Cancer Cell Resistance against the Clinically Investigated Thiosemicarbazone COTI-2 Is Based on Formation of Intracellular Copper Complex Glutathione Adducts and ABCC1-Mediated Efflux

Article abstract of DOI:10.1021/acs.jmedchem.0c01277

COTI-2 is a novel anticancer thiosemicarbazone in phase I clinical trial. However, the effects of metal complexation (a main characteristic of thiosemicarbazones) and acquired resistance mechanisms are widely unknown. Therefore, in this study, the copper and iron complexes of COTI-2 were synthesized and evaluated for their anticancer activity and impact on drug resistance in comparison to metal-free thiosemicarbazones. Investigations using Triapine-resistant SW480/Tria and newly established COTI-2-resistant SW480/Coti cells revealed distinct structure-activity relationships. SW480/Coti cells were found to overexpress ABCC1, and COTI-2 being a substrate for this efflux pump. This was unexpected, as ABCC1 has strong selectivity for glutathione adducts. The recognition by ABCC1 could be explained by the reduction kinetics of a ternary Cu-COTI-2 complex with glutathione. Thus, only thiosemicarbazones forming stable, nonreducible copper(II)-glutathione adducts are recognized and, in turn, effluxed by ABCC1. This reveals a crucial connection between copper complex chemistry, glutathione interaction, and the resistance profile of clinically relevant thiosemicarbazones.

Full text of DOI:10.1021/acs.jmedchem.0c01277

This is an open access article published under a Creative Commons Attribution (CC-BY)
License, which permits unrestricted use, distribution and reproduction in any medium,
provided the author and source are cited.
pubs.acs.org/jmc
Article
Cancer Cell Resistance Against the Clinically Investigated
Thiosemicarbazone COTI‑2 Is Based on Formation of Intracellular
Copper Complex Glutathione Adducts and ABCC1-Mediated Efflux
#
#
́
́
Julia H. Bormio Nunes, Sonja Hager, Marlene Mathuber, Vivien Posa, Alexander Roller, Eva A. Enyedy,
Alessia Stefanelli, Walter Berger, Bernhard K. Keppler, Petra Heffeter,* and Christian R. Kowol*
Cite This: https://dx.doi.org/10.1021/acs.jmedchem.0c01277
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: COTI-2 is a novel anticancer thiosemicarbazone in
phase I clinical trial. However, the effects of metal complexation (a
main characteristic of thiosemicarbazones) and acquired resistance
mechanisms are widely unknown. Therefore, in this study, the
copper and iron complexes of COTI-2 were synthesized and
evaluated for their anticancer activity and impact on drug resistance
in comparison to metal-free thiosemicarbazones. Investigations
using Triapine-resistant SW480/Tria and newly established COTI-
2-resistant SW480/Coti cells revealed distinct structure−activity
relationships. SW480/Coti cells were found to overexpress
ABCC1, and COTI-2 being a substrate for this efflux pump. This was unexpected, as ABCC1 has strong selectivity for glutathione
adducts. The recognition by ABCC1 could be explained by the reduction kinetics of a ternary Cu-COTI-2 complex with glutathione.
Thus, only thiosemicarbazones forming stable, nonreducible copper(II)-glutathione adducts are recognized and, in turn, effluxed by
ABCC1. This reveals a crucial connection between copper complex chemistry, glutathione interaction, and the resistance profile of
clinically relevant thiosemicarbazones.
Scheme 1. Chemical Structures of the Clinically
Investigated Triapine, DpC, and COTI-2 as well as the
Terminally Unsubstituted Derivative COTI-NH2
INTRODUCTION
■
α-N-Heterocyclic thiosemicarbazones represent an important
class of metal-chelating agents, owing to their N,N,S-donor
ligand set.1 Both, the free ligands and their metal complexes
(e.g., Cu, Fe, Zn, Ga, Ru, etc.) have been widely investigated as
antibacterial, antiviral, and anticancer agents.2 Concerning
their antitumor properties, they were originally developed as
iron chelators. However, they are not just iron chelators, such
as, e.g., desferrioxamine (DFO) that sequesters mainly
extracellular iron due to its strong iron(III)-binding ability.
Instead, thiosemicarbazones are considered as “iron-interact-
ing” drugs influencing diverse iron-dependent biological
pathways due to their ability to form highly stable complexes
with both iron(II) and iron(III) ions.3,4 Also, the interaction
with cellular copper ions has been associated with their
mechanism of action, at least for a part of the known
anticancer thiosemicarbazones.5,6
With regard to clinical development, 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (Triapine, Scheme 1) is the
most prominent representative. Triapine, whose main target is
considered to be the iron-dependent ribonucleotide reduc-
tase,3,4 has been evaluated in multiple phase I and II clinical
trials against a number of different cancer types.7 Recently,
also, a clinical phase III study was initiated with Triapine in
combination with cisplatin and radiation therapy in cervical or
vaginal cancer patients (study number NCT02595879,
clinicaltrials.gov). Based on the clinical success of Triapine,
there is also interest to develop other thiosemicarbazones with
improved anticancer activity, novel modes of action, and
Received: July 22, 2020
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
© XXXX American Chemical Society
J. Med. Chem. XXXX, XXX, XXX−XXX
A

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
different drug resistance profiles. Thus, several reports were
published recognizing that α-N-heterocyclic thiosemicarba-
zones with terminally disubstituted NH2 groups display highly
increased anticancer activity in the nanomolar range in cell
culture.8,9 Noteworthy, this thiosemicarbazone subclass is
characterized by a higher copper(II) complex stability and
additional modes of action such as induction of paraptotic cell
death.6,10 One of these thiosemicarbazones, namely, di-2-
pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone
(DpC; Scheme 1), entered a phase I clinical trial in 2016
(study number NCT02688101).11,12 At the same time, another
highly active but structurally more divergent thiosemicarba-
zone, 4-(pyridine-2-yl)-N-([(8E)-5,6,7,8-tetrahydroquinolin-8-
ylidene]amino)piperazine-1-carbothioamide, also known as
COTI-2 (Scheme 1), was developed by Cotinga Pharmaceut-
icals as a third-generation representative. COTI-2 has entered
a phase Ib/IIa clinical trial for the treatment of gynecologic
malignancies in 2015 (study number NCT02433626).13
Biological investigations revealed that COTI-2 possesses high
efficacy against multiple cancers both in vitro (also in the
nanomolar range) and in vivo. Moreover, COTI-2 seems to
differ in its mode of action from other thiosemicarbazones, as it
has been reported to restore the functionality (and thus tumor
suppressor activity) of mutated p53.14,15
RESULTS AND DISCUSSION
■
Syntheses and Chemical Characterization. COTI-2
was obtained in a three-step procedure,21 starting with the
piperazine insertion to thiocarbonyldiimidazole (Supporting
Information, Scheme S1). Reaction with hydrazine hydrate
generated the respective COTI-2 thiosemicarbazide, which was
condensed with 6,7-dihydroquinoline-8-one yielding COTI-2.
COTI-NH2 was obtained by condensation of the ketone with
the appropriate commercially available N4-NH2 thiosemicarba-
zide.22
The NMR spectroscopy analysis revealed that the two
compounds are present in a different number of isomers, a
phenomenon well-known for thiosemicarbazones.23,24 Three
different types of isomers are distinguished: (i) the E-
configuration regarding the CN bond, (ii) the respective
Z-isomer, and (iii) a zwitterionic form called E′-isomer
(structures for COTI-2 see Scheme 2). The 1H NMR
Scheme 2. Different Possible Isomers of COTI-2. The Z-
and E′-Isomers Have Strongly Downfield Shifted NH
Rresonance NMR Signals due to the Presence of
Intramolecular Hydrogen Bonds
However, until now, no studies on the interaction of COTI-
2 with cellular metal ions, such as iron and copper, have been
performed. Additionally, it is unknown whether the anticancer
activity of COTI-2 is influenced by acquired drug resistance to
other thiosemicarbazones. This is of interest, as our previous
investigations revealed that small structural changes, which are
associated with a shift into the nanomolar range of activity, also
result in a distinctly altered drug resistance profile. In more
detail, while Triapine was found to be a weak substrate for the
ABC transporter efflux protein P-glycoprotein (P-gp,
ABCB1),16 terminally dimethylated α-N-heterocyclic thiose-
micarbazones are transported by the multidrug resistance
protein 1 (MRP1, ABCC1) but not by ABCB1.17 This is of
interest, as ABCC1 is an efflux pump, which usually transports
drugs in a glutathione (GSH)-dependent manner (e.g., in the
form of GSH adducts or by a co-transport mechanism).18−20
However, although GSH depletion strongly enhanced the
anticancer activity of these previously tested nanomolar-active
derivatives, no GSH conjugate formation by incubation with
GSH-S-transferase could be observed.17
The aim of this study was, on the one hand, to synthesize
the first metal complexes of COTI-2 with the biologically
relevant ions, iron(III) and copper(II), together with the
Triapine-like nonsubstituted (COTI-NH2) representative as a
reference (Scheme 1). On the other hand, these compounds
were subsequently tested for their biological activity in
chemosensitive and thiosemicarbazone-resistant SW480 cells,
including the newly generated COTI-2-resistant SW480/Coti
model. Based on these studies, we could show that the
resistance of SW480/Coti cells is due to overexpression of
ABCC1 and strongly associated with the thiosemicarbazone
copper complex chemistry. In more detail, in the presence of
copper(II), COTI-2 forms stable, nonreducible ternary GSH
copper(II) complexes, which are, in turn, substrates for
ABCC1. This indicates that the specific copper chemistry of
thiosemicarbazones not only affects their mode of action but
also their resistance profile.
spectrum of COTI-NH2 in DMSO-d6 showed only one type
of isomer, whereas in case of COTI-2, two isomers could be
observed. Interestingly, for both compounds and all isomers,
the chemical shifts of the hydrazonic NH protons were in the
range of 14.3−15.5 ppm. Consequently, we assigned them to
the Z- and E′-isomers, which both possess intramolecular
hydrogen bonds, explaining the strong downfield shift. In
contrast, the E-isomer (without a hydrogen bond) usually has
an NH signal resonance at around 10−11 ppm.23,24 Recently,
we could separate the E-and Z-isomers of a Triapine
derivative9 and investigated their cytotoxicities; however, no
significant differences were observed. Therefore, we do not
expect an influence of the different isomers on the biological
characteristics of the COTI-2 compounds.
The calculated log P values25 for COTI-2 and COTI-NH2
are +2.89 and +1.43, respectively, indicating high lipophilicity,
especially for COTI-2.
