Microwave assisted synthesis, X-ray crystallography and DFT calculations of selected aromatic thiosemicarbazones

Article abstract of DOI:10.1016/j.molstruc.2012.11.050

Series of four benzaldehyde thiosemicarbazones has been synthesized under microwave irradiation and characterized structurally by means of infrared and NMR spectroscopies and mass spectrometry. Their crystal structures were determined by single crystal X-

Full text of DOI:10.1016/j.molstruc.2012.11.050

Journal of Molecular Structure 1037 (2013) 63–72
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Microwave assisted synthesis, X-ray crystallography and DFT calculations
of selected aromatic thiosemicarbazones
a
Maciej Serda a, Jan G. Małecki b, Anna Mrozek-Wilczkiewicz a, Robert Musioł a, , Jarosław Polanski
⇑
´
a Department of Organic Chemistry, Institute of Chemistry, University of Silesia, 9th Szkolna St., 40-006 Katowice, Poland
b Department of Crystallography, Institute of Chemistry, University of Silesia, 9th Szkolna St., 40-006 Katowice, Poland
h i g h l i g h t s
"
"
"
"
We have synthesized 4 thiosemicarbazones as possible anticancer agents.
Their structures were studied by X-ray, DFT calculations, NMR, MS and IR.
H-bonding, planarity distortion and electronic properties are discussed.
These features may influence their biological activity.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 June 2012
Received in revised form 4 October 2012
Accepted 23 November 2012
Available online 13 December 2012
Series of four benzaldehyde thiosemicarbazones has been synthesized under microwave irradiation and
characterized structurally by means of infrared and NMR spectroscopies and mass spectrometry. Their
crystal structures were determined by single crystal X-ray analysis followed by DFT calculations. Partial
charges on the molecular surface and dipole moments of the structures were calculated. Crystal struc-
tures are stabilized by intramolecular hydrogen bonding and stacking interactions. Studied compounds
are interesting as antiproliferative and antifungal agents acting through interactions with iron. Thus pre-
sented results may be useful in design new more active or specific structures.
Keywords:
Microwave synthesis
Aromatic thiosemicarbazones
Crystal structure
Ó 2012 Elsevier B.V. All rights reserved.
DFT calculations
1. Introduction
atoms within the same ligand multiplying coordination modes
and affects the properties of ligands and complexes [12,13]. On
Thiosemicarbazones are a class of compounds of great pharma-
ceutical value. Probably most important potential use of them is
connected with their anticancer activity [1–3]. Although they have
been reported also as potent antimicrobial, antiviral and anticon-
vulsant agent [4–6,7], there are plethora of different mechanisms
according of which the thiosemicarbazones exerting the influence
on biological systems. Four different mechanisms of action for anti-
cancer activity of such compounds has been suggested, as reactive
oxygen species (ROS) formation [1,8] inhibition of cellular iron up-
take and mobilization of iron stored within the cells [1,9], and inhi-
bition of ribonucleotide reductase [10]. Different or non-specific
mechanisms may be drawn for antifungal or antimicrobial activity.
Apparently this variety has common prerequisite laying beneath.
Thiosemicarbazones are especially effective iron chelators through
their N,N,S-donor character [11]. The presence of multiple donor
the other hand due to these unique structural features thiosemi-
carbazones exist in equilibrium of various tautomers or conform-
ers which greatly affect their chelating ability [14,15]. Detailed
knowledge on this phenomena is the requirement for design new
tailor made molecules.
In our laboratory some works on novel active thiosemicarba-
zone based chelators are running. During this project we utilize
microwave assisted synthesis for facilitating the wet chemistry
part of work. This methodology gives an opportunity to synthesize
large libraries of compounds which could be tested in vitro. The
advantages of this method are short reaction time, absence of cat-
alyst or toxic solvents, higher yield and selectivity. Purification
process could be also simplified as less side products are obtained
(no column chromatography is required) [16,17].
Nevertheless more detailed on arrangement and specificity of
the complexes with various metals, hydrogen bonding formation
are needed. In our current work, four stable thiosemicarbazones
were synthesized using microwave irradiation (Fig. 1). These
⇑
Corresponding author. Tel.: +48 032 3591514; fax: +48 032 2599978.
E-mail address: robert.musiol@us.edu.pl (R. Musioł).
0022-2860/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2012.11.050

64
M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
products were characterized by IR spectroscopy, (Fig. 2) mass spec-
trometry and 1H and 13C NMR spectroscopy. The crystal structures
of 1, 2, 3 and 4 (Fig. 1) were determined by single crystal X-ray
structural analysis, followed by DFT calculations.
