Anticancer activities of manganese-based photoactivatable CO-releasing complexes (PhotoCORMs) with benzimidazole derivative ligands

Article abstract of DOI:10.1007/s11243-017-0136-x

Carbon monoxide is an important signaling molecule which is produced by heme oxygenase-1. CO shows antiproliferative activity against cancer cells; hence, activation of HO-1 is a significant inhibition strategy against tumor formation and survival of cancer cells. In this work, manganese-based CO-releasing molecules (CORMs) were designed and synthesized to inhibit breast cancer cell proliferation. Human invasive ductal breast cancer cells (MCF-7) were treated with the synthesized CORMs to investigate the effect of the complexes on breast cancer survival under UV light. In vitro experiments indicated that the complexes inhibited breast cancer cell proliferation, and further, the antiproliferative effects were increased under UV light. Thus, these novel CORMs may provide a drug template for the treatment of invasive ductal breast cancer.

Full text of DOI:10.1007/s11243-017-0136-x

Transit Met Chem
DOI 10.1007/s11243-017-0136-x
Anticancer activities of manganese-based photoactivatable
CO-releasing complexes (PhotoCORMs) with benzimidazole
derivative ligands
1
2
•
Elvan Ustu¨n Aykut Ozgu¨r Ku¨bra A. Cos¸kun2 Serpil Demir Du¨s¸u¨nceli3
¨
¨
¨
•
•
•
Ismail Ozdemir Yusuf Tutar4
3
˙
•
Received: 2 January 2017 / Accepted: 17 March 2017
Ó Springer International Publishing Switzerland 2017
Abstract Carbon monoxide is an important signaling
molecule which is produced by heme oxygenase-1. CO
shows antiproliferative activity against cancer cells; hence,
activation of HO-1 is a significant inhibition strategy
against tumor formation and survival of cancer cells. In this
work, manganese-based CO-releasing molecules (CORMs)
were designed and synthesized to inhibit breast cancer cell
proliferation. Human invasive ductal breast cancer cells
(MCF-7) were treated with the synthesized CORMs to
investigate the effect of the complexes on breast cancer
survival under UV light. In vitro experiments indicated that
the complexes inhibited breast cancer cell proliferation,
and further, the antiproliferative effects were increased
under UV light. Thus, these novel CORMs may provide a
drug template for the treatment of invasive ductal breast
cancer.
Introduction
Carbon monoxide is a colorless and odorless gas that, when
inhaled, enters the bloodstream and replaces the oxygen on
hemoglobin to form carboxyhemoglobin and is therefore a
threat to human health. Notwithstanding, CO is endoge-
nously produced during cellular metabolism, primarily
from the degradation of heme by the heme oxygenase (HO)
enzyme system [1, 2]. Although CO was long thought to be
only a waste product of this process, it is now widely
accepted that CO is formed endogenously for cytoprotec-
tion against tissue injury/dysfunction [3, 4].
HO catalyzes the rate-limiting step in heme degradation,
leading to the generation of equimolar amounts of iron,
biliverdin and CO [1, 2]. HO not only regulates important
biological processes including oxidative stress, inflamma-
tion, apoptosis, cell proliferation, fibrosis and angiogenesis,
but also has antiinflammatory, antioxidant and antiapop-
totic effects [5–7]. HO exists in two distinct isoforms, an
inducible (HO-1) and a constitutive form (HO-2) [8]. The
role of HO-1 in angiogenesis was first analyzed by Abra-
ham et al. [9], who showed that overexpression of HO-1 in
endothelial cells enhances their proliferation and confirmed
that HO-1 promotes endothelial cell cycle progression.
HO-1 was also found to protect against neuron damage,
inflammation, atherosclerosis, and cardiovascular diseases
[10, 11]. It is crucial for wound healing and neovascular-
ization of ischemic heart and peripheral muscles, but can
have obvious detrimental results in diseases in which
angiogenesis is not desirable, such as cancer [12–16]. High
levels of HO-1 may promote tumor cell survival, hindering
the effectiveness of anticancer therapies; conversely, inhi-
bition of HO has been shown to enhance tumor regression
in animal models, suggesting that the HO-1 pathway may
be a therapeutic target in carcinogenesis [12–20]. These
Electronic supplementary material The online version of this
article (doi:10.1007/s11243-017-0136-x) contains supplementary
material, which is available to authorized users.
