Synthesis, Raman, FT-IR, NMR spectroscopic characterization, antimicrobial activity, cytotoxicity and DNA binding of new mixed aza-oxo-thia macrocyclic compounds

Article abstract of DOI:10.1016/j.ejmech.2009.07.003

Series of new mixed aza-oxo-thia macrocyclic ligands 1,9(2,6)-ditriazina-2,8,10,16-tetraaza-3,7,11,15-tetraoxo-5,13-dithia-cyclohexadecaphan-1⁴,9⁴-diphenyl (L₁); 1,10(2,6)-ditri azina-2,9,11,18-tetraaza-3,8,12,17-tetraoxo-

Full text of DOI:10.1016/j.ejmech.2009.07.003

European Journal of Medicinal Chemistry 44 (2009) 4681–4689
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, Raman, FT-IR, NMR spectroscopic characterization, antimicrobial
activity, cytotoxicity and DNA binding of new mixed aza-oxo-thia
macrocyclic compounds
,
b
b
c
c
c
d
Naz M. Aghatabay a , A. Bas¸ , A. Kircali , G. S¸en , M.B. Yaziciog˘lu , F. Gu¨cin , B. Du¨lger
*
a Department of Chemistry, Fatih University, Bu¨yu¨kçekmece, Istanbul 34500, Turkey
b Department of Chemistry, Koc University, Sariyer, 34450 Istanbul, Turkey
c Department of Biology, Fatih University, Bu¨yu¨kçekmece, Istanbul 34500, Turkey
d Department of Biology, Canakkale Onsekiz Mart University, Canakkale, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of new mixed aza-oxo-thia macrocyclic ligands 1,9(2,6)-ditriazina-2,8,10,16-tetraaza-3,7,11,15-tet-
raoxo-5,13-dithia-cyclohexadecaphan-14,94-diphenyl (L1); 1,10(2,6)-ditri azina-2,9,11,18-tetraaza-3,8,12,
17-tetraoxo-5,6,14,15-tetrathia-cyclooctadecaphan-14,104-diphenyl (L2); 1,11(2,6)-ditriazina-2,10,12,20-tet-
Received 24 July 2008
Received in revised form
2 June 2009
Accepted 9 July 2009
Available online 16 July 2009
raaza-3,9,13,19-tetraoxo-6,16-dithia-cyclocosa-phan-14,114-diphenyl
(L3);
1,12(2,6)-ditriazina-2,11,13,
22-tetraaza-3,10,14,21-tetraoxo-6,7,17,18-tetrathia-cyclodocosaphan-14,124-diphenyl (L4) were synthesised.
The structural features of the compounds have been studied by elemental analyses, Mass, FT-Raman, FT-IR,
1H and 13C NMR spectroscopy. The antimicrobial activities of the ligands were evaluated using disk
diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC)
dilution method, against several bacteria and yeast cultures. The obtained results from both methods were
assessed in side-by-side comparison with commercial antibacterial and antifungal agents. In most cases,
the compounds show strong antifungal activity in the comparison tests. Cytotoxic activities of the ligands
against two different human cancer cell lines, stomach (23132/87) and lung (A549) were determined by
MTT assay. DNA fragmentation assay tested cell lines were used to analyze the DNA ladder formation
which is a characteristic of apoptotic cell death. The binding of the ligands with calf thymus DNA (CT-DNA)
has also been investigated by absorption spectroscopy.
Keywords:
Apoptotic
CT-DNA
Cytotoxic
FT-Raman
Inhibitory
Macrocyclic
Yeast cultures
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
possibilities to construct novel supramolecular assemblies that are
capable of performing various specific molecular functions [8–11].
The design and synthesis of macrocyclic compounds having
hetero-multi-donor centre have constituted one of the largest areas
of research mainly as a result of the roles played by these mixed
multi-function materials in: organic and coordination chemistry as
ligands, medicinal chemistry as antimicrobials, antitumors and
material applications, including dyes and photography [1–7]. Due
to structural impact preference, some macrocyclic derivatives play
important and fundamental biological functions in nature such as
enzymatic actions, photosynthesis, storage and transport of oxygen
in mammalian and other respiratory biological systems. In this
respect the macrocycles having various donor centres offer exciting
For instance, the precise molecular recognition between these
ligands and their guests, mostly transition metal ions or biomole-
cules such as nucleic acids, proteins provide a good opportunity for
studying key aspects of supramolecular chemistry, which are also
significant in a variety of disciplines including bioorganic chem-
istry, biocoordination chemistry, biology, medicine and related
science and technology [12–17]. Chemically, multi-donor ligands
and particularly mixed donor atoms of these ligands are important
because of great availability as ligands due to the presence of
several potential donor centres and their flexibility to bind with
biomolecules or to coordinate with various metal ions. The coor-
dination geometry and properties of most transition metal
complexes with numerous hemo and hetero macrocyclic ligands
have been studied [18–23]. Among these, the N–S donor macro-
cycles also have theoretical interest, as they are capable of
* Corresponding author. Tel.: þ90 212 866 3300; fax: þ90 212 866 3402.
E-mail addresses: natabay@fatih.edu.tr, natabay@yahoo.com (N.M. Aghatabay).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.003

4682
N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
furnishing an environment of controlled geometry and ligand field
strength such as the Irving–Williams series of stability and hard–
soft acid–base principle of Pearson–Parr [24–26].
