In vitro anticancer active cis-Pt(II)-diiodido complexes containing 4-azaindoles

Article abstract of DOI:10.1007/s00775-019-01643-8

4-Azaindole (1H-pyrrolo[3,2-b]pyridine; 4aza) and its N1-alkylated derivative N1-isopropyl-4-azaindole (1-(propan-2-yl)-1H-pyrrolo[3,2-b]pyridine; ip4aza) have been used for the preparation of the cis-diiodido-platinum(II) complexes cis-[Pt(4aza)₂I₂] (1), cis-[PtI₂(ip4aza)₂] (2), cis-[Pt(4aza)I₂(NH₃)] (3) and cis-[PtI₂(ip4aza)(NH₃)] (4). The prepared complexes were thoroughly characterized (e.g., multinuclear NMR spectroscopy and ESI mass spectrometry) and their in vitro cytotoxicity was assessed at human ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R) and colon carcinoma (HT-29) cell lines, where they showed, in some cases, significantly higher activity than the used reference-drug cisplatin. The results of in vitro cytotoxicity testing at the A2780 and A2780R cells indicated that alkylation of the 4-azaindole moiety at the position of the N1 atom had a positive biological effect, because the ip4aza-containing complexes 2 and 4 showed significantly (p < 0.005) higher cytotoxicity than 4aza-containing analogues 1 and 3. The resistance factors (A2780R/A2780 model) equalled 0.8–1.4, indicating the ability of complexes 1–4 to overcome the acquired resistance of the A2780 cells against cisplatin. Complexes 1 and 2 revealed low toxicity against primary culture of human hepatocytes. The flow cytometry studies of the A2780 cell cycle modification showed that complexes 1–4 induce different cell cycle perturbations as compared with cisplatin, thus suggesting a different mechanism of their antitumor action.

Full text of DOI:10.1007/s00775-019-01643-8

JBIC Journal of Biological Inorganic Chemistry
https://doi.org/10.1007/s00775-019-01643-8
ORIGINAL PAPER
In vitro anticancer active cis‑Pt(II)‑diiodido complexes containing
4‑azaindoles
Pavel Štarha · Zdeněk Trávníček1 · Ján Vančo · Zdeněk Dvořák
1
1
1
Received: 1 November 2018 / Accepted: 27 December 2018
©
Society for Biological Inorganic Chemistry (SBIC) 2019
Abstract
4
-Azaindole (1H-pyrrolo[3,2-b]pyridine; 4aza) and its N1-alkylated derivative N1-isopropyl-4-azaindole (1-(propan-
2
-yl)-1H-pyrrolo[3,2-b]pyridine; ip4aza) have been used for the preparation of the cis-diiodido-platinum(II) complexes
cis-[Pt(4aza) I ] (1), cis-[PtI (ip4aza) ] (2), cis-[Pt(4aza)I (NH )] (3) and cis-[PtI (ip4aza)(NH )] (4). The prepared com-
2
2
2
2
2
3
2
3
plexes were thoroughly characterized (e.g., multinuclear NMR spectroscopy and ESI mass spectrometry) and their in vitro
cytotoxicity was assessed at human ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R) and colon
carcinoma (HT-29) cell lines, where they showed, in some cases, signiꢀcantly higher activity than the used reference-drug
cisplatin. The results of in vitro cytotoxicity testing at the A2780 and A2780R cells indicated that alkylation of the 4-azain-
dole moiety at the position of the N1 atom had a positive biological eꢁect, because the ip4aza-containing complexes 2 and 4
showed signiꢀcantly (p<0.005) higher cytotoxicity than 4aza-containing analogues 1 and 3. The resistance factors (A2780R/
A2780 model) equalled 0.8–1.4, indicating the ability of complexes 1–4 to overcome the acquired resistance of the A2780
cells against cisplatin. Complexes 1 and 2 revealed low toxicity against primary culture of human hepatocytes. The ꢂow
cytometry studies of the A2780 cell cycle modiꢀcation showed that complexes 1–4 induce diꢁerent cell cycle perturbations
as compared with cisplatin, thus suggesting a diꢁerent mechanism of their antitumor action.
Keywords Platinum(II) complexes · 4-Azaindole · Iodido · Cytotoxicity · In vitro
Introduction
pharmacologically prospective representatives reported in
the literature in past few years [4], such as phenanthriplatin
([PtCl(NH ) (Ph)]Cl; Ph = phenanthridine) [5]. However,
Several platinum(II) complexes are clinically used for the
treatment of various cancer types [1]. Among them, the cis-
diammine-dichloridoplatinum(II) complex (cisplatin, cis-
3
2
it has to be stated here that it is not only platinum, whose
complexes have been studied for their anticancer activity,
because there is a plenty of other platinum-group metal com-
plexes [e.g., 6, 7] or other transition metal complexes [8, 9],
which have been recently reported as highly cytotoxic and
prospective from the pharmacological point of view.
[
PtCl (NH ) ]) [2], approved in 1978 by FDA for the clini-
2 3 2
cal use, still represents the crucial anticancer drug applied
for the treatment of cancer patients receiving chemotherapy
[
3]. Besides the clinical use of the platinum-based drugs [1],
topicality and perspective of development and bioanalysis
of novel platinum(II) complexes are underlined by several
In general, 7-azaindoles are frequently used as suitable
N-donor ligand for various types of complexes of many
transition metals [10]. To date, several research teams
have been dealt with platinum(II) complexes contain-
ing 7-azaindole (1H-pyrrolo[2,3-b]pyridine) and its C- or
N-derivatives (7aza) as N-donor carrier-ligands [11–19].
Some concrete examples of such complexes which have
been prepared and bioanalysed in last couple of years are
as follows: cis-[Pt(7aza) Cl ] [11–13], cis/trans-[Pt(7aza)
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00775-019-01643-8) contains
supplementary material, which is available to authorized users.
*
Zdeněk Trávníček
zdenek.travnicek@upol.cz
2
2
1
Biologically Active Complexes and Molecular Magnets,
Regional Centre of Advanced Technologies and Materials,
Faculty of Science, Palacký University in Olomouc,
Šlechtitelů 27, 783 71 Olomouc, Czech Republic
Cl (NH )] [14, 15], [Pt(7aza) (cbdc)] [16], [Pt(7aza) (ox)]
2 3 2 2
[17], [Pt(7aza) (mal)] and cis-[Pt(7aza) (dec) ] [18], and
2
2
2
cis-[Pt(7aza) I ] [19]; cbdc=cyclobutane-1,1-dicarboxylato,
2
2
Vol.:(0
1
123456789)
3

JBIC Journal of Biological Inorganic Chemistry
ox=oxalato, dec=decanoato and mal=malonato. Among
With respect to the above-mentioned high in vitro cyto-
toxicity of platinum(II) complexes containing 7-azain-
doles, it was of great interest to investigate whether the
platinum(II) complexes containing 4-azaindole will aꢁect
the cell viability of selected human cancer cell lines as
well. Since complexes cis-[Pt(7aza) I ] represent the most
them, the cis-[PtCl (7aza) ] complexes showed considerably
2
2
higher in vitro cytotoxicity against a panel of human cancer
cell lines than cisplatin [13, 20, 21], high anticancer activ-
ity in vivo [20] and relevant diꢁerences in several processes
connected with their mechanism of action as compared
with cisplatin [13, 20, 22]. However, in vitro cytotoxicity
of the mentioned cis-[Pt(7aza) Cl ] complexes was in many
2
2
cytotoxic structural type of platinum(II) agents containing
7-azaindoles [19], we chose this structural type of com-
pounds (i.e., diiodido complexes) for the present study as
well, with the aim to ꢀnd out how a relatively small struc-
tural change (i.e., N1 substitution by isopropyl) of 4aza may
inꢂuence the resulting cytotoxicity of the ꢀnal complexes,
as it can be deduced from the literature data reported for a
couple of platinum(II) dichlorido complexes cis-[Pt(7aza)
Cl (NH )] (RA ~ 0.4) [14] and cis-[PtCl (Me7aza)(NH )]
2
2
cases surpassed by their diiodido analogues. For example,
1
the best-performing chlorido complex cis-[Pt(7aza ) Cl ]
2
2
1
(
7aza =4-bromo-7-azaindole) was highly eꢁective against
the human A2780 ovarian carcinoma, HOS osteosarcoma,
MCF7 breast carcinoma and HeLa cervix carcinoma cell
lines, with RA~5.7, 5.6, 6.7 and 4.3, respectively [21]; RA
(
relative activity) = IC (cisplatin)/IC (complex). How-
50 50
2
3
2
3
ever, the RA values obtained for the analogical diiodido
(RA ~ 0.6) [15], both studied at the A2780 cells (72 h
exposure) and referenced against cisplatin; Me7aza = N1-
methyl-7-azaindole (1-(methyl)- 1H-pyrrolo[2,3-b]pyri-
dine). Next motivation can be associated with the previous
1
complex cis-[Pt(7aza ) I ] were markedly higher (ca. 8.8,
2
2
4
7.3, 17.9, and 8.0, respectively) than for the complex cis-
1
[
Pt(7aza ) Cl ] [19]. Furthermore, the diiodido complexes
2
2
n
were also found to be anticancer eꢁective in vivo and their
mechanism of action seems to be diꢁerent from their chlo-
rido analogues as well as from cisplatin [23, 24].
ꢀndings that complexes cis-[PtI (L )(NH )], containing
2
3
1
5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine (L ) [32] or
2
L = 2-(4-chlorophenyl)-8-aminoimidazo[1,2-a]pyridin-
Although complexes containing 7-azaindole or its deriv-
atives are well known in the literature [10], 4-azaindole
3-yl]-N,N-di-n-propylacetamide [33] were reported as more
in vitro cytotoxic than cisplatin.
(
1H-pyrrolo[3,2-b]pyridine; 4aza) or its derivatives (in any
In the present study, diiodido platinum(II) complexes
protonation state) have been used as ligands of transition
metal complexes only in two works [25, 26]. In particular,
complex cis-[Ru(4aza)(bpy) Cl]PF containing electroneu-
cis-[Pt(4aza) I ] (1), cis-[PtI (ip4aza) ] (2), cis-[Pt(4aza)
2
2
2
2
I (NH )] (3) and cis-[PtI (ip4aza)(NH )] (4) containing
2
3
2
3
4-azaindole (4aza) or its derivative N1-isopropyl-4-azain-
dole (1-(propan-2-yl)-1H-pyrrolo[3,2-b]pyridine; ip4aza)
have been prepared (Fig. 1) and their purity and composi-
tion were studied by relevant techniques. Complexes 1–4
were tested for their in vitro cytotoxicity at the human ovar-
ian carcinoma (A2780), cisplatin-resistant ovarian carci-
noma (A2780R) and colon carcinoma (HT-29) cell lines.
The A2780 cells were chosen for the additional studies
of the crucial processes (cellular accumulation, cell cycle
2
6
−
tral 4aza [25], and Na [Fe(4aza ) ]·2Solv (Solv = tetrahy-
2
4
drofuran (THF) or CH CN) containing deprotonated 4aza
3
[
26], have been prepared. It is also noteworthy, that variously
substituted 4-azaindole derivatives exhibit high biological
activity of diꢁerent type, such as antitubercular activity [27,
2
8], p21-activated kinase inhibition activity [29], HIV-1
attachment inhibition activity [30] or cytotoxic and protein
kinase inhibition activity [31].
Fig. 1 Synthetic pathways for
the preparation of a N1-iso-
propyl-4-azaindole (1-(pro-
pan-2-yl)-1H-pyrrolo[3,2-b]
pyridine; ip4aza) given
with the atom numbering
scheme, and b platinum(II)
complexes cis-[Pt(4aza) I ]
2
2
(
1), cis-[PtI (ip4aza) ] (2),
2 2
cis-[Pt(4aza)I (NH )] (3) and
2
3
cis-[PtI (ip4aza)(NH )] (4)
2
3
containing 4-azaindole (4aza)
or ip4aza
1
3

