Synthesis, crystal structure, and cytotoxic activity of novel cyclic systems in [1,2,4]thiadiazolo[2,3-a]pyridine benzamide derivatives and their copper(ii) complexes

Article abstract of DOI:10.1039/c3dt52905c

Three N-(pyridine-2-ylcarbamothioyl)benzamide derivatives were synthesized by the reaction of potassium thiocyanate, benzoyl chloride, and 2-amino pyridine derivatives in one pot. The obtained derivatives were oxidized using copper(ii) chloride. During the oxidation, two hydrogen atoms were removed, cyclization of the derivatives occurred, and finally, three new N-(2H-[1,2,4]thiadiazolo[2,3- a]pyridine-2-ylidene)benzamide derivatives were produced. Coordination of these three new derivative ligands to the copper(ii) ion resulted in the formation of three new complexes: dichlorobis(N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2- ylidene)benzamide)copper(ii), dichlorobis(N-(7-methyl-2H-[1,2,4]thiadiazolo[2,3- a]pyridine-2ylidene)benzamide)copper(ii), and dichlorobis(N-(5-methyl-2H-[1,2,4] thiadiazolo[2,3-a]pyridine-2-ylidene)benzamide)copper(ii). All the synthesized products were characterized by IR, ¹H NMR, and ¹³C NMR spectroscopies. Crystal structures of the obtained N-(pyridine-2- ylcarbamothioyl)benzamide derivatives, N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine- 2-ylidene)benzamide derivatives, and complexes were determined using X-ray single-crystal diffraction; the positions of atoms, bond lengths, bond angles, and dihedral angles were also determined. In all complexes, the coordination of two large monodentate ligands and two chloride anions to the copper(ii) ion resulted in the formation of a stable planar geometry around the central ion. Three N-(pyridine-2-ylcarbamothioyl)benzamide derivatives, three N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-ylidene)benzamide derivatives, and three complexes were evaluated for their cytotoxicity against five human cancer cell lines (breast cancer cell line MDA-MB-231, neuroblastoma cell line SK-N-MC, prostate adenocarcinoma cell line LNCap, nasopharyngeal epidermoid carcinoma cell line KB, and liver cancer cell line HEPG-2) using an in vitro analysis. The N-(pyridine-2-ylcarbamothioyl)benzamide derivatives showed no cytotoxic activity, whereas the N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-ylidene) benzamide derivatives and their complexes showed significant cytotoxicity, especially against MDA-MB-231 and LNCap cell lines. The complexes demonstrated smaller IC₅₀ values than N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2- ylidene)benzamide derivatives. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3dt52905c

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Synthesis, crystal structure, and cytotoxic activity
of novel cyclic systems in [1,2,4]thiadiazolo[2,3-a]-
pyridine benzamide derivatives and their copper(II)
complexes†
Cite this: Dalton Trans., 2014, 43,
7945
Forogh Adhami,*a Maliheh Safavi,b Maryam Ehsani,a Sussan K. Ardestani,c
Franziska Emmerlingd and Farzaneh Simyaria
Three N-(pyridine-2-ylcarbamothioyl)benzamide derivatives were synthesized by the reaction of potass-
ium thiocyanate, benzoyl chloride, and 2-amino pyridine derivatives in one pot. The obtained derivatives
were oxidized using copper(II) chloride. During the oxidation, two hydrogen atoms were removed, cycli-
zation of the derivatives occurred, and finally, three new N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-
2-ylidene)benzamide derivatives were produced. Coordination of these three new derivative ligands to
the copper(II) ion resulted in the formation of three new complexes: dichlorobis(N-(2H-[1,2,4]thiadiazolo-
[2,3-a]pyridine-2-ylidene)benzamide)copper(II), dichlorobis(N-(7-methyl-2H-[1,2,4]thiadiazolo[2,3-a]pyri-
dine-2ylidene)benzamide)copper(II), and dichlorobis(N-(5-methyl-2H-[1,2,4]thiadiazolo[2,3-a]pyridine-
2-ylidene)benzamide)copper(II). All the synthesized products were characterized by IR, 1H NMR,
and 13C NMR spectroscopies. Crystal structures of the obtained N-(pyridine-2-ylcarbamothioyl)benz-
amide derivatives, N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-ylidene)benzamide derivatives, and com-
plexes were determined using X-ray single-crystal diffraction; the positions of atoms, bond lengths, bond
angles, and dihedral angles were also determined. In all complexes, the coordination of two large mono-
dentate ligands and two chloride anions to the copper(II) ion resulted in the formation of a stable planar
geometry around the central ion. Three N-(pyridine-2-ylcarbamothioyl)benzamide derivatives, three
N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-ylidene)benzamide derivatives, and three complexes were eval-
uated for their cytotoxicity against five human cancer cell lines (breast cancer cell line MDA-MB-231, neu-
roblastoma cell line SK-N-MC, prostate adenocarcinoma cell line LNCap, nasopharyngeal epidermoid
carcinoma cell line KB, and liver cancer cell line HEPG-2) using an in vitro analysis. The N-(pyridine-2-
ylcarbamothioyl)benzamide derivatives showed no cytotoxic activity, whereas the N-(2H-[1,2,4]thiadia-
zolo[2,3-a]pyridine-2-ylidene)benzamide derivatives and their complexes showed significant cytotoxicity,
especially against MDA-MB-231 and LNCap cell lines. The complexes demonstrated smaller IC50 values
than N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-ylidene)benzamide derivatives.
Received 15th October 2013,
Accepted 26th February 2014
DOI: 10.1039/c3dt52905c
www.rsc.org/dalton
1. Introduction
Cancer, as a major health disorder, is responsible for more
than 20% of the world’s mortality. Therefore, development of
processes for the prevention and treatment of cancer is becom-
ing more important than ever. Medicinal chemistry, with a
long history of improvement of drug families to treat cancer,
has succeeded in its goal of developing efficient anticancer
agents for the treatment of various cancers.
Benzamide derivatives and their substitutes, with diverse
and interesting structures, have been shown to be pharmaco-
logically active in the treatment of different diseases such as
cancer.1–4 Metoclopramide (MCA) as a benzamide derivative
has been reported to enhance the effects of cisplatin and
aDepartment of Chemistry, Shahr-ray Branch, Islamic Azad University, PO Box
18155-144, Tehran, Iran. E-mail: fadhami@gmail.com; Fax: +982155229297;
Tel: +982155229200
bBiotechnology Department, Iranian Research Organization for Science and
Technology, Tehran, Iran
cInstitute of Biochemistry and Biophysics, Department of Biochemistry, University of
Tehran, PO Box 13145-1384, Tehran, Iran
dBAM Federal Institute for Materials Research and Testing, Berlin, Germany
†Electronic supplementary information (ESI) available. CCDC 711636, 713327,
714500–714503 and 962672. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c3dt52905c
This journal is © The Royal Society of Chemistry 2014
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ionizing radiation on xenografted human squamous cell carci- against different human cancer cell lines. For this purpose,
nomas of the head and neck region.5,6 Further, several benz- three carbamothioylbenzamide derivatives were synthesized
amide compounds synthesized from anacardic acid, with a and used as starting materials. Reactions of the synthesized
cytotoxic effect on a HeLa cell line of wild type, have been derivatives with copper(II) chloride yielded three new thiadiazo-
reported to have anticancer and anti-inflammatory properties; lobenzamide derivatives and their copper(II) complexes. All
examples include 2-isopropoxy-6-pentadecyl-N-pyridin-4-yl- these compounds were examined and screened by in vitro ana-
benzamide, 2-ethoxy-N-(3-nitrophenyl)-6-pentadecylbenzamide, lysis for their cytotoxic activity to inhibit the proliferation of
and
2-ethoxy-6-pentadecyl-N-pyridin-4-ylbenzamide,
with five human cancer cell lines: MDA-MB-231, SKNMC, LNCap,
respective IC50 values of 11.02, 13.55, and 15.29 μM.7,8 Cyano- KB, and HEPG-2.