Raman spectra were obtained from powdered samples placed in
a Pyrex tube using the Bruker RFS 100/S spectrometer in the range
4000–20 cmꢀ1. The 1064 nm line, provided by a near infrared
Nd:YAG air-cooled laser was used as excitation line. The output laser
power was set to 180–200 mW. Routine 1H (400 MHz) and 13C
(100 MHz) spectra were recorded at ambient temperature in
Metal ions are required for many critical functions in humans.
Metal ions can also induce toxicity in humans, classic examples
being heavy metal poisons such as mercury, antimony and lead.
Toxicity can arise from excessive quantities of either an essential
metal, possibly the result of a metabolic deficiency, or a nonessen-
tial metal due to contamination or misuse. Detection and detoxi-
fication of the toxic metals on humans and as well as environment
are one of the most challenging and interesting area of research.
Treatment of both as acute and chronic exposure can be studied by
chelation therapy [27–31]. Macrocycles having several sulphur
donor atoms are critical for soft heavy metal detoxification, because
sulphur atoms in its various forms are the primary binding site of
many toxic metals [25,26].
The synthesis of these ligands by cycloaddition reactions
between amines is well documented [15,32–35]. Recently, atten-
tion has been directed towards the use of polydentate aromatic
nitrogen heterocycles [36,37]. One possible application of these
materials is in generating supramolecular arrays, which embody
additional functional groups that are capable of metal complexa-
tion. This would result in a metallo-ligand framework with possible
potential as novel antimicrobial or therapeutic agents. In this paper,
we describe the synthesis and characterization of mixed multi-
dentate macrocycles from 2,4-diamino-6-phenyl-1,3,5-triazine,
using 5–8 atom linker dicarboxylic acids. Our principal focus is on
the synthesis, spectroscopical characterization and antimicrobial
activities due to variation of the macrocycles cavity shape.
DMSO-d6. Chemical shifts (d) are expressed in units of parts per
million relative to TMS. The analytical data, Mass, NMR and physical
properties are summarized for each experiment and the prominent
vibrational spectral data are presented in Table 1.
2.1.1. Synthesis
2.1.1.1. 1,9(2,6)-Ditriazina-2,8,10,16-tetraaza-3,7,11,15-tetraoxo-5,13-
dithia-cyclohexadecaphan-14,94-diphenyl
(L1). Centrifuged
hot
ethanolic solution (90 cm3, absolute) of 2,4-diamino-6-phenyl-
1,3,5-triazine (1.25 g, 10 mmol) was mixed with hot ethanolic
solution of (10 cm3) of 2,2-thiodiacetic acid (1.50 g, 10 mmol) in the
presence of few drops of concentrated HCl. The solution mixture
was refluxed for several hours at 85–90 ꢁC. The white crystalline
solid product was formed, which was filtered, washed with cold
EtOH and dried under vacuum. The dried solid product was then
suspended in dry toluene and the mixture was refluxed using
a Dean–Stark trap for 4 h, before the mixture was cooled down,
which was then filtered, washed with light petroleum ether and
dried under vacuum (1.54 g, 64%). m.p.230 ꢁC. Found (calculated)
(L1), [C26H22N10O4S2]: C, 52.1 (51.8); H, 3.9 (3.7); N, 22.9 (23.3);
S, 11.0 (10.6). MS (EI) m/z (%): 602 (8). 1H NMR, dH 3.37 (s, 8H, CH2);
6.76 (s, 4Hg); 7.47 (m, 4Hf); 8.25 (d, 2Hh, J ¼ 7.2 Hz); 9.0 (br, 4H, NH).
13C{1H}NMR, dC 33.99 (4C, CH2); 128.15 (4Cf, Ph); 128.58 (4Cg, Ph),
131.51 (2Ch, Ph); 137.49 (2Ce, Ph); 167.86 (4Cc, t-ring); 170.68 (2Cd,
t-ring); 171.44 (4C, CO).
2. Experimental protocols
The other ligands were prepared in a similar manner to the
ligand (L1) and the results are presented as following:
2.1. Chemistry
2.1.1.2. 1,10(2,6)-Ditriazina-2,9,11,18-tetraaza-3,8,12,17-tetraoxo-5,
6,14,15-tetrathia-cycloocta-decaphan-14,104-diphenyl (L2). Hot etha-
nolic solution (90 cm3, absolute) of 2,4-diamino-6-phenyl-1,3,5-
triazine (1.25 g, 10 mmol) was reacted with ethanolic solution of
(10 cm3) of 2,2-dithiadiacetic acid (1.82 g, 10 mmol) in the presence
of few drops of HCl. The white crystalline solid was treated in
a Dean–Stark trap (1.80 g, 67%). m.p.197–200 ꢁC. Found (calculated)
(L2) 2H2O, [C26H26N10O6S4]: C, 44.9 (44.4); H, 3.9 (3.7); N, 20.2
(19.9); S, 17.9 (18.2). MS (EI) m/z (%): 666 (9), 436 (10), 241(12), 188
(100). 1H NMR, dH 3.68 (s, 8H, CH2); 6.79 (s, 4Hg); 7.50 (m, 4Hf); 8.26
All chemicals and solvents were reagent grade and were used as
purchased without further purification except toluene (Na/benzo-
phenone under N2). Melting points were determined using an Elec-
tro-thermal 9100 melting-point apparatus. Analytical data were
obtained with a Thermo Finnigan Flash EA 1112 analyser. UV–vis
spectral measurements of the DNA binding studies were carried out
on UV-1700 PharmaSpec Shimadzu spectrophotometer. Mass (EI)
analyses were carried out in positive ion modes using a Thermo
Finnigan LCQ Advantage MAX LC/MS/MS Spectrometer. FT-IR spectra
were recorded as KBr pellets on a Jasco FT/IR-600 Plus-Spectrometer.