¨
& Elvan Ustu¨n
elvanustun77@gmail.com
1
Department of Chemistry, Faculty of Art and Science, Ordu
University, 52200 Ordu, Turkey
2
Department of Bioengineering, Faculty of Natural Sciences
and Engineering, Gaziosmanpas¸a University, 60100 Tokat,
Turkey
3
˙
¨ ¨
Department of Chemistry, Faculty of Science, Inonu
University, 44280 Malatya, Turkey
4
Division of Biochemistry, Department of Basic
Pharmaceutical Sciences, Faculty of Pharmacy, Cumhuriyet
University, 58140 Sivas, Turkey
123

Transit Met Chem
HO-1-dependent processes are due, at least in part, to CO.
The beneficial effects of CO have been demonstrated in
cell culture and animal models of some diseases [4, 21–23].
Several studies have demonstrated the antitumor activity of
CO, as a byproduct of HO-1. Dulak et al. [3, 4] have
reported that CO inhibits proliferation of human pancreatic
cancer cell lines in a dose-dependent manner. Also,
Schatzschneider et al. [24] have analyzed photoinitiated
cytotoxicity against the human colon cancer cell line HT29
by the complex [Mn(CO)3(tpm)]PF6. Lee et al. [25] have
found that treatment with [Ru(CO)3Cl2]2, a CO-releasing
molecule (CORM), reduced the growth of human MCF-7
and MDA-MB-231 breast cancer cells.
Results and discussion
Synthesis and characterization
The free ligands used to synthesize these complexes (L1–
1
L5) were characterized by H NMR, 13C NMR, IR and
elemental analysis, as detailed in the Experimental sec-
tion. The NCHN hydrogens of the free ligands show sin-
glets between 8.02 and 7.40 ppm in the 1H NMR, while the
NCHN carbons show resonances between 143.2 and
144.2 ppm in the 13C NMR spectra. The benzyl CH2 signal
1
is seen between 5.25 and 5.38 ppm in the H NMR and
43.1–48.9 ppm in the 13C NMR.
Organometallic compounds can target DNA and protein
macromolecules specifically in tumors, so reducing side
effects [26]. Organometallic compounds of Mn were syn-
thesized and tested in vitro through cell cytotoxicity and
myoglobin assay in order to elucidate novel CORMs [27].
A fundamental consideration with CO-releasing systems
is safe and controlled delivery of CO for therapeutic
applications. This issue can be resolved by employing a
photoinduced releasing system that allows one to control
the location, timing and dosage of the therapeutic agent.
These kinds of dark–stable, light-sensitive and potentially
CO-releasing complexes are called photoCORMs (pho-
toactivatable CO-releasing molecules). Since the first
photoCORM, namely [Mn2(CO)10], was reported by Mot-
terlini et al. [28], several research groups have exploited
this strategy. Schatzschneider et al. [29, 30] have suc-
cessfully developed [Mn(CO)3(R-tpm)]? complexes
[tpm = tris(pyrazolyl)methane], coupled to target mole-
cules such as peptides and SiO2 nanoparticles, without
altering the photochemical CO-release properties of the
metal complexes.
The complexes were synthesized by stepwise ligand
addition to pre-synthesized Mn(CO)3(bpy)Br (Scheme 1).
The complexes were characterized by 1H NMR, 13C NMR, IR
andLC–MS. Inthe1H NMRspectrumofcomplex3, signalsat
7.71 ppm (t), 8.27 ppm (t), 8.53 ppm (d) and 9.30 ppm
(d) with the expected multiplicities and integrals are assigned
to bpy. A singlet at 6.52 ppm is assigned to the hydrogen atom
situated between the nitrogen of the benzimidazole moiety in
1
complex 3 (Fig. S1). A H COSY NMR experiment was
carried out on complex 3 to help in the assignment of the
proton chemical shifts. The sets of cross peaks of the bipyridyl
and benzimidazole derivative ligands can clearly be observed
(Fig. S2). The 1H NMR spectra of the other complexes were
similar, as detailed in the Experimental section.
NCHN signals between 159.4 and 112.5 ppm in the 13
C
NMR spectra of the complexes are very expected for the
aromatic groups. The benzyl CH2 groups of the ligands
show signals between 55.0 and 43.5 ppm (Fig. S3 for 3).
