Synthesis, characterization, biological screenings and interaction with calf thymus DNA as well as electrochemical studies of adducts formed by azomethine [2-((3,5-dimethylphenylimino)methyl)phenol] and organotin(IV) chlorides

Article abstract of DOI:10.1016/j.poly.2012.03.048

Novel azomethine adducts of Sn(IV) have been synthesized by the reaction of R_nSnCl_4-n (n = 1-3, R = CH₃, n-C₄H₉, C₆H₅, C₆H ₁₁) with 2-((3,5-dimethylphenylimino)methyl)phenol (HL). The products were characterized by elemental analysis, FT-IR, ₁H, ₁₃C and ₁₁₉Sn NMR spectroscopy. Triphenyltin(IV) chloride [2-((3,5-dimethylphenylimino)methyl)phenol] (3) was also characterized by single crystal analysis. Spectroscopic and crystal data suggest that in all the complexes the ligand acts as a monodentate neutral molecule and is coordinated through oxygen to the tin atom forming pentacoordinated tin species. The phenolic hydrogen within the ligand is transferred to the imine nitrogen atom due to the coordination of oxygen with tin after complex formation. The ligand and its complexes have been screened for their biological activities, including DNA interaction, enzymatic, antibacterial, antifungal and cytotoxicity studies. Moreover, the electrochemical behavior of the synthesized compounds was also studied and the results obtained evidenced their irreversible oxidation.

Full text of DOI:10.1016/j.poly.2012.03.048

Polyhedron 40 (2012) 19–31
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, characterization, biological screenings and interaction with calf
thymus DNA as well as electrochemical studies of adducts formed by
azomethine [2-((3,5-dimethylphenylimino)methyl)phenol] and organotin(IV)
chlorides
a
a,
⇑
a
b
a
Muhammad Sirajuddin , Saqib Ali , Ali Haider , Naseer Ali Shah , Afzal Shah ,
b
Muhammad Rashid Khan
a
Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Department of Biochemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel azomethine adducts of Sn(IV) have been synthesized by the reaction of R SnCl
4ꢀn
(n = 1–3, R = CH3,
n
Received 27 January 2012
Accepted 16 March 2012
Available online 11 April 2012
n-C
4
H
9
, C
6
H
5
, C
6
H
11) with 2-((3,5-dimethylphenylimino)methyl)phenol (HL). The products were character-
H, 13C and 119Sn NMR spectroscopy. Triphenyltin(IV) chloride [2-((3,5-
ized by elemental analysis, FT-IR,
1
dimethylphenylimino)methyl)phenol] (3) was also characterized by single crystal analysis. Spectroscopic
and crystal data suggest that in all the complexes the ligand acts as a monodentate neutral molecule and is
coordinated through oxygen to the tin atom forming pentacoordinated tin species. The phenolic hydrogen
within the ligand is transferred to the imine nitrogen atom due to the coordination of oxygen with tin after
complex formation. The ligand and its complexes have been screened for their biological activities, includ-
ing DNA interaction, enzymatic, antibacterial, antifungal and cytotoxicity studies. Moreover, the electro-
chemical behavior of the synthesized compounds was also studied and the results obtained evidenced
their irreversible oxidation.
Keywords:
Azomethine
Organotin adduct
Proton transfer
Biological activity, Cyclic voltammetry
Ó 2012 Elsevier Ltd. All rights reserved.
1
. Introduction
prevent attack by microorganisms, and as insecticides and fungi-
4 9 3 2
cides; [(C H ) Sn] O and the water-soluble tributyl mesylimide
Metal complexes have a massive impact on medicine. They are
are widely used as wood preservatives. Several organotin com-
pounds are used commercially as hydrochloric acid scavengers in
polyvinyl chloride preparation, and dibutyltin laurate is now a
well-established highly effective and universal stabilizer for rigid
and flexible PVCs. Organotin species provide excellent protection
to the PVC under high thermal stress and confer crystal clarity to
the finished article. In relatively recent years, it has been reported
that RSn(SCH -COOC H ) compounds inhibit dehydrochlorina-
2 8 17 3
tion reactions by exchanging their anionic SR-moiety with the Cl
atoms in the polymer [5].
attractive candidates for the design of new anticancer agents. Cis-
platin, carboplatin and oxaloplatin were the first clinically used
antitumor drugs. Although platinum based treatment is efficient
for several types of solid tumors, their effectiveness is limited by
serious nephrotoxicity, myelotoxicity, peripheral neuropathy and
tumor resistance. To overcome their clinical problems, a large
number of new non-platinum-based anticancer complexes have
been screened. Some complexes containing Ti, V, Mo and Ru took
the lead in connection with good antitumor activity and remark-
ably low toxicity. Some diorganotin(IV) complexes were found to
have even better activity than cisplatin, which made them suitable
candidates for possible clinical use [1].
The chemistry of organotin compounds is gaining paramount
attention on account of their interesting structural features [2–4],
schizonticidal, antimalarial and fungicidal activities and their po-
tential as agricultural biocides. Organotin compounds have a range
of applications, including their use as boat paint additives, to
Caution: Nowadays some of these compounds are being banned
because of their environmental concerns.
Azomethine ligands are considered as ‘‘privileged ligands’’ due
to their ease of preparation and use as fluorogenic agents, pesti-
cides, herbicidal and blocking agents [6]. Coordination of azometh-
ines to organotin(IV) compounds has received considerable
attention with respect to their structural variation and potential
applications in medicinal chemistry and biotechnology [7–9].
Azomethines in the neutral and deprotonated forms react with
organotin(IV) halides, and the complexes formed have been re-
ported to exhibit different modes of coordination [10–17], which
may be the cause of their wide applications.
⇑
Corresponding author. Tel.: +92 51 90642130; fax: +92 51 90642241.
E-mail address: drsa54@yahoo.com (S. Ali).
0
277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2012.03.048