Table 1
Prominent IR and Raman bands for the (L1–L4) compounds.
Com.
FT-IR (cmꢀ1
)
Raman (cmꢀ1
3100, 3069 (C–H)ar, 2972, 2921
1601 (C–C)ar, 1552 (C–N)tr, 1497 [
1400, 1181, 1028, 1000, 776 ((C–H)ar, 678, 621 n
)
(L1)
3371
1673
n
(N–H), 3326
(C]O), 1592
n
(N–H), 3178 [
n
n
(CO) þ
(C–C)ar, 1535 [
(N–H) þ
((N–H) þ (C]O)], 687
d
(N–H)], 3034
(C–N], 1513
((C–C)ar, 621
n
(C–H)ar, 2980
(C–N)tr, 1390, 1361–1273
(C–S), 592, 463.
n
(C–H),
n
n
(C–H), 1681
(CO) þ (N–H)], 1466,
(C–S), 95.
n(C]O),
n
d
n
n
n
n
n
d
[n(C–N) þ
d
((N–H)], 896, 780 [
u
f
u
n
u
(L2)
(L3)
(L4)
3467
(C–H), 1686,
1396, 1296–1261 [
780 ((C–C)ar, 715, 630
n
(O–H), 3414
(C]O), 1648
(C–N) þ
(C–S), 586, 470.
n
(N–H), 3322 [
(C–C)ar, 1570 [
((N–H)], 913, 817 [
n
(CO) þ
d
(N–H)], 3127
(N–H) þ (C–N], 1530
((N–H) þ (C]O)],
n
(C–H)ar, 2937, 2833
3129, 3071
n
(C–H)ar, 2998, 2937
(C]O), 1601 (C–C)ar, 1527 [
(C–N)tr, 1404, 1195, 1156, 1024, 1003, 913,
(C–S), 573, 505 (S–S), 263, 118.
n
(C–H), 1701
n(C]O),
n
n
n
d
n
n(C–N)tr
,
1651
1504
n
n
n
n
(CO) þ (N–H)],
d
n
d
u
f
u
n
784
u((C–H)ar, 681, 631
n
n
3478
2975
1565
n
n
n
(O–H), 3440
(C–H), 1685
(C–N)tr, 1394–1261 [
n
(N–H), 3332, 3215 [
(C]O), 1628 (C–C)ar, 1591 [
(C–N) þ ((N–H)], 1193, 908, 822
((C–C)ar, 709, 621 (C–S), 553, 460.
n
(CO) þ
d
(N–H)], 3073
n(C–H)ar
,
3198, 3081, 3069
1602 (C–C)tr, 1538
1182, 1163, 1004, 781
n
(C–H)ar, 2936, 2916
(C–N)tr, 1495 [
(CO) þ
((C–H)ar, 676, 616 n
n
(C–H), 1646
(N–H)], 1399,
(C–S), 395, 97, 70.
n(C]O),
n
n
d
(N–H) þ (C–N],
n
n
n
n
d
n
d
u
[u((N–H) þ
f
(C]O)], 785
u
n
3486
n
(O–H),
n
(N–H), 3319 [
(C]O), 1673
(C–N)tr, 1399–1203 [
689, 630 (C–S).
n
(CO) þ
(C]O), 1644
(C–N) þ ((N–H)], 1062, 820
d
(N–H)], 3123
n
(C–H)ar, 2965, 2925
(N–H) þ (C–N],
((N–H), 773 ((C–C)ar
3064,
1684
n
(C–H)ar, 2968, 2956, 2927, 2912
(C]O), 1622, 1605 (C–C)tr, 1578
(N–H)], 1404, 1362, 1003, 778
(C–S), 612, 601, 568, 508
n
(C–H), 1708
n
(C]O),
n(C–H), 1703
n
n
n
(C–C)ar, 1568 [
d
n
n
n
n(C–N)tr, 1504
1533
n
n
d
u
u
,
[
n
(CO) þ
d
u((C–H)ar, 682,
n
628
n
n(S–S), 487, 130, 90.
n, stretching;
d
, bending; , out-of-plane wagging; ar, aromatic ring; tr, triazine ring.
u

N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
4683
L4
L3
(L4)
(L3)
L2
(L2)
L1
(L1)
3500
3000
2500
2000
1500
1000
500 250
3500
3000
1500
1000
500
Raman shift (cm-1)
Wavenumber (cm-1)
Fig. 2. FT-Raman spectra of (L1–L4) in the 3500–60 cmꢀ1 region.
Fig. 1. FT-IR spectrum of (L1–L4) ligands in the 3600–2600/1800–400 cmꢀ1 regions.