(acrylamido)benzamide has been reported to exhibit antitumor
activity against the breast carcinoma cell line.9 In addition,
cyano(phenyl)(hexahydroquinoline-2-yl)benzamide has shown
2. Experimental
2.1. General procedure
potent cytotoxicity against hepatic cancer cells.10 Halogen
derivatives of oxoindolin-3-ylideneaminobenzamide have been
reported to exhibit moderate cytotoxic activity against the
human breast adenocarcinoma cell line.11 Cytotoxic activity
has also been reported for a series of N-(thiophen-2-yl)benz-
amide derivatives employed to control the V600E BRAF kinase
Benzoyl chloride, potassium thiocyanate, 2-aminopyridine,
4-methyl-2-aminopyridine, 6-methyl-2-aminopyridine, copper
(II) chloride dehydrate, and all the used solvents were supplied
by Merck. All chemical compounds were used without further
purification. A few experiments were performed using the
microwave oven Samsung Trio, CE117ADV (230 V, 50 HZ).
Melting points were determined by Electro Thermal IA 9000
Series. FT-IR spectra were obtained with a Nicolet 5SXC
mutation.12
N-Acetyldinaline[4-(acetylamino)-N-(2-amino-
phenyl)benzamide] has also demonstrated a wide spectrum
of antitumor activities in preclinical models.13,14 N-(2-Amino-
phenyl)-4-[N-(pyridine-3-ylmethoxycarbonyl)aminomethyl]benz-
amide (MS275), a synthetic benzamide derivative, has been
known for its proapoptotic properties, depending on tissue/
cell type, doses, or incubation time.15 Three benzamide deriva-
tives of oxo-thiazolidinbenzamide have also been found to
exhibit antitumor activity against Dalton’s lymphoma ascites
(DLA) cells.16
A few studies have indicated that metal-based drugs show
more cytotoxic activity against cancer cell lines than their
ligands.17–19 These effects have led to an increased interest in
the preparation of these compounds and complexes, to study
their pharmaceutical and medicinal properties.20–24 Among
the metal-based drugs that are used as anticancer agents,
copper complexes have attracted more attention due to the
role of copper as an essential element in human life.25,26 The
series of R-heterocyclic Cu(II) thiosemicarbazonato complexes
(Cu(TSC)Cl) have exhibited more cytotoxic activity against two
breast cancer cell lines, SK-BR-3 and MCF-7, than their free
ligands.27
1
spectrometer, using KBr (4000–400 cm−1) pellets. The H and
13C NMR spectra were recorded on a Bruker Advance 500
spectrometer at room temperature in acetone-d6 and DMSO-d6,
using TMS as an internal reference. Elemental analysis of C,
H, and N was performed on a Fligan Flash 1112 elemental ana-
lyzer. The quantity of Cu in the complexes was defined by the
Varain Spectra 200-Rapid atomic absorption spectroscopy.
Diffraction data for crystals of carbamothioylbenzamide
derivatives (compounds
2 and 3), thiadiazolobenzamide
derivatives (ligands O1, O2, and O3), and complexes C2 and
C3 were collected on a Bruker AXS SMART diffractometer at
room temperature using Mo Kα radiation (λ = 0.71073 Å)
monochromatized by a graphite crystal. Data reduction was
carried out using the Bruker AXS SAINT and SADABS
packages.29 The structure was solved by direct methods and
refined by full-matrix least-squares calculations using
SHELX.30 An empirical absorption correction (Ψ-scan) was
applied. All non-hydrogen atoms were refined anisotropically.
The positions of hydrogen atoms were calculated corres-
ponding to their geometrical conditions and refined using a
riding model. Isotropic displacement parameters of hydrogen
atoms were derived from the parent atoms.
Moreover, copper complexes, including 2-oxo-1,2-dihydro-
quinoline-3-carbaldehyde(4′-methylbenzoyl)hydrazine,
have
shown higher cytotoxic activity against human cervical cancer
cells (HeLa) and human laryngeal epithelial carcinoma cells
(HEp-2) than their ligands; for example, the ligand (4′-methyl-
benzoyl)hydrazone (H2L) exhibited lower IC50 values of 127
5 μM against HeLa cell line and 145 4 μM against HEp-2 cell
2.2. Synthesis and characterization of carbamothioyl-
benzamide derivatives: compounds 1, 2, and 3
line compared to the respective IC50 values of 28.3 1.6 and Compounds 1 (N-(pyridine-2-ylcarbamothioyl)benzamide), 2
9.3 1.2 μM of its copper(II) complexes, square pyramidal (N-(4-methylpyridine-2-ylcarbamothioyl)benzamide), and
3
complex [Cu(H2L)Cl2]·2H2O with copper(II) chloride and (N-(6-methylpyridine-2-ylcarbamothioyl)benzamide) were pre-
square planar complex [Cu(HL)NO3]·DMF with copper(II) pared according to the reported protocol, which was modified
nitrate.28
in our laboratory.31 According to the modified protocol,
Considering the increasing importance of benzamide com- benzoyl chloride (0.5 mmol) and potassium thiocyanate
pounds and their copper complexes as anticancer agents, this (0.5 mmol) were mixed in acetonitrile (10 cm3) at room temp-
study aimed to synthesize and characterize novel benzamide erature. The mixture was stirred for 30 min and a 2-aminopyri-
derivatives and their metal complexes with cytotoxic activity dine derivative (0.5 mmol) was gradually added. The mixture
7946 | Dalton Trans., 2014, 43, 7945–7957
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was warmed to 50–60 °C for 4–8 h, incubated at room tempera-
FTIR data (KBr, cm−1): ν 3213m (NH), 2923w, 1703m
ture for 24 h, and finally filtered. The precipitate was purified (CvO), 1675s, 1567s, 1524m (CvN + CvC(py)), 1486m (CvN
by recrystallization from the solution of ethanol and acetone + CvC(py)), 1381s, 1345m (CvS), 1298s, 1260s (CvS), 1156m
(1 : 1), and the potassium chloride that remained undissolved (C–N), 1068m, 885m (CvS), 753m, 712s, 688w (py), and 569w.
was separated.
Compounds 2 and 3 were also prepared using microwaves H, 4.83%; N, 15.49%; Found: C, 60.04%; H, 3.90%; N, 14.30%.
as an energy source (2: 90 W, 5 min; 3: 360 W, 10 min) and 2.2.3. Compound 3: N-(6-methylpyridine-2-yl-carba-
Elemental analysis calculated for C14H13N3OS: C, 61.97%;
acetonitrile as a solvent. The ratio of reactants was the same as mothioyl)benzamide. Yellow powder, yield: MWI 53.8%,
that in the modified reported protocol. Identification and Reflux 70.4%, MP 142–144 °C.
characterization of synthesized compounds 1, 2, and 3 were
1H NMR (acetone-d6, 500 MHz): δ 2.49 (s, 3H, CH3), 7.14 (d,
conducted by melting point, elemental analysis, IR spec- 1H, H2), 7.77 (s, 1H, H3), 8.76 (s, 1H, H4), 8.29 (d, 2H, H9 and
troscopy, 1H NMR spectroscopy, 13C NMR spectroscopy, and 13), 7.60 (dd, 2H, H10 and 12), 7.71 (t, 1H, H11), 10.13 (s, 1H,
X-ray analysis (Scheme 1).
