Rapid synthesis, characterization, anticancer and antimicrobial activity studies of substituted thiadiazoles and their dinucleating ligand metal complexes

Article abstract of DOI:10.1007/s00044-011-9778-y

Synthesis of 2,5-disubstituted thiadiazoles was accomplished via a conventional method as well as microwave irradiation method. These substituted thiadiazoles were diazotized and coupled with 2,4-pentanedione (AcAc), ethylcyanoacetate (ECA) and malanodinitrile (MN) to get dinucleating ligands. The ligands were isolated, characterized and condensed with Ni (II), Cu (II) and Ru(III) chlorides. These compounds were screened on HL-60 Human leukemia cell Line and U-937 Lymphoma cell lines for anticancer activities. The antimicrobial activity of the ligands and their complexes against bacteria and fungi was also investigated. The effect of metal on the ligand activity is discussed. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9778-y

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2548–2556
DOI 10.1007/s00044-011-9778-y
ORIGINAL RESEARCH
Rapid synthesis, characterization, anticancer and antimicrobial
activity studies of substituted thiadiazoles and their dinucleating
ligand metal complexes
•
•
•
Anjali Jha Y. L. N. Murthy U. Sanyal
G. Durga
Received: 14 April 2011 / Accepted: 30 July 2011 / Published online: 18 August 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Synthesis of 2,5-disubstituted thiadiazoles was
accomplished via a conventional method as well as
microwave irradiation method. These substituted thiadiaz-
oles were diazotized and coupled with 2,4-pentanedione
(AcAc), ethylcyanoacetate (ECA) and malanodinitrile
(MN) to get dinucleating ligands. The ligands were iso-
lated, characterized and condensed with Ni (II), Cu (II) and
Ru(III) chlorides. These compounds were screened on
HL-60 Human leukemia cell Line and U-937 Lymphoma
cell lines for anticancer activities. The antimicrobial
activity of the ligands and their complexes against bacteria
and fungi was also investigated. The effect of metal on the
ligand activity is discussed.
Introduction
Cancer is one of the leading causes of death in the world.
With an improved understanding of the genes and path-
ways responsible for cancer initiation and progression,
cancer drug development has undergone a paradigm
change in recent years, from predominantly cytotoxic
agent-based therapy to therapy aimed at molecular and
genetic targets. Several five-membered aromatic systems
having three hetero atoms at symmetrical position have
been studied because of their interesting physiological
properties (Hui et al., 2002 and Rudolph et al., 2001). It is
also well established that various derivatives of 1,2,4-tria-
zole and 1,3,4-thiadiazole exhibit a broad spectrum of
pharmacological properties (Clerici, 2002). Thiazole
derivatives are present in many natural and synthetic
products displaying a wide range of activities, such as anti-
bacterial (Tsuji and Ishikawa, 1994), antifungal (Sharma,
1967), antitumor (Crim John et al., 1967; Modi et al.,
1970), anticancer (Gulsory and Guzeldemirci, 2007), anti-
inflammatory and (Sharma et al., 2009) activities. In
addition, 1,3,4-thiadiazoles exhibit diverse biological
activities possibly due to the presence of =N–C–S moiety
(Holla et al., 2003). 2-aminothiazoles are well explored as
useful clinical agents, and some of the derivatives of thi-
azoles have shown inhibition toward herpes simplex virus
(Spector et al., 1998). 2-Aminothiazole can also be used as
a thyroid inhibitor in the treatment of hyperthyroidism.
Many studies (Crowe, 1988; Chohan et al., 2006) have
described the biological role of metal chelation and have
established the fact that anticancer drugs exhibit increased
anticancer activity when administrated as their metal che-
lates. The connected metal centers in such molecules may
involve different functions such as oxygen transport, DNA
inhibition, enzymatic activity, electron transfer and locking
Keywords 2,5-disubstituted thiadiazoles ꢀ Dinucleating
ligands ꢀ Ni(II) ꢀ Cu(II) and Ru(II) complexes ꢀ
Anticancer activity ꢀ Antimicrobial activity
A. Jha (&)
Department of Chemistry, GIS, GITAM University,
Rushikonda, Visakhapatnam 530045, India
e-mail: anjalimanishjha@yahoo.com
Y. L. N. Murthy
Department of Organic Chemistry and FDW,
Andhra University, Visakhapatnam 530004, India
U. Sanyal
Chitaranjan National Cancer Institute, Kolkata, India
G. Durga
Department of Chemistry, St Josephs College for Women,
Visakhapatnam 530004, india
123

Med Chem Res (2012) 21:2548–2556
2549
Synthesis of 2-amino-5-methyl/ethyl thiadiazole
geometry. All these observations related to the essential
role of metal linkages attracted our attention to commence
a systematic research program to investigate the factors
responsible for this relationship.