JBIC Journal of Biological Inorganic Chemistry
perturbation) connected with their high antiproliferative
activity at these human cancer cells.
to 50 °C and 5 molar equiv. of KI was added (166 mg).
The reaction mixture was stirred at 50 °C for 10 min, pro-
viding the solution of K [PtI ]. After that, the solution
2
4
was cooled to ambient temperature and the stoichiomet-
ric amount of 4aza (0.4 mmol, 47 mg) dissolved in 1 mL
of MeOH was poured in. The obtained yellow suspension
was stirred for 2 h, then the solid was collected by ꢀltration
and washed with water (2 × 0.5 mL), MeOH (2 × 0.5 mL),
and DEE (2 × 1 mL). The yellow solid product was dried
in desiccator under the reduced pressure (30 min) and
the yield was ca. 75% (calculated to K [PtCl ]). Anal.
Experimental
Materials
K PtCl was purchased from Precious Metals Online, while
2
4
other chemicals (4aza, NaH, MgSO , AgNO , KI, KCl,
4
3
2
-bromopropane, NH (25% water solution), HCl (35%
3
2
4
water solution), cisplatin, reduced glutathione (GSH),
l-cysteine (CysH), cytochrome c from bovine heart (Cytc),
hen egg-white lysozyme (HEWL), guanosine monophos-
phate disodium salt hydrate (GMP), solvents (MeOH, diethyl
ether (DEE), EtOH, THF, ethyl acetate, DMF, DMSO and
n-octanol) and deuterated solvents for NMR experiments
Calc. for PtC H N I : C, 24.5; H, 1.8; N, 8.2; Found:
14 12 4 2
C, 24.9; H, 1.7; N, 8.0%. ESI+ MS (MeOH, m/z): 707.8
+
(calc. 708.9; 30%; {[Pt(4aza) I ] + Na} ), 675.9 (calc.
2
2
+
676.0; 40%; [Pt(4aza) I] ), 558.0 (calc. 558.0; 100%;
3
+
[Pt(4aza) I] ), 430.1 (calc. 430.1; 20%; {[Pt(4aza) ]–
2
2
+
+
H} ), 119.2 (calc. 119.0; 40%; {(4aza)+ H} ). ESI– MS
−
(
CDCl , DMF-d , D O) were supplied by VWR Interna-
(MeOH, m/z): 811.9 (calc. 811.8; 100%; [Pt(4aza) I ] ),
3
7
2
2 3
−
tional (Stříbrná Skalice, Czech Republic), Sigma-Aldrich
693.8 (calc. 693.7; 10%; [Pt(4aza)I ] ), 683.9 (calc.
3
−
(
Prague, Czech Republic), Lach-Ner (Neratovice, Czech
683.9; 10%; {[Pt(4aza) I ]–H} ), 576.0 (calc. 575.7;
2
2
−
Republic) and Litolab (Chudobín, Czech Republic).
20%; [PtI ] ), 566.0 (calc. 565.8; 25%; {[Pt(4aza)I ]–
3
2
−
1
H} ). H NMR (DMF-d , 25 °C, ppm): 11.89 (s, N1–H),
7
Synthesis
8.94 (d, J = 5.5 Hz, C5–H), 7.93 (s, C2–H), 7.88 (d,
1
3
J = 8.3 Hz, C7–H), 7.52 (s, C3–H), 7.14 (s, C6–H).
C
N1‑isopropyl‑4‑azaindole (ip4aza)
NMR (DMF-d , 25 °C, ppm): 146.4 (C7a), 144.5 (C5),
7
1
31.1 (C2), 130.6 (C3a), 121.0 (C7), 117.4 (C6), 103.6
1
5
NaH (1.2 mmol, 48 mg) and 4aza (1.0 mmol, 118 mg) were
poured into the reaction ꢂask, which was closed with a rub-
ber septum and repeatedly evacuated and ꢀlled with nitrogen
gas. After that, 5 mL of THF was slowly poured in to start
the reaction (− 20 °C). After 15 min of stirring at − 20 °C,
(C3). N NMR (DMF-d , 25 °C, ppm): 200.6 (N4), N1—
7
1
95
not detected. Pt NMR (DMF-d , 60 °C, ppm): -3186.
7
−
1
FTIR (ν
cm ): 3284s, 3071m, 2894w, 2706w, 1569w,
ATR
1496w, 1480m, 1417vs, 1273vs, 1353s, 1327s, 1209m,
1131w, 1095w, 1059m, 888w, 792w, 760vs, 625w.
2
-bromopropane (1.5 mmol, 183 µL) was added to this mix-
ture, which reacted in microwave synthesizer at 150 °C for
Complex cis‑[PtI (ip4aza) ] (2) Complex 2 was prepared by
2
2
1
h. After cooling to ambient temperature, the mixture was
the same protocol as described for complex 1, but starting
from 0.1 mmol K [PtCl ] and using ip4aza instead of 4aza.
centrifuged to separate NaBr and supernatant, which was
2
4
dried (MgSO ), ꢀltered, and evaporated on a rotary evapo-
The reaction mixture was stirred for 24 h and the obtained
yellow solid was collected by ꢀltration, washed with water
(1×0.5 mL), MeOH (1×0.5 mL), and DEE (2×0.5 mL),
and dried in desiccator under the reduced pressure (30 min).
The yield was ca. 45% (calculated to K [PtCl ]). Anal.
4
rator. The obtained brown oily product was puriꢀed using
ꢂash silica gel chromatography with the mixture of ethyl
acetate and NH (100:1, v/v) and using the thin-layer chro-
3
matography (TLC) control. The product (ip4aza; Fig. 1) was
2
4
obtained as brown–yellow oil after evaporation of solvents
Calc. for PtC H N I : C, 31.2; H, 3.1; N, 7.3; Found: C,
20 24 4 2
1
on a rotary evaporator. H NMR (CDCl , 25 °C, ppm): 8.46
31.3; H, 3.3; N, 7.0%. ESI+MS (MeOH, m/z): 802.0 (calc.
3
+
(
d, J=4.4 Hz, C5–H), 7.81 (d, J=8.1 Hz, C7–H), 7.55 (d,
802.0; 100%; {[Pt(ip4aza) I ]+CH OH+H} ), 791.9
2
2
3
+
J = 2.9 Hz, C2–H), 7.20 (dd, J= 8.4, 4.8 Hz, C6–H), 6.83
(calc. 792.0; 5%; {[Pt(ip4aza) I ]+Na} ), 642.0 (calc.
2 2
+
(
d, J=2.9 Hz, C3–H), 4.70 (sep, J=6.6 Hz, C8–H), 1.58 (d,
642.1; 85%; [Pt(ip4aza) I] ), 551.1 (calc. 551.1; 10%;
2
1
3
+
J=7.3 Hz, C9–H ). C NMR (CDCl , 25 °C, ppm): 144.6
[Pt(ip4aza) Cl] ), 514.2 (calc. 514.2; 5%; {[Pt(ip4aza) ]–
6
3
2
2
+
+
(
C7a), 140.8 (C5), 129.2 (C2), 128.7 (C3a), 118.4 (C7),
H} ), 161.2 (calc. 161.1; 10%; {(ip4aza)+H} ). ESI– MS
1
5
−
1
1
15.9 (C6), 101.3 (C3), 47.9 (C8), 22.7 (C9). N NMR
(MeOH, m/z): 735.4 (calc. 735.8; 30%; [Pt(ip4aza)I ] ). H
3
(
(
CDCl , 25 °C, ppm): 293.2 (N4), 124.9 (N1). ESI+ MS
NMR (DMF-d , 25 °C, ppm): 8.99 (d, J=5.9 Hz, C5–H),
3
7
+
MeOH): 161.2 (calc. 161.1 for {(ip4aza)+H} ).
8.10 (d, J=7.3 Hz, C7–H), 7.54 (m, 2H, C2–H), 7.33 (bs,
2
H, C3–H), 7.19 (m, 2H, C6–H), 4.83 (sep, J=7.3 Hz,
−
1
Complex cis‑[Pt(4aza) I ] (1) The solution of K [PtCl ]
C8–H), 1.48 (d, J=5.8 Hz, C9–H ). FTIR (ν
cm ):
2
2
2
4
6
ATR
(
0.2 mmol, 83 mg) in 1 mL of deionized water was heated
3091w, 2971w, 2927w, 1608w, 1566w, 1494s, 1426vs,
1
3

