In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline-piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline-piperazine hybrids L(1-3) were prepared in situ and isolated as six ruthenium and osmium complexes [(η(6)-p-cymene)M(L(1-3))Cl]Cl, where L(1) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L(2) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L(3) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV-vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl-[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure-activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline-piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents.
In this study, the indoloquinoline backbone and piperazine were combined to prepare indoloquinoline-piperazine hybrids and their ruthenium- and osmium-arene complexes in an effort to generate novel antitumor agents with improved aqueous solubility. In addition, the position of the metal-binding unit was varied, and the effect of these structural alterations on the aqueous solubility and antiproliferative activity of their ruthenium- and osmium-arene complexes was studied. The indoloquinoline-piperazine hybrids L(1-3) were prepared in situ and isolated as six ruthenium and osmium complexes [(η(6)-p-cymene)M(L(1-3))Cl]Cl, where L(1) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-2-N-amine, M = Ru ([1a]Cl), Os ([1b]Cl), L(2) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-4-N-amine, M = Ru ([2a]Cl), Os ([2b]Cl), L(3) = 6-(4-methylpiperazin-1-yl)-N-(pyridin-2-yl-methylene)-11H-indolo[3,2-c]quinolin-8-N-amine, M = Ru ([3a]Cl), Os ([3b]Cl). The compounds were characterized by elemental analysis, one- and two-dimensional NMR spectroscopy, ESI mass spectrometry, IR and UV-vis spectroscopy, and single-crystal X-ray diffraction. The antiproliferative activity of the isomeric ruthenium and osmium complexes [1a,b]Cl-[3a,b]Cl was examined in vitro and showed the importance of the position of the metal-binding site for their cytotoxicity. Those complexes containing the metal-binding site located at the position 4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative activity. The results provide important insight into the structure-activity relationships of ruthenium- and osmium-arene complexes with indoloquinoline-piperazine hybrid ligands. These studies can be further utilized for the design and development of more potent chemotherapeutic agents.
The search for more
effective ruthenium complexes and organoruthenium compounds as chemotherapeutic
agents with fewer side effects than currently clinically applied platinum
compounds continues to attract attention.[1−8] In fact, the use of biologically active ligands in combination with
ruthenium is an option to achieve synergistic effects.[9] The effect of metal coordination on their antiproliferative
activity, alteration of their pharmacological properties, is an area
of study that requires further investigation.Paullones or indolo[3,2-d]benzazepines (Figure 1, top left
and top middle), initially predicted to exhibit a cyclin-dependent
kinase (CDK) inhibitory profile similar to that of flavopiridol,[10,11] were later shown to act as protein kinase inhibitors,[12,13] targeting glycogen synthase kinase-3β and mitochondrial malate
dehydrogenase.[14,15] However, their clinical use was
hampered by their limited aqueous solubility. One way to potentially
overcome this problem was the coordination of these organic compounds
to metal ions. Complexes with gallium(III),[16,17] ruthenium,[18] copper(II),[19] as well as organoruthenium(II) and organoosmium(II) compounds
have been synthesized, and due to their improved aqueous solubility,
their antiproliferative activity has been investigated.[20−24] In fact, interesting structure–activity relationships have
been established. For example, replacement of the seven-membered azepine
ring in modified paullones by a six-membered pyridine ring has led
to another class of biologically active compounds, namely, indolo[3,2-c]quinolines (Figure 1, top right
and bottom).
Figure 1
Original paullone, also named indolo[3,2-d]benzazepin-6-one (top left),[11] modified
paullone (top middle),[23] and modified indolo[3,2-c]quinolines with specific atom positions labeled (top right
and bottom)[27−29] as ligands for ruthenium(II) and osmium(II).