MP: 226–227 °C
HR-MS(EI): 246.9745 (calc. for C8H7Cl2N3S: 246.9738)
IR: 3391, 3251 (
C@N), 1470 (dCH/NH), 1291 (
1069 ( NH), 1031 ( ring), 938 (dCH), 868, 823 (
UV–Vis (methanol; log
m
NH), 3155 (
mPhH), 2990 (mCH), 1593 (dNH2), 1540
(m
m
NAC/dNH), 1129 (
mN–N), 1109 (
xNH),
q
m
mCS), 625 (dNACAN).
e): 324.0 (4.00), 238.0 (3.61), 204.0 (3.94)
2. Experimental section
2.1.2. (E)-2-(2,3-dichlorobenzylidene)-N-ethylhydrazinecarbothioamide
Yield: 67%
2.1. Synthesis
1H NMR (d6-DMSO, 400 MHz, ppm): 11.70(s, 1H, NH), 8.70(m,
1H, NH), 8.49(s, 1H, CH), 8.31(d, J = 7.9 Hz, 1H, ArH), 7.67(d,
J = 7.9 Hz, 1H, ArH), 7.41(t, J = 7.9 Hz, 1H, ArH), 3.70–3.52(m, 2H,
CH2), 1.16(t, J = 7.0 Hz, 3H, CH3).
Equimolar quantities of an appropriate thiosemicarbazide and
benzaldehyde derivative were mixed and 2 drops of glacial acetic
acid were added. The resulting mixture was heated in a microwave
reactor at 85 °C for 10 min (max microwave power 50 W). After
cooling, the precipitated solid were filtered and washed with ether
and crystallized from ethanol. The melting point were recorded on
Optimelt automated system from SRS and are uncorrected. High
resolution mass spectroscopy were measured on Finnigan MAT95
spectrometer.
13C NMR (d6-DMSO, 100 MHz, ppm): 177.4, 137.6, 134.4, 132.5,
131.5, 131.3, 128.5, 126.02, 38.9, 14.7
MP: 213–214 °C
HR-MS(EI):275.0054(calc. for C10H11Cl2N3S: 275.0051)
IR: 3349 (
1520 (dCH), 1452 (dCH/NH), 1416 (dCH3), 1311 (dCNH), 1264 (mNAC
dNH), 1160 ( N–N), 1108 ( NH), 1042 ( NH), 929, 906 (dNCS), 782
CS), 737, 704 (ring), 626 (dNACAN). UV–Vis (methanol; log ):
323.0 (3.96), 237.0 (3.67), 204.0 (3.91).
mNH), 3142 (mPhH), 2983 (mCH), 1584 (mC@N), 1542 (mC–N),
/
m
x
q
2.1.1. (E)-2-(2,3-dichlorobenzylidene)hydrazinecarbothioamide
Yield: 78%
(m
e
1H NMR (d6-DMSO, 400 MHz, ppm): 11.69 (bs, 1H, NH), 8.49(s,
1H, CH), 8.36(s, 1H, NH), 8.30(d, J = 8.0 Hz, 1H, ArH), 8.18(bs, 1H,
NH), 7.66(d, J = 7.9 Hz, 1H, ArH), 7.38(t, J = 8.0 Hz, 1H, ArH).
13C NMR (d6-DMSO, 100 MHz, ppm): 178.8, 138.2, 134.2, 132.6,
131.6, 131.2, 128.5, 126.5.
2.1.3. (E)-2-(3,4-dichlorobenzylidene)hydrazinecarbothioamide
Yield: 85%
1H NMR (d6-DMSO, 400 MHz, ppm): 11.56 (s, 1H, NH), 8.28 (s,
1H, NH), 8.24 (d, J = 1.7 Hz, 1H, ArH), 8.00 (s, 1H, CH), 7.72 (dd,
J = 8.4, 1.8 Hz, 1H, ArH), 7.64 (d, J = 8.3 Hz, 1H, ArH).
13C NMR (d6-DMSO, 100 MHz, ppm):178.7, 139.9, 135.6, 132.3,
132.3, 131.2, 128.7, 128.2.
MP: 205–206 °C
IR: 3395, 3256 (
C@N), 1471 (dCH/NH), 1291 (
1065 ( NH), 1031 ( ring), 938 (dCH), 870, 823 (
UV–Vis (methanol; log ): 320.0 (4.01), 239.0 (3.63), 203.0 (3.92)
m
NH), 3154 (
m
PhH), 2992 (
m
CH), 1596 (dNH2), 1538
N-N), 1100 ( NH),
CS), 625 (dNACAN).
