛,˛,˛-2-(2,6-
bis(4-methyl piperazine-1-yl-methyl)-4-iminomethyl
phenol)phenyl] porphyrin [(˛4-TNH2PP)L]Cu9
Compound [(␣4-TNH2PP)L] (0.5 g, 0.25 mmol) was dissolved in
250 ml of DMF and Cu(OAc)2·2H2O (0.56 g, 2.2 mmol) was added to
this mixture, with N2 passed over the reaction mixture. The mixture

654
K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
(%): C, 53.68; H, 5.45; N, 10.37; Ni, 16.30. UV–vis [CH2Cl2, ꢁmax/nm
(log ε)] 432 (5.10) (Soret band), 549 (4.31), 594 (3.69), (Q-band),
Fluorescence (CH2Cl2, ꢁmax/nm) 660,731, Selected IR (KBr, ꢀ/cm−1
1620 ꢀ(CH N–), 1463 ꢀ(–N–CH3).
)
2.6. [(˛4-TNH2PP)L]Co9
Analytical data for C144H176N24O28Co9 Formula weight:
3220.04 Calculated (%): C, 53.71; H, 5.50; N, 10.43; Co, 16.47. Found
(%): C, 53.61; H, 5.42; N, 10.32; Co, 16.35. UV–vis [CH2Cl2, ꢁmax/nm
(log ε)] 430 (5.03) (Soret band), 548 (4.26), 591 (3.98) (Q-band),
Fluorescence (CH2Cl2, ꢁmax/nm) 658, 729 Selected IR (KBr, ꢀ/cm−1
1622 ꢀ(CH N–), 1462 ꢀ(–N–CH3).
)
2.7. Antibacterial activity
2.7.1. Antibacterial assay
The sterile petri plates were prepared by pouring 20 ml of
nutrient agar medium for all the bacteria and allowed to solidify.
Then the petri plates containing the solidified media were spread
with inoculum of 0.1 ml of logarithmic phase bacteria at a den-
sity adjusted to a 0.5 McFarland turbidity standard. A sterile cotton
swab was used to spread by streaking the organisms all over the
surface of the sterile solidified medium and allowed to dry for
about 5 min. Then each impregnated discs were placed at differ-
ent areas on the surface of inoculated agar medium and incubated
for 37 ◦C for 24 h in an incubator. Then, the resulting diameter of the
growth inhibition halos caused by the picket-fence porphyrin com-
plexes was measured after 24 h of incubation. The inhibition zones
found around the discs were evaluated in millimeters. The diam-
eters of the zone of inhibition produced by the compounds were
compared with the standard antibiotics Streptomycin (50 ␮g/disc).
The experiments were performed at three times to minimize the
error.
Fig. 2. Synthesis of [(␣4-TNH2PP)L].
was refluxed for 12 h and the solvent removed under reduced pres-
sure. The residue was chromatographed by using dichloromethane
as eluent, and crystallized from CH2Cl2-light petroleum. Yield.
0.39 g (Fig. 3). Analytical data for C144H176N24O28Cu9. Formula
weight: 3261.53. Calculated (%): C, 53.02; H, 5.43; N, 10.30; Cu,
17.53. Found (%): C, 52.92; H, 5.33; N, 10.16. Cu, 17.42. UV–vis
[CH2Cl2, ꢁmax/nm (log ε)] 437 (5.02) (Soret band), 552 (4.08), 596
(3.97) (Q-band), Fluorescence (CH2Cl2, ꢁmax/nm) 661,729 Selected
IR (KBr, ꢀ/cm−1) 1618 ꢀ(CH N–), 1460 ꢀ(–N–CH3).
Complexes [(␣4-TNH2PP)L4]Ni and[(␣4-TNH2PP)L4]Co were syn-
thesized by following the above procedure by using Ni(OAc)2·4H2O
and Co(OAc)2·4H2O.