2
0
M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
2
The interaction of organometallic compounds with DNA is of
a geometric area of 0.071 cm , a Pt wire counter electrode and a
saturated calomel reference electrode (SCE), in a one-compartment
interest for therapeutic treatment as these molecules recognize
specific DNA sequences, alter the local DNA structure, inhibit
access to the activator or repressor protein and ultimately affect
the gene-expression process [18,19]. Most of the chemotherapeu-
tic drugs are DNA-targeted. The binding ability of organotin com-
pounds with DNA depends on the coordination number and
nature of the groups bonded to the central tin atom. Small mole-
cules bind to DNA through covalent or non-covalent interactions.
Such binding can take place either with the nitrogenous bases or
negatively charged oxygen of the phosphate backbone of DNA [20].
In the current article we report the synthesis of an azomethine
ligand, [2-((3,5-dimethylphenylimino)methyl)phenol], and its
complexes with organotin(IV) compounds. The synthesized ligand
and complexes were characterized by various techniques, includ-
ing FT-IR, NMR, elemental analysis and single crystal analysis.
Biological activities including the interaction with DNA, an enzy-
matic study, antibacterial, antifungal and cytotoxicity of the
synthesized ligand and its organotin(IV) complexes were studied.
The drug-DNA binding parameters of the synthesized compounds
were calculated. The electrochemical behavior of the synthesized
compounds was also studied.
electrochemical cell. The GCE was cleaned by polishing with 1 lm
alumina paste and rinsed with water before each experiment.
1 mM stock solutions of 1, 2 and 3 were prepared in 80% ethanol.
Compound 3 was used as a representative example of the organo-
tin(IV) complexes of HL. All the experiments were carried out at
room temperature (25 ± 1 °C).
2.2. Synthesis
2.2.1. Synthesis of the ligand, 2-((3,5-dimethylphenylimino)methyl)
phenol (HL)
Stoichiometric amounts of 3,5-dimethylamine and salicylalde-
hyde (5 mmol of each) were added to freshly dried toluene. The
mixture was refluxed for 3–4 h and the water that formed was
removed by using a Dean and Stark apparatus. The solvent was re-
moved by using a rotavapor and the product was obtained in the
form of an oily yellow viscous liquid. The chemical reaction is
shown in Scheme 1.
Yield: 80%; Molecular formula: C15
H
15NO; Mol. wt.: 225;
ꢀ1
Physical state: Liquid; IR (cm
3
Ar. C@C), 2865–3017 (broad) (OH), 1572 (CO); H NMR (CDCl ,
) m: 1664 (C@N), 1404 and 1605
1
(
0
0
ppm): 2.42 (s, 6H, H1, H1 ), 6.99 (s, 1H, H3), 6.96 (s, 2H, H4, H4 ),
8.37 (s, 1H, H6), 7.40–7.45 (m, 3H, H10, H11, H12), 7.08–7.11 (d,
2
. Experimental
3
1
1
13
H9, J[ H– H] = 8.7 Hz), 13.48 (s, 1H, Nꢁ ꢁ ꢁH–O); C NMR (CDCl
3
,
0
0
2.1. Materials and methods
ppm): 21.4 (2C, C1, C1 ), 139.1 (2C, C2, C2 ), 128.7 (1C, C3), 119
0
(
2C, C4, C4 ), 148.4 (1C, C5), 161.3 (1C, C6), 117.3 (1C, C7), 162.3
The reagents (CH
3
)
3
SnCl, (n-C
4
H
9
)
3
SnCl, (C
6
H
5
)
3
SnCl, (C
6
H
11
)
3
(1C, C8), 132.3 (1C, C9), 119.3 (1C, C10), 133 (1C, C11), 113.1 (1C,
C12); Molar Conductance (Km, Scm mol ): 15 at 25 °C; Solubil-
2
ꢀ1
SnCl, (C )SnCl and salicylaldehyde, 3,5-dimethylaniline were ob-
4
H
9
3
tained from Aldrich (USA) and were used without further purifica-
tion. All the solvents purchased from E. Merck (Germany) were
dried before use according to literature procedures [21]. The melting
points were determined in a capillary tube using a Gallenkamp (UK)
electrothermal melting point apparatus. IR spectra in the range
ity: Chloroform, Ethanol, DMSO, Acetone.
2.2.1.1. General procedure for the synthesis of the organotin(IV)
complexes of HL. Complexes 1–5 were prepared by dissolving
1.125 g (5 mmol) of HL in 60 mL freshly dried toluene, followed
ꢀ
1
4
000–100 cm were obtained on a Thermo Nicolet-6700 FT-IR
by the addition of 5 mmol R
3 3 4 9 6 5
SnCl (R = CH (1), n-C H (2), C H
spectrophotometer. Microanalysis was done using a Leco CHNS
(3), C 11 (4) and C SnCl (5). The reaction mixture was stirred
6
H
4
H
9
3
1
13
9
32 apparatus. H and C NMR were recorded on a Bruker-
00 MHz FT-NMR spectrometer, using CDCl as an internal refer-
H (CDCl ) = 77]. Chemical shifts are gi-
) = 7.25 and 13C (CDCl
ven in ppm and coupling constant (J) values are given in Hz. The
multiplicities of the signals in the H NMR spectra are given with
and refluxed for about 6–7 h. The contents of the flask were then
cooled and filtered. The filtrate was rotary evaporated to get the
desired product, which was recrystallized in the mixed solvent of
chloroform and n-hexane (4:1). The general chemical reaction is
shown in Scheme 2.
3
3
ence [
1
3
3
1
the chemical shifts (s = singlet, d = doublet, t = triplet, q = quartet,
A trial experiment in the presence of triethylamine was also
performed for a comparison of the product with the one prepared
without triethylamine. The ligand was taken in freshly dried tolu-
ene, to which a stoichiometric amount of triethylamine was added,
and the mixture was stirred and refluxed for about 30 min. Then a
stoichiometric amount of the organotin compound was added and
mixture was again stirred and refluxed for 6–7 h. The contents of
the flask were allowed to settle overnight and then they were fil-
tered and rotary evaporated to get the desired product. In the
NMR spectrum some extra peaks of triethylamine were observed
when compared with the product without triethylamine. From this
it was concluded that triethylamine has no role in the adduct
formation.
quint = quintet, m = multiplet). 1 Sn NMR spectra were recorded
19
6
at 298 K on samples redissolved in DMSO-d with a 400 MHz JEOL
ECS instrument. The measurements were recorded at a working fre-
quency of 149.4 MHz and the chemical shifts were referenced to
4
Me Sn as an external standard. The absorption spectra were mea-
sured on a Shimadzu 1800 UV–Vis spectrophotometer. Viscosity
was measured by an Ubbelohde viscometer at room temperature.
The electrical conductance was measured on a WTW Series Inolab
Cond 720. The X-ray diffraction data were collected on a Bruker
SMART APEX CCD diffractometer, equipped with a 4 K CCD detector
set 60.0 mm from the crystal. The crystals were cooled to 100 ± 1 K
using a Bruker KRYOFLEX low temperature device and intensity
measurements were performed using graphite monochromated
Mo Ka
radiation from a sealed ceramic diffraction tube (SIEMENS).
2.2.1.2. Trimethyltin(IV)chloride [2-((3,5-dimethylphenylimino)methyl)
Generator settings were 50 kV/40 mA. The structure was solved by
Patterson methods and an extension of the model was accomplished
by direct methods using the program DIRDIF or SIR2004. Final
refinement on F was carried out by full-matrix least squares tech-
niques using SHELXL-97, a modified version of the program PLUTO
phenol] (1). Yield: 83%; Molecular formula: C18
Wt.: 424.5; Anal. Calc. for C18 24ClNOSn: C, 50.92; H, 5.70; N, 3.30.
Found: C, 51.10; H, 5.90; N, 3.60%. IR (cm
and 1605 (Ar. C@C), 1536 (CO), 2865–3020 (broad, hydrogen bonded
H24 ClNOSn; Mol.
H
ꢀ
1
) m: 1619 (C@N), 1408
2
1
3
OH), 546 (Sn–C), 454 (Sn–O), 303 (Sn-Cl); H NMR (CDCl , ppm):
0
0
(
preparation of illustrations) and the PLATON package. Voltammetric
experiments were carried out using a Autolab running with GPES
.9 software, Eco-Chemie, Utrecht, the Netherlands. Measurements
were carried out using a glassy carbon working electrode (GCE) with
2.42 (s, 6H, H1, H1 ), 6.99 (s, 1H, H3), 6.96 (s, 2H, H4, H4 ), 8.63 (s,
1H, H6), 7.40–7.45 (m, 3H, H10, H11, H12), 7.08–7.11 (d, 1H, H9,
l
3
1
1
1
2
117/
4
J[ H– H] = 9 Hz), 13.52 (s, 1H, N–Hꢁ ꢁ ꢁO), 0.71 (s, SnMe), J[
19 1 13
0
Sn– H, (57, 59 Hz)]; C NMR (CDCl
3
, ppm): 21.4 (2C, C1, C1 ),

