Important cytotoxic and cytostatic effects of new copper(i)-phosphane compounds with N,N, N,O and N,S bidentate ligands

Article abstract of DOI:10.1039/c8dt01653d

A family of six phosphane Cu(i) complexes bearing N,N, N,O and N,S bidentate ligands was synthesized. All the compounds were fully characterized by classical analytical and spectroscopic methods, and five of them were also characterized by X-ray diffraction studies. All the compounds exhibit high cytotoxicity against the human breast cancer cell line MCF7 with IC₅₀ values far lower than those found for cisplatin, a current chemotherapeutic in clinical use. Compounds 1 and 3 induce cell cycle arrest in the G2/M phase and cell death by apoptosis. The cytotoxic and cytostatic effects of these compounds on MCF7 cells suggest that they are suitable for further in vivo studies with breast cancer models.

Full text of DOI:10.1039/c8dt01653d

View Article Online
View Journal
Dalton
Transactions
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: T. Morais, Y.
Jousseaume, M. F. Minas da Piedade, C. Rodrigues, A. Fernandes, F. M. Marques, M. V. Brito and M. H.
Garcia, Dalton Trans., 2018, DOI: 10.1039/C8DT01653D.
This is an Accepted Manuscript, which has been through the
Volume 45 Number
1 7 January 2016 Pages 1–398
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
Accepted Manuscripts are published online shortly after
An international journal of inorganic chemistry
www.rsc.org/dalton
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

Please do not adjust margins
Dalton Transactions
Page 1 of 12
View Article Online
DOI: 10.1039/C8DT01653D
Journal Name
ARTICLE
Important cytotoxic and cytostatic effects of new Copper(I)-
phosphane compounds with NN, NO and NS bidentate ligands
Tânia S. Morais*a,b, Yann Jousseaumea,b, M. Fátima M. Piedadeb,c, Catarina Roma-Rodriguesd,
Received 00th January 20xx,
Accepted 00th January 20xx
Alexandra R. Fernandesd, Fernanda Marquese, Maria J. Villa de Brito*a,b, M. Helena Garciaa,b
DOI: 10.1039/x0xx00000x
A family of six phosphane Cu(I) complexes bearing NN, NO and NS bidentate ligands was synthesized. All the compounds
were fully characterized by classical analytical and spectroscopic methods and five of them were also characterized by X-ray
diffraction studies. All the compounds exhibit high cytotoxicity against the human breast cancer cell line MCF7 with IC50
values far lower than those found for cisplatin, a current chemotherapeutic in clinical use. Compounds 1 and 3 induce cell
cycle arrest in G2/M phase and cell death by apoptosis. The cytotoxic and cytostatic effect of these compounds on MCF7
cells suggest that they are suitable for further in vivo studies with breast cancer models.
www.rsc.org/
ligands and donor atoms bound to the metal ion8-10. In this frame,
several classes of copper complexes containing phosphanes,
imidazoles, thiosemicarbazones and carbenes have been identified
Introduction
Copper is an essential micronutrient involved in fundamental life
processes. It readily cycles between Cu(I) and Cu(II), which makes it
redox active in biological systems. This redox-cycling ability is vital for
as potential anticancer agents8, 11-17
.
Some of these promising copper complexes can be effective
anticancer agents as much as platinum-based drugs, with the
advantage of reduced toxicity toward normal cells and can even
potentially overcome resistance associated with platinum drugs. For
this particular process of chemoresistance, copper transporters play
a central role. Certain copper coordination compounds can modulate
the copper homeostasis across the copper transporters and hence
cooper to act as
a key component of many important
metalloenzymes and metalloproteins with important roles in
biological functions such as enzyme activity, cell signalling and
oxygen transport1-3
.
Copper has been recognized as a controlling factor for a variety of
processes related to cancer development and progression,
particularly in cancer growth, angiogenesis and metastasis4, 5
re-sensitize cancer cells to platinum drugs 8, 18-20
.
.
Despite the different classes of copper coordination compounds
synthesized and characterized, few data are available concerning the
mechanisms underlying their anticancer activity. Nevertheless, the
reported data suggest that copper compounds display mechanisms
of action mainly based on the ability to induce apoptosis, oxidative
stress, inhibition of proteasome activity and non-apoptotic forms of
programmed cell death6, 21-25
Elevated levels of copper and oxidative stress have been found in
cancer conditions, thus providing an opportunity to exploit these
differences for the development of cancer treatment strategies6.
Additionally, altered copper metabolism can also constitute a
promising target for cancer therapy7.
The vast array of information on copper properties and mode of
action in several biological systems, together with the assumption
that endogenous metals may be less toxic, led to the development of
a novel generation of copper coordination compounds for cancer
therapy with properties generally determined by the nature of
In the scope of copper coordination compounds we recently
reported our studies on a family of copper(I)-phosphane complexes
of general formula [Cu(dppe)L][BF4], where dppe
=
1.2-
bis(diphenylphosphino)ethane and represents
L
a bidentate
heteroaromatic ligand with N-donor atoms. These complexes
bearing dppe phosphane ligands showed marked cytotoxic activity
against A2780 and MCF7 cancer cells at submicromolar
a. Centro de Química Estrutural, Faculdade de Ciências, Universidade de Lisboa,
Portugal.
b. Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade de
Lisboa, Portugal
concentrations 26
.
c. Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa,
Portugal
Our ongoing research interest on this field led us to keep on the
exploitation of related copper(I) compounds in a systematic fashion,
focusing on the design and synthesis of new complexes involving
coordination of various ligands to a similar metal-phosphane
fragment so as to assess the effect of individual structural changes
on the cytotoxic properties of the compounds. Thus, in this paper we
d. UCBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Portugal
e. Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico,
Universidade de Lisboa, Portugal
E-mail: tsmorais@ciencias.ulisboa.pt (T.S. Morais); mjbrito@ciencias.ulisboa.pt
(M.J. Villa de Brito)
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 2 of 12
ARTICLE
Journal Name
NMR Spectroscopic studies
View Article Online
DOI: 10.1039/C8DT01653D
NMR characterization of all complexes was carried out by recording
1H, 13C{1H}, 31P{1H} and 2D experiments (COSY, HSQC and HMBC). The
1H and 13C NMR spectra of complexes 1-3 and 5 and 6 in acetone-d6
or CDCl3 at room temperature displayed well resolved resonances
which were fully assigned (see Experimental section and atom
labelling given in Scheme 1) using those 2D methods.
In contrast, the 1H NMR signals of complex 4 were very broad in the
dpp domain at room temperature. This observation is in accordance
with our previous results which showed that the dpp ligand, (2,3-
bis(2,3-pyridyl)pyrazine), is quite flexible when coordinated to
copper(I), even if the other N,N moiety is chelated to a second metal
fragment such as {RuCp(PPh3)}26 and prompted us to undertake a VT-
NMR study of this compound to assess its fluxional behavior. The
effect of cooling an acetone-d6 solution of 4 is shown in Figure S1 in
Supplementary Information. As the temperature was lowered, the
1
dpp region in the H NMR spectra became more resolved, showing
that one conformer was dominant. The set of 1D and 2D experiments
performed at -50ºC permitted full assignment of the 1H and 13C
resonances of the major conformer (see Experimental section). A
second set of signals was also detected, with much lower intensity
than the dominant peaks (~0.08:1.0), indicating that one
conformation is clearly favored for this compound. Evidence of the
presence of two conformers in similar ratio proportions, detected by
VT-NMR studies of Cu(I) complexes with flexible ligands, has already
report the synthesis and characterization of new Cu(I) complexes of
general formula [Cu(PPh3)2L][BF4] where L represents different NN,
NO or NS bidentate systems. The cytotoxic activity, the ability to
induce apoptosis and cell cycle arrest was also evaluated in MCF7
breast human cell line.
been reported26, 27
.