2.2.1. Compound 1: N-(pyridine-2-yl-carbamothioyl)benz-
N2H), and 13.29 (s, 1H, N3H).
13C NMR (acetone-d6, 125 MHz): δ 23.55 (CH3), 151.39 (C1),
amide. Light yellow powder, yield: reflux 40%, MP 138–139 °C. 121.06 (C2), 138.29 (C3), 112.66 (C4), 158.05 (C5), 177.86 (C6),
1H NMR (acetone-d6, 500 MHz): δ 8.95 (s, 1H, H1), 7.28 (d, 168.25 (C7), 133.75 (C8), 129.13 (C9 and 13), 128.67 (C10 and
1H, H2), 7.74 (s, 1H, H4), 8.46 (d, 2H, H9 and 13), 8.10 (dd, 12), and 132.69 (C11).
2H, H10 and 12), 7.90 (t, 1H, H11), 13.36 (s, 1H, N2H), and
10.36 (s, 1H, N3H).
FTIR data (KBr, cm−1): ν 3192w (NH), 2930w, 1713s (CvO),
1610m, 1557s, 1529s (CvN + CvC(py)), 1493m (CvN +
13C NMR (acetone-d6, 125 MHz): 149.00 (C1), 121.76 (C2), CvC(py)), 1443s, 1414m, 1380m, 1333s (CvS), 1239s (CvS),
138.02 (C3), 115.86 (C4), 152.13 (C5), 178.16 (C6), 168.36 (C7), 1212m, 1176vs, 1195m, 1159s (C–N), 1084w, 1068m, 1023w,
133.76 (C8), 129.14 (C9 and 13), 128.68 (C10 and 12), and 999w, 969w, 899m (CvS), 793s, 747, 701s, 684m (py), 662s,
132.66 (C11).
and 516s.
Elemental analysis calculated for C14H13N3OS: C, 61.97%;
FTIR data (KBr, cm−1): ν 3277m (NH), 2923w, 1703m, 1667s
(CvO), 1557s (CvN + CvC(py)), 1524m, 1486m (CvN + H, 4.83%; N, 15.49%, found: C, 62.45%; H, 4.08%; N, 14.70%.
CvC(py)), 1381s, 1350m (CvS), 1298s, 1248s (CvS), 1156m,
1061m (C–N), 847m (CvS), 753m, 712s, 694w (py), and 569w.
Elemental analysis calculated for C14H13N3OS: C, 61.16%;
H, 3.55%; N, 16.46%; Found: C, 61.04%; H, 4.27%; N, 16.89%.
2.2.2. Compound 2: N-(4-methylpyridine-2-yl-carbamothioyl)-
benzamide. Light yellow powder, yield: MWI 63.5%, Reflux
2.3. Synthesis and characterization of novel [1,2,4]-
thiadiazolo[2,3-a]pyridinebenzamide derivatives: ligands
O1, O2, and O3
Copper(II) chloride (0.084 g, 0.5 mmol) in 3 cm3 ethanol was
42.2%, MP 140–142 °C.
1H NMR (acetone-d6, 500 MHz): δ 2.29 (s, 3H, CH3), 8.29 (s, added to each of the solutions of compounds 1, 2, and 3
1H, H1), 7.10 (d, 1H, H2), 8.80 (s, 1H, H4), 8.08 (d, 2H, H9 and (0.5 mmol) in 10 cm3 acetone. The reaction was heated at
13), 7.59 (dd, 2H, H10 and 12), 7.70 (t, 1H, H11), 13.36 (s, 1H, 55 °C and stirred for 5 h. Precipitates, obtained as the products
N2H), and 10.36 (s, 1H, N3H).
of oxidative cyclization (ligands O1, O2, and O3), were filtered,
13C NMR (acetone-d6, 125 MHz): δ 21.33 (CH3), 149.11 (C1), and yellowish-orange or yellowish-green powders were separ-
1
123.18 (C2), 149.90 (C3), 116.80 (C4), 152.70 (C5), 177.86 (C6), ated. These precipitates were characterized by CHN and IR, H
168.74 (C7), 134.21 (C8), 129.61 (C9 and 13), 129.13 (C10 and NMR, and 13C NMR spectroscopies (Scheme 1).
12), and 133.18 (C11).
2.3.1. Ligand O1: N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-
2-ylidene)benzamide. Yellowish-white powder, yield 98%, MP
251 °C.
1H NMR (DMSO-d6, 500 MHz): δ 9.16 (d, 1H), 7.66 (dd, 1H),
7.49 (dd, 1H), 8.95 (d, 1H), 8.23 (d, 2H, H9 and H13), 7.57 (dd,
2H, H10 and H12), and 8.16 (t, 1H, H11).
13C NMR (DMSO-d6, 125 MHz): δ 139.97 (C1), 118.85 (C2),
137.74 (C3), 117.85 (C4), 155.30 (C5), 173.34 (C6), 174.86 (C7),
133.14 (C8), 128.68 (C9 and C13), 128.72 (C10 and C12), and
131.16 (C11).
FTIR data (KBr, cm−1): ν 3026w, 2825w, 1644s (CvO),
1624m (CvN), 1600m, 1578m (CvN), 1556s, 1529s (CvC +
CvN(py)), 1468s (CvC + CvN(py)), 1310m, 1287s, 1253w,
1107m (C–N), 1089m, 1026w, 921m, 888m, 771m, 703s (C–S),
and 662w (py).
Elemental analysis calculated for C13H9N3OS: C, 61.16%; H,
3.55%; N, 16.46%, found: C, 61.04%; H, 4.27%; N, 16.89%.
Scheme 1 Schematic of synthesis of O1, O2, O3, C1, C2, and C3.
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2.3.2. Ligand O2: N-(7-methyl-2H-[1,2,4]thiadiazolo[2,3-a]-
13C NMR (DMSO-d6, 125 MHz): δ 139.45 (C1), 117.26 (C2),
pyridine-2-ylidene)benzamide. Yellow powder, yield 99%, MP 133.14 (C3), 117.12 (C4), 155.04 (C5), 172.33 (C6), 174.35 (C7),
256 °C.
134.32 (C8), 128.15 (C9 and C13), 128.10 (C10 and C12), and
1H NMR (DMSO-d6, 500 MHz): δ 2.45 (s, CH3), 8.90 (s, 1H), 131.02 (C11).
7.24 (d, 1H), 7.67 (s, 1H), 8.22 (d, 2H, H9 and H13), 7.54 (dd,
2H, H10 and H12), and 7.61 (t, 1H, H11).
FT-IR data (KBr, cm−1): ν 3062w, 2835w, 1653s (CvO),
1621m (CvN), 1610m, 1588m (CvN), 1566s, 1523s (CvC +
13C NMR (DMSO-d6, 125 MHz): δ 21.04 (CH3), 151.41 (C1), CvN(py)), 1481s (CvC + CvN(py)), 1310m, 1287s, 1255w,
119.02 (C2), 133.29 (C3), 117.62 (C4), 155.56 (C5), 175.84 (C6), 1166m (C–N), 1089m, 1026w, 921m, 868m, 751m, 719s (C–S),
176.69 (C7), 133.43 (C8), 128.58 (C9), 128.61 (C10), and 132.45 and 674w (py).