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
21
H C
H31'C
3
2
'
4'
4
9
10
Toluene, Reflux
6
5
7
NH2
O
C
H
3
N
CH
11
- H O
2
2
8
12
H C
HO
H31C
3
HO
Scheme 1. Structural representation of for the formation of azomethine ligand (HL).
1
'
1'
3
H C
H C
3
2
'
4'
4
9
4'
10
1
0
2'
9
5
6
CH
7
6
Reflux
5
7
N
11
R SnCl
3
3
N
H
C
11
3
1
H C
6-7hrs
H
2
8
2
8
1
2
4
12
1
HO
H
3
C
O
3
R = Me, n-Bu, Ph, Cy
Cl
Sn
R
R
R
Scheme 2. General structural representation of the organotin(IV) complexes of HL.
0
0
3J[117/119Sn–13
(63 Hz)]), 130.49 (Cd, 4J[117/119Sn–13Cd,
1
1
(
39.1 (2C, C2, C2 ), 128.7 (1C, C3), 119.1 (2C, C4, C4 ), 148.3 (1C, C5),
61.3 (1C, C6), 117.3 (1C, C7), 162.2 (1C, C8), 132.3 (1C, C9), 119.3
(C
(12.8 Hz)]) (SnPh)};
ductance (
c
,
C
c
,
1
2
19
Sn NMR (DMSO, ppm): ꢀ219; Molar Con-
1
117/119
ꢀ1
1C, C10), 133 (1C, C11), 113.2 (1C, C12), 21 (s, SnCH
3
,
J[
K
m, Scm mol ): 18 at 25 °C; Solubility: chloroform,
1
3
119
Sn–
Conductance (
Ca
, (206.3 Hz)]);
Sn NMR (DMSO, ppm): ꢀ222.6; Molar
ethanol, DMSO, acetone.
2
ꢀ1
K
m, Scm mol ): 16 at 25 °C; Solubility: chloroform,
ethanol, DMSO, acetone.
2
.2.1.5. Tricyclohexyltin(IV)chloride [2-((3,5-dimethylphenylimino)
methyl)phenol] (4). Yield: 78%; Molecular formula: C33 48 ClNOSn;
Mol. wt.: 628.9; m.p: 108–109 °C; Anal. Calc. for C33 48ClNOSn: C,
63.02; H, 7.69; N, 2.23. Found: C, 63.05; H, 7.66; N, 2.26%; IR (cm
: 1620 (C@N), 1441 and 1606 (Ar. C@C), 1542 (CO), 2844–2913
H
2
.2.1.3. Tri-n-butyltin(IV)chloride [2-((3,5-dimethylphenylimino)methyl)
phenol] (2). Yield: 80%; Molecular formula: C27 42 ClNOSn; Mol.
: 1621 (C@N), 1413
and 1606 (Ar. C@C), 1540 (CO), 2852–3025 (broad, hydrogen
H
ꢀ1
H
)
ꢀ1
wt.: 550.79; Physical state: liquid; IR (cm
)
m
m
(broad, hydrogen bonded OH), 562 (Sn–C), 455 (Sn–O), 319 (Sn–
Cl); H NMR (CDCl
6.84 (s, 2H, H4, H4 ), 8.64 (s, 1H, H6), 7.41–7.46 (m, 3H, H10,
H11, H12), 6.93–6.96 (d, H9, J[ H– H] = 7.8 Hz), 13.29 (s, 1H, N–
1
1
0
bonded OH), 562 (Sn–C), 455 (Sn–O), 319 (Sn–Cl); H NMR (CDCl
ppm): 2.41 (s, 6H, H1, H1 ), 6.97 (s, 1H, H3), 6.95 (s, 2H, H4, H4 ),
3
0
,
3
, ppm): 2.29 (s, 6H, H1, H1 ), 6.86 (s, 1H, H3),
0
0
3
1
1
8
1
H
SnCH
1
.63 (s, 1H, H6), 7.38–7.43 (m, 3H, H10, H11, H12), 7.06–7.09 (d,
3
1
1
0
H, H9, J[ H– H] = 8.4 Hz), 13.46 (s, 1H, N–Hꢁ ꢁ ꢁO), {1.81–1.84 (t,
Hꢁ ꢁ ꢁO), 1.79–1.89 (t, 1H, H
a
), 1.53–1.68 (m, 4H, Hb, Hb ), 1.53–
0
13
a
), 1.68–1.76 (quint, Hb), 1.34–1.48 (m, H
c
), 0.98–1.03 (t, Hd),
, ppm): 21.3 (2C, C1, C1 ),
1.68 (quint, 4H, H
c, H
c
), 1.20–1.32 (m, 2H, Hd); C NMR (CDCl
3
,
1
3
0
0
0
2
CH CH
2 2
CH
3
0
}; C NMR (CDCl
3
ppm): 21.3 (2C, C1, C1 ), 139.1 (2C, C2, C2 ), 128.6 (1C, C3), 119
(2C, C4, C4 ), 148.4 (1C, C5), 161.2 (1C, C6), 118.9 (1C, C7), 162.2
0
0
39.1 (2C, C2, C2 ), 128.7 (1C, C3), 119.3 (2C, C4, C4 ), 148.4 (1C,
C5), 161.3 (1C, C6), 119 (1C, C7), 162.2 (1C, C8), 132.3 (1C, C9),
(1C, C8), 132.2 (1C, C9), 119.3 (1C, C10), 133 (1C, C11), 117.2 (1C,
1
117/119
1
117/119
13
1
Sn–
2
CH
19.3 (1C, C10), 133 (1C, C11), 117.2 (1C, C12), {17.6 (C
a
, J[
, (23 Hz)]),
, (63 Hz)]), 13.7 (Cd) (SnCH CH CH
m,
C12), {33.8 (C
a
), J[
Sn– Ca, (302, 316 Hz)]; 31.1 (Cb),
1
3
2
117/119
13
2
117/119
13
117/119
3
117/119
13
C
a
, (336, 322 Hz)]) 27.9 (Cb, J[
Sn–
C
a
J[
26.8 (Cd), 4J[
ppm): -223.5; Molar Conductance (
Solubility: chloroform, ethanol, DMSO, acetone.
Sn– Cb, (65 Hz)]; 28.8 (C
c
), J[
Sn– Cc, (15 Hz)];
3
117/119
13
13
119
6.9 (C
)};
c
,
J[
Sn–
C
a
2
2
2
Sn– Cd, (8 Hz)] (SnCy)};
Sn NMR (DMSO,
1
19
2
ꢀ1
3
Sn NMR (DMSO, ppm): ꢀ220; Molar Conductance (
K
K
m, Scm mol ): 15 at 25 °C;
2
ꢀ1
S cm mol ): 19 at 25 °C; Solubility: chloroform, ethanol, DMSO,
acetone.
2
.2.1.6. Butyltin(IV)trichloride [2-((3,5-dimethylphenylimino) methyl)
phenol] (5). Yield: 78%; Molecular formula: C19 NOSn; Mol.
wt.: 507.47; m.p.: 102–103 °C; Anal. Calc. for C19 NOSn: C,
44.97; H, 4.77; N, 2.76. Found: C, 44.99; H, 4.74; N, 2.69%. IR
2
.2.1.4. Triphenyltin(IV)chloride [2-((3,5-dimethylphenylimino)methyl)
phenol] (3). Yield: 78%; Molecular formula: C33 30ClNOSn; Mol.
wt.: 610.76; m.p: 84–85 °C; Anal. Calc. for C33 30ClNOSn: C,
4.90; H, 4.95; N, 2.29. Found: C, 64.93; H, 4.93; N, 2.30%. IR
H
24Cl
3
H
H24Cl
3
H
ꢀ1
6
(cm
) m: 1643 (C@N), 1446 and 1589 (Ar C@C), 1551 (CO),
ꢀ1
(
cm
)
m: 1641 (C@N), 1428 and 1607 (Ar. C@C), 1536 (CO),
3078–3353 (broad, hydrogen bonded OH), 585 (Sn–C), 454 (Sn–
1
0
2
853–3064 (broad, hydrogen bonded OH), 565 (Sn–C), 453 (Sn–
3
O), 302 (Sn–Cl); H NMR (CDCl , ppm): 2.28 (s, 6H, H1, H1 ), 6.97
1
0
0
O), 303 (Sn–Cl); H NMR (CDCl
3
0
, ppm): 2.4 (s, 6H, H1, H1 ), 6.99
(s, 1H, H3), 6.81 (s, 2H, H4, H4 ), 8.63 (s, 1H, H6), 7.38–7.43 (m,
3
1
1
(
s, 1H, H3), 6.94 (s, 2H, H4, H4 ), 8.64 (s, 1H, H6), 7.37–7.43 (m,
3H, H10, H11, H12), 7.09–7.12 (d, H9, J[ H– H] = 8.4 Hz), 13.46
), 1.76–1.86 (quint, Hb), 1.33–
H, H10, H11, H12), 7.07–7.09 (d, 1H, H9, 3J[ H– H] = 8.1 Hz),
1
1
(s, 1H, N–Hꢁ ꢁ ꢁO), {1.56–1.60 (t, H
a
3
1
13
13
3.39 (s, 1H, N–Hꢁ ꢁ ꢁO), 7.46–7.80 (m, 15H, SnPh); C NMR (CDCl
3
,
1.46 (m, H
c
), 0.84–0.88 (t, Hd), SnCH
2
CH
2
CH
2
CH
3
0
}; C NMR
0
0
0
ppm): 21.3 (2C, C1, C1 ), 139.1 (2C, C2, C2 ), 128.6 (1C, C3), 119 (2C,
C4, C4 ), 148.5 (1C, C5), 161.2 (1C, C6), 118.9 (1C, C7), 162.2 (1C,
C8), 132.2 (1C, C9), 119.3 (1C, C10), 133 (1C, C11), 117.3 (1C,
C12), {139.2 (C
(CDCl
3
, ppm): 21.4 (2C, C1, C1 ), 139.9 (2C, C2, C2 ), 128.6 (1C,
C3), 119.2 (2C, C4, C4 ), 140.5 (1C, C5), 160.2 (1C, C6), 117.9 (1C,
C7), 168.7 (1C, C8), 131.7 (1C, C9), 117.9 (1C, C10), 133 (1C, C11),
0
0
), 136.2 (Cb, 2J[
117/119
Sn– Cb, (48.8 Hz)]), 129.2
13
115.8 (1C, C12), {25.6 (C
a
), 36.2 (Cb, J[
2
117/119
Sn– Cb, (43 Hz)]),
13
a