The well-defined proton signals observed for all the compounds also
confirm that no oxidation to Cu(II) occurred under the experimental
conditions.
The 31P{1H} spectra of the new Cu(I) complexes show the usual broad
signal (1/2= 26–330 Hz) in the range 3.0 – 1.1 ppm, downfield from
the parent compound [Cu(PPh3)2(NCMe)2][BF4], ( -0.9 ppm)
reflecting a decrease of electron density in the phosphorus atoms as
the NCMe ligands are replaced by better acceptor ones.
Results and discussion
Synthesis and Characterization of Cu(I) compounds
Six new Cu(I) complexes of general formula [Cu(PPh3)2(L)][BF4] were
prepared in high yields (82-92 %), through substitution of the labile
acetonitrile ligands on the parent complex [Cu(PPh3)2(NCMe)2][BF4]
by bidentate ligands L, namely 2-(2-pyridyl)benzo[b]thiophene (pbt),
1, 2- benzoylpyridine (bopy), 2, di(2-pyridyl)ketone (dpk), 3, 2,3-
bis(2-pyridyl)pyrazine (dpp), 4, 2,2’-bipyridine (2,2’-bipy), 5 and 2,2′-
bipyridine-4,4′-dicarboxylic acid (dcbipy), 6. The reactions were
carried out in THF, dichloromethane or methanol solutions at room
temperature (Scheme 1). The compounds were recrystallized, at
room temperature, by slow diffusion of diethyl ether in
dichloromethane solutions of the compounds.
The new complexes were fully characterized by FTIR and NMR
spectroscopic methods (1H, 13C{1H}, 31P{1H}). The elemental analyses
were in accordance with the proposed formulations. Compounds 1,
2, 3, 4 and 5 were also characterized by single crystal X-ray diffraction
techniques. The FTIR spectra of all the compounds in KBr pellets
present the characteristic C-H bands of aromatic rings in the region
3040 – 3100 cm-1, the C=C vibrations appear at 1400 – 1590 cm-1 and
the C-H vibrations in the 690 - 700 cm-1 range. The B-F vibrations of
-
the BF4 anion were observed at 1095-1057 cm-1 (very strong and
broad bands) and ~690 cm-1. Compounds 2, 3 and 6 also presented
the C=O vibrations in the range 1626 – 1720 cm-1 as expected.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 3 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
View Article Online
DOI: 10.1039/C8DT01653D
Table 1 Electronic spectra data for complexes 1 – 6, precursor and free ligands, in dichloromethane solutions.
max (nm)
max (nm)
( M-1cm-1)
Compound
Compound
( M-1cm-1)
[Cu(PPh3)2(NCMe)2][BF4]
259 (sh)
279 (sh)
[Cu(PPh3)2(pbt)][BF4] (1)
258 (sh)
279 (sh)
pbt
─
262 (sh)
319 (22200)
315 (40200)
333 (sh)
328 (sh)
[Cu(PPh3)2(bopy)][BF4] (2)
[Cu(PPh3)2(dpk)][BF4] (3)
272 (38800)
417 (3300)
─
270 (35000)
426 (2120)
265 (30700)
326 (sh)
bopy
dpk
266 (14300)
354 (225)
244 (11900)
277 (11200)
360 (206)
252 (20900)
288 (27900)
─
[Cu(PPh3)2(dpp)][BF4] (4)
dpp
385 (3310)
[Cu(PPh3)2(2,2’-bipy)][BF4] (5) 283 (sh)
367 (3310)
256 (sh)
2,2’-bipy
244 (15730)
282 (22104)
insoluble
[Cu(PPh3)2(dcbipy)][BF4] (6)
dcbipy
311 (9100)
405 (2810)
changes were observed either on the number of peaks displayed or
on their δ values (Figure S2 in Supplementary Information).
other Cu(I)-(PPh3) complexes17, and show a slight shift to higher The stability of the new complexes in 1%DMSO/DMEM + GlutaMAX-
These 31P resonances are similar to previously reported values for
frequencies relatively to the Cu(I) dppe analogues that we previously I™ cellular medium was also evaluated by UV–vis spectroscopy.
reported26.
Changes observed in UV–vis spectrum of the complexes over 24 h
were insignificant, indicating that these complexes are air-stable in
this solution (Figure S3 in Supplementary Information).
Electronic absorption spectroscopy
The electronic absorption spectra of the six new synthesized copper
Crystal structures of complexes 1,2, 3, 4 and 5
complexes were recorded in ~10-4 to 10-6
M solutions of
dichloromethane and the corresponding maximum absorption bands Single crystals of complexes 1, 2, 3, 4 and 5 suitable for SCXRD were
are indicated in Table 1. For comparison, also the electronic spectra obtained by slow diffusion of diethyl ether in dichloromethane
of the free ligands were obtained in the same experimental solution of the corresponding complexes. The X-ray single crystal
conditions.
data of all compounds were analyzed in the temperature range 160
– 150 K to prevent disorder of the molecules. The molecular
structures of the compounds are presented in Figure 2 and selected
bond lengths and angles are displayed in Table 2.
All the studied Cu(I) complexes showed intense absorption bands in
the UV region, in the range of 230 – 330 nm, attributed to π-π*
electronic transitions occurring in the coordinated ligands of the
inorganic fragment {Cu(PPh3)2}+. In addition to these transitions, one The five compounds crystallize in triclinic crystal system, space group
metal to ligand charge transfer (MLCT) band was found, for P -1, with a cationic complex molecule and a BF4 as a counter anion
complexes 2 - 6, placed in the visible region between 360 and 430 in the asymmetric unit; complex 2 co-crystalizes with CH2Cl2. The
nm. These high values of energy found for MLCT bands were also copper(I) ion presents two coordinated triphenylphosphanes and the
observed in other Cu(I) compounds with dppe and N,N bidentate ligand L in a highly distorted tetrahedral geometry. This
heteroaromatic rings26. The absorption spectra found for compound distortion arises from the “bite” of the bidentate ligand, N(1)-Cu(1)-
5 is in agreement with those published by Andrés-Tomé et al. for the S(1) 77.76(5)° for 1, N(1)-Cu(1)-O(1) 76.55(9)° in 2 and N(1)-Cu(1)-
same compound28. Figure 1 typifies the behavior of this set of N(2) 92.17(11)°, 79.18(15)°, 79.64(9)° for 3, 4 and 5 respectively,
compounds.
much smaller than the angle of the perfect tetrahedron, 109.4°.
However, the bite angle of the dpk ligand, in compound 3, is larger
than the others as it forms a six-membered ring with the metal
center while the others form a five membered metallacycle. Also the
Stability studies in DMSO and DMSO/DMEM
The stability of the complexes in DMSO-d6 (deuterated form of the P(1)-Cu(1)-P(2) angles (129.37(2)°, 125.96(4)°, 123.14(3)°, 123.76(5)°
1
co-solvent used in the biological assays) was studied by H and 31P and 124.91(3)°), for complexes 1, 2, 3, 4, and 5, respectively) are
1
NMR spectroscopy over 24 h. In both, H and 31P NMR spectra, no larger due to the steric effects caused by the bulky PPh3 ligands.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 4 of 12
ARTICLE
Journal Name
View Article Online
DOI: 10.1039/C8DT01653D
Table 2 Selected Bonds (Å) and Bonds Angles (°) of the cations [Cu(PPh3)2(L)]+ (L= pbt, 1; bopy, 2; dpk, 3; dpp, 4; 2,2’bipy, 5).