2.8. Determination of minimum inhibitory concentration (MIC)
Minimum inhibitory concentration (MIC) determination was
performed by serial tube dilution technique. The picket-fence por-
phyrin complexes were dissolved in 2 ml DMF to obtain stock
solution having a concentration of 512 ␮g/ml. In serial dilution
technique, 1 ml of prepared stock solution was transferred to a test
tube containing 1 ml of nutrient broth medium to give a concen-
tration of 256 ␮g/ml, from which 1 ml was transferred to another
test tube containing 1 ml nutrient broth medium to give a concen-
tration of 128 ␮g/ml and so on up to a concentration of 2 ␮g/ml.
After the preparation of suspensions of the test organisms (108
organism per ml), 1 drop of suspension (0.02 ml) was added to
each broth dilution. After 18 h incubation at 37 ◦C, the tubes were
then examined for the growth. The MIC of the extract was taken
as the lowest concentration that showed no growth. Growth was
observed in those tubes where the concentration of the extract was
below the inhibitory level and the broth medium was observed
turbid (cloudy). The solvent DMF and Streptomycin were used as
negative and positive control, respectively.
2.5. [(˛4-TNH2PP)L]Ni9
Analytical data for C144H176N24O28Ni9. Formula weight:
3217.88 Calculated (%): C, 53.74; H, 5.50; N, 10.44; Ni, 16.41. Found
2.9. Antifungal activity
The antifungal activity of the picket-fence porphyrin complexes
were tested by disc diffusion method against the two pathogenic
fungi Aspergillus niger and Candida albicans at a concentration of
50 ␮g/disc and 100 ␮g/disc.
2.9.1. Preparation of fungal inoculum
The fungal culture C. albicans was inoculated on Sabouraud
Fig. 3. Synthesis of [(␣4-TNH2PP)L]M9 (M = Cu, Ni, Co).
broth during 24 h at 35 ◦C. The inoculate absorbance was estab-

K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
655
Fig. 4. IR spectrum of [(␣4-TNH2PP)L].
lished between 0.08 and 0.10 AU (equivalent to 0.5 McFarland
108 CFU/ml) adding sterile Sabouraud broth, before incorporating
the yeast (ꢁ = 530 nm). The inoculum of fungal culture A. niger was
prepared from 5- to 15-day-old culture grown on PDA medium.
The petri plates were covered with 10 ml of distilled water and the
conidia were scraped using sterile spatula. The spore density of
each inoculum was adjusted with spectrophotometer (ꢁ = 530 nm)
to obtain a final concentration of approximately 104 spores/ml.
The sterile petri plates were prepared by pouring 20 ml of potato
dextrose agar medium for A. niger and Sabouraud agar medium
for C. albicans cultures and allowed to solidify. Then the petri
plates containing the solidified media were spread with inoculum
of 0.1 ml of logarithmic phase bacteria and fungal cultures at a den-
sity adjusted to a 0.5 McFarland turbidity standard. A sterile cotton
swab was used to spread by streaking the organisms all over the
surface of the sterile solidified medium and allowed to dry for about
5 min. Then the each impregnated discs were placed at different
areas on the surface of inoculated agar medium and incubated for
37 ◦C for 24 h in an incubator. Then, the resulting diameter of the
growth inhibition halos caused by the different metalloporphyrins
was measured after 24 h of incubation C. albicans and after 48 h for
A. niger. The inhibition zones found around the discs were evaluated
in millimeters. All the assays were carried out in triplicates.
fixed in 70% methanol. The cell DNA was stained with 0.5 mg/ml
propidium iodide in PBS for 15 min and destained in PBS.
