New BI and TRI-Thiazole copper (II) complexes in the search of new cytotoxic drugs against breast cancer cells

Article abstract of DOI:10.1016/j.ica.2020.119622

New thiazolyl derivatives (BT and TT) and their copper (II) complexes [Cu₂Cl₂(BT)₂] (Cu-BT) and [Cu₄ClO₂(TT)₂]PF₆?3.5H₂O (Cu-TT) were synthesized and characterized by elemental analysis, ¹H NMR and ¹³C NMR, HRMS, X-ray diffraction, IR and UV–Vis spectroscopies. The crystal structure of Cu-BT shows the formation of a dinuclear complex where each copper(II) center is bonded to two thiazol N atoms, from different BT ligands, one deprotonated amide N atom, an O atom from the ester terminal groups and a chlorine atom. The structure found for Cu-TT is a positively charged tetranuclear moiety containing two deprotonated TT ligands, a chlorine anion, two hydroxide anions acting as bridges between the copper centers and a water molecule. The cytotoxic activity of both copper complexes was evaluated on metastatic breast cancer cell lines, characterized for its rapidly dividing behavior. Both, Cu-BT and Cu-TT, show higher cytotoxic activity against these tumor cells than free BT and TT and also than cisplatin. In addition, we found that both complexes interact with DNA. Consistently, they also show cytotoxicity against a rapidly dividing non-tumor cell line, although with higher IC_50, being such interaction and selectivity an indicator of the possible coexistence of more than one mechanism of action.

Full text of DOI:10.1016/j.ica.2020.119622

InorganicaChimicaActa508(2020)119622
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
New BI and TRI-Thiazole copper (II) complexes in the search of new
cytotoxic drugs against breast cancer cells
Natalia Alvareza, Francesca Vellutia, Florencia Guidalib, Gloria Serrab, M. Gabriela Kramerc,d
,
Javier Ellenae, Gianella Facchina, Laura Scaroneb,⁎, María H. Torrea,⁎
a Química Inorgánica (DEC), Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo, Uruguay
b Química Farmacéutica (DQO), Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo, Uruguay
c Química Orgánica (DQO), Facultad de Química, Universidad de la República, Gral. Flores 2124, Montevideo, Uruguay
d Espacio de Ciencia y Tecnología Química, Sede Tacuarembó, Universidad de la República, Campus Interinstitucional, Ruta 5 km 386, Tacuarembó, Uruguay
e Laboratorio de Cristalografía, Instituto de Física de São Carlos, Universidade de São Paulo, C.P. 369, 13560 São Carlos (SP), Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Copper complexes
Thiazoles
X-ray diffraction
Cytotoxicity
Breast cancer
New thiazolyl derivatives (BT and TT) and their copper (II) complexes [Cu2Cl2(BT)2] (Cu-BT) and
[Cu4ClO2(TT)2]PF6⋅3.5H2O (Cu-TT) were synthesized and characterized by elemental analysis, 1H NMR and 13
C
NMR, HRMS, X-ray diffraction, IR and UV–Vis spectroscopies. The crystal structure of Cu-BT shows the for-
mation of a dinuclear complex where each copper(II) center is bonded to two thiazol N atoms, from different BT
ligands, one deprotonated amide N atom, an O atom from the ester terminal groups and a chlorine atom. The
structure found for Cu-TT is a positively charged tetranuclear moiety containing two deprotonated TT ligands, a
chlorine anion, two hydroxide anions acting as bridges between the copper centers and a water molecule. The
cytotoxic activity of both copper complexes was evaluated on metastatic breast cancer cell lines, characterized
for its rapidly dividing behavior. Both, Cu-BT and Cu-TT, show higher cytotoxic activity against these tumor cells
than free BT and TT and also than cisplatin. In addition, we found that both complexes interact with DNA.
Consistently, they also show cytotoxicity against a rapidly dividing non-tumor cell line, although with higher
IC50, being such interaction and selectivity an indicator of the possible coexistence of more than one mechanism
of action.
1. Introduction
One of these strategies is the synthesis of metal complexes with
active ligands, looking for enhanced activity and improved selectivity
Breast cancer is the most prevalent malignancy in women and in its
metastatic state, the second most common cause of mortality [1].
Moreover, the global expansion of cancer is increasing dramatically in
the world. By 2030, the annual number of new cancer diagnoses is
projected to be 21 million worldwide, with 17 million patients dying
every year [2].
Although the existence of many available antitumor agents, the high
toxicity of most part of them and the emergence of resistance make it
necessary to investigate new drugs. Due to this fact the research in
cancer chemotherapy is a very important issue and it has changed along
the centuries following different strategies.
or to use the metal ions as carriers of active ligands inside the tumor
cells. These studies have become of interest since the discovery of a
series of complexes with cytotoxic activity like cisplatin and the next
platinum drug generations, copper (II) antineoplastic compounds
(Casiopeínas®), ruthenium and gold complexes, among others [4–7].