(C11).
Elemental analysis calculated for C28H22Cl2CuN6O2S2: C,
FTIR data (KBr, cm−1): ν 3025w, 2916w, 1662w (CvO), 52.09%; H, 3.43%; N, 13.03%; Cu, 9.85% Found: C, 51.67%; H,
1623m (CvN), 1617s, 1595m (CvN), 1543w, 1515s (CvC + 4.20%; N, 13.56%; Cu, 9.40%.
CvN(py)), 1480s (CvC + CvN(py)), 1447s, 1430w, 1370s,
2.4.2. Complex C2: dichlorobis(N-(7-methyl-2H-[1,2,4]thia-
1327w, 1266s, 1237w, 1186m, 1146s (C–N), 1069w, 1025m, diazolo[2,3-a]pyridine-2-ylidene)benzamide) copper(II). Jasper
897m, 859m, 813m, 791m, 722s (C–S), and 659w (py).
Elemental analysis calculated for C14H11N3OS: C, 62.44%;
H, 4.11%; N, 15.60%, found: C, 62.97%; H, 3.90%; N, 16.09%.
green crystals, yield 56%.
1H NMR (DMSO-d6, 500 MHz): δ 2.49 (s, CH3), 8.91 (d, 1H,
H1), 7.24 (d, 1H, H2), 7.66 (s, 1H, H4), 8.22 (d, 2H, H9 and 13),
2.3.3. Ligand O3: N-(5-methyl-2H-[1,2,4]thiadiazolo[2,3-a]- 7.54 (dd, 2H, H10 and 12), and 7.60 (t, 1H, H11).
pyridine-2-ylidene)benzamide. Yellow powder, yield 99%, MP
13C NMR (DMSO-d6, 125 MHz): δ 20.85 (CH3), 151.18 (C1),
253 °C.
118.81 (C2), 133.24 (C3), 117.42 (C4), 155.48 (C5), 175.05 (C6),
1H NMR (DMSO-d6, 500 MHz): δ 2.78 (s, CH3), 7.27 (d, 1H), 176.66 (C7), 133.08 (C8), 128.16 (C9 and 13), 128.13 (C10 and
7.59 (s, 1H), 7.71 (d, 1H), 8.21 (d, 2H, H9 and H13), 7.53 (dd, 12), and 132.24 (C11).
2H, H10 and H12), and 8.01 (t, 1H, H11).
FT-IR data (KBr, cm−1): ν 3035w, 2921w, 1659s (CvO),
13C NMR (DMSO-d6, 125 MHz): δ 19.76 (CH3), 145.08 (C1), 1628s (CvN), 1590m (CvN), 1523s (CvC + CvN(py)), 1481m
115.62 (C2), 138.57 (C3), 116.64 (C4), 155.32 (C5), 174.58 (C6), (CvC + CvN(py)), 1444s, 1365sm, 1329m, 1267m, 1237w,
176.24 (C7), 132.90 (C8), 129.25 (C9), 128.23 (C10), and 132.10 1159w (C–N), 1118w, 1027m, 897w, 852w, 805s, 717s (C–S),
(C11).
FTIR data (KBr, cm−1): ν 3048w, 1650s (CvO), 1621s
677m (py), 584m, 522w, 457w, and 433vw.
Elemental analysis calculated for C28H22Cl2CuN6O2S2: C,
(CvN), 1588w, 1567s, 1561s (CvN), 1526s (CvC + CvN(py)), 49.96%; H, 3.29%; N, 12.48%; Cu, 9.40% Found: C, 48.80%; H,
1487w (CvC + CvN(py)), 1444m, 1389w, 1378s, 1329s, 4.50%; N, 11.98%; Cu, 10.04%.
1288m, 1277s, 1170m (C–N), 1065w, 905m, 796m, 721m (C–S),
710m, and 655w (py).
2.4.3. Complex C3: dichlorobis(N-(5-methyl-2H-[1,2,4]thia-
diazolo[2,3-a]pyridine-2-ylidene)benzamide) copper(II). Jasper
Elemental analysis calculated for C14H11N3OS: C, 62.44%; green crystals, yield 57%.
H, 4.11%; N, 15.60%, found: C, 61.97%; H, 4.83%; N, 15.23%.
1H NMR (DMSO-d6, 500 MHz): δ 2.80 (s, CH3), 7.35 (d, 1H,
H2), 7.63 (dd, 1H, H3), 7.81 (d, 1H, H4), 8.20 (d, 2H, H9 and
13), 7.54 (dd, 2H, H10 and 12), and 8.07 (t, 1H, H11).
2.4. Synthesis and characterization of complexes C1, C2, and
C3
13C NMR (DMSO-d6, 125 MHz): δ 19.62 (CH3), 145.29 (C1),
Complexes C1, C2, and C3 were synthesized via the reaction of 117.20 (C2), 131.48 (C3), 115.71 (C4), 154.78 (C5), 172.18 (C6),
copper(II) chloride dihydrate and respective ligands O1, O2, 174.60 (C7), 138.99 (C8), 128.13 (C9 and 13), 128.16 (C10 and
and O3. Copper(II) chloride dihydrate (0.084 g, 0.5 mmol) in 12), and 132.46 (C11).
5 cm3 ethanol was added gradually to the solutions of ligands
FT-IR data (KBr, cm−1): ν 3006w, 2923w, 1652m (CvO),
O1 (0.128 g, 0.5 mmol), O2 (0.135 g, 0.5 mmol), and O3 1621m (CvN), 1594m (CvN), 1540m (CvC + CvN(py)),
(0.135 g, 0.5 mmol) in 8 cm3 acetone. The mixtures were 1489m (CvC + CvN(py)), 1445s, 1378s, 1332s, 1280m,
heated at 55 °C and stirred for 18 h. The obtained mixture was 1240m, 1169m (C–N), 1098w, 1064m, 1009vw, 906m, 797m,
filtered; after 5 days at room temperature, the complexes, in 723s (C–S), 680m (py), 542vw, and 478vw.
the form of jasper green crystals, could be isolated from the fil-
Elemental analysis calculated for C28H22Cl2CuN6O2S2: C,
trate. The crystals were characterized by CHN and IR, 1H NMR, 49.96%; H, 3.29%; N, 12.48%; Cu, 9.40%, found: C, 49.05%;
and 13C NMR spectroscopies; all these spectroscopic analyses H, 4.30%; N, 13.08%; Cu, 8.96%.
indicated a common formula [Cu(O)2Cl2] for all obtained com-
plexes (Scheme 1).
For single-crystal X-ray diffraction experiments, suitable
crystals of compounds 2 and 3 were obtained by recrystalliza-
2.4.1. Complex C1: dichlorobis(N-(2H-[1,2,4]thiadiazolo- tion of their ethanol and acetone (1 : 1) solutions. Suitable
[2,3-a]pyridine-2-ylidene)benzamide) copper(II). Jasper green crystals of ligands O1, O2, and O3 were obtained by re-
crystals, yield 54%.
crystallization of their methanol and acetonitrile (1 : 1)
1H NMR (DMSO-d6, 500 MHz): δ 9.18 (d, 1H), 7.71 (dd, 1H), solutions. The isolated jasper green crystals of complexes
7.47 (dd, 1H), 7.90 (d, 1H), 8.22 (d, 2H, H9 and 13), 7.56 (dd, C2 and C3 were used in X-ray diffraction experiments
2H, H10 and 12), and 8.14 (t, 1H, H11).