JBIC Journal of Biological Inorganic Chemistry
1
1
272vs, 1368 m, 1306 m, 1205 m, 1129w, 1101w, 1070w,
014w, 792w, 760vs, 725w, 607w.
by water (1 mL), MeOH (2×0.5 mL) and DEE (2×1 mL),
and dried in desiccator under the reduced pressure (2 h).
The yield of the complex 3 was ca. 40% (calculated to
K[PtCl (NH )]). Anal. Calc. for PtC H N I : C, 14.4; H,
Complex cis‑[Pt(4aza)I (NH )] (3) A key intermediate
2
3
3
3
7
9
3 2
K[PtI (NH )] was prepared by the modiꢀcation of the
1.6; N, 7.2; Found: C, 14.8; H, 1.6; N, 7.5%. ESI+ MS
3
3
+
formerly reported protocols [33, 34]. In particular, KI
(MeOH, m/z): 574.9 (calc. 575,0; 5%; [Pt(NH )(4aza) I] ),
3
2
+
(
5.4 mmol, 900 mg) was added to the solution of K [PtCl ]
557.9 (calc. 558.0; 30%; [Pt(4aza) I] ), 457.0 (calc. 457.0;
2
4
2
+
(
0.9 mmol, 375 mg) in 3 mL of distilled water at 50 °C.
10%; [Pt(NH )(H4aza)I] ), 440.0 (calc. 439.9; 15%;
3
+
+
After 10 min of stirring at this temperature, connected with
the colour change to dark red, the mixture was cooled down
[Pt(4aza)I] ), 430.1 (calc. 430.1; 45%; {[Pt(4aza) ]–H} ),
2
+
312.1 (calc. 312.0; 5%; {[Pt(4aza)]–H} ), 119.2 (calc.
+
to the laboratory temperature and 600 µL of NH /water
119.0; 100%; {(4aza) + H} ). ESI– MS (MeOH, m/z):
3
−
solution was added. After 30 min of stirring at ambient tem-
582.8 (calc. 582.9; 15%; {[Pt(NH )(4aza)I ]–H} ), 565.9
3
2
−
perature, the yellow solid of cis-[PtI (NH ) ] was ꢀltered
(calc. 565.8; 35%; {[Pt(4aza)I ]–H} ), 491.9 (calc. 491.9;
2
3 2
2
−
oꢁ, washed with distilled water (1 mL), EtOH (2×1 mL),
and DEE (2×1 mL), and dried under vacuum (1 h). The
yield was ca. 90% (calculated to K [PtCl ]).
90%; {[Pt(NH )(4aza)ClI]–H} ), 475.0 (calc. 474.9; 50%;
3
−
1
{[Pt(4aza)ClI]–H} ). H NMR (DMF-d , 25 °C, ppm):
7
11.98 (s, N1–H), 8.62 (d, J = 5.1 Hz, C5–H), 8.07 (d,
2
4
The complex cis-[PtI (NH ) ] (0.82 mmol, 400 mg) was
J=8.1 Hz, C7–H), 7.97 (bs, C2–H), 7.32 (bs, C3–H), 7.25
2
3 2
1
3
dissolved in 50 mL of distilled water at 80 °C and 2 molar
(dd, J=8.1, 5.9 Hz, C6–H), 4.33 (bs, N10–H ). C NMR
3
equiv. of AgNO (280 mg) was added. After 20 min of stir-
(DMF-d , 25 °C, ppm): 147.0 (C7a), 146.3 (C5), 132.0
3
7
1
5
ring of the reaction mixture in the dark (under aluminium
(C2), 131.7 (C3a), 121.8 (C7), 118.3 (C6), 102.9 (C3).
N
foil), the formed precipitate was ꢀltered oꢁ and the obtained
NMR (DMF-d , 25 °C, ppm): 202.3 (N4), 135.9 (N1), –46.8
7
195
ꢀ
ltrate was concentrated to the volume of ca. 5 mL. An
(N10–H ). Pt NMR (DMF-d , 60 °C, ppm): − 2548. FTIR
3 7
−
1
excess of KCl (13.3 mmol, 990 mg) was added to the con-
centrated solution at 0 °C, leading to the immediate precipi-
tation of the yellow solid of cis-[PtCl (NH ) ] (cisplatin),
(ν
cm ): 3559w, 3252vs, 3106, 2712w, 2598w, 1594w,
ATR
1567w, 1497s, 1417vs, 1274s, 1355w, 1326 m, 1207w,
1098w, 1060w, 889w, 762vs.
2
3 2
which was collected after 15 min of stirring at 0 °C. The
product was washed by EtOH (3 mL) and DEE (2×3 mL),
and dried under vacuum (1 h). The yield was ca. 60% (cal-
culated to cis-[PtI (NH ) ]).
Complex
cis‑[PtI (ip4aza)(NH )]
(4) The
complex
2
3
K[PtCl (NH )] (0.15 mmol, 54 mg) was dissolved in
3
3
1.2 mL of distilled water and the stoichiometric amount of
KI (0.45 mmol, 75 mg) was added, providing the red solu-
tion of K[PtI (NH )]. A slight excess of ip4aza (0.16 mmol,
2
3 2
The complex cis-[PtCl (NH ) ] (cisplatin; 0.5 mmol,
2
3 2
1
50 mg) was suspended in 8 mL of the HCl/H O mixture
2
3
3
of solvents (1:2, v/v) and heated while stirring at 120 °C
for 8 h. After cooling to laboratory temperature, the unre-
acted cisplatin was removed by ꢀltration and the orange
filtrate was concentrated under reduced pressure. The
obtained red solid was dissolved in MeOH (2 mL), ꢀltered
and solvent was removed, which was repeated three times
and led to the dark red NH [PtCl (NH )] intermediate.
26 mg), dissolved in 1.2 mL of MeOH, was added to the water
solution of K[PtI (NH )] and the mixture was stirred over-
3
3
night, leading to the formation of yellow solid of complex
4. The product was removed by ꢀltration, washed by water
(0.5 mL), MeOH (2×0.5 mL) and DEE (2×1 mL), and dried
in desiccator under the reduced pressure (2 h). The yield of
complex 4 was ca. 20% (calculated to K[PtCl (NH )]). Anal.
4
3
3
3
3
NH [PtCl (NH )] was dissolved in 3 mL of MeOH, cooled
Calc. for PtC H N I : C, 19.2; H, 2.4; N, 6.7; Found: C,
4
3
3
10 15 3 2
to 0 °C and excess KOH (1.1 mmol, 60 mg) was added.
19.1; H, 2.4; N, 7.1%. ESI+MS (MeOH, m/z): 659.0 (calc.
+
The yellow solid of K[PtCl (NH )] was collected after
659.1; 20%; [Pt(NH )(ip4aza) I] ), 642.0 (calc. 642.1;
3
3
3
2
+
1
0 min of stirring at 0 °C, washed by EtOH (acidiꢀed by
100%; [Pt(ip4aza) I] ), 531.2 (calc. 531.2; 5%; {[Pt(NH )
2
3
+
HCl; 1 mL) and DEE (2×1 mL), and dried under vacuum
(ip4aza) ]–H} ), 514.2 (calc. 514.2; 5%; {[Pt(ip4aza) ]–
2 2
+
+
(
1 h). The yield of K[PtCl (NH )] was ca. 55% (calculated
H} ), 161.2 (calc. 161.1; 30%; {(ip4aza)+H} ). ESI– MS
3
3
−
to cis-[PtCl (NH ) ]).
(MeOH, m/z): 735.4 (calc. 735.8; 30%; [Pt(ip4aza)I ] ),
2
3 2
3
−
The complex K[PtCl (NH )] (0.15 mmol, 54 mg) was
661.9 (calc. 661.9; 30%; {[Pt(NH )(ip4aza)ClI ] ), 570.2
3
3
3
2
−
1
dissolved in 1.5 mL of distilled water and KI (0.45 mmol,
(calc. 569.9; 50%; {[Pt(NH )(ip4aza)Cl I] . H NMR
3 2
7
5 mg) was added and the mixture was stirred at the labora-
(DMF-d , 25 °C, ppm): 8.64 (d, J=5,1 Hz, C5–H), 8.26
7
tory temperature until turned red, indicating the formation
of the complex K[PtI (NH )]. Then the solution of 4aza
(d, J=8.8 Hz, C7–H), 8.11 (d, J=2.9 Hz, C2–H), 7.34
(d, J=2.9 Hz, C3–H), 7.28 (dd, J=8.4, 5.5 Hz, C6–H),
3
3
(
0.30 mmol, 35 mg) in 1.5 mL of MeOH was poured in
4.95 (sep, J=6.6 Hz, C8–H), 4.33 (bs, N10–H ), 1.56 (d,
3
−
1
and the reaction mixture was stirred for 24 h. The resulting
J=6.6 Hz, C9–H ). FTIR (ν
cm ): 3332 m, 3221 s,
6
ATR
amber solid (complex 3) was collected by ꢀltration, washed
3173 s, 3093 s, 1495 s, 2971 s, 2928 s, 2871w, 1612w,
1
3