One interesting pharmacophoric group is the thiazolyl moiety.
Several studies have shown that molecules with this residue produce
different pharmacological properties. The [1]thiazoles and benzathia-
zoles are found in a wide variety of bioactive molecules and natural
products [8] and show antimicrobial, antiprotozoal, analgesic, anti-in-
flammatory, antipyretic and cytotoxic properties, among others [9–12].
Antiproliferative drugs like tiazofurin and bleomycins, and their
related analogues, have pointed out the importance of thiazole moiety
for anticancer drug design. For example, Bleomycins are used to treat
several types of cancers, like squamous cell carcinomas, malignant
To date, a very large number of compounds with cytotoxic activ-
ities, both natural and synthetic, were identified, providing useful in-
formation that allow the researchers to redesign new drugs and to pose
alternative strategies [3].
⁎ Corresponding authors.
E-mail addresses: laurito@fq.edu.uy (L. Scarone), mtorre@fq.edu.uy (M.H. Torre).
https://doi.org/10.1016/j.ica.2020.119622
Received 14 February 2020; Received in revised form 20 March 2020; Accepted 21 March 2020
Availableonline24March2020
0020-1693/©2020ElsevierB.V.Allrightsreserved.

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
lymphomas and testicular cancers [8]. The bis-thiazole moiety of
bleomicins seems to bind to DNA and several researchers have studied
this interaction [13–15] including with metal ions [16]. Dasatinibis
another example of thiazole derivatives used in the treatment of cancer
and also the new drug SNS-032, currently in clinical trials [17]. On the
other hand, from a chemical point of view, the presence of N and S
donor atoms in the heterocyclic ring of thiazoles gives a versatile co-
ordination ability with transition metal ions and suggestthat their bio-
logical function might involve the binding of metal ions inside the or-
ganisms.
Therefore, the development of thiazolyl coordination compounds
with different metal ions has attracted considerable interest [18–22]. In
particular, among the transition metals, copper is an essential metal
that play a vital role in many biochemical processes and also the or-
ganisms have different detoxification pathways to get rid of copper
excess. Consequently, the development of new copper compounds with
antiproliferative activity is interesting given that the detoxification
mechanisms could result in a decrease of secondary toxic effects, often
seen in metallodrugs [23].
Encouraged by these antecedents and considering our previous ex-
perience in the development of biological interesting thiazoles [24–26]
and copper complexes [27–31] presenting cytotoxic activity against
different tumor cell lines, in this paper we report the synthesis and
characterization of two new thiazole derivatives and the corresponding
copper complexes.
The cytotoxic activity of free ligands and copper complexes com-
pared to cisplatin, a widely used antineoplastic platinum-complex, was
evaluated on human (MCF-7 and MDA-MB-231) and mouse (4T1)
breast cancer metastatic cell lines. Such tumor cells were chosen be-
cause metastatic breast cancer represents the worst clinical scenario
and it is usually associated with resistance to current chemotherapies
[3233]. In addition, 4T1 breast cancer cell line would allow posterior
evaluation of any new treatment in a clinically relevant animal model
of metastatic breast cancer [34]. With the aim of studying some aspects
of the mechanism of action and selectivity, the complex/DNA interac-
tion and the cytotoxic behavior was evaluated also against 293T cells,
which exemplify a non-tumor and fast-dividing condition.
Fig. 1. bi (BT) and tri (TT) thiazolyl ligands.
UV–Vis spectra were carried out with a Shimadzu UV 1603 spec-
trophotometer, using 1 cm path length quartz cells.
Melting points of ligands were measured using a Fisher-Johns
Melting Point Apartus.
Single crystal X-ray diffraction data was obtained at 298(2) and
100(2) on an Enraf-Nonius FR590 Kappa-CCD diffractometer using
grafite monochromated MoKα radiation (0,71073 Å) for Cu-BT (see
Fig. 1) and at 100(2) on a Bruker D8 Venture using multilayer mirror
monochromated CuKα radiation (1.54178 Å) for Cu-TT (see Fig. 1).
Bruker AXS Collect software was used for data collection and the HKL
Denzo-Scalepack program suite for data reduction of Cu-BT, whereas
the APEX 2 software suite was used for Cu-TT and cell now was used to
resolve twinning. Multi-scan absorption corrections were applied in all
cases [36]. Cu-BT structure was solved by direct methods using SIR-92
[37] and Cu-TT by intrinsic phasing with SHELXT, all structures were
refined with SHELXL within SHELXle using the full-matrix least-squares
on F2 method [38,39].
Molecular structure graphics were prepared in Mercury [40]. All
structures presented positional disorder which was treated with the part
instruction in SHELX until model convergence.