2.10.2. Morphologic changes of apoptotic cells
After treatment with different concentrations of picket-fence
porphyrin complexes, the cell morphology, where the nuclei
were stained with propidium iodide was determined by light
microscopy. In all, five different fields were randomly selected for
counting at least 500 cells. The percentage of apoptotic cells was
calculated for each experiment. Cells designated as apoptosis were
those that displayed the characteristic morphological features of
apoptosis, including cell volume shrinkage, chromatin condensa-
tion and the presence of membrane-bound apoptotic bodies. For
each experiment, 500 cells were counted. The cells in apoptosis
were calculated by the formula: % of apoptotic cells = Number of
apoptotic cells/Total number of cells × 100.
2.11. Statistical analysis
The experiment was repeated three times and the results were
statistically analyzed using ANOVA.
3. Results and discussion
3.1. IR spectral analysis
2.10. Cytotoxicity of picket-fence porphyrin complexes by
apoptotic method
The FT-IR spectrum of the symmetric ligand shows a broad
band around 3446 cm−1 due to the presence of phenolic OH group.
Fig. 4 shows the FT-IR spectrum of the [(␣4-TNH2PP)L]. The band
around 1458 cm−1 is due to the presence of N–CH3 group. In picket-
2.10.1. Cytotoxicity assay
The picket-fence porphyrin complexes were dissolved in DMSO
and different concentrations of 0.5, 5.0, and 10.0 ␮M were prepared
forcytotoxicity assay. Themorphologicalchanges ofthe cancer cells
treated with different concentrations of porphyrin complexes were
studied at different time intervals during incubation period for 6,
12 and 24 h.
fence porphyrin, the C C stretching, imino band peak at 1603 cm−1
,
1642 cm−1 and the bands at 1458, 1358, 1088, and 965 cm−1 have
been found to be sensitive to metal substitutions and were, there-
fore, assigned to the porphyrin skeletal modes.
The various cancer cells (1 × 105 cells) in 35 mm sterile dispos-
able petriplates were seeded and grown at 37 ◦C in 5% Minimal
Essential Medium (MEM), supplied with different concentration of
porphyrin complexes (0.5, 5.0, and 10.0 ␮M) and control (without
porphyrin complexes) and incubated at 37 ◦C for 6, 12 and 24 h.
After the incubation period, the medium was decanted and the cells
were washed with phosphate buffered saline (PBS) at pH 7.4 twice,
3.2. Mass spectral analysis
The FAB mass spectra were carried out for the picket-fence por-
phyrin complexes. The FAB mass spectra of [(␣4-TNH2PP)]L is shown
in Fig. 5. This compound [(␣4-TNH2PP)]L shows a molecular ion peak
at m/z = 1988. The spectra show some prominent peaks correspond-

656
K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
Fig. 5. Mass spectrum of [(␣4-TNH2PP)L].
ing to the various fragments of the complexes. The appearance of
many peaks is due to the presence of isotopic atom.
Table 1 Compared to TPP, the Soret bands of the new compounds
were slightly red shifted, which indicated that the introduction of
the new groups to the meso-phenyl of the porphyrin moiety did
not greatly change the energy of S1 [20,21]. Table 1 highlights the
electronic absorption wavelengths of porphyrin free bases and
porphyrin complexes. It is observed that the porphyrin free bases
absorb at shorter wavelengths than the corresponding complexes.
The UV–visible absorption spectrum of the highly conjugated
porphyrin complexes exhibits an intense feature (extinction coef-
3.3. Electronic spectral analysis
The absorption spectra of picket-fence porphyrins in
dichloromethane show the typical Soret and Q-bands charac-
teristic of porphyrin derivatives and their metal-complex are
shown in Fig. 6a and b. The absorption maxima are summarized in
Fig. 6. (a) & (b) Absorption spectrum of [(␣4-TNH2PP)L]Cu9.

K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
657
Table 1
Absorption spectral data of picket-fence porphyrin complexes.