The metal complexes of COTI-2 were obtained by reaction
with the respective metal salts. For the iron(III) complex (Fe-
COTI-2), the reaction of COTI-2 with Fe(NO3)3·9H2O in
methanol resulted in a complex with 1:2 metal-to-ligand ratio,
with two neutral ligands and consequently a +3 charge
[Fe(HL)2](NO3)3 (HL = COTI-2, Scheme 3). This
composition is different from what is usually observed for
the iron(III) complex with Triapine or similar thiosemicarba-
zones, in which both ligands are deprotonated and the complex
has a +1 charge ([Fe(L)2]+).8,26,27 For the copper(II) complex
(Cu-COTI-2), the ligand was reacted with CuCl2·2H2O,
yielding a complex with 1:1 metal-to-ligand ratio, with a
neutral thiosemicarbazone and two chlorido ligands
B
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Scheme 3. Chemical Structure of the Iron(III) and
Copper(II) Complex of COTI-2
([Cu(HL)Cl2], Scheme 3). This composition was also
observed for, e.g., the Triapine copper(II) complex.28 All
proposed structures were confirmed by mass spectrometry and
elemental analyses.
X-Ray Crystal Structures. For COTI-2 and Cu-COTI-2,
crystals were obtained by slow evaporation of the mother
liquor, which were suitable for analysis by single-crystal X-ray
̅
diffraction. COTI-2 crystalized in the triclinic P1 space group.
Selected bond distances and angles are quoted in the legend of
Figure 1A. The COTI-2 crystal adopted the zwitterionic E′-
isomeric form (in agreement with one isomer observed in the
NMR spectrum) in which an intramolecular hydrogen bond is
observed between N1, S1, and the hydrogen H−N2. Notably,
in this case, N2 is protonated and not N3, as usually assumed
and drawn in the typical thiosemicarbazone structures
(Scheme 1). The piperazine moiety is in a chair configuration,
while the cyclohexyl of the tetrahydroquinoline moiety is in a
half-chair configuration. Comparing the crystal structure of
COTI-2 with the crystal structure of COTI-NH2 reported in
literature,29 COTI-NH2 crystalized in the P21/n space group
and adopted the Z-isomeric form. Most bond angles and
distances are comparable. However, a major difference is the
length of the C−S bond, which is shorter in the case of COTI-
NH2 (1.683 Å) compared to COTI-2 (1.717 Å). This can
nicely be explained by the thione form of the sulfur atom in the
case of COTI-NH2 and the zwitterionic thiol for COTI-2.
For Cu-COTI-2, two different crystal structures were
obtained, one deprotonated (from the synthesis without
HCl), named Cu-COTI-2a, and the other one protonated
(from the synthesis with HCl), named Cu-COTI-2b. Notably,
both crystal structures differ from the bulk material, where
COTI-2 is neutral (HL) with two chlorido ligands for each
copper center (Scheme 3). Two bridging chlorido ligands are
observed between the copper(II) centers for both structures
(Figure 1B,C), a structural mode which has already been
reported for other copper(II) thiosemicarbazone com-
plexes.30−32 In both crystal structures, the copper(II) ions
have a coordination number of five, being in a square-
pyramidal geometry, where N1, N2, S1 and Cl1 are the base of
the pyramid (forming the asymmetric unit of the complex),
and the Cl′ from another unit is in the axial position (for Cu-
COTI-2a, 2.749 Å vs 2.256 Å for Cu-Cl1; for Cu-COTI-2b,
2.720 Å vs 2.245 Å for Cu-Cl1). Most of the bond lengths and
angles are comparable between the deprotonated complex and
protonated form (quoted in the legend of Figure 1). However,
for example, the C10−S1 bond length of Cu-COTI-2a at 1.754
Å is distinctly longer compared to Cu-COTI-2b at 1.706 Å.
This nicely fits to the expected elongated C10−S1 bond of the
deprotonated ligand and the greater double-bond character in
the case of Cu-COTI-2b due to the protonated N3 (Figure 1).
Figure 1. ORTEP plots of (A) COTI-2, (B) Cu-COTI-2a, and (C)
Cu-COTI-2b with atom numbering. Ellipsoids are drawn at the 50%
probability level. Selected bond lengths (Å) and bond angles (°) for
(A) COTI-2 (bond precision for C−C single bond is 0.0030 Å): C8−
N2 1.300(9), N2−N3 1.354(3), N3−C10 1.351(8), C10−S 1.717(1),
C10−N4 1.361(4); N1−C9−C8 116.3, N2−N3−C10 112.0. For (B)
Cu-COTI-2a (bond precision for C−C single bond is 0.0030 Å), two
different types of Cu−Cl bonds occur: Cu1−Cl1 2.255(8) Å, Cu1−
Cl1′ (1−X,−Y,−Z) 2.748(6) Å. Further selected bond lengths (Å)
and bond angles (°): N1−Cu 2.029(9) Å, N2−Cu 1.961(3), S1−Cu
2.268(2) Å, Cu···Cu 3.52; Cl−Cu1−Cl′ 91.2, Cl−Cu−S 97.8, N1−
Cu−N2 80.9, N2−Cu−S 84.2, N1−Cu1−Cl 96.3. For (C) Cu-
COTI-2b (bond precision for C−C single bonds is 0.0121 Å), the
main residue disorder is 39%. The counter ion [CuCl4]−2 position is
shifted, and solvent (H2O) and the disordered second part were
omitted for clarity. Two different types of Cu−Cl bonds occur: Cu1−
Cl1 2.245(3) Å (light red shaded) and Cu1−Cl1′ (1−X,1−Y,1−Z)
2.719(8) Å (light orange shaded). Green-shaded areas visualize
protonated nitrogen positions. Further selected bond lengths (Å) and
bond angles (°): N1−Cu 2.022(7) Å, N2−Cu 1.958(8), S1−Cu
2.260(7) Å, Cu···Cu 3.42; Cl−Cu1−Cl′ 93.3, Cl−Cu−S 95.8, N1−
Cu−N2 80.5, N2−Cu−S 84.8, N1−Cu1−Cl 98.1.
The influence of the protonated N3 can be also observed in
the C10−N3 bond, which is 1.328 Å for Cu-COTI-2a and
1.376 Å for Cu-COTI-2b. In the crystal structure of Cu-COTI-
2b, two [CuCl4]2− counter ions are present, which compensate
the +4 charge from [Cu2Cl2(H2L)]4+ (with H2L = protonated
COTI-2). Also, conformational differences are observed for the
piperazine moiety of the complexes; for Cu-COTI-2a, the
piperazine is in a chair-conformation, while for Cu-COTI-2b, it
is in a half-chair conformation (Figure S1). In case of the
respective Triapine complex, the binding mode to copper(II)
is different, with some intermolecular Cu−S bonds forming a
C
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 2. Resistance of SW480/Tria and SW480/Coti cells against Triapine and COTI-2. Anticancer activity of the drugs was tested by the MTT
viability assay after 72 h drug incubation in SW480 vs the resistant sublines. Mean standard deviation (SD) was derived from triplicates of one
representative experiment out of three.
Table 1. Anticancer Activity (IC50 Values after 72 h) of the Indicated Thiosemicarbazones and Metal Complexes in SW480 and
Thiosemicarbazone-Resistant SW480/Coti and SW480/Tria Colon Cancer Cells
a
a
b
a
b
compound
SW480 (IC50, μM)
SW480/Coti (IC50, μM)
relative resistance
SW480/Tria (IC50, μM)
relative resistance
COTI-2
Triapine
COTI-NH2
Fe-COTI-2
Cu-COTI-2
0.56 0.16
0.82 0.18
7.85 0.92
9.32 3.29
0.04 0.01
9.51 3.11
0.81 0.08
4.39 0.56
7.13 2.91
0.48 0.05
18.9*
1.0n.s.
0.6***
0.7n.s.
0.41 0.16.
>50
>20
3.62 0.80
0.04 0.01
0.8n.s
>61
>2.5
0.3**
1.1n.s.
c
12.0**
a
IC50 values were calculated from concentration−response curves. Values are given as mean SD of three independent experiments performed in
b
triplicates. Differences in sensitivity calculated by dividing the IC50 values of the resistant subline by those of the parental line. ***p ≤ 0.001, **p
≤ 0.01, *p ≤ 0.05; n.s. (not significantly different), calculated by one-sample t-test. Taken from ref.9
c
chain structure instead of the bridging chlorido ligands for
COTI-2 (even though the distances and angles are very
similar).28 Notably, such dimeric structures with a metal-to-
ligand ratio of 1:1 are usually only present in the solid state and
dissociate in aqueous solution at room temperature into the
respective monomers.
activity (14-fold less active than COTI-2). That terminally
unsubstituted derivatives like Triapine and COTI-NH2 show
much lower cytotoxic activity, it is in line with previous
publications on other thiosemicarbazones8,9,35,36 and demon-
strates the strong impact of N4-terminal modification on the
anticancer activity of thiosemicarbazones. Noteworthy, also,
the thiosemicarbazone resistance profile was strongly affected
by the N4-terminal alterations. In detail, while SW480/Coti
cells were 18.9-fold resistant against COTI-2 compared to the
parental cells, Triapine was not affected by the acquired COTI-
2 resistance, and SW480/Coti cells displayed even collateral
sensitivity to COTI-NH2. On the opposite, SW480/Tria cells
were cross-resistant against COTI-NH2 but not COTI-2. This
indicates that a terminal NH2 moiety is important for
resistance of SW480/Tria but not of SW480/Coti cells.
In case of the metal complexes of COTI-2, complexation of
iron significantly reduced its activity by 19-fold in parental
SW480 cells. This is in agreement to a previous report,8 which
showed a 3-fold decreased activity of the iron(III) complex of
Triapine (in 41M and SK-BR-3 cells) compared to the metal-
free ligand. The lower activity upon iron complexation could
be explained by a decreased uptake of the complexes (maybe
due to their positive charges, which impairs cell membrane
crossing). With regard to the resistance profile, SW480/Coti
cells were not cross-resistant to Fe-COTI-2, while SW480/Tria
cells even showed collateral sensitivity. This could indicate that
the remaining activity of COTI-2 against SW480/Coti is based
on an intracellularly formed iron complex, which is not
recognized by the mechanisms underlying the drug resistance
of the COTI-2-resistant subclone (vide infra). In general, the
collateral sensitivity of the ABCB1-overexpressing SW480/Tria
Activity against Chemosensitive and Thiosemicarba-
zone-Resistant Cancer Cells. We have recently reported on
an SW480 subline with acquired resistance to Triapine
(SW480/Tria, Figure 2 left), which was characterized by
upregulation of ABCB116 and the loss of phosphodiesterase
4D.33 To investigate the resistance profile against COTI-2 and
test the cross-resistance against its metal complexes, COTI-
NH2 and Triapine, in the course of this study, a new cell line
with acquired resistance to COTI-2 was established. Notably,
there is already one report in literature that COTI-2 did not
induce resistance in the human lung cancer cell lines DMS-
153, SHP-77, and A549.34 However, in this study, the lung
cancer cells were exposed only to a short-term treatment (four
rounds in 4 days) of COTI-2. Consequently, we chose a
different selection approach, and the resistant cells, termed
SW480/Coti, were generated by continuous exposure of
chemonaive SW480 cells to increasing concentrations of
COTI-2 over a period of approximately one year. At that
time, SW480/Coti cells displayed a 19-fold resistance
compared to parental SW480 cells (Figure 2 right, Table 1).