(Table 1).
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Table 1 Crystallographic refinement data for compounds 2 and 3, ligands O1, O2, and O3 and complexes C2, and C3
2
3
O1
O2
O3
C2
C3
Empirical
formula
Formula weight
T (K)
Wavelengths (Å)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
α (°)
β (°)
C
14H13N3OS
C14H13N3OS
C13H9N3OS
C14H11N3OS
C14H11N3OS
C28H22Cl2CuN6O2S2 C28H22Cl2CuN6O2S2
271.33
294(2)
0.71073
Monoclinic
P21/c
12.018(4)
5.2813(16)
21.723(6)
—
97.502(4)
—
1367.0(7)
4
271.33
294(2)
255.29
273(2)
0.71073
Monoclinic
P21/c
6.255(3)
18.109(7)
10.221(4)
—
95.525(6)
—
269.32
273(2)
0.71073
Monoclinic
P21/n
11.678(6)
3.995(2)
26.939(15)
—
98.965(9)
—
269.32
296(2)
673.07
294(2)
673.07
294(2)
0.71073
Monoclinic
P21/n
7.201(3)
18.487(8)
10.441(5)
—
108.926(7)
—
1314.9(10)
4
0.71073
Monoclinic
P21/c
12.7107(7)
18.0290(9)
11.5181(6)
—
101.980(3)
—
2582.0(2)
8
0.71073
Triclinic
P1
0.71073
Monoclinic
P21/n
ˉ
8.158(5)
8.426(5)
11.183(6)
78.716(8)
76.857(8)
71.514(8)
703.6(7)
1
13.947(4)
14.750(5)
13.894(4)
—
98.561 (5)
—
2827.1(14)
4
1.579
γ (°)
V (Å3)
1152.4(8)
4
1.471
1241.4(12)
4
1.366
Z
D (g cm−3
)
1.318
0.232
1.371
0.241
1.386
0.245
1.589
μ (mm−1
)
0.270
0.251
1.154
1.149
Rint
0.0589
0.0507
99.6 (θ =
26.45°)
R1 = 0.0384,
wR2 = 0.0892
R1 = 0.0681,
wR2 = 0.0969
714501
0.0462
99.4 (θ =
27.56°)
R1 = 0.0361,
wR2 = 0.0735 wR2 = 0.1286
R1 = 0.0845, R1 = 0.0767,
wR2 = 0.0833 wR2 = 0.1604
711636 713327
0.0507
99.3 (θ =
23.94°)
R1 = 0.0502,
0.0824
99.2 (θ =
24.76°)
R1 = 0.0385,
wR1 = 0.0972
R1 = 0.0607,
wR2 = 0.1060
962672
0.0449
97.8 (θ = 29.49°)
0.0582
99.7 (θ = 28.25°)
Completeness to θ 95.2 (θ =
(%)
Final R indices
[I > 2σ(I)]
R indices (all
data)
30.36°)
R1 = 0.0417,
wR2 = 0.1051
R1 = 0.1109,
wR2 = 0.1231
714500
R1 = 0.0406,
wR1 = 0.0973
R1 = 0.0568,
wR2 = 0.0973
714502
R1 = 0.0420,
wR1 = 0.0945
R1 = 0.0813,
wR2 = 0.1008
714503
CCDC
2.5. Cell culture and in vitro cytotoxicity assay
3. Results and discussion
3.1. Spectroscopic studies
Five human cell lines, the breast cancer cell line MDA-MB-231,
the neuroblastoma cell line SK-N-MC, the prostate adeno-
carcinoma cell line LNCap, the nasopharyngeal epidermoid Infrared spectroscopy was used to study the solid-state pro-
carcinoma cell line KB, and the liver cancer cell line HEPG-2, perties of all synthesized compounds, ligands, and complexes.
were obtained from the National Cell Bank of Iran (NCBI, No absorption bands characteristic of the NH groups were
Iran). The cells were grown in RPMI-1640 medium sup- observed for ligands O1, O2, and O3, because compounds 1, 2,
plemented with 10% heat-inactivated fetal calf serum (from and 3 were oxidized by copper(II) and lost two hydrogen atoms
GibcoeBRL, UK), 100 mg cm−3 streptomycin, and 100 U cm−3 of the NH groups. In addition, complexes C1, C2, and C3,
penicillin at 37 °C/95% rh/5% CO2. In vitro cytotoxic activities including the coordinated ligands, did not indicate any
of the test compounds 1, 2, 3, O1, O2, O3, C1, C2, and C3 were absorption bands for the NH groups. During oxidation, the
investigated in comparison with standard chemotherapy drugs sulfur atom was joined to the nitrogen atom of the pyridine
doxorubicin (DOX) and etoposide, using a modified MTT-dye ring, forming a five-membered ring.
reduction assay.32 Briefly, cultures in the exponential growth
The presence of the CvN bond, after oxidation and cycliza-
phase were trypsinized and diluted in a complete growth tion, was confirmed by the absorption bands at 1624 and
medium to a total cell count of 5 × 104 cells cm−3. Then 195 × 1578 cm−1 (O1), 1632 and 1592 cm−1 (O2), 1621 and
10−3 cm3 of the suspension was added to the wells of a sterile 1561 cm−1 (O3), 1630 and 1588 cm−1 (C1), 1628 and 1590 cm−1
96-well plate (NUNC, Denmark) and allowed to attach over- (C2), and 1621 and 1594 cm−1 (C3).33
1
night. After plating, 5 × 10−3 cm3 of a serial concentration of
The H NMR spectroscopy characterized the structural pro-
every compound was added. The plates were incubated for perties of the sample in solution. The chemical shifts of 13.36
48 h. After incubation, 200 × 10−3 cm3 of 0.5 mg cm−3 solution and 10.36 ppm (1), 13.36 and 10.36 ppm (2) and 10.13 and
of MTT (Sigma-Aldrich, Steinheim, Germany) was added to 13.29 ppm (3) correspond to the hydrogen atoms in NH bonds.
each well and the plate was again incubated for 4 h. Then the Differences in the chemical shifts for each compound revealed
culture medium was replaced with 100 × 10−3 cm3 of DMSO, the extent of intramolecular hydrogen bonding in N2H, N2H
and the absorbance was measured at 492 nm. Each experiment and N3H (13.36, 13.36, and 13.29 ppm), which caused the
was carried out in triplicate for each concentration. DOX and movement of resonances to downfield in compounds 1, 2 and
etoposide were used as positive controls for cytotoxicity, and 3.34–36 The absence of resonances above 10 ppm in O1, O2, O3,
the tumor cells cultured in 200 × 10−3 cm3 of complete C1, C2, and C3 confirmed the removal of hydrogen atoms
medium were applied as controls for cell viability. For each from NH groups.34,37–39
compound, the concentration causing 50% inhibition in cell
growth (IC50 value) was calculated from the concentration ences in the chemical shifts of compounds 1, 2, and 3; ligands
The data of 13C NMR spectra showed no significant differ-
response curves using regression analysis.