(m
m
NAC/dNH), 1129 (
m
m
x
q
m
e
2.1.4. (E)-2-(4-bromobenzylidene)-N-ethylhydrazinecarbothioamide
Yield: 78%
1H NMR (d6-DMSO, 400 MHz, ppm): 11.47 (s, 1H, NH), 8.61 (t,
J = 5.8 Hz, 1H, NH), 8.02 (s, 1H, CH), 7.77 (d, J = 8.5 Hz, 2H, ArH),
7.61 (d, J = 8.5 Hz, 2H, ArH), 3.63–3.56(m, J = 7.0 Hz, 2H, CH2),
1.15 (t, J = 7.1 Hz, 3H, ArH).
13C NMR (d6-DMSO, 100 MHz, ppm):177.2, 140.9, 134.1, 132.0,
129.6, 123.4, 38.8, 15.0.
1
2
MP: 199–200 °C
HR-MS(EI): 284.9944 (calc. for
284.9935)
IR: 3363 (
1522 (dCH), 1485 (dCH/NH), 1397 (dCNH), 1293 (
N), 1104 ( NH), 1081 ( NH), 1066 ( NH), 1006 (
816 ( CS), 623 (dNACAN). UV–Vis (methanol; log ): 330 (sh), 319.5
C10H12BrN3S exact mass:
m
NH), 3139 (
m
PhH), 2980 (
m
CH), 1589 (
NAC/dNH), 1158 (mN–
ring), 921 (dCH),
mC@N), 1538 (mC–N),
m
x
x
q
m
m
e
(3.98), 233.0 (3.65), 218 (sh), 202.5 (3.91)
2.2. DFT calculations
The calculations were carried out by using Gaussian09 [18] pro-
gram. The DFT/B3LYP [19,20] method was used for the geometry
optimization and electronic structure determination. The calcula-
tions were performed using the polarization functions for all
atoms: 6-31Gꢀꢀ [21] – carbon, nitrogen, oxygen, halogens (Cl, Br)
and hydrogen. The contribution of a group to a molecular orbital
was calculated using Mulliken population analysis. GaussSum 2.2
[22] was used to calculate group contributions to the molecular
orbitals and to prepare the density of states (DOS). The DOS spectra
were created by convoluting the molecular orbital information
3
4
Fig. 1. Structures of synthesizes benzaldehyde thiosemicarbazones.

M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
65
with Gaussian curves of unit height and FWHM (Full Width at Half
Maximum) of 0.3 eV. The electronic spectra were calculated by the
time-dependent density functional (TDDFT) [23] method based on
the optimized geometries in the singlet states.
Center in Gliwice, Poland. The cells were grown as monolayer cul-
tures in 75 cm2 flasks (Nunc) in Dulbecco’s modified Eagle’s med-
ium supplemented with 12% fetal bovine serum (Gibco-BRL),
100 lg/mL of gentamicin, 100 lg/mL of streptomycin and 100 IU/
mL of crystalline penicillin (Polfa). Cells were cultured under stan-
dard conditions at 37 °C in a humidified atmosphere at 5% CO2.
Briefly, in cellular proliferation assays, 24 h before addition of the
chelators, the cells were seeded in 96-well plates (3 ꢁ 103 cells/
well). The assay was performed following a 72 h incubation with
varying concentrations of the agents. Each compound was tested
in triplicate in a single experiment with each experiment being re-
peated three times. After a 72 h incubation with the investigated
2.3. Crystal structure determination and refinement
The colorless crystals of 1, 2, 3 and 4 were mounted in turn on
an Xcalibur, Atlas, Gemini ultra-Oxford Diffraction automatic dif-
fractometer equipped with a CCD detector for data collection.
X-ray intensity data were collected with graphite monochromated
Mo Ka radiation (k = 0.71073 Å) at temperature of 295.0(2) K, with
compounds, 20
(100 L) and incubated for 1 h at 37 °C. The optical densities of
the samples were analyzed at 490 nm. Results were expressed as
a percentage of the control. The inhibitory concentration (IC50
lL of MTS solution was added to each well
x
scan mode. Ewald sphere reflections were collected up to
l
2h = 50.10. The unit cell parameters were determined from least-
squares refinement at the setting angles of 1262, 1944 and 1798
strongest reflections for compounds 1, 2 and 3 respectively. Details
of crystal data and refinement are gathered in Table 1. During the
data reduction, the decay of the correction coefficient was taken
into account. Lorentz, polarization, and numerical absorption cor-
rections were applied. The structures were solved by direct meth-
od. All the non-hydrogen atoms were refined anisotropically using
full-matrix, least-squares technique on F2. All the hydrogen atoms
were found from the difference of the Fourier synthesis after four
cycles of anisotropic refinement, and refined as ‘‘riding’’ on the
adjacent atom with individual isotropic temperature factor equal
to 1.2 times the value of equivalent temperature factor of the par-
ent atom, with geometry idealization after each cycle. The Olex2
[24] and SHELXS97, SHELXL97 [25] programs were used for all
the calculations. Atomic scattering factors were those incorporated
in the computer programs.
