Synthesis, Structure, and Cytotoxicity of a New Sulphanyl-Bridged Thiadiazolyl-Saccharinate Conjugate: The Relevance of S???N Interaction

Article abstract of DOI:10.1002/chem.201705319

Reports showing that the copper concentration is considerably higher in neoplasms than in normal tissues prompted the need to develop selective copper chelators. We disclosed recently that some N-linked tetrazole-saccharinates bind selectively to copper, forming complexes that are highly cytotoxic towards cancer cells. Because tetrazole-saccharinates are photolabile, due to the photoreactivity of tetrazoles, we proposed thiadiazolyl-saccharinates as an alternative. Herein we describe the synthesis, structure, and monomeric photochemistry of a sulphanyl-bridged thiadiazolyl-saccharinate, 3-[(5-methyl-1,3,4-thiadiazol-2-yl)sulphanyl]-1,2-benzothiazole 1,1-dioxide (MTSB). The monomeric structure, charge density analysis, and characteristic infrared spectrum of MTSB were investigated theoretically, using quantum chemical calculations, and also experimentally, using matrix-isolation infrared spectroscopy. The crystal structure was investigated by combining X-ray crystallography with infrared and Raman spectroscopies. Results show that the structure of isolated MTSB is similar to that found in the crystal, with an S???N interaction clearly contributing to the structure of the molecule and of the crystal. Matrix irradiation revealed a high photostability of MTSB, compared to parent tetrazole-saccharinates and to the 5-methyl-1,3,4-thiadiazole building block, emphasizing the photostabilizing effect of the saccharyl system. Finally, in vitro toxicity assays of MTSB showed a copper concentration-dependent toxicity against cancer cells, without affecting normal cells. In particular, MTSB was most effective towards the hepatic (HepG2), neuroblastoma (SH-SY5), and lymphoma cell lines (U937). Thus, MTSB represents a promising lead for cancer chemotherapy based on chelating agents.

Full text of DOI:10.1002/chem.201705319

A Journal of
Accepted Article
Title: Synthesis, Structure and Cytotoxicity of a Novel Sulphanyl-
Bridged Thiadiazolyl-Saccharinate Conjugate. The Relevance of
S…N Interaction
Authors: Lilia Cabral, Elisa Brás, Marta Henriques, Catia Marques, Luís
Frija, Luísa Barreira, José António Paixão, Rui Fausto, and
Maria Lurdes Cristiano
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To be cited as: Chem. Eur. J. 10.1002/chem.201705319
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10.1002/chem.201705319
Chemistry - A European Journal
Synthesis, Structure and Cytotoxicity of a Novel Sulphanyl-Bridged Thiadiazolyl-
Saccharinate Conjugate. The Relevance of S…N Interaction
e
L. I. L. Cabral,a,b E. M. Brás,c M. S. C. Henriques,d C. Marques,b L. M. T. Frija, L.
Barreira,a,b J. A. Paixão,d R. Fausto,c,* and M. L. S. Cristianoa,b,*
a Department of Chemistry and Pharmacy, F.C.T., University of Algarve, P-8005-039 Faro (Portugal), b Center
c
of Marine Sciences, CCMar, University of Algarve, P-8005-039 Faro (Portugal), CQC, Department of
d
Chemistry, University of Coimbra, P-3004-535 Coimbra (Portugal), CFisUC, Department of Physics,
University of Coimbra, P-3004-516 Coimbra (Portugal), e CQE, Centro de Química Estrutural, IST, University
of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon (Portugal).
*Corresponding authors: R. Fausto, M. L. S. Cristiano
E-mail: rfausto@ci.uc.pt ; mcristi@ualg.pt
Abstract
Reports showing that the copper concentration is considerably higher in neoplasms than in normal
tissues prompted the need to develop selective copper chelators. We disclosed recently that some N-
linked tetrazole-saccharinates bind selectively to copper, forming complexes that are highly cytotoxic
towards cancer cells. Because tetrazole-saccharinates are photolabile, due to the photoreactivity of
tetrazoles, we proposed thiadiazolyl-saccharinates. Herein we describe the synthesis, structure and
monomeric photochemistry of a sulphanyl-bridged thiadiazolyl-saccharinate, 3-[(5-methyl-1,3,4-
thiadiazol-2-yl)sulphanyl]-1,2-benzothiazole 1,1-dioxide (MTSB). The monomeric structure, charge
density analysis and infrared spectroscopy of MTSB were investigated theoretically, using quantum
chemical calculations, and also experimentally, using matrix-isolation infrared spectroscopy. The
crystal structure was investigated by combining X-ray crystallography with infrared and Raman
spectroscopies. Results show that the structure of isolated MTSB is similar to that found in the crystal,
with a S…N interaction clearly contributing to the structure of the molecule and of the crystal. Matrix
irradiation revealed a high photostability of MTSB, compared to parent tetrazole saccharinates and to
the 5-methyl-1,3,4-thiadiazole building block, emphasizing the photostabilizing effect of the saccharyl
system. The toxicity of MTSB was assessed in vitro, towards 6 cancer cell lines and non-tumoral
cells. Results have clearly shown that MTSB is toxic towards cancer cells at concentrations that do
not affect normal cells, the toxicity being dependent on copper concentration. In particular, MTSB
revealed to be most effective towards the hepatic (HepG2), neuroblastoma (SH-SY5) and lymphoma
cell lines (U937). Thus, in addition to its structure predictability and photostability, MTSB is safe
towards normal cells and cytotoxic towards cancer cells, thus representing a promising lead for cancer
chemotherapy based on chelating agents.
Keywords: Saccharinate-based copper chelators; Thiadiazol-sacharin sulphanyl-bridged ligands;
Structural studies; S…N interactions; Photostability; Selectivity; Cu(II)-dependent cytotoxicity.
1
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1. Introduction
Coordination chemistry is very relevant in crucial areas, such as catalysis, supramolecular
chemistry, molecular magnetism, optical materials, environmental remediation and
medicine.1-4 Multidentate ligands may be viewed as the touchstone in coordination chemistry,
because their structure and chemical nature determines the reactivity and function of
coordination structures. In designing ligands, the binding affinity and selectivity towards a
specific metal are instrumental features to consider, especially if applications in medicinal
chemistry are envisaged, where structure-based design must keep pace with the increasing
demand for solutions that provide higher efficacy, while reducing toxicity.
The design of selective ligands for copper has attracted attention is recent years. While
this metal is involved in a variety of vital redox processes in the human body, alterations of
its normal homeostasis are known to promote toxicity through induced formation of reactive
oxygen species (ROS) that may target lipids, proteins or nucleic acids.5-7
It has been reported that the proliferation of cancer cells requires a higher abundance of
Cu(II) than that of corresponding normal cells.8,9 Additionally, it was proposed that the
apoptotic properties of Elesclomol, a drug candidate currently under clinical evaluation for
cancer chemotherapy, arise from its coordination to copper(II), leading to a complex that
promotes formation of ROS in cancer cells.10 This evidence prompted the development of
new copper chelators that can avidly capture copper ions in neoplasms, depriving them from
Cu(II). If, in addition, the redox active copper complexes formed exhibit cytotoxicity,
ultimately leading to harmful reactive oxygen species (ROS) in cancer cells, then the copper
chelators could inhibit tumor growth by a dual mode of action, acting as powerful tools for
cancer chemotherapy. In view of these potential applications, the mandatory properties of
such copper chelators are neglectable cytotoxicity, high binding affinity and selectivity
towards Cu(II), in addition to chemical stability, drug-like properties and easy access through
chemical synthesis.