Subsequently, the impact of the N4-terminal modification of
COTI-2 and metal complexation on the anticancer activity and
resistance profile was assessed by comparing IC50 values in the
parental SW480 cells to the ones in SW480/Tria and SW480/
Coti cells (Table 1 and Figure S2).
In the sensitive SW480 cells, the anticancer activity of
COTI-2 was in the upper nanomolar range with an IC50 value
of 0.56 μM, slightly lower than that of Triapine with an IC50 of
0.82 μM. In contrast, COTI-NH2 displayed distinctly weaker
cells to Fe-COTI-2 is interesting; as in a recent report of
37
́
Szakacs et al. the iron complex of the 8-hydroxyquinoline-
based iron chelator and ribonucleotide reductase inhibitor
NSC297366 turned out to be an ABCB1 substrate, which
D
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 3. Cell cycle arrest and cell death induction by COTI-2 in SW480 and SW480/Coti cells after 24 h treatment. (A) The percentage of cells
in G2/M phase was analyzed by staining ethanol-fixed cells with propidium iodide (PI) followed by flow cytometry. Mean SD was derived from
three independent experiments. (B) Nucleic morphology was analyzed by DAPI staining of methanol/acetone (1:1)-fixed cells. Mitotic cells are
indicated with an arrow (scale bar: 50 μm). (C) Cell death induction was analyzed by AV and PI stain followed by flow cytometry. Mean SD was
derived from three independent experiments. (D) Representative microscopy images of paraptotic vesicle formation (arrow) in SW480 and
SW480/Coti cells after 24 h treatment with 1 μM COTI-2 (scale bar: 50 μm). (E) Percentage of vacuolated cells (counted in Image J). Mean
SD was derived from three independent experiments. Significance between cell lines (asterix between bars) was calculated in (A) and (E) by two-
way ANOVA and the Sidak’s multiple comparison test. Significant difference to the control group (indicated by the asterisk above bar) was
calculated in (A) and (C) by one-way ANOVA and the Dunnett’s multiple comparison test (p < 0.05 *, p < 0.01 **, p < 0.001 ***, p < 0.0001
****).
resulted in the hypothesis that the collateral sensitivity is based
on ABCB1-mediated iron efflux. This indicates that, although
these two compound classes share certain aspects of their
modes of action, the mechanisms underlying the collateral
sensitivity of ABCB1-overexpressing cells against certain iron
chelators and thiosemicarbazones might be even more
complex.
In contrast to iron, complexation with copper led to a 14-
fold higher anticancer activity compared to the metal-free
COTI-2 in the chemosensitive SW480 cells. In general, this
enhanced sensitivity is in good agreement with previous data
from others and our group.6,38 In these studies (although no
significant difference was observed in the IC50 values after
72 h), shorter incubation times (3 or 24 h) or very toxic drug
concentrations (IC90 values) revealed a marked increase in
activity of the copper complexes of nanomolar-active
thiosemicarbazones such as Me2NNMe2, DpC, Dp44mT,
PTSC.6,38 This supports the current hypothesis that the
copper complex plays an important role in the mode of action
of these nanomolar-active thiosemicarbazones.4,10,39,40 Regard-
ing the resistance profile, similar to the metal-free ligand, Cu-
COTI-2 was 12-fold less active in SW480/Coti cells compared
to parental cells, but it was not affected by the resistance of
SW480/Tria cells. This indicates that, in contrast to the iron
complex, Cu-COTI-2 is recognized by similar resistance
mechanisms as the metal-free ligand. Taken together, the cell
viability investigations revealed that the acquired resistance
against COTI-2 is distinctly different from that of Triapine and
metal complexation can result in strong changes of the
resistance profile.
To gain more insights into the mode of resistance of the
SW480/Coti cells, the impact of COTI-2 on cell cycle
distribution (Figure 3A,B and Figure S3) and cell death levels
of SW480 versus SW480/Coti was investigated. In agreement
with the comparable growth curves of both parental and
resistant cells, the cell cycle distribution without treatment was
E
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 4. ABC transporter expression of the investigated SW480 cell clones. (A) Protein expression of ABC transporters was investigated in
membrane-enriched fractions of the indicated cell lines by Western blotting. GADPH and β-actin were used as loading controls and A549 as
positive control for ABCC1 and ABCG2 expression. Borders between cell lines indicate the rearrangement of original lanes of the same blot. (B)
Immunofluorescence staining of ABCC1 (green) in SW480 and SW480/Coti cells. Cells were transferred to microscopy slides by cytospin and
fixed with 4% PFA. DAPI (blue) and rhodamine-conjugated wheat-germ agglutinin (red) were used as counterstains for nuclei and membranes,
respectively. (Scale bar: 50 μm).
Figure 5. Influence of ABCC1 expression on the activity of COTI-2. Anticancer activity of COTI-2 was tested in GLC-4 and GLC-4/adr lung
cancer cells by the MTT assay after 72 h drug incubation. Vincristine, a known ABCC1 substrate, was used as a reference drug. Values given are
means SD of three experiments performed in triplicates. The insert shows a Western blot of membrane-enriched protein fractions confirming the
overexpression of ABCC1 in GLC-4/adr compared to parental GLC-4 cells.
rather similar between SW480 and SW480/Coti cells (Figure
S3A). Drug treatment led to a significant reduction of G2/M
phase cells in comparison to untreated controls in SW480 but
not in the resistant subline (Figure 3A). In SW480/Coti cells,
only at the highest COTI-2 concentration of 10 μM, changes
in cell cycle distribution (shift of cells from G0/G1 into S-
phase) were observed (Figure 3A and Figure S3B). Preliminary
microscopy analysis indicated that the observed reduction in
the G2/M population is associated with a loss of the mitotic
cell faction (Figure 3B).
With regard to cell death induction, annexin V (AV)/
propidium iodide (PI) stains (Figure 3C) showed that, after
24 h treatment, most of the parental cells were already in a
late-phase cell-death (AV+/PI+) state, while SW480/Coti cells
were still in the early phase of cell death (AV+/PI−). In
addition, also the induction of paraptotic cell death, measured
by the increase of cytoplasmic vesicles, was less pronounced in
the resistant cell model (Figure 3D,E). Overall, these
observations point out that SW480/Coti cells are characterized
by a less pronounced and delayed onset of COTI-2 activity,
suggesting a resistance mechanism affecting the intracellular
drug levels.
SW480/Coti Cells Express High Levels of the ABC
Transporter Efflux Pump ABCC1. From previous studies,
we know that resistance development against anticancer
thiosemicarbazones is often associated with upregulation of
ABC transporter efflux pumps.16,17,33 Consequently, SW480/
Coti and SW480/Tria cells were assessed on the protein level
for upregulation of the most frequently resistance-associated
ABC transporters (ABCB1, ABCC1, ABCC2, and ABCG2)
(Figure 4). In line with literature,16 SW480/Tria cells showed
an upregulation of ABCB1, while the expression levels of
ABCC1 and ABCC2 were decreased compared to parental
SW480 cells. In contrast, SW480/Coti cells upregulated
ABCC1 in concert with a downregulation of ABCB1 and
ABCC2 (Figure 4A). In both resistant and the parental SW480
cells, protein levels of ABCG2 were too low for detection. The
expression and membrane localization of ABCC1 in SW480/
F
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Coti were also confirmed by immunofluorescent staining
(Figure 4B). Moreover, previous studies on parental SW480
cells showed that Triapine treatment stimulated ABCB1
expression already after short-term incubation (24 h),16 so
similar experiments were also performed with SW480/Coti
cells. In contrast to Triapine, short-term incubation with
COTI-2 was not sufficient for the stimulation of ABCC1
expression (Figure S4), indicating that the ABCC1 expression
is not due to an unspecific stress response. This is also in line
with the report on the human lung cancer cell lines DMS-153,
SHP-77, and A549 in which short-term treatment of four
rounds during 4 days was not able to induce COTI-2
resistance.34
Identification of COTI-2 and Cu-COTI-2 as ABCC1
Substrates. Our previous investigations16 revealed that
Triapine is only a weak ABCB1 substrate, and thus,
upregulation of the transporter was not the major factor
underlying the resistance in SW480/Tria cells. Therefore, we
investigated if COTI-2 is indeed an ABCC1 substrate. To do
so, as a first step, we assessed the activity of all compounds
(COTI-2, its metal complexes, COTI-NH2 and Triapine) in a
known ABCC1-overexpressing cell model (GLC-4/adr) and
compared the IC50 values to the ones of parental GLC-4 cells
(Figure 5 left, Table 2, and Figure S5). As confirmation of the
functional ABCC1 in this cell model, the known ABCC1
substrate vincristine was used (Figure 5 right).
to efficiently reverse the resistance of SW480/Coti cells against
COTI-2 and the known ABCC1 substrate vincristine (Figure
6). In contrast, COTI-NH2 and Triapine were not affected in
their activity by the addition of the modulator drugs, further
supporting the hypothesis that they are not transported by
ABCC1 expressed by SW480/Coti cells.
Role of GSH in the Resistance of SW480/Coti Cells
against COTI-2, COTI-NH2, and Triapine. ABCC1 is known
for its high phase II conjugate substrate specificity. Thus, the
protein has been shown to efflux various neutral and anionic
hydrophobic compounds and products of phase II drug
metabolism, including many GSH and glucuronide conju-
gates.18 Moreover, contrary to other transport proteins
involved in cancer-related multidrug resistance (MDR), the
activity of ABCC1 is related to the GSH content of cells.19 To
investigate the role of GSH in the resistance of SW480/Coti
cells, buthionine sulphoximine (BSO), an inhibitor of the de
novo synthesis of GSH, was utilized.41 SW480 and SW480/
Coti cells were pretreated with BSO for 18 h before the
addition of COTI-2, COTI-NH2, or Triapine for 72 h after
which cell viability was measured by MTT (Figure 7). Indeed,
BSO resensitized SW480/Coti cells against the anticancer
activity of COTI-2. Moreover, these experiments revealed that
SW480/Coti cells were distinctly more sensitive to BSO
treatment than the parental SW480 cells (up to 40% reduction
in the cell number in SW480/Coti cells compared to 20% in
SW480 cells at 25 μM BSO). Interestingly, BSO treatment had
a strong sensitizing effect on cells treated with COTI-NH2,
which was similar in SW480 and SW480/Coti cells. This effect
seems to be independent from the ABCC1 expression of the
SW480/Coti cells and indicates to a specific protective
mechanism of GSH against this new derivative but not
COTI-2. Taken together, these experimental findings show
that GSH synthesis is crucial for SW480/Coti resistance and
transportation of COTI-2 by ABCC1.