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M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
2
7.1 (C
c
,
3J[117/119Sn–13
C
c
,
(90 Hz)]), 13.5 (Cd) (SnCH
2
CH
2
DF = Dilution factor, 18.5 = Millimolar extinction coefficient of p-
nitrophenol at 405 nm. By the addition of inhibitor the activity of
the enzyme decreased, and at higher concentrations it was almost
completely inhibited in some cases, as shown in Fig. 8.
1
19
CH CH )};
2
3
Sn NMR (DMSO, ppm): ꢀ220.8; Molar Conductance
2
ꢀ1
(K
m, Scm mol ): 17 at 25 °C; Solubility: chloroform, ethanol,
DMSO, acetone.
2.2.2. DNA interaction study by UV–Vis spectroscopy
2.2.4. Antibacterial assay
CT-DNA (20 mg) was dissolved in double deionized water (pH
The synthesized ligand and its organotin(IV) complexes were
tested against six bacterial strains; two Gram-Positive [Micrococcus
luteus (ATCC10240) and Staphylococcus aureus (ATCC6538)] and
four Gram-negative [Escherichia coli (ATCC15224), Enterobactor
aerogenes (ATCC13048), Bordetella bronchiseptica (ATCC4617) and
Klebsiella pneumoniae (MTCC618)]. The agar well-diffusion method
was used for the determination of antibacterial activity [27]. Broth
7
(
2
.0) and kept at 4 °C for less than 4 days. The nucleotide to protein
N/P) ratio of ꢂ1.9 was obtained from the ratio of absorbance at
60 and 280 nm (A260/A280 = 1.9), indicating that the DNA was suf-
ficiently free from protein [22]. The DNA concentration was deter-
mined via absorption spectroscopy, using the molar absorption
ꢀ
ꢀ
1
ꢀ1
coefficient of 6600 M cm (260 nm) for CT-DNA [23], and was
4
found to be 2.97 ꢃ 10 M. Then from this stock solution 10, 15,
culture (0.75 mL) containing ca. 10 colony forming units (CFU) per
6
2
0, 25 and 30 M working solutions were prepared by the dilution
l
mL of the test strain was added to 75 mL of nutrient agar medium
method. The compounds were dissolved in 80% ethanol at a con-
centration of 0.392 mM. The UV absorption titrations were per-
formed by keeping the concentration of the compound fixed
while varying the DNA concentration. Equivalent solutions of
DNA were added to the complex and reference solutions to elimi-
nate the absorbance of DNA itself. Compound–DNA solutions were
allowed to incubate for 30 min at room temperature before the
measurements were made. Absorption spectra were recorded
using cuvettes of 1 cm path length at room temperature.
at 45 °C, mixed well, and then poured into a 14 cm sterile petri
plate. The media was allowed to solidify, and 8 mm wells were
dug with a sterile metallic borer. Then a DMSO solution of the test
sample (100 lL) at 1 mg/mL was added to the respective wells.
DMSO served as a negative control, and the standard antibacterial
drugs Roxyithromycin (1 mg/mL) and Cefixime (1 mg/mL) were used
as positive controls. Triplicate plates of each bacterial strain were
prepared, which were incubated aerobically at 37 °C for 24 h. The
activity was determined by measuring the diameter of the zone
showing complete inhibition (mm).
2
2
.2.3. Enzymatic activity
.2.3.1. Assay of alkaline phosphatase inhibition. The inhibition of
2.2.5. Antifungal assay
alkaline phosphatase was assayed by monitoring the rate of hydro-
lysis of p-nitrophenyl phosphate (pNPP) at 25 °C in 0.1 M Na CO
NaHCO (sodium carbonate–bicarbonate) buffer (pH 10.1) [24,25].
Antifungal activity against five fungal strains [Fusarium monili-
2
3
–
formis, Aspergillus niger, Fusarium solani, Mucor species and Aspergil-
lus fumigatus] was determined by using the Agar tube dilution
method [27]. Screw caped test tubes containing Sabouraud dex-
trose agar (SDA) medium (4 mL) were autoclaved at 121 °C for
3
The enzyme catalyzes the hydrolysis of phosphate monoesters,
resulting in the formation of inorganic phosphate and an alcohol.
The identity of the alcohol varies depending on the specific phos-
phatase. The assay of alkaline phosphatase activity takes advantage
of the fact that the enzyme is non-specific and utilizes the non-bio-
logical substrate p-nitrophenyl phosphate (colourless) to give the
yellow coloured p-nitrophenol upon hydrolysis, which helps to
monitor the reaction, as shown in Scheme 3 [25].
Stock solutions of 50 lM inhibitors (compounds) in 1 mL DMSO
were prepared at room temperature. The buffer and substrate were
mixed in 1:4 ratios to make the reagent solution. Then from the re-
agent solution 2000 lL (2 mL) was taken in the cell, to which 40 lL
enzyme and varying concentrations of the inhibitor were added.
The activity of the alkaline phosphatase in the presence and ab-
sence of inhibitor was measured spectrophotometrically. The
release of the yellow coloured p-nitrophenol chromophore was
monitored at 405 nm wavelength. Enzyme activity has been ex-
1
5 min. The tubes were allowed to cool at 50 °C and non-solidified
SDA was loaded with 66.6 L of compound from the stock solution
12 mg/mL in DMSO) to make a 200 g/mL final concentration. The
l
(
l
tubes were then allowed to solidify in a slanting position at room
temperature. Each tube was inoculated with a 4 mm diameter
piece of inoculum from seven days old fungal culture. The media
supplemented with DMSO and Turbinafine (200 lg/mL) were used
as a negative and a positive control, respectively. The tubes were
incubated at 28 °C for 7 days and growth was determined by mea-
suring the linear growth (mm) and growth inhibition was calcu-
lated with reference to growth in the control as shown in the
equation below.
ꢀ
ꢁ
Linear growth in test sampleðmmÞ
Linear growth in controlðmmÞ
% Growth inhibition ¼ 100ꢀ
ꢃ100
pressed as the
lM of p-nitrophenol released per min for 5 min
for each concentration of the inhibitor, and then their average va-
lue was taken. The inhibition of the enzyme by the inhibitor was
calculated by the following formula [26]:
2.2.6. Cytotoxicity
Cytotoxicity was studied by the brine-shrimp lethality assay
method [27,28]. Brine-shrimp (Artemia salina) eggs were hatched
in artificial sea water (3.8 g sea salt/L) at room temperature
(22–29 °C). After two days these shrimps were transferred to vials
containing 5 mL of artificial sea water (10 shrimps per vial) with
Units=mL enzyme
À
Á
D
A405 nm
min Test
D A405 nm
min Blank
ꢀ
total VmLðreagent þ enzyme þ inhibitorÞ ꢃ DF
¼
1
8:5 ꢃ VmL of enzyme taken
1
0, 100 and 1000 lg/mL final concentrations of each compound ta-
ken from their stock solutions of 12 mg/mL in DMSO. After 24 h,
O
O
P
Alkaline
phosphatase
O N
OH+ HO
P
O
O N
O
O + H
2
O
2
2
O
O
p-nitrophenyl phosphate
p-nitrophennol
yellow colored)
(
colorless)
(
Scheme 3. The hydrolysis of p-nitrophenyl phosphate as catalyzed by alkaline phosphatase.