)
was defined as the compound concentration necessary to reduce
the absorbance to 50% of the untreated control.
3. Results and discussions
3.1. Crystal structures
The studied compounds crystallize in monoclinic P21/c and tri-
clinic P1 space groups for compounds 1, 2 and 3, 4 respectively.
The molecular structures of the thiosemicarbazones derivatives
are displayed as ORTEP representations in Fig. 1. The atomic num-
bering scheme of all studied molecules was chosen to be identical.
The main part of the investigated structures is the thiosemicarba-
zone skeleton NNC(S)N substituted by benzyl moiety bonded to
terminal nitrogen in @NANHA group and ethyl group at nitrogen
atom of C(S)NA. The phenyl rings are substituted by chlorine in
the positions 2, 3 and 3, 4 in the molecules of 1, 2 and 3 respec-
tively. In the case of compound 4 the para position of benzene is
substituted by bromine. The crystallographic data and the details
2.4. Antiproliferative activity
The human colon cancer cell line HCT116 with wild type p53
were obtained from Maria Sklodowska-Curie Memorial Cancer
Table 1
Selected details of the experimental diffraction data collections and refinements.
1
2
3
4
Empirical formula
Formula weight
C8H7Cl2N3S
248.14
C10H11Cl2N3S
276.18
C8H7Cl2N3S
248.14
C10H12BrN3S
286.20
Temperature (K)
Crystal system
Space group
Unit cell dimensions
295.0(2) K
Monoclinic
P21/c
295.0(2) K
Monoclinic
P21/c
295.0(2) K
Triclinic
P ꢂ 1
295.0(2) K
Triclinic
P ꢂ 1
a (Å)
b (Å)
c (Å)
a
12.7143(9)
5.6203(3)
16.0382(9)
90
109.046(7)
90
1083.34(12)
4
1.521
6.2674(12)
15.346(3)
13.304(2)
90
90.017(15)
90
1279.5(4)
4
1.434
5.8447(3)
7.7316(5)
12.6901(7)
96.330(5)
95.412(4)
106.992(5)
540.22(5)
2
6.5767(5)
7.8801(5)
11.8484(9)
83.220(6)
87.389(5)
89.906(6)
609.12(8)
2
b
c
Volume (Å3)
Z
Calculated density (Mg/m3)
Absorption coefficient (mmꢂ1
F(000)
1.519
0.756
252
1.555
3.518
288
)
0.753
504
0.647
568
Crystal dimensions (mm)
h range for data collection (°)
Index ranges
0.27 ꢁ 0.22 ꢁ 0.07
3.39–25.04
ꢂ14 6 h 6 15
ꢂ6 6 k 6 6
ꢂ19 6 l 6 19
8212
0.26 ꢁ 0.04 ꢁ 0.03
3.42–25.05
ꢂ7 6 h 6 7
ꢂ18 6 k 6 18
ꢂ15 6 l 6 15
8774
0.19 ꢁ 0.13 ꢁ 0.06
3.42–25.04
ꢂ6 6 h 6 6
ꢂ9 6 k 6 9
ꢂ15 6 l 6 15
4967
0.31 ꢁ 0.12 ꢁ 0.07
3.47–25.05
ꢂ7 6 h 6 7
ꢂ9 6 k 6 8
ꢂ14 6 l 6 14
4547
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
1921 [R(int) = 0.0296]
1921/0/127
1.082
2264 [R(int) = 0.0637]
2264/0/146
1.059
1903 [R(int) = 0.0248]
1903/0/139
1.052
2151 [R(int) = 0.0277]
2151/0/145
1.045
Final R indices [I > 2
r
(I)]
R1 = 0.0328
wR2 = 0.0815
R1 = 0.0418
wR2 = 0.0865
0.235 and ꢂ0.271
R1 = 0.0586
wR2 = 0.1403
R1 = 0.1066
wR2 = 0.1690
0.341 and ꢂ0.375
R1 = 0.0471
wR2 = 0.1133
R1 = 0.0699
wR2 = 0.1234
0.440 and ꢂ0.367
R1 = 0.0398
wR2 = 0.0768
R1 = 0.0738
wR2 = 0.0865
0.335 and ꢂ0.333
R indices (all data)
Largest diff. peak and hole

66
M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
10.25° in 3 and 25.15° in compound 4, which show distortion of
planarity. All bond distances and angles as well as the distortion
of planarity are consistent with those in similar compounds found
in Cambridge Structural Database (CSD; ConQuest v. 1.14; 2012).