JBIC Journal of Biological Inorganic Chemistry
1
1
566w, 1495 s, 1431vs, 1302vs, 1274vs, 1205 m, 1128w,
101w, 1067w, 1015w, 789w, 762vs, 726w.
solution of KI by the overnight stirring. Stock solutions were
prepared by ultrasonication (5 min) and shaking (30 min) of
1
µmol of complexes 1–4 in 20 mL of OSW. After that, the
General methods
mixtures were centrifuged (5 min, 11,000 rpm) and super-
natants were collected. 5 mL of this solution was added to
5 mL of WSO and shaken at ambient temperature for 2 h.
After that, the mixture was centrifuged and aqueous layer
was separated carefully and the Pt concentration was deter-
mined by ICP-MS (the obtained value was corrected for the
Elemental analysis was performed by Flash 2000 CHNS Ele-
mental Analyser (Thermo Scientiꢀc). Electrospray ioniza-
tion mass spectrometry (ESI–MS; methanol solutions) was
carried out with LCQ Fleet ion trap spectrometer (Thermo
Scientiꢀc; QualBrowser software, version 2.0.7) in both the
adsorption eꢁects). logP=log([Pt]WSO/[Pt]OSW ) equation
a
1
positive (ESI+) and negative (ESI–) ionization modes. H
was used for the partition coeꢃcient calculation; [Pt]OSW
b
1
3
1
1
1
13
and C NMR spectroscopy, and H– H gsCOSY, H– C
and [Pt]OSW stands for the Pt concentration before and
a
1
13
gsHMQC and H– C gsHMBC two dimensional correlation
after partition, respectively, and [Pt]WSO=[Pt]OSW –[Pt]
b
experiments were performed on the CDCl (for ip4aza) or
OSW .
3
a
DMF–d (complexes 1–4) solutions at 300 K using JEOL
7
1
JNM-ECA 600II spectrometer at 600.00 MHz (for H NMR)
Cell culture and cell‑based studies
1
3
and 150.9 MHz (for C NMR); gs = gradient selected,
COSY=correlation spectroscopy, HMQC=heteronuclear
multiple quantum coherence, HMBC=heteronuclear mul-
The human ovarian carcinoma cell line (A2780), cisplatin-
resistant human ovarian carcinoma cell line (A2780R) and
human colon carcinoma cell line (HT-29) were cultured
according to the supplier’s (European Collection of Cell
Cultures, ECACC) instructions, using RPMI-1640 medium
supplemented with 10% of fetal calf serum, 1% of 2 mM
glutamine and 1% penicillin/streptomycin. The 96-well cul-
ture plate with the primary culture of human hepatocytes
1
13
tiple bond coherence. H and C NMR spectra were cali-
1
brated against the residual DMF H NMR (8.03, 2.92 and
1
3
2
.75 ppm) and C NMR (163.2, 34.9 and 29.8 ppm) sig-
1
nals. The splitting of proton resonances in the reported H
spectra is deꢀned as s=singlet, d=doublet, dd=doublet of
doublets, sep=septet, m=multiplet and bs=broad signal.
1
15
H– N gsHMBC experiments were carried out at natural
(
Hep) was purchased from Biopredic Intl. and cultured
abundance and calibrated against the residual signals of
according to the suppliers´ instructions using the chemi-
cally deꢀned medium consisting of a mixture of William’s
E and Ham’s F-12 (1:1, v/v). All cell lines were grown as
1
15
195
DMF adjusted to 8.03 ppm ( H) and 104.7 ( N) ppm. Pt
spectra were measured on the DMF-d solutions and they
7
were calibrated against K [PtCl ]. Note: complexes 2 and 4
2
6
adherent monolayers at 37 °C and 5% CO in a humidiꢀed
1
2
were characterized only by H NMR, due to the low yields
atmosphere. The 50 mM stock solutions of complexes 1–4
obtained. FTIR spectroscopy was performed using Nexus
were prepared by dissolving of their appropriate amount in
−1
6
70 FT-IR spectrometer (ThermoNicolet) at 400–4000 cm
5
00 µL of DMF. Cisplatin was involved in the testing for
(
attenuated total reꢂectance (ATR) technique. Simultane-
ous thermogravimetry and diꢁerential scanning calorimetry
TG/DSC) were performed using TG/DSC thermal analyser
comparative purposes.
(
STA449 F1 (Netzsch); ceramic crucible, air/He atmosphere
In vitro cytotoxicity testing
(
(
60/40 mL/min), from laboratory temperature to 900 °C
5.0 °C/min).
The cultured A2780, A2780R, and HT-29 cells were seeded
to 96-well culture plates. The A2780, A2780R, HT-29, and
Hep cells were pre-incubated in drug-free media at 37 °C
for 24 h and then treated for next 24 h at 37 °C with dif-
ferent concentrations of complexes 1–4 and cisplatin (note:
only with complexes 1 and 2 for the Hep cells) prepared
from their stock solutions by a dilution with the appropriate
medium. After 24 h drug exposure, the supernatants were
removed and the cells were washed with drug-free PBS fol-
lowed by staining with MTT dye (3-(4,5-dimethylthiazol-
Solution behaviour
The appropriate amount of complexes 1–4 for 1 mM solu-
tions was dissolved in 120 µL of DMF-d and 480 µL of
7
1
D O was added. H NMR spectra were recorded at diꢁer-
2
ent time points over 48 h (300 K, JEOL JNM-ECA 600II).
1
The obtained H NMR spectra were calibrated against the
residual signal of D O found at 4.85 ppm [35].
2
2
-yl)-2,5-diphenyltetrazolium bromide). The cell viability
Hydrophobicity studies (logP)
was determined using the spectrophotometric evaluation
of the formed formazan (from MTT dye) concentration
(540 nm, Inꢀnite 200 PRO microplate reader, Tecan Group
Ltd., Männedorf, Switzerland).
Octanol-saturated water (OSW) and water-saturated octanol
(
WSO) were prepared from n-octanol and 0.2 M water
1
3

JBIC Journal of Biological Inorganic Chemistry
The data were expressed as the percentage of viability,
where 100% and 0% represent the treatments with negative
Mass spectrometry studies of interactions
with sulfur‑containing biomolecules and model
proteins
(
0.1% DMF in medium) and positive (1% Triton X-100)
controls, respectively. The data from the cancer cells
were acquired from three independent experiments (con-
ducted in triplicate) using cells from diꢁerent passages.
The resulting IC values (µM) were calculated from the
The interactions of selected complex 2 with biomolecules
(sulfur-containing agents l-cysteine (CySH) and reduced
glutathione (GSH) and model proteins cytochrome c from
bovine heart and hen egg-white lysozyme) were studied by
means of a mass spectrometric studies involving the mix-
tures of complex 2 and serum levels of CySH (290 µM) and
GSH (6 µM), and complex 2 and model proteins (3 mg/mL),
respectively. The evaluation of the interacting species was
done according to the previously published procedure [36]
using a LCQ Fleet ion trap spectrometer (Thermo Scientiꢀc;
QualBrowser software, version 2.0.7) in positive ionization
mode (ESI+) and the spectra of mixtures containing proteins
were deconvoluted using Promass for Xcalibur ver. 3.0, rev
10 software (Novatia LLC, Newtown, PA, USA) producing
the neutral mass spectra, allowing us to deduce the interact-
ing intermediates.
5
0
viability curves and the results are presented as arithmetic
mean ± standard deviation (SD).
Cellular accumulation
The cultured A2780 cells were seeded in 6-well culture
6
plates (1 × 10 cells per well) and incubated overnight
(
37 °C and 5% CO in a humidiꢀed incubator). After that
2
the cells were treated with the IC concentrations of com-
5
0
plexes 1–4. The treatment was followed by washing with
PBS (2 × 2 mL) and the washed cells were harvested by
the trypsin treatment, collected and centrifuged in PBS.
The supernatants were discarded after centrifugation and
the obtained pellets were immediately digested in 250 µL
of nitric acid (overnight, 70 °C; TMS-200 Thermo Shaker
Incubator, Hangzhou Allsheng Instruments Co., Ltd.,
Hangzhou, China) to give the fully homogenized solu-
tions. The solutions were diluted with 2.25 mL of water
and the Pt content was determined by inductively coupled
plasma mass spectrometry (ICP-MS) using the 7700× ICP-
MS device (Agilent Technologies). The obtained values
were corrected for adsorption eꢁects. The experiments
were conducted in duplicate. The untreated A2780 cells
were used as a negative control and the A2780 cells treated
by the IC concentration of cisplatin were involved for
Statistical analysis
An ANOVA test was used for statistical analysis with the
values of p < 0.05 (*), 0.01 (**) and 0.005 (***) consid-
ered to be statistically signiꢀcant. QC Expert 3.2 Statistical
software (TriloByte Ltd.) was used to perform the analysis.
Results
Chemistry
5
0
comparative purposes.
The intermediates NH [PtCl (NH )], K[PtCl (NH )], and
4
3
3
3
3
K[PtI (NH )] were prepared using the slightly modiꢀed
3
3
procedures [33, 34], while the ꢀnal platinum(II) complexes
Cell cycle analysis
cis-[Pt(4aza) I ] (1), cis-[PtI (ip4aza) ] (2), cis-[Pt(4aza)
2
2
2
2
I (NH )] (3) and cis-[PtI (ip4aza)(NH )] (4) were prepared
2
3
2
3
6
The A2780 cells (1.0×10 per well) were pre-incubated in a
six-well plate for 24 h, as described above. Complexes 1–4
were added at their IC concentrations. After 24 h, the cells
as described in the Experimental section (Fig. 1) [37]. Com-
plexes 1–4 were only negligibly soluble in water, methanol
(MeOH), ethanol (EtOH) or acetone, but they showed good
solubility in N,N-dimethylformamide (DMF) and dimethyl
sulfoxide (DMSO). Purity of complexes 1–4 was conꢀrmed
by a combination of elemental analysis (<0.5% diꢁerences
between the calculated and found percentage of C, H and
5
0
were harvested by trypsinization, washed with PBS, cen-
trifuged and stained for 30 min by a propidium iodide (PI)
supplemented with RNase A and 0.1% (v/v) Triton X-100
(
25 °C). After that, the DNA content was assessed using
1
ꢂow cytometry detecting emission at 617 nm after excita-
tion at 535 nm (CytoFlex, Beckman Coulter). The obtained
data were analysed using CytExpert™ software (Beckman
Coulter). The A2780 cells treated by cisplatin (IC con-
N) and H NMR.
ESI+ mass spectra of the cis-[PtI (naza) ] complexes
2
2
+
1 and 2 contained the peaks of the {[PtI (naza) ] + Na}
2
2
+
and [PtI(naza) ] species; naza = 4aza or ip4aza. In the
5
0
2
−
centration) were involved for comparative purposes and the
untreated A2780 cells were used as a negative control. All
experiments were carried out in triplicate (three samples
ESI– mass spectra, the peaks of the [Pt(naza)I ] species
3
showed with appropriate m/z value and isotopic distribu-
tion for both complexes 1 and 2. The peaks of several spe-
5
−
counting ca. 3×10 cells).
cies (e.g., {[Pt(4aza)I (NH )]–H} for 3), containing both
2
3
1
3