(d, 2Hh, J ¼ 9.4 Hz); 9 (br, 4H, NH). 13C{1H} NMR, dC 41.55 (4C, CH2);
128.53 (4Cf, Ph); 128.99 (4Cg, Ph); 131.91 (2Ch, Ph); 137.89 (2Ce, Ph);
168.26 (4Cc, t-ring); 171.04 (2Cd, t-ring); 171.41 (4C, CO).
2.2. Pharmacology
The antimicrobial activities are evaluated against Gram positive
(Staphylococcus aureus ATCC 6538, Bacillus cereus ATCC 7064,
Mycobacterium smegmatis CCM 2067, Listeria monocytogenes ATCC
15313, Micrococcus luteus La 2971) and Gram-negative (Escherichia
coli ATCC 11230, Klebsiella pneumoniae UC57, Pseudomonas aerugi-
nosa ATCC 27853, Proteus vulgaris ATCC 8427, Enterobacter aero-
genes ATCC 13048) bacteria and the yeast cultures (Candida albicans
ATCC 10231, Kluyveromyces fragilis NRRL 2415, Rhodotorula rubra
DSM 70403, Debaryomyces hansenii DSM 70238 and Hanseniaspora
guilliermondii DSM 3432) using both the disk diffusion and the
dilution methods.
2.1.1.3. 1,11(2,6)-Ditriazina-2,10,12,20-tetraaza-3,9,13,19-tetraoxo-6,16-
dithia-cyclocosa-phan-14,114-diphenyl (L3). Hot ethanolic solution
(90 cm3, absolute) of 2,4-diamino-6-phenyl-1,3,5-triazine (1.25 g,
10 mmol) was reacted with ethanolic solution of (10 cm3) of 3,3-
thiodipropionic acid (1.78 g, 10 mmol) in the presence of few drops
of HCl. The white crystalline solid was treated in a Dean–Stark trap
(2.34 g, 82%). m.p. 210 ꢁC. Found (calculated) (L3) (H2O)2,
[C30H34N10O6S2]: C, 51.3 (51.9); H, 5.2 (4.9); N, 2.7 (20.2); S 9.5 (9.2).
MS (EI) m/z (%): 658 (not detected), 360, 229. 1H NMR, dH 2.51
(t, J ¼ 6.8 Hz, 8H, CH2–CO); 2.70 (t, J ¼ 6.8 Hz, 8H, S–CH2) 6.76 (s,
4Hg); 7.47 (m, 4Hf); 8.26 (d, 2Hh, J ¼ 7.2 Hz); 10 (br, 4H, NH). 13C{1H}
NMR, dC 26.41 (4C, S–CH2); 34.55 (4C, CH2–CO); 127.66 (4Cf, Ph);
128.08 (4Cg, Ph); 130.99 (2Ch, Ph); 137.04 (2Ce, Ph); 167.41 (4Cc,
t-ring); 170.23 (2Cd, t-ring); 173.01 (4C, CO).
2.2.1. Methods
2.2.1.1. Disk diffusion. Sterilised antibiotic discs (6 mm) were used
following the literature procedure [38,39]. Fresh stock solutions of
the ligand and the complex were prepared in DMSO according to
the needed concentrations for experiments. To ensure that the
solvent had no effect on bacterial growth, a control test was per-
formed with test medium supplemented with DMSO as the same
procedures as used in the experiments. All the bacteria were
incubated at 30 ꢁC for 24 h in Nutrient Broth. The yeasts were
incubated in Malt Extract Broth for 48 h. The discs injected with
solutions were placed on the inoculated agar and incubated at 35 ꢁC
(24 h) and at 25 ꢁC (72 h) for bacteria and yeast, respectively. On
each plate an appropriate reference antibiotic disc was applied
depending on the test microorganisms. In each case triplicate tests
were performed and the average was taken as the final reading.
2.1.1.4. 1,12(2,6)-Ditriazina-2,11,13,22-tetraaza-3,10,14,21-tetraoxo-6,7,
17,18-tetrathia-cyclo-docosaphan-14,124-diphenyl (L4). Hot etha-
nolic solution (90 cm3, absolute) of 2,4-diamino-6-phenyl-1,3,5-
triazine (1.06 g, 8.5 mmol) was reacted with ethanolic solution of
(10 cm3) of 3,-dithiadipropionic acid (1.78 g, 8.5 mmol) in the
presence of few drops of HCl. The off white crystalline solid was
treated in a Dean–Stark trap (1.77 g, 70%). m.p.165–166 ꢁC. Found
(calculated) (L4)(H2O), [C30H32N10O5S4]: C, 48.7 (48.6); H, 4.7 (4.3);
N, 19.3 (18.90); S, 17.5 (17.3). MS (EI) m/z (%): 722 (12). 1H NMR, dH
2.64 (t, J ¼ 6.8 Hz, 8H, CH2–CO); 2.91 (t, J ¼ 6.8 Hz, 8H, S–CH2) 6.81
(s, 4Hg); 7.50 (m, 4Hf); 8.27 (d, 2Hh, J ¼ 9.9 Hz); 10 (br, 4H, NH).