Reaction of the Cu-COTI Complexes with GSH. While
these results demonstrate that GSH plays a role in drug export
of COTI-2, it is not clear how a GSH adduct can be formed, or
more general, how can a thiosemicarbazone be pumped by
ABCC1 in a GSH-dependent manner? We recently inves-
tigated a possible direct conjugation of GSH to a nanomolar-
active thiosemicarbazone (2-acetylpyridine 4,4-dimethylthiose-
micarbazone, KP1550) using the enzyme gluathione-S-trans-
ferase.17 However, no adduct formation could be observed in
these studies, prompting us to the hypothesis that the
thiosemicarbazone could be effluxed in co-transport with
unconjugated GSH. A different explanation would be the
generation of a GSH adduct via the intracellularly formed
copper-thiosemicarbazone complex and its transport by
ABCC1. Thus, we investigated the reaction of the copper(II)
complexes of COTI-2, COTI-NH2, and Triapine with GSH
spectrophotometrically at pH 7.4 in aqueous solution under
anaerobic conditions. The complexes besides Cu-COTI-2 were
prepared in situ by mixing the metal ion and ligand at the 1:1
ratio. Notably, copper(II) complexes of α-N-pyridyl thiosemi-
carbazones bearing an N,N,S− coordination mode are generally
highly stable at physiological pH even at fairly low
concentrations.4,6
Table 2. Differences in Viability of GLC-4 and ABCC1-
Overexpressing GLC-4/adr Cells Treated with COTI-2,
COTI-NH2, Fe-COTI-2, Cu-COTI-2, and Triapine for 72 h
GLC-4
(IC50, μM)
GLC-4/adr
relative
resistance
ABCC1
c
a
a
b
compound
(IC50, μM)
substrate
COTI-2
Triapine
COTI-NH2
Fe-COTI-2
Cu-COTI-2
0.03 0.01
0.65 0.09
3.64 0.39
0.79 0.07
0.02 0.01
0.48 0.15
0.57 0.05
3.65 0.41
0.80 0.11
0.06 0.02
17.3*
0.9n.s.
1.0n.s.
1.0n.s.
3.2*
YES
NO
NO
NO
YES
a
IC50 values were calculated from concentration−response curves
measured by the MTT assay. Values are given as mean SD of two
or three independent experiments, in triplicates. Relative resistance
b
was calculated by dividing the IC50 values of the resistant GLC-4/adr
by those of the parental GLC-4 cells. *p ≤ 0.05; n.s., not significantly
c
different; calculated by one-sample t-test. Proposed substrate
assignment based on data of GLC-4 and SW480 models.
Indeed, the ABCC1-overexpressing GLC-4/adr cells were
17.3-fold resistant to COTI-2 in comparison to the parental
GLC-4 cells. In addition, also, Cu-COTI-2 showed (in
agreement with the resistance of SW480/Coti cells) reduced
activity in GLC-4/adr cells, suggesting them as potential
ABCC1 substrates (Table 2). In contrast, COTI-NH2, Fe-
COTI-2, and Triapine were not affected by the multidrug
resistance of GLC-4/adr cells.
To confirm that the resistance of SW480/Coti cells is based
on enhanced (ABCC1-mediated) drug efflux, it was tested
whether the respective resistance against COTI-2 could be
reversed by combination with the known ABC transporter
modulators verapamil and cyclosporine A (CSA). The
modulators alone (at the used concentrations) did not impact
on the growth of neither SW480 nor SW480/Coti cells to a
relevant extent (less than 15% reduction in cell number, data
not shown). Indeed, the addition of verapamil or CSA was able
Compared to the metal-free ligands, in all copper complexes,
a larger increase in absorbance was observed in the wavelength
range ∼360−460 nm due to the S → Cu charge transfer band
(Figure 8; free ligands in dashed lines, copper complexes
without GSH in red). Immediately after the addition of GSH,
G
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 6. Impact of ABC transporter modulators (1 μM CSA and 10 μM verapamil) on the anticancer activities of vincristine, COTI-2, COTI-
NH2, and Triapine in the SW480/Coti cells in comparison to the parental cells. Vincristine was used as a positive control of an ABCC1 substrate.
Viability was measured by the MTT assay after 72 h of combined drug treatment. Mean SD was derived from triplicates of one representative
experiment out of three. Values are given normalized to their respective controls. Thus, modulator controls were set to 1.
for all complexes, a small shift of the λmax value around 380−
400 nm could be observed (Figure 8, bold black lines). This
can be explained by the formation of a mixed-ligand complex
with GSH via the coordination of the thiolate at the fourth
coordination site of the copper(II), as also reported for similar
complexes.6,42,43 For Cu-COTI-2, this was also proven by mass
spectrometry measurements, clearly indicating the respective
GSH-Cu-COTI-2 complex at m/z = 735 (Figure S6). This
GSH-adduct formation was similar for all copper(II)
complexes, but over time, a very different behavior was
observed. In case of Cu-COTI-NH2, the absorbance of the S
→ Cu charge transfer band decreased, while it was increased at
the λmax of the free ligand (Figure 8A). This can be explained
by a reduction of the metal center and release of the
thiosemicarbazone from copper(I) with the formation of a
copper(I)-GSH complex (due to the high excess of GSH, we
cropped the spectra at wavelengths <310 nm, where GSH and
its Cu(I) complex absorb). However, the redox reaction was
far from being complete after the 6 h measurement time. In
contrast, in case of Cu-COTI-2, after the initial changes due to
the GSH addition, the spectra were almost unchanged within
6 h (Figure 8B). For the reference complex Cu-Triapine
(Figure 8C), the reduction was even distinctly faster than for
Cu-COTI-NH2 with a nearly complete reduction and release
of free Triapine already after 20 min.6 For comparison, the
changes of the absorbance values at the λmax of the S → Cu
charge transfer band were plotted for all investigated
complexes as a function of time (Figure 8D). This clearly
shows that the redox reaction is very fast in case of Cu-
Triapine and moderate for Cu-COTI-NH2, while it does not
take place at a measurable level with Cu-COTI-2. This is in
line with very recent GSH reduction data of copper(II)
complexes of other thiosemicarbazones, where terminally
unsubstituted thiosemicarbazones also showed much faster
reduction kinetics than their respective disubstituted deriva-
tives.6 In addition, we investigated the same reaction of the
H
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 7. Impact of GSH synthesis inhibition by BSO on the anticancer activity of COTI-2, COTI-NH2, and Triapine in SW480 and SW480/Coti
cells. BSO was added 18 h before drug treatment, and viability was measured by MTT after 72 h of drug treatment. Mean SD was derived from
triplicates of one representative experiment out of three.
Cu-COTI-2 complex with GSH in cell culture medium
(RPMI-1640) as a solvent instead of HEPES buffer (Figure
S7). Again, the addition of GSH led to a shift in λmax according
to the formation of the mixed-ligand complex with GSH. Over
time, just small changes could be observed indicating very high
redox stability also in the presence of high excess amino acids
(up to 2 mM) and alkaline earth metals (∼400 μM Mg(II) and
Ca(II)).
When we compare the copper complex reduction kinetics in
the presence of GSH with the resistance profiles, we clearly see
that the GSH-Cu(II)-TSC complexes of COTI-NH2 and
Triapine, which are easily reduced together with dissociation of
the thiosemicarbazone ligand, are no ABCC1 substrates. In
contrast, the very stable GSH-Cu(II)-TSC complexes of
COTI-2 and preformed Cu-COTI-2 are nonreducible and
are affected by ABCC1 transportation. This is in line with the
hypothesis that the GSH-Cu(II)-TSC adduct is the species
transported by ABCC1. The observation that Fe-COTI-2 did
not react with GSH (data not shown) explains why the COTI-
resistant cells were not resistant against this complex and it is
no ABCC1 substrate.
free thiosemicarbazone ligands to determine the effect of metal
complex formation on the anticancer activity and drug
resistance profile of COTI-2. To this end, the anticancer
activity was tested in chemosensitive and thiosemicarbazone-
resistant SW480 cells, including the newly generated COTI-2-
resistant SW480/Coti model. Distinct differences in the
resistance profile of the individual thiosemicarbazones and
metal complexes were observed. The two terminally non-
substituted ligands COTI-NH2 and Triapine as well as the
iron(III) complex of COTI-2 were not affected by the ABCC1-
based resistance of the SW480/Coti cells. In turn, COTI-NH2
was a substrate for the resistance of the Triapine-selected
SW480/Tria cells (which are characterized by ABCB1
overexpression and the loss of phosphodiesterase 4D).16,33 In
contrast, the copper complex of COTI-2 resembled COTI-2 in
its resistance profile being an ABCC1 substrate.
Noteworthy, ABCC1 is an efflux pump known for its
substrate specificity for GSH and other conjugates of the phase
II metabolism.18 In addition, it has been repeatedly reported
that copper complex formation plays an important role in the
anticancer activity of nanomolar-active thiosemicarbazones
such as DpC or Dp44mT.4,39,40,44 This prompted us to the
hypothesis that the GSH adduct formed with the copper(II)
complexes of the thiosemicarbazones43 is the species exported
by ABCC1. Indeed, the copper complex of COTI-2 forms a
stable and nonreducible adduct with GSH. There is increasing
evidence that the specific copper chemistry of certain
CONCLUSIONS
■
Here, in the presented study, the first COTI-2 metal complexes
were synthesized using the physiologically relevant metal ions,
iron(III) and copper(II). Subsequently, these new compounds
were biologically investigated in comparison to several metal-
I
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 8. Time-dependent changes of the UV−vis spectra of the (A) Cu-COTI-NH2, (B) Cu-COTI-2, and (C) Cu-Triapine complex (30 μM) in
the presence of 300 equiv of GSH at pH 7.4 in aqueous solution under anaerobic conditions. (T = 25 °C; pH = 7.40 (50 mM HEPES)). Dashed
lines represents the metal-free ligand and red lines the Cu-TSC complex without GSH. Red arrows indicate the change after the addition of GSH,
black arrows after incubation with GSH for up to 360 min and violet arrows after oxygen exposure. (D) Time-dependent absorbance changes at the
λmax of the S → Cu charge transfer band for the Cu-COTI-2 (circle solid), Cu-COTI-NH2 (triangle up solid), and Cu-Triapine complex (times)
(30 μM) in the presence of 300 equiv of GSH at pH 7.4 in aqueous solution under anaerobic conditions (T = 25 °C; pH = 7.40 (50 mM HEPES)).
scheme of the compounds, see Supporting Information Figures S8
and S9.
Synthesis of N′-(6,7-Dihydroquinolin-8(5H)-ylidene)-4-(pyridine-
2-yl)piperazine-1-carbothioamide (COTI-2). The compound was
prepared in three steps (see Scheme S1) according to the patent
US8034815B2.21
thiosemicarbazones distinctly affects their biological activity
(strongly enhanced cytotoxicity based on disruption of the
cellular thiol homeostasis and induction of paraptotic cell
death).6,10 However, this is the first report that this is also true
for their drug resistance profile by offering a mechanistic
explanation on how such compounds (which as metal-free
ligands do not interact with GSH) are recognized and are in
turn transported by ABCC1. It can be expected that this also
impacts on the pharmacological behavior of this compound
class, as ABCC1 is of high importance in the phase II
metabolism of the healthy body.18 Moreover, the findings of
this work are essential for considerations of cancer treatment
strategies with thiosemicarbazones, as drug resistance is still
the major obstacle for successful therapy especially at the late
stage of the disease. Thus, knowledge on the resistance
mechanisms allows the selection of both, an appropriate
patient collective and drug candidates for combination therapy.