Substituted aminothiadiazole was prepared by conventional
method as well as MW irradiation as outlined in Scheme 1.
A mixture of carboxylic acid (acetic acid/propionic acid)
(0.01 mol) and thiosemicarbazide (0.012 mol) in the
presence of conc. H2SO4 (15 ml) was heated under reflux
on water bath for 6 h, following the reported method
(Pandey et al., 2003).
Recently, organic transformation accelerated under sol-
vent-free microwave irradiation condition has gained
popularity due to many advantages, like enhanced reaction
rates, high yields, improved selectivity and eco-friendly
reaction condition in tune with green chemistry (Lindstrom
et al., 2001; Lin et al., 2004; Mistry and Desai, 2006).
Enthused by the interesting biological properties of metal
complexes of NNS ligands and their potential cytotoxic
and antimicrobial activities (Dholakiya and Patel, 2004), it
was worthwhile to synthesize some new 2,5-disubstituted
thiadiazoles by conventional as well as microwave-assisted
(MW) method. These amines were further diazotized and
condensed with active methylene groups to form dinucle-
ating ligands. These ligands were thereafter condensed
with metal chlorides. Ligands and metal complexes were
characterized by various spectral methods and were sub-
jected to activity studies. Copper complexes showed
greater activity in antimicrobial studies while ruthenium
complexes were found to be better anticancerous
compounds.
Same ratio of carboxylic acid (acetic acid/propionic
acid) and thiosemicarbazide in conc. H2SO4 (15 ml) was
irradiated in microwave for 8 min. The resulting reaction
mixture obtained from both the methods was cooled to
room temperature and neutralized with ammonia solution.
A dark brown solid that separated out was filtered and
washed repeatedly with water. It was then recrystallized
from ethanol. The substituted aminothiadiazoles were
characterized by various spectral methods.
0
L1 : (C3H5N3S): Yield (%): Conventional: 50, Micro-
wave: 70; mp: 110°C; IR (mmax, KBr, cm-1): 3345, 3245
(NH2); 2987, 2941 (CH3); 2640 (CS);1555 (C=N of the
1
ring), NMR(DMSO-d6dppm) H: 6.9 (NH2); 2.1 (CH3);
13C: 168.45, 153.16 (C of thiadiazole ring in vicinity of
NH2 and in vicinity of CH3, respectively); 20.1 CH3;
Analysis: Calculated C, 31.30; H, 4.34; N, 36.52; Found:
C, 31.28; H, 4.39; N, 36.60.
0
L2 : (C4H7N3S): Yield (%): Conventional: 55, Micro-
Experimental
wave: 78; mp: 115°C; IR (mmax, KBr, cm-1): 3318, 3240
(NH2); 2987, 2941 (CH3); 2644 (CS); 1555 (C=N of the
Materials
1
ring), NMR(DMSO-d6dppm) H: 7.6 (NH2); 3.2 (CH2);
1.8 (CH3); 13C: 168.45, 159.16 C of the thiadiazole ring in
vicinity of NH2 and in vicinity of (CH2); 31.2 CH2, 20.1
CH3; Analysis: Calculated C, 37.20; H, 5.42; N, 32.55;
Found: C, 37.28; H, 5.39; N, 32.58.
Reagents such as thiosemicarbazide, acetic acid, propionic
acid, 2,4-pentanedione(AcAc), ethylcyanoacetate(ECA),
malanodinitrile(MN) and metal chlorides were purchased
from Across Ltd. and used as it is. All the solvents were of
analytical grade and were distilled before use.
Synthesis of dinucleating ligands
Measurements
0
0
A solution of substituted amino-thiadiazoles (L1 and L2 ;
0.1 mol in separate reactions) in 10 ml conc. HCl was
cooled between –5° and –10°C. To this solution, cold
NaNO2 solution (1.5 g in 10 ml H2O) was added drop wise
while stirring constantly (Scheme 2). The resulting reac-
tion mixture was stirred for another 30 min. To this dia-
zonium salt solution, a cold solution of acetyl acetone/
Melting points are uncorrected. IR spectra were recorded
on Thermo Nicolet FTIR spectrophotometer at Andhra
University, Visakhapatnam, and 1 H NMR and 13C spectra
were taken on JEOL Model AL 400 NMR at RRL, Bhu-
baneswar, in DMSO-d6 using TMS as internal reference.