3
Compound
1
2
4
5
Bond lengths (Å)
Cu(1) – N(1)
Cu(1) – X
2.0303(17)
2.6902(6)
2.2507(6)
2.2509(6)
2.050(3)
2.211(2)
2.076(3)
2.099(3)
2.2796(9)
2.2836(9)
2.078(4)
2.104(4)
2.099(2)
2.055(2)
2.2569(8)
2.2593(7)
Cu(1) – P(1)
Cu(1) – P(2)
2.2423(11)
2.2442(11)
2.2474(14)
2.2459(14)
Angles (°)
X – Cu(1) – N(1)
P(2) – Cu(1) – P(1)
N(1) – Cu(1) – P(1)
N(1) – Cu(1) – P(2)
P(1) – Cu(1) – X
P(2) – Cu(1) – X
77.76(5)
129.37(2)
116.39(5)
113.17(5)
106.55(2)
93.08(2)
76.55(9)
125.96(4)
116.53(8)
112.17(8)
105.06(7)
107.66(6)
92.17(11)
123.14(3)
115.06(8)
103.38(8)
105.53(8)
113.43(8)
79.18(15)
123.76(5)
117.33(12)
109.34(12)
104.33(11)
113.93(11)
79.64(9)
124.91(3)
113.19(6)
107.90(6)
109.64(7)
112.53(6)
Nevertheless these angles along with the bond lengths involving the coordinated to metal centre. The structure of this cation with the
copper atom are in the range of values found for this family of cations [NO2]- anion was already described by Navarro et al.30, no significant
[Cu(I)(PPh3)2L]+ (where L is a bidentate ligand)20, 21. The pbt ligand in differences being observed in the bond lengths and angles of the
complex 1, is not planar; the plane of the pyridyl ring and the plane complexes with the two counter-ions.
of the benzothiophenyl rings form an angle of 28°, while the free
In the packing of these compounds, the intermolecular interactions
ligand is almost planar with an angle of 7.7°29. In compound 2, the
found are of the type C-H···F and C-H···. Compounds 2, 3 and 4 form
non-planarity of the bidentante ligand is caused by distortion of the
phenyl ring attached to the O=C bond making an angle of 47.3°
chains of cations through C-H··· interactions between phenyl –
phenyl rings of the phosphines and phenyl and pyridyl of the
between the two rings, while in the free ligand the same dihedral
ligand is much larger, ca 61°. This is due to complexation of the ligand
to the copper that restrains stereochemically the ligand. The two six
membered rings in the bidentate ligand of compound 3 make an
bidentate ligand (C-H··· range of 3.1 – 3.5 Å). These cations are also
surrounded by 3, 5 and 5 anions, respectively, that form interactions
C-H···F ranging from 2.4 Å in compound 4 to 2.9 Å in compound 3.
Compound 3 has an additional interaction of the type C-H···O (2.6 Å)
angle of 17° and the carbonyl group that binds the two rings is almost between the carbonyl group and a phenyl of the phosphine.
coplanar with the ring that contains the N1 atom. In this case, the
Each cation of compound 1 has five other cations and five BF4- anions
in its environment. The cations interact with each other via C-H···
angle between the two rings is much smaller than the one in the free
ligand (55.8°). The cation of complex 4 also presents a non-planar
interactions (3.2 Å) between two bidentate ligands and among a
bidentate ligand (dpp) bonded to the copper center. In this ligand,
bidentate ligand and a phenyl group of the phosphine. A C-H···
the planes of the rings linked by the C5-C6 bond have an angle of
interaction between two phenyl rings of the phosphine (3.1 Å) has
35.9°, and the planes of the two pyridyl rings make an angle of 44°.
also been detected.
The non-planarity of the ligand in the cation of complex 4 is slightly
smaller than that in the free ligand were the dihedral angles between
the three rings are all equal with a value of 42.2°. However, in the
The anions of 1 display C-H···F interactions with the cations with
intermolecular distances in the range of 2.4 to 2.6 Å.
The packing of compound 5 comprises one cation surrounded by 4
other cations and 5 anions. C-H··· interactions are evident between
a pyridyl ring of the bidentate ligand and a phenyl ring of PPh3 and
also between two phenyl rings of the phosphines (3.2 and 3.4 Å). The
interactions of the cations with the anions (C-H···F) are in the range
of 2.4 to 2.6 Å.
cation of the similar complex with
a bidentate phosphine,
[Cu(dppe)(dpp)]BF4]26, the dihedral angles between the planes of the
rings joined by C5-C6 bond are even smaller (24.8°) and the mean
plane of the two rings joined by C5-C6 bond with the aromatic ring
that is linked by C9-C10 bond form an angle of 59.8°. In complex 5,
the angle between the rings of the bidentate ligand is ca. 17°,
although in the free ligand the two rings are totally coplanar. This
difference in the angles between the two planes of the N,N
coordinated ligand and the one of the free ligands is due to the
stereochemical restraint imposed by the two bulky phosphines
Compounds 1 and 5 had a more planar ligand than the other three
compounds, leading to a more freely stereochemical environment
around the cation, assembling more molecules around each other.
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 5 of 12
View Article Online
DOI: 10.1039/C8DT01653D
Journal Name
ARTICLE
Biological studies in human tumor cells
Cytotoxicity
the same magnitude order as those found for other copper(I)-
triphenylphosphane complexes with heteroaromatic ligands for the
same cells17, 32
.
The cytotoxic potential of compounds 1 - 6 and their precursor in
MCF7 adenocarcinoma cells lines was evaluated by the MTT assay to
assess cell viability and survival. As observed in Table 3 all complexes
including the precursor [Cu(PPh3)2(NCMe)2][BF4] are active in the
breast adenocarcinoma cancer cells at 24h incubation with IC50
values varying from 6.6 to 18 µM.
Since the free ligands did not show cytotoxic effect it is possible to
conclude that the cytotoxicity of these complexes was in part due to
the coordinated triphenylphosphine coligands. Despite this, by
substituting the phosphane fragment PPh3 by dppe in this
complexes, as we previously reported, a remarkable improvement of
about ten times in the cytotoxic activity was found26.
The activity of the free ligands was also evaluated using the same
experimental conditions in MCF7 cells at 24 h incubation. Within the
concentration range of 1 - 200 µM the free ligands were not active Apoptosis evaluation
with the exception of pbt that displayed an IC50 value of 55 ± 7.7 µM.