The Cu-BT structure showed no temperature dependence, only a
slight compression of the cell was observed with no symmetry mod-
ifications, therefore all discussion on this paper will be made on the
model obtained for the crystals measured at 100 K.
All non-hydrogen atoms were refined using anisotropic displace-
ment parameters. Carbon bonded hydrogen atoms were stereo-
chemically position and refined isotropically using the riding-model,
thermal parameters set at 1.2 times the Ueq of the C atom they are
bonded to.
2. Experimental
Amino and water hydrogen atoms were found in the difference
Fourier map, positionally fixed and their thermal parameters set to 1.2
times the Ueq of the nitrogen atom for the amine and 1.5 times the Ueq
of the oxygen atom for the water molecules.
2.1. Materials and general procedures
The solvents used in the synthesis of thiazoles were purified ac-
cording to literature procedures [35]. All reactions were carried out in
dry, freshly distilled solvents under anhydrous conditions unless
otherwise stated.
Summary of the crystallographic data, experimental details and
refinement results are listed in Table 1
Flash column chromatography was carried out with Silica gel 60
(J.T. Baker, 40 µm average particle diameter). All reactions and chro-
matographic separations were monitored by TLC, conducted on
0.25 mm Silica gel plastic sheets (Macherey/Nagel, Polygram_SIL G/UV
254). TLC plates were analyzed under 254 nm UV light, iodine vapor, p-
hydroxybenzaldehyde spray or ninhydrine spray. Yields are reported
for chromatographically and spectroscopically (1H and 13C NMR) pure
compounds.
2.2. Synthesis of thiazolyl derivatives
General procedure for ethyl ester hydrolysis: An aqueous KOH
(10%) solution was added to an ester solutionin THF. The reaction
mixture was stirred at room temperature until reagent disappearance
was confirmed by TLC. Aqueous HCl 1 M was added until pH 4 and the
solution was extracted with EtOAc. The organic layer was dried over
MgSO4 and concentrated in vacuo to afford the acid.
The chemicals and starting materials in the synthesis of complexes
were of analytical grade, and used without further purification.
Elemental analyses were performed with a Thermo Scientific Flash
2000 analyzer, using V2O5 as catalyst.
2-methylthiazole-4-carboxilic acid (2a): Compound 2a was pre-
pared following general procedure for ester hydrolisis from 1a. The acid
was employed without further purification.
General procedure for amide bond formation: HBTU (1.2 eq),
DIPEA (2.2 eq) and 4-DMAP (0.2 eq) were added to a stirred solution of
the respective amine (1.0 eq) and acid (1.0 eq.) under N2 atmosphere in
dry CH2Cl2 at 0 °C. The resulting mixture was stirred at room tem-
perature overnight. Then was filtered through celite, washed with
CHCl3 and evaporated in vacuo. The crude was redissolved in EtOAc,
washed with 5% HCl and then with saturated solution of NaHCO3, dried
with MgSO4, filtered and evaporated in vacuo. The crude was purified
by flash chromatography using the corresponding eluent to give the
1H NMR and 13C NMR spectra were recorded on Bruker Avance
DPX- 400 for the ligands. Chemical shifts are related to TMS as an in-
ternal standard.
High resolution mass spectra (HRMS) were obtained on a micro Q-
TOF (ESI) (Bruker Daltonics).
IR spectra of ligands and complexes were recorded on a Shimadzu
IR-Prestige 21 spectrophotometer spectrophotometer using KBr pellet
technique, in the range between 4000 and 200 cm−1
.
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N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Table 1
Crystal data and structure refinements Cu-BT and Cu-TT complexes.