The CH3 substituents on the benzyl groups of the benz-
imidazole are observed at 55.1 ppm for 2; 18.8 and
20.6 ppm for 3; 14.5 and 20.0 ppm for 4; and 15.5, 16.6
and 16.9 ppm for 5. The 13C NMR data are also detailed in
the Experimental section.
Research into new CORMs has attracted considerable
interest due to the favorable results obtained to date. In this
study, we synthesized [Mn(CO)3(bpy)L]X-type CORMs
[bpy = 2,2-bipyridyl, X = PF6 or trifluoromethanesul-
fonate (OTf); L = N-benzylbenzimidazole, N-(4-methyl-
benzyl)benzimidazole, N-(2,4,6-trimethylbenzyl)benzimi-
dazole, N-(2,3,5,6-tetramethylbenzyl)benzimidazole or
N-(2,3,4,5,6-pentamethylbenzyl) benzimidazole] and char-
IR spectra of both possible stereoisomers of the com-
plexes should have three CO bands due to the CS point
group. The IR spectra of complexes 1 and 4 are compatible
with this prediction, but the unobserved third band of
complexes 2, 3 and 5 could be obscured by the second broad
carbonyl band (Fig. S4). Bands at 1604 s cm-1 for benz-
imidazoles are assigned to C–N. Features at 1261, 1229,
1146 and 1030 cm-1 are assigned to OTf in complexes 1, 2
and 5, while bands at 829 and 822 cm-1 are assigned to P-F
stretching frequencies for complexes 3 and 4, respectively.
The spectra of all of the complexes LC–MS were con-
sistent with expectations. The strongest peaks are attributed
to M-PF6 and to M-OTf species.
1
acterized these complexes by H NMR, 13C NMR, IR and
LC–MS. The CO-releasing properties of the complexes
were analyzed by myoglobin assays, and their anticancer
activities were tested on human invasive ductal breast
cancer cell line (MCF-7) using 5-fluorouracil (5-FU) as a
control. Our results suggest that these complexes are
promising candidate CORMs in cancer treatment.
123

Transit Met Chem
PF6
OTf
OTf
OTf
PF6
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
OC
OC
OC
OC
OC
OC
OC
OC
OC
Mn
Mn
Mn
Mn
Mn
OC
N
CO
CO
CO
CO
CO
1
2
3
4
5
Scheme 1 Fac-manganese(I) tricarbonyl bipyridyl complexes with benzimidazole derivative ligands
All of the complexes displayed broad maxima in the
374–379 nm range and the extinction coefficients were
calculated according to the Lambert–Beer Law. Addition-
ally, the complexes except 3 and 4 gave weak shoulders at
320–323 and 277–285 nm, whose extinction coefficients
were also calculated (Table S1).
ligands present per molecule. All the myoglobin assay
measurements were taken for 45 min. The CO-releasing
properties of the complexes 1–5 are given in Table 1.
One of the aims of this study to gain insight into the
dependence of the CO photorelease from complexes with
different numbers of methyl groups on the ligands. We
conceived that boosting electron donation through
increasing numbers of methyl groups could induce electron
density on the metal and so strengthen Mn–CO p-back-
bonding. However, complex 1 released 1.4 of 3 carbonyls
(47%), while complex 5 which has five methyl substituents
released 2.2 of 3 carbonyls (73%). With increasing num-
bers of methyl substituents on the benzyl moiety of the
benzimidazole ligand, both the equivalent of released CO
and MbCO concentration also increase (Table 1). These
results can be explained by the stabilities of the product
complexes with fewer carbonyl ligands. On the other hand,
if the CO-releasing properties are considered according to
half-life, there is no steady alteration depending on the
number of methyl substituents (Table 1).
Myoglobin assays
CO-release from a coordination complex can be realized in
several ways [28, 29]. Photoinduced CO-release from
dark–stable metal–carbonyl complex prodrugs, as used in
this study, is an important option, since many metal car-
bonyl complexes are sensitive to UV–visible light [30–34].
Complexes 1–5 were first dissolved in DMSO, and the
solutions were kept in the absence of light for 4 h to
confirm their stability. They were then irradiated with a
366-nm UV lamp in 10-min intervals. Their absorptions
were measured at selected wavelengths (510 nm, isobestic
point; 540 nm, Mb–CO; 557 nm, deoxy-Mb; 577 nm, Mb–
CO) in the dark for 16 h (960 min) with solutions of the
complexes [15 lM in 0.1 M PBS at pH = 7.4 in the
presence of myoglobin (60 lM) and sodium dithionite
(10 mM)] under argon atmosphere. All five complexes
showed good dark stability over 16 h, with only negligible
spectral changes.