13C{1H} NMR, dC 33.51 (4C, S–CH2); 34.00 (4C, CH2–CO); 128.17 (4Cf,
Ph); 128.64 (4Cg, Ph); 131.67 (2Ch, Ph); 137.17 (2Ce, Ph); 167.75 (4Cc,
t-ring); 170.80 (2Cd, t-ring); 173.29 (4C, CO).
2.2.1.2. Dilution. Screening for antibacterial and antifungal activi-
ties was carried out by preparing a broth micro dilution, following
the procedure outlined in Manual of Clinical Microbial [40]. All the

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N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
lowest concentrations of antimicrobial agents that result in
complete inhibition of microorganisms were represented as (MIC)
g mLꢀ1. In each case triplicate tests were performed and the
m
results are expressed as means.
2.2.1.3. Cytotoxicy. The cytotoxic effect of the (L1–L4) ligands against
23132/87 (stomach adenocarcinoma, human) and A549 (non-small
cell lung adenocarcinoma, human) were assayed by MTT method
[41]. The cells were plated at 37 ꢁC for 24 h on 96-well plates
(Grainer) at a density of 3.103–5.103 cells per well, with RPMI 1640
medium in a humidified atmosphere (5% CO2). The RPMI 1640
medium was supplemented with 10% (v/v) fetal bovine serum (FBS),
S−S 508
C−S 630
C−S 621
(L4)
(L3)
4 mM L-glutamine and antibiotics (penicillin/streptomycin). Freshly
prepared ligands (DMSO) were further diluted in complete culture
medium and the final volume of DMSO in the culture medium was
adjusted to 0.5% (v/v). The seeded cells were exposed to 0.1, 1, 10,
100
37 ꢁC in humidified incubator with 5% CO2 at a final volume of
150 L. After incubation,15 L MTT solutions was added to each well
and incubated for further 4 h. The MTT solution then was carefully
removed and dissolved by adding 200 L (DMF 50%, SDS 5%)
aqueous solution and analyzed at 570 nm in a microplate reader.
mM concentrations of the test compounds for 24 and 48 h at
S−S 505
m
m
C−S 631
m
Cytotoxicity values were determined using 100ꢂ(OD570treated
/
cells
(L2)
(L1)
OD570untreated cell) equation.
C−S 621
2.2.1.4. DNA fragmentation. Tested cell lines were used to analyze
the DNA ladder formation which is a characteristic of apoptotic cell
death [42]. The cells (10 ꢂ 104) were plated on 6 well plates and
exposed on the (L1–L4) ligands (100 mM) for 24 h. After the incu-
900
800
700
600
500
400
300
200
bation the cell lines were collected and washed with PBS (phos-
phate-buffered saline). The DNA was extracted with the Nucleospin
Tissue kit. The extracts were run on 1.5% agarose gel. The products
were visualized under UV-light with Bio-Rad Gel Doc gel scanner
system.
Raman shift (cm-1)
Fig. 3. FT-Raman spectra of (L1–L2) compounds in the 950–160 cmꢀ1 region.
bacteria were incubated and activate at 30 ꢁC for 24 h inoculation
into Nutrient Broth, and the yeasts were incubated in Malt Extract
Broth for 48 h. The compounds were dissolved in DMSO
(2 mg mLꢀ1) and then diluted using caution adjusted Mueller
Hinton Broth (Oxoid). Two-fold serial concentrations of the
compounds were employed to determine the (MIC) ranging from
2.2.1.5. Hoechst 33258 staining [43]. The cells that were cultured on
6 well plates and treated with the compound (100 mM) for 24 h.
After the treatment, cells were washed with PBS (3 times) and fixed
with ice cold methanol for 10 min. Following fixation, cells were
incubated with Hoechst 33258 (5 mg mLꢀ1) for 5 min and then
destained with double-distilled water for further 5 min and were
examined by fluorescence microscopy (CarlZeis).
200 m m
g mLꢀ1 to 1.56 g mLꢀ1. Cultures were grown at 37 ꢁC (20 h)
and the final inoculation (inoculums) was approximately
106 cfu mLꢀ1. Test cultures were incubated at 37 ꢁC (24 h). The
g
f
c
g
f
h
e
d
c
a
a
CO
d
h
e
180 175 170 165 160 155 150 145 140 135 130 125
ppm
44 43 42 41 40 39 38 37 36
Fig. 4. 13C{1H} NMR spectrum of (L2) ligand.

N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
4685
DMSO-d6
a
b
g
c
f
e
d
h
a
g
c
f
b
a
b
CO
d
130 129 128 127
34
33
h
e
170 160 150 140 130 120 110 100 90
ppm
80
70
60
50
40
30
Fig. 5. 13C{1H} NMR spectrum of (L4) ligand.
2.2.1.6. DNA Binding UV–visible. Calf thymus DNA was purchased
from Worthington Biochemical Corp. and prepared in ddH2O at
a concentration of 60 mg/mL. Purity of CT-DNA was determined by
comparing UV absorbance ratios at 260 and 280 nm (1.8:1), indi-
cating that the DNA was sufficiently free of protein. The absorption
involved in the production of genetic material, preventing the
bacteria from reproducing); Tetracyclines exerting their antimi-
crobial effect the inhibition of protein synthesis; Nystatin (binding
to sterols in the fungal cellular membrane altering the permeability
to allow leakage of the cellular contents and destroying the
fungus); Ketoconazole (inhibiting the growth of fungal organisms
by interfering with the formation of the fungal cell wall) and Clo-
trimazole (interfering with their cell membranes and causing
essential constituents of the fungal cells leakage). Mueller Hinton
media, Nutrient Broth and Malt Extract Broth are purchased from
Difco and yeast extracts is obtained from Oxoid.
spectra of the DNA solution (250
mM) were obtained in the absence
and presence of increasing amounts of the ligands [0–500
mM].