To understand if the decrease of MCF7 tumor cells viability in the
The cytotoxic effect of the triphenylphosphane (PPh3) was also
assessed and found negligible. With exception of complexes 1, 3 and
5 the substitution of the acetonitrile ligands on the parent complex
by the bidentate ligands did not result in any considerable
improvement of activity. Comparing compounds 5 and 6 the
introduction of two carboxylic groups in the 2,2’-bipy ligand resulted
in a less active compound. This difference in the activity could be
related to the fact that in solution compound 6 has a double negative
charge in the carboxylic groups which could hinder the cellular
uptake31. With the same experimental conditions, all the compounds
are much more active than cisplatin, the antitumor drug in clinical
use. The IC50 values found for the compounds here presented are of
presence of the compounds is due to the induction of apoptosis, cells
were treated with the IC50 concentration (Table 3) of the precursor,
compounds 1, 3 and 5 and respective solvent (DMSO) as vehicle
control and DNA staining with Hoechst 33258 was accessed via
fluorescence microscopy. Compounds 1, 3 and 5 were chosen due to
their lower relative IC50 in MCF7 cells compared to the precursor and
compounds 2 and 4. Staining the cell nuclear DNA with Hoechst
allows to infer the presence of apoptotic events, including
condensation of the chromatin, fragmentation of the nucleus and
the presence of the so called apoptotic vesicle33. Hoechst staining
revealed an increased number of nucleus with condensed chromatin
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 6 of 12
ARTICLE
Journal Name
Table 3 IC50 values found for compounds (1 – 6), precursor and cisplatin in the breast
adenocarcinoma MCF7 cells (24 h, 37 °C).
a careful examination of the cell cycle progression ofVcieowmAprtiocluenOdnslin1e
and 3 shows a slight increase of the % cells in G2/M from 12h to 24h
DOI: 10.1039/C8DT01653D
with subsequent decrease of the % cells in S and G0/G1 phases
(Figure 4). These results suggest that treatment of MCF7 breast
adenocarcinoma cancer cells with precursor and compounds 1 and 3
result in similar a cell cycle profiles, with a delay in cell cycle
progression over time and, arrest in G2/M phase. Unlike the others,
compound 5 shows a high cytostatic effect, causing cell cycle arrest
after 24h, where 91% of the cells are at S phase. The cytotoxic (at low
micromolar range) and the cytostatic capability of these compounds,
particularly of compound 5, suggest that they can be suitable for
further in vivo studies aiming their potential translation towards
breast cancer therapy.
Compound
IC50 (µM)
[Cu(PPh3)2(NCMe)2][BF4]
[Cu(PPh3)2(pbt)][BF4] (1)
[Cu(PPh3)2(bopy)][BF4] (2)
[Cu(PPh3)2(dpk)][BF4] (3)
[Cu(PPh3)2(dpp)][BF4] (4)
[Cu(PPh3)2(2,2’-bipy)][BF4] (5)
[Cu(PPh3)2(dcbipy)][BF4] (6)
Cisplatin
13.4 ± 5.3
6.6± 2.2
11.6 ± 3.9
7.6 ± 3.2
17.7 ± 5.5
6.7 ± 2.5
13.9 ± 5.0
59.0 ± 12.0
Experimental Section
General procedures
and the presence of apoptotic vesicles in cells exposed to compounds
1, 3 and 5 relative to the vehicle control and precursor (Figures 3A
and 3B). These results suggest that the substitution of NCMe groups
by pbt, dpk and 2,2’-bipy ligands in [Cu(PPh3)2(L)]+ result in an
increase of MCF7 apoptosis (Figure 3B). The similar cytotoxicity of
compounds 1, 3 and 5 (Table 3) agrees with the apoptosis results.
However, these results do not exclude that other non-apoptotic cell
death mechanisms might also occur.
All starting reagents and solvents were obtained from standard
chemical suppliers. All manipulations involving air-free syntheses
were carried out under dinitrogen atmosphere using Schlenk
techniques and the solvents used were dried by standard methods35.
Starting materials [Cu(NCMe)4][BF4] and [Cu(NCMe)2(PPh3)2][BF4]
were prepared following the methods described in the literature36,
37. NMR spectra were recorded on a Bruker Avance 400 spectrometer
(1H NMR at 400.13 MHz, 13C NMR at 100.6 MHz, 31P NMR at 161.97
MHz) at room temperature except when stated otherwise. Chemical
shifts () are reported in parts per million (ppm) using CD3CN, CDCl3
Cell cycle analysis
The cytostatic potential of the compounds was inferred via
propidium iodide (PI) staining after MCF7 cells synchronization and
incubation for 6h, 12h and 24h in the presence of the precursor and
compounds 1, 3 and 5 at their IC50. DMSO was used as vehicle
control, and Doxorubicin was used as positive control of cell cycle
arrest on G2/M phase34. While treatment of samples with
doxorubicin (DOXO) resulted in cell cycle arrest after 12h incubation,
or (CD3)2CO as solvent. H and 13C chemical shifts were measured
1
relative to solvent peaks considering internal Me4Si (0 ppm) and the
31P NMR was externally referenced to 85% H3PO4. Abbreviations: s =
singlet, d = doublet, t = triplet, m = multiplet, q = quartet, sept =
septet, m = multiplet, c = complex, br = broad, J = coupling constant.
Figure 3 Morphological changes in the nucleus of MCF7 cells exposed to the precursor [Cu(PPh3)2(NCMe)2][BF4] and compounds 1, 3 and 5. A) Nucleus of MCF7
stained with Hoechst 33258 for visualization of apoptotic events, including chromatin condensation and nucleus fragmentation (white arrows). Cells were
stained after 6h treatment with culture medium (DMEM supplemented with 10 % FBS, antibiotics and non-essential amino-acids) supplemented with A1: 0.3%
(v/v) DMSO (vehicle control), A2: precursor [Cu(PPh3)2(NCMe)2][BF4] (at the IC50), A3: compound 1 (at the IC50), A4: compound 3 (at the IC50), A5: compound 5
(at the IC50). Images were obtained using an AXIO Scope (Carl Zeiss. Oberkochen Germany) and respective software. B) % of apoptotic MCF7 cells after 6h
treatment with vehicle control, precursor and compounds 1, 3 and 5. Three random fields with at least 30 nuclei were selected for analysis. * p-Value < 0.05
relative to apoptosis events in MCF7 cells treated with vehicle control (DMSO). *** p-Value < 0.05 relative to apoptosis events in MCF7 treated with precursor.
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 7 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
acquisition, integration and handling were performed using a PC with
View Article Online
DOI: 10.1039/C8DT01653D
the software package EAGER-200 (Carlo Erba Instruments).
Electronic spectra (range 220-900 nm) were recorded at room
temperature on a Jasco V-660 spectrometer.
Syntheses of Cu(I) complexes[Cu(PPh3)2(L)][BF4] (1 - 6):
General procedure
The Cu(I) complex salts 1 - 6 were prepared according to the
following general synthetic procedure: the desired ligand (0.5 mmol)
was added to a stirred solution of [Cu(PPh3)2(NCMe)2][BF4] (0.5
mmol) in acetonitrile or THF or methanol (20 mL) and the mixture
was stirred overnight at room temperature. After evaporation of the
solvent under vacuum, the products were washed with n-hexane
(2×10 mL) and recrystallized from dichloromethane/diethyl ether,
affording crystalline products.
Data for [Cu(PPh3)2(pbt)][BF4] (1): solvent: THF; white crystals;
yield: 85%.
1H NMR [CDCl3, 400.13 MHz] /ppm: 8.30 (d, 5.2 Hz, 1H, H-1), 8.13
(t, 7.6 Hz, 1H, H-3), 8.05 (d, 8.0 Hz, 1H, H-4), 7.81 (d, 6.6 Hz, 1H, H-9),
7.54 (s, 1H, H-7), 7.48 - 7.45 (m*, 1H, H-2), 7.43 (s br*, 3H, H-10, H-
11, H-12), 7.40 (t, 7.5 Hz, 6H, H-p PPh3), 7.20 (t, 7.5 Hz, 12H, H-m
PPh3), 7.01 (s br, 12H, H-o PPh3). 13C{1H} NMR [CDCl3, 100.61 MHz]
/ppm: 151.9 (C-5), 149.3 (C-1), 141.1 (C-8), 140.5 (C-6), 140.3 (C-3),
1
137.7 (C-13), 133.2 (C-o PPh3), 130.9 (C-p PPh3), 130.5 (t, JC-P =19.0
Hz, C-ipso PPh3), 129.3 (C-m PPh3), 126.8 and 126.4 (C-10 and C-11),
125.8 (C-9), 125.4 (C-7), 125.3 (C-2), 123.9 (C-4) 122.7 (C-12). 31P{1H}
NMR [CDCl3 161.97 MHz] /ppm: 1.1 (br). FT-IR[KBr, cm-1]: 3100-
3040 cm-1 (C-H, phenyl rings), 1590-1450 cm-1 (C=N,C=C, phenyl
rings), 1057 and 690 cm-1 (B-F, BF4), 700-690 cm-1 (C-H, phenyl rings).