Cell proliferation assays
The antiproliferative activities of the complexes against the
MCF-7 cell line were assayed by XTT cell proliferation.
The complexes and 5-FU were incubated at different
concentrations (200, 100, 50, 25, 12.5 and 6.125 lM) with
breast cancer cells for 24 h in the dark. MCF-7 cells were
treated with the compounds and irradiated with UV light at
366 nm for 10 min. The complexes were dissolved in
DMSO and diluted in Dulbecco’s modified Eagle’s med-
ium (DMEM). Control cells were treated with DMEM
containing 0.1% DMSO. All five CORMs demonstrated
effective anticancer activities, and the IC50 values of the
complexes and 5-FU as control are given in Table 2.
The in vitro results indicated that these CORMs have
significant anticancer potency and were able to kill MCF-7
cells in the absence of UV light. Moreover, breast cancer
cell viability was found to decrease with photoactivation.
Myoglobin assay is principally based on following the
transformation of deoxymyoglobin to carbonmonoxy-
myoglobin after the addition of CORM, using UV–Vis
spectroscopy. Carbonmonoxy-myoglobin has two absorp-
tion maxima at 540 and 577 nm, while deoxymyoglobin
has only one maximum at 557 nm. Hence, release of CO
can be quantified using these spectral changes (Fig. S5). It
is also known that irradiation of MbFe(II) under the same
conditions without the manganese tricarbonyl complex in
the dark does not lead to any spectral changes [26].
Total released CO, CO equivalents and half-life (t1/2
)
were determined by UV–visible spectroscopy at 1-min
intervals with a 366-nm UV lamp. The t1/2 in this study is
defined as the time taken to release 50% of the total CO
123

Transit Met Chem
Table 1 CO-releasing data for
complexes 1–5
Complexes
Concentration of MbCO (lM)
Half-life t1/2 (min)
Equivalent of CO released
1
2
3
4
5
18.6
20.2
23.0
25.8
32.9
9.5
11.4
13.9
3.9
1.4
1.4
1.5
1.7
2.2
8.7
distilled after refluxing over metallic sodium or phospho-
rous pentoxide for 3–4 days. IR spectra were recorded on
solid samples with a Shimadzu IRAffinity-1 ATR spec-
trometer. Band intensities are marked as strong (s), med-
ium (m), weak (w) or shoulder (sh). NMR spectra were
recorded on a Bruker Ultra Shield 300 MHz spectrometer.
Chemical shifts d in ppm indicate a downfield shift relative
to tetramethylsilane (TMS) and were referenced relative to
the solvent signals. Coupling constants J are given in Hertz.
Absorption spectra were measured using a Shimadzu UV-
1800 spectrophotometer equipped with quartz cuvettes
(d = 1 cm). Elemental analyzes (C, H and N) were
obtained using a CHNS-932 (LECO) instrument. LC–MS
was carried out on an Agilent 1100 Series instrument. All
chemicals were purchased from Sigma-Aldrich and used
without further purification. The MCF-7 cell line was
obtained from the American Type Culture Collection,
USA. Dulbecco’s modified Eagle’s medium (DMEM) was
obtained from Merck. Heat-inactivated fetal bovine serum
was obtained from Life Technologies. Trypsin–EDTA, L-
glutamine–penicillin–streptomycin and phosphate-buffered
saline (PBS) solutions were obtained from Sigma-Aldrich.
The XTT cell proliferation kit was purchased from Applied
Chem.
Table 2 IC50 values of CORMs and 5-FU against MCF-7 cell line
Complexes
IC50 (lM)
IC50/UV (lM)
5-FU
13.25
51.93
22.89
3.22
9.45
2.91
12.25
1.79
1.43
\1
1
2
3
4
5
17.32
6.49
In general, the number of methyl substituents on the ben-
zene ring of the benzimidazole skeleton significantly
enhanced the anticancer and CO-releasing activities of
these complexes, especially under conditions of photoac-
tivation. Complex 3 had the lowest IC50 values, which can
be attributed to the number of methyl groups and associ-
ated steric effects. In general, CO-release is linearly cor-
related to UV-treated MCF-7 cell line cytotoxicity;
although complex 2 is an exception to this correlation. It is
possible that the para-substituent in complex 2 may perturb
electron distribution adversely with respect to UV irradia-
tion. Nevertheless, complex 2 displayed similar cytotoxic
effects to 5-FU, while the other four complexes all show
better activities than 5-FU.