O1, O2, and O3, and complexes C1, C2, and C3. The position
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Table 2 Selected bond lengths (Å) and angles (°) in compounds 1, 2, and 3
Bond length (Å)
131
2
3
Bond angle (°)
131
2
3
N1–C5
N2–C5
N2–C6
N3–C6
N3–C7
S1–C6
O1–C7
C7–C8
1.325(5)
1.417(4)
1.329(4)
1.382(4)
1.388(4)
1.658(3)
1.220(4)
1.470(5)
1.329(2)
1.403(2)
1.336(2)
1.383(2)
1.382(2)
1.653 (2)
1.220(2)
1.476(3)
—
1.330(2)
1.407(2)
1.358(2)
1.367(2)
1.392(2)
1.662 (2)
1.201(2)
1.491(2)
—
C1–N1–C5
N1–C5–N2
C5–N2–C6
N2–C6–S1
N3–C6–S1
N2–C6–N3
C6–N3–C7
O1–C7–N3
O1–C7–C8
N3–C7–C8
117.0(4)
109.9(3)
114.7(3)
126.8(3)
118.6(3)
114.7(3)
128.5(3)
121.5(3)
121.7(3)
116.8(3)
116.13(18)
109.76(16)
132.88(15)
127.22(14)
118.60(12)
114.18(15)
129.06(15)
121.35(18)
121.48(16)
117.17(16)
117.78(15)
118.45(15)
131.70(14)
118.98(13)
126.21(13)
114.78(14)
128.32(15)
122.68(16)
122.23(16)
115.08(15)
—
—
of the methyl group had no effect on the chemical shift. bonding, N3–H3⋯N1 (2.664 Å and 140.3°). In the unit cell of
During oxidative cyclization, the conversion of the CvS bond compound 2, two weak intermolecular hydrogen bonds (N3–
to the C–S bond in O1, O2, O3, C1, C2, and C3 decreased the H3⋯S1) connect two molecules together and form a centro-
chemical shift of carbon to upfield and increased the reson- symmetric dimer in the solid state (Fig. 3). In contrast, in the
ance of the CvO bond to downfield.40
unit cell of compound 3, three weak intermolecular hydrogen
bonds (N2–H2⋯S1 and C2–H2⋯O1) bind the molecules
(Fig. 4).
3.2. Crystal structure
3.2.2. Ligands O1, O2, and O3. Oxidative cyclization of
compounds 1, 2, and 3 changed their bond lengths and
resulted in the formation of an N–S bond (N of the pyridine
ring) in ligands O1, O2, and O3. The N–S bond and changed
bond lengths were confirmed by measuring the bond lengths
and angles of ligands O1 (ESI, Fig. S2†), O2, and O3 (Fig. 5
and 6, Table 3). The alkyl group on the pyridine ring had no
effect on the bond lengths and angles. The planar structure of
ligands caused by conjugated double bonds and the nonbond-
ing electron pair of the nitrogen atom (N3) was confirmed by
the torsion angles (ESI, Table S1†). The obtained data indi-
cated that ligands O1 and O2 have similar structures.
Relevant crystallographic information of O1, O2, O3, C1, C2
and C3 has been summarized in Table 1.
3.2.1. Compounds 2 and 3. The similar bond lengths and
angles in compounds 2 and 3 (Table 2) indicated that the posi-
tion of the substituted alkyl group on the pyridine ring has no
effect on bond lengths and angles (Fig. 1 and 2).36 The crystal
structure of previously reported compound 1 (ESI, Fig. S1†)31
is similar to that of compound 2 (Fig. 1).
The strong intramolecular hydrogen bonding, N2–H2⋯O1
(2.597 Å and 143.3°), in compound 2 shifted the resonance of
the hydrogen to downfield. However, the chemical shift in
compound 3 is caused by the strong intramolecular hydrogen
In the unit cell of ligand O1, strong intramolecular inter-
actions exist between sulfur and oxygen atoms (S1–O1,
2.168 Å), whereas weak intermolecular interactions exist
between sulfur and carbon atoms (S1–C7, Å 3.445 Å). Several
weak intermolecular hydrogen interactions (C1–H1⋯O1,
3.408 Å; C4–H4⋯N2, 3.466 Å; and C11–H11⋯N4, 3.363 Å)
connect the molecules to each other and form a centro-
symmetric crystal lattice (ESI, Fig. S3†).
Fig. 1 Molecular structure of compound 2 (30% probability level).
Fig. 3 View of the unit cell of compound 2 with hydrogen bonding
(1: x, y, z; 2: −x, 1/2 + y, 1/2 − z; 3: −x, −y, −z; 4: x, 1/2 − y, 1/2 + z).
Fig. 2 Molecular structure of compound 3 (30% probability level).
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3.331 Å). Each molecule of dimers is connected again to the
third molecule of other dimers by intermolecular hydrogen
bonding (C4–H4⋯N2, 3.367 Å) (Fig. 7).
The obtained data indicated that the unit cell of ligand O1
is similar to that of ligand O2.
Strong intramolecular S⋯O bonding exists among different
molecules in the unit cell of ligand O3 (S1⋯O1, 2.210 Å and
S2⋯O2, 2.140 Å) (Fig. 8). In addition, π–π weak interactions
(3.625 Å in ligand O1, 3.995 Å in ligand O2, and 3.640 Å in
ligand O3) are observed between two phenyl rings or between
a phenyl and a pyridine ring.
Ligands O1, O2, and O3, derivatives of [1,2,4]thiadiazolo-
[2,3-a]pyridine benzamide, reported in our work are the first
benzamide derivatives that could be produced by oxidative
cyclization reaction of N-(pyridine-2-ylcarbamothioyl)benz-
amide compounds using the copper(II) ion as an oxidant.
Planar ligands with structures similar to those of ligands O1,
O2, and O3 have not yet been reported in the literature.
The already published compounds containing [1,2,4]thia-
diazolo[2,3-a]pyridine, which were produced by oxidative cycli-
zation reaction of N-pyridyl-thioureas, are neither a benzamide
derivative nor similar to our ligands O1, O2, and O3.41,42
Although 2,5-disubstituted oxazoles43 and furoxan44 have
been reported to be produced by oxidative cyclization using
the copper(II) ion as an oxidant, contrary to our products, they
are not able to react as ligands and cannot connect to copper
ions to form complexes.
Fig. 4 View of the unit cell of compound 3 with hydrogen bonding (1: x,
y, z; 2: 1/2 − x, 1/2 + y, 1/2 − z; 3: −x, −y, −z; 4: 1/2 + x, 1/2 − y, 1/2 + z).
As reported by Che et al.,38,45 the reaction of N-(pyridine-2-
ylcarbamothioyl)benzamide (compound 1) with the copper(II)
ion resulted in the formation of a polymeric tetrahedral
complex directly, whereas in our study, this reaction produced
the ligand O1 in addition to complex C1. The ligand O1 could
be isolated and was tested for its cytotoxic effect.
Fig. 5 Molecular structure of ligand O2 (30% probability level).
3.2.3. Complexes C2 and C3. Connection of ligands O2
and O3 to the copper(II) ion via their nitrogen atoms resulted
in the formation of complexes C2 and C3.46 In both com-
plexes, each copper ion with a coordination number of four
was surrounded by two chloride ions and two ligands. Bond
angles of about 90° with very small distortion resulted in the
formation of a square planar structure around the copper ion.
The bond lengths and angles around the central ion were
similar in both C2 and C3 complexes (Fig. 9 and 10, Table 4;
for more detailed information: ESI Table S2†).
The unit cell of complex C2 has a triclinic crystal system,
ˉ
with z = 1 and space group P1. In this unit cell, weak π–π inter-
actions between the phenyl rings (average 3.858 Å) of two com-
plexes led to the formation of the crystal lattice (Fig. 11).