2.2.1.7. Biological data. Standardised samples of Penicillin-g
blocking the formation of bacterial cell walls, rendering bacteria
unable to multiply and spread; Ampicillin (penetrating and pre-
venting the growth of Gram-negative bacteria); Cefotaxime (used
against most Gram-negative enteric bacteria); Vancomycin (acting
by interfering with the construction cell walls in bacteria), Oflox-
acin (entering the bacterial cell and inhibiting DNA-gyrase, which is
3. Results and discussion
3.1. Vibrational spectra
O
O
The present vibrational spectra can be discussed in terms of
three characteristic wave regions: 3570–2850 cmꢀ1 corresponding
NH
N
X
HN
N
n(H2C)
n(H2C)
(CH2)n
(CH2)n
to
aromatic characteristic modes,1800–800 cmꢀ1 belongs to
amides, (C–N)tr,
(N–H) and 750–500 cmꢀ1 frequencies regions due
to the (C–S) and (S–S) characteristic stretching modes, which are
n(H–O–H) lattice water,
n(N–H) amides and
n(C–H) aliphatic and
N
N
N
N
n(CONH–)
n
d
n
n
clearly assignable in the Raman spectra. Prominent Raman and IR
frequency data values and their some assignments are presented in
Table 1. Appearance of a strong broad band in the region 3490–
3380 cmꢀ1 in the IR spectra may be due to the presence of lattice
water (H–O–H) (antisymmetric and symmetric), which was also
confirmed by elemental analysis [44]. These modes are either very
NH
HN
X
O
O
Fig. 6. General proposed structure of the ligands: (L1), n ¼ 1, X ¼ S; (L2), n ¼ 1, X ¼ S–
S;(L3), n ¼ 2, X ¼ S; (L4), n ¼ 2, X ¼ S–S.

4686
N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
Table 2
In vitro antimicrobial activity of the (L1–L4) compounds and the standard reagents (inhibition zone mm).
Microorganisms/compounds
(L1)
(L2)
(L3)
(L4)
P10
AMP
CTX
VA
OFX
TE
NY
KET
CLT
Escherichia coli
10.0
12.0
13.0
14.0
14.0
12.0
10.0
10.0
9.0
15.0
17.0
16.0
20.0
18.0
10.0
14.0
15.0
17.0
18.0
11.0
10.0
10.0
11.0
19.0
18.0
19.0
20.0
21.0
10.0
10.0
13.0
13.0
12.0
11.0
9.0
10.0
11.0
16.0
16.0
19.0
19.0
18.0
10.0
12.0
16.0
15.0
17.0
13.0
11.0
10.0
11.0
21.0
21.0
19.0
22.0
22.0
18
13
18
8
10
14
15
10
36
–
12
16
14
10
16
12
21
12
32
–
10
12
13
54
18
14
11
16
32
–
22
13
22
10
20
18
20
26
34
–
30
24
28
44
28
30
32
30
28
–
28
26
30
34
26
25
24
28
22
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Staphylococcus aureus
Klebsiella pneumoniae
Bacillus cereus
Micrococcus luteus
Proteus vulgaris
Mycobacterium smegmatis
Listeria monocytogenes
Pseudomonas aeruginose
Kluyveromyces fragilis
Rhodotorula rubra
Candida albicans
Hanseniaspora guilliermondii
Debaryomyces hansenii
–
–
–
20
18
18
21
16
21
16
22
24
14
15
18
16
22
18
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
P10, Penicillin G (10 Units); AMP, Ampicillin 10
Ketoconazole 20 g; CLT, Clotrimazole 10 g.
mg; CTX, Cefotaxime 30
m
g; VA, Vancomycin 30
m
g; OFX, Ofloxacin 5
m
g; TE, Tetracycline 30
m
g; NY, Nystatin 100
m
g; KET,
m
m
weak or not active in Raman spectra. The bands corresponding to
free carboxylic acid (–COOH) and amine (–NH2) groups are not
observed in the IR spectra of the (L1–L4) ligands, which suggests
complete condensation of the amine groups of 2,4-diamino-6-
phenyl-1,3,5-triazine with corresponding dicarboxylic acids. The
bands observed in the regions 3300 and 3150 cmꢀ1 in the IR spectra
are assignable to amide A and amide B vibration respectively. The
amide A is almost caused by the NH stretching vibration, this mode
of vibration is not depend on the backbone conformation but is very
sensitive to the strength of a hydrogen bonding. The amide B is
originates from a Fermi resonance between the first overtone of
amide II and the N–H stretching vibration [45,46]. Due to non-
planar characteristic behaviour of the compounds in the solid state,
the bands could be observed as multiple signals. These lines have
extremely weak Raman intensity, particularly in the solid form (Figs.
in the Table 1 [45,46]. The bending vibrations for
spectra are observed in the region 1640 cmꢀ1 (Figs. 1 and 2).