Elemental analysis (%) found: C, 66.5; H, 4.5; N, 1.5; S, 4.0; Calc. for
C49CuH39NSP2BF4 (886.20): C, 66.41; H, 4.44; N, 1.58; S, 3.62. * Partial
overlap
Data for [Cu(PPh3)2(bopy)][BF4] (2): solvent: THF; red crystals;
yield:89%.
1H NMR [CDCl3, 400.13 MHz] /ppm: 8.56 (d, 4.8 Hz, 1H, H-1), 8.30
(t, 7.6 Hz, 1H, H-3), 8.12 (d, 7.8 Hz, 1H, H-4), 7.88 – 7.85 (m, 1H, H-2),
7.69 (t, 7.3 Hz, 1H, H-10), 7.62 (d, 7.7 Hz, 2H, H-8 and H-12), 7.54 (t,
7.8 Hz, 2H, H-9 and H-11), 7.38 (t, 7.3 Hz, 6H, H-p PPh3), 7.23 (t, 7.6
Hz, 12H, H-m PPh3), 7.15 (s br, 12H, H-o PPh3). 13C{1H} NMR [CDCl3,
100.61 MHz] /ppm: 195.3 (C-6), 150.7 (C-1), 148.2 (C-5), 139.9 (C-
2
3), 134.6 (C-7), 134.4 (C-10), 133.3 (t, JC-P =6.1 Hz, C-o PPh3), 131.1
(t, 1JC-P =17.4 Hz, C-ipso PPh3), 130.7 (C-p PPh3 + C-2 + C-4), 130.3 (C-
8 + C-12), 129.2 (br, C-m PPh3), 129.1 (C-9 + C-11). 31P{1H} NMR [CDCl3
161.97 MHz] /ppm: 1.8 (br). FT-IR [KBr, cm-1]: 1626 cm-1 (C=O),
2D NMR experiments (COSY, HSQC, HMBC) were used for specific
assignments. Infrared spectra (4000-250 cm-1) were recorded in a
Thermo Nicolet 6700 spectrophotometer with KBr, only significant
bands being cited in the text. Elemental analyses were obtained at
our laboratories (Laboratório de Análises, at Instituto Superior
3100-3040 cm-1 (C-H, phenyl rings), 1500-1420 cm-1 (C=N,C=C
,
phenyl rings), 1094 and 699 cm-1 (B-F, BF4), 700-690 cm-1 (C-H, phenyl
rings). Elemental analysis (%) found: C, 66.9; H, 4.6; N, 1.6; Calc. for
C48CuH39NOP2BF4 (858.13): C, 67.18; H, 4.58; N, 1.63.
Técnico), using
a Fisons Instruments EA1108 system. Data
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 8 of 12
ARTICLE
Journal Name
Data for [Cu(PPh3)2(dpk)][BF4] (3): solvent: THF; brown crystals;
yield: 82%.
7.5 Hz, 12H, H-m PPh3), 7.07 (s br, 12H, H-o PPh3), 4.08V(ieswbArr,tiOcleHO).n1li3nCe
NMR [CDCl3, 100.61 MHz] /ppm: 165.8 (C-6), 152.3 (C-5), 150.2 (C-
DOI: 10.1039/C8DT01653D
1), 141.1 (C-3), 133.1 (C-o PPh3), 131.6 (br, C-ipso PPh3), 130.7 (br, C-
p PPh3), 129.2 (br, C-m PPh3), 126.3 (C-2), 123.4 (C-4). 31P{1H} NMR
[CDCl3, 161.97 MHz] /ppm: 3.1 (br). FT-IR [KBr, cm-1]: 3100-3040 cm-
1H NMR [(CD3)2CO, 400.13 MHz] /ppm: 8.65 (d, 4.6 Hz, 2H, H-1), 8.52
(d, 8.0 Hz, 2H, H-4), 8.22 (td, 7.8 and 1.4 Hz, 2H, H-3), 7.72 (m, 2H, H-
2), 7.47 (t, 7.4 Hz, 6H, H-p PPh3), 7.33 (t, 7.5 Hz, 12H, H-m PPh3), 7.25
(t, 8.5 Hz, 12H, H-o PPh3). 13C{1H} NMR [(CD3)2CO, 100.61 MHz]
/ppm: 189.1 C-6), 153.0 (C-5), 151.0 (C-1), 139.9 (C-3), 134.1 (d, 2JC-
P =14.1 Hz, C-o PPh3), 132.4 (d, 1JC-P =31.8 Hz, C-ipso PPh3), 131.4 (C-p
1
(C-H, phenyl rings), 1720 cm-1 (C=O), 1580-1430 cm-1 (C=C, phenyl
rings), 1380 cm-1 (C=N), 1056 and 694 cm-1 (B-F, BF4), 700-690 cm-1
(C-H, phenyl rings). Elemental analysis (%) found: C, 63.1; H, 4.1; N,
3.0; Calc. for C48CuH38N2O4P2BF4 (919.12): C, 62.72; H, 4.17; N, 3.05.
3
PPh3), 130.0 (d, JC-P =8.6 Hz, C-m PPh3), 129.8 (C-2), 129.5 (C-4).
31P{1H} NMR [(CD3)2CO, 161.97 MHz] /ppm: 1.0 (br). FT-IR [KBr, cm-
1]: 1667 cm-1 (C=O), 3100-3040 cm-1 (C-H, phenyl rings), 1500-1400
X-ray crystallography
cm-1 (C=C, phenyl rings), 1312 cm-1 (C=N), 1058 and 698 cm-1 (B-F
,
Crystals of 1, 2, 3, 4 and 5, suitable for X-ray diffraction study were
mounted on a loop with Fomblin© protective oil. X-ray diffraction
data were collected on a Bruker AXS-KAPPA APEX II diffractometer
with graphite-monochromated radiation (Mo Kα, λ = 0.71073 Å) at
150 K. Crystals of compound 4 had poor quality and diffracting
power, resulting in a low theta full value. The X-ray generator was
operated at 50 kV and 30 mA, and the X-ray data collection was
monitored by the APEX238 program. Empirical absorption correction
using SADABS39 was applied and data reduction was done with
SMART and SAINT programs40. Data collection and refinements
details are listed in Table 4. SHELXS41 were used for structure
solution, and SHELXL42 was used for full matrix least-squares
refinement on F2. Both programs are included in the package of
programs WINGX-Version 2014.143. Non-hydrogen atoms were
refined anisotropically. All the hydrogen atoms were inserted in
calculated positions and allowed to refine in the parent carbon atom.
The hydrogen bonds and intermolecular interactions were calculated
by PLATON44. The graphical representations were prepared using
MERCURY 3.845.
BF4), 700-690 cm-1 (C-H, phenyl rings). Elemental analysis (%) found:
C, 65.5; H, 4.6; N, 2.9; Calc. for C47CuH38N2OP2BF4 (859.11): C, 65.71;
H, 4.46; N, 3.26.