The electronic spectra of the ligand and the complexes
were recorded in DMSO solution using a Shimadzu
UV-1700 spectrophotometer. ESR spectra were taken on
Varian E 112 at room temperature and as well as liquid
nitrogen temperature using DPPH as standard at SAIF, IIT
Chennai. Microwave irradiation was carried out using a
domestic microwave oven. Elemental analysis was carried
out at Micro Analytical centre at Andhra University. The
HPLC was recorded using Shimadzu LC 6A with Shim-
pack silica gel column.
Conc H2SO4
N
N
RCOOH + NH2CSNHNH2
NH2
S
R
(a) Reflux,6 hrs
(b) MW,8min
Scheme 1 Synthesis of 2-amino-5-methyl/ethyl thiadiazole
123

2550
Med Chem Res (2012) 21:2548–2556
R'
Scheme 2 Synthesis of
dinucleating ligands
R''
H2C
N
N
NaNO2/HCl
N N
S
N N
S
R'
+
N
R
R
°
S
NCl-
R
N=N--HC
-5 to -10 C
NH2
NaOAc
R''
°
20 C
N N
S
R'
R''
R
N--N=C
H
Where R=CH3 for L1,L2,L3
or R=CH2CH3forL4,L5L6
R' & R''=COCH3=L1&L4
R'=CN & R''=COOC2H5=L2&L5
R' & R''=CN=L3&L6
ethylcyanoacetate/malanodinitrile (each 0.1 mol) contain-
ing sodium acetate (5 g) in water (10 ml) was added.
Corresponding mixtures were further stirred for 3 h at 20°C
and then refrigerated overnight. The solids thus obtained
were filtered and washed several times with water and
ethanol and then dried in vacuum. The crude products were
crystallized in EtOH. The ligands (L1–L6) showed satis-
factory elemental analysis and were further characterized
NMR(DMSO-d6dppm) 1H: 5.8 (NH); 1.8 (CH3 of AcAc);
2.7 & 1.2 (CH2 & CH3 of thiadiazole); 13C: 208(CO);
169.45, 159.1 (C of thiadiazole ring); 55.2 (C of AcAc);
32.2, 15.6 (CH2, CH3 of thiadiazole ring); 22.4 (CH3 of
AcAc). Analysis: Calculated C, 45.00; H, 5.0; N, 23.33;
Found: C, 45.05; H, 5.06; N, 23.29.
L5: (C9H11N5SO2): Yield 52%, mp: 182°; IR (mmax,
KBr, cm-1): 3308(NH); 2987, 2941 (CH3); 2232 (C:N);
1680 (C=O); 1555 (C=N of the ring) NMR(DMSO-
d6dppm)1H: 5.3 (NH); 2.8, 1.8 (CH2 & CH3 of ECA); 2.4
& 1.1 (CH2 & CH3 of thiadiazole); 13C: 208 (CO); 169.45,
159.1 (C of thiadiazole ring); 110.1 (C:N of ECA); 58.2
(C of ECA); 39.2, 22.4 (CH2 & CH3 of ECA); 15.6 (CH3 of
thiadiazole ring). Analysis: Calculated C, 42.68; H, 4.34;
N, 27.66; Found: C, 42.65; H, 4.36; N, 27.69.
1
by IR and H and 13C NMR spectra.
L1: (C8H10N4SO2): Yield 65%, mp: 147°C; IR (mmax,
KBr, cm-1): 3310 (NH); 2987, 2941 (CH3); 1680 (C=O);
1
1555 (C=N of the ring), NMR(DMSO-d6dppm) H: 5.4
(NH); 2.2 (CH3 of AcAc); 1.7 (CH3 of thiadiazole ring);
13C: 210 (CO); 168.45, 158.16 (C of the thiadiazole ring);
56.2 (C of AcAc); 22.4 (CH3 of AcAc); 15.6 (CH3 of
thiadiazole ring). Analysis: Calculated C, 42.47; H, 4.42;
N, 24.77; Found: C, 42.51; H, 4.39; N, 24.72.
L6: (C7H6N6S): Yield 67%, mp: 210°C; IR (mmax, KBr,
cm-1): 3305 (NH); 2985, 2940 (CH3); 2544 (CS); 2228
(C:N); 1555 (C=N of the ring); NMR(DMSO-
d6dppm)1H: 5.5 (NH); 2.9, 1.5 (CH2 & CH3 of thiadia-
zole); 13C: 169.45, 159.1 (C of thiadiazole ring); 118.2
(C:N of MN); 57.8 (C of MN); 33.1 & 18.6 (CH2 & CH3
of thiadiazole ring). Analysis: Calculated C, 40.77; H, 2.91;
N, 40.77; Found: C, 40.75; H, 2.95; N, 40.72.