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
23
the number of surviving shrimps was counted. Data was analyzed
with a biostat 2009 computer program (Probit analysis) to deter-
mine LD50 values.
to which the chemical shift of the proton is very sensitive [35]. A
notable feature of the H NMR spectra of the complexes is the
1
broadening of the phenolic proton peak due to the weakening of
the O–H bond and strengthening of the C–N–H hydrogen bond,
and so the interaction of the proton signal with the electric qua-
druple moment of N [36]. No change in the signal attributable to
the imine proton (HC@N) in the spectra of both the ligand and
complexes indicates that the N atom is not coordinated to tin(IV).
3
. Results and discussion
The azomethine, 2-((3,5-dimethylphenylimino)methyl)phenol
(
HL) was synthesized from the 1:1 condensation of 3,5-dimethyl-
n
No J(Sn, H) couplings were observed for the n-Bu group and Sn
amine and salicylaldehyde. The structural formula with the tauto-
meric equilibrium is shown in Scheme 4. The new complexes were
1
atom due to the complex nature of the protons. The H NMR spec-
trum of 1 showed a singlet at 0.71 ppm (9H) for CH
3
–Sn, having a
prepared by the reaction of R
n 3 4 9
SnCl4ꢀn (n = 1–4, R = CH , n-C H ,
2
119
1
J( Sn– H) value (59 Hz) in the range for pentacoordinated tri-
C H
6 5
and C 11) with HL in toluene. The composition of the new
6
H
2
119
1
1
119
13
methyltin(IV) compounds. The J( Sn– H) and J( Sn– C) het-
eronuclear coupling values can be used to calculate C–Sn–C
angles by using the Lockhart–Manders equations [37,38]. Substitu-
compounds was confirmed by their analytical data and the sug-
gested structures were recognized by spectroscopic investigations.
The factors behind the selection of azomethines were their ability
to exist in keto-enol forms, establish amine-imine equilibria and
the presence of a hydrogen bond in the six membered chelate ring.
In common solvents, the azomethines exist in appreciable amounts
in the ketoenamine (ketamine) form II [29].
2
119
1
tion of the J( Sn– H) coupling constant in the equation gives a
value of 111.62° for the Me–Sn–Me angle of Me SnClHL.
3
1
3
The characteristic resonance peaks in the C NMR spectra of
1
3
3
the complexes were recorded in CDCl . In the C NMR spectrum
of the ligand, the peaks at 161.3 and 162.2 ppm belong to the azo-
methine C-6 and C-8 carbon atoms, respectively. There is no signif-
icant change in the chemical shifts of the carbons in the 13C NMR
spectra of the complexes compared with the ligand, showing ad-
duct formation. The C chemical shift of the ipso-carbon of the
3 3
SnPh moiety is around 139.2 ppm in CDCl solution, which indi-
3
.1. IR spectra
In the infrared spectrum of the free ligand HL, no band was ob-
ꢀ1
13
served in the region (3200–3500 cm ), attributable to the stretch-
ing vibration of free NH or OH groups, indicating intramolecular
hydrogen bonding in the ligand. Instead, these bands were shifted
to lower frequencies and overlapped with
2
of the adducts 1–5, which points to the fact that the ring formed
by the intramolecular hydrogen bond in the ligand is retained in
these adducts. Weak broad bands appearing in the range 2865–
cates a five-coordinated triphenyltin centre [39]. Coordination of
the tin atom in organotin compounds has been related to the
m
(C–H) in the range
ꢀ1
1
119
13
1
119
13
865–3017 cm . The same behavior was observed in the spectra
J( Sn– C) coupling constants. The J( Sn– C) coupling con-
stants for the synthesized compounds are given in Table 1. In the
2
119
13
case of the butyl derivatives (compounds 2 and 5), J( Sn– C)
is smaller than J( Sn– C) because J in space can couple more
3
119
13
3
ꢀ
1
2
3
017 cm for the free ligand are assigned to
m
(O–H) and
m
(N–H),
easily to Sn atom than J [40,41].
The 119Sn chemical shift values give information about the envi-
ronment around the tin atom. All spectra were recorded in DMSO
overlapping with (C–H). These bands were found to shift slightly
to higher frequency due to changes in hydrogen bonding and the
coordination of the phenolic oxygen atom with tin [30,31]. The
shift of a strong band occurring at 1664 cm , attributable to
m
1
19
(coordinating solvent). The d( Sn) shifts not only depend upon
ꢀ1
the electron-releasing power of the alkyl and aryl groups, but also
m
1
(C@N) in the free ligand, to lower frequency, 1619 (1), 1621 (2),
641 (3), 1620 (4) and 1643 cm (5), indicate adduct formation
n
on the nature of X in R SnX4ꢀn, as the electron-releasing power of
the alkyl group increases or the electronegativity of X decreases,
ꢀ1
and proton transfer from the phenolic oxygen atom to the imine
nitrogen atom [17]. Further comparison with the spectrum of the
free ligand reveals that the band observed at 1572 cm , due to
the tin atom becomes progressively more shielded and the
1
19
d( Sn) value moves to higher field [40]. A very important prop-
ꢀ
1
119
erty of the
Sn chemical shift is that an increase in the coordina-
CO, shifts to a lower frequency, 1536 (1 and 3), 1540 (2), 1542
tion number of tin from four to five, six or seven usually produces a
large upfield shift of d( Sn). Holecek et al. and others suggested
ꢀ1
119
(4) and 1551 cm (5), providing evidence of the participation of
oxygen in bonding with tin and so weakening of the C@O bond
the d values from +200 to ꢀ60 for four coordinated, ꢀ90 to ꢀ190
[
5
32]. The appearance of new bands observed in the region 546–
for five-coordinated and ꢀ210 to ꢀ400 ppm for six coordinated
ꢀ1
ꢀ1
65 cm
(m
Sn–C) and 453–455 cm
(m
Sn–O), indicate complex
tin atoms relative to tetra-methyltin as a Refs. [42–45].
ꢀ1
119
formation [33,34]. The bands at 302–319 cm were assigned to
The
Sn NMR spectra of all the complexes show only a sharp
the Sn–Cl bond [35].
singlet, indicating the formation of a single species. However, these
values are strongly dependent on the nature and orientation of the
organic groups bonded to tin. The shifts observed in complexes can
be explained quantitatively in terms of an increase in electron den-
sity on the tin atom as the coordination number increases. The
3.2. NMR spectra
1
The H NMR spectrum of the ligand showed the signal of the hy-
1
19
droxyl proton at 13.48 ppm. A pronounced downfield shift is
Sn chemical shift values measured in coordinating solvent for
clearly due to strong intramolecular O–Hꢁ ꢁ ꢁN hydrogen bonding,
the synthesized compounds shows penta-coordination around
CH
3
CH3
N
H
CH
N
CH
H
O
CH3
O
CH3
I
II
Scheme 4. Possible tautomeric forms for HL.

2
4
M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
Table 1
C–Sn–C) angles (°) based on NMR parameters of selected organotin(IV) derivatives.
(
Compound 1J(119Sn–13C)
Hz)
2J(119Sn–13C)
(Hz)
2
J(119Sn– H)
1
Angle (°)
(
(Hz)
h(1J)
h(2J)
1
2
3
4
5
206.3
336
63
315.8
–
–
23
–
–
40
59.1
95
112
–
–
–
–
111.6 106.2
82.3
104.5
–
–
–
106.4
H C
3
N
H
C
H
H C
O
3
Cl
Sn
R
R
R
3
Fig. 1. PLATON diagram of Ph SnClHL.
Scheme 5. Structure suggested for the coordination of HL to Sn(IV).
numbering scheme is shown in Fig. 1, while crystal data, selected
bond distances and angles are given in Tables 2 and 3, respectively.
The complex exists as a monomer. The coordination around the tin
centre in the complex is defined by three phenyl groups, one oxy-
gen atom of the phenolic group and one chlorine atom. The geom-
etry around the Sn atom can be determined by the value of
tin, with a trigonal bipyrimidal geometry. It can be inferred from
the results that the geometry around tin in the solid and solution
state is the same.
It is concluded that in all the complexes, HL seems to act as a
monodentate neutral ligand and coordinates to tin through the
oxygen atom, while an intramolecular hydrogen bond still exists
between O and N (Scheme 5).
s
= (b ꢀ
is the next largest basal angle. The
square pyramid (a = b = 180°) and unity for a perfect trigonal
pyramidal geometry ( = 120°) [46]. The value (b = Cl1–Sn1–
O1 = 176.49° and = C–Sn1–C7 = 126.73(8)°) is equal to 0.83,
a
)/60, where b is the largest basal angle around Sn and
a
s
value is zero for a perfect
a
s
3.3. Crystal structure
a
indicating a distorted trigonal bipyramidal geometry around the
tin atom. The three phenyl groups are located on the basal plane,
occupying the equatorial positions, and the more electronegative
O and Cl atoms occupy the axial positions of the trigonal bipyramid
The molecular structure of triphenyltin(IV)chloride [2-((3,5-
dimethylphenylimino)methyl)phenol] with the crystallographic
[
47]. The phenolic oxygen–tin bond distance, 2.3244(14) Å, corre-
sponds to a much weaker interaction, and a similar value of
.35 Å has been reported for triphenyltin compounds [48]. The hy-
Table 2
3
Crystal data and structure refinement parameters for Ph SnClHL (3).
2
Formula
C
33
H
30ClN
835.76
monoclinic
2 2
O Sn
droxyl H atom is involved in an intramolecular interaction with the
imine N atom. Selected bond lengths and bond angles are given in
Table 3. Details of the hydrogen bonds are given in Table 4. In the
case of the free ligand, the phenolic H atom is attached to the phe-
nolic O atom (enol or imine form) and forms a hydrogen bonding
with the imine N atom, while in the case of the complex, the pheno-
lic H atom is transferred to the imine N atom (keto or amine form)
and forms a hydrogen bond with the phenolic O atom, as shown in
the crystal structure. In the free ligand the N@CH (N2–C42) bond
length is 1.274(3) Å, showing double bond character, while in the
complex this bond length is increased to 1.300(3) Å (N1–C25),
showing single bond character. Also the O2–C48 bond length in
the free ligand is 1.369(3) Å, while in the complex this bond length
is decreased to 1.312(2) Å (O1–C19), showing double bond charac-
ter, i.e., the keto form. Thus all this information shows that in the
case of the free ligand, the imine form is dominant, while in the case
of the adduct, the amine form is dominant. The molecular packing
structure is shown in Fig. 2.
Formula weight
Crystal system
Space group
a (Å)
b (Å)
c (Å)
P21/n (no. 14)
17.1699(5)
10.6551(3)
22.5311(7)
90
91.729(1)
90
4120.1(2)
4
1.348
0.725
1720
yellow prisms/
a
(°)
b (°)
(°)
c
3
V (Å )
Z
d (gcmꢀ3)
) (mm 1
ꢀ
)
l
(Mo K
a
F(000)
Crystal habit/size (mm)
0
.36 ꢃ 0.25 ꢃ 0.23
T (K)
296 (2)
Radiation (Å) (Mo K
h Minimum–maximum (°)
Total reflections
Total unique data, Rint
Observed data [I > 0.0
a
)
0.71073
2.44–27.86
9794
36279, 9794, 0.0342
7413
r
(I)]
Nref, Npar
9794, 485
2
2
2
3.4. UV–Vis spectroscopy
w = 1/[
r
(Fo) +(0.0342P) + 1.1488P] where
2
2
P = [(Fo) + 2(Fc) ]/3
R, wR , S
2
0.0322, 0.0700, 1.008
0.002, 0.00
ꢀ0.398, 0.317
The interaction of azomethine (HL) and its organotin(IV) com-
plexes with DNA were examined by UV–Vis spectroscopy in order
to get some information about their mode of interaction and bind-
Maximum and average shift/error
Minimum and maximum residual Density
3
(
e/Å )
ing strength. The effect of varying the concentration (10–35
lM) of
Goodness-of-fit
1.008
DNA on the electronic absorption spectra of 0.392 mM of HL and