Picket-Fence
porphyrins
Soret band ꢁ (nm)
(log ε)
Q-band ꢁ (nm) (log ε)
[(␣4-TNH2PP)L]
419 (4.92)
514 (3.88), 552 (3.28),
591 (3.10), 647 (2.98)
552 (4.08), 596 (3.97)
549 (4.31), 594 (3.69)
548 (4.26), 591 (3.98)
[(␣4-TNH2PP)L]Cu9
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
437 (5.02)
432 (5.10)
430 (5.03)
ficient > 200.000) at about 420–440 nm (Soret band), followed by
several weaker absorptions (Q bands) at higher wavelengths (from
450 to 700 nm). While variations of the peripheral substituents on
the porphyrin ring often cause minor changes to the intensity and
wavelength of the absorption features, protonation of two of the
inner nitrogen atoms or the insertion/change of metal atoms into
the macrocycle usually strongly change the visible absorption spec-
trum. Absorption spectrum of the picket-fence porphyrin of copper
complex is shown in Fig. 6.
Fig. 7. Fluorescence spectra of [(␣4-TNH2PP)L]Cu9.
The absorption spectra of copper porphyrin complexes reveal a
number of interesting characteristics. Typically [(␣4-TNH2PP)L]Cu9
exhibits a strong Soret band at 437 nm and two weaker Q bands
located at 552 and 596 nm. Copper tetraarylporphyrins with o-
phenyl substituents in CH2Cl2 usually give typical absorption
spectra, with one very intense B band at 420–440 nm. [(␣4-
TNH2PP)L]Cu9 exhibits a similar absorption pattern; however, both
the B and Q band regions are located at longer wavelengths. The
meso-substituted groups of picket-fence porphyrins show an addi-
tional 15 nm shift. It is also noted that significant band broadening
and spectral shift are observed when Mannich base ligands are
substituted at the outer amino positions.
Fig. 8. Emission decay profile of [(␣4-TNH2PP)L]Cu9.
3.5. Electrochemical studies
All of the picket-fence porphyrin complexes show the reduc-
tion leading to the ␲-anion radical and oxidations leading to
the ␲-cation radical [22–27]. Cyclic voltammetry of the picket-
fence porphyrin complex (Fig. 9) in CH2Cl2 containing 0.1 M TBAP
(supporting electrolyte) are examined. The redox potentials are
summarized in Table 3. As shown in table, the reversible redox
processes are observed for H2TPP. Comparing the data of H2TPP
with the other free bases indicates that the amination at the
o-phenyl positions causes a negative shift in the half-wave poten-
tials due to the electron-donating properties of amino groups, in
parallel with the substituent effects on the formal potentials for
tetra(o-aryl)porphyrins studied by Kadish and Morrison [28]. The
one-electron oxidation process was also established by differential
pulse voltammetry [29]. [(␣4-TNH2PP)L]Ni9 and [(␣4-TNH2PP)L]Cu9
3.4. Fluorescence spectral analysis
The fluorescence emission spectra of the porphyrin Schiff bases
excited at their Soret bands are shown in Fig. 7. Wavelengths of
major emission bands of the freebase and metal derivatives of
each picket-fence porphyrins are given in Table 2. The spectra of
this porphyrin complexes show prominent bands appearing in the
region of 650–660 nm. There is also a less intense band with its
maximum appearing in the wavelength region 700–730 nm for
these porphyrins. The case is similar with all other porphyrins in
that each picket-fence porphyrins shows a red-shifted broad band
with considerable quenching of fluorescence in comparison with
the corresponding unsubstituted tetra-aryl porphyrin. It is to be
noted that the unsubstituted porphyrin, in each case, does not
show any major change with respect to a change of solvent. Direct
excitation of picket-fence porphyrins (Soret bands) in degassed
dichloromethane gave strong fluorescence bands at about 660 and
730 nm due to the porphyrin units.
The time-correlated single-photon counting (TCSPC) technique
was used to determine the singlet excited state fluorescence life-
times (Table 2) for picket-fence porphyrin complexes. The emission
decay profile of picket-fence porphyrin complex is shown in Fig. 8.