Nevertheless some subtle differences may be found when certain
geometrical parameters are examined. The C@S bond lengths in
the molecules are slightly different (ꢃ0.01 Å) in the 1, 2 and 3, 4
compounds, indicating the slightly more double bond character
of thione in the compounds 2 and 4. Additionally thiosemicarba-
zone 4, containing a Br atom, are much more distorted when com-
pared with the Cl substituted compounds and the distortion is
connected with impact of bromine substituent on the electronic
density distribution in this compound. In the structures of the
compounds the thione sulfur atoms S(1) are trans to the azometine
nitrogen atoms N(1). The thiosemicarbazones have NAH groups
capable of acting as H-donors and sulfur and nitrogen atoms
capable acting as H-acceptors in hydrogen bonds. In the structures
of the compounds the N(3)AH(3)AN(1) intramolecular hydrogen
bonds exist. Moreover the compounds
1
and
2
have
C(7)AH(7)ꢄ ꢄ ꢄCl(1) intramolecular bond (see Table 2). Besides the
NAHꢄ ꢄ ꢄS intermolecular bonds form centrosymmetric dimmers
and one-dimensional network in a plane as is presented on the
Fig. 2. Experimental and calculated IR spectra of compound 3.
Fig. 3. In the structure of compound 4 the
p-stacking interaction
between the benzene rings is visible. The distance between the
molecules in positions (x, y, z) and (1 ꢂ x; 1 ꢂ y; 2 ꢂ z) of 3.720 Å
and shift distance 0.424 Å indicates rather week interactions. In
the compounds 2 and 4 with aliphatic substituent at N(3) atom
some interaction between ethyl group and benzene ring exist as
shown Fig. 4. The distances in the T-shaped interaction are in the
range 2.89–3.20 Å which suggest strong CAHꢄ ꢄ ꢄCbenz interaction.
of the X-ray analysis of 1, 2, 3 and 4 are given in Table 1 and se-
lected bond lengths and angles with hydrogen bonds in Table 2.
In the compounds the AC@NANACA chain separating the dou-
ble-bond S atom and the benzene ring adopts an extended zigzag
conformation. The angles between the planes of phenyl ring and
thiosemicarbazone part (NNC(S)N) are 12.33° in 1, 4.53° in 2,
Table 2
Selected bond lengths and angles for compounds 1, 2, 3 and 4 with hydrogen bonds (Å and °).
Bond lengths (Å)
1
2
3
4
Exp
Calc
Exp
Calc
Exp
Calc
Exp
Calc
C@S
1.688(2)
1.271(3)
1.368(2)
1.346(2)
1.312(2)
1.675
1.290
1.352
1.377
1.349
1.675(5)
1.282(4)
1.362(4)
1.360(5)
1.311(5)
1.470(6)
1.736(4)
1.729(4)
1.678
1.291
1.349
1.385
1.344
1.461
1.747
1.751
1.694(3)
1.268(4)
1.372(3)
1.341(3)
1.306(4)
1.675
1.289
1.352
1.377
1.349
1.675(3)
1.272(4)
1.364(3)
1.358(4)
1.313(4)
1.446(4)
1.884(3)
1.679
1.289
1.352
1.384
1.344
1.461
1.914
N(1)AC(7)
N(1)AN(2)
N(2)AC(8)
N(3)AC(8)
N(3)AC(9)
X(1)AC
1.723(2)
1.730(2)
1.751
1.747
1.726(4)
1.729(3)
1.743
1.746
Cl(2)AC
Bond angles (°)
C(7)AN(1)AN(2)
C(8)AN(1)AN(2)
N(1)AC(7)AC(1)
N(3)AC(8)AN(2)
N(3)AC(8)AS(1)
N(2)AC(8)AS(1)
116.41(16)
119.96(16)
120.11(18)
117.16(18)
123.78(15)
119.07(14)
117.5
122.3
121.4
115.1
124.9
119.9
117.5(3)
120.2(3)
119.4(4)
115.4(4)
125.1(3)
119.5(3)
117.7
122.3
121.3
115.1
125.8
119.0
115.6(2)
120.3(2)
122.4(3)
118.0(3)
123.2(2)
118.8(2)
117.7
122.1
122.2
115.1
124.9
119.1
116.8(3)
119.2(3)
121.1(3)
116.5(3)
123.7(3)
119.8(3)
117.9
122.3
122.6
115.1
125.8
119.1
Hydrogen bonds
DAHꢄ ꢄ ꢄA
d(DAH)
d(Hꢄ ꢄ ꢄA)
d(Dꢄ ꢄ ꢄA)
<(DHA)
1
N(3)AH(3A)ꢄ ꢄ ꢄN(1)
C(7)AH(7)ꢄ ꢄ ꢄCl(1)
2
0.86
0.93
2.27
2.64
2.628(2)
3.028(2)
104.8