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
25
Table 3
(ꢂ1.2 nm) was observed with increasing concentrations of DNA.
These spectral characteristics are indicative of their binding to
DNA. Hyperchromic and hypochromic effects are the spectral
features of DNA concerning its double-helix structure. These spec-
tral change processes reflect the changes in the conformation and
structure of DNA after the complex binds to it [50,51]. Hypochro-
mism results from the contraction of the helix of DNA, as well as
from the conformational changes of DNA, while hyperchromism
3
Selected bond lengths (Å) and bond angles (°) for Ph SnClHL (3).
Bond lengths
Sn1–O1
Sn1–C1
Sn1–C7
Sn1–C13
O1–C19
O2–C48
N1–C25
Bond angle
O1–Sn1–C1
O1–Sn1–C7
O1–Sn1–C13
C1–Sn1–C7
C1–Sn1–C13
C7–Sn1–C13
Sn1–O1–C19
2.3244(14)
2.139(2)
2.144(2)
2.133(2)
1.312(2)
1.369(3)
1.300(3)
N1–C26
1.424(3)
1.4200 (16)
1.416(3)
1.416(3)
1.381(3)
1.419(3)
1.274(3)
N2–C34
C19–C20
C19–C24
C1–C2
C24–C25
N2–C42
results from damage to the double-helical structure of DNA. In
⁄
intercalation after binding to DNA, the
p
orbital of the binding li-
87.45(6)
86.32(6)
86.89(7)
126.73(8)
114.97(8)
117.42(8)
127.86(12)
Cl1–Sn1–O1
Cl1–Sn1–C13
Cl1–Sn1–C7
Cl1–Sn1–C1
N1–C25–C24
N1–C26–C31
C1–C6–C5
176.49(4)
96.58(6)
91.59(6)
91.56(6)
123.8(2)
117.2(2)
120.8(3)
gand could couple with the
p orbital of base pairs in the DNA. The
⁄
coupling
p
orbital is partially filled by electrons, thus decreasing
the transition probabilities, and hence resulting in the hypochrom-
icity [52]. These results suggest that HL and its organotin(IV)
complexes interact with DNA via the intercalation mode of interac-
tion, since only a hypochromic effect is observed, without any
significant changes of shifts in the spectral profiles, which is the
indication of a weak interaction with DNA [53]. The stability of
the binding properties of the complexes studied towards DNA
was examined by taking spectra after 24 and 48 h, and the same
results were obtained.
Table 4
Hydrogen-bond geometries (Å, °) for Ph
3
SnClHL (3).
D–Hꢁ ꢁ ꢁA
N1–H1ꢁ ꢁ ꢁO1
N2–H2ꢁ ꢁ ꢁO2
D–H
Hꢁ ꢁ ꢁA
1.86(2)
1.7400
Dꢁ ꢁ ꢁA
2.603(2)
D–Hꢁ ꢁ ꢁA
142(2)
142(2)
Based upon the variation in absorbance, the association/binding
constants of these complexes with DNA were determined accord-
ing to the Benesi–Hildebrand equation, Eq. (1) [49]:
0.87(2)
0.9400
2.5989(15)
Symmetry codes: (i) 1 ꢀ x, 1 ꢀ y, ꢀz; (ii) ꢀ1/2 + x, 3/2 ꢀ y, 1/2 + z; (iii) 3/2 ꢀ x, 1/
+ y, 1/2 ꢀ z.
2
A
e
HꢀG
e
HꢀG
1
0
G
G
¼
þ
ꢃ
ð1Þ
A ꢀ A
0
e
ꢀ
e
G
e
ꢀ
e
G
K½DNAꢄ
the organotin(IV) complexes is shown in Figs. 3–6. With an
increasing concentration of DNA, the absorption bands of the com-
plexes were affected, resulting in the tendency of hypochromism.
The strong absorption of these compounds in the near UV region
where K is the association/binding constant, A and A are the absor-
bances of the drug and its complex with DNA, respectively, and eG
0
and eH–G are the absorption coefficients of the drug and the drug–
DNA complex, respectively. The association constants were ob-
tained from the intercept-to-slope ratios of A /(A ꢀ A ) versus 1/
(
319–330 nm) is attributed to the long-living triplet excited state
of the aromatic system [49]. The binding of HL, 1, 2 and 3 to
DNA caused hypochromism. slight bathochromic shift
o
o
[DNA] plots. The same value of the binding constant was calculated
for both the peaks (i.e. 319–324 and 252–253 nm) in all spectra. The
A
3
Fig. 2. The molecular packing of Ph SnClHL viewed along the b-axis. The dotted lines showing the intramolecular H-bonding.

2
6
M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
Fig. 3. Absorption spectra of 0.392 mM HL in the absence (a) and presence of 10
l
M (b), 15
lM (c), 20 lM (d), 25 lM (e) and 30 lM (f) DNA. The arrow direction indicates
increasing concentrations of DNA. The inside graph is the plot of A
adduct.
o
/(A ꢀ A
o
) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the HL–DNA
Fig. 4. Absorption spectra of 0.392 mM (CH
indicates increasing concentrations of DNA. The inside graph is the plot of A
CH SnClHL–DNA adduct.
3
)
3
SnClHL in the absence (a) and presence of 10
l
M (b), 15
lM (c), 20 lM (d), 25 lM (e) and 30 lM (f) DNA. The arrow direction
o
/(A ꢀ A
o
) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the
(
3 3
)
4
4
binding constant values for HL, 1, 2 and 3 are 5.7 ꢃ 10 , 6.1 ꢃ 10 ,
respectively. The negative values of
of these compounds with DNA is a spontaneous process.
DG show that the interaction
4
4
ꢀ1
0
5
.6 ꢃ 10 and 3.8 ꢃ 10 M respectively. The Gibb s free energy
(DG) was determined from Eq. (2)
3
.5. Viscometric studies
D
G ¼ ꢀRT InK
ð2Þ
ꢀ1
ꢀ1
where R is general gas constant (8.314 JK mol ) and T is the
temperature (298 K). The Gibb s free energy (
HL, 1, 2 and 3 are ꢀ27.13, ꢀ27.30, ꢀ27.10 and ꢀ26.10 kJ mol
The viscometric technique is an effective tool in clarifying the
0
D
G) values for the
mode of interaction of small molecules with DNA. The viscosity of
DNA is sensitive to DNA length change, therefore its measurement
ꢀ1
,