Original paullone, also named indolo[3,2-d]benzazepin-6-one (top left),[11] modified
paullone (top middle),[23] and modified indolo[3,2-c]quinolines with specific atom positions labeled (top right
and bottom)[27−29] as ligands for ruthenium(II) and osmium(II).Although specific kinase inhibition
has not been documented for indolo[3,2-c]quinolines,[25] tree extracts containing indolo[3,2-b] and indolo[3,2-c]quinolines have a long
history of usage in traditional African medicine.[26] The main difference compared to the paullones is the full
conjugation of the heterocyclic system, leading to the planarity of
the indoloquinoline backbone. This resulted in different physicochemical
properties and a marked alteration in the biological activity of these
indoloquinolines.[23] It was discovered that
the modified paullones containing an ethylenediamine binding moiety
(Figure 1, top middle) formed ruthenium- and
osmium-arene complexes that were more stable compared to their indoloquinoline
counterparts[23] (Figure 1, top right). However, the complexes with indoloquinolines
were approximately 1 order of magnitude more cytotoxic.[23]Interestingly, replacement of the sp3-hybridized nitrogen donor atoms with sp2-hybridized
nitrogens present in the formylpyridine–azine modified indoloquinolines
(Figure 1, bottom left) afforded complexes
that do not dissociate with liberation of the indoloquinoline ligand
under biological conditions.[27,28] These studies enabled
elucidation of the effects of substituents at positions 2 and 8 of
the indoloquinoline backbone (Figure 1, bottom
left) on their biological activity.[27,28]Further
structural diversity was achieved through synthetic procedures involving
the reduction of a nitro group at position 2 of the indoloquinoline
backbone. This created a potential metal-binding site via condensation
of the emerging NH2 group with 2-formylpyridine. This,
in combination with the preservation of the lactam group, resulted
in a new class of paullone analogues (Figure 1, bottom middle).[29] This group was of
particular interest since the lactam group of the metal-free paullones
was shown to be essential for efficient CDK inhibition.[11] Their resulting ruthenium and osmium indoloquinoline
complexes exhibited lower in vitro antiproliferative
activity.[29] In fact, their efficacy was
2 orders of magnitude lower when compared to complexes with ligands
coordinated via a similar binding site at position 6 of the backbone.[29] Furthermore, one osmium complex with the ligand
shown in Figure 1 (bottom right) demonstrated
antitumor activity in vivo.[29] Intriguingly, this complex showed activity not only after intraperitoneal
injection, but also when administered orally.[29]We were interested in combining bioactive substructures, namely,
that of the indoloquinoline backbone with a piperazine heterocycle,
to improve the water solubility of the indoloquinoline moiety. Moreover,
piperazine is frequently found in the structures of biologically active
compounds in a large spectrum of therapeutic areas.[30−33] Some of these agents, such as
Ciprofloxacin, Ofloxacin, Posaconazole, Enoxacin, Eperezolid, and
Piperacillin, are currently used successfully in clinical therapy.[34−36] Metal-based derivatives of biologically active compounds containing
this heterocyclic nucleus as a pharmacophore have been also reported.[37−41] An efficient approach to the synthesis of water-soluble biologically
active compounds is the attachment of piperazine or a modified piperazine
heterocycle to lipophilic pharmacophore moieties.[42−44] Further modulation
of hydrophilic/lipophilic balance can be achieved by introducing different
substituents to the distant N atom on the piperazine ring.[45]Furthermore, it was also of interest to
vary the metal-binding unit position and to study the effect of these
changes on the aqueous solubility and antiproliferative activity of
the ruthenium- and osmium-arene complexes.Herein, we report
the in situ synthesis and characterization of three
isomeric indoloquinoline–methyl-piperazine conjugates with
the location of the metal-binding site at position 2, 4, or 8 of the
indoloquinoline scaffold. These compounds were isolated as six water-soluble
ruthenium- or osmium-arene complexes of the general formulas [(η6-p-cymene)M(L1–3)Cl]+ (Chart 1). The effects of the metal-binding
unit position and the identity of the metal (Ru vs Os) on the antiproliferative
activity of isomeric ruthenium- and osmium-arene complexes with indoloquinoline–piperazine
hybrids have been elucidated. The results obtained provide an important
insight into the structure–activity relationships of ruthenium-
and osmium-arene complexes with indoloquinoline–piperazine
hybrids and can be utilized further for the design and development
of more effective chemotherapeutic agents.
Chart 1
Ruthenium- and Osmium-Arene
Complexes with Indoloquinoline–Piperazine Hybrid Ligands Modified
at Position 2, 4 or 8
Experimental Section
Materials
2-Aminobenzylamine,
phosphorus oxychloride, 1-methylpiperazine, and iron powder were purchased
from Sigma-Aldrich (St. Louis, MO), while isatin, 5-nitroisatin, and
2-pyridinecarboxaldehyde were purchased from Acros (Geel, Belgium).
All chemicals, as well as the synthesized precursors, were used without
further purification. 2-Amino-5-nitrobenzylamine hydrochloride was
synthesized according to a procedure published elsewhere.[46] The free amine was obtained by the dissolution
of the hydrochloride in a minimal amount of water, addition of 4 equiv
of aqueous ammonia, and collection of the formed precipitate by filtration.