Saccharin (3-oxo-1,2-benzisothiazole 1,1-dioxide),11 the oldest artificial sweetener
known, and related saccharinates (known as benzisothiazoles), are generally stable and non-
toxic compounds and are also known to act as bridging ligands in coordination chemistry.3,12
Additionally, the saccharyl system is easily linked to other heterocycles by using 3-chloro-
1,2-benzisothiazole 1,1-dioxide (2; Scheme 1) as the saccharyl building block, providing easy
access to a plethora of saccharinate-based conjugates that can be screened as multidentate
ligands.
2
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We recently developed saccharinate-based molecules where the saccharyl system is
linked to a tetrazolyl moiety (1; Scheme 1; R= tetrazolyl), studied their structure and
properties13-15 and uncovered their potential as multidentate nitrogen ligands in coordination
chemistry. Interestingly, it was found that some of the N-linked tetrazole-saccharinates
investigated act readily as selective Cu(II) chelators, over Fe(II) and Ca(II).16 The chelators
and corresponding copper complexes were scrutinized regarding their toxicity towards
various cancer cell lines and also towards non-tumoral cells. Quite surprisingly, the ligands
did not show toxicity but the corresponding copper complexes revealed high toxicity towards
all tumor cell lines tested, with IC50 values at the low micromolar range of concentrations.16
From these preliminary results we selected one compound, 2-methyltetrazole saccharinate,
for deeper investigations of anti-proliferative activity, analysis of cell cycle distribution,
apoptosis induction and oxidative stress levels.
Scheme 1
Because tetrazoles are known to be photolabile (unlike saccharins, known to be relatively
photostable)17,18 we proposed to develop saccharinate-based conjugates incorporating other
heterocycles (Scheme 1) and, considering the properties of thiadiazoles,19 thiadiazolyl
saccharinates appeared to be good candidates.
Herein we describe the synthesis, structure and monomeric photochemistry of 3-[(5-
methyl-1,3,4-thiadiazol-2-yl)sulfanyl]-1,2-benzothiazole 1,1-dioxide (MTSB), a sulphanyl-
bridged thiadiazolyl-saccharinate. The monomeric structure, charge density analysis and
infrared spectroscopy of MTSB were investigated theoretically, using quantum chemical
calculations, and also experimentally, using matrix-isolation infrared spectroscopy. The
crystal structure was investigated by combining X-ray crystallography with infrared and
Raman spectroscopies. The photostability of matrix-isolated MTSB was evaluated. Finally,
its toxicity was assessed in vitro, towards a range of cancer cell lines and non-tumoral cells.
3
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2. Results and discussion
2.1. Synthesis of MTSB
For a convergent synthesis of MTSB, the saccharyl (2) and thiadiazole (4) building blocks
were coupled through nucleophilic displacement of the chloride anion from 2 by the thiol
functionality of 4 (Scheme 2). The reaction proceeded smoothly, affording the crystalline
product in 60 % yield. 3-Chloro-1,2-benzisothiazole 1,1-dioxide (2) was prepared by
halogenation of saccharin, as previously described.17
Scheme 2
2.2. Investigation of the structure of monomeric MTSB
2.2.1. Quantum chemical calculations for the isolated molecule
A deep understanding of the structure of MTSB is instrumental for predicting and
interpreting its reactivity and properties. As such, monomeric MTSB was investigated
theoretically, to determine its preferred conformation. The structures were calculated using
Gaussian 09,20 at the DFT level of theory, with the B3LYP hybrid functional21,22 and the 6-
311++G(d,p) and 6-311++G(3df,3pd) basis sets.23,24 In searching for the relevant minima of
the isolated molecule of the studied compound, the potential energy profile for internal
rotation about the S12–C13 bond (Figure 1) was calculated using the smaller basis set. The
more extended basis set was then used to confirm the nature of the initially identified minima
and for calculation of the geometrical parameters and vibrational spectra, as well as for
undertaking the charge density analysis. The latter was performed according to the theory of
Atoms in Molecules (AIM)25 and the Wiberg bond order analysis,26,27 using the Multiwfn
program (version 3.2).28
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22
17
23
12
16
15
5
13
6
4
3
19
20
18
24
9
7
14
2
21
8
25
1
11
10
I
II
Figure 1. (left) Global minimum on the studied potential energy surfaces of MTSB, I. (right) Higher
energy minimum (note that a structure symmetrically-equivalent to the one represented in the figure
exists; see text), as predicted by the calculations performed with the smaller 6-311++G(d,p) basis set;
II collapses to the global minimum I upon optimization with the larger 6-311++G(3df,3pd) basis set.
2.2.2. Conformational search and geometry of the minimum energy conformation of
MTSB
The preliminary conformational search was undertaken at the B3LYP/6-311++G(d,p)
level of approximation. Structures with the N2–C3–S12–C13 fragment in the trans
conformation were not taken into account since they are highly-sterically-hampered due to
the close proximity of the two ring moieties (thiadiazole and benzothiazole). The calculated
potential energy profile for internal rotation about the S12–C13 bond, shown in Figure 2,
shows 3 minima: a global minimum (I) for C3–S12–C13–N17 equal to 180o and two symmetry-
o
related higher energy minima (II, II’) for C3–S12–C13–N17 equal to 112.3o (and 247.7 )
(Figures 1,2). The higher energy minima stay 2.33 kJ mol–1 above the conformational ground
state, and are predicted to be separated from this latter by a very small energy barrier (0.09 kJ
mol–1). The calculated barrier separating the two symmetrically-equivalent minima amounts
to 26.24 kJ mol–1, the transition state structure corresponding to a C3–S12–C13–N17 dihedral
angle of 0o.
The very small energy barrier separating II/II’ from the global minimum (I) appeared not
realistic and further calculations were performed using the larger 6-311++G(3df,3pd) basis
set. Indeed, when re-optimized with the more extended basis set, the higher energy minima
collapse to the global minimum.
The calculated geometrical parameters for I are provided in Table S1 (Electronic
Supplementary Information; ESI). The most relevant structural feature of the minimum
energy conformation of MTSB is the close proximity of the S14 and N2 atoms, since the
S14…N2 distance is only 2.896 Å, which is considerably shorter than the sum of the van der
Walls radii of N and S (N: 1.55 Å, S: 1.80 Å; N+S = 3.35 Å).
5
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30
25
20
15
10
5
B3LYP/6-311++G(d,p)
II
II'
I
0
0
60
120
180
240
300
360
C3-S12-C13-N17 (º)
Figure 2. B3LYP/6-311++G(d,p) calculated potential energy profile for internal rotation about the
S12–C13 bond for MTSB. When reoptimized using the larger 6-311++G(3df,3pd) basis set, the higher
energy minima (II, II´) collapse to the minimum energy conformer (I).