L2: (C8H9N5SO2): Yield 55%, mp: 155°C; IR (mmax,
KBr, cm-1): 3308 (NH); 2985, 2940 (CH3); 2540 (CS);
2230 (C:N); 1687 (C=O); 1555 (C=N of the ring);
1
NMR(DMSO-d6dppm) H: 5.5 (NH); 2.8, 1.8 (CH2 &
CH3 of ECA); 1.3 (CH3 of thiadiazole); 13C: 208 (CO);
169.45, 159.1 (C of thiadiazole ring); 105.2 (C:N of
ECA); 55.2 (C of ECA); 38.2 (CH2), 20.4 (CH3 of ECA);
15.6 (CH3 of thiadiazole ring). Analysis: Calculated C,
40.16; H, 3.76; N, 29.28; Found: C, 40.11; H, 3.69; N,
29.22.
General procedure for the preparation of metal complexes
An ethanolic solution of metal salts (2 mmol each
NiCl2ꢀ6H2O, CuCl2ꢀH2O and RuCl3ꢀ3H2O) (10 ml) was
added to the respective solution of ligands (L1–L6; 1 mmol
each in 10 ml of ethanol) while stirring constantly (Mishra
and Jha, 1996) followed by the addition of 2–3 drops of
Et3N. The resulting solutions were then boiled under reflux
while monitoring the progress of reaction periodically by
TLC (thin layer chromatography) in 10% ethyl acetate and
n-hexane. After 3 h, the reaction mixtures were cooled to
room temperature and refrigerated overnight. The solids
thus obtained were filtered and washed successively with
ethanol and ether and dried in vacuum. The complexes
L3: (C8H9N6S): Yield 60%, mp: 158°C; IR (mmax, KBr,
cm-1): 3306 (NH); 2985, 2940 (CH3); 2538 (CS); 2226
(C:N); 1555 (C=N of the ring); NMR(DMSO-d6dppm)
1H: 5.4 (NH); 1.2 (CH3 of thiadiazole); 13C: 169.45, 159.1
(C of thiadiazole ring); 115.4 (C:N of MN); 55.2 (C of
MN); 15.6 (CH3 of thiadiazole ring). Analysis: Calculated
C, 37.50; H, 2.08; N, 43.75; Found: C, 37.54; H, 2.11; N,
43.72.
L4: (C9H12N4SO2): Yield 68%, mp: 165°C; IR (mmax,
KBr, cm-1): 2987, 2941 (CH3); 2542(CS); 2238 (CN);
2238 (CN); 1685(C=O); 1555 (C=N of the ring);
123

Med Chem Res (2012) 21:2548–2556
2551
Table 1 Analytical and physical data of complexes
Sl. no.
Compounds
Color/yield%
Found(Cacld)%
g
–
g\
A RT (LNT)
k
k
C
H
N
1
[Ni2(L1)Cl3(H2O)5]
[Cu2(L1)Cl3(H2O)5]
[Ru2(L1)Cl3(H2O)5]
[Ni2(L2)Cl3(H2O)5]
[Cu2(L2)Cl3(H2O)5]
[Ru2(L2)Cl3(H2O)5]
[Ni2(L3)Cl3(H2O)5]
[Cu2(L3)Cl3(H2O)5]
[Ru2(L3)Cl3(H2O)5]
[Ni2(L4)Cl3(H2O)5]
[Cu2(L4)Cl3(H2O)5]
[Ru2(L4)Cl3(H2O)5]
[Ni2(L5)Cl3(H2O)5]
[Cu2(L5)Cl3(H2O)5]
[Ru2(L5)Cl3(H2O)5]
[Ni2(L6)Cl3(H2O)5]
[Cu2(L6)Cl3(H2O)5]
[Ru2(L6)Cl3(H2O)5]
Dark green
69
17.72
3.68
10.34
–
–
(17.78)
17.50
(3.70)
3.59
(10.37)
10.15
2.
Dark green
61
2.241
(2.322)
–
2.023
(2.144)
–
165
(160)
–
(17.46)
15.40
(3.63)
3.24
(10.18)
8.94
3.
Dark brown
72
(15.37)
17.39
(3.20)
3.47
(8.96)
12.64
4.
Dark green
65
–
–
–
(17.36)
17.10
(3.43)
3.35
(12.66)
12.48
5.
Dark green
60
2.232
(2.332)
–
2.123
(2.116)
–
162
(165)
–
(17.06)
15.09
(3.37)
2.95
(12.44)
10.94
6.
Dark brown
75
(15.05)
14.27
(2.98)
2.72
(10.98)
16.56
7.