Myoglobin assay
Conclusions
Stock solutions of the complexes for myoglobin assays
were prepared in DMSO. PBS (0.1 M, pH = 7.4), 100 mM
sodium dithionite (100 lL), 15 lM carbonyl complex and
60 lM myoglobin were combined in a cuvette to give a
total volume of 1000 lL. Solutions were degassed by
bubbling with argon at each step of the procedure. Horse
muscle myoglobin solution prepared in PBS (0.1 M,
pH = 7.4) was reduced to deoxymyoglobin by addition of
a solution of sodium dithionite in PBS (0.1 M, pH = 7.4).
Irradiation was made with a 365-nm CAMAG UV lamp at
1-min intervals during the initial 20 min and then contin-
ued at 5-min intervals until no further difference in MbCO
concentration was observed. The final solution was placed
5 cm in front of the UV lamp. All irradiation experiments
were carried out in triplicate. Solutions were freshly pre-
pared for the dark stability and photoactivation experi-
ments. Dark stability spectra were collected automatically
We have synthesized and characterized five manganese
carbonyl complexes of general formula Mn(CO)3(bpy)L,
with benzimidazole derivative ligands. Our studies into the
CO-releasing and anticancer activities of these manganese
CORMs gave promising results, and suggest that they may
find applications in the effective treatment of invasive
ductal breast carcinoma.
Experimental section
Materials and methods
All reactions were carried out under argon using standard
Schlenk and vacuum techniques. Solvents were freshly
123

Transit Met Chem
for the required period of time by the spectrometer
software.
(CH2C6H2(CH3)3), 19.5 and 21.0 (CH2C6H2(CH3)3). Anal.
Calc. for C17H18N2 (250.34): C, 81.56; H, 7.25; N, 11.19.
Found: C, 81.60; H, 7.27; N, 11.14%.
Preparation of L1–L5
N-(2,3,5,6-tetramethylbenzylbenzimidazole) (L4)
Benzimidazole (10 mmol) was added dropwise to a solu-
tion of dried sodium hydride (10 mmol) in THF (50 mL) in
an air-evacuated Schlenk flask. When gas outflow finished,
the required alkyl chloride (10.1 mmol) (benzyl chloride
for L1; 4-methylbenzyl chloride for L2; 2,4,6-trimethyl-
benzyl chloride for L3; 2,3,5,6-tetramethylbenzyl chloride
for L4; and 2,3,4,5,6-pentamethylbenzyl chloride for L5)
was added. The final solution was stirred for a day at room
temperature and then refluxed for 3 h at 70 °C. Solvent was
then evaporated under vacuum. The pure benzimidazole
derivative ligand was obtained upon recrystallization from
CH2Cl2/Et2O.
Yield: 2.43 g (92%). 1H NMR (400 MHz, CDCl3): d
(ppm) = 7.45 (s, 1H, NCHN), 7.88–7.84 (m, 1H, NC6H4
N), 7.58–7.55 (m, 1H, NC6H4N), 7.43–7.33 (m, 2H, NC6
H4N), 7.11 (s, 1H, CH2C6H(CH3)4), 5.33 (s, 2H, CH2C6
H(CH3)4), 2.31 and 2.18 (s, 12H, CH2C6H(CH3)4-2,3,5,6).
13C NMR (100.5 MHz, CDCl3): d (ppm) = 43.9 (CH2C6
H(CH3)4), 143.2 (NCHN), 141.5, 133.9, 122.6, 120.1,
113.7 and 109.7 (NC6H4N), 134.7, 132.7, 129.6 and 123.1
(CH2C6H(CH3)4), 20.5 and 15.5 (CH2C6H(CH3)4). Anal.
Calc. for C18H20N2 (264.36): C, 81.78; H, 7.63; N, 10.60.
Found: C, 81.75; H, 7.67; N, 10.57%.