The structural relevance of S…N contacts has been reported long ago by Fausto, Teixeira-
Dias and Carey,29 in a study on N-formylglycine dithio acid, where a 5-membered ring
involving an S…N contact also exists in the most stable conformer of the compound. For this
compound, the calculated S…N distance was found to be 2.898 Å, in good agreement with the
X-ray data for the distance between the corresponding atoms in a series of crystalline N-
acylglycine dithio esters used as models for enzyme-substrate complexes of cysteine
proteases,30-32 which range from 2.846 to 2.930 Å. In all those compounds, as in MTSB, the
N atom points to the S–R (R= H or C) moiety, with the N…S–R angle being larger than
154.4o (163.5o for MTSB), thus matching the Rosenfield’s et al.33 criterion for a nucleophilic
nitrogen approaching an electrophilic sulphur. According to the literature,29,31,34 the S…N
contact is mainly a -hole chalcogen type interaction (see Figure S1, ESI) involving nitrogen
and sulphur orbitals properly oriented spatially. The detailed analysis of the S14…N2
interaction in MTSB is presented in the next section, based on both the Atoms in Molecules
(AIM) theory25 and the Wiberg bond order analysis.26,27
2.2.3. The S14…N2 interaction as described by the analysis of the electron charge density
(AIM and Wiberg bond order analyses)
Analyses of the charge distribution in the MTSB molecule and of the topological
properties of the electron density and the Laplacian of the electron density allowed us to
understand some of the fundamental structural characteristics of the molecule, in particular
the impact of the S14…N2 contact on the structure.
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A simple and elegant way to estimate the relative strengths of the different bonds in a
molecule is to use the AIM theory approach25 and evaluate the value of the charge density at
the bond critical points of the molecule, then correlating these values with the bond strengths.
In AIM topology analysis language, the points where the gradient norm of a given real space
function value is zero (except at infinity) are called critical points (CP). When the electron
density function () is considered, besides the critical points where all three eigenvalues of
Hessian matrix of the function are negative, which correspond to local maxima and are nearly
coincident with the nuclei, there are three other types of critical points: (i) the bond critical
points (BCP), associated to two negative eigenvalues of the Hessian matrix (second-order
saddle points), and that appear between attractive atom pairs; (ii) the ring critical points
(RCP), exhibiting only one negative eigenvalue of Hessian matrix (first-order saddle point)
and that appear in the center of a ring; and (iii) the cage critical points (CCP), where none of
the eigenvalues of the Hessian matrix of the electron density function are negative (local
minimum) and that are located in the center of a cage system.
The values of real space functions at BCP have great physical significance. For example,
the value of  and the sign of the Laplacian of the electron density (2) at a BCP are closely
related to bonding strength and bonding type, respectively.35,36 The maximal gradient path
linking BCP and associating two local maxima of density is termed as “bond path”, which
reveals the atomic interaction path for all kinds of bonding. The collection of bond paths is
known as molecular graph, which provides an unambiguous definition of the molecular
structure.25
Figure 3 shows the molecular graph for MTSB, where the BCP and RCP are shown,
together with the bond paths. The values of the electron density function () and of the
Laplacian of the electron density (2) at the BCP and RCP are provided in Table 1, together
with the calculated bond lengths and Wiberg bond orders.26,27
Figure 3. Molecular graph for MTSB, built from the 6-311++G(3df,3pd) calculated electron density,
showing the positions of the CPs (BCP shown in orange; RCP in yellow) and the bond paths,
including those relative to the S14…N2 contact, whose bond paths are highlighted in orange. Note also
the existence of a RCP associated with the S12–C13–S14…N2–C3 fragment.
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Figure 4. Plots showing the correlations between atomic distances, Wiberg bond orders, and the
values of the electron density at the associated BCP.
8
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Table 1. Values of the electron density function (ρ) and of the Laplacian of the electron density (2 ρ)
at the BCP and RCP, calculated equilibrium bond distances and Wiberg bond orders.a
Pair of
Bond
Pair of
Bond
atoms
ρ
2 ρ
Distance order
atoms
ρ
2 ρ
Distance
order
C18-H19
C18-H20
C18-H21
C15-C18
N16-C15
N16-N17
S14-C15
C13-N17
S14-C13
S12-C13
C3-S12
C3-N2
0.277 –0.932
0.274 –0.910
0.274 –0.910
0.255 –0.617
0.369 –1.003
0.354 –0.654
0.202 –0.348
0.373 –1.055
0.200 –0.343
0.193 –0.337
0.202 –0.375
0.376 –0.965
0.214 –0.469
0.260 –0.619
1.087
1.090
1.090
1.491
1.300
1.361
1.742
1.299
1.736
1.760
1.739
1.283
1.692
1.487
1.433
1.433
0.91
0.91
0.91
1.11
1.88
1.49
1.32
1.91
1.31
1.23
1.32
1.96
0.97
1.04
1.89
1.89
C5-H22
C5-C4
C9-S1
C4-C9
C5-C6
C8-C9
C6-H23
C6-C7
0.283 –0.982
0.308 –0.853
0.200 –0.367
0.312 –0.862
0.308 –0.855
0.311 –0.870
0.284 –0.985
0.308 –0.859
0.306 –0.848
0.284 –0.991
0.284 –0.985
1.081
1.388
1.785
1.388
1.392
1.379
1.081
1.393
1.394
1.080
1.081
2.868
0.86
1.39
0.83
1.36
1.46
1.45
0.88
1.46
1.45
0.87
0.88
0.15
C7-C8
C8-H25
C7-H24
S14...N2
RThiadiazole
RThiazole
RPhenyl
RS12 13 14
0.017
0.043
0.038
0.022
30.012
0.054
0.291
0.236
0.162
0.054
S1-N2
C3-C4
O10-S1
O11-S1
0.293
0.293
0.969
0.969
…
N C
C
S
2
a Values of the electron density of Laplacian at the CP are in atomic units; bond distances are in Å.
Figure 5. Contour map of the molecular charge density (ρ) in the molecular plane of MTSB, and the
trajectories of the corresponding gradients. Atoms are indicated by the dark orange circles; the points
marked with the blue and orange circles correspond to BCP and RCP. The two unique gradient paths
which originate at infinity or at a ring critical point and terminate at a bond critical point define the
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boundary between a pair of bonded atoms, in the considered plane. Contour values (in atomic units) in
this figure are 2 x 10n, 4 x 10n and 8 x 10n, where n starts at –3 and increases in steps of unity.