JBIC Journal of Biological Inorganic Chemistry
N-donor ligands (i.e., NH and naza) were detected in the
147.0 ppm for 3) ↔ H5 (δ=8.94 ppm for 1 and 8.62 ppm
15
3
mass spectra of the sterically hindered complexes 3 and 4.
for 3) long-range correlation peak. The N NMR signal of
the N4 nitrogen atom shifted considerably as a consequence
of the formation of complexes 1 (Δδ = –92.6 ppm) and 3
(Δδ=–90.9 ppm), suggesting that this atom represents the
The FTIR spectra showed the peaks of the ν(C–H) (ca.
ar
−
1
−1
3
1
100 cm ), ν(C–C) (ca. 1600 cm ) and ν(C–N) (ca.
ar
ar
−
1
420 cm ) vibrations of the 4-azaindole ring. In addi-
tion, the FTIR spectra of complexes 3 and 4 contained the
coordination site of the used 4aza ligand involved in the
−
1
15
peaks of ν(NH ) at 3252 and 1326 cm (for 3) and 3221
structure of complexes 1 and 3; Δδ=δ
– δ . The N
3
complex
4aza
−
1
and 1302 cm (for 4) [38]. The spectra of complexes 2
and 4 (containing ip4aza) showed the intensive peaks of the
NMR signal of the NH ligand showed at − 46.8 ppm in the
3
1
15
H– N HMBC spectrum of complex 3 (Fig. S6). No new
−
1
1
ν(C–H)
vibrations at 2971 and 2927 cm (for 2), and at
signals were detected in the H NMR spectra of complexes
aliph
−
1
2
971 and 2928 cm (for 4).
The simultaneous TG/DSC analysis proved the represent-
ative complex 1 as unsolvated and thermally stable up to ca.
10 °C. Behind this temperature, a gradual thermal decom-
1–4 dissolved in DMF-d after 48 h of standing at ambient
7
temperature, suggesting their stability in the used solvent.
1
95
The Pt NMR signals were detected at − 3186 ppm for
1
complex 1 and at − 2548 ppm for complex 3 (Fig. S7).
position was observed and ꢀnished at ca. 550 °C without
the formation of thermally stable intermediates (69.2/68.0%
calcd./found weight loss; calculated to PtO). The thermal
decomposition was accompanied on the DSC curve by the
exothermic eꢁects centred at ca. 220, 430 and 545 °C (Fig.
S1).
Solution behaviour and hydrophobicity
Complexes 1 and 2 were hydrolytically stable in the 20%
DMF-d /80% D O mixture of solvents, because no new
7
2
1
signals showed in the obtained H NMR spectra even after
72 h of standing at ambient temperature (Fig. S8); note:
DMF ensured the suꢃcient solubility of low water soluble
1
The H NMR spectra of complexes 1–4 contained the
signals of aromatic C–H hydrogen atoms at 7.14–8.94 ppm
1
1
(
Fig. 2, S2 and S3). In addition, the N1–H broad signal was
complexes for H NMR experiments. The H NMR spec-
tra of complexes 3 and 4 showed a new set of naza sig-
nals (Fig. S9). For example, the representative C5–H signal
was detected in the fresh solutions at 8.73 ppm (for 3) and
8.72 ppm (for 4), while a new C5–H signal observed during
the standing of complexes 3 and 4 at 8.63 ppm (for 3) and
8.62 ppm (for 4). Because the C5–H signal of free H4aza
showed at 8.43 ppm in the 20% DMF-d /80% D O mixture
detected in the spectra of complexes 1 and 3 containing
unsubstituted 4-azaindole, while the spectra of complexes 2
and 4 (containing ip4aza) showed the signals of the isopro-
pyl substituent (ca. 4.9 ppm and 1.5 ppm) of the N1 nitrogen
atom. The presence of the NH ligand in the structure of the
3
sterically hindered complexes 3 and 4 was conꢀrmed by the
broad signal at 4.33 ppm (for both complexes).
7
2
1
3
15
1
The C (Fig. S4 and S5) and N (Fig. S6) NMR spectra
were recorded only for the representative 4aza-containing
complexes 1 and 3. The individual aromatic carbon atoms
of solvents, the observed H NMR spectral changes of com-
plexes 3 and 4 (as discussed above) can be assigned to the
hydrolysis of these complexes.
1
13
of the C–H groups were clearly assigned using the H– C
HMQC correlation experiments, while the quaternary C3a
The logP studies were not possible to realize owing to
a limited solubility of complexes 1 and 2 in the solvent
system used (OSW). Complexes 3 and 4 were found to be
hydrophilic with similar logP values (− 0.78, and − 0.81,
respectively).
1
13
and C7a carbons were reꢀned in the H– C HMBC spec-
tra, as exempliꢀed by the C7a (δ = 146.4 ppm for 1 and
In vitro cytotoxicity and cellular accumulation
Complexes 1–4 were studied for their in vitro cytotoxicity
against three human carcinoma cell lines [ovarian carcinoma
(
A2780), its cisplatin-resistant variant (A2780R) and colon
carcinoma (HT-29)], while their cellular accumulation was
studied at the representative A2780 cells only. The obtained
results are summarized in Table 1 (together with cisplatin
for comparative purposes).
The cis-[PtI (naza) ] complexes 1 and 2 signiꢀcantly
2
2
(
p<0.005) exceeded in vitro cytotoxicity of their sterically
hindered cis-[PtI (naza)(NH )] analogues 3 and 4 against
1
2
3
Fig. 2 H NMR spectra of the representative complexes 1 (bottom)
the A2780 and A2780R cells. Within the pairs of the cis-
and 4 (top); DMF-d solutions. See Fig. 1 for the structural formulas
7
and atom numbering scheme
[PtI (naza) ] (1 and 2) and cis-[PtI (naza)(NH )] (3 and 4)
2
2
2
3
1
3

JBIC Journal of Biological Inorganic Chemistry
Table 1 The in vitro
Complex
HT-29
A2780
7.7± 1.2***
3.8± 0.8***
26.1± 3.0
8.9± 0.8*** 12.1± 0.5
26.7± 1.8 > 50.0
A2780R
RF
Cellular accumulation
cytotoxicity (IC ± SD; μM)
50
and cellular accumulation (pmol
cis-[Pt(4aza) I ] (1)
4.2± 1.0
4.9± 1.0
5.8± 0.4
8.0± 0.6
> 50.0
8.9± 0.1
2.9± 0.3
32.4± 2.0
1.2
0.8
1.2
1.4
–
43.0± 2.1
592.3± 4.4
19.8± 0.2
78.5± 3.6
86.0± 2.0
2
2
6
Pt/10 A2780 cells) assessed
cis-[PtI (ip4aza) ] (2)
2
2
for complexes 1–4 and cisplatin
cis-[Pt(4aza)I (NH )] (3)
(
for comparative purposes)
2
3
cis-[PtI (ip4aza)(NH )] (4)
2
3
Cisplatin
Cytotoxicity was studied at the human HT-29 colon carcinoma, A2780 ovarian carcinoma, and A2780R
cisplatin-resistant ovarian carcinoma cells (24 h exposure time, MTT assay), with the signiꢀcant diꢁer-
ences between the obtained IC values of complexes 1–4 against cisplatin given as *** p<0.005. Cellular
5
0
accumulation was studied at the A2780 cells
RF resistance factor (calculated as IC (A2780R)/IC (A2780))
5
0
50
complexes containing the same naza ligands, the ip4aza-
containing complexes 2 and 4 were signiꢀcantly (p<0.005)
more in vitro cytotoxic at the A2780 and A2780R cells
than their analogues (1 and 3) containing the unsubstituted
(Table 1). Complexes 1–3 were signiꢀcantly more cytotoxic
(p < 0.05) than the least eꢁective complex 4. Similarly as
in the A2780 cells, the cis-[PtI (naza) ] complexes 1 and 2
2
2
exceeded the in vitro cytotoxicity of their ammine-contain-
4
-azaindole. Complexes 1, 2, and 4 exhibited signiꢀcantly
ing cis-[PtI (naza)(NH )] analogues 3 and 4 at the HT-29
2
3
(
p<0.005) higher cytotoxic potency at the A2780 cells than
cells.
cisplatin. Cellular accumulation at the A2780 cells was
Complex 1 and 2 were tested for their toxicity at the pri-
mary culture of human hepatocytes (Hep). Complex 1 did
not aꢁect the viability of the Hep cells up to the highest
tested concentration (IC >100 µM), while the toxicity of
markedly higher for (a) the ip4aza-containing complexes (2,
4
), and (b) for the complexes with two azaindoles involved
in their structure (1, 2), meaning that Pt of the highest-
active complex 2 was accumulated signiꢀcantly more than
observed for other studied complexes (Table 1).
5
0
complex 2 equalled IC =38.4 µM.
5
0
The resistance factors, calculated as IC (A2780R)/
5
0
IC (A2780) (see Table 1), indicated that complexes 1–4
Cell cycle analysis
5
0
are able to circumvent the acquired resistance of the A2780
human ovarian carcinoma cells against the therapeutic action
of cisplatin.
The ꢂow cytometry studies of the cell cycle perturbation
were performed at the A2780 cells treated by the IC con-
5
0
The in vitro cytotoxicity of complexes 1–4 against the
HT-29 cells ranged from the IC value of 4.2–8.0 μM,
centrations of complexes 1–4 and cisplatin for compara-
tive purposes, and stained with propidium iodide (PI). The
results are depicted in Fig. 3 and summarized in Table S1.
5
0
and was considerably higher as compared with cisplatin
Fig. 3 Left: the selected results of the ꢂow cytometry studies of the
cell cycle modiꢀcation induced at the A2780 human ovarian carci-
noma cells (stained with PI/RNase) by the equitoxic (IC ) concen-
the populations in the cell cycle phases observed at the A2780 cells
treated with complexes 1–4 and cisplatin (for comparative purposes),
depicted together with the negative control (untreated cells); see
Table S1 for the complete data given as arithmetic mean± SD
5
0
trations of complexes 1–4 and cisplatin (for comparative purposes),
given together with the negative control (untreated cells). Right:
1
3