Synthesis of 1H-Imidazol-1-yl-(4-(pyridin-2-yl)piperazin-1-yl)-
methanethione (1a). Thiocarbonyldiimidazole (0.89 g, 5.0 mmol, 1
equiv) was dissolved in dichloromethane (30 mL), and N-(2-
pyridyl)piperazine (762 μL, 5.0 mmol, 1 equiv) was added. The
solution was stirred overnight, and on the next day, was washed three
times with water. The organic phase was dried over magnesium sulfate
and evaporated. The resulting yellow oil of 1a was dried in vacuum.
1
Yield: 1.36 g (99%). H NMR (500.10 MHz, DMSO-d6): δ = 2.77
(m, 4H, piperazine), 3.38 (m, 4H, piperazine), 6.70 (ddd, J = 0.6 Hz,
J = 4.9 Hz, J = 7.0 Hz, 1H, CHpy), 6.84 (d, J = 8.5 Hz, 1H, CHpy),
7.06 (dd, J = 1.0 Hz, J = 1.3 Hz, 1H, imidazole), 7.56 (dd, J = 1.3 Hz,
1H, imidazole), 7.59 (ddd, J = 1.8 Hz, J = 7.0 Hz, J = 8.5 Hz, 1H,
CHpy), 8.08 (dd, J = 1.0 Hz, J = 1.0 Hz, 1H, imidazole), 8.15 (ddd, J =
0.6 Hz, J = 1.8 Hz, J = 4.9 Hz, 1H, CHpy).
Synthesis of 4-(Pyridin-2-yl)-piperazine-1-carbothioamide (1b).
1a (1.36 g, 5.0 mmol, 1 equiv) was dissolved in ethanol (20 mL), and
hydrazine hydrate was added (267 μL, 5.5 mmol, 1.1 equiv). The
mixture was stirred under reflux for 2 h, and a white solid of 1b was
formed, which was filtered off, washed with cold ethanol, and dried in
EXPERIMENTAL SECTION
■
Materials and Methods. All solvents and reagents were obtained
from commercial suppliers and used without further purification.
Elemental analyses were performed on a Perkin Elmer 2400 CHN
elemental analyzer at the Microanalytical Laboratory of the University
of Vienna and are within 0.4%, confirming >95% purity.
Electrospray ionization (ESI) mass spectra were recorded on a
Bruker amaZon SL ion trap mass spectrometer in positive mode by
direct infusion. High-resolution mass spectra were measured on a
Bruker maXis UHR ESI time of flight mass spectrometer. One-
1
vacuum. Yield: 0.77 g (65%). H NMR (500.10 MHz, DMSO-d6): δ
= 3.53 (m, 4H, piperazine), 3.85 (m, 4H, piperazine), 4.84 (br, 1H,
NH), 6.66 (ddd, J = 0.6 Hz, J = 5.0 Hz, J = 7.0 Hz, 1H, CHpy), 6.83
(d, J = 8.6 Hz, 1H, CHpy), 7.55 (ddd, J = 2.0 Hz, J = 7.0 Hz, J = 8.6
Hz 1H, CHpy), 8.12 (ddd, J = 0.6 Hz, J = 2.0 Hz, J = 5.0 Hz, 1H,
CHpy), 9.35 (s, 1H, NH2), 10.23 (s, 1H, NH2).
Synthesis of N′-(6,7-Dihydroquinolin-8(5H)-ylidene)-4-(pyridine-
2-yl)piperazine-1-carbothioamide (COTI-2). 1b (0.77 g, 3.26 mmol,
1 equiv) was suspended in ethanol (25 mL), and 6,7-dihydroquino-
line-8-one was added (0.48 g, 3.26 mmol, 1 equiv). The mixture was
stirred under reflux for 20 h, resulting in a yellow solid, which was
filtered off, washed with cold ethanol and diethyl ether, and dried in
vacuum. Yield: 1.01 g (85%). Elemental analysis: Calcd. for
C19H22N6S (%): C, 62.27; H, 6.05; N, 22.90; S, 8.75. Found (%):
1
dimensional H NMR spectra of the precursors were recorded on a
Bruker Avance III 500 MHz spectrometer at 298 K. One- and two-
1
dimensional H NMR and 13C NMR spectra of the final products
were recorded on a Bruker Avance III 600 MHz spectrometer at
298 K. For 1H NMR spectra, the solvent residual peak was taken as an
internal reference (s = singlet, d = doublet, t = triplet, quint = quintet,
dd = doublet of doublets, ddd = doublet of doublet of doublets, br =
broad signal, m = multiplet, py = pyridine). For the NMR numbering
J
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
C, 61.87; H, 5.93; N, 22.58; S, 8.61. MS in ACN/MeOH +1% H2O
filtered off, washed with cold methanol and diethyl ether, and dried in
vacuum. Yield: 0.28 g (72%). Elemental analysis: Calcd. for
[CuCl2(C19H22N6S)] (%): C, 45.56; H, 4.43; N, 16.78; S, 6.40.
Found (%): C, 45.25; H, 4.41; N, 16.47; S, 6.14. ESI-MS in ACN/
MeOH +1% H2O (positive): m/z 428.09, [CuL]+; 464.05, [Cu-
(HL)Cl]+; 891.07 [Cu2L2Cl]+ (Figure S11). The synthesis was
repeated without HCl, yielding the same complex. Green crystals were
obtained by slow evaporation of the mother-liquor from both
syntheses, with and without HCl.
X-Ray Diffraction Analysis. The X-ray intensity data were
measured on a Bruker D8 Venture diffractometer equipped with a
multilayer monochromator, MoKα for COTI-2 and CuKα for Cu-
COTI-2, an INCOATEC micro focus sealed tube, and an Oxford
cooling system. The structures were solved by direct methods.
Nonhydrogen atoms were refined with anisotropic displacement
parameters. Hydrogen atoms were inserted at calculated positions and
refined with a riding model. The list of software used can be found in
the Supporting information. Experimental data and CCDC-codes
(available online at http://www.ccdc.cam.ac.uk/conts/retrieving.
html) can be found in Table S1. Crystal data, data collection
parameters, and structure refinement details are in Tables S2−S7.
Mass Spectroscopic Studies of the Reaction of Cu-COTI-2 and
GSH. A stock solution of Cu-COTI-2 (1 mM in DMF) was diluted
1:1 with an ammonia acetate buffer (50 mM, pH 6.85). GSH was
freshly dissolved in the same buffer and mixed 1:1 with the previously
prepared Cu-complex solution, resulting in an excess of GSH (3
equiv). The sample was kept at r.t. for 15 min and subsequently
measured by mass spectroscopy using an amaZon speed ETD. For the
reference measurement, the same solution without the addition of
GSH was used.
Cell Culture. The following human cell lines and resistant sublines
were used: the colon carcinoma cell line SW480 (from American
Type Culture Collection, Manassas, VA), and the Triapine-resistant
line SW480/Tria;24 the small cell lung carcinoma GLC-4, and its
ABCC1-overexpressing adriamycin-resistant subline GLC-4/adr
(from Dr. deVries, Groningen, The Netherlands). The ABC
transporter expression levels of GLC-4/adr were assessed by Western
blotting of membrane-enriched fractions. SW480 cells were grown in
minimal essential medium with 10% fetal bovine serum (FBS), while
GLC-4 cells were grown in RPMI-1640 with 10% FBS.
The newly established SW480/Coti-resistant cell line was
generated by continuous exposure of SW480 cells to increasing
concentrations of COTI-2 (starting point 250 nM, end point 1360
nM) over a period of approximately one year. COTI-2 was
administered to the cells once a week at the day after passage when
cells had attached to the cell culture flasks.
1
(positive): m/z 367.60, [HL + H]+; 389.6, [HL + Na]+. H NMR
(600 MHz, DMSO-d6; for the numbering scheme see Figure S8):
Major isomer (91%) δ = 1.98 (quint, 3J = 6.1 Hz, 2H, CH2, H6), 2.90
3
3
(t, J = 6.1 Hz, 2H, CH2, H7), 3.09 (t, J = 6.5 Hz, 2H, CH2, H5),
3
3.56 (t, J = 5.2 Hz, 4H, CH2, H12,13), 4.10 (s, 4H, CH2, H11,14),
6.66 (dd, 4J = 5.2 Hz, 3J = 6.9 Hz, 1H, CHpy, H16), 6.85 (d, 3J = 8.5
3
3
Hz, 1H, CHpy, H18), 7.45 (dd, J = 4.5 Hz, J = 7.7. Hz, 1H, CHpy,
H2), 7.56 (ddd, 4J = 1.9 Hz, 3J = 7.1 Hz, 3J = 8.3 Hz, 1H, CHpy, H17),
7.78 (d, 3J = 7.7 Hz,1H, CHpy, H3), 8.13 (dd, 4J = 1.3 Hz, 3J = 4.8 Hz,
4
3
1H, CHpy, H19), 8.59 (dd, J = 1.3 Hz, J = 4.5 Hz, 1H, CHpy, H1),
14.57 (s, 1H, NH, N3). 13C NMR (151 MHz, DMSO-d6): δ = 20.2
(CH2, C6), 26.3 (CH2, C5), 27.2 (CH2, C7), 44.4 (CH2, C12,13),
47.9 (CH2, C11,14), 107.1 (Cpy, C18), 113.1 (Cpy, C16), 125.4 (Cpy,
C2), 136.4 (Cq, C4), 137.6 (Cpy, C17), 137.8 (Cpy, C3), 148.3 (Cq,
C9), 148.2 (Cpy C1), 143.3 (CN, C8), 147.6 (Cpy, C19), 158.8
1
(Cq, C15), 184.0 (CS, C10). H NMR (600 MHz, DMSO-d6):
Minor isomer (9%) δ = 1.95 (2H, CH2, H6′), 2.73 (2H, CH2, H7′),
2.92 (2H, CH2, H5′), 3.69 (4H, CH2, H12,13′), 6.81 (1H, CH,
H18′), 7.50 (1H, CHpy, H2′), 7.89 (1H, CHpy, H3′), 8.69 (1H, CHpy,
H1′), 15.53 (1H, NH, N3′). 13C NMR (151 MHz, DMSO-d6): δ =
22.1 (CH2, C6′), 28.8 (CH2, C5′), 33.4 (CH2, C7′), 43.7 (CH2,
C12,13′), 106.8 (Cpy, C18′), 113.0 (Cpy, C16′), 124.3 (Cpy, C2′),
137.3 (Cq, C4′), 145.6 (Cpy C1′), 143.9 (CN, C8′), 158.4 (Cq,
C15′), 179.8 (CS, C10′). Because of the small amount of second
isomer, not all proton and carbon atoms could be assigned and no
coupling constants were calculated. Crystals were obtained by slow
evaporation of the mother liquor.