Dark green
65
–
–
–
(14.23)
13.99
(2.76)
2.74
(16.60)
16.32
8.
Dark green
58
2.233
(2.323)
–
2.112
(2.210)
–
160
(162)
–
(13.96)
12.22
(2.71)
2.40
(16.29)
14.25
9.
Dark brown
70
(12.19)
19.51
(2.37)
3.99
(14.22)
10.15
10.
11.
12.
13.
14.
15.
16.
17.
18.
Dark green
68
–
–
–
(19.49)
19.20
(3.97)
3.92
(10.10)
9.96
Dark green
60
2.242
(2.322)
–
2.022
(2.114)
–
165
(160)
–
(19.16)
16.94
(3.90)
3.47
(9.93)
8.79
Dark brown
72
(16.91)
19.09
(3.44)
3.74
(8.77)
12.37
Dark green
61
–
–
–
(19.05)
18.76
(3.70)
3.67
(12.34)
12.17
Dark green
59
2.222
(2.343)
–
2.102
(2.204)
–
160
(160)
–
(18.73)
16.60
(3.64)
3.26
(12.14)
10.76
Dark brown
70
(16.57)
16.19
(3.22)
3.10
(10.74)
16.19
Dark green
66
–
–
–
(16.15)
15.84
(3.07)
3.08
(16.15)
15.90
Dark green
58
2.231
(2.302)
–
2.110
(2.116)
–
165
(165)
–
(15.86)
13.92
(3.02)
2.58
(15.86)
13.91
Dark brown
72
(13.89)
(2.60)
(13.89)
showed satisfactory elemental analysis and were further
characterized by various spectral methods (Table 1).
Drug stock solutions were prepared in DMSO [20 mg of
compound/1 ml of HYBRIMAX grade, Sigma, USA] to
obtain working stock solutions. These were serially tenfold
diluted with complete sterile growth medium (RPMI-1640
medium with 2 Mm glutamine, containing 10% fetal
bovine serum and 1% antibiotics [10000 U penicillin/ml
and 10,000 lg streptomycin/ml] prior to use to obtain
different drug concentrations. Four clinical drugs such as
Cis-Platinum, BCNU, Hydroxyurea and 5-fluoro uracil
were included as standards. About 100 ll of cell
Study of anticancer activity
The anticancer screening of dinucleating ligands of
substituted thiadiazoles and their metal complexes were
done at Chitaranjan National Cancer Institute (CNCI),
Kolkata, against HL-60 Human leukemia cell line and
U-937 Lymphoma cell lines received from NCCS, Pune.
123

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Med Chem Res (2012) 21:2548–2556
Table 2 In vitro cytotoxicity results of synthesized compounds
Table 3 Antifungal activity of synthesized compounds at concen-
tration of 1 lg/ml
Compounds
IC50 value (lM)
Compound
Fungi
AF
Leukemia HL-60
Lymphoma U-937
PE
LT
RS
L1
23.01
22.05
28.12
16.50
14.55
1.95
24.67
19.34
Inactive
17.96
15.11
4.93
L1
12
13
16
17
11
9
18
12
17
16
17
18
22
17
15
16
19
17
14
14
21
11
16
17
23
12
16
16
22
17
NA
35
6
4
L2
L1–Ni
L1–Cu
L1–Ru
L2
16
19
19
7
11
14
12
NA
11
15
14
NA
NA
14
15
6
L3
L1–Ni
L1–Cu
L1–Ru
Cis–Platinum
BCNU
5-FU
L2–Ni
L2–Cu
L2–Ru
L3
15
21
18
8
7.0
3.2
12
13
12
12
15
14
10
13
18
15
12
12
19
17
12
11
20
18
NA
31
30.50
266.0
Inactive
12.3
4.7
Hydroxyurea
115.0
L3–Ni
L3–Cu
L3–Ru
L4
18
19
18
7
IC50 = the cytotoxic dose at 50%, i.e. the concentration needed to
reduce the growth of cancer cells by 50%. Generally, IC50 \ 5.0
means compound is strongly active, IC50 5.0–10.0 means moderately
active and IC50 [ 25.0 means the compound is inactive
L4–Ni
L4–Cu
L4–Ru
L5
17
22
15
10
16
23
16
15
18
23
17
NA
NA
11
14
12
NA
11
17
14
NA
11
18
13
NA
29
suspension from the stock 0.2 9 105 HL-60/U-937 was
added to 96-well cell culture plates. Ten microliters of drug
solutions of different concentrations was added to respec-
tive wells followed by the addition of the medium (90 ll)
in triplicate (total volume 200 ll/well). All vehicle controls
contained the same concentration of DMSO. Plates were
incubated for 96 h at 37°C, 5% CO2/95% air with
humidity. After removal of 100 ll of media from each
well, 10 ll of a 5 mg/ml solution of MTT in Dulbecco’s
PBS (GIBCO, BRL) was added to each well and the plate
was incubated for 4 h at 37°. Subsequently, 100 ll of acid
isopropanol solution (0.04 N HCl in isopropanol) was
added to the wells to dissolve formed formazan crystals.