106.1
N(2)AH(2)AS(1) #2
N(3)AH(3)AN(1)
C(7)AH(7)ꢄ ꢄ ꢄCl(1)
3
0.86
0.86
0.93
2.57
2.22
2.65
3.396(4)
2.610(5)
3.046(4)
161.8
107.5
106.1
N(2)AH(2)AS(1)
N(3)AH(3A)AN(1)
N(3)AH(3B)AS(1) #1
4
0.86
0.80(3)
0.86(3)
2.56
2.32(3)
2.53(3)
3.369(2)
2.650(4)
3.377(3)
156.2
105(2)
169(3)
N(2)AH(2)AS(1) #3
N(3)AH(3)AN(1)
0.86
0.86
2.56
2.22
3.390(3)
2.613(4)
163.5
107.8
X denotes Cl or Br.
Symmetry transformations used to generate equivalent atoms: #1: 1 ꢂ x, 1 ꢂ y, 2 ꢂ z; #2: 2 ꢂ x, 2 ꢂ y, 1 ꢂ z; #3: 2 ꢂ x, 1 ꢂ y, 3 ꢂ z.

M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
67
Fig. 3. The molecular structures of compounds 1–4 with anisotropic displacement parameters shown at the 50% probability level.
3.2. Infrared spectra characterization
3.3. NMR spectra
Infrared spectra of the compounds are similar and present char-
acteristic bands due to moiety of studied molecules. The NH
stretching vibration on the IR spectra of the compounds present
strong bands in the range of 3349–3395 cmꢂ1. In the spectra of
compounds 1 and 3 the bands are split into two bands with max-
ima at 3395, 3391 cmꢂ1 and 3256, 3251 cmꢂ1 due to symmetric
and antisymmetric stretching vibrations of NH2 groups. The dNH2
bands in 1 and 3 are presented at 1593 cmꢂ1 and 1596 cmꢂ1
respectively. The NH rock and wagging modes are observed in
As the aromatic thiosemicarbazones can be considered as Schiff
bases they could exist as E and Z isomeric forms or as a mixture of
both isomers. In some cases the presence of selected isomer,
mainly Z isomer, could be promoted by intramolecular hydrogen
bonding, especially connected with pyridine rings [26,27]. In other
cases the steric hindrance is a main factor in equilibrium between
isomers. The most important signal in the process of describing the
correct stereoisomer is that connected with NH group. Compounds
possessing the Z isomer generally exhibit the NH signal in the 14–
15 ppm range, whereas those having an E form exhibit the signal in
the 9–12 ppm range. The 1H NMR of all synthesized compounds
shows a singular peak around 11.50 ppm relative to the NH signal
which indicates E isomer which is additionally confirmed by X-ray
structure and DFT calculations.
the region 1109–1081 cmꢂ1and 1069–1042 cmꢂ1
. The out-of-
plane wagging of NH is moderately active with a band in the re-
gion of 625 cmꢂ1. The bands with maxima close to 1129 cmꢂ1 in
spectra of compounds 1, 3 and 1160 cmꢂ1 in the case of 2 and 4
are assigned to NAN stretching mode. The IR spectra of the
reported thiosemicarbazones show bands in the region 1538–
1589 cmꢂ1 as stretching C@N groups. The CAN stretching
vibration coupled with the dNH is active in the region 1264–
1291 cmꢂ1 in studied compounds. The mCS bands have maxima at
870, 823 cmꢂ1 and 782, 816 cmꢂ1 in compounds 1, 2, 3 and 4
respectively. The infrared spectra present bands connected with
vibrations of aryl and alkyl parts of the thiosemicarbazone mole-
cules. Fig. 2 presents, as example the experimental and calculated
spectra of compound 3.
3.4. Quantum chemical calculations
To get an insight in the electronic structures and of the com-
pounds, the calculations in the DFT method at the B3LYP/6-31Gꢀꢀ
level were carried out. Before the calculations of electronic struc-
tures of the studied compounds, their geometries were optimized
in singlet states using the DFT method with the B3LYP functional.