Synthesis of 2-(6,7-Dihydroquinolin-8(5H)-ylidene)hydrazine-1-
carbothioamide (COTI-NH2). This compound was synthesized by a
procedure from ref22 however, slightly modified to avoid column
chromatography.
Hydrazinecarbothioamide (0.37 g, 4.0 mmol, 1 equiv) was
suspended in isopropanol (10 mL) at 80 °C. Then, 6,7-dihydroquino-
line-8-one (0.59 g, 4.0 mmol, 1 equiv) was added, and the mixture
was stirred under reflux for 5 h. A pale-yellow solid was formed, which
was filtered off, washed with cold isopropanol, and dried in vacuum.
Yield: 0.65 g (74%). Elemental analysis: Calcd. for C10H12N4S (%): C,
54.52; H, 5.49; N, 25.43; S, 14.55. Found (%): C, 54.53; H, 5.48; N,
25.35; S, 14.59. MS in ACN/MeOH +1% H2O (positive): m/z
243.0675, [HL + Na]+. 1H NMR (600 MHz, DMSO-d6; for the
3
numbering scheme see Figure S9): δ = 1.93 (quint, J = 6.2 Hz, 2H,
3
CH2, H6), 2.69 (m, 2H, CH2, H7), 2.91 (t, J = 6.0 Hz, 2H, CH2,
H5), 7.47 (dd, 3J = 4.7 Hz, 3J = 7.7. Hz, 1H, CHpy, H2), 7.85 (dd, 3J =
4
7.8 Hz, J = 1.5 Hz, 1H, CHpy, H3), 7.96 (s, 1H, NH2, N4), 8.37 (s,
4
3
1H, NH2, N4), 8.61 (dd, J = 1.5 Hz, J = 4.8 Hz, 1H, CHpy, H1),
14.28 (s, 1H, NH, N3). 13C NMR (151 MHz, DMSO-d6): δ = 22.2
(CH2, C6), 28.9 (CH2, C5), 33.4 (CH2, C7), 124.4 (Cpy, C2), 137.7
(Cq, C4), 138.6 (Cpy, C3), 139.2 (CN, C8), 145.9 (Cpy C1), 148.7
(Cq, C9), 178.2 (CS, C10). No isomerization in DMSO solution
was observed for this compound.
Viability Assays. Cells were seeded (2 × 104 cells/well for SW480,
SW480/Coti, GLC-4 and GLC-4/adr; 3 × 104 cells/well for SW480/
Tria) in 100 μL/well in 96-well plates and allowed to attach for 24 h
at 37 °C and 5% CO2. Compounds were diluted in DMSO and then
further diluted in a growth medium (DMSO concentration < 1%).
Drug dilutions were added in 100 μL/well, with the final
concentrations of 0, 0.005, 0.01, 0.05, 0.1, 1, 5, 10, and 20 μM,
depending on the compound and cell line. For modulator studies,
compounds were added in a 50 μL growth medium and modulators
(verapamil and CSA) in another 50 μL medium. For glutathione
synthesis inhibition, BSO was added 18 h before drug treatment. After
drug treatment, cells were incubated for 72 h at 37 °C and 5% CO2.
The proportion of viable cells was determined by 3-(4,5-
dimethylthiazole-2-yl)-2,5-diphenyltetrazolium assay (MTT) follow-
ing the manufacturer’s recommendations (EZ4U, Biomedica, Vienna,
Austria). Anticancer activity was expressed as IC50 values (drug
concentrations inducing 50% reduction of cell survival in comparison
to the control) calculated from full dose−response curves using
GraphPad Prism software.
Synthesis of the Iron(III) Complex of COTI-2 (Fe-COTI-2). COTI-2
(0.20 g, 0.54 mmol, 2 equiv) was suspended in methanol (10 mL)
and stirred at 50 °C. Next, a solution of iron(III) nitrate nonahydrate
(0.13 g, 0.30 mmol, 1.1 equiv) in methanol (2 mL) was added
dropwise. A dark green solution was formed, which was stirred under
reflux for 2 h. The solvent volume was reduced by evaporation under
reduced pressure, and the solution was cooled at −20 °C overnight.
On the next day, a dark brown-greenish solid was formed, which was
filtered off, washed with cold methanol, and dried in vacuum. Yield:
37%. Elemental analysis: Calcd. for [Fe(C19H22N6S)2](NO3)3·3H2O
(%): C, 44.36; H, 4.90; N, 20.42; S, 6.23. Found (%): C, 44.61; H,
4.48; N, 20.08; S, 6.17. ESI-MS in ACN/MeOH +1% H2O (positive):
m/z 393.60, [FeL(HL)]2+; 786.24, [FeL2]+ (Figure S10).
Synthesis of the Copper(II) Complex of COTI-2 (Cu-COTI-2).
Copper(II) chloride dihydrate (0.17 g, 1.0 mmol, 1.1 equiv) was
dissolved in methanol (10 mL) at 40 °C, and concentrated HCl (134
μL) was added. Next, a suspension of COTI-2 (0.30 g, 0.8 mmol, 1
equiv) was added dropwise to the copper(II) solution. A dark green
solid was formed. The mixture was stirred for 2 h, and the solid was
Growth Curve. Cells were seeded (5 × 104 cells/mL) in 500 μL in
12-wells. After indicated incubation times at 37 °C and 5% CO2, cells
were trypsinized and counted. After every 24 h, 400 μL of the growth
medium was added to ensure sufficient growth stimulation.
K
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Western Blot Analysis. Protein lysates of membrane-enriched
fractions were prepared, separated by SDS-PAGE (7.5% and 10%
gels), and transferred onto a polyvinylidene difluoride membrane for
Western blotting as described previously.16,17 The following primary
antibodies were used: anti-ABCB1 monoclonal mouse C219 (Signet,
Dedham, USA), dilution: 1:100; anti-ABCG2 monoclonal mouse
MAB4146 (Chemicon, Temicola, USA), 1:500; anti-ABCC1
monoclonal mouse MON9017 (Synbio), 1:1000; anti-ABCC2
monoclonal mouse C250 (Alexis Corp., Lausen, Switzerland), 1:50;
anti-β-actin monoclonal mouse AC-15 (Sigma Aldrich), 1:1000; anti-
GAPDH monoclonal rabbit D16H11 (Cell Signaling. Germany).
Secondary, horseradish peroxidase-labeled antibodies that were anti-
rabbit monoclonal mouse (sc-2357, Santa Cruz Biotechnology,
Austria) and anti-mouse polyclonal goat (A0168, Merck, Germany)
were used in working dilutions of 1:10,000.
Flow Cytometry. For cell death analysis, cells were stained with
AV/PI, and for cell cycle analysis, cells were fixed and stained with PI,
as previously described.10 Briefly, 2 × 105 SW480 or SW480/Coti
cells/well were seeded in 6-wells and treated with 0.1, 1, and 10 μM of
COTI-2 for 24 h. Then, cells were trypsinized and for cell death
analysis stained with allophycocyanine (APC)-labelled AV (BD
Biosciences) and PI (0.01 mg/mL, Sigma). For cell cycle analysis,
collected cells were fixed in ice-cold 70% ethanol, treated with RNase
(0.2 mg/mL, Sigma), and DNA was stained using PI. Stained cells
were measured using a flow cytometry (FACS Calibur, Becton
Dickinson, CA, USA) and analyzed using Cell Quest Pro software.
Cell Death Analysis by DAPI Staining. Cells were seeded (2 × 105
cells/well) in 6-wells and left to recover for 24 h. Then, indicted
treatment was added for 24 h after which cells were collected by
trypsinization and transferred to microscopic slides by centrifugation
with the cytocentrifuge Thermo Scientific Cytospin 4 (400 rpm, 5
min). After drying, cells were fixed on −20 °C with a precooled
methanol/aceton (1:1) mixture for 10 min and dyed with DAPI (1
μg/mL) for another 10 min. For microscopy, cells were mounted with
Vectashield (H-100, Vector Laboratories, Inc., CA, USA).
was used directly, while the other complexes were prepared in situ by
mixing the equimolar solutions of the metal ion and ligand. The pH of
all the solutions was adjusted to 7.40 by 50 mM HEPES buffer. The
stock solutions of the reducing agent and complexes were freshly
prepared every day.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01277.
■
sı
Scheme of the synthesis of COTI-2; overlay of crystal
structures of Cu-COTI-2a/b; concentration−response
curves in SW480, SW480/Coti, and SW480/Tria cells;
growth curves of SW480 and SW480/Coti cells;
Western blot of short-term exposition of COTI-2 in
SW480 cells; concentration−response curves in GLC-4
and GLC-4/adr cells; mass spectra of Cu-COTI-2 after
incubation with GSH; UV−vis spectra of COTI-2 with
GSH in HEPES vs RPMI1640; NMR numbering
schemes for COTI-2 and COTI-NH2; mass spectra of
Fe-COTI-2 and Cu-COTI-2; and details of X-ray data
collection and refinement for COTI-2 and Cu-COTI-
2a/b (PDF)
Molecular formula strings (CSV).
AUTHOR INFORMATION
Corresponding Authors
■
Petra Heffeter − Institute of Cancer Research, Medical
University of Vienna, Vienna 1090, Austria; Research Cluster
“Translational Cancer Therapy Research”, Vienna 1090,
Austria; Email: petra.heffeter@meduniwien.ac.at
Christian R. Kowol − Institute of Inorganic Chemistry,
Faculty of Chemistry, University of Vienna, Vienna 1090,
Austria; Research Cluster “Translational Cancer Therapy
Research”, Vienna 1090, Austria; orcid.org/0000-0002-
8311-1632; Email: christian.kowol@univie.ac.at
Microscopy. Cells were seeded 2 × 204/well in 24-well plates and
left to recover for 24 h. Then, cells were treated with COTI-2 with
indicated concentrations. After 24 h, microscopy images were taken
with a Nikon eclipse Ti-e fluorescence microscope with differential
interference contrast and a sCMOS pco.edge camera. Images were
processed and analyzed by ImageJ. Cells with vacuoles were counted
and given in percent of all attached cells. For immunofluorescence
imaging of ABCC1, cells were transferred to microscopy slides using a
cytocentrifuge (Cytospin 4, Thermo Scientific, USA) at 400 rpm for 5
min. After drying, the cells were fixed with 4% PFA for 15 min at
room temperature and blocked with 20% fetal calf serum in PBS for
1 h. Cells were stained with primary anti-ABCC1 monoclonal mouse
MON9017 (Synbio) (1:50 in 1% bovine serum albumin in PBS) and
secondary anti-mouse conjugated to AlexaFluor488 (Thermo Fisher,
1:200 in 1% bovine serum albumin in PBS). After washing,
counterstain was performed with DAPI (1 μg/mL) and wheat germ
agglutinin (WGA) conjugated to Rhodamine (10 μg/mL, Vector
Laboratories, USA) for 10 min. Vectashield (H-100, Vector
Laboratories, Inc., CA, USA) was used for mounting, and cells were
imaged at the confocal Zeiss LSM 700 Olympus microscope (Carl
Zeiss AG, Oberkochen, Germany) and processed with ImageJ.