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
27
Fig. 5. Absorption spectra of 0.392 mM (C
indicates increasing concentrations of DNA. The inside graph is the plot of A
SnClHL–DNA adduct.
4
H
9
)
3
SnClHL in the absence (a) and presence of 10
l
M (b), 15
l
M (c), 20
l
M (d), 25
l
M (e) and 30
l
M (f) DNA. The arrow direction
o
/(A ꢀ A
o
) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the
4 9 3
(C H )
Fig. 6. Absorption spectra of 0.392 mM (C
indicates increasing concentrations of DNA. The inside graph is the plot of A
SnClHL–DNA adduct.
6 5 3
H ) SnClHL in the absence (a) and presence of 10 lM (b), 15 lM (c), 20 lM (d), 25 lM (e) and 30 lM (f) DNA. The arrow direction
o
/(A ꢀ A
o
) vs. 1/[DNA] for the determination of the binding constant and Gibb’s free energy of the
6 5 3
(C H )
upon addition of a compound is often considered the least ambigu-
ous and most critical method to clarify the interaction mode of a
compound with DNA and provides reliable evidence for the interca-
lative binding mode. Relative viscosity measurements have proved
to be a reliable method for the assignment of the mode of binding
compounds to DNA. In the case of classic intercalation, DNA base
pairs are separated in order to host the bound compound, resulting
in the lengthening of the DNA helix and subsequently increased
DNA viscosity. On the other hand, the binding of a compound exclu-
sively in DNA grooves by means of partial and/or non-classic inter-
calation, under same conditions, (e.g., netropsin, distamycin),
causes a bend or kink in the DNA helix, reducing its effective length

2
8
M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
was markedly decreased by increasing the concentration of HL and
its organotin(IV) complexes, and it was almost completely lost at
high concentration. The remarkable activity of the ligand may be
due to the OH group, which can play an important role in the enzy-
matic activity [59]. Among the studied azomethine and its organo-
tin(IV) complexes, the highest activity was exhibited by the
3
Ph Sn(IV) derivative. The lower activity of the organotin(IV)
complexes (except for triphenyltin(IV)) is most probably be due
to the formation of a stable bond upon complexation between
the tin and oxygen atoms. The different behavior of the triphenyl-
tin(IV) complex can be due to weaker interactions of ligand with
tin because of the bulky phenyl groups, thereby regulating the for-
mation of the R Sn+(IV) moiety [60] which plays a key role in the
3
inhibition of the alkaline phosphatase enzyme. The inhibition pro-
file is shown in Fig. 8.
Fig. 7. Effects of the increasing amount of HL, and compounds 2 and 3 on the
ꢀ
4
relative viscosity of CT-DNA at 25 ± 0.1 °C. [DNA] = 2.97 ꢃ 10 M.
3.7. Antimicrobial activity and cytotoxicity
0
In vitro biological screening tests of the synthesized ligand and
and, as a result, the DNA solution s viscosity is decreased or remains
its organotin(IV) complexes were carried out for antibacterial
activity. The experiment was performed in triplicate using the agar
well-diffusion method. Roxyithromycin and Cefixime were used as
positive controls. The criteria for activity is based on the zone of
inhibition (mm); an inhibition zone of more than 20 mm shows
significant activity, for an 18–20 mm zone the inhibition activity
is good, 15–17 mm is low, and below 11–14 mm is non-significant
activity. The antibacterial study demonstrates that all compounds,
except the ligand and compound 1, have activity toward the tested
bacteria. Complex 3, showed significant activity against three path-
ogenic strains, S. aureus, M. luteus and B. bronchiseptica, 5 showed
significant activity against four strains, S. aureus, M. luteus, E. coli
and B. bronchiseptica, where the activity exhibited against E. coli
is better than Cefixime, used as positive control. Complex 2 showed
low to good activity against all pathogenic strains. Complex 4
showed non-significant to good activity against all tested strains.
The results are shown in Table 5.
unchanged, i.e. groove binders and electrostatic interaction do not
lengthen the DNA molecules [54–57].
A series of solutions were made with a constant concentration
ꢀ4
of DNA (2.97 ꢃ 10 M) and varying the concentration of com-
pounds HL, 1, 2 and 3. All of the samples were allowed to equili-
brate for 10 min prior to the flow time measurement. The values
1
/3
1/3
of the relative viscosity (
for all samples, where t, t
ity of the DNA solution with and without compound respectively.
g
o
/
,
g
g
o
)
were calculated from (t/t
g
o
o
)
ratio
and represent the time and viscos-
1
/3
(g/g
o
)
was plotted versus the [compound]/[DNA] ratio (Fig. 7),
which shows that the viscosity increases upon the addition of
increasing amounts of the compounds, therefore the intercalation
mode is selected for the interaction of HL, 1, 2 and 3 with DNA.
3.6. Enzymatic study
The effect of various concentrations of HL and its organotin(IV)
complexes on the activity of the enzyme Alkaline Phosphatase EC
.1.3.1 was studied for the hydrolysis of pNPP. Alkaline Phospha-
tases catalyze the transfer of phosphate groups to water (hydroly-
sis) or alcohol (transphosphorylation) using a wide variety of
phosphomonoesters and are characterized by a high pH optima
and a broad substrate specificity [58]. From the study, we have ob-
served that the presence of HL and its organotin(IV) complexes re-
sults in the deactivation of the enzyme. The activity of the enzyme
The synthesized ligand and its organotin(IV) were also sub-
jected to antifungal activity tests against five fungal strains [Alter-
naria species, A. niger, F. solani, Mucor species and A. fumigatus] by
using the Agar tube dilution method. The results are shown in
Table 5. Terbinafine was used as a standard drug in this assay.
The criteria for activity is based on percent growth inhibition;
more than 70% growth inhibition was considered as significant
activity, 60–70% inhibition activity was good, 50–60% inhibition
activity was moderate, while below 50% inhibition activity was
3
Fig. 8. Bar graphical representation of the inhibition of ALP by HL and its organotin(IV) complexes.