The plate was read in a microplate reader at 540 nm.
Curvefit software was used to calculate the IC50 value. The
results are shown in Table 2.
L5–Ni
L5–Cu
L5–Ru
L6
L6–Ni
L6–Cu
L6–Ru
DMSO (control)
STD
AF-Aspergillus flavus, PE-Penicillium expansum, LT-Lasiodiplodia
theobroma, RS-Rhizoctonia solani, NA-not active, STD-ampicillin
with 50 ll of different investigated compounds. The solvent
DMSO, used for reconstituting the solvent for diluting the
compounds, was similarly analyzed for control. The plates
were incubated at 37°C for 24 h. The above procedure is
adopted also for fungal assays, and the medium is potato
dextrose agar (instead of nutrient agar) and incubated at
27°C for 48 h. The zone of inhibition was measured with a
Hi Antibiotic Zone Scale in mm, and the experiment was
carried out in duplicate. The results are shown in Tables 3,
4.
Study of antimicrobial assay
The dinucleating ligands (L1–L6) and their metal com-
plexes were examined for antimicrobial assay against
eight bacteria [BS-Bacillus subtilis, EC-Ervinia carotovora,
PV-Pseudomonas vulgaris,
E
coli-Escherichia coli,
EF-Enterococcus faecalis, SF-Streptococcus faecalis,
KP-Klebsiella pneumoniae and ML-Micrococcus luteus]
and four fungi [AF-Aspergillus flavus, PE-Penicillium ex-
pansum, LT-Lasiodiplodia theobroma and RS-Rhizoctonia
solani] using the well diffusion method (Odds, 1989). Two
hundred milliliters of nutrient agar growth medium was
dispensed into sterile conical flasks; these were then inoc-
ulated with 20 ll of cultures mixed gently and poured into
sterile petri dish. After setting a borer with 6 mm diameter
was properly sterilized by flaming and used to make three
uniform wells in each petri dish. The wells were loaded
Results and discussions
Chemistry
To reduce the reaction time and for better yields, substi-
tuted aminothiadiazole was prepared by conventional
123

Med Chem Res (2012) 21:2548–2556
2553
The solid-state IR spectra of the 2,5-disubstituted thi-
adiazoles and free ligands were compared. The peaks for
NH2 of thiadiazoles disappeared, and peaks at *3,300,
*1,685 cm-1 in ligands L1, L2, L4 and L5 have been con-
sidered to arise from mNH and mC=O (Mishra et al., 1997)
vibrations, respectively, whereas a peak observed in the
range *2,640–2,620 is assigned to C–S of the ligands.
Furthermore, strong peaks observed at *2,230 in the
spectrum of L2, L3, L5 and L6 have been attributed to mC:N
(Mishra and Jha, 1996). In the spectra of complexes, mNH
peak disappeared while a new peak at 1,458–1,466 cm-1
assigned to N=N appeared, indicating that complexes exist
Table 4 Antibacterial activity of synthesized compounds at con-
centration of 1 lg/ml
Compound
Bacteria
BS EC PV Ecoli EF SF KP ML
L1
7
6
7
8
8
7
8
NA
10
13
11
NA
9
L1–Ni
L1–Cu
L1–Ru
L2
12
14
13
8
10
12
10
NA
10
13
10
9
10
14
13
7
11
12
11
7
10
12
10
6
9
11
10
8
12
9
7
L2–Ni
L2–Cu
L2–Ru
L3
11
15
12
10
11
14
11
8
8
8
11
16
13
9
10
14
12
6
11
13
10
8
12
11
11
12
6
13
11
7
1
in diazo form which is further supported by their H/13C
NMR data. The lowering of mC=O from 1,680–1,690 to
1,644–1,655 cm-1 in the IR of metal complexes was
indicative of metal ligand coordination. However, the peak
observed in the range 1,548–1,518 cm-1 in the spectra of the
complexes with all ligands were assigned to coordinated
mC=N vibration of the ring (Tennant and Vevers, 1976).