The vibrational frequency of
(R–S–S–R) (R ¼ Alkyl) is observed
in the region 520–480 cmꢀ1 [47]. In the Raman spectra,
(C–S) and
(S–S) modes for the (C–S–C) and (C–S–S–C) moieties are very
characteristic giving rise to a medium to strong stretching bands in
the wave region 710–570 and 530–500 cmꢀ1, respectively [47,48].
The medium to strong bands at 508 of (L2) and 505 cmꢀ1 of (L4)
d(H–O–H) in IR
n
n
n
clearly show characteristic
n(S–S) stretching modes for these
macrocycles (Fig. 3). The assignments are supported by the fact that
the other relevant bands at this region remain almost unchanged
among these compounds. No clear-cut assignments are possible for
n
(C–S) modes, but bands at ca. 530–520 cmꢀ1 could be assignable
for these particular vibrations (Fig. 3). Because of the non-polar
character of the (C–S) and (S–S) modes, their counterpart in IR are
quite weak (Table 1, Fig. 1).
1 and 2). The pure characteristic n(CH) modes of aliphatic groups are
observed in the wave region 2930–2820 cmꢀ1 in both Raman and IR
spectra as expected. The peptide bond formation could also be
confirmed due to the observance of two major bands in the
frequency regions 1700–1650 cmꢀ1 (amide I) and 1590–1540 cmꢀ1
(amide II). The amide I band is the most intensive absorption band
particularly in the IR spectrum and it is almost fully governed by the
stretching vibration of the C]O mode with a very small contribu-
tion of the C–N groups. Amide II is more complex than amid I, which
derives mainly from in-plane N–H bending vibration with certain
3.2. Nuclear magnetic resonance
The 1H NMR spectra of the (L1–L4) ligands in DMSO-d6 do not
give any signal corresponding to primary amine –NH2 (at 6.8 ppm)
and carboxylic acid –COOH (ca. 11 ppm), instead they show broad
bands in the region 10–9 ppm corresponding to amide (4H)
protons. The low field position as well as broadness of NH protons
could be attributed to the combination of deshielding via weak
hydrogen bonding due to amide and sulphur atoms. As expected no
significant changes are observed for the phenyl protons chemical
shifts compared with starting material. Two singlets at 3.37 and
3.68 ppm are attributed to the methylene protons of S–CH2–CO
groups for the (L1) and (L2) ligands, respectively. Two triplets are
observed for the (L3) and (L4) ligands at 2.70, 2.51 and 2.91,
2.64 ppm for the methylene protons of S–CH2–CH2–CO (8H) and
S–S–CH2–CH2–CO (8H) groups, respectively. Considering the slight
stronger deshielding characteristic of sulphur atoms compared
with amide groups, the slight higher chemical shift values 2.70 (L3)
and 2.91 (L4), may be assignable to the methylene protons that are
adjacent to the sulphur atoms [49].
No significant changes are expected for the phenyl and triazine
carbon chemical shift values due to the cyclic formation. The
phenyl-triazine carbon atom chemical shifts are observed approx-
imately at: 170–171 (Cd), 67–168 (Cc), 137–138 (Ce), 131–132 (Ch),
128–129 (Cg), 128 (Cf) [50]. The signals in the 171–173 ppm regions
are assignable to CO of the CO–NH amide group. Similarly two
singlets at 33.75 and 40.97 ppm are attributed to methylene
carbons of S–CH2–CO group for (L1) and (L2) ligands, respectively.
Two singlets are appeared for each of the (L3) and (L4) ligands at
26.41, 34.55 and 33.51, 34.0 ppm for methylene carbons of S–CH2–
contribution of n(C–N) groups. Amide III and IV are very complex
bands resulting from a mixture of several coordinate displacements.
These modes are dominated by the out-of-plane motions particu-
larly (N–H) wagging and (C]O) deformation vibrations as reported
Table 3
.In vitro antimicrobial activity (MIC,
m
g mLꢀ1) of the (L1–L4) compounds.
Microorganisms/Compounds (L1)
(L2)
(L3)
(L4)
GEN NYS
Escherichia coli
25
12.5
12.5
6.25
12.5
25
12.5
6.25
3.125 12.5
3.125 12.5
25
25
12.5
25
12.5
6.25
6.25
6.25
6.25
25
6,25
6.25
25
–
–
–
–
Staphylococcus aureus
Klebsiella pneumoniae
Bacillus cereus
Micrococcus luteus
–
Proteus vulgaris
12.5
25
25
50
6.25
6.25
6.25
1.56
3.125
25
25
50
25
25
6.25
6.25
3.125
1.56
3.125
12.5
6.25
12.5
12.5
6.25
–
–
–
–
–
–
–
–
–
Mycobacterium smegmatis
Listeria monocytogenes
Pseudomonas aeruginose
Kluyveromyces fragilis
Rhodotorula rubra
Candida albicans
Hanseniaspora guilliermondii
Debaryomyces hansenii
25
25
25
25
25
25
1.56
1.56
6.25
6.25
3.125
3.125
12.5
3.125
3.125
1.56
3.125
3.125
1.56
1.56
1.56
GEN, Gentamycin; NYS, Nystatin.