Ethanol was dried over molecular sieves (3 Å), and tetrahydrofuran
(THF) was dried by using a standard procedure (Na/benzophenone).[47]
General Synthetic Procedure (A) for the Synthesis
of Complexes [1b]Cl, [2b]Cl, and [3a,b]Cl
The corresponding 2-, 4-, or
8-amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline
(1 equiv) and the respective metal-p-cymene dimer,
[RuCl(μ-Cl)(η6-p-cymene)]2 or [OsCl(μ-Cl)(η6-p-cymene)]2 (0.9 equiv), were suspended in dry ethanol.
2-Pyridinecarboxaldehyde (0.9 equiv) was added, and the reaction mixture
was stirred under an argon atmosphere, whereupon a clear solution
was obtained within a few minutes. After stirring at room temperature
for 20 h, the solution was filtered through a GF-3 fiber filter and
then added dropwise into dried diethyl ether over sodium sulfate (approximately
400 mL of diethyl ether per 3 mL of solution). The precipitate formed
was filtered off and dried in vacuo at 45 °C.
One-dimensional
(1D) 1H and 13C NMR and two-dimensional (2D) 1H–1H COSY, 1H–1H TOCSY, 1H–1H ROESY or 1H–1H NOESY, 1H–13C
HSQC, and 1H–13C HMBC NMR spectra were
recorded on two Bruker Avance III spectrometers at 500.32 or 500.10
(1H), and 125.82 or 125.76 (13C) MHz, respectively,
by using DMSO-d6 as a solvent at room
temperature and standard pulse programs. 1H and 13C shifts are quoted relative to the solvent residual signals. The
atom numbering scheme used for NMR assignments is depicted in Supporting Information, Scheme S1. Electrospray
ionization mass spectrometry (ESI-MS) was carried out with a Bruker
Esquire 3000 instrument, and the samples were dissolved in methanol.
Elemental analyses were performed at the Microanalytical Laboratory
of the University of Vienna with a PerkinElmer 2400 CHN Elemental
Analyzer (PerkinElmer, Waltham, MA). Hydrolysis studies were undertaken
on a ThermoFisher Dionex UltiMate 3000 HPLC (Waltham, MA) device.
The column compartment was kept at 298 K. A Hypersil Gold C18 Reversed
phase Silica Column (250 × 4.6 mm, 5 μm particle size,
ThermoFisher; Waltham, MA) equipped with a guard column was used.
The complexes were dissolved in either pure deionized water or phosphate
buffer at pH 7.40 so that the final concentrations were 100 mM or
75 μM, respectively. Samples were automatically injected into
the HPLC, and separation of the hydrolysis products was achieved by
applying gradient elution (5–80% or 10–50% acetonitrile
in 15 mM aqueous formic acid). Detection and quantification of the
peaks was conducted using the ThermoFisher Dionex DAD-3000RS UV detector
(Waltham, MA) at a wavelength of 300 nm and the Chromeleon 7 software
package (ThermoFisher Dionex, Sunnyvale, CA). In the case of [2b]Cl, further identification of the peaks was done by collecting
the main fractions and analyzing them using a Bruker micrOTOF-Q2 ESI-MS
instrument.