JBIC Journal of Biological Inorganic Chemistry
The treatment of the A2780 cells by complexes 1, 2,
and 4 induced the cell cycle perturbation connected with a
decrease of the G /G cell cycle phase population (39–58%
by the appearance of the peaks in the ESI+ MS spectrum
(see Fig. 4) of the mixture measured after 48 h. These
+
signals are assignable to the species [Pt(ip4aza)(Cys)]
0
1
+
for 1, 2, and 4, and 72% for negative control) while the G /M
(m/z = 475.22), [Pt(ip4aza) (Cys)] (m/z = 635.16) and
2
2
+
cell cycle phase populations were higher for the samples
treated by complexes 1, 2, and 4 (27–45%), as compared
with the untreated cells (16%; Fig. 3 and Table S1). The
most profound diꢁerences in the A2780 cell cycle were
induced by the ip4aza-complexes 2 and 4. Within the A2780
[Pt(ip4aza) (Cys) + CH OH] (m/z = 667.22). No peaks
2
3
representing the interaction species with the reduced glu-
tathione (GSH) were found in the spectra.
Complex 2 showed higher affinity towards the
cytochrome c, with whom it formed both the 1:1 and 1:2
adducts, than towards the second-used model protein
HEWL (only 1:1 adduct was observed) (see Fig. 5). The
mass diꢁerence in all adducts of 768 or 769 Da corre-
sponds to the whole molar equivalent of complex 2, which
means that there can be two alternative modes of interac-
tion of complex 2 with both proteins. The ꢀrst one involves
an electrostatic interaction, while the second one is associ-
ated with the formation of coordination bond between the
platinum atom and appropriate donor atom from the pro-
tein, while the released iodide anion form an electrostatic
interaction with positively charged surface of the protein.
cells treated by complex 2, high sub-G cell cycle phase
1
population was observed. On the other hand, the G /M cell
2
cycle phase population was markedly higher for complex
4
, as compared with complexes 1–3, as well as with con-
ventional cisplatin. Relevant diꢁerences were also observed
between the biological eꢁect of cisplatin (48% S cell cycle
phase population) and complexes 1–4 (10–14% S cell cycle
phase populations).
Mass spectrometry studies of interactions
with sulfur‑containing biomolecules and model
proteins
The mass spectrometric studies revealed the moderate
reactivity of the representative complex 2 with l-cysteine
(
CySH; a ꢀnal concentration of 290 µM), as conꢀrmed
Fig. 4 The ESI+MS spectrum
of the solution of complex 2
in MeOH: H O (1:1, v/v) after
2
4
8 h of incubation at labora-
tory temperature (a) and the
ESI+MS spectrum of the
mixture containing complex 2
(
at the ꢀnal concentration of
1
0 µM), l-cysteine (at the ꢀnal
concentration of 290 µM) and
reduced glutathione (at the ꢀnal
concentration of 6 µM) after
4
8 h of incubation at laboratory
temperature (b). The identi-
ꢀ
ed species corresponding to
hydrolytic intermediates are
noted in green and the species
corresponding to intermediates
involving CySH or GSH are
noted in orange colour
1
3

JBIC Journal of Biological Inorganic Chemistry
characterized. The syntheses of complexes 1 and 2 started
from K [PtCl ] through the key intermediate K [PtI ] [37],
2
4
2
4
whose stoichiometric reaction with 4aza and ip4aza led to
complexes 1 and 2, respectively, as described for example
for similar diiodido platinum(II) complexes containing
7
-azaindole and its various C-derivatives [19]. The synthe-
ses of complexes 3 and 4 also started from K [PtCl ] and
2
4
they were conducted through the cis-[PtCl (NH ) ] (cispl-
2
3 2
atin), NH [PtCl (NH )], K[PtCl (NH )] and K[PtI (NH )]
4
3
3
3
3
3
3
intermediates, similarly as described previously [33, 34].
Finally, the reactions of K[PtI (NH )] with either 4aza or
3
3
ip4aza provided complexes 3 and 4, respectively (Fig. 1).
1
Concerning the H NMR spectroscopy, the preparation
of ip4aza led to the disappearance of the N1–H signal of
4
aza (due to deprotonation) and detection of two new sig-
nals at 4.70 ppm and 1.58 ppm (due to alkylation), clearly
assignable to the isopropyl substituent of ip4aza [42]. Simi-
lar diꢁerences were observed also for the pairs of the cis-
[PtI (naza) ] (1 and 2) and cis-[PtI (naza)(NH )] (3 and 4)
Fig. 5 The neutral mass spectra obtained by the deconvolution of the
ESI+MS spectra of the mixtures containing complex 2 (at the ꢀnal
concentration of 10 µM) and one of the model proteins, cytochrome c
or HEWL at the ꢀnal concentration of 3 mg/mL. The depicted mass
diꢁerences correspond to the formation of 1:1 and 1:2 adducts with
cytochrome c (upper panel) and 1:1 adduct with lysozyme (lower
panel)
2
2
2
3
complexes containing 4aza (for 1 and 3) or ip4aza (for 2 and
4
) ligands. The NH ligand of complexes 3 and 4 showed for
3
both complexes at 4.33 ppm, which is consistent with similar
complexes of the cis-[PtI (L)(NH )] type, where L stands for
2
3
pyrimidine or 5,7-diphenyl-1,2,4-triazolo[1,5-a]pyrimidine
1
195
Discussion
(L ) [32, 43]. The positions of the Pt NMR signals in
the spectra of complexes 1 and 3 (Fig. S6) correlated well
1
The platinum(II) complexes containing 7-azaindole and its
derivatives (7aza) were recently reported by us and others
as highly in vitro cytotoxic agents at various human can-
cer cell lines [11–22, 39–41]. Among these complexes of
various structural types (e.g., dichlorido [13–15], diiodido
with the formerly reported complexes cis-[PtI2(L )2] and cis-
1
[PtI2(L )(NH3)] containing 5,7-diphenyl-1,2,4-triazolo[1,5-
1
a]pyrimidine (L ) [32].
The cis-[PtI2(naza)2] complexes 1 and 2 are hydrolyti-
cally stable in 20% DMF-d7/80% D2O, while the sterically
hindered complexes 3 and 4 hydrolysed under the used
experimental conditions. Hydrolysis of complexes 3 and 4
is not connected with a release of the naza ligand, because
[
19], oxalato [17] or cyclobutane-1,1-dicarboxylato [16])
the diiodido complexes cis-[Pt(7aza) I ] were the high-
2
2
est active ones, exceeding the cytotoxic activity of their
various analogues including the dichlorido complexes and
showing some pharmacological advantages over their ana-
logues and cisplatin (e.g., diꢁerent cell cycle perturbation
or diꢁerent interaction with relevant biomolecules). Bear-
ing this in mind, we decided to study two structural types,
cis-[PtI (naza) ] (1, 2) and cis-[PtI (naza)(NH )] (3, 4), of
1
the positions of the H NMR signals were diꢁerent for the
new signals in the spectra of complexes 3 and 4, and free
naza. In general, hydrolytic stability of platinum(II) diiodido
complexes depends on the N-donor ligand involved in their
structures. In particular, the cis-[PtI2(NH3)2] complex, an
iodido analogue of cisplatin, readily hydrolysed at physi-
ological pH which was connected with a release of both the
halogenido ligands (similarly as for cisplatin) [44]. The
hydrolysis rate was somehow slower for cis-[PtI2(NH3)2]
as compared with cisplatin. However, a release of the NH3
ligand at slightly acidic pH was also observed in the same
work for cis-[PtI2(NH3)2], indicating its diꢁerent solution
2
2
2
3
platinum(II) diiodido complexes containing 4-azaindole
(
4aza) or its N1-alkylated derivative ip4aza. Design of this
series of compounds allowed us to evaluate, whether (a)
the platinum(II) complexes containing 4-azaindoles show
the in vitro cytotoxicity against the selected human cancer
cell lines, as recently reported for similar complexes with
1
7
-azaindoles, (b) the replacement of the NH ligand by naza
behaviour from cisplatin. The cis-[Pt(7aza )2I2] complex
3
1
aꢁects the in vitro cytotoxicity of the studied complexes, and
(7aza = 4-bromo-7-azaindole), representing platinum(II)
(
c) the N1-alkylation of the 4-azaindole moiety aꢁects the
diiodido complexes containing 7-azaindoles, showed high
hydrolytic stability, because only ca. 10% hydrolysed in the
used 10% DMF-d7/90% D2O mixture of solvents after 48 h
of standing at ambient temperature [19]. Concerning the
hydrolysis of complex 2, the ESI+ MS studies uncovered
in vitro cytotoxicity of complexes containing such ligands.
A series of four diiodido platinum(II) complexes of the
general formulas cis-[PtI (naza) ] (1, 2) and cis-[PtI (naza)
2
2
2
(
NH )] (3, 4) (Fig. 1) was prepared and thoroughly
3
1
3