N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
4687
L
L
L
L
1
2
3
4
104
100
96
L
L
L
L
100
96
1
2
3
4
92
92
88
88
0
0,1
1
10
100
0
0.1
1
10
100
Concentration (µM, 24 h)
Concentration (µM, 24 h)
106
102
98
L
1
104
100
96
L
L
L
2
3
4
L
1
L
2
L
L
3
4
92
94
88
90
0
0,1
1
10
100
0
0,1
1
10
100
Concentration (µM, 48 h)
Concentration (µM, 48 h)
Fig. 7. Effect of the ligands on the viability of 23132/87 and A549 cell lines (0–100
mM, 24, 48 h).
CH2–CO and S–S–CH2–CH2–CO residues, respectively. Considering
the weaker deshielding characteristic of sulphur atoms in
comparison with amide group on the carbon chemical shift values,
the lower ppm values 26.41 (L3) and 33.51 (L4), may be assignable
to the methylene carbons that are close to the sulphur atoms (Figs.
4 and 5) [51]. In conclusion, presented generic structure in Fig. 6 is
in best accord with the experimental data obtained from the
analytical, vibrational and nuclear magnetic resonance spectra.
values of compared antibiotic and antifungal reagents are listed in
Tables 2 and 3. All the compounds tested exhibit strong or
moderate antimicrobial activity. Of all the test compounds at
tempted, (L2) and (L4) showed slightly higher activities against
most Gram positive than Gram negative bacteria, but all
compounds show strong activity on the yeast cultures. The MIC
values in Table 3 indicate that all the compounds tested exhibit
moderate to strong antimicrobial activity on the tested
microorganisms.
Once again the data indicate that the (L2) and (L4) macrocycles
have slightly stronger activity against some Gram positive bacteria
3.3. Biological activity
such as Bacillus cereus (L2 ¼ 3.125 and L4 ¼ 6.25
m
g mLꢀ1) and
3.3.1. Antimicrobial activity
The results concerning in vitro antimicrobial activities of the
macrocycles together with the inhibition zone (mm) and (MIC)
Micrococcus luteus (L2 ¼ 3.125 and L4 ¼ 6.25
m
g mLꢀ1) compared
with Gentamycin on these microorganisms 25 and 6.26 m ,
g mLꢀ1
Fig. 8. Hoechst nuclear staining of A549 cells with (100
mM, 24 h) a, untreated; (b–e) with (L1–L4), respectively; f, treated DMSO.

4688
N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
Fig. 9. Hoechst nuclear staining of 23132/87 cells with (100 mM, 24 h) a, untreated; (b–e) with (L1–L4), respectively; f, treated DMSO.
respectively (Table 3). Similarly these two compounds show same
active against Gram positive than Gram negative bacteria. This
conception seems partially true, but in this study, the compounds
are relatively active against on both types of the bacteria and as well
as strongly active against yeasts, which may indicate a broad-
spectrum affect. The results of our study indicate that the
compounds have the potential to generate novel antimicrobial
properties by displaying moderate to high affinities for most of the
receptors particularly against yeast cultures.
activity with Gentamycin against Gram negative Klebsiella bacteria
(6.25
against the yeast cultures. For instance, all the compounds tested
g mLꢀ1) against Hansenias-
m
g mLꢀ1). All the tested compounds show very strong activity
showed superior activity (MIC ¼ 1.56
m
pora guilliermondii culture compared with Nystatin antifungal
agent. Once more the data indicate that the (L2) and (L4) macro-
cycles have stronger activity against yeast cultures Kluyveromyces
fragilis (1.56
comparison with Nystatin antifungal agent, showing MIC values
6.25 and 12.50
g mLꢀ1 on the same micro-organisms, respectively.
m m
g mLꢀ1) and Debaryomyces hansenii (1.56 g mLꢀ1) in
3.3.2. Cytotoxicity
m
The pattern of cytotoxic activity of the ligands is similar to each
other and indicates that none of the ligand shows significant
cytotoxic effect on the tested cell lines. Cytotoxicity ranges from
0 to 14% for both of the cell lines, indicating a high percentage of the
cell lines capability to survive (86–100%). There was a fluctuation in
the survival of the cells lines (86–100%) in a dose-independent
manner (Fig. 7). In certain cases the survival rates seems increased
suggesting an attempt of the cell to develop a defence mechanism
The inhibition activity of the compounds seems to be governed
in certain degree by the percentage amount of the sulphur presence
in the compounds, because the dithio (L2) and (L4) macrocycles
seems indicate more activity against most micro-organisms,
compared to the monothio macrocycles. Furthermore, in classifying
the antibacterial activity as Gram positive or Gram negative, it
would generally be expected that a much greater number would be
MM
C
1
2
3
4
NC
C
5
6
7
8
NC
Fig. 10. Gel electrophoresis lines (1–4) 23132/87 cells treated (L1–L4) (100 mM, 24 h); lines (5–8) A549 cells treated (L1–L4) (100 mM, 24 h); MM, 100 bp molecular marker; NC, DNA
extracted from cells treated with DMSO (24 h); C, untreated cells.

N.M. Aghatabay et al. / European Journal of Medicinal Chemistry 44 (2009) 4681–4689
4689
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We would like to extend our gratitude to the Fatih University
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P50020702.
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