Table 2
Fluorescence lifetime measurements of picket-fence porphyrin complexes.
Picket-Fence
porphyrins
Fluorescence
maxima ꢁ (nm)
Fluorescence lifetimes
ꢂ1 (ns)
ꢂ2 (ns)
[(␣4-TNH2PP)L]
660,720
661, 729
660, 731
658, 729
1.89
1.43
1.35
1.75
3.01
2.97
2.88
2.75
[(␣4-TNH2PP)L]Cu9
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
Fig. 9. Cyclic voltammogram of [(␣4-TNH2PP)L]Cu9.

658
K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
Table 3
4.3. Antifungal activity
Electrochemical data of picket-fence porphyrin complexes.
The growth of fungi, including yeasts, is also inhibited by the
non-iron metalloporphyrins of the present invention. Non-iron
metalloporphyrins effective in the methods and compositions of
the protoporphyrin IX containing gallium, gadolinium, indium,
ruthenium, manganese, zinc, magnesium and chromium ions,
desirably gallium, indium or manganese. Some porphyrin deriva-
tives are already used in the treatment of certain noninfectious
diseases in humans [35].
The antifungal activities of the picket-fence porphyrins and their
complexes were quantitatively assessed by the presence or absence
of inhibition zones and zone diameters are shown in Table 6. Results
of antifungal activity showed that the picket-fence porphyrin com-
plexes inhibited the growth of the two tested pathogenic fungi, A.
niger and C. albicans.
Picket-Fence porphyrins
Oxidation
Reduction
Ep1a (V)
Ep1c (V)
[(␣4-TNH2PP)L]
0.70
0.83
0.80
0.89
−0.81
−0.91
−1.03
−0.90
[(␣4-TNH2PP)L]Cu9
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
exhibited the reversible oxidations at less positive potentials. It is
noted that the redox potential difference between oxidation is sig-
nificantly decreased from [(␣4-TNH2PP)L]Ni9 to [(␣4-TNH2PP)L]Cu9.
4. Biological studies
In the present investigation, picket-fence porphyrins and their
complexes showed moderate activity against C. albicans and they
showed remarkable inhibition zones at 100 ␮g/ml concentration.
The results of studies on antifungal activity of the complexes con-
firm the presence of inhibitors. This study provides an insight
for exploring the antifungal bioactives for better management of
fungal pathogens. The different antimicrobial activity of the com-
plexes indicated their different mechanism of biocidal property
and further studies are required to explore the exact mechanism
of antibacterial potency. It was concluded that among the tested
complexes, the results of MIC experiment did not depend on the
nature of the solvent in which metalloporphyrins were dissolved.
4.1. Antibacterial activity
Stojiljkovic et al. discovered that some non-iron metallopor-
phyrins have a potent and broad antibacterial activity [30].
In the present study, the antibacterial activity was done by disc
diffusion method and then we determined the minimum inhibitory
concentration values for the picket-fence porphyrin complexes to
estimate their potentiality against the tested human pathogenic
bacteria are shown in Table 4. We also determined the antifungal
activity of the new complexes of porphyrins against two human
pathogenic fungi to evaluate their antifungal property.
The tetra (o-hydroxyphenyl) porphine, which was studied as a
mixture of atropisomers in the PC6 plasma cell tumor implanted in
mice; this porphyrin was an effective sensitizer in vivo, although
significant cutaneous photosensitivity was also observed [31]. They
are believed to be an excellent source of biologically active com-
pounds. This study gives credence to its ethnopharmacological use
as a remedy to treat infections and diseases caused by the organ-
isms.