Additionally, the lower value by *15–20 cm-1 for C:N
observed in the complexes with ligands L2, L3, L5 and L6
further supported the coordination with metal ions. This
lowering of C:N, C=O, C=N of the ring upon complexation
could be understood in view of the participation of their
p-electrons in coordination with metal ions (Nakamoto,
2009). The bands at 550–450 cm-1 have been attributed to
mM–N, mM–O and mM–Cl bonds; however, some overlap-
ping bands of ligands are also present.
NA 10
L3–Ni
L3–Cu
L3–Ru
L4
NA
10
9
9
10
11
10
8
9
8
NA NA
12
11
6
11
12
8
10
9
9
9
5
10
11
8
7
8
L4–Ni
L4–Cu
L4–Ru
L5
10
13
10
9
NA 11
9
NA NA NA NA
10 NA
NA 11
11
9
13
11
12
8
8
9
NA NA
NA NA
9
6
5
7
6
L5–Ni
L5–Cu
L5–Ru
L6
9
9
11
14
9
9
NA 11
9
10
12
11
6
17
12
11
11
15
11
11
10
7
14
12
9
14
13
7
14
12
8
17
13
7
NA
8
L6–Ni
L6–Cu
L6–Ru
7
8
9
9
10
17
11
9
10
9
16
11
17
12
15
12
16
13
13
12
1
The H and 13C NMR spectra of the 2,5-disubstituted
thiadiazoles and free ligands were compared. The NH2
peaks at 6.9 and 7.6 ppm for methyl and ethyl thiadiazole
were changed to NH *5.5 ppm for all the ligands and
indicative of diazotization and further condensation with
active methylene groups, viz. AcAc, ECA and MN. This
was further supported by the presence of peaks at
*2.2–2.9 for CH2 and *1.5–1.9 for CH3. The 13C values
for all the ligands (L1 & L6) fitted well for the corre-
sponding hydrazone form (Scheme 2). The poor solubility
and paramagnetic nature of the metal complexes restricted
us from recording their good NMR spectra. However, the
1H NMR spectra of the Ni complexes showed no peaks due
to OH or NH protons, which indicated the coordination of
deprotonated ligand with the metal ions.
DMSO (control) NA NA NA NA
STD 30 31 33 28
NA NA NA NA
NA 30 29 32
AF-Aspergillus flavus, PE-Penicillium expansum, LT-Lasiodiplodia
theobroma, RS-Rhizoctonia solani, BS-Bacillus subtilis, EC-Ervinia
carotovora, PV-Pseudomonas vulgaris
E coli-Escherichia coli, EF-Enterococcus faecalis, SF-Streptococcus
faecalis
KP-klebsiella pneumoniae, ML-Micrococcus luteus; NA-not active;
STD-ampicillin
method as well as MW irradiation method. We had reduced
the reaction time considerably in minutes (MW) from
hours in conventional method. Identical TLC and 99%
HPLC purity were found for the product obtained from
both the methods. These substituted thiadiazoles were
diazotized and coupled with active methylene groups to get
dinucleating ligands. These ligands were further condensed
with metal chlorides and characterized by various spectral
methods. All the compounds were air stable. The ligands
were soluble in organic solvents such as ethanol, acetoni-
trile, dimethylformamide (DMF) and dimethyl sulfoxide
(DMSO) while complexes were soluble in DMF and
DMSO only. Analytical data as well as other physical
properties of the complexes are listed in Table 1.
Electronic spectral data of synthesized compounds were
recorded in DMSO. The Cu (II) complexes exhibited two
low-energy weak bands at 15,151–15,873 cm-1 and a strong
high-energy band at 30,255–30,420 cm-1. The low-energy
band in this position typically is expected for an octahedral
configuration and may be assigned to 10Dq corresponding to
2
the transition 2Eg ? T2g (Ballhausen, 1962; Lever,
1984),which was further supported by their ESR values. The
strong high-energy band, in turn, is assigned to metal ?
ligand charge transfer. Ni(II) complexes showed d–d bands
123

2554
Med Chem Res (2012) 21:2548–2556
Fig. 1 Powder ESR spectra of
[Cu2(L1)Cl3(H2O)5] at LNT
in the regions 24,390–25,000, 16,528–16,667 and 12,987–
13,333 cm-1. These are assigned to the spin-allowed
and O, O & N, O donors on the other could not be iden-
tified at this stage .