Table 2. AIM atomic charges for isolated MTSB molecule obtained from the theoretical calculations,
and multipole and integration atomic charges obtained from the experimentally determined electron
densities for crystalline MTSB.a
Atomic charge
Atomic charge
Atom
AIM
Multipole Integration
Atom
AIM
Multipole
Integration
S1
2.671
–1.084
0.534
–0.038
0.023
–0.003
–0.012
0.039
–0.097
–1.224
–1.224
0.421
0.74(5)
–0.19(6)
–0.21(6)
–0.04(5)
0.02(6)
0.237
–0.16
0.05
–0.02
0.02
S14
0.521
0.424
–0.625
–0.601
–0.008
0.079
0.042
0.042
0.062
0.066
0.056
0.071
0.34(4)
–0.16(6)
–0.10(6)
–0.06(6)
0.03(8)
0.07(4)
0.07(4)
0.07(4)
0.08(8)
0.08(8)
0.17(7)
0.16(8)
0.068
0.03
–0.09
–0.06
0.04
0.05
0.06
0.06
0.02
0.09
0.13
0.13
N2
C3
C4
C5
C6
C7
C8
C9
O10
O11
S12
C15
N16
N17
C18
H19
H20
H21
H22
H23
H24
H25
–0.04(7)
0.06(6)
0.03
–0.01
–0.03
–0.06
–0.31
–0.30
0.057
–0.03
–0.08(7)
–0.17(6)
–0.44(3)
–0.44(3)
0.24(4)
C13
0.314
–0.20(6)
a
Charges are in atomic units (units of e). The MTSB molecules in the studied crystal of the compound have a
non-planar skeleton, so that the pairs of atoms O10/O11 and H20/H21 are not symmetrically related.
Figure 4 presents plots that relate the bond lengths, the bond orders calculated according
to the Wiberg’s method,26,27 and the values of the electron density at the associated BCP.
Figure 5 shows the contour map of the molecular charge density (ρ) in the molecular plane of
MTSB, together with the trajectories of the corresponding gradients, including those
delimiting the basins associated with the different atoms. Atomic AIM charges (obtained by
integration of the charge density within a given basin) are shown in Table 2.
The most relevant result presented in Figures 3,4 and Table 1 is the presence of a BCP
between S14 and N2, which is a clear indication of existence of an attractive interaction
between these two atoms. Also significant is the location of a RCP defining the S12–C13–
S14…N2–C3 quasi-ring, which does also stress the attractive character of the S14…N2 contact.
The value of the electron density at a BCP correlates with the strength of the bond or
attractive interaction between atoms, and this is clearly seen in Figure 4 (mid and bottom
panels), which show plots of ρ (BCP) vs. the atom distances and Wiberg bond-orders.
Interestingly, the value of ρ at the BCP associated with the S14…N2 contact obeys the general
trends exhibited by the values related with the BCP defining the covalent bond in the
molecule, following the tendency previously described by Espinosa et al.,36 who noticed that
there are no discontinuities in the description of this type of correlation when both strong
shared-shell (SS) and weak close-shell (CS) interactions are considered. Also, the value of ρ
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at the BCP associated with the S14…N2 contact is similar to those found for a BCP associated
to an H-bond in H-bonded systems,37 a result that also stresses the structural relevance of the
S14…N2 interaction in the studied molecule.
The values of 2 ρ at the BCP can be correlated with the type of bonding: strong shared-
shell (SS) covalent interactions are generally characterized by a considerable charge
concentration near the BCP and the Laplacian of the electron density function at the critical
point is negative; conversely, for weak closed-shell (CS) interactions a situation of charge
depletion at the BCP occurs, and the Laplacian is positive.36,37 For SS covalent bonds, values
of ρ (in atomic units) of about 0.230 are typical of single bonds while those of ca. 0.340 are
typical of double bonds.38 These trends are clearly evidenced in Table 1, with the Laplacian
being positive for the S14…N2 contact and negative for all covalent bonds, except for the
specific case of the S=O bonds, where the Laplacian at the BCP is positive. Although, as
expected, for the S=O bonds the electron density at the BCP is large, the fact that the
Laplacian is positive indicates that the charge is concentrated in the separate atomic basins
rather than in the internuclear region. Such type of interaction has been termed “intermediate”
between a shared and a closed-shell interaction.37 In an “intermediate”-type interaction, the
BCP is located close to the nodal surface of the Laplacian and, consequently, the bonded
maximum in the valence shell charge concentration of sulphur lies within the atomic basin of
oxygen. This, in turn, is an indication of the charge transfer from sulphur to oxygen, and the
integrated AIM atomic charges of sulphur and oxygen confirm this finding (Table 2). Note,
though, that the transfer of charge is not an indication of an ionic interaction, as ρ at the BCP
has the value of a shared interaction (see Table 1), but rather reflects the polarity of the bond.
Similar trends have been reported before for the SO linkage in sulphinylamines and
sulphinylhydrazines.39
The characterization of the RCP in terms of their ρ, 2 ρ, and integrated total energy
density values provides information on the level of electron delocalization of each of the
rings in the molecule (thiadiazole, thiazole and phenyl) and also of the S12–C13–S14…N2–C3
quasi-ring. The integrated total energy density values for these rings (in atomic units x103)
are, respectively, 7.49, 6.34, 7.54 and 2.47, ordering the rings by increased electron
delocalization character as phenyl ≈ thiadiazole > thiazole >> S12–C13–S14…N2–C3 quasi-ring.
These results are in agreement with the expectations: the phenyl and thiadiazole rings are
aromatic, while the electron delocalization is intermediate in the substituted thiazole ring and
negligible in the S12–C13–S14…N2–C3 quasi-ring. The relative values of both ρ and 2 ρ at the
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RCP accompany the trend of the corresponding energy densities (see Table 1), but in this
case the fact that the phenyl ring has a different number of atoms has to be taken into
account, since the values of ρ and 2 ρ also reflect the extent of steric strain of the ring,
increasing with this property.40 In this regard, the position of the phenyl ring in the series
defined based on the values of ρ and 2 ρ is affected by the fact that it is considerably less
strained. In general, the electron delocalization insights extracted from the AIM topological
analysis are in line with the calculated bond-orders for the bonds of the different rings shown
in Table 1.
2.3. Structure and vibrational spectra of matrix-isolated MTSB monomers
The infrared spectrum of MTSB isolated in solid argon is presented in Figure 6, where it
can be compared with the simulated spectrum generated from the B3LYP/6-
311++G(3df,3pd) calculated data (see also Table S2, ESI, with proposed band assignments).
In the simulated spectrum, the bands are represented by Lorentzian functions centered at the
calculated wavenumbers and with a full-width-at-half-maximum of 2 cm-1. No scaling was
applied to the wavenumbers. Note that the calculated intensities correspond, in the simulated
spectrum, to the areas below the peaks, so that the peak intensities do not correspond to the
values shown in Table S2. The chosen theoretical method and basis set have been shown41 to
provide vibrational frequencies in very good agreement with the experimental data (without
requiring scaling), in particular for the vibrations of the -SO2- fragment, usually difficult to
describe.
From Figure 6, it can indeed be concluded that the B3LYP/6-311++G(3df,3pd)
calculations are able to predict properly the IR spectrum of MTSB. On the other hand,
preliminary calculations performed with the smaller 6-311++G(d,p) basis set were found to
strongly underestimate the frequencies for the modes localized in the -SO2- fragment, in
particular the two stretching vibrations and the scissoring and wagging modes. These results
follow the trends described in previous studies41,42 where the infrared spectra of other
molecules bearing the -SO2- moiety were discussed. It has been shown that the use of highly-
polarized basis sets is necessary to provide the wavefunction the required flexibility to
account properly for properties (including vibrational frequencies) of hypervalent sulphur
containing molecules.