4.4. Cytotoxic activity of picket-fence porphyrin complexes by
apoptosis in cancer cells
4.4.1. The morphological characteristics of apoptotic cells
Apoptosis is a process of gene-mediated programmed cell death
essential for the elimination of unwanted cells in various bio-
logical systems and is the key mechanism of chemotherapeutic
agents. There are several characteristic cellular and biochemical
hallmarkers of apoptotic cell death, including oligonucleosomal
DNA fragmentation [36], nucleus condensation, DNA laddering, and
PARP-1 cleavage [37,38]. The sequence of changes in cellular mor-
phology of apoptosis can be summed up as (1) cell shrinkage, (2)
condensation, margination and fragmentation of chromatin and (3)
retention of cytoplasmic organelle structure, but loss of positional
interrelationships of organelles.
The potential application of picket-fence porphyrins and their
complexes as a new class of anticancer drugs with higher cytotoxi-
city and fewer side effects than existing metal anticancer drugs has
been eagerly explored recently.
All the complexes of picket-fence porphyrins showed cyto-
toxic activity positively. At 0.5 and 5.0 ␮M concentration, most
of the cells were arrested during cell division and the cell nuclei
became condensed and segmented after 12 h incubation which
4.2. Minimum inhibitory concentration measurements
The determination of the MIC [32] involves a semi-quantitative
test procedure, which gives an approximation to the least concen-
tration of an antimicrobial needed to prevent microbial growth.
The method displays tubes of growth broth containing a test level
of preservative, into which an inoculum of microbes was added. The
end result of the test was the minimum concentration of antimi-
crobial (test materials) which gave a clear solution, i.e., no visual
growth [33,34].
The MIC values of the complexes of Picket-Fence porphyrins
against all the tested bacteria were very small ranging from 4 to
32 ␮g/ml for tested bacteria which indicated that these metallo-
porphyrins are most active against the tested bacteria. The MIC
values of the complexes against B. subtilis, S. aureus, E. coli and K.
pneumoniae were shown in Table 5.
Table 4
Antibacterial activity of picket-fence porphyrin complexes (50 and 100 ␮g/disc) and standard Streptomycin (50 ␮g/disc) by disc-diffusion method.
S. No.
Picket-Fence porphyrins
Concentration
Diameter of the inhibition zone (in mm)
(␮g/ml)
B. subtilis
S. aureus
E. coli
K. pneumoniae
[(␣4-TNH2PP)L]Cu9
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
1
2
3
50
100
50
100
50
100
50
0
15
23
11
22
12
13
25
0
15
26
12
23
14
15
26
0
10
20
13
18
11
13
26
0
17
25
15
23
13
18
27
0
4
5
Streptomycin (positive control)
Negative control

K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
659
Table 5
Minimum inhibitory concentration (MIC) values of picket-fence porphyrin complexes and standard Streptomycin by dilution method.
S. No.
Picket-Fence porphyrins
Minimum inhibitory concentration (␮g/ml)
B. subtilis
S. aureus
E. coli
K. pneumoniae
1
2
3
4
5
[(␣4-TNH2PP)L]Cu9
32
32
64
2
32
32
64
2
32
32
64
2
32
32
64
2
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
Streptomycin (positive control)
Negative control
0
0
0
0
Table 6
Antifungal activity of picket-fence porphyrin complexes (50 and 100 ␮g/disc) and standard Nystatin (50 ␮g/disc) by disc-diffusion method.
S. No.
Picket-Fence porphyrins
Concentration (␮g/ml)
Diameter of the inhibition zone (in mm)
A. niger
C. albicans
[(␣4-TNH2PP)L]Cu9
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
1
2
3
50
100
50
100
50
6
18
7
19
4
4
8
6
9
2
100
50
0
10
23
0
6
28
0
4
5
Nystatin (positive control)
Negative control
is the indication of apoptosis. The results from light microscope
displayed morphological abnormality of the cells after treatment
with the metalloporphyrins. On the contrary, the untreated cells
(control) did not show these apoptotic characteristics. Most of the
condensed and segmented nuclei degraded after 24 h incubation
(Fig. 10).