Identification code
Cu-BT 298 K
Cu-BT 100 K
Cu-TT
Empirical formula
Formula weight
Temperature
C22H20Cl2Cu2N6O6S4
790.68
298(2) K
C22H20Cl2Cu2N6O6S4
790.68
100(2) K
C60H66Cl2Cu8F12N20O29P2S12
2785.20
100(2) K
Wavelength
0.71073 Å
0.71073 Å
1.54184 Å
Crystal system
Space group
Triclinic
P-1
Triclinic
P-1
Triclinic
P1
Unit cell dimensions
a = 11.1441(2) Å
b = 11.2582(2) Å
c = 13.0039(2) Å
α = 106.5400(10)°
β = 90.8690(10)°
γ = 102.6770(10)°
1520.60(5) Å3
2
a = 10.51(2) Å
b = 10.63(3) Å
c = 11.54(6) Å
α = 106.93(13)°
β = 91.86(10)°
a = 10.6888(3) Å
b = 14.6062(3) Å
c = 17.8915(5) Å
α = 71.1060(10)°
β = 82.4990(10)°
γ
= 102.47(8)°
γ = 70.1230(10)°
Volume
Z
Density (calculated)
Absorption coefficient
F(0 0 0)
Crystal size
Theta range for data collection
Index ranges
1198(7) Å3
2484.67(11) Å3
2
1
1.727 Mg/m3
1.897 mm−1
2.192 Mg/m3
1.861 Mg/m3
2.408 mm−1
5.930 mm−1
796
796
1394
0.482 × 0.109 × 0.102 mm3
3.001 to 25.073°
−12 ≤ h ≤ 13, −13 ≤ k ≤ 13,
−15 ≤ l ≤ 15
33,677
0.482 × 0.109 × 0.102 mm3
3.189 to 29.117°
−13 ≤ h ≤ 13, −13 ≤ k ≤ 13,
−15 ≤ l ≤ 15
24,509
0.092 × 0.070 × 0.051 mm3
2.611 to 74.652°
−13 ≤ h ≤ 13, −16 ≤ k ≤ 18, 0 ≤ l ≤ 22
Reflections collected
9919
Independent reflections
Completeness to
5363 [R(int) = 0.0605]
99.20%
5602 [R(int) = 0.0512]
99.70%
9919 [R(int) = 0.0492]
98.6%
theta = 25.242°
Absorption correction
Max. and min. transmission
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices [I > 2sigma(I)]
R indices (all data)
Semi-empirical from equivalents
0.995 and 0.750
5363/0/387
Semi-empirical from equivalents
0.918 and 0.786
5602/0/424
Semi-empirical from equivalents
0.7538 and 0.6086
9919/3/1336
1.040
1.031
1.037
R1 = 0.0491, wR2 = 0.1262
R1 = 0.0738, wR2 = 0.1443
0.732 and −0.756 e.Å−3
R1 = 0.0430, wR2 = 0.1105
R1 = 0.0579, wR2 = 0.1190
1.244 and −0.860 e.Å−3
R1 = 0.0514, wR2 = 0.1270
R1 = 0.0662, wR2 = 0.1352
1.048 and −0.651 e.Å−3
Largest diff. peak and hole
amide.
appeared and were washed with cold water and dried at room tem-
perature. Adequate single crystals for SCXRD were obtained in both
cases.
The stoichiometries of both complexes were: [Cu2Cl2(BT)2] and
[Cu4Cl(OH)2(TT)2H2O]PF6⋅3.5H2O.
Ethyl 2-(2-methylthiazole-4-carboxamido)thiazole-4-carboxylate (BT):
Compound BT was prepared following general procedure for amide
bond formation from 1b and 2a. Y = 90%. Solid. M.p. 166–169 °C.
Rf = 0.33 (EtOAc:hexane, 1:1),1H NMR (400 MHz, CDCl3) δ 1.43 (t,
J = 7.1 Hz, 3H), 2.75 (s, 3H), 4.44 (q, J = 7.1 Hz, 2H), 7.91 (s, 1H),
8.16 (s, 1H), 10,73 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 14.4, 19.2,
61.5, 122.5, 125.8, 142.2, 147.0, 157.3, 158.6, 161.5, 167.0. HRMS m/
zcalcd for C11H11N3NaO3S2 ([M + Na]+) 320.0134, found 320.0183.
IR film ν(cm−1) 3109, 2924, 1716, 1666, 1624, 1535, 1234, 1211,
1173, 1096.
2-(2-methylthiazole-4-carboxamido) thiazole-4-carboxylic acid (3):
Compound 3 was prepared following general procedure for ester hy-
drolisis from BT. The acid was employed without further purification.
Ethyl 2-(2-(2-methylthiazole-4-carboxamido)thiazole-4-carboxamido)
thiazole-4-carboxylate (TT): Compound TT was prepared following
general procedure for amide bond formation from 1b and 3. Y = 66%.
The analytic results were:
[Cu2(BT)2Cl2] (code Cu-BT): Yield: 25%. Anal. calcd.(%) for
C22H20Cl2Cu2N6O6S4.
C, 33,38; H, 2.62; N, 10.57; S,16.51.Calcd.: C, 33.44; H, 2.54; N,
10.63; S, 16.23. λmax(DMSO)(nm): 421 (sh)(dimeric structure) [41],
772. IR (KBr pellet, cm−1): 1701(s) (νas(C]O) amide),1684(sh) (νas(C]
O)ester), 1638(s) (νs(C]O) ester), 1631(s) (νs(C]O)amide).
[Cu4Cl(OH)2(TT)2H2O]PF6⋅3·.5H2O(code Cu-TT) Yield: 22%. Anal.
calcd. for C60H66Cl2Cu8F12N20O29P2S12
.