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The spectrum of MTSB is dominated by the bands originating predominantly in the -SO2-
fragment and the C3–S12 bond of the bridging group. The most intense band in the spectrum
is observed as a site-split band with maxima at 1193/1190/1189/1188 cm-1 (calculated 1192
cm-1) and is assigned to the SO2 symmetric stretching. The SO2 anti-symmetric stretching
vibration is predicted at 1379 cm-1 and also observed as a multiplet with major maximum at
1374 cm-1, while the SO2 scissoring (SO2) and wagging (wSO2) modes are predicted at 598
and 566 cm-1, respectively, and observed at 586 and 563 cm-1. According to the calculations,
two vibrations presenting intense IR absorptions have significant contributions from the
C3–S12 bond stretching of the bridging group. These modes are predicted at 1239 cm-1 (also
with a significant contribution from the C3–C4 stretching) and 1005 cm-1, and have
experimental counterparts with major maxima observed at 1237/1236 and 994 cm-1,
respectively.
0.9
B3LYP/6-311++G(3df,3pd) (I)
0.6
0.3
0.0
1600
1400
1200
1000
800
600
0.9
Ar matrix (15 K)
0.6
0.3
0.0
1600
1400
1200
1000
800
600
Wavenumber / cm -1
Figure 6. (Top) Simulated IR spectrum built based on the B3LYP/6-311++G(3df,3pd) calculated data
for I. (Bottom) IR spectrum of the as-deposited argon matrix of MTSB (15 K).
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The features with main maxima observed at 1532/1528 and 779/764 cm-1 (this latter
showing a complex band structure spreading within the position of the two main maxima) are
assigned to C3=N2 and to mixed modes with major contributions from C13–S14, S1–N2 and
S1–C9 stretching coordinates, and have been predicted at 1565 and 776/774 cm-1,
respectively, in good agreement with the experimental values. The complex structure
observed in the band at lower frequency is also related to the contribution to this feature
originated in another, lower intensity mode predicted to occur in the same spectral region and
presenting also major contributions from the S1–N2 and S1–C9 coordinates (see Table S2).
The C13–S12 stretching mode is predicted at 1081 cm-1 and observed at 1090 cm-1. The other,
less intense vibrations have also been predicted well by the calculations, including the N16–
N17 stretching mode of the thiadiazole ring (calculated: 1114 cm-1; observed 1118 cm-1). The
global percent error in frequencies for all bands observed experimentally is as low as 2.6%,
which clearly demonstrates the general quality of the theoretical predictions and ensures a
high degree of confidence to the proposed assignments (see Table S2).
2.4. Assessment of the photostability of matrix-isolated MTSB monomers
The matrix-isolated MTSB was subjected to UV irradiation, performed in situ with
broadband UV light produced by a 500 W Hg(Xe) lamp set up to provide an output of 200 W
at the sample. During the irradiation experiments, the Hg(Xe) lamp was fitted with a water
filter to absorb heat.
The compound was found to be rather photostable, with only 35-40 % of the initial
material being consumed after prolonged (≈3 h) irradiation. The effects of UV irradiation on
matrix-isolated 5-methyl-1,3,4-thiadiazole-2-thione (5MTT), the parent compound of the
thiadiazole fragment of MTSB, has been studied by Rostkowska et al..43 Those authors
reported observation of tautomerization of the most stable thione tautomeric form to a less
stable thiol tautomer of the compound upon irradiation at  > 295 nm. We studied the
photochemistry of 5MTT using the same irradiation conditions used for MTSB and noticed
that, under these conditions, the compound is promptly photolysed (consumption was
achieved after ca. 30 min of irradiation) to CS2, which shall be formed together with N2 and
ethylene. The increased photostability observed for MTSB, when compared to its 5MTT
precursor, then comes in line with recent observations44 that the benzisothiazole ring bound to
other 5-membered heterocycles (e.g., tetrazoles) acts as a photo-stabilizer, at least in
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conditions of matrix-isolation. Noteworthy also is the observation that the sulphanyl-bridged
thiadiazolyl-saccharinate MTSB appears to be considerably more photostable than the
tetrazole saccharinates previously investigated.45
2.5. X-ray structure of the crystalline compound
The molecular structure of MTSB, obtained by single-crystal XRD at 120 K, is shown in
Figure 7, and determined bond lengths, valence and dihedral angles are provided in Table S1,
where they can be compared with the geometrical parameters obtained theoretically for the
isolated MTSB molecule. As it can be seen in Table S1, the experimentally determined bond
lengths and valence angles are in very good agreement with those calculated for the isolated
molecule, with the exception of the S1=O10 bond, which is found to be somewhat longer than
the second S=O bond (S1=O11) and the DFT calculated value. Note that in the isolated
molecule these two bonds are equal due to symmetry (the molecule belongs to the Cs point
group), but in the crystal the symmetry is broken and they become different. Interestingly, as
described below in details, the O10 atom is involved in relevant intermolecular interactions
(including two H-bond-like interactions of C−H…O type), while O11 does not participate in
any similar interaction. The intermolecular interactions involving O10 explain the observed
elongation of the S1=O10 bond compared to the S1=O11 bond, and the observed deviation in
relation to the estimated value for the isolated MTSB molecule.
Figure 7. ORTEP drawing of the MTSB molecule showing that anisotropic displacement ellipsoids
(drawn at the 50 % probability level) at 120 K.
In the crystal, the 5 and 6-membered rings of the fused benzothiazole group are almost
coplanar, the angle between the least-squares plane of the two rings being 1.10(6)º. The
thiadiazole 5-membred ring makes an angle of 9.71(5)º with the least squares plane of the 9-
membered benzothiazole fused rings, showing that the molecule main skeleton is not planar
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in the crystalline state. There is, in fact, a rotation of the thiadiazole ring along the C13−S12
bond, as shown by the torsion angle S14−C13−S12−C3 of −11.14(8)º.
As found for the isolated molecule, the distance between the S14 and N2 atoms
[2.8452(16) Å] in the crystal is also significantly shorter than the sum of the atomic van der
walls radii [3.35 Å], which indicates that the S14…N2 interaction is still present in the crystal,
in spite of the non-planarity of the molecule.
Packing of the molecules in the crystal appears to be dictated by weak intermolecular
…
interactions of the form C−H…O, C−H…N, S=O…Cg and Cg Cg, Cg being the gravity centre
of the 휋 electron cloud of an aromatic ring, as shown in Table 3 and Figures 8 and 9. Atom
O10 acts as a proton acceptor of two C−H…O weak intermolecular hydrogen bonds and is also
involved in one S=O…Cg interaction, whereas atom O11 does not participate in such
interactions. This explains the already noticed asymmetry of the measured S=O bond lengths,
S=O11 being longer [1.4798(19) Å] than S=O10 [1.4469(19) Å]. Furthermore, this favourable
packing asymmetry may also be the cause of the rotation of the thiadiazole ring, along the
C13−S12 bond, out of the plane of the benzothiazole moiety.