Data for [Cu(PPh3)2(dpp)][BF4] (4): solvent: THF; yellow crystals;
yield: 91%.
o
1H NMR [(CD3)2CO, 400.13 MHz], T= -50 C,/ppm: 8.89 (d, 4.8 Hz,
1H, H-1), 8.76 (s br, 1H, H-7), 8.68 (d, 2.3 Hz, 1H, H-8), 8.53 (d, 4.0 Hz,
1H, H-14), 8.23 (s br, 2H, H-11 and H-12), 7.85 (t, 7.8 Hz, 1H, H-3),
7.68 – 7.65 (m, 1H, H-13), 7.46 – 7.40 (c, 7H, H-2 and H-p PPh3), 7.38
(d, 8.1 Hz, 1H, H-4), 7.30 (t, 7.5 Hz, 12H, H-m PPh3), 7.19 – 7.15 (m,
12H, , H-o PPh3). 13C NMR [(CD3)2CO, 100.61 MHz], T= -50 oC,/ppm:
156.5 (C-10), 155.0 (C-9), 152.7 (C-5), 150.8 (C-1), 150.1 (C-14), 147.6
(C-6), 145.2 (C-8), 142.5 (C-7), 139.0 (C-12), 137.9 (C-3), 133.7 (t, 2JC-P
1
=7.6 Hz, C-o PPh3), 132.5 (t, JC-P = 17.4 Hz, C-ipso PPh3), 131.1 (C-p
PPh3), 129.7 (t, 3JC-P = 4.7 Hz, C-m PPh3), 128.8 (C-4), 126.8 (C-2), 126.0
(C-13), 125.3 (C-11). 31P{1H} NMR [(CD3)2CO, 161.97 MHz], T= -50
oC,/ppm: 2.2 (br). FT-IR [KBr, cm-1]: 3100-3040 cm-1 (C-H, phenyl
rings), 1588-1435 cm-1 (C=C, phenyl rings), 1398 cm-1 (C=N), 1095 and
690 cm-1 (B-F, BF4), 700-690 cm-1 (C-H, phenyl rings). Elemental
analysis (%) found: C, 61.7; H, 4.2; N, 5.5; Calc. for
C49CuH40N4P2BF4·0.8CH2Cl2 (965.11): C, 61.98; H, 4.34; N, 5.81.
Biological assays
Cell lines and culture conditions
Human MCF7 (breast) cancer cells were grown in 25 cm2 culture
flasks as adherent monolayers in complete culture medium DMEM +
GlutaMax I containing 10% heat-inactivated fetal bovine serum (FBS)
and 1% antibiotics. Cultures were maintained at 37°C, 5% CO2 in a
Data for [Cu(PPh3)2(2,2’-bipy)][BF4] (5): solvent: acetonitrile; red
crystals; yield: 92%.
1H NMR [(CD3)2CO, 400.13 MHz]/ppm: 8.62 (s br, 2H, H-1), 8.62 (d humidified atmosphere using a CO2 incubator (Heraeus, Germany).
br, 7.6 Hz, 2H, H-4), 8.19 (t br, 2H, H-3), 7.44 (tt, 7.4 and 7.1 Hz, 6H, Cell media and supplements, phosphate buffer saline (PBS) and
H-p PPh3), 7.30 (t, 7.7 Hz, 12H, H-m PPh3), 7.18 (d br, 7.0 Hz, 12H, H- tripsin-EDTA were obtained from Gibco, Invitrogen (Thermo Fisher
o PPh3). 13C NMR [(CD3)2CO, 100.61 MHz] /ppm: 152.9 (C-5), 150.9 Scientific, USA).
(C-1), 140.0 (C-3), 134.0 (C-o PPh3), 133.0 (br, C-ipso PPh3), 131.1 (C-
p PPh3), 129.8 (br, C-m PPh3), 127.2 (C-2), 123.9 (C-4). 31P{1H} NMR
[(CD3)2CO, 161.97 MHz] /ppm: 2.1 (br). FT-IR [KBr, cm-1]: 3100-3040
MTT assay
cm-1 (C-H, phenyl rings), 1589-1435 cm-1 (C=C, phenyl rings), 1315 Cytotoxicity was measured by the MTT assay26. Briefly, cells were
cm-1 (C=N), 1049 and 694 cm-1 (B-F, BF4), 700-690 cm-1 (C-H, phenyl cultured until approx. 80% confluence in culture flasks and were
rings). Elemental analysis (%) found: C, 66.4; H, 4.5; N, 3.3; Calc. for detached by trypsin. Then 20000 cells/well were seeded into 96-well
C46CuH38N2P2BF4 (831.1): C, 66.48; H, 4.61; N, 3.37.
plates with culture medium and incubated for 24 h. Compounds
were first solubilized in DMSO (5 mM) and then in medium through
serial dilutions from 50 μM to 0.01 μM. After treatment with the
compounds the cell medium was replaced by 200 μL (0.5 mg/ml) of
Data for [Cu(PPh3)2(dcbipy)][BF4] (6): solvent: methanol; yellow
crystals; yield: 87%.
1H NMR [CDCl3, 400.13 MHz]/ppm: 9.34 (s br, 2H, H-4), 8.45 (d, 5.2 diphenyltetrazolium bromide) in PBS and further incubated for 3 h at
Hz, 2H, H-1), 8.00 (d, 2H, H-2), 7.37 (t, 7.4 Hz, 6H, H-p PPh3), 7.21 (t, 37 °C. The resulting formazan product were solubilized in DMSO and
an MTT solution (MTT
=
3-(4,5-Dimethylthiazol-2-yl)-2,5-
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 9 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
absorbance measured at 575 nm. Each experiment was repeated at The IC50 values were calculated from concentration–response curves
View Article Online
least twice, and each concentration tested with at least six replicates. using the GraphPad Prism software (vs. 5.0).DOI: 10.1039/C8DT01653D
Table 4 Data collection and structure refinement parameters for [Cu(PPh3)2(pbt)][BF4] (1), [Cu(PPh3)2(bopy)][BF4] (2), [Cu(PPh3)2(dpk)][BF4] (3), [Cu(PPh3)2(dpp)][BF4] (4) and
[Cu(PPh3)2(2,2’-bipy)][BF4] (5)
3
Compound
Empirical formula
1
2
4
5
C50.86H41.72BCl1.72CuF4N
4P2
C49H39BCuF4NP2S
C48H39BCuF4NOP2
C47H38BCuF4N2OP2
C47H40BCl2CuF4N2P2
859.08
150(2)
Formula Weight
T (K)
886.16
160(2)
858.09
150(2)
994.07
916.00
150(2)
150(2)
0.71073
Triclinic
Wavelength (Å)
0.71073
0.71073
0.71073
0.71073
Crystal System
Space Group
a (Å)
Triclinic
P-1
10.8372(5)
Triclinic
P-1
10.987(5)
Triclinic
P-1
11.952(2)
Triclinic
P-1
10.5640(14)
P-1
11.1690(7)
13.8156(9)
15.0939(9)
83.627(2)
69.883(2)
71.108(2)
2069.2(2)
2
b (Å)
11.9688(6)
17.1523(7)
90.664(2)
105.748(2)
101.455(2)
2093.57(17)
2
13.145(6)
16.023(7)
96.187(19)
94.340(19)
106.60(2)
2191.0(18)
2
13.949(2)
14.710(3)
100.285(8)
98.437(9)
95.128(8)
2370.1(7)
2
12.4697(16)
17.463(2)
102.070(6)
97.123(6)
103.401(6)
2152.0(5)
2
c (Å)
 (°)
 (°)
 (°)
Volume (Å3)
Z
Calculated density (Mgm-3)
Absorption Coefficient (mm-1)
F (000)
1.406
1.301
1.393
1.414
1.379
0.662
0.702
0.624
0.697
0.760
884
912
884
1020
940
3.122 to 26.425
2.450 to 25.697
2.855 to 26.765
1.494 to 25.753
1.213 to 26.201
Range for data collection (°)
Limiting indices
13 ≤ h ≤ 13
-14 ≤ k ≤ 14
-13 ≤ h ≤ 13
-15 ≤ k ≤ 15
-14 ≤ h ≤ 14
-17 ≤ k ≤14
-14 ≤ h ≤ 14
-17 ≤ k ≤ 17
-13 ≤ h ≤ 12
-15 ≤ k ≤ 15
-21 ≤ l ≤ 20
28011 / 8536
-19 ≤ l ≤ 19
28081 / 8005
19 ≤ l ≤ 19
27745 / 8765
-17 ≤ l ≤ 17
29051 / 8451
-20 ≤ l ≤ 21
26721 / 8186
Reflections collected/ unique