C, 25.58; H, 2.45; N, 10.03; S, 14.10 Calcd.:
C 25.85, H 2.37, N 10.05, S 13,79 λmax(DMSO)(nm): 726. IR (KBr
pellet, cm1):1712(m) (νas(C]O) amide),1622(s) (νs(C]O) ester) and (νs
(C]O)amide).
Solid.M.p. 225–229. Rf
=
0.34 (EtOAc: hexane, 2:1), 1H NMR
(400 MHz, CDCl3) δ 1.44 (t, J = 7.1 Hz, 3H), 2.86 (s, 3H), 4.45 (q,
J = 7.1 Hz, 2H), 7.92 (s, 1H), 8.04 (s, 1H), 8.22 (s, 1H), 10.41 (s, 1H),
10.54 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 14.4, 19.2, 61.4, 121.2,
122.5, 126.2, 142.1, 142.3 146.7, 157.3, 157.4, 158.5, 158.7, 161.4,
167.4. HRMS m/zcalcd for C15H13N5NaO4S3 ([M + Na]+) 446.0022,
found 446.0025. IR film ν(cm−1) 3421, 2928, 1624, 1539, 1230, 1092.
2.4. DNA binding constant: UV absorption titration experiments
The intrinsic binding constant (Kb) of the compounds to isolated
calf thymus DNA were determined using absorption titration mea-
surements and the Benesi-Hildebrand equation. In the latter the con-
stant is calculated as the slope to the intercept ratio of the [complex]/
Aobs as a function of 1/[DNA] plot [42–46]. In all cases the con-
centration of the complexes was kept constant at 10–15 μM in 5 mM
buffer Tris/HCl, pH = 7.5 and 50 mM of NaCl while adding Calf
Thymus-DNA (CT-DNA) to final concentrations 0 to 250 μM.
The stock solution of CT-DNA gave an absorbance at 260 to 280
ratio (A260/A280) of 1.8–1.9, consistent with the DNA being suffi-
ciently free of protein [46]. It was kept at 4 °C and used within 1 day.
DNA concentration was measured from the intensity of the 260 nm
2.3. Synthesis of copper complexes
Copper complexes with bi-thiazoles were obtained mixing metha-
nolic solutions containing 2.7 mg (0.01 mmol) of BT and 2.0 mg of
CuCl2·2H2O (0.01 mmol) for the synthesis of Cu-BT and 2.5 mg of TT
and 2 mg of CuCl2·2H2O for the synthesis of Cu-TT.
The resulting green solutions were stirred for 30 min at room
temperature and the solvent was slowly evaporated. Green crystals
3

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
absorption band using the extinction coefficient value of
ε260 = 6600 M−1 cm−1. The organic ligands and their corresponding
copper(II) complexes in buffer solution were stable in the assay con-
ditions.
(25% for Cu-BTa and 22% for Cu-TT) due to the fact that we bet on the
highest purity for subsequent characterization and not performance.
The electronic spectra in DMSO of both complexes did not have changes
during 24 h, showing that the solvent did not affect the coordination
environment.
2.5. Cytotoxic activity
3.2. Crystallographic structure description
The cytotoxic activity of ligands and copper complexes were de-
terminate against breast cancer cell lines MDA – MB – 231 and MCF-7
(human metastatic breast adenocarcinoma) and 4T1 (murine metastatic
breast cancer) and non-tumoral 293T (immortalized human kidney
cells) obtained from the American Type Culture Collection (ATCC).
MCF-7, 4T1 and 293T cells were grown in Dulbecco’s Modified Eagle
Medium (DMEM) and MDA – MB– 231 cells were grown in RPMI
Medium containing L-Glutamine and supplemented with 10% Fetal
Bovine Serum. Cells were incubated at 37 °C on 5% CO2 atmosphere
and removed enzymatically from flasks using 0.01% Trypsin-EDTA
solution. Cu-complexes solutions were prepared by dissolving the
complexes in dimethylsulphoxide (DMSO) to a concentration of 1 mM.
All solutions were sterilized by filtration. When required, 1/10 dilutions
were done in sterile PBS 1×. In all cases, a blank containing only sol-
vent was carried out as control and a blank with copper salt was per-
formed previously in the same conditions. For cytotoxic evaluation,
15.000 to 20.000 cells were grown in 96-well plates for 48 h containing
solutions of the Cu-complexes (final concentrations ranging from 1 to
20 μM). Due to the limited solubility of the complexes, 20 μM was the
higher concentration of complex able to be tested without DMSO in-
terference (no > 1% final concentration), since DMSO is toxic at higher
concentration. Cell viability in response to the complexes was de-
termined by colorimetric assay using Cell Counting Kit – 8 (Sigma
Aldrich). The amount of the formazan dye generated by the activity of
dehydrogenases in cells is directly proportional to the number of living
cells, which was measured at 450 nm. All assays were done in triplicate.