For each compound a frequency calculation was carried out,

68
M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
Fig. 3. (continued)
verifying that the optimized molecular structure obtained corre-
sponds to energy minimum, thus only positive frequencies were
expected. In general, the predicted bond lengths and angles are
in a good agreement with the values based on the X-ray crystal
structure data, and the general trends observed in the experimen-
tal data are well reproduced in the calculations.
moment. The high dipole moment can be explained by stronger
electrodonating effects of chloride substituents in 2, 3 positions
in phenyl ring than 3,4 ones (1 vs 3) and influence of alkyl moiety
in thiourea part of molecule (1 vs 2).
The electronic structures of the compounds are similar each
other. The partial density-of-states (DOS) in terms of Mulliken pop-
ulation analysis were calculated using the GaussSum program.
They provide a pictorial representation of MOs compositions and
their contributions to chemical bonding. The DOS diagram for com-
pound 2 as example is shown in Fig. 7. The DOS plot mainly pre-
sents the composition of the fragment orbitals contributing to
the molecular orbitals. The molecules of studied compounds were
divided into the aromatic, thiosemicarbazone and aliphatic frag-
ments. The energies of highest occupied and lowest virtual molec-
ular orbitals of the compounds are negative and close to ꢂ6.15 eV
(HOMO) and ꢂ2.50, eV (LUMO) for 1, 3 and ꢂ6.03, ꢂ5.96 and
ꢂ2.41, ꢂ2.22 for 2, 4 respectively. Therefore the less negative en-
ergy levels and slightly bigger HOMO–LUMO gap in compound 4
suggest strongest acceptor affinity of the compound compared
with the others. Moreover the values of HOMO–LUMO gaps vary
between 3.62 eV to 3.74 eV which points the compounds as hard
bases. As one can see from the Fig. 6 the HOMO and HOMO-1 orbi-
tals are quasi-degeneracy of the energetic levels and composed
The atomic charge calculations can give a feature for the reloca-
tion of the electron density of the compounds, but the local con-
centration and local depletion of electron charge density allow us
to determine whether the nucleophile or electrophile can be at-
tracted. Fig. 6 presents the plots of the electrostatic potentials for
the studied compounds. The isoelectronic contours are plotted at
0.05 a.u. (31 kcal/mol). The color code of these maps is in the range
between 0.05 a.u. (deepest red) to 0.005 a.u. (deepest blue) in all
compounds, where blue indicates the strongest attraction and
red indicates the strongest repulsion. Regions of negative potential
are usually associated with the lone pair of electronegative atoms.
The negative potential in the studied compounds wraps the sulfur
and nitrogen atoms and as one can see in Fig. 5, negative potentials
on halogen atoms in the studied compounds are considerably
lower than the ones on S or N atoms. The dipole moments of the
compounds, calculated in metanolic solutions are also presented
in Fig. 5 and one can see that the compound 2 has highest dipole

M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
69
1
3
Fig. 4. A view of part of the crystal structures of compounds 1 and 3 in which the intra- and inter-molecular hydrogen bonds produced the one-dimensional polymeric chain.
from the phenyl ring and NNCS part of carbazone. The LUMO is also
localized on the phenyl (65%) with admixture of pꢀ orbitals of
carbazone (35%).
1 to 4 respectively. In the case of compound 1 in the Z isomers
the CACl bonds are elongated by about 0.07 Å while the XAC bonds
in the compounds 2–4 are longer in the Z isomers by about 0.01 Å.
The C@S bond lengths in the Z isomers are also longer by 0.03 Å
compared with E forms. Generally the E forms of the compounds
are more stable than Z isomers but taking into account its chelating
ability the compound 4 seems to be the best chelator.
Due to similar electronic structures of the compounds theirs
electronic spectra are also very similar. The electronic spectra were
calculated with the TD-DFT method with methanol as solvent in
the polarizable continuum model (PCM) at B3LYP/6-31Gꢀꢀ level.
The first experimental band with maximum near 320 nm is con-
nected with the HOMO ? LUMO and H ꢂ 1 ? LUMO transitions.
In the bands with maxima close to 230 nm the transitions between
H ꢂ 4 ? LUMO (70%) and HOMO ? L + 3 (64%) were calculated.