Crystallographic Structure Determination
X-ray diffraction measurements were performed on a Bruker X8 APEXII
CCD diffractometer. Single crystals were positioned at 35, 40, and
35 mm from the detector, and 969, 790, and 2330 frames were measured,
each for 60, 90, and 20 s over 1° scan width for [1aH]Cl2·EtOH·2H2O, [2aH]Cl2·1.5PrOH·0.5H2O, and [2a]Cl·0.5EtOH·H2O, respectively. The data were processed using SAINT software.[48] Crystal data, data collection parameters, and
structure refinement details are given in Table 1. The structures were solved by direct methods and refined by full-matrix
least-squares techniques. Non-hydrogen atoms were refined with anisotropic
displacement parameters. H atoms were inserted in calculated positions
and refined with a riding model. The following computer programs and
hardware were used: structure solution, SHELXS-97; structure refinement, SHELXL-97;[49] molecular diagrams, ORTEP.[50]
Table 1
Crystal Data and Details of Data Collection for [1aH]Cl2·EtOH·2H2O, [2aH]Cl2·1.5PrOH·0.5H2O, and [2a]Cl·0.5EtOH·H2O
compound
[1aH]Cl2·EtOH·2H2O
[2aH]Cl2·1.5iPrOH·0.5H2O
[2a]Cl·0.5EtOH·H2O
empirical formula
C38H49Cl3N6O3Ru
C40.5H52Cl3N6O2Ru
C37H43Cl2N6O1.5Ru
fw
845.25
862.30
767.74
space group
P21/n
P21/c
Pc
α, Å
19.2712(14)
15.2896(5)
10.0291(4)
b, Å
7.8687(5)
19.5424(6)
9.4460(4)
c, Å
25.5459(19)
15.3917(5)
19.2958(7)
β, deg
91.724(4)
113.677(2)
93.479(2)
V [Å3]
3872.0(5)
4211.8(2)
1824.62(13)
Z
4
4
2
λ [Å]
0.710 73
0.710 73
0.710 73
ρcalcd, g cm–3
1.450
1.360
1.397
cryst size, mm3
0.12 × 0.08 × 0.02
0.25 × 0.13 × 0.03
0.15 × 0.10 × 0.04
T [K]
150(2)
100(2)
100(2)
μ, mm–1
0.657
0.604
0.615
R1a
0.0519
0.0480
0.0362
wR2b
0.1137
0.1422
0.0922
GOFc
1.084
1.058
1.036
R1 = Σ||F0| – |Fc||/Σ|F0|.
wR2 = {Σ[w(F02 – Fc2)2]/Σ[w(F02)2]}1/2.
GOF = {Σ[w(F02 – Fc2)2]/(n – p)}1/2, where n is the number
of reflections and p is the total number of parameters
refined.
R1 = Σ||F0| – |Fc||/Σ|F0|.wR2 = {Σ[w(F02 – Fc2)2]/Σ[w(F02)2]}1/2.GOF = {Σ[w(F02 – Fc2)2]/(n – p)}1/2, where n is the number
of reflections and p is the total number of parameters
refined.
Cell Culture
Human
SK-N-MC neuroepithelioma cells were obtained from the American Type
Culture Collection (ATCC; Manassas, VA). The SK-N-MC cell type was
grown as described previously.[51]
MTT Assay
The effect of complexes [1a,b]Cl–[3a,b]Cl on the cellular proliferation of SK-N-MC
cells was examined using the 1-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
(MTT) assay by standard techniques.[52,53] Human SK-N-MC
cells were chosen for these studies as the antiproliferative effect
of metal complexes has been extensively examined in this cell type.[52−54] MTT reduction is proportional to viable cell counts using SK-N-MC
cells.[51] SK-N-MC cells were seeded at 1.5
× 104 cells/well in 96-well microtiter plates in a
medium containing the compounds (0–50 μM) and human diferric
transferrin (Tf) at 1.25 μM ([Fe] = 2.5 μM). Control samples
contained Tf (1.25 μM) in the absence of the compounds. Notably,
Tf is utilized as it is the physiological iron donor and is required
for cellular proliferation.[51] It is essential
to add to the media when assessing the antiproliferative activity
of the ligands used as positive controls, namely, desferrioxamine
(DFO), di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT),
and Doxorubicin.[61] The cells were incubated
at 37 °C for 72 h, after which 10 μL of MTT solution (stock
solution: 5 mg/mL) was added to each well and incubated for 2 h at
37 °C. After cell solubilization using 100 μL of 10% SDS–50%
isobutanol in 0.01 M HCl, the plates were read at 570 nm using a scanning
multiwell spectrophotometer. The inhibitory concentration (IC50) was defined as the concentration of the compound necessary
to reduce the absorbance to 50% of the untreated control.
Statistical
Analysis
Results are expressed as mean ± standard deviation
(SD). Statistical comparisons were performed using Prism v6 (GraphPad
Software, Inc., La Jolla, CA) implementing a one-way ANOVA with a
Bonferroni posthoc test and were considered statistically significant
when p < 0.05.