Figure 8. Crystal packing of the MTSB molecules, showing the C–H…O, C–H…N interactions as
dashed lines.
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Figure 9. Detail of the interactions (depicted as dashed lines) in the crystal packing between the 휋
electron clouds of the aromatic rings.
Table 3. Weak intermolecular interactions in the crystal structure of MTSB.a
D–H…A
D–H
H…A
D…A
∠D–H…A
…
i
C7–H24 O10
1.10(3)
1.10(4)
2.55(3)
2.56(4)
3.410(2)
3.591(2)
3.510(2)
134.5(14)
154.9(14)
163.0(14)
…
ii
C8–H25 O10
…
iii
C18 –H21 N17
1.011(18)
2.531(19)
Cg(I)…Cg(J)
Cg(I)…Cg(J)
CgI_perp
CgJ_perp
Cg(1)…Cg(2)iv
Cg(2)…Cg(3)iv
4.0605(9)
3.9155(7)
3.7518(5)
3.5183(5)
3.5068(5)
-.6930(5)
Y–X…Cg
X…Cg
Y…Cg
∠Y–X…Cg
S1–O10 …Cg(2)v
3.8639(17)
5.2826(8)
160.60(9)
a
Distances in Å; angles in degrees. Symmetry codes: i = x,3/2−y, −1/2+z; ii =1−x,1−y,-z ; iii = −x,
−1/2+y,1/2−z ; iv = −x,2−y,-z; v =1−x, −1/2+y,1/2+z. Cg(1): S1–N2–C3–C4–C9; Cg(2): S14–C13–N17–
N16–C15; Cg(3): C4–C5–C6–C7–C8–C9; CgI_perp = perpendicular distance of Cg(I) on ring J.
As the XRD data collected at low temperature is of very high quality, the difference
electron density map obtained from the observed scattering amplitudes and those calculated
using a spherical atom model showed clearly a build-up of positive residual density along the
chemical bonds. Thus, we have undertaken another structural refinement using scattering
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amplitudes from aspherical atoms based on a multipole expansion formalism of the charge
density. Technical details of such multipole refinement are given in the Experimental Section.
The dynamical deformation density map calculated from the refined multipole model is
depicted in Figure 10, where it can be compared with that obtained using the B3LYP/6-
311++G(3fd,3dp) calculated data for the isolated molecule. As it can be noticed, the two
differential maps show similar patterns, noticeably also in the S14…N2 contact region, where a
significant positive residual density between the S and N atoms can be clearly observed.
Furthermore, the analysis of the critical points of the experimental electron density confirmed
the presence of a bond critical point between S14 and N2, as found in the BCP analysis of the
DFT density for the isolated molecule. This result confirms the structural relevance of the
intramolecular S14…N2 contact, even in the crystalline phase, where intermolecular packing
forces are present. Atomic charges derived from the multipole model (see Table 2) are in
qualitative agreement with those derived from the DFT calculations for the isolated molecule,
the S atoms bearing a positive charge, and the N and O atoms being negatively charged.
Figure 10. (left) Dynamical deformation density map calculated from the refined multipole model,
projected on the C4–C5–C6 plane, showing the bonding electron density distribution. (right)
B3LYP/6-311++G(3fd,3dp) calculated data for the isolated molecule (molecular skeleton
plane).
2.6. Infrared and Raman spectroscopy of the MTSB crystal
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The infrared (ATR) and Raman spectra of MTSB in the neat crystalline phase are given
in Figure 11, together with the simulated spectra built based on the calculated vibrational data
for the minimum energy configuration of the isolated MTSB molecule. The proposed
assignments are given in Table S2.
The most relevant observation extracted from the data shown in Figure 11 is that the
calculated spectra (both IR and Raman) for the isolated molecule of MTSB reproduce rather
reasonably the experimental spectra of the neat crystal of the compound. This means that the
intramolecular vibrational potential is not significantly perturbed by the intermolecular
interactions which exist in the crystal. Such conclusion is in agreement with the X-ray
structure data that, as pointed out above, revealed weak C–H…O or C–H…N intermolecular
contacts as the major interactions present in the crystal. It is also consistent with the relatively
o
low melting point of the compound (190-192 C), temperature at which the compound also
starts to decompose.
Raman temperature variation studies were conducted, from room temperature to ~200 oC.
Only minor spectral changes were observed (very slight band broadening) before degradation
of the compound started, indicating that no phase transitions occur in the whole range of
temperatures probed.
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21
14
7
6-311++G(3df,3pd)
0
1600 1400 1200 1000 800
600
400
200
0.9
0.6
0.3
0.0
IR (experimental)
1600 1400 1200 1000 800
600
400
200
15
10
5
Raman (experimental)
0
1600 1400 1200 1000 800
600
400
200
6-311++G(3df,3pd)
0.9
0.6
0.3
0.0
1600 1400 1200 1000 800
600
400
200
Wavenumber or Raman Shift / cm -1
Figure 11. Vibrational spectra for neat crystalline MTSB (room temperature). (top to bottom panels):
B3LYP/6-311++G(3df,3pd) calculated infrared and Raman spectra, respectively, for the minimum
energy configuration of the isolated molecule (simulated using Lorentzian functions with fwhm of 10
and 5 cm-1, respectively; for the used method to convert the Raman activities extracted from the
Gaussian 09 output to the Raman intensities, see;45-47 Raman intensities were normalized to unity
intensity of the band at 1403 cm–1); (middle panels): experimental ATR and Raman spectra of the
compound.
We conclude from these findings that the structure of MTSB is similar in the monomeric
state and in the crystal state. The structural stability lands predictability when MTSB is
considered as bridging ligand in the design of coordination structures.
2.7. In vitro cytotoxicity of MTSB
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The cytotoxicity of MTSB was evaluated in vitro against 6 cancer derived cell lines
(HepG2, HeLa, SH-SY5Y, N9, THP-1 and U937) and a non-tumoral cell line (S17).
Following recent findings6 showing that the physiological concentration of Cu(II) in
neoplasms range from 30 to around 50 µM our experiments were conducted in the presence
of 50 µM Cu(II). The cytotoxicity of MTSB against each cancer cell line, expressed as the
half maximal inhibitory concentration (IC50), and the corresponding selectivity index,
calculated with respect to the non-tumoral cell line (S17), are provided in Table 4.
Table 4. Cytotoxicity of MTSB against the tumor derived cell lines HepG2, HeLa, SH-SY5Y, N9,
THP-1 and U937 and the non-tumoral cell line (S17), expressed as IC50 (µM). The confidence
intervals are presented in brackets. The selectivity index (SI) was calculated with reference to the cell
line S17.