The IC50 was estimated from the dose-response curves using the free R
statistical software.
A search done at the CSD database (September 2019) gives a total of
22 crystal structures containing thiazole rings. When a metal is added
the number drops to 18, including from synthesis and structure reports
of tungsten [50] and vanadate [51] complexes, iron(II) and nickel(II)
complexes studied for their innovative magnetic properties [5253] to
cadmium(II)-bithiazole complexes as precursors for the obtention of
CdS nanoparticles [54]. So far there is only one crystal structure re-
ported containing a copper(II) complex with a coordinated thiazole ring
(refcode: 1112016). So far, it can be highlighted that the structure of
thiazole containing molecules and their coordination chemistry has not
been thoroughly studied. In this context, this work contributes to the
understanding of the latter.
3.2.1. Cu-BT
Cu-BT crystallized as a binuclear copper complex containing two
deprotonated BT ligands and two chlorine anions completing the pen-
tacoordinatedcopper(II) centers.
The copper centers present highly distorted geometries, using factor
tau as a measure of the geometry distortion, the values obtained were
0.92 and 0.77 for Cu1 and Cu2, respectively [55]. Given that 0 stands
for a perfect square pyramidal geometry and 1 for a perfect trigonal
bipyramid, it can be concluded that both copper centers adopt trigonal
bipyramid geometries. This geometry is stabilized by the presence of
intramolecular CeH·····Cl interactions with distances around 2.8 Å be-
tween a hydrogen atom from a terminal methyl group in the thiazole
unit and the chlorine anion bonded to each copper center. Fig. 4 shows
the ORTEP diagram of [Cu2(BT)2Cl2].
Both copper centers are bonded to a carbonyl amide oxygen and a
thiazole nitrogen atom from one ligand, a deprotonated amide nitrogen
and other thiazole nitrogen atom from the second ligand, and the fifth
position is occupied by a chlorine anion.
3. Results and discussion
3.1. Ligand and complex synthesis
As expected, copper binds rather to nitrogen than sulfur, leaving the
sulfur atom of the thiazole ring available to participate in inter-
molecular interactions with oxygen and nitrogen atoms from other
molecules as observed in intramolecular interactions in macro-
molecules. SeO and SeN interactions are similar in energy to hydrogen
bonds. These interactions have been deeply studied in terms of crystal
engineering and molecular modelling as modulators of drug selectivity
[56].
Cu-BT units pack in a diagonal fashion creating zig-zag molecular
chains through CeS·····O interactions with an angle of 166.5° and a
distance of 3.152 Å as shown in Fig. 5.
Along the crystal the zig-zag chains bind to each other by means of
CeS·····N interactions with an angle of 115.6° and a distance of 3.242 Å
(figure not shown).
The thiazolyl derivatives synthesized and used as ligands in this
work are shown in Fig. 1.
The synthetic strategy to obtain these ligands is shown in Figs. 2 and
3.
The synthetic strategy started with the preparation of thiazole 1a
and 1b by Hantszch’sthiazole synthesis [47] using ethyl bromopiruvate
and acetylthioamide (1a) or thiourea (1b), respectively. Then, ester
hydrolysis of thiazole1a allowed us to obtain carboxilic acid 2a. The bi-
thiazole amide BT was prepared fromamine thiazole1b and thiazole
acid 2a employing HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetra-
methyluronium hexafluorophosphate) as coupling reagent following
the group experience [24–26,48,49].
Finally, ester hydrolysis of bi-thiazole amide BT rendered bi-thia-
zole acid 3 as precursor of the third ligand tri-thiazoleTT prepared from
thiazole amine 1b and HBTU as coupling reagent.
3.2.2. Cu-TT
Both ligands were obtained with good yield (90% for BTa and 66%
for TT). On the contrary, the copper complex yields complexes were low
In the case of Cu-TT the structure found is a positively charged
tetranuclear moiety containing two deprotonated TT ligands, a chlorine
Fig. 2. Synthetic pathways to obtain BT.
4

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Fig. 3. Synthetic pathways to obtain TT.
anion, two hydroxide anions acting as bridges between the copper
centers and a water molecule. Fig. 6 shows one Cu-TT moiety in the unit
cell with atom numbering scheme for the donor atoms in the structure.
All copper centers are pentacoordinated and present distorted geo-
metries, τ factor was calculated giving values of 0.53, 0.26, 0.54 and
0.28 for Cu 1, 2, 3 and 4, respectively. The most distorted ones corre-
spond to the more distant copper atoms that have a carbonyl oxygen
oriented towards them, with a distance of around 2.9 Å, which if nearer
would complete an hexacoordinated center with distorted octahedral
geometry.
intermolecular interactions in this case.