Spectrophotometric Kinetic Measurements on the Reduction of
the Copper(II) Complexes by GSH. The redox reaction of the
copper(II) complexes of COTI-2, COTI-NH2, and Triapine with
GSH was studied at 25.0 0.1 °C on an Agilent Cary 8454 diode
array spectrophotometer using a special, tightly closed tandem cuvette
(Hellma Tandem Cell, 238-QS). The reactants were separated until
the reaction was triggered. Both isolated pockets of the cuvette were
completely deoxygenated by bubbling a stream of argon for 10 min
before mixing the reactants. Spectra were recorded before and then
immediately after the mixing, and changes were followed until no
further absorbance change was observed, and then O2 was passed
through the samples. One of the isolated pockets contained GSH and
its concentration was 9.0 mM, and the other contained the copper(II)
complex at 30 μM concentration. The copper(II) complex of COTI-2
Authors
Julia H. Bormio Nunes − Institute of Inorganic Chemistry,
Faculty of Chemistry, University of Vienna, Vienna 1090,
Austria; Inorganic Chemistry Department, Institute of
Chemistry, University of Campinas - UNICAMP, Campinas,
São Paulo 13083-970, Brazil; orcid.org/0000-0001-
8113-6679
Sonja Hager − Institute of Cancer Research, Medical
University of Vienna, Vienna 1090, Austria; Research Cluster
“Translational Cancer Therapy Research”, Vienna 1090,
Austria
Marlene Mathuber − Institute of Inorganic Chemistry, Faculty
of Chemistry, University of Vienna, Vienna 1090, Austria
́
Vivien Posa − Department of Inorganic and Analytical
Chemistry, Interdisciplinary Excellence Centre and MTA-
SZTE Lendület Functional Metal Complexes Research
Group, University of Szeged, Szeged H-6720, Hungary
Alexander Roller − Institute of Inorganic Chemistry, Faculty
of Chemistry, University of Vienna, Vienna 1090, Austria
́
Eva A. Enyedy − Department of Inorganic and Analytical
Chemistry, Interdisciplinary Excellence Centre and MTA-
SZTE Lendület Functional Metal Complexes Research
Group, University of Szeged, Szeged H-6720, Hungary;
orcid.org/0000-0002-8058-8128
Alessia Stefanelli − Institute of Cancer Research, Medical
University of Vienna, Vienna 1090, Austria
L
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Walter Berger − Institute of Cancer Research, Medical
University of Vienna, Vienna 1090, Austria; Research Cluster
“Translational Cancer Therapy Research”, Vienna 1090,
Austria; orcid.org/0000-0003-0014-1658
Bernhard K. Keppler − Institute of Inorganic Chemistry,
Faculty of Chemistry, University of Vienna, Vienna 1090,
Austria; Research Cluster “Translational Cancer Therapy
Research”, Vienna 1090, Austria
REFERENCES
■
(1) Lobana, T. S.; Sharma, R.; Bawa, G.; Khanna, S. Bonding and
structure trends of thiosemicarbazone derivatives of metals-An
overview. Coord. Chem. Rev. 2009, 253, 977−1055.
(2) Beraldo, H.; Gambino, D. The wide pharmacological versatility
of semicarbazones, thiosemicarba-zones and their metal complexes.
Mini-Rev. Med. Chem. 2004, 4, 31.
(3) Yu, Y.; Kalinowski, D. S.; Kovacevic, Z.; Siafakas, A. R.; Jansson,
P. J.; Stefani, C.; Lovejoy, D. B.; Sharpe, P. C.; Bernhardt, P. V.;
Richardson, D. R. Thiosemicarbazones from the old to new: Iron
chelators that are more than just ribonucleotide reductase inhibitors.
J. Med. Chem. 2009, 52, 5271−5294.
(4) Heffeter, P.; Pape, V. F. S.; Enyedy, E. A.; Keppler, B. K.;
Szakacs, G.; Kowol, C. R. Anticancer thiosemicarbazones: chemical
properties, interaction with iron metabolism, and resistance develop-
ment. Antioxid. Redox Signaling 2019, 30, 1062−1082.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01277
Author Contributions
#J.H.B.N. and S.H. contributed equally to the main findings of
this manuscript.
Notes
(5) Lovejoy, D. B.; Jansson, P. J.; Brunk, U. T.; Wong, J.; Ponka, P.;
Richardson, D. R. Antitumor Activity of Metal-Chelating Compound
Dp44mT Is Mediated by Formation of a Redox-Active Copper
Complex That Accumulates in Lysosomes. Cancer Res. 2011, 71,
5871−5880.
The authors declare no competing financial interest.
∇P.H. and C.R.K. have shared last authorship.
ACKNOWLEDGMENTS
́
(6) Hager, S.; Pape, V. F. S.; Posa, V.; Montsch, B.; Uhlik, L.;
■
́
́
Szakacs, G.; Toth, S.; Jabronka, N.; Keppler, B. K.; Kowol, C. R.;
Enyedy, E. A.; Heffeter, P. High copper complex stability and slow
reduction kinetics as key parameters for improved activity, paraptosis
induction and impact on drug-resistant cells of anticancer
thiosemicarbazones. Antioxid. Redox Signaling 2020, 33, 395−414.
(7) Miah, A. B.; Harrington, K. J.; Nutting, C. M. Triapine in clinical
practice. Eur. J. Clin. Med. Oncol. 2010, 2, 1−6.
This work was supported by the Austrian Science Fund (FWF)
grant P31923 (to C.R.K. and P.H.). J.H.B.N. would like to
̈
thank Osterreichischer Austauschdienst (OeAD) for her Ernst
Mach Grantworldwide. Furthermore, we thank the bilateral
OeAD project HU 02/2020. A.S. was employed by the Doc-
Funds project “International PhD program translational
oncology” (IPPTO) by the FWF, and S.H. was funded by
the DOC Fellowship of the Austrian Academy of Sciences.
This work was supported by National Research, Development
and Innovation Office-NKFIA through project FK 124240 and
Ministry of Human Capacities, Hungary grant, TKP-2020. We
thank Jennifer Hsu for establishing the COTI-resistant
SW480/Coti cells.
(8) Kowol, C. R.; Trondl, R.; Heffeter, P.; Arion, V. B.; Jakupec, M.
A.; Roller, A.; Galanski, M.; Berger, W.; Keppler, B. K. Impact of
Metal Coordination on Cytotoxicity of 3-Aminopyridine-2-carbox-
aldehyde Thiosemicarbazone (Triapine) and Novel Insights into
Terminal Dimethylation. J. Med. Chem. 2009, 52, 5032−5043.
(9) Kowol, C. R.; Miklos, W.; Pfaff, S.; Hager, S.; Kallus, S.; Pelivan,
́
K.; Kubanik, M.; Enyedy, E. A.; Berger, W.; Heffeter, P.; Keppler, B.
K. Impact of Stepwise NH2-Methylation of Triapine on the
Physicochemical Properties, Anticancer Activity, and Resistance
Circumvention. J. Med. Chem. 2016, 59, 6739−6752.
(10) Hager, S.; Korbula, K.; Bielec, B.; Grusch, M.; Pirker, C.;
Schosserer, M.; Liendl, L.; Lang, M.; Grillari, J.; Nowikovsky, K.;
ABBREVIATIONS LIST
■
ABC, ATP-binding cassette; ABCB1 (=P-gp), ATP-binding
cassette transporter B1; ABCC1 (=MRP1), ATP-binding
cassette transporter C1; ABCC2, ATP-binding cassette
transporter C2; ABCG2, ATP-binding cassette transporter
G2; ACN, acetonitrile; APC, allophycocyanine; AV, annexin
V; BSO, buthionine sulfoximine; COTI-2, 4-(pyridine-2-yl)-N-
([(8E)-5,6,7,8-tetrahydroquinolin-8-ylidene]amino)-
piperazine-1-carbothioamide; CSA, cyclosporine A; DAPI,
4′,6-diamidin-2-phenylindol; DFO, desferrioxamine; DMF,
dimethylformamide; DMSO, dimethyl sulfoxide; Dp, di-2-
pyridyl; Dp44mT, di-2-pyridylketone-4,4-dimethyl-3-thiosemi-
carbazone; DpC, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-
thiosemicarbazone; ESI, electrospray ionization; FBS, fetal
bovine serum; GADPH, glycerinaldehyd-3-phosphat-dehydro-
genase; GSH, glutathione; HEPES, 2-[4-(2-hydroxyethyl)-
piperazin-1-yl]ethanesulfonic acid; MDR, multidrug resistance;
MeOH, methanol; MRP1 (=ABCC1), multidrug resistance
protein 1; MS, mass spectrometry; MTT, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear
magnetic resonance; PBS, phosphate buffered saline; PFA,
paraformaldehyde; PI, propidium iodide; PTSC, pyridine-2-
carboxaldehyde N4,N4-dimethyl-thiosemicarbazone; P-gp, P-
glycoprotein; SD, standard deviation; SDS-PAGE, sodium
dodecyl sulfate polyacrylamide gel electrophoresis; TSC,
thiosemicarbazone; UV, ultraviolet; UV−vis, UV−visible;
WGA, wheat germ agglutinin
́
Pape, V. F. S.; Mohr, T.; Szakacs, G.; Keppler, B. K.; Berger, W.;
Kowol, C. R.; Heffeter, P. The thiosemicarbazone Me2NNMe2
induces paraptosis by disrupting the ER thiol redox homeostasis
based on protein disulfide isomerase inhibition. Cell Death Dis. 2018,
9, 1052.
(11) Guo, Z.-L.; Richardson, D. R.; Kalinowski, D. S.; Kovacevic, Z.;
Tan-Un, K. C.; Chan, G. C.-F. The novel thiosemicarbazone, di-2-
pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC),
inhibits neuroblastoma growth in vitro and in vivo via multiple
mechanisms. J. Hematol. Oncol. 2016, 9, 98.
(12) Seebacher, N. A.; Richardson, D. R.; Jansson, P. J. A
mechanism for overcoming P-glycoprotein-mediated drug resistance:
novel combination therapy that releases stored doxorubicin from
lysosomes via lysosomal permeabilization using Dp44mT or DpC.
Cell Death Dis. 2016, 7, e2510−e2510.
(13) Salim, K. Y.; Maleki Vareki, S.; Danter, W. R.; Koropatnick, J.
COTI-2, a new anticancer drug currently under clinical investigation,
targets mutant p53 and negatively modulates the PI3K/AKT/mTOR
pathway. Eur. J. Cancer 2016, 69, S19.
(14) Lindemann, A.; Patel, A. A.; Tang, L.; Liu, Z.; Wang, L.; Silver,
N. L.; Tanaka, N.; Rao, X.; Takahashi, H.; Maduka, N. K.; Zhao, M.;
Chen, T.-C.; Liu, W.; Gao, M.; Wang, J.; Frank, S. J.; Hittelman, W.