IC50 (MTSB + 50 µM Cu2+) (µM)
SI
75.1
(72.9 – 77.4)
16.0
(13.6 – 19.0)
7.62
(6.28 – 9.25)
8.46
(7.61 – 9.40)
33.4
(29.7 – 37.6)
119
(115 – 124)
39.9
S17
HepG2
HeLa
SH-SY5Y
N9
4.68
9.86
8.87
2.25
0.63
1.88
THP-1
U937
(30.3 – 52.6)
Results show that, in the presence of 50 µM of Cu(II), MTSB exhibits toxicity towards all
cell lines tested, with IC50 values ranging from ca. 8 to 120 µM. From the results, MTSB
appears to be more toxic (lower IC50) to adherent (HepG2, HeLa, SH-SY5Y and N9) than to
non-adherent (THP-1 and U937) cell lines. For the range of cell lines tested, MTSB exhibited
the highest toxicity towards HeLa and SH-SY5Y cells (IC50 of 7.62 and 8.46 µM,
respectively) and also the highest selectivity index (SI of 9.86 and 8.46, respectively,
compared to the S17 cell line).
To understand the dependence of MTSB cytotoxicity on Cu(II) concentration, another set
of experiments were conducted to determine the IC50 for Cu(II) towards each cell line, using
as fixed concentration of MTSB the IC50 value previously determined for the fixed Cu(II) of
50 µM and shown in Table 4. The results obtained for this set of experiments are presented in
Table 5.
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Table 5. Cytotoxicity of Cu(II), expressed as IC50 (µM), towards the cancer derived cell lines HepG2,
HeLa, SH-SY5Y, N9, THP-1 and U937 and the non-tumoral cell line S17, in the presence of MTSB
at the fixed concentration corresponding to the IC50 values shown in Table 4. Values in brackets
represent the confidence interval.
IC50 (Cu2+) (µM)
Concentration of MTSB (µM)
38.5
(32.5 – 45.6)
21.5
(17.8 – 25.9)
75.8
(60.9 – 94.3)
31.4
(29.8 – 33.1)
85.3
(78.0 – 93.2)
45.3
(41.9 – 48.9)
26.5
S17
75.1
16.0
7.62
8.46
33.4
119
HepG2
HeLa
SH-SY5Y
N9
THP-1
U937
39.9
(24.2 – 29.0)
Results presented in Table 5 show that MTSB remains toxic for 5 cell lines, at Cu(II)
concentrations lower than 50µM. That was the case for the hepatic line (HepG2; 21.5) and
neuroblastoma (SH-SY5Y; 31.4), both adherent cancer cell lines, and for the non-adherent
cell lines of monocytic leukemia (THP-1; 45.3) and hystiocytic lymphoma (U937; 26.5).
These values fall within the range of Cu(II) concentrations found in neoplasms. In this sense,
MTSB appears to be most effective towards hepatic, neuroblastoma and lymphoma, for
which the least combination of Cu(II) and MTSB concentrations can be used to obtain a 50%
reduction in cell viability after 72 h of exposure. For the monocytic leukemia THP-1, a 50%
reduction in cell viability would require a 119 µM concentration of MTSB.
For the non-tumor-derived cell line S17 a 50 % reduction in cell viability is achieved with
concentrations of MTSB and Cu(II) of 75.1 and 38.5, respectively. However, it is worth
noting that, in non-tumor tissues, the normal concentration of Cu(II) were found to be less
than 10 µM. Thus, we can argue that MTSB is toxic towards a range of cancer cells at
concentrations that do not affect normal cells.
3. Conclusions
In the present study, the synthesis and the detailed structural characterization of 3-[(5-
methyl-1,3,4-thiadiazol-2-yl)sulphanyl]-1,2-benzothiazole 1,1-dioxide (MTSB) were
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addressed. In addition, the photochemical stability of the matrix-isolated compound has been
investigated. The relevant regions of the potential energy surface of the molecule were
scrutinised and a detailed charge density analysis of the identified most stable conformation
performed based on the AIM theory and Wiberg’s bond order methods. The structural
relevance of the intramolecular N…S interaction in MTSB was stressed. In situ matrix UV-
irradiation revealed a high photostability of MTSB, compared to parent tetrazole
saccharinates and to the 5-methyl-1,3,4-thiadiazole building block, emphasizing the
photostabilizing effect of the saccharyl system.
The infrared spectrum of matrix-isolated (argon matrix) MTSB was recorded and
interpreted taking into account results obtained using quantum chemical calculations at the
B3LYP/6-311++G(3df,3pd) level of approximation.
The crystal structure of the compound was also investigated by X-ray crystallography and
infrared and Raman spectroscopies. It was concluded that in the crystal the molecules of
MTSB assume a conformation similar to that predicted theoretically to be the most stable one
for the isolated molecule situation, which was also proved to be the one present in the argon
matrix. The S…N interation was also found to play a relevant role to the structure of the
molecule in the crystal.
Finally, the toxicity of MTSB was evaluated in vitro, towards 6 cancer cell lines and non-
tumoral cells. The results showed that MTSB is toxic towards cancer cells at concentrations
that do not affect normal cells, the compound being particularly effective towards the hepatic
(HepG2), neuroblastoma (SH-SY5) and lymphoma cell lines (U937).
4. Experimental
4.1. Chemicals and routine analytical equipment; general information
All chemicals (analytical grade) were used as purchased from commercial sources. When
required, solvents (technical grade) were freshly distilled from appropriate drying agents
before use.
Melting points were recorded on a Stuart Scientific SMP3 melting point apparatus and are
uncorrected. Mass spectra were obtained on a VG 7070E mass spectrometer by electron
ionization (EI) at 70 eV. NMR spectra were obtained on a Bruker AM-400 spectrometer,
using TMS as the internal reference (δ = 0.0 ppm).
4.2. Synthesis of ligands
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Preparation of 3-chloro-1,2-benzisothiazole 1,1-dioxide (2): Saccharyl chloride
(3-chloro-1,2-benzisothiazole-1,1-dioxide, (2)) was obtained from saccharin (10.2 g; 56
mmol) and phosphorus pentachloride (14.0 g; 66 mmol), heated at 180 ºC, using a procedure
reported previously:17 colorless needles (7.00 g; 63 % yield); m.p. 143-145 ºC; IR (KBr,
1
cm-1): 1724, 1654, 1603 (C=C), 1346 (SO2), 775 (Ar-H) and 692 (C–Cl); H NMR (400
MHz, CDCl3): 7.85 (m, 4H); MS (EI): m/z 201 [M]+.
Preparation of 3-[(5-methyl-1,3,4-thiadiazol-2-yl)sulfanyl]-1,2-benzothiazole 1,1-dioxide
(MTSB): A mixture of anhydrous 5-methyl-1,3,4-thiadiazole-2-thiol (4) (0.65 g; 4.92 mmol)
and 3-chloro-1,2-benzisothiazole 1,1-dioxide (2) (1.0 g, 4.97 mmol) in dry THF (40 mL) was
stirred at 60 ºC, under a nitrogen atmosphere. The reaction was monitored by TLC (ethyl
acetate/hexane; 4:6). After 24 h, the solvent was evaporated under reduced pressure and the
remaining solid was dried under vacuum at room temperature. Crystallization from
acetone/THF (2:1) yielded the required product as yellow crystals (1.0 g; 60 % yield); m.p.
1
190-192 ºC. H NMR (400 MHz, CDCl3):  8.04-7.99 (m, 2H), 7.95-7.86 (m, 2H), 2.79 (s,
3H); MS (EI): m/z 298 [M]+.