Tetranuclear copper(II) moieties with oxo and hydroxo bridges have
been extensively studied for technological applications regarding their
tunable magnetic properties since they present
a range of anti-
ferromagnetic [57,58] and ferromagnetic coupling effects [59,60].
More recently butterfly-like tetranuclear copper(II) clusters, as the one
presented on this article, have been studied for anion exchange induced
catalysis [61].
On the other hand, they have been rarely studied for biological
applications, one close example would be the tetranuclear copper(II)
with water bridging molecules and phenanthroline ligands reported by
Gao, et al. which presents apoptotic effect on HeLa cells with cytotoxic
specificity and potent cancer cell inhibitory rate [62].
Cu1 bonds to two thiazole nitrogen atoms from different ligands, a
deprotonated amide nitrogen atom a hydroxide anion acting as bridge
between with Cu2 and Cu4 and an oxygen atom from a water molecule,
completing the fifth position.
Cu2 bonds to two hydroxide anions acting as bridges between Cu2
and Cu1 and Cu3, a deprotonated amide nitrogen atom, a thiazole ni-
trogen atom and an oxygen from a water molecule.
3.3. Complex-DNA interaction
Cu3 bonds has a similar coordination scheme than Cu1, the only
difference being that instead of the fifth position being completed by an
oxygen from a water molecule it is completed by a chlorine anion.
Cu4 has a similar coordination scheme than Cu2, with the exception
that the water molecule completing the pentacoordination is a chlorine
anion.
The presented analysis is for one of the molecules in the unit cell.
The second molecule in the asymmetric unit does not present significant
differences compared to the one presented and is consequently not
discussed separately in this paper.
Fig. 7 shows, as an example, the titration measurements of Cu-BT
with increasing DNA concentration, as referenced in point 2.4.
The intrinsic binding constant (Kb) of the compounds to isolated
calf thymus DNA was calculated as the slope to the intercept ratio of the
[complex]/Aobs as a function of 1/[DNA] plot.
To quantitatively determine the DNA binding strength of the com-
plexes, their intrinsic binding constants (Kb) were calculated as the
slope to the intercept ratio of the [complex]/Aobs as a function of 1/
[DNA]. Table 2 shows these results.
The obtained values were all around 1 × 103, which is a value
typically found for molecules that bind DNA as groove binding agents
such as Rhodamine B [63] and netropsin [64] and lower than that’s of
classical intercalators such as ethidium bromide (1.4 × 106) [65].
The predominant intermolecular interactions present in this struc-
ture include electrostatic attraction between the complex cationic
specie and tetrafluorofosfate anion and predominantly classic hydrogen
bonding (OeH·····O and OeH·····Cl). There are no sulfur mediated
Fig. 4. ORTEP diagram with 50% probability ellipsoids for the asymmetric unit of [Cu2(BT)2Cl2] (100 K) (ester disorder is not shown for clarity purposes) with atom
names and numbering scheme for the coordination sphere.
5

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Fig. 5. Molecular zig-zag chain array for Cu-BT guided by CeS·····O interactions along the axis of the crystal.
Fig. 6. ORTEP diagram with 50% probability ellipsoids for one Cu-TT moiety in the unit cell.
Fig. 7. Absorption titration measurements of Cu-BT being r = [DNA]/[compound]
6

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Table 2
DNA binding constants (Kb).
between 0.9 and 1.2 for Cu-TT and 1.3–1.8 for Cu-BT. These data in-
dicate the possible existence of a secondary mechanism of action that
may be able to distinguish between tumor and non-tumor cells, al-
though primary death may be caused by the DNA interaction of di-
viding cells. This behavior is relevant, given that the majority of the
side effects of conventional chemotherapies occur in non-tumor cells
with high division rate [66]. Therefore, counting on new drugs that
have greater cytotoxic activity in tumor cells rather than in high-divi-
sion non-tumor cells would be beneficial since they could have lower
side effects in vivo.
Compound
Kb (×103 M−1
)
BT
TT
Cu-BT
Cu-TT
3.1
2.2
2.0
1.7
3.4. Cytotoxic activity against metastatic breast cancer cells
Fig. 10 indicate that both cooper complexes induce cell death. Here,
the morphology of MCF-7 cells treated for 48 hs with ligands (BT and
TT) or complexes (Cu-BT and Cu-TT) are shown as a representative
example of the observation of all cell lines tested. Confluent live fusi-
form cells look attached to the plate in the case of BT and TT in the all
the concentration tested, as well as in Cu-BT (2,5 and 5 μM) and Cu-TT
(2,5 μM). On the other hand, death cells treated with 5,10 and 15 μM of
Cu-TT or 15 μM of Cu-BT, look spread out and round shaped. In the case
of Cu-BT at 10 μM, about 50% of the treated cells look alive (fusiforms),
while the other 50% look round and death. These observations coincide
with data presented in Figs. 8 and 9.