[R(int) = 0.0391]
[R(int) = 0.0356]
[R(int) = 0.0703]
[R(int) = 0.0385]
[R(int) = 0.0276]
Completeness to 

99.7%
97.2%
93.8 %
95.9%

Data/ restraints/ parameters
Goodness-on-fit on F2
8536 / 0 / 532
1.080
8004/ 0 / 463
1.144
8765 / 0 / 523
1.035
8451 / 31 / 542
1.025
8186 / 0 / 532
0.991
R1 = 0.0396,
wR2 = 0.1039
R1 = 0.0482,
wR2 = 0.1078
R1 = 0.0670,
wR2 = 0.1944
R1 = 0.1019,
wR2 = 0.2119
R1 = 0.0574,
wR2 = 0.1362
R1 = 0.0840,
wR2 = 0.1528
R1 = 0.0861,
wR2 = 0.2325
R1 = 0.1155,
wR2 = 0.2478
R1 = 0.0432,
wR2 = 0.1288
R1 = 0.0571,
wR2 = 0.1413
Final R indices [I>2(I)]
R indices (all data)
Largest diff. peak and hole (eÅ)-3 0.906 and -0.624
1.357 and -1.058
0.790 and -0.551
2.182 and -1.176
1.605 and -0.658
Apoptosis evaluation by Hoechst 33258 labelling
7.5µg/mL Hoechst 33258 (LifeTechnologies). Cells were washed 3
times with PBS, fixed for 20 min with Formaldehyde 4%, washed
again 3 times with PBS, and placed on top of a glycerol drop in a
microscope slide. At least five images with 30 cells were obtained and
analyzed using the AxioImager D2 fluorescence microscope (Zeiss)
and corresponding software. Results are represented as mean ± SEM
of three independent experiments. Statistical analysis consisting in
one-way ANOVA and t-test analysis, was performed using GraphPad
Prism software version 6.01.
MCF7 cells were plated in 24-wells plate containing a sterilized
coverslip at 50,000 cells/well. Culture medium was removed 24 h
after platting and replaced with 500 µL of fresh medium containing
[Cu(PPh3)2(NCMe)2][BF4] or derived compounds 1, 3 or 5 at IC50
concentration (Table 3), or 0.3% (v/v) DMSO (vehicle control). After
6h incubation at 37°C, 5% (v/v) CO2 and 99% (v/v) relative humidity,
the medium was removed and cells washed twice with phosphate
buffer saline (PBS). Cell DNA was then stained for 15 min at 37°C with
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 10 of 12
ARTICLE
Journal Name
Cell cycle analysis
Acknowledgements
View Article Online
DOI: 10.1039/C8DT01653D
Cells were seeded in a 6-well plate at 200,000 cells/well and
synchronized in early S-phase using a thymidine double block, as
previously described46. After the second block, the medium was
replaced with fresh medium containing each compound, 1 µM
Doxorubicin (control of cell cycle arrest) or 0.3 % (v/v) DMSO (vehicle
control). Cells were collected by trypsinization with Tryple Express
(LifeTechnologies) after 0 h, 6 h, 12 h and 24 h incubation at 37°C, 5
% (v/v) CO2 and 99 % (v/v) relative humidity. After a centrifugation at
650 xg for 5 min at 4 °C, pelleted cells were washed with 1 mL cold
PBS and centrifuged at 3,000 xg for 5 min at 4 °C. Cells were then
suspended in 100 µL cold PBS, submitted to an up-and-down
pipetting to assure complete cell disaggregation and added drop by
drop in a 1 mL cold ethanol solution 80 % (v/v). After at least 12 h
incubation at 4 °C, cell suspension was centrifuged at 5,000 xg for 10
min at 4 °C, ethanol was completely removed, cells were treated for
30 min with an RNase 50 µg/mL solution at 37 °C, and propidium
iodide was then added to a final concentration of 25 µg/mL. Data was
collected using an Attune acoustic focusing cytometer (ThermoFisher
Scientific) and analyzed using FCS express flow cytometry vs 6 (De
Novo Software).
We thank the Fundação para a Ciência e Tecnologia (FCT) for the
financial support (UID/QUI/00100/2013 (CQE),
UID/Multi/04378/2013 (UCIBIO) and UID/Multi/04349/2013
(C2TN/IST)). Unidade de Ciências Biomoleculares Aplicadas – UCIBIO
also thank the co-financing by the ERDF under the PT2020
Partnership Agreement (POCI-01-0145-FEDER-007728). Tânia S.
Morais
thanks
FCT
for
her
post-doctoral
grant
(SFRH/BPD/93513/2013).
Notes and references
1.
K. G. Daniel, R. H. Harbach, W. C. Guida and Q. P. Dou,
Front. Biosci., 2004, 9, 2652-2662.
2.
D. S. Kalinowski, C. Stefani, S. Toyokuni, T. Ganz, G. J.
Anderson, N. V. Subramaniam, D. Trinder, J. K. Olynyk, A.
Chua, P. J. Jansson, S. Sahni, D. J. Lane, A. M. Merlot, Z.
Kovacevic, M. L. Huang, C. S. Lee and D. R. Richardson,
Biochim. Biophys. Acta, 2016, 1863, 727-748.
A. Hordyjewska, L. Popiolek and J. Kocot, Biometals, 2014,
27, 611-621.
3.
4.
L. D. D'Andrea, A. Romanelli, R. Di Stasi and C. Pedone,
Dalton Trans., 2010, 39, 7625-7636.
Statistical analysis
5.
G. Crisponi, V. M. Nurchi, D. Fanni, C. Gerosa, S. Nemolato
and G. Faa, Coord. Chem. Rev., 2010, 254, 876-889.
A. Gupte and R. J. Mumper, Cancer Treat Rev, 2009, 35, 32-
46.
J. Wachsmann and F. Peng, World J. Gastroenterol., 2016,
22, 221-231.
All data were expressed as mean ± SEM from at least three
independent experiments. Statistical significance was evaluated
using the Student’s t-test; p < 0.05 was considered statistically
significant.
6.
7.
8.
C. Marzano, M. Pellei, F. Tisato and C. Santini, Anticancer
Agents Med. Chem., 2009, 9, 185-211.
9.
S. Tardito and L. Marchio, Curr. Med. Chem., 2009, 16,
1325-1348.
F. Tisato, C. Marzano, M. Porchia, M. Pellei and C. Santini,
Conclusions
A family of copper(I) complexes with N,N-, N,O- and N,S-bidentate
heteroaromatic ligands have been synthesized and fully
characterized.
10.
11.
12.
Med. Res. Rev., 2010, 30, 708-749.
C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato and C.
Marzano, Chem. Rev., 2014, 114, 815-862.
Z. Zhang, Y. Gou, J. Wang, K. Yang, J. Qi, Z. Zhou, S. Liang,
H. Liang and F. Yang, Eur. J. Med. Chem., 2016, 121, 399-
409.