The unit cell of complex C3 has a monoclinic crystal system,
with z = 4 and space group P21/n. Two medium intramolecular
hydrogen bondings, C14–H14A⋯S1 (3.104 Å) and C14B–
H14D⋯S1B (3.093 Å), were observed in complex C3 (Fig. 12).
Complexes C2 and C3 are characterized by large monoden-
Fig. 6 Molecular structure of ligand O3 (30% probability level).
In the unit cell of ligand O2, the sulfur and oxygen atoms tate ligands and a stable square planar structure.
(2.156 Å) are connected by strong intramolecular bonding. The
Most reported copper(II) ion complexes have a tetrahedral,
binding of two molecules over intermolecular hydrogen square bipyramidal, or octahedral geometry.47–49 Copper(II) ion
bonding creates centrosymmetric dimer (C1–H1⋯O1, complexes having a square planar structure have rarely been
a
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Table 3 Selected bond lengths (Å) and angles (°) in ligands O1, O2, and O3
Bond length (Å)
O1
O2
O3
Bond angle (°)
O1
O2
O3
N1–S1
N1–C5
N2–C5
N2–C6
S1–C6
N3–C6
N3–C7
O1–C7
S1–O1
1.7623(16)
1.357(2)
1.352(2)
1.311(2)
1.7637(19)
1.339(2)
1.338(2)
1.264(2)
2.1678(15)
1.768(3)
1.267(4)
1.346(4)
1.333(4)
1.776(3)
1.325(4)
1.346(4)
1.267(4)
2.156(3)
1.7736(17)c
1.365(3)b
1.347(3)a
1.319(3)c
1.762(2)c
1.348(3)c
1.345(3)d
1.259(3)d
2.141(2)
C1–N1–C5
C5–N1–S1
N1–C5–N2
N1–S1–C6
C5–N2–C6
N2–C6–S1
N3–C6–S1
N2–C6–N3
C6–N3–C7
O1–C7–N3
O1–C7–C8
N3–C7–C8
122.85(17)
111.54(13)
115.48(17)
85.96(9)
110.90(16)
116.11(15)
120.83(15)
123.05(17)
112.52(16)
122.48(19)
119.84(17)
117.67(17)
121.0(3)
112.3(2)
114.7(3)
122.57(18)b
111.56(14)b
115.34(18)b
85.69(9)
110.98(19)
116.42(16)
120.78(16)
122.8(2)
86.07(15)
112.2(3)
114.8(3)
120.6(2)
124.6(3)
113.0(3)
121.7(3)
120.2(3)
118.1(3)
113.39(19)
122.4(2)
120.8(2)
116.8(2)
a First molecule in the ortep O3. b C1 in O3 = C5 in O1 and O2; C13 in O3 = C5 in O1 and O2. c C2 in O3 = C6 in O1 and O2. d C3 in O3 = C7 in O1
and O2.
Fig. 7 Unit cell of ligand O2 with hydrogen bonding (1: x, y, z; 2: 1/2 − x, 1/2 + y, 1/2 − z; 3: −x, −y, −z; 4: 1/2 + x, 1/2 − y, 1/2 + z).
Fig. 8 Unit cell of ligand O3 with hydrogen bonding (1: x, y, z; 2: −x, y
+ 1/2, 1/2 − z; 3: −x, −y, −z; 4: x, 1/2 − y, z + 1/2).
Fig. 9 Molecular structure of complex C2 (30% probability level).
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Fig. 11 π–π interaction between phenyl rings of two C2 complexes
(1: x, y, z; 2: x, −y, −z).
copper(II) ion and a stable square planar structure, with a trans
geometric configuration, was formed. As shown in Scheme 2,
the core structure of monodentate ligands O1, O2, and O3 is
more complex than the monodentate ligand reported by
Ahmadi et al. The large scale of the monodentate ligands, their
planar surface and the position of the cycles in them protect
the square planar structure of complexes C2 and C3 against
reactions with other ligands or solvent molecules and the
copper–copper interaction. This led to the excellent stability of
the novel square planar structure of complexes C2 and C3.
Fig. 10 Molecular structure of complex C3 (30% probability level).
reported; most of these complexes are generated by the
binding of bi- or more dentate ligands to the copper(II)
ion.50–54 The complex [Cu(C6H6N3S2)2] with a square planar
structure was reported to be formed by the binding of the
bidentate ligand thiophene-2-carbaldehydethiosemicarbazone,
to the copper(II) ion52 and the square planar complex [Cu(N-(2-
methylpyridyl)benzenesulfonylamidate)2] by the binding of the
bidentate ligand N-(2-methylpyridyl)benzenesulfonylaide to
the copper(II) ion.53
Formation of a square planar structure through binding of
monodentate ligands to the copper(II) ion has rarely been poss-
ible. The square planar structure of the complex [Cu(2-amino-
5-bromopyridine)2(Cl)2], as reported by Ahmadi et al.,55 was
formed by the binding of 2-amino-5-bromopyridine, a mono-
dentate ligand, to copper(II) chloride. In complexes C2 and C3,
two large monodentate ligands were coordinated to the
3.3. In vitro cytotoxicity studies
The cytotoxic activities of compounds 1, 2, and 3; ligands O1,
O2, and O3; and complexes C1, C2, and C3 against
MDA-MB-231, SK-N-MC, LNCap, KB, and HEPG-2 human
cancer cell lines were determined compared to references DOX
and etoposide. Etoposide and DOX are standard chemotherapy
drugs that have been widely used to treat different types of
cancers, such as neuroblastoma, soft tissue and bone sarco-
mas, breast carcinoma, ovarian carcinoma, lung cancer,
thyroid carcinoma, and gastric carcinoma.56,57 Since no benz-
amide-based copper(II) complex with an anticancer activity was
Table 4 Selected bond lengths (Å) and angles (°) in complexes C2 and C3
Bond length (Å)
C2
C3
Bond angle (°)
C2
C3
Cu1–N2
Cu1–N2#1/2B
Cu1–Cl1
Cu1–Cl1#1/Cl2
S1–C6
S1–N1
S1–O1
N1–C5
N1–C1
N2–C6
N2–C5
1.988(2)
1.988(2)
2.2637(14)
2.2637(14)
1.754(2)
1.795(2)
2.124(2)
1.361(3)
1.364(3)
1.333(3)
1.362(3)
1.328(3)
1.345(3)
1.990(2)
1.996(2)
2.2356(10)
2.2544(10)
1.747(3)
1.809(2)
2.076(2)
1.353(3)
1.368(3)
1.331(3)
1.363(3)
1.331(3)
1.344(3)
N2–Cu1–N2#1/2B
N2–Cu1–Cl1
N2#1/2B–Cu1–Cl1
N2–Cu1–Cl1#1/Cl2
N2#1/2B–Cu1–Cl1#1
Cl1–Cu1–Cl1#1/Cl2
C6–S1–N1
C6–S1–O1
N1–S1–O1
C7–O1–S1
C5–N1–C1
C5–N1–S1
C1–N1–S1
C6–N2–C5
C6–N2–Cu1
C5–N2–Cu1
C6–N3–C7
180.0
168.33(9)
91.92(7)
91.37(7)
89.74(7)
88.55(7)
171.94(4)
85.60(13)
80.43(12)
165.97(10)
106.36(18)
122.6(3)
112.13(19)
125.2(2)
112.4(2)
91.10(6)
88.90(6)
88.90(6)
91.10(6)
180.0
85.53(10)
79.71(9)
165.22(7)
105.68(15)
121.4(2)
112.92(14)
125.63(15)
112.73(18)
117.29(14)
129.87(15)
111.69(19)
119.4(2)
N3–C6
N3–C7
120.98(19)
126.64(19)
110.8(2)
C2–C1–N1
117.5(3)
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Fig. 12 Unit cell of complex C3 with hydrogen bonding (1: x, y, z; 2: −x, 1/2 + y, 1/2 − z; 3: −x, −y, −z; 4: x, 1/2 + y, 1/2 − z).