The cytotoxic activity of the two copper complexes, compared to the
free ligand and cisplatin, was evaluated against metastatic breast cancer
cell lines (MCF-7, MDA-MB-231 and 4T1), which are characteristic for
its fast division rate. Figs. 8 and 9 show that both Cu-BT and Cu-TT
complexes have cytotoxic effect against all tumor cell lines, while the
free ligands BT and TT were inactive at concentrations ranging up to
20 μM. This behavior is according to the metal coordination through
polar groups of ligands that might increase the lipophilicity and affect
the permeation of the cells. In all the cases, Cu-TT was more active than
Cu-BT and, according to the graph shape, a more acute dynamics of cell
death was induced with Cu-TT compared to Cu-BT. Moreover, Cu-BT
and Cu-TT were more active than cisplatin. In addition, we found that
both complexes have cytotoxic effect on fast dividing non-tumor cells
(293-T). This data agrees with a mechanism of action that involves a
DNA interaction, as shown above, where the DNA damage and/or DNA
replication disturbance would trigger cell death. Interestingly, Cu-TT
and Cu-BT showed slightly higher IC50 on non-tumoral 293T cell line
4. Conclusions
Two new ligands with bi and tri thiazole groups and the corre-
sponding copper complexes with interesting structural and biological
performance, are presented.
Both ligands and their copper (II) complex, were synthesized and
well characterized by different analytical and spectroscopic methods.
(Table 3), being the selectivity index (S.I. = IC50293T/IC50tumoral cells
)
Fig. 8. Viability of MDA-MB-231 (abbreviated as MDA), MCF-7, 4T1 and 293T in response to increasing concentrations of BT (▴) and Cu-BT (●).
7

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Fig. 9. Viability of MDA-MB-231 (abbreviated as MDA), MCF-7, 4T1 and 293T in response to increasing concentrations of TT (▴) and Cu-TT(●).
The IC50 of these copper complexes were lower compared to cisplatin,
Table 3
IC50(μM) for BT, TT, Cu-BTa, Cu-TT on tumor (MDA-MB-231, MCF-7 and 4T1)
and non-tumor (293T) cell lines.
reinforcing their high activity. In addition, we found that both com-
plexes interact with DNA. Consistently, they also show cytotoxicity
against a rapidly dividing non-tumoral cell line, although with higher
IC50, being such interaction and selectivity an indicator of a possible
coexistence of more than one mechanism of action.
In order to deepen the knowledge about the antitumor activity and
systemic toxicity we consider that both complexes are promising to be
evaluated in animal models of cancer.
Compound
MDA-MB-231
MCF-7
4T1
293T
BT
TT
Cu-BT
Cu-TT
Cisplatin*
> 20
> 20
9
> 20
> 20
10
3.7
> 20
> 20
> 20
12.5
3.7
> 20
> 20
16
3.5
> 20
3
> 20
> 20
* IC50 for cisplatin: 72, 72 and 75 for MDA-MB-231, 4T1 and MCF-7 respec-
tively [28].
CRediT authorship contribution statement
Natalia Alvarez: Investigation. Francesca Velluti: Investigation.
Florencia Guidali: Investigation. Gloria Serra: Investigation. M.
Gabriela Kramer: Investigation. Javier Ellena: Supervision. Gianella
Facchin: Investigation. Laura Scarone: Writing - original draft. María
H. Torre: Writing - review & editing.
The copper complexes presented a relevant crystallographic structure
showing the flexibility of thiazole ligands.
Cu-BT crystallized as a binuclear copper complex containing two
deprotonated BT ligands and two chlorine anions completing the pen-
tacoordinated copper(II) centers. The copper centers present highly
distorted geometries stabilized by the presence of intramolecular
CeH···Cl interactions.
Declaration of Competing Interest
Cu-TT presented a tetranuclear moiety containing two deprotonated
TT ligands, a chlorine anion, two hydroxide anions acting as bridges
between the copper centers and a water molecule.
Due to the fact that the structure of thiazole containing molecules
and their coordination chemistry has not been thoroughly studied, this
work represents a chemical antecedent and could lead to a new line of
research.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
We thank Dr. Hugo Naya (Institut Pasteur de Montevideo) for his
contribution in the use of R statistical software. We also thank
PEDECIBA Química and PECECIBA Biología (Uruguayan research
In the biological tests against tumor cell lines we found that both,
Cu-BT and Cu-TT, show higher cytotoxic activity than the free ligands.
8

N. Alvarez, et al.
InorganicaChimicaActa508(2020)119622
Fig. 10. Morphology of MCF-7 cells after treatment with BT, TT, Cu-BT and Cu-TT at the indicated concentrations. Optical microscopy, magnification 200×.
funding Programs) for their financial support.
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