N-benzylbenzimidazole (L1)
N-(2,3,4,5,6-pentamethylbenzylbenzimidazole) (L5)
Yield: 1.98 g (95%). 1H NMR (400 MHz, CDCl3): d
(ppm) = 8.02 (s, 1H, NCHN), 7.86 (d, 1H, J = 7.2 Hz
NC6H4N), 7.19–7.40 (m, 8H, NC6H4N and CH2C6H5),
5.38 (s, 2H, CH2C6H5). 13C NMR (100.5 MHz, CDCl3): d
(ppm) = 48.9 (CH2C6H5), 143.7 (NCHN), 143.2, 35.4,
33.9, 129.1, 128.3, 127.1, 123.2, 122.4, 120.3, 110.1
(NC6H4N and CH2C6H5). Anal. Calc. for C14H12N2
(208.26): C, 80.74; H, 5.81; N, 13.45. Found: C, 80.78; H,
5.78; N, 13.48%.
Yield: 2.39 g (86%). 1H NMR (400 MHz, CDCl3): d
(ppm) = 7.40 (s, 1H, NCHN), 7.30–7.37 (m, 2H, NC6H4
N), 7.52–7.54 (m, 1H, NC6H4N), 7.82–7.84 (m, 1H, NC6
H4N), 5.31 (s, 2H, CH2C6(CH3)5), 2.21, 2.27 and 2.32 (s,
15H, CH2C6(CH3)5). 13C NMR (100.5 MHz, CDCl3): d
(ppm) = 144.2 (NCHN), 141.9, 134.1, 133.4, 122.2, 120.4
and 109.5 (NC6H4N), 136.2, 133.5 and 122.7 (CH2C6
(CH3)5), 44.3 (CH2C6(CH3)5), 16.5, 16.8 and 17.2 (CH2
C6(CH3)5). Anal. Calc. for C14H12N2 (278.39): C, 81.97; H,
7.97; N, 10.06. Found: C, 81.94; H, 7.92; N, 10.01%.
N-(4-methylbenzylbenzimidazole) (L2)
Preparation of complexes 1–5
Yield: 2.05 g (92%). 1H NMR (400 MHz, CDCl3): d
(ppm) = 7.84 (s, 1H, NCHN), 7.75–7.78 (m, 1H, NC6H4
N), 6.85–7.25 (m, 7H, NC6H4N and CH2C6H4CH3), 5.26
(s, 2H, CH2C6H4CH3), 2.23 (s, 3H, CH2C6H4CH3). 13C
NMR (100.5 MHz, CDCl3): d (ppm) = 48.9 (CH2C6H5),
143.2 (NCHN), 110.6, 114.8, 119.3, 123.5, 123.9, 125.9,
126.7, 128.1, 128.8, 131.0, 132.3, 136.2 (NC6H4N and
CH2C6H4CH3), 21.4 (CH2C6H4CH3). Anal. Calc. for
C15H14N2 (222.29): C, 81.05; H, 6.35; N, 12.60. Found: C,
81.00; H, 6.33; N, 12.67%.
The complexes were prepared by similar methods.
Mn(CO)3(bpy)Br (100 mg, 0.27 mmol) was added to a
solution of AgOTf (82.2 mg, 0.320 mmol) in acetone
(10 mL). Precipitated AgBr was filtered out with celite,
and the ligand (0.32 mmol) was added to the filtrate after
stirring for a day at room temperature. Acetone was
evaporated under vacuum, and KPF6/methanol solution
(10 mL) was added. The resulting orange precipitate was
filtered off and washed with 5 mL cold methanol followed
by 10 mL cold diethyl ether. There was no need to change
anion for 1, 2 and 5; these complexes precipitated directly
and were filtered off and washed with 5 mL cold methanol
followed by 10 mL cold diethyl ether. At all steps of the
synthesis, all glassware was protected from light by
wrapping in aluminum foil.
N-(2,4,6-trimethylbenzylbenzimidazole) (L3)
Yield: 2.06 g (82%). 1H NMR (400 MHz, CDCl3): d
(ppm) = 7.43 (s, 1H, NCHN), 7.29–7.35 (m, 2H, NC6H4
N), 7.45–7.47 (m, 1H, NC6H4N), 7.81–7.83 (m, 1H, NC6
H4N) 6.97 (s, 2H, CH2C6H2(CH3)3), 5.25 (s, 2H, CH2C6
H2(CH3)3), 2.34 and 2.25 (s, 9H, CH2C6H2(CH3)3). 13C
NMR (100.5 MHz, CDCl3): d (ppm) = 43.1 (CH2C6H2
(CH3)3), 144.1 (NCHN), 141.7, 134.2, 122.8, 122.2, 120.4,
and 109.5 (NC6H4N), 138.8, 137.8, 129.6 and127.1
[Mn(CO)3(bpy)(L1)]OTf (1)
1
Yield: 136 mg (78%). H NMR (300 MHz, DMSO-D6): d
(ppm) = 5.36 (s, 2H, NCH2C6H5), 9.48 (s, 1H, NCHN),
123

Transit Met Chem
8.60–6.92 (m, 16H, NCH2C6H5, NC10H8N and N2C6H5).