Fig. 13 IC50 values of O1, O2, O3, C1, C2, and C3 against the breast
cancer cell line MDA-MB-231 (SD = 2.00%).
Scheme 2 Comparison of monodentate ligand reported by Ahmadi
et al. to monodentate ligands O1, O2, and O3.
available commercially, etoposide and DOX were used as posi-
tive references. Although compounds 1, 2, 3 did not exhibit
any cytotoxic activity, such activity was demonstrated by
ligands O1, O2, and O3 and complexes C1, C2, and C3.
The IC50 values of ligands O1, O2, and O3 and complexes
C1, C2, and C3 for all five cancer cell lines are shown in
Fig. 13–17. IC50 values of all ligands and complexes against all
used cell lines are presented in Fig. 18.
The complexes showed higher cytotoxic activities than the
ligands. This could be attributed to the presence of two
ligands and the copper ion in each complex that synergistically
increased the cytotoxicity.20,28
Fig. 14 IC50 values of O1, O2, O3, C1, C2, and C3 against the neuro-
blastoma cell line SK-N-MC (SD = 3.00%).
Compared to the other ligands and complexes, complex C2
showed the lowest IC50 value and the highest cytotoxicity
against all used cell lines, with the exception of the breast
cancer cell line MDA-MB-231. This complex with an IC50 value
of 3.44 0.01 µM showed the most significant inhibitory effect
on the growth of the prostate adenocarcinoma cell line LNCap.
The inhibition effect was comparable to that of the reference
standard DOX and significantly stronger than that of
etoposide. In addition, ligand O2 as a part of complex C2
showed a lower IC50 value and a higher cytotoxicity against
LNCap, KB, and HEPG-2 human cancer cell lines than the
other ligands. This could be due to the different position of
the methyl group in this ligand than that in the other ligands.
Complex C3 with an IC50 value of 5.46 0.66 µM showed
the highest cytotoxic activity against the breast cancer cell
7954 | Dalton Trans., 2014, 43, 7945–7957
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Fig. 15 IC50 values of O1, O2, O3, C1, C2, and C3 against the prostate
adenocarcinoma cell line LNCap (SD = 3.00%).
Fig. 18 Comparison of IC50 values of O1, O2, O3, C1, C2, and C3
against the human cancer cell lines: MDA-MB-231,
LNCap, KB, HEPG-2.
SK-N-MC,
N-benzyl-2-(2-cyano-3-(3,4-dimethoxyphenyl)acrylamido)benzamide
(IC50 9.90 μg mL−1) and 2-(2-cyano-3-(3,4-dimethoxyphenyl)-
acrylamido)-N-(2-hydroxyethyl)benzamide (IC50 7.95 μg mL−1);
and C, 4-(5-fluoro-2-oxoindolin-3-ylideneamino)benzamide
(IC50 > 100 μM), 4-(2-oxo-5-(trifluoromethoxy)indolin-3-ylidene-
amino)benzamide (IC50 27.2 7.1 μM), and 4-(5, 7-dichloro-2-
oxoindolin-3-ylideneamino)benzamide (IC50 65.86 8.1 μM).
The cytotoxic activity of our ligands O1, O2, and O3 against the
breast cancer cell line MDA-MB-231 was in a range of 13.92
1.15 to 20.10 4.93 μM (O1, IC50 17.46 0.58 μM; O2, IC50
20.10 4.93 μM; and O3, IC50 13.92 1.15 μM).
Fig. 16 IC50 values of O1, O2, O3, C1, C2, and C3 against the nasophar-
yngeal epidermoid carcinoma cell line KB (SD = 3.70%).
For the ligand H2PDIMAla and its complex [Cu2(dpq)2(PDI-
MAla)(H2O)2](ClO4)2CH3OH, the IC50 values have been
reported to be >160 and 2.7 μM, respectively, against the
human prostate cancer cell line PC3.25 The IC50 values of
complex C2 and its ligand O2, which showed the highest cyto-
toxic activity against the human prostate cancer cell line PC3,
were about 3.44 0.01 and 12.21 2.56 μM, respectively.
4. Conclusion
The reaction of 2-amino pyridine derivatives, potassium thio-
cyanate, and benzoyl chloride in one pot produced the follow-
ing N-(pyridine-2-ylcarbamothioyl)benzamide derivatives:
Fig. 17 IC50 values of O1, O2, O3, C1, C2, and C3 against the liver
cancer cell line HEPG-2 (SD = 1.70%).
N-(pyridine-2-ylcarbamothioyl)benzamide,
N-(4-methylpyri-
dine-2-ylcarbamothioyl)benzamide, and N-(6-methylpyridine-
2-ylcarbamothioyl)benzamide. Oxidation of the obtained
derivatives using copper(II) chloride as an oxidant resulted in
the removal of two hydrogen atoms, cyclization of the com-
pounds, and finally, formation of three new N-(2H-[1,2,4]thia-
diazolo[2,3-a]pyridine-2-ylidene)benzamide derivatives with
planar structure: N-(2H-[1,2,4]thiadiazolo[2,3-a]pyridine-2-
ylidene)benzamide, N-(7-methyl-2H [1,2,4]thiadiazolo[2,3-a]-
pyridine-2-ylidene)benzamide, and N-(5-methyl-2H [1,2,4]thia-
diazolo[2,3-a]pyridine-2-ylidene)benzamide.
line MDA-MB-2. The inhibition effect of this complex was
approximately six times higher than that of the reference stan-
dard etoposide.
Some benzamide derivatives have been shown to possess
cytotoxic activity. Different kinds of benzamide derivatives (A,3
B,9 and C11) showing cytotoxic activity against the breast
cancer cell line MCF7 have been reported. Their IC50 values,
ranging from 27.2 7.1 to >100 μM, are as follows: A, N-(2-{4-
[(3,7-dimethylocta-2,6-dien-1-yl)oxy]phenyl}ethyl)benzamide
(IC50 80.6 4.0 μM) and N-(2-{4-[(3,7-dimethyl-4-acetyl-octa-2,6-
These planar ligands were coordinated to the copper(II) ion,
and three new complexes C1, C2, and C3, each consisting of
dien-1-yl)oxy]phenyl}ethyl)benzamide (IC50
> 100 μM); B,
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two large monodentate ligands and two chlorine anions, were
synthesized. These new complexes were neutral, having a rare
square planar geometry around the copper ion. Due to the
planar surface and the large scale of the monodentate ligands,
the square planar structures of complexes C1, C2, and C3 are
well protected and very stable.
All obtained compounds, ligands, and complexes were sub-
jected to an in vitro analysis using an MTT-based assay to
define their cytotoxic activity against MDA-MB-231, SK-N-MC,
LNCap, KB, and HEPG-2 human cancer cell lines.
N-(Pyridine-2-yl-carbamothioyl)benzamide derivatives did
not exhibit any cytotoxic activity. Significant cytotoxic activity
could be observed by ligands O1, O2, and O3 and complexes
C1, C2, and C3. The complexes exhibited higher cytotoxic
activities in comparison with the ligands.
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