3.3. Matrix experiments
The cryogenic matrices were prepared by sublimation of the compound, using a
o
homemade mini-oven (T ~120 C) placed within the vacuum chamber of the cryostat, and
mixing the resulting vapors with argon N60 in an approximate 1:1000 solute/matrix molar
ratio.
The infrared spectra were recorded in a Thermo Nicolet 6700 Fourier transform infrared
(FTIR) spectrometer (resolution 0.5 cm-1), equipped with a Ge/KBr beam splitter and a
deuterated triglycine sulfate (DTGS) detector.
UV irradiations were performed in situ, through an outer quartz window (cut-off ~200
nm) of the cryostat, with broadband UV light produced by a 500 W Hg(Xe) lamp (Newport,
Oriel Instruments) set up to provide an output of 200 W at the sample. During the performed
irradiation experiments, the Hg(Xe) lamp was fitted with a water filter (8 cm long) to absorb
heat.
3.4. X-ray crystallography
Single crystal X-ray diffraction (XRD) data were collected on a Bruker APEXII CCD
diffractometer using graphite monochromatized Mo Kα radiation (λ = 0.71073 Å). The data
collection was performed at 120(2) K using a cooled nitrogen-gas stream in laminar flow.
24
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10.1002/chem.201705319
Chemistry - A European Journal
The single-crystal specimen was plate-shaped with dimensions 0.33×0.13×0.07 mm3. A
monoclinic unit cell (a = 8.3068(6), b = 9.3966(7), c = 15.6916(11) Å, β = 104.065(3)º), was
first derived from indexing the Bragg spots of the first 36 CCD frames and it was further
refined by least-squares from the positions of 5825 measured Bragg spots at the end of the
data-collection. Systematic absences pointed to the monoclinic P21/c space-group, and this
assignment was confirmed from the structure solution. Data reduction and a multi-scan
absorption correction were performed with the SAINT and SADABS48 suite of programs.
The crystallographic structure was solved by direct methods using SHELXT-2014/5.49
Refinements using spherical atomic scattering factors were carried out with the SHELXL-
2016/6 package.50 All refinements were made by full-matrix least-squares on F2 with
anisotropic displacement parameters for all non-hydrogen atoms. Hydrogen atoms were
placed at calculated idealized positions and refined as riding using SHELXL-2016/6 default
parameters. The refined structural model gave a final R1 factor of 0.0232 for 2481 reflections
of I > 2σ and Rall of 0.0260 for all 2726 reflections and 164 parameters. A refinement of the
structural model using aspherical scattering factors, based on a Hansen-Coppens multipole
expansion formalism51 were carried out with XD2006.52 Slater-type radial functions were
used. All non hydrogen atoms were refined with na expansion up to l = 3 (octupoles), the
hydrogen atoms were refined up to dipoles (l = 1). A set of k contraction-expansion
parameters for each atomic species, multipole population parameters, and anisotropic
displacement parameters (isotropic for H atoms) were refined. A charge neutrality constraint
was imposed and apropriate restrictions on the multipole parameters for an assumed Cs
symmetry of the electron density of the aromatic rings. In addition, atoms with equivalent
chemical environments were assumed to have the same deformation electron density. The
multipole and radial k parameters of non-H atoms were refined using 2481 reflections with
I > 2σ, giving a R1 factor of 0.0124 and Rall of 0.0163 for all 2726 reflections and 406
parameters.
Crystallographic data for the structure in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 1536536.
Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road,
Cambridge CB2 1EZ, UK, (fax: +44-(0)1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
3.5. IR and Raman spectroscopy of crystalline MTSB
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10.1002/chem.201705319
Chemistry - A European Journal
ATR infrared spectra were collected with 2 cm-1 spectral resolution (126 scans), in a
ThermoNicolet IR300 FTIR spectrometer, equipped with a Smart Orbit ATR accessory, a
DTGS detector and a Ge/KBr beam splitter.
Raman spectra were obtained using a Horiba LabRam HR Evolution micro-Raman
system, with excitation at 632.8 nm from a He-Ne laser, spectral resolution 4.0 cm-1, 50x
magnification objective, acquisition time of 60 s, 10 accumulations, and laser power ~17
mW. Calibration was done using the Si wafer band. The wavenumber accuracy is better than
0.5 cm-1 in the spectral region of interest. In all cases, the automatic focusing option was
o
used. The Raman temperature variation studies (0.1 C resolution) were undertaken by
coupling a Linkam THMS600/720 stage (controlled by a PE95/T95 system controller) with
the Raman spectrometer.
3.6. Cell viability assays
Cell viability was determined against four cell lines of adherent tumors: human
hepatocellular carcinoma cell line (HepG2), human epitheloid cervix carcinoma (HeLa),
human neuroblastoma cell line (SH-SY5Y) and mouse microglia cell line (N9); and two cell
lines of non-adherent tumors: human monocytic leukaemia (THP-1) and human histiocytic
lymphoma (U937). The non-tumoral cell line of mouse bone marrow stroma (S17) was used
to assess the selectivity of the compound towards tumoral cells.
Exponentially growing S17, HepG2 and HeLa cells were plated in 96-well tissue plates at
a density of 5×103 cells/well while SH-SY5Y, N9, THP-1 and U937 were seeded at a density
of 10×103 cells/well. All the plates were incubated for 24 h. Compounds were then applied at
various concentrations (0-250 µM) for 72 h. Control cells were treated with DMSO at the
highest concentration used in test wells (0.5 %) plus copper solution at 50 µM, and cell
viability was determined by the MTT colorimetric assay.53 Briefly, 2 hours prior to the end of
the incubation period 20 μL of MTT (5 mg mL-1 in PBS) were added to each well and further
incubated at 37 °C. Then, 150 μL of DMSO was added to each well in order to dissolve the
formazan crystals and absorbance was measured at 590 nm (Biotek Synergy 4).
For the evaluation of the copper concentration effect on MTSB cytotoxicity, cells were
treated with the MTSB at the concentrations correspoding to its IC50 (previously determined)
and copper(II) at various concentrations (0-200 µM). Results were expressed in terms of IC50
(μM). All experiments were run in triplicate.
IC50 values were calculated by sigmoidal fitting of the data in the GraphPad Prism V 5.0
program (GraphPad Software, La Jolla, CA, USA).
26
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10.1002/chem.201705319
Chemistry - A European Journal
Acknowledgements
This investigation has been performed within the Projects PTDC/QEQ-QFI/3284/2014 –
POCI-01-0145-FEDER-016617 and PTDC/MAR-BIO/4132/2014, funded by the Portuguese
“Fundação para a Ciência e a Tecnologia” (FCT) and FEDER/COMPETE 2020-EU.
CCMAR in funded by FCT, through the project UID/Multi/04326/2013. The Coimbra
Chemistry Centre (CQC) and the Coimbra Physics Centre are supported by FCT, through the
projects UI0313/QUI/2013 and UID/FIS/04564/2016, respectively, also co-funded by
FEDER/COMPETE 2020-EU.
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Chemistry - A European Journal
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