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
29
considered as non-significant. The results show that the synthe-
sized organotin(IV) complexes in general have more activity than
the ligand, except in few cases. Complex 2 shows 100% growth
inhibition against all tested fungal strains, like the reference drug
Terbenafine. Similarly complexes 3 and 4 showed significant inhib-
iting activity against all strains, but less than compound 2. Com-
plexes 1 and 5 showed no to good activity against the tested
strains.
The antimicrobial activity of the azomethines expressed a
considerable enhancement on coordination with the metal ions.
This increase in the activity may be due to the fact that their struc-
tures mainly possess an additional C@N bond. It has been reported
earlier that azomethines with nitrogen and oxygen donor systems
inhibit enzyme activity since the enzymes, which require these
groups for their activity, appear to be especially more susceptible
to deactivation by metal ions on coordination.
(vi) Formation of a hydrogen bond through the azomethine
group with the active centers of various cellular constitu-
ents, resulting in interference with normal cellular processes
[63].
(vii) Inhibition of the replication of DNA by interacting with the
enzyme prosthetic group (non-protein group) [64].
The cytotoxicity was studied by the brine-shrimp lethality
method and the results are summarized in Table 6. The LD50 data
show that all the tested compounds, even the ligand, are toxic with
LD50 values in the range 0.42–44.55
reference drug MS-222 (Tricaine Methanesulfonate) with an LD50
value of 4.3 g/mL. Complexes 1, 3 and 5 were the most toxic as
lg/mL in comparison to the
l
compared to the tested compounds and reference drugs.
3.8. Conductometric study
Though the exact biochemical mechanism is not completely
understood, the mode of action of antimicrobials may involve var-
ious targets in the microorganisms. These targets include the
following:
The conductance of the synthesized ligands and complexes in
2
ꢀ1
80% ethanol at 25 °C falls in the range of 15–19 Scm mol , sug-
gesting their non-electrolytic nature [65]. This shows that they
are stabile and hence do not undergo ionization.
(
i) The higher activity of the metal complexes may be due to
the different properties of the metal ions upon chelation.
The polarity of the metal ions will be reduced due to the
overlap of the ligand orbitals and partial sharing of the posi-
tive charge of the metal ion with donor groups. Thus, chela-
tion increases the penetration of the complexes into the lipid
membranes and the blockage of metal binding sites in the
3.9. Electrochemical characterization
The redox behaviour of 0.5 mM 1, 2 and 3 was studied by cyclic
ꢀ1
voltammetry (CV) at 100 mVs scan rate in a N2 saturated solu-
tion of 80% ethanol. The CV’s were initially started at +0.00 V and
recorded between potential limits of +1.50 and ꢀ1.00 V (Fig. 9A).
On the 1st voltammogram, started in the positive direction, one
oxidation peak was observed showing that compound 3 is oxidiz-
able under these conditions. No corresponding peak was observed
in the reverse scan, which indicates that the oxidation product of
compound 3 is not reducible. The same response was also shown
by 1 and 2. The absence of a peak on the cathodic part of the vol-
tammograms indicates the stability of the Sn(IV) oxidation state in
all cases. Therefore, for the oxidation of these complexes, CV’s were
always recorded in the positive going direction from 0.00 to 1.00 V.
The first scan CV’s of 0.5 mM 1, 2 and 3 (shown in Fig. 9B) reg-
istered single irreversible oxidation peaks at 0.73, 0.60 and 0.78 V.
The anodic peaks of these complexes are attributed to the oxida-
tion of the –N@CH– group. The attribution is strengthened by the
appearance of irreversible anodic peak in the same CV region of
guanine due to the oxidation of the –N@CH– group, as reported
by earlier electrochemists [65,66]. The higher oxidation potential
enzymes of the microorganisms [61].
0
(
ii) Tweedy s chelation theory predicts that chelation reduces
the polarity of the metal atom, mainly because of partial
sharing of its positive charge with donor groups and possible
electron delocalization over the entire ring. This conse-
quently increases the lipophilic character of the chelates,
favoring their permeation through the lipid layers of the
bacterial membrane [62].
iii) Interference with the synthesis of cellular walls, causing
damage that can lead to altered cell permeability character-
istics or disorganized lipoprotein arrangements, ultimately
resulting in cell death.
(
(
iv) Deactivation of various cellular enzymes that play a vital
role in the metabolic pathways of these microorganisms.
v) Denaturation of one or more cellular proteins, causing the
normal cellular processes to be impaired.
(
Table 5
Antimicrobial Activity of HL and its organotin(IV) compounds.
Compound no.
Antibacterial
Antifungal
Average zone of inhibition(mm) inhibition ± SD
Mean value of percent growth inhibition ± SD
S. aureus
K. pneumonia M. luteus
E. aerogenes E. coli
B. brochiseptica A. Flavus
F. solani A. niger
Mucor sp. A. fumigatus
HL
1
2
3
4
5
–a
–
–
–
–
–
–
–
–
–
–
–
–
91 ± 0.5 62 ± 0.4 60 ± 0.4
–
35 ± 0.24
100
100
–
100
79 ± 0.6 59 ± 0.5
100 100
10 ± 0.9
100
100
100
19.5 ± 0.2 15 ± 0.23
20 ± 0.21
24 ± 0.21
16 ± 0.26
24 ± 0.26
35 ± 0.26
14.5 ± 0.2
16 ± 0.22
14 ± 0.27
19 ± 0.25
20 ± 0.25
19 ± 0.24
17 ± 0.12
17 ± 0.02
30 ± 0.21
26 ± 0.20
19 ± 0.24
21 ± 0.24
18 ± 0.19
21 ± 0.27
35 ± 0.3
30 ± 0.23
15 ± 0.25
22 ± 0.22
36 ± 0.42
15 ± 0.34
12 ± 0.3
15 ± 0.75
26 ± 0.23
99 ± 0.5 98 ± 0.4 98 ± 0.5
95 ± 0.23 80 ± 0.8 95 ± 0.2 99 ± 0.1
25 ± 0.69
–
69 ± 0.2 35 ± 0.6
55 ± 0.19
b
⁄d
⁄
⁄
⁄
⁄
⁄
⁄
⁄
Roxythromycin
b
⁄
⁄
Cefixime
22 ± 0.02
21 ± 0.020
30 ± 0.024 16 ± 0.025
22 ± 0.024 26 ± 0.02
Terbinafinec
⁄
⁄
⁄
⁄
⁄
⁄
100
100
100
100
100
a
b
c
Show no activity.
Reference drug, Roxythromycin and Cefixime 1 mg/ml for antibacterial activity.
Reference drug, Terbinafine for antifungal activity.
No activity. SD = Standard deviation. % inhibition of fungal growth = 100 ꢀ g
d
t
/g
c
ꢃ 100 where g
t c
= linear growth in test (mm) and g = linear growth in vehicle control
(
mm).

3
0
M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
Table 6
diffusion-controlled irreversible system |Epa ꢀ Epa/2| = 47.7/(
where is the anodic charge transfer coefficient and n the number
of electrons in the rate-determining step [67], n = 0.50 was
a
a n),
Cytotoxicity of HL and its organotin(IV) compounds.
a
a
Compound
No. of shrimps killed out of 30 per dilutiona
LD50b,c
a
a
calculated. The peak current in amperes for an irreversible system
1
000
l
g/ml
100
l
g/ml
10 lg/ml
5
1/2
1/2 1/2
is given by Ipc(A) = 2.99 ꢃ 10 n (
a
a
n) A [O]
1
D
O
m
where n is
is the
HL
1
2
3
4
5
29
30
30
30
30
30
0
24
27
30
27
30
24
0
3
25
25
23
19
22
0
44.55
0.42
5.05
1.46
8.24
1.89
the number of electrons transferred during the oxidation,
m
2
number of scans, A is the electrode area in cm , D
O
is the diffusion
2
ꢀ1
ꢀ3
coefficient in cm s , [O]
1
is the concentration in mol cm and
m
ꢀ1
1/2
is in Vs [68]. By plotting Ipc versus
m
, assuming n = 1 for 3 the
ꢀ6
2 ꢀ1
value of D
O
was obtained as 3.17 ꢃ 10 cm s . Similarly the
Vehicle control
values of the diffusion coefficient for 1 and 2 were calculated as
a
b
c
ꢀ6
ꢀ6
2 ꢀ1
Against brine-shrimps (in vitro).
Data is based on mean value of three replicates each of 10, 100 and 1000
Compared to standard drug MS-222 (Tricaine methanesulfonate) with LD50
g/mL.
8
.58 ꢃ 10 and 5.33 ꢃ 10 cm s , respectively. The lower D
O
l
g/mL.
value of 3 corresponds to its slower mobility due to its greater
molecular mass.
value of 4.30
l
The cyclic voltammetric behavior of 0.5 mM 3 in the absence
and presence of 50 lM DNA at a bare GCE is shown in Fig. 9D.
The voltammogram of 3 in the presence of DNA registered a de-
crease in peak current. The rationale behind the diminution in peak
current is the probable decrease in the concentration of free 3 due
to the formation of a macromolecular 3-DNA complex. The interac-
tion of 3 with DNA is expected to cause an alteration in the DNA
copying machinery that may stop the proliferation of cancerous
cells. Similar behavior was observed for 1 and 2. The weak current
signals and noise could not allow for quantitative measurements.
of 3 indicates that it is difficult to oxidize as compared to 1 and 2.
The abstraction of an electron from the imine group is made diffi-
cult by the involvement of the lone pair of electrons of N in hydro-
gen bonding with the hydrogen of the phenolic group. The result is
also supported by crystallography, NMR and IR spectroscopy. The
higher peak current of 1 can be linked to its greater mobility as
its molecular mass is lower than that of 2 and 3. On recording suc-
cessive scans in the same solution, without cleaning the electrode
surface, a decrease in the peak currents of all the complexes was
observed. The 1st and 2nd scan CV’s of 0.5 mM 1 are shown in
Fig. 9C. The decrease in peak currents can be explained by the
adsorption of 1 oxidation products at the GCE surface, which low-
ers the available electrode surface area. CVs were also obtained at
different scan rates in a N saturated solution of 0.5 mM 3. With
2
the increase in scan rate, a slight shift of the peak potential to more
positive values was observed.
The difference between the peak potential Epa and the potential
at peak half height Epa/2 was |Epa ꢀ Eapa/2| ꢂ95 mV. Since for a
4. Conclusion
The ligand 2-((3,5-dimethylphenylimino)methyl)phenol and its
organotin(IV) complexes were successfully synthesized and charac-
terized. The ligand was treated with different organotin(IV) chlo-
rides to form the corresponding complexes. It may be concluded
that the ligand coordinates through oxygen to the Sn atom, leading
to the formation of a five coordinated tin species. The synthesized
compounds show moderate antimicrobial and cytotoxic activities.
Fig. 9. (A) 1st scan CV of 0.5 mM 3 in 80% ethanol at 50 mV/s. (B) 1st scan CV’s of 0.5 mM 2 ( ), 2 ( ) and 3 ( ) in 80% ethanol at 100 mV/s. (C) 1st scan ( ) and 2nd scan
) CV’s of 0.5 mM 1 80% ethanol at 100 mV/s. (D) CVs of 0.5 mM of 3 in the absence ( ) and presence ( ) of 50 M DNA at 100 mV/s.
(
l

M. Sirajuddin et al. / Polyhedron 40 (2012) 19–31
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31
The conductance of the ligand and its complexes was recorded in
[
[
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1
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ꢀ1
of UV–Vis spectroscopy, viscometry and cyclic voltammetry
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[
Crystallographic data for the structure reported in this paper
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