The highest energy band at 203 nm is contributed from the transi-
tions between H ꢂ 2 ? L + 3 (59%) and H ꢂ 2 ? L + 4 (54%). Taking
into account the electronic structures of the compounds it is not
3.5. Biological activity determination
The antiproliferatrive activity of the all studied compounds
have been measured in MTS assay against human colon cancer
cells (HCT116), with normal expression of apoptotic gene p53
(wild type). Compounds 1–3 appeared to be moderately active
wonder that the p-aromatic electrons are engaged into transitions.
with activity: 1. IC50 = 41.5 1.7
IC50 = 46.0 0.9 M. Compound 4 can be regarded as inactive due
to IC50 exceeding the level 60 M and its solubility in the testing
lM; 2. IC50 = 58.5 0.2 lM; 3.
In the longest wavelength band transitions from the thiosemicar-
l
bazone to phenyl pꢀ play role and at higher energy bands the
l
pPh
!
pꢀPh are visible.
medium. Although the limited number of structures make it
impossible to draw more general conclusions some interesting re-
marks can be done. At a first glance can be seen that free amino
group N(3)H2 is crucial for the activity. On the other hand one
bromine atom instead of two chlorine deprived the activity of
compound 4. Furthermore activity can be correlated somewhat
with the bond length of C@S group (Table 2). Two most active
Additionally some conformational analyses are performed. Tak-
ing into account the possible biological activity of these com-
pounds especially as metal chelators their conformational
stability effects were estimated. The energies of Z isomers (formed
by rotation about N(2)AC(8) bonds are higher by 142 kcal/mol,
138 kcal/mol, 111 kcal/mol and 25 kcal/mol form compounds

70
M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
2
4
Fig. 5. The stacking interactions in structures of 2 and 4.
compounds 1 and 3 have the lowest bond lengths (1.675 Å) com-
pound 2 which is less active has length 1.678 Å and the inactive
4 has the highest length 1.679 Å. These observations seems to be
in contrary of the stated above high potency ability but are in
agreement with our former results [28] where we show that ability
to mobilize Fe ions from cells is the most important factor.
Synthetic method that is proposed allows obtaining various
thisemicarbazones from readily available cheap chemicals in sim-
ple one step procedure. The yields were satisfactory exceeding
75%. From the synthetic point of view the most positive aspect of
the proposed method is short time and thus relatively low pres-
ence of side products. Synthesized compounds were pure (HPLC
purity over 98%) after crystallization.
4. Conclusions
As expected all compounds are E isomers according to 1H NMR
spectroscopy. However all presented compounds are very conge-
neric their crystal structures and DFT calculations suggest some
subtle differences. Due to important biological activity of thiosem-
icarbazone these observations may be important for medicinal
chemist. For thiourea part of the structure the substitution pattern
at N(3) amine group is crucial e.g. C@S is shortened for 2 and 4.
In present work we obtained by microwave assisted synthesis
four new thisemicarbazones based on benzaldehyde. Structures
of these compounds have been studied by means of NMR spectros-
copy, single crystal X-ray diffraction method. Their electronic prop-
erties were calculated in DFT method.

M. Serda et al. / Journal of Molecular Structure 1037 (2013) 63–72
71
µ= 4.91 D
µ= 4.10 D
1
3
µ= 7.06 D
µ= 4.66 D
2
4
Fig. 6. Plots of the electrostatic potentials for the 1–4 compounds. The isoelectronic contours are plotted at 0.005 a.u. (3.1 kcal/mol).
chelates. Our results on biological activity of the studied
compounds will hopefully elucidate the impact of abovementioned
features on their possible mechanism of action.
Appendix A. Supplementary data
CCDC 857492 (1), CCDC 825813 (2), CCDC 825885 (3), and CCDC
825886 (4) contain the supplementary crystallographic data for
this paper. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cam-
bridge Crystallographic Data Centre, 12, Union Road, Cambridge
CB2 1EZ, UK; fax: +44 1223 336033).
Acknowledgments
M.S. was supported by a TWING fellowship and NCN Grant
(DEC-2011/01/N/NZ4/01166).
The GAUSSIAN09 calculations were carried out in the Wrocław
Centre for Networking and Supercomputing, WCSS, Wrocław,
Poland (http://www.wcss.wroc.pl Grant Number 18).
Fig. 7. The partial density-of-states plot diagram for compound 2.
Electronegativity of S(1) atom plays vital role in chelation of metal
ions by thiosemcarbazones – the base of their mechanism of
antiproliferative and anticancer action. Although all NAH are good
H-bond donors and N, S are H-bond acceptors there are differences
in dipole moments of the studied compounds. Structure 3 quite
unexpectedly has the largest moment significantly exceeding the
moments of 1, 2 and 4. On the other hand 1 and 2 have intramolec-
ular hydrogen bonds which can prevent the formation of stable
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