JBIC Journal of Biological Inorganic Chemistry
only the formation of a deiodinated hydroxido-intermediate
analogue of cisplatin, had RA ~ 3.4 against the IGROV-1
with the proposed composition [Pt(ip4aza) (OH)+2H O+
ovarian carcinoma cells (Trypan blue assay, 24 h expo-
2
2
+
2
CH OH] after 48 h of incubation.
sure) [44], while the complex cis-[Pt(7aza ) I ] showed
3
2 2
2
On the other hand, the interaction studies involving
sulfur-containing biomolecules, l-cysteine and reduced
glutathione conꢀrmed the moderate reactivity of complex
RA ~16.5 against the A2780 cells (7aza = 2-methyl-4-
chloro-7-azaindole; MTT assay, 24 h exposure time) [19].
2
The complex cis-[PtI (L )(NH )] exhibited ca. 2-fold
2
3
2
towards the normal serum levels of l-cysteine, as dem-
lower activity (RA ~ 0.5) at the A2780 cells than cisplatin
onstrated by the emergence of new peaks in the ESI+ MS
spectrum (see Fig. 4). Moreover, the interaction studies
involving the model proteins cytochrome c and lysozyme
(MTT assay, 72 h exposure time) [33].
Similar cytotoxicity of complexes 1–4 at both the A2780
and A2780R cells resulted in the resistance factors (RF) of
ca. 0.8–1.4 (Table 1), indicating their ability to overcome
the acquired resistance of the human cancer cells against
cisplatin. This has to be highlighted especially for the best-
performing complex 2 showing unusually low RF~0.8. The
(
see Fig. 5) revealed the ability of complex 2 to form the
adduct containing up to 2 molecules of complex 2 per one
molecule of cytochrome c and the formation of 1:1 adduct
with lysozyme. This ꢀnding may indicate that the nature of
the interaction between complex 2 and model proteins could
involve either pure electrostatic interaction or the covalent
bonding of the deiodinated species accompanied at the same
time by the electrostatic interaction of the released iodide
anions with cationic parts of the protein.
2
complex cis-[Pt(7aza ) I ], containing 7-azaindole-based
2
2
N-donor ligand, was also somehow more in vitro cytotoxic
at the resistant variant (IC =1.0 μM) than at the cisplatin-
5
0
sensitive A2780 cells (IC =1.7 μM), meaning that RF was
5
0
ca. 0.6 [19]. Similar trend of in vitro cytotoxicity (RF=0.8)
The in vitro cytotoxicity of complexes 1–4 was screened
at three human cancer cell lines, carefully chosen to rep-
resent the cisplatin-sensitive cells (A2780), cells with
acquired resistance to cisplatin (A2780R) and cells known
to be intrinsically resistant to cisplatin (HT-29) (Table 1).
Complex 2 was signiꢀcantly (p<0.01) more cytotoxic than
complex 1, and complex 4 exceeded signiꢀcant (p<0.005)
potency of complex 3 against the A2780 cells. Similarly, the
ip4aza-containing complexes 2 and 4 exceeded the cytotoxic
activity of their 4aza-containing analogues 1 and 3, respec-
tively, at the A2780R cells. This suggests that alkylation of
was also reported for the above-mentioned sterically hin-
2
dered complex cis-[PtI (L )(NH )] [33].
2
3
Complexes 1, 2, and 4 exhibited comparable in vitro
cytotoxicity against the cisplatin-resistant HT-29 and cis-
platin-sensitive A2780 cells, while complex 3 was mark-
edly more potent at the HT-29 cells than at the A2780 ones
(Table 1). All complexes 1–4 were considerably more cyto-
toxic at the HT-29 cells than cisplatin. Although the RA
values could not be calculated, it can be stated that com-
plexes 1 and 2 are more than 10-times more cytotoxic at the
HT-29 colon carcinoma cells than cisplatin. The RA value
of the complexes cis-[PtI (NH ) ] and [Pt(dach)I ] equalled
4
-azaindole moiety aꢁected the in vitro cytotoxicity of both
2
3 2
2
types of the studied complexes in a positive way at both the
cisplatin-sensitive and -resistant human ovarian carcinoma
cells (i.e., A2780, A2780R). Further, the results also proved
that complexes 1 and 2 are more in vitro cytotoxic than their
sterically hindered analogues 3 and 4 against the A2780 and
ca. 1.4 and 0.6 at the HT-29 cells (Trypan blue assay, 24 h
exposure; dach = (1R,2R)-cyclohexane-1,2-diamine) [46].
A representative of the platinum(II) diiodido complexes
2
containing 7-azaindoles, cis-[Pt(7aza ) I ], was several
2
2
times more in vitro cytotoxic (IC =0.4 μM) than cisplatin
5
0
A2780R cells, indicating that the replacement of the NH
(IC >90 μM) at the Caco-2 human colon carcinoma cells
3
50
ligand by naza has a positive impact on the resulting cyto-
(MTT assay, 24 h exposure time) [19]. Further, the complex
3
toxic potency. The opposite trend was reported for a pair of
trans-[Pt(ach)I (L )] showed higher activity (IC =1.5 µM)
2
50
1
1
the cis-[PtI (L ) ] (IC = 67 μM) and cis-[PtI (L )(NH )]
against the HT-29 cells than cisplatin (IC > 100 µM;
2
2
50
2
3
50
(
IC =7 μM) complexes studied at the A549 non-small lung
72 h exposure, MTT assay); ach = cyclohexanamine,
50
3
carcinoma cells [32].
L =1,3-dimethyl-N-boc-O-methylhistidin-2-ylidene [47].
Concerning a comparison with other platinum(II)
iodido complexes studied at the human ovarian carci-
noma cells, the relative activity (RA; see Introduction) of
the best-performing complex 2 equalled ca. 7.0, meaning
approximately 7-fold higher cytotoxicity as compared with
cisplatin at the used A2780 cells. The cis-[PtI (ipam) ]
Complexes 1 and 2 showed a pharmacologically prospec-
tive selectivity towards the cancer cells over the non-can-
cerous ones. In particular, toxicity of the best-performing
complex 2 was approximately one order of magnitude lower
at the primary culture of human hepatocytes than at the used
cancer cells. Although these results indicated higher toxicity
of complex 2 than determined for cisplatin under the same
experimental conditions (IC >75 µM), it has to be high-
2
2
complex, where ipam stands for isopropylamine, was
studied at several human cancer cell lines including the
A2780 cells, where this agent markedly exceeded cispl-
atin (RA ~ 7.3; sulforhodamide B (SRB) assay, 48 h expo-
sure time) [45]. The complex cis-[PtI (NH ) ], an iodido
5
0
lighted that both complexes 1 and 2 are much less eꢁective
against the Hep cells than their recently reported analogues
containing 7-azaindole-based ligands, which showed the
2
3 2
1
3

JBIC Journal of Biological Inorganic Chemistry
IC values of 3.8–11.8 µM at the Hep cells (MTT assay,
culture of human hepatocytes (Hep) was used; IC > 100
5
0
50
2
4 h exposure time) [19].
μM (1) and IC =38 μM (2)] has to be pointed out as a sig-
5
0
The studies of cellular accumulation by the A2780 cells
niꢀcant improvement from the recently reported analogues
containing various 7-azaindole derivatives (toxicity at Hep
cells with IC ≈ 4–12 μM). The obtained results indicated
showed good correlation between cytotoxicity and amount
of platinum uptaken by the used cells (Table 1). In par-
ticular, the best-performing complex 2 was markedly (one
order of magnitude) more accumulated by the used cells than
complexes 1, 3, and 4, as well as than cisplatin. It is also
noteworthy that complex 4 and cisplatin showed diꢁerent
cytotoxicity while their cellular accumulation was compara-
ble, and on the other hand, complex 3 and cisplatin showed
diꢁerent cellular accumulation while their potency at the
A2780 cells was of the same level. On the other hand, the
correlation of cellular accumulation and logP (or cytotoxic-
ity and logP) is not possible, because both the complexes are
comparably hydrophilic, but their cellular accumulation and
cytotoxicity diꢁer markedly.
5
0
that 4-azaindole and its derivatives have to be taken into
account as suitable ligands for development of prospective
platinum anticancer complexes, because complexes 1–4
exceeded the cytotoxicity of conventional cisplatin at the
used human cancer cells. Further, the alkylation of 4-azain-
dole moiety as well as the replacement of the NH ligand by
3
4-azaindole-based ligand had a positive eꢁect on the in vitro
cytotoxicity and cellular accumulation within a series of the
studied complexes.
Acknowledgements The authors gratefully thank the National Pro-
gram of Sustainability I (LO1305) of the Ministry of Education, Youth
and Sports of the Czech Republic and Palacký University in Olomouc
The cytotoxic effect of complexes 1, 2, and 4 at the
A2780 cells was connected with the profound cell cycle
changes, as compared with the results obtained by the ana-
logical experiments for the untreated cells (negative control)
as well as for the cells treated by the equipotent concentra-
tion of cisplatin, implying diꢁerent mechanism of action
of these diiodido platinum(II) complexes and cisplatin
(
a Grant No. PrF_2018_011) for ꢀnancial support. The authors also
thank Ms. Kateřina Kubešová and Ms. Marta Rešová for performing
the in vitro cytotoxicity experiments and preparation of the samples for
cellular accumulation and ꢂow cytometry experiments, Dr. Bohuslav
Drahoš for recording the ESI–MS and NMR spectra, Dr. Alena Klani-
cová for performing FTIR spectroscopy and Mrs. Pavla Richterová for
carrying out elemental analyses. The authors also thank Mr. Ivan Saksa
for his help with the syntheses of the studied compounds.
(
Fig. 3 and Table S1). However, the relevant diꢁerences
were observed also in the individual cell cycle phase popu-
lations of the ip4aza-containing complexes 2 (high sub-G
1
References
population) and 4 (high G /M population). This indicates
2
that the structural type of the studied complexes could be
decisive for the cell cycle progress of the cells treated by
such complexes. Importantly, the cell cycle perturbation
induced by complexes 1, 2, and 4 at the A2780 cells was
diꢁerent than in the case of the platinum(II) diiodido com-
plexes containing 7-azaindoles [19], 9-deazahypoxanthine
derivatives [48] or sterically hindered complexes contain-
ing 1-(cyclohexylmethyl)-3-methylimidazol-2-ylidene and
G-quadruplex-binding pyridodicarboxamide [49], which,
similarly to the herein studied complex 3, did not modify
the cell cycle of the A2780 human cancer cells.
1
2
3
.
.
.
Kelland L (2007) Nat Rev Cancer 7:573–584
Rosenberg B, van Camp L, Krigas T (1965) Nature 205:698–699
World Health Organization (2017) WHO model list of essential
medicines. WHO, Geneva
4
5
.
.
Johnstone TC, Suntharalingam K, Lippard SJ (2016) Chem Rev
1
16:3436–3486
Park GA, Wilson JJ, Song Y, Lippard SJ (2012) Proc Natl Acad
Sci USA 109:11987–11992
6. Li JJ, Tian M, Tian Z, Zhang S, Yan C, Shao C, Liu Z (2018) Inorg
Chem 57:1705–1716
7
8
9
.
.
.
Yang GJ, Zhong HJ, Ko CN, Wong SY, Vellaisamy K, Ye M, Ma
DL, Leung CH (2018) Chem Commun 54:2463–2466
Štarha P, Trávníček Z, Dvořák Z (2018) Chem Commun
5
4:9533–9536
Mukherjee N, Podder S, Mitra K, Majumdar S, Nandi D,
Chakravarty AR (2018) Dalton Trans 47:823–835
Conclusions
1
1
0. Zhao SB, Wang S (2010) Chem Soc Rev 39:3142–3156
1. Harrison RC, McAuliꢁe CA, Friedman ME (1984) Inorg Chim
Acta 92:43–46
In the present work, we synthesized four new cis-diiodido-
platinum(II) complexes of two structural types, either con-
taining two 4-azaindole-based ligands (complexes 1 and
1
1
2. Štarha P, Marek J, Trávníček Z (2012) Polyhedron 33:404–409
3. Štarha P, Trávníček Z, Popa A, Popa I, Muchová T, Brabec V
(
2012) J Inorg Biochem 115:57–63
2
) or ammine complexes with only one 4-azaindole-based
1
1
4. New EJ, Roche C, Madawala R, Zhang JZ, Hambley TW (2009)
J Inorg Biochem 103:1120–1125
ligand (complexes 3 and 4). The in vitro cytotoxicity of com-
plexes 1–4 was investigated at three cell lines with diꢁerent
sensitivity towards cisplatin (A2780, A2780R, and HT-29).
5. Pracharova J, Saltarella T, Radosova Muchova T, Scintilla S,
Novohradsky V, Novakova O, Intini FP, Paciꢀco C, Natile G, Ilik
P, Brabec V, Kasparkova J (2015) J Med Chem 58:847–859
Besides high in vitro cytotoxicity (IC ≈ 3–9 μM) of the
5
0
16. Štarha P, Trávníček Z, Dvořák Z, Radošová-Muchová T,
Prachařová J, Vančo J, Kašpárková J (2015) PLoS One
studied complexes 1 and 2 containing 4-azaindole-based
1
0:e0123595
ligands, their low toxicity at non-cancerous cells [primary
1
3