It is indicated that with the increase of picket-fence por-
phyrin complexes concentration from 0.5 to 5.0 ␮M, porphyrin
complexes induced increased cell death through apoptosis. With
further increase of porphyrin complexes concentration from
5.0 to 10.0 ␮M, the porphyrin complexes induced cell death
through apoptosis decreased dramatically. In the present study,
we observed that at low to medium concentration (0.5–5.0 ␮M),
the efficacy of porphyrin complexes was quite dependent on the
specific cell type (Table 7).
With increasing concentrations, there is a gradual increase in
the formation of bundles of microtubules. Consequently, at a very
low concentration (0.5 ␮M), metalloporphyrins only block a small
portion of cells in the G2/M phase by inducing the formation of
ball-shaped aggregations of condensed chromosomes containing
one or more asters of microtubules. With the increasing concen-
tration to 5.0 ␮M, the portion of ball-shaped spindles increased
dramatically. This is coincident with the enhanced inhibition on
DNA synthesis, the increase of the cell apoptosis and the first phase
of viable cell decrease. With the further increase of the metallopor-
phyrins concentration from 5.0 to 10.0 ␮M, the proportion of the
ball-shaped spindles only increased slightly. This is the coincident
with the unchanged percentages of the cells in apoptosis. How-
ever, at 5 ␮M concentration range, most of the cells were prevented
from entering S phase and cell necrosis increase dramatically. We
Fig. 10. Cytotoxicity of [(␣4-TNH2PP)L]Cu9 on cancer cell lines (H 116) at different. concentrations and time intervals.

660
K. Rajesh et al. / Spectrochimica Acta Part A 77 (2010) 652–660
Table 7
Cytotoxic activity of picket-fence porphyrin complexes against human cancer cell lines by apoptotic method.
S. No.
1
Picket-Fence porphyrins
Concentration (␮M)
Apoptotic cell (%)
BT 220 (Breast)
H 116 (Human colon)
Int 407 (Human intestine)
HL 251 (Human lung)
[(␣4-TNH2PP)L]Cu9
0
0 a
0 a
0 a
0 a
0.5
5.0
10.0
12.8 0.1 c
38.3 0.6 g
39.8 4.6 g
15 0.6 c
37.3 0.3 g
38.7 1.2 h
16.9 3.6 d
34.6 0.4 e
36.3 1.2 f
15.3 1.2 d
39.6 3.8 f
40.3 0.8 g
[(␣4-TNH2PP)L]Ni9
[(␣4-TNH2PP)L]Co9
2
3
0
0 a
0 a
0 a
0 a
0.5
5.0
10.0
12.3 0.5 e
33.4 1.2 f
38.3 1.2 g
16.3 0.2 c
32.8 1.1 f
36.3 3.5 g
13.6 4.6 c
34.6 3.4 e
39.3 0.5 g
14.5 0.6 c
36.6 4.8 e
38.8 3.5 f
0
0 a
0 a
0 a
0 a
0.5
5.0
10.0
1.8 1.1 b
16.6 0.6 d
20.9 0.6 e
2.5 4.6 b
17.3 3.3 d
19.7 1.2 e
3.6 4.6 b
14.6 3.4 c
16.3 0.5 d
4.5 0.08 b
18.3 0.6 d
19.8 0.5 d
Different letters besides figures in each column indicate statistically significant at P = 0.05.
observed that the efficacy of metalloporphyrins at low to medium
concentration (0.5–10 ␮M) was quite dependent on the specific
cell type. Further experiments to understand the molecular mech-
anisms underlining the differences would be greatly important
to guide the clinical application of metalloporphyrins. Additional
studies are required to understand the molecular basis for this
differential response to enhance the effectiveness of metallopor-
phyrins in the treatment of patients with malignant disease.
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Acknowledgements
The authors are grateful for the financial supports from the
University Grants Commission, New Delhi, India. The authors
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