13C NMR (300 MHz, DMSO-D6):
(ppm) = 47.9
(NCH2C6H(CH3)4), 54.9 (NCH2C6H(CH3)4), 112.5
(NCHN), 142.5, 141.8, 133.9, 133.6, 133.1, (NCH2C6
H(CH3)4), 132.3, 129.0, 124.3, 124.2 (NC6H4N), 154.5,
154.3, 140.4, 127.7, 123.7 (NC10H8N). LCMS: m/z 559.2
[M-PF6]?. IR (cm-1, ATR): m = 1446.6, 1516.0 (s, C–H),
1608.6 (s, C–N), 2036.8, 1932.7 (s, CO).
d
(NCH2C6H5), 145.9 (NCHN), 155.0, 140.4, 128.7, 127.8,
127.1 (N2C10H8), 154.8, 141.3, 135.4, 133.0 (NCH2C6H5),
128.0, 124.0, 117.1, 112.5 (NC6H4N). LCMS: m/z 503.1
[M-OTf]?. IR (cm-1, ATR): m = 1442.7, 1519.9 (s, C-H),
1604.8 (s, C–N), 2033.0, 1944.2, 1925.0 (s, CO).
[Mn(CO)3(bpy)(L5)]OTf (5)
[Mn(CO)3(bpy)(L2)]OTf (2)
1
Yield: 166 mg (86%). H NMR (300 MHz, DMSO-D6) d
1
Yield: 145 mg (82%). H NMR (300 MHz, DMSO-D6) d
(ppm) = 1.66 (s, 6H, NCH2C6H2(CH3)5), 2.15 (s, 6H,
NCH2C6H2(CH3)5), 2.34 (s, 3H, NCH2C6H2(CH3)3), 5.24
(s, 2H, NCH2C6H2(CH3)3), 5.89 (s, 1H, NCHN), 7.73-7.64
(m, 4H, NC6H4N), 8.25 (t, J = 7.4, 2H, NC10H8N, 5/50),
7.54–7.42 (m, 2H, NC10H8N, 4/40), 8,48 (d, J = 7.8, 2H,
NC10H8N, 3/30), 9,17 (d, J = 5.2, 2H, NC10H8N, 6/60). 13C
NMR (300 MHz, DMSO-D6) d (ppm) = 15.5 (NCH2C6
(CH3)5), 16.6 (NCH2C6(CH3)5), 16.9 (NCH2C6(CH3)5),
112.6 (NCHN), 117.2 (NCH2C6(CH3)5) 135.8, 135.1,
133.6, 132.7, 132.6, 132.5 (NCH2C6(CH3)3), 142.1, 142.0,
126.2, 124.4, 124.3 (NC6H4N) 154.5, 154.3, 140.3, 127.7,
(ppm) = 5.68 (s, 2H, NCH2C4H4Cl), 3.75 (s, 3H, NCH2-
C4H4CH3), 9.49 (d, J = 4.0, 2H, NCH2C4H4CH3), 8.60 (d,
J = 6.0, 2H, NCH2C4H4CH3), 7.30–6.79 (m, 6H, NC6H4N
and NC10H8N), 7.99 (s, 1H, NCHN), 8.32 (t, J = 6.5, 2H,
NC10H8N), 7.84 (t, J = 5.5, NC10H8N), 7.79 (d, J = 4.0,
NC10H8N). 13C NMR (300 MHz, CDCl3) d (ppm) = 49.5
(NCH2C6H4CH3), 55.1 (NCH2C6H4CH3), 159.4 (NCHN),
154.9, 140.3, 130.9, 130.0, 128.7 (N2C10H8), 154.7, 142.0,
132.8, 127.7, 124. 0 (NCH2C6H4CH3), 126.6, 125.5, 114.3,
113.9 (NC6H4N). LCMS: m/z 517.4 [M-OTf]?. IR (cm-1
,
ATR): 1442.6, 1473.6 (s, C–H), 1604.8 (s, C–N), 2040.7,
1932.7 (s, CO).