JBIC Journal of Biological Inorganic Chemistry
1
1
1
2
7. Štarha P, Trávníček Z, Popa I, Dvořák Z (2014) Molecules
34. Battle AR, Choi R, Hibbs DE, Hambley TW (2006) Inorg Chem
45:6317–6322
1
9:10832–10844
8. Štarha P, Trávníček Z, Pazderová L, Dvořák Z (2016) J Inorg
Biochem 162:109–116
35. Gottlieb HE, Kotlyar V, Nudelman A (1997) J Org Chem
62:7512–7515
9. Štarha P, Vančo J, Trávníček Z, Hošek J, Klusáková J, Dvořák Z
36. Massoud SS, Louka FR, Ducharme GT, Fischer RC, Mautner FA,
Vančo J, Herchel R, Dvořák Z, Trávníček Z (2018) J Inorg Bio-
chem 180:39–46
(
2016) PLoS One 11:e0165062
0. Štarha P, Hošek J, Vančo J, Dvořák Z, Suchý P Jr, Popa I,
Pražanová G, Trávníček Z (2014) PLoS One 9:e90341
37. Rochon FD, Buculei V (2004) Inorg Chim Acta 357:2218–2230
38. Malik M, Wysokiński R, Zierkiewicz W, Helios K, Michalska D
(2014) J Phys Chem A 118:6922–6934
2
2
1. Štarha P, Dvořák Z, Trávníček Z (2015) PLoS One 10:e0136338
2. Muchová T, Prachařová J, Štarha P, Olivová R, Vrána O, Benešová
B, Kašpárková J, Trávníček Z, Brabec V (2013) J Biol Inorg Chem
39. Ruiz J, Rodríguez V, de Haro C, Espinosa A, Pérez J, Janiak C
(2010) Dalton Trans 39:3290–3301
1
8:579–589
2
2
3. Štarha P, Vančo J, Trávníček Z (2019) Coord Chem Rev
40. Zamora A, Rodríguez V, Cutillas N, Yellol GS, Espinosa A,
Samper KG, Capdevila M, Palacios Ó, Ruiz J (2013) J Inorg Bio-
chem 128:48–56
3
80:103–135
4. Messori L, Cubo L, Gabbiani C, Alvarez-Valdes A, Michelucci
E, Pieraccini G, Rios-Luci C, Leon LG, Padron JM, Navarro-
Ranninger C, Casini A, Quiroga AG (2012) Inorg Chem
41. Pracharova J, Radosova Muchova T, Dvorak Tomastikova E, Intini
FP, Paciꢀco C, Natile G, Kasparkova J, Brabec V (2016) Dalton
Trans 45:13179–13186
5
1:1717–1726
2
5. Sá DS, Fernandes AF, Silva CDS, Costa PPC, Fonteles MC,
Nascimento NRF, Lopes LGF, Sousa EHS (2015) Dalton Trans
42. Colley HE, Muthana M, Danson SJ, Jackson LV, Brett ML, Har-
rison J, Coole SF, Mason DP, Jennings LR, Wong M, Tulasi V,
Norman D, Lockey PM, Williams L, Dossetter AG, Griꢁen EJ,
Thompson MJ (2015) J Med Chem 58:9309–9333
43. Rochon FD, Titouna H (2010) Inorg Chim Acta 363:1679–1693
44. Marzo T, Pillozzi S, Hrabina O, Kasparkova J, Brabec V,
Arcangeli A, Bartoli G, Severi M, Lunghi A, Totti F, Gabbiani C,
Quiroga AG, Messori L (2015) Dalton Trans 44:14896–14905
45. Messori L, Casini A, Gabbiani C, Michelucci E, Cubo L, Rios-
Luci C, Padron JM, Navarro-Ranninger C, Quiroga AG (2010)
ACS Med Chem Lett 1:381–385
4
4:13633–13640
2
2
6. Przyojski JA, Kiewit ML, Fillman KL, Arman HD, Tonzetich ZJ
2015) Inorg Chem 54:9637–9645
(
7. Chatterji M, Shandil R, Manjunatha MR, Solapure S, Ramachan-
dran V, Kumar N, Saralaya R, Panduga V, Reddy J, Prabhakar KR,
Sharma S, Sadler C, Cooper CB, Mdluli K, Iyer PS, Narayanan S,
Shirude PS (2014) Antimicrob Agents Chemother 58:5325–5331
8. Shirude PS, Shandil RK, Manjunatha MR, Sadler C, Panda M,
Panduga V, Reddy J, Saralaya R, Nanduri R, Ambady A, Rav-
ishankar S, Sambandamurthy VK, Humnabadkar V, Jena LK,
Suresh RS, Srivastava A, Prabhakar KR, Whiteaker J, McLaughlin
RE, Sharma S, Cooper CB, Mdluli K, Butler S, Iyer PS, Naray-
anan S, Chatterji M (2014) J Med Chem 57:5728–5737
2
2
46. Cirri D, Pillozzi S, Gabbiani C, Tricomi J, Bartoli G, Stefanini M,
Michelucci E, Arcangeli A, Messori L, Marzo T (2017) Dalton
Trans 46:3311–3317
47. Schmitt F, Donnelly K, Muenzner JK, Rehm T, Novohradsky
V, Brabec V, Kasparkova J, Albrecht M, Schobert R, Mueller T
(2016) J Inorg Biochem 163:221–228
9. Lee W, Crawford JJ, Aliagas I, Murray LJ, Tay S, Wang WR,
Heise CE, Hoeꢂich KP, La H, Mathieu S, Mintzer R, Ramas-
wamy S, Rouge L, Rudolph J (2016) Bioorg Med Chem Lett
48. Vančo J, Trávníček Z, Křikavová R, Gáliková J, Chalupová M,
Dvořák Z (2017) J Photochem Photobiol B Biol 173:423–433
49. Betzer JF, Nuter F, Chtchigrovsky M, Hamon F, Kellermann G,
Ali S, Calméjane MA, Roque S, Poupon J, Cresteil T, Teulade-
Fichou MP, Marinetti A, Bombard S (2016) Bioconj Chem
27:1456–1470
2
6:3518–3524
3
3
0. Wang T, Yang Z, Zhang ZX, Gong YF, Riccardi KA, Lin PF,
Parker DD, Rahematpura S, Mathew M, Zheng M, Meanwell NA,
Kadow JF, Bender JA (2013) Bioorg Med Chem Lett 23:213–217
1. Pin F, Buron F, Saab F, Colliandre L, Bourg S, Schoentgen F, Le
Guevel R, Guillouzo C, Routier S (2011) Med Chem Commun
2
:899–903
Publisher’s Note Springer Nature remains neutral with regard to
3
3
2. Łakomska I, Fandzloch M, Popławska B, Sitkowski J (2012) Spec-
trochim Acta A 91:126–129
jurisdictional claims in published maps and institutional aꢃliations.
3. Margiotta N, Denora N, Ostuni R, Laquintana V, Ander-
son A, Johnson SW, Trapani G, Natile G (2010) J Med Chem
5
3:5144–5154
1
3