3
3
3
3
transitions A2g(F) ? T2g(F), A2g(F) ? T1g(F) and
Three ligands L1, L2 and L3 and nickel, copper and
ruthenium complexes of ligand L1 were assayed for their
cytotoxicity against HL-60 Human leukemia cell line and
U-937 Lymphoma cell line received from NCCS, Pune, at
the CNCI, Kolkata. The cytotoxic activity data are shown
in Table 2 as IC50 value, i.e., the concentration that inhibits
cell replication by 50% relative to control. Free ligands are
rather less active in most of the cases. However, upon
complexation, their activities enhanced significantly in
almost all cases. Ruthenium complex of ligand L1 found to
be most active against Leukemia HL-60 followed by
Lymphoma U-937 cell line, while nickel and copper
complexes of same ligand also showed considerable
activity against these tumor cell lines.
3
3A2g(F) ? T1g(P), respectively, and in accordance with
their well-defined octahedral configuration (Mohammed,
2001). The band at 29,815–30,335 cm-1 was assigned to
metal ? ligand charge transfer. Three to four bands
appeared in the region 15,625–40,000 cm-1 in the elec-
tronic spectrum of Ru complexes. The two spin-allowed
1
1
1
1
transitions, A1g ? T1g (20,450 cm-1) and A1g ? T2g
(18,800 cm-1), and a weak spin forbidden transition
1A1g ? T1g (15,625 cm-1) are assigned to octahedral con-
3
figuration of Ru(II) complexes (Karvembu and Natarajan,
2002). A strong intensity band at 37,700 cm-1 is probably
due to charge transfer transition (T2g ? p*). The nature of
the electronic spectra is similar to that observed for other
octahedral Ru(II) complexes (Mishra and Sinha, 2002;
Prasad et al., 2011).
A comparative study of the ligands and their complexes
(MIC values showed in Tables 3 and 4, graphical repre-
sentation in Figs. 2, 3) indicated that all the complexes
exhibited higher antibacterial activity than the free ligands.
The ESR parameters for the Cu (II) complexes of all
ligands at room temperature (RT) as well as liquid nitrogen
temperature (LNT) are given in Table 1. The powder-state
spectra of the complex [Cu2(L)Cl3(H2O)5] at RT and LNT
showed four equally spaced lines as expected for Cu (II)
ion showing g|| [ g\ with more clarity in LNT spectra
(Fig. 1). The values are supportive of octahedral geometry
around metal ion. Basic spectral characteristics at both
temperatures are the same with slightly better resolution
(Coucouvanis and Fackler Jr., 1967) at LNT. The half-field
signal was not observed in any of these spectra, indicating
that there is no Cu–Cu interaction between the complexes,
hence supportive of dinuclear complexes. Additionally, the
14N super hyperfine splitting showing five signals separated
at 8G could easily be seen in the spectra of the complexes
in solution state at both temperatures (Kumar et al., 2001).
However, at LNT this splitting is better resolved showing
two nitrogens around Cu (II) ions but the difference
between the two coordination environments in the complex
with L1, L2,L4 and L5 having N, N donor on the one side
AF
PE
LT
20
RS
15
10
5
0
L1 N C R L2 N C R L3 N C R L4 N C R L5 N C R L6 N C R
Compounds
Fig. 2 Antifungal activity of synthesized compounds. N=Ni, C=Cu,
R=Ru complexes
123

Med Chem Res (2012) 21:2548–2556
2555
aminothiadiazole, which can be a viable alternative of
conventional synthesis. The synthetic protocol has the
inherent potential for future drug synthesis. A new series of
mixed ligand complexes of nickel, copper and ruthenium
were synthesized, and their octahedral geometry was
inferred from their spectral data. Ru complexes showed
promising activity against both the cell lines, while Ni and
Cu complexes showed moderate activity. A comparative
study of the MIC values of the ligands and their complexes
indicates that complexes exhibit higher antimicrobial
activity than the free ligands. However, Cu complexes
showed more activity against almost all bacteria and fungi.
In view of the structural formula of the complexes that
exhibit anticancer as well as antimicrobial activity, metal
moiety may play a significant role. From the results, it is
also clear that these compounds would be better used in
drug development to combat bacterial and fungal infec-
tions. Ru complexes may be better used as anticancer
compounds.
BS
EC
PV
ECOLI
EF
SF
KP
ML
18
16
14
12
10
8
6
4
2
0
L1 N
C R L2 N C R L3 N C R L4 N C R L5 N C R L6 N C R
Compounds
Fig. 3 Antibacterial activity of synthesized compounds. N = Ni,
C = Cu, R = Ru complexes
OH2
Cl
Cl
OH2
M
OH2
R'
R''
Cl
N
N
Acknowledgments Anjali Jha is thankful to the Department of
Science and Technology (DST), Delhi, for financial support.
M
N
C
R
N
OH2
S
OH2
Where R=CH3 for L1,L2,L3
or R=CH2CH3forL4,L5L6
R' & R''=COCH3=L1&L4
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