123.7 (NC10H8N). LCMS: m/z 573.3 [M-OTf]?. IR (cm-1
,
ATR): 1446.6, 1473.6 (s, C–H), 1608.6 (s, C–N), 2033.0,
1936.5 (s, CO).
[Mn(CO)3(bpy)(L3)]PF6 (3)
Cell culture and growth
1
Yield: 141 mg (76%). H NMR (300 MHz, DMSO-D6) d
(ppm) = 1.76 (s, 6H, NCH2C6H2(CH3)3), 2.33 (s, 3H,
NCH2C6H2(CH3)3), 7.43–7.36 (m, 2H, NC6H4N), 7.53 (d,
J = 8.0, NC6H4N), 7.93 (d, J = 8.0, NC6H4N), 5.23 (s,
2H, NCH2C6H2(CH3)3), 6.52 (s, 1H, NCHN), 6.62 (s, 2H,
NCH2C6H2(CH3)3), 7.71 (t, J = 7.0, 2H, NC10H8N, 5/50),
8,27 (t, J = 9.0, 2H, NC10H8N, 4/40), 8,53 (d, J = 8.0, 2H,
NC10H8N, 3/30), 9,30 (d, J = 5.5, 2H, NC10H8N, 6/60). 13C
NMR (300 MHz, DMSO-D6) d (ppm) = 18.7 (NCH2C6
H2(CH3)3), 20.5 (NCH2C6H2(CH3)3), 43.5 (NCH2C6H2
(CH3)3), 112.3 (NCHN), 143.3, 141.7, 138.1, 137.0, 133.4,
129.2, 127.6, 126.4, 124.2, 123.9, 123.9, 117.1 (NCH2C6
H2(CH3)3), 154.6, 154.3, 140.3, 137.4, 129. 3 (NC10H8N).
LCMS: m/z 545.1 [M-PF6]?. IR (cm-1, ATR): 1446.6,
1473.6 (s, C–H), 1600.9 (s, C–N), 2036.8, 1952.0, 1928.8
(s, CO).
MCF-7 cells were cultured in DMEM supplemented with
10% heat-inactivated fetal bovine serum, 1% L-glutamine,
100 IU mL-1 penicillin and 10 mg mL-1 streptomycin in
75 cm2 polystyrene flasks. Cells were cultivated at 37 °C
in a humidified atmosphere with 5% CO2.
XTT cell proliferation assay
MCF-7 cells were cultured in Dulbecco’s modified Eagle
medium (DMEM) supplemented with 10% FBS (heat-in-
activated fetal bovine serum), 1% L-glutamine,
100 IU mL-1 penicillin and 10 mg mL-1 streptomycin in
75 cm2 polystyrene flasks. Cells were cultivated at 37 °C
in a humidified atmosphere with 5% CO2.
The anticancer activities of the manganese CORMs
were determined using the 2,3-bis[2-methoxy-4-nitro-5-
sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) cell
proliferation assay. Approximately 10 9 104 MCF-7 cells
(final volume of 200 ll) were seeded in 96-well flat-bottom
ELISA plates and incubated overnight at 37 °C in a 5%
CO2 incubator. Wells were treated with different concen-
trations of the complexes and then incubated at 37 °C in
5% CO2 for 48 h. XTT solution was then applied to each
well and the plates were incubated at 37 °C for 4 h. Optical
densities of the plates was measured using an ELISA
reader at 450 nm.
[Mn(CO)3(bpy)(L4)]PF6 (4)
1
Yield: 164 mg (87%). H NMR (300 MHz, DMSO-D6) d
(ppm) = 1.66 (s, 6H, NCH2C6H(CH3)4), 2.18 (s, 6H,
NCH2C6H(CH3)4), 5.28 (s, 2H, NCH2C6H(CH3)4), 6.12 (s,
1H, NCHN), 7.07 (s, 1H, NC6H4N), 7.48–7.67 (m, 2H,
NC10H8N), 7.71–7.67 (m, 3H, NC6H4N), 7.98 (d, J = 8.4,
1H, NC6H4N), 8.26 (t, J = 7.8, 2H, NC10H8N), 8.51 (d,
J = 7.8, 2H, NC10H8N), 9.22 (d, J = 4.8, 2H, NC10H8N).
13C NMR (300 MHz, DMSO-D6) d (ppm) = 14.5, 20.0
123

Transit Met Chem
Acknowledgements This work by Scientific and Technological
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