N. Mukherjee, S. Podder, S. Banerjee, S. Majumdar, D.
Nandi and A. R. Chakravarty, Eur. J. Med. Chem., 2016, 122,
497-509.
C. Marzano, M. Pellei, D. Colavito, S. Alidori, G. G. Lobbia,
V. Gandin, F. Tisato and C. Santini, J. Med. Chem., 2006, 49,
7317-7324.
V. Gandin, M. Porchia, F. Tisato, A. Zanella, E. Severin, A.
Dolmella and C. Marzano, J. Med. Chem., 2013, 56, 7416-
7430.
M. Wehbe, A. W. Y. Leung, M. J. Abrams, C. Orvig and M. B.
Bally, Dalton Trans., 2017, 46, 10758-10773.
W. Villarreal, L. Colina-Vegas, G. Visbal, O. Corona, R. S.
Corrêa, J. Ellena, M. R. Cominetti, A. A. Batista and M.
Navarro, Inorg. Chem., 2017, 56, 3781-3793.
D. Denoyer, S. Masaldan, S. La Fontaine and M. A. Cater,
Metallomics, 2015, 7, 1459-1476.
All the compounds were active in vitro against MCF7 human breast
adenocarcinoma cancer cells with IC50 values in the micromolar
range. Interestingly, all the compounds are far more active in the
breast cancer cells than the metallodrug cisplatin. The introduction
of two carboxyl substituents in the 2,2’-bipy ligand resulted in a loss
of activity that can be related to the double negative charge acquired
by the compound in solution which could hinder the cellular uptake.
Compounds 1 and 3 induces MCF7 apoptotic cell death and a cell
cycle arrest in G2/M phase. Compound 5, unlike the others, showed
a high cytostatic effect, causing cell cycle arrest at the S phase. The
cytotoxic and cytostatic effects exhibited by these compounds
suggest that additional in vivo studies should be performed in order
to validate their potential action as anticancer agents towards breast
cancer.
13.
14.
15.
16.
17.
18.
19.
Conflicts of interest
There are no conflicts to declare.
C. X. Zhang and S. J. Lippard, Curr Opin Chem Biol, 2003, 7,
481-489.
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Page 11 of 12
Dalton Transactions
Please do not adjust margins
Journal Name
ARTICLE
20.
21.
22.
23.
T. Pivetta, V. Lallai, E. Valletta, F. Trudu, F. Isaia, D. Perra, E. 46.
Pinna and A. Pani, J. Inorg. Biochem., 2015, 151, 107-114.
F. R. Kona, D. Buac and M. B. A, Curr. Cancer Drug Targets,
2011, 11, 338-346.
V. Gandin, M. Pellei, F. Tisato, M. Porchia, C. Santini and C.
Marzano, J. Cell. Mol. Med., 2012, 16, 142-151.
A. S. Mendo, S. Figueiredo, C. Roma-Rodrigues, P. A.
Videira, Z. Ma, M. Diniz, M. Larguinho, P. M. Costa, J. C.
Lima, A. J. Pombeiro, P. V. Baptista and A. R. Fernandes, J.
Biol. Inorg. Chem., 2015, 20, 935-948.
P. M. Borralho, B. T. Kren, R. E. Castro, I. B. Moreira da Silva,
C. J. Steer and C. M. P. Rodrigues, FDEOBIS: 1J0.,.12003099/C, 28D76T0, 61665839D-
6700.
View Article Online
24.
25.
Z. Ma, B. Zhang, M. F. C. Guedes da Silva, J. Silva, A. S.
Mendo, P. V. Baptista, A. R. Fernandes and A. J. L.
Pombeiro, Dalton Trans., 2016, 45, 5339-5355.
K. Czerwinska, B. Machura, S. Kula, S. Krompiec, K. Erfurt,
C. Roma-Rodrigues, A. R. Fernandes, L. S. Shul'pina, N. S.
Ikonnikov and G. B. Shul'pin, Dalton Trans., 2017, 46, 9591-
9604.
26.
27.
J. Lopes, D. Alves, T. S. Morais, P. J. Costa, M. F. M. Piedade,
F. Marques, M. J. Villa de Brito and M. Helena Garcia, J.
Inorg. Biochem., 2017, 169, 68-78.
S. Keller, A. Pertegas, G. Longo, L. Martinez, J. Cerda, J. M.
Junquera-Hernandez, A. Prescimone, E. C. Constable, C. E.
Housecroft, E. Orti and H. J. Bolink, J. Mater. Chem., 2016,
4, 3857-3871.
28.
29.
30.
I. Andres-Tome, J. Fyson, F. Baiao Dias, A. P. Monkman, G.
Iacobellis and P. Coppo, Dalton Trans., 2012, 41, 8669-
8674.
D. E. Janzen, S. R. Doherty, D. G. VanDerveer, L. M. Hinkle,
D. A. Benefield, H. M. Vashi and G. J. Grant, J. Organomet.
Chem., 2014, 755, 47-57.
M. Navarro, O. A. Corona, T. Gonzalez and M. V. Capparelli,
Acta Crystallogr. sec E, 2008, 64, m533-534.
31.
32.
E. Frohlich, Int. J. Nanomed, 2012, 7, 5577-5591.
V. Gandin, F. Tisato, A. Dolmella, M. Pellei, C. Santini, M.
Giorgetti, C. Marzano and M. Porchia, J. Med. Chem., 2014,
57, 4745-4760.
33.
34.
O. Kepp, L. Galluzzi, M. Lipinski, J. Yuan and G. Kroemer,
Nat. Rev. Drug Discov., 2011, 10, 221-237.
N. K. Verma, M. P. Purohit, D. Equbal, N. Dhiman, A. Singh,
A. K. Kar, J. Shankar, S. Tehlan and S. Patnaik, Bioconjug.
Chem., 2016, 27, 2605-2619.
35.
36.
37.
D. D. Perrin, W. L. F. Amarego and D. R. Perrin, Purification
of Laboratory Chemicals, Pergamon, New York, 2nd edn.,
1980.
G. J. Kubas, B. Monzyk and A. L. Crumbliss, in Inorg. Synth.,
John
Wiley
&
Sons,
Inc.,
2007,
DOI:
10.1002/9780470132500.ch18, pp. 90-92.
J. Díez, S. Falagán, P. Gamasa and J. Gimeno, Polyhedron,
1988, 7, 37-42.
38.
39.
APEX2 v2009.3-0, Bruker AXS: Madison, WI, 2009.
SABADS: Program for Empirical Absorption Correction,
University of Gꢀttingen: Gꢀttingen, Germany, 1996.
SAINT, Area-detector Integration Software (Version 6.28A),
Brucker AXS Inc., Madinson, WI, 2001.
40.
41.
42.
G. Sheldrick, Acta Crystallogr. Sec. A, 2008, 64, 112-122.
SHELXL-97: Program for the Refinement of Crystal
Structure, University of Gottingen, Germany, 1997.
L. Farrugia, J. Appl. Crystallogr., 1999, 32, 837-838.
A. Spek, Acta Crystallogr. sec D, 2009, 65, 148-155.
C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P.
Shields, R. Taylor, M. Towler and J. van de Streek, J. Appl.
Crystallogr., 2006, 39, 453-457.
43.
44.
45.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Dalton Transactions
Page 12 of 12
View Article Online
DOI: 10.1039/C8DT01653D
Table of contents
Synthesis of new phosphane-Cu(I) complexes with bidentate ligands displaying
cytotoxic and cytostatic effects that make them attractive as anticancer agents.