N.; Mills, G. B.; Myers, J. N.; Osman, A. A. COTI-2, a novel
thiosemicarbazone derivative, exhibits antitumor activity in HNSCC
through p53-dependent and -independent mechanisms. Clin. Cancer
Res. 2019, 25, 5650−5662.
(15) Salim, K. Y.; Vareki, S. M.; Danter, W. R.; Koropatnick, J.
COTI-2, a novel small molecule that is active against multiple human
M
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
cancer cell lines in vitro and in vivo. Oncotarget 2016, 7, 41363−
41379.
special emphasis on their interactions with DNA. Dalton Trans. 2015,
44, 6140−6157.
(16) Miklos, W.; Pelivan, K.; Kowol, C. R.; Pirker, C.;
Dornetshuber-Fleiss, R.; Spitzwieser, M.; Englinger, B.; van
Schoonhoven, S.; Cichna-Markl, M.; Koellensperger, G.; Keppler, B.
K.; Berger, W.; Heffeter, P. Triapine-mediated ABCB1 induction via
PKC induces widespread therapy unresponsiveness but is not
underlying acquired triapine resistance. Cancer Lett. 2015, 361,
112−120.
(17) Heffeter, P.; Pirker, C.; Kowol, C. R.; Herrman, G.;
Dornetshuber, R.; Miklos, W.; Jungwirth, U.; Koellensperger, G.;
Keppler, B. K.; Berger, W. Impact of terminal dimethylation on the
resistance profile of α-N-heterocyclic thiosemicarbazones. Biochem.
Pharmacol. 2012, 83, 1623−1633.
(18) Robey, R. W.; Pluchino, K. M.; Hall, M. D.; Fojo, A. T.; Bates,
S. E.; Gottesman, M. M. Revisiting the role of ABC transporters in
multidrug-resistant cancer. Nat. Rev. Cancer 2018, 18, 452−464.
(19) Wiese, M.; Stefan, S. M. The A-B-C of small-molecule ABC
transport protein modulators: From inhibition to activationa case
study of multidrug resistance-associated protein 1 (ABCC1). Med.
Res. Rev. 2019, 39, 2031−2081.
(20) Cole, S. P. C.; Deeley, R. G. Transport of glutathione and
glutathione conjugates by MRP1. Trends Pharmacol. Sci. 2006, 27,
438−446.
(33) Miklos, W.; Heffeter, P.; Pirker, C.; Hager, S.; Kowol, C. R.;
van Schoonhoven, S.; Stojanovic, M.; Keppler, B. K.; Berger, W. Loss
of phosphodiesterase 4D mediates acquired triapine resistance via
Epac-Rap1-Integrin signaling. Oncotarget 2016, 7, 84556−84574.
(34) Maleki Vareki, S.; Salim, K. Y.; Danter, W. R.; Koropatnick, J.
Novel anti-cancer drug COTI-2 synergizes with therapeutic agents
and does not induce resistance or exhibit cross-resistance in human
cancer cell lines. PLoS One 2018, 13, 1−17.
(35) Yuan, J.; Lovejoy, D. B.; Richardson, D. R. Novel di-2-pyridyl−
derived iron chelators with marked and selective antitumor activity: in
vitro and in vivo assessment. Blood 2004, 104, 1450−1458.
(36) Trondl, R.; Flocke, L. S.; Kowol, C. R.; Heffeter, P.; Jungwirth,
́
U.; Mair, G. E.; Steinborn, R.; Enyedy, E. A.; Jakupec, M. A.; Berger,
W.; Keppler, B. K. Triapine and a More Potent Dimethyl Derivative
Induce Endoplasmatic Reticulum Stress in Cancer Cells. Mol.
Pharmaceutics 2014, 85, 451−459.
́ ́
(37) Cserepes, M.; Turk, D.; Toth, S.; Pape, V. F. S.; Gaal, A.; Gera,
̈
́
́
́
M.; Szabo, J. E.; Kucsma, N.; Varady, G.; Vertessy, B. G.; Streli, C.;
́
́
Szabo, P. T.; Tovari, J.; Szoboszlai, N.; Szakacs, G. Unshielding
Multidrug Resistant Cancer through Selective Iron Depletion of P-
Glycoprotein−Expressing Cells. Cancer Res. 2020, 80, 663−674.
(38) Jansson, P. J.; Sharpe, P. C.; Bernhardt, P. V.; Richardson, D. R.
Novel Thiosemicarbazones of the ApT and DpT Series and Their
Copper Complexes: Identification of Pronounced Redox Activity and
Characterization of Their Antitumor Activity. J. Med. Chem. 2010, 53,
5759−5769.
(21) Danter, W. R.; Brown, M.; Lepifre, F. Compounds and method
for treatment of cancer. Patent US8034815B2. 2011.
(22) Lemke, T. L.; Shek, T. W.; Cates, L. A.; Smith, L. K.; Cosby, L.
A.; Sartorelli, A. C. Synthesis of 5,6-dihydro-8(7H)-quinolinone
thiosemicarbazones as potential antitumor agents. J. Med. Chem. 1977,
20, 1351−1354.
(39) Park, K. C.; Fouani, L.; Jansson, P. J.; Wooi, D.; Sahni, S.; Lane,
̂
́
D. J. R.; Palanimuthu, D.; Lok, H. C.; Kovacevic, Z.; Huang, M. L. H.;
Kalinowski, D. S.; Richardson, D. R. Copper and conquer: copper
complexes of di-2-pyridylketone thiosemicarbazones as novel anti-
cancer therapeutics. Metallomics 2016, 8, 874−886.
(23) Kowol, C. R.; Eichinger, R.; Jakupec, M. A.; Galanski, M.;
Arion, V. B.; Keppler, B. K. Effect of metal ion complexation and
chalcogen donor identity on the antiproliferative activity of 2-
acetylpyridine N,N-dimethyl(chalcogen)semicarbazones. J. Inorg.
Biochem. 2007, 101, 1946−1957.
(40) Ishiguro, K.; Lin, Z. P.; Penketh, P. G.; Shyam, K.; Zhu, R.;
Baumann, R. P.; Zhu, Y.-L.; Sartorelli, A. C.; Rutherford, T. J.; Ratner,
E. S. Distinct mechanisms of cell-kill by triapine and its terminally
dimethylated derivative Dp44mT due to a loss or gain of activity of
their copper(II) complexes. Biochem. Pharmacol. 2014, 91, 312−322.
(41) Drew, R.; Miners, J. O. The effects of buthionine sulphoximine
(BSO) on glutathione depletion and xenobiotic biotransformation.
Biochem. Pharmacol. 1984, 33, 2989−2994.
̂
(24) Pessoa, M. M. B.; Andrade, G. F. S.; Paoli Monteiro, V. R.;
Temperini, M. L. A. 2-Formylpyridinethiosemicarbazone and methyl
derivatives: Spectroscopic studies. Polyhedron 2001, 20, 3133−3141.
(25) ChemAxon Instant J Chem/MarvinSketch; ChemAxon Ltd.:
Budapest, Hungary, 2012.
(26) Richardson, D. R.; Sharpe, P. C.; Lovejoy, D. B.; Senaratne, D.;
Kalinowski, D. S.; Islam, M.; Bernhardt, P. V. Dipyridyl
Thiosemicarbazone Chelators with Potent and Selective Antitumor
Activity Form Iron Complexes with Redox Activity. J. Med. Chem.
2006, 49, 6510−6521.
(27) Kowol, C. R.; Berger, R.; Eichinger, R.; Roller, A.; Jakupec, M.
A.; Schmidt, P. P.; Arion, V. B.; Keppler, B. K. Gallium(III) and
Iron(III) Complexes of α-N-Heterocyclic Thiosemicarbazones: Syn-
thesis, Characterization, Cytotoxicity, and Interaction with Ribonu-
cleotide Reductase. J. Med. Chem. 2007, 50, 1254−1265.
(28) Kowol, C. R.; Heffeter, P.; Miklos, W.; Gille, L.; Trondl, R.;
Cappellacci, L.; Berger, W.; Keppler, B. K. Mechanisms underlying
reductant-induced reactive oxygen species formation by anticancer
copper(II) compounds. J. Biol. Inorg. Chem. 2012, 17, 409−423.
(29) Korp, J. D.; Bernal, I.; Lemke, T. L. Crystal and molecular
structure of (Z)-5,6-dihydro-8(7H)quinolinone thiosemicarbazone. J.
Cryst. Mol. Struct. 1978, 8, 115−126.
(42) Santoro, A.; Calvo, J. S.; Peris-Díaz, M. D.; Kręzel, A.; Meloni,
̇
G.; Faller, P. The Glutathione/Metallothionein System Challenges
the Design of Efficient O2-Activating Copper Complexes. Am. Ethnol.
2020, 132, 7904−7909.
̀
(43) Santoro, A.; Vileno, B.; Palacios, O.; Peris-Díaz, M. D.; Riegel,
̇
G.; Gaiddon, C.; Kręel, A.; Faller, P. Reactivity of Cu (ii)−, Zn (ii)−
and Fe (ii)−thiosemicarbazone complexes with glutathione and
metallothionein: from stability to dissociation to transmetallation.
Metallomics 2019, 11, 994−1004.
(44) Kalinowski, D. S.; Stefani, C.; Toyokuni, S.; Ganz, T.;
Anderson, G. J.; Subramaniam, N. V.; Trinder, D.; Olynyk, J. K.;
Chua, A.; Jansson, P. J.; Sahni, S.; Lane, D. J. R.; Merlot, A. M.;
Kovacevic, Z.; Huang, M. L. H.; Lee, C. S.; Richardson, D. R. Redox
cycling metals: Pedaling their roles in metabolism and their use in the
development of novel therapeutics. Biochim. Biophys. Acta, Mol. Cell
Res. 2016, 1863, 727−748.
(30) Joseph, M.; Kuriakose, M.; Kurup, M. R. P.; Suresh, E.;
Kishore, A.; Bhat, S. G. Structural, antimicrobial and spectral studies
of copper(II) complexes of 2-benzoylpyridine N(4)-phenyl thiosemi-
carbazone. Polyhedron 2006, 25, 61−70.
́
(31) Beshir, A. B.; Guchhait, S. K.; Gascon, J. A.; Fenteany, G.
Synthesis and structure-activity relationships of metal-ligand com-
plexes that potently inhibit cell migration. Bioorg. Med. Chem. Lett.
2008, 18, 498−504.
(32) Saswati; Chakraborty, A.; Dash, S. P.; Panda, A. K.; Acharyya,
R.; Biswas, A.; Mukhopadhyay, S.; Bhutia, S. K.; Crochet, A.; Patil, Y.
P.; Nethaji, M.; Dinda, R. Synthesis, X-ray structure and in vitro
cytotoxicity studies of Cu(I/II) complexes of thiosemicarbazone:
N
https://dx.doi.org/10.1021/acs.jmedchem.0c01277
J. Med. Chem. XXXX, XXX, XXX−XXX