Results and Discussion
These studies examined the effects of attachment of the piperazine
heterocycle to the indoloquinoline scaffold and of varying the position
of the same metal-binding site on the indoloquinoline backbone. This
resulted in the generation of three different structural isomers of
the indoloquinoline–piperazine hybrid. The effect of the metal-binding
site position on the antiproliferative activity of the resultant ruthenium
and osmium complexes [1a,b]Cl–[3a,b]Cl was examined. The ruthenium and osmium
complexes were assembled in situ by reacting the
corresponding indoloquinoline–piperazine hybrid precursors 7a–c containing an amino group at the
2-, 4-, or 8-position of the indoloquinoline scaffold with 2-pyridinecarboxaldehyde.Attempts to isolate the pure indoloquinoline–piperazine
hybrids in the absence of ruthenium(II) or osmium(II) failed. Thus,
the compounds L1–3 and their resultant ruthenium
and osmium complexes [1a,b]Cl–[3a,b]Cl were formed in a single reaction mixture in situ. This suggests that the indoloquinoline–piperazine
hybrids were stabilized through their coordination with the ruthenium(II)-
or osmium(II)-arene moieties. The combination of the indoloquinoline
and piperazine substructures and the introduction of amino groups
at the three different positions were accomplished as shown in Scheme 1.
Scheme 1
Synthetic Pathways to Prepare the Isomeric Indoloquinoline-Piperazine
Hybrids 7a, 7b, and 7c
Reagents and conditions: (i)
POCl3, Ar, 110 °C, 24 h; (ii) Fe (powder), AcOH, EtOH,
35 °C, 3–4 h, ultrasound bath; (iii) 1-methylpiperazine,
Ar, 130 °C, 24 h; (iv) MeOH, 10% Pd/C, H2, RT, 24
h.
Synthetic Pathways to Prepare the Isomeric Indoloquinoline-Piperazine
Hybrids 7a, 7b, and 7c
Reagents and conditions: (i)
POCl3, Ar, 110 °C, 24 h; (ii) Fe (powder), AcOH, EtOH,
35 °C, 3–4 h, ultrasound bath; (iii) 1-methylpiperazine,
Ar, 130 °C, 24 h; (iv) MeOH, 10% Pd/C, H2, RT, 24
h.The synthesis of 2-amino-6-chloroindolo[3,2-c]quinoline 6a was performed as described previously.[29] Briefly, 5-nitroisatin and 2-aminobenzylamine
were reacted in glacial acetic acid in a manner similar to the unsubstituted
isatin.[55] The resulting 2-nitroindolo[3,2-c]quinoline-6-one 4a was chlorinated using
POCl3 to give 5a, again under conditions similar
to those applied for the synthesis of the unsubstituted congener.[56] Reduction of the nitro group was carried out
using an adaption[29] of the procedure published
by Gamble et al. to afford 6a.[57] 7-Nitroisatin was easily obtained by adaption of the procedure used
for the synthesis of 2-nitroaniline (see Supporting
Information for detail).[58] The 4-amino
isomer 6b was prepared by following the same synthetic
route used for the synthesis of 6a. Application of the
same synthetic scheme failed to yield 6c in acceptable
yield and purity. However, starting from isatin and 2-amino-5-nitrobenzylamine,
obtained by neutralization of its hydrochloride salt[46] with aqueous ammonia, resulted in the synthesis of 8-nitro-indolo[3,2-c]quinoline-6-one 4c in low yield but acceptable
purity. Subsequent chlorination of 4c yielded 6-chloro-8-nitro-indolo[3,2-c]quinoline 5c. In the final step, the imidoyl
chloride 6a or 6b was reacted with 1-methylpiperazine,
yielding the corresponding 2- or 4-amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline 7a or 7b, respectively.
To obtain 7c, compound 5c was allowed to
react with 1-methylpiperazine to yield 6c, followed by
the reduction of the nitro group at position 8 by using H2 and Pd/C as shown in Scheme 1.Further
condensation reactions of the indoloquinoline–piperazine hybrids 7a–7c with 2-pyridinecarboxaldehyde in
the presence of [RuCl(μ-Cl)(η6-p-cymene)]2 or [OsCl(μ-Cl)(η6-p-cymene)]2 afforded the chelating bidentate
ligands isolated as metal-arene complexes [1a,b]Cl–[3a,b]Cl shown in Chart 1. Note that, because of the enhanced aqueous solubility
of two series of isomeric complexes [1a]Cl–[3a]Cl and [1b]Cl–[3b]Cl,
the purification method had to be modified. Microcrystalline osmium
complexes [1b]Cl–[3b]Cl as well as
ruthenium complex [3a]Cl could be obtained with high
purity by dropwise addition of the reaction mixture to large amounts
of diethyl ether. Prior drying of diethyl ether over Na2SO4 was required to avoid the formation of a sticky oil.
In the case of the ruthenium complexes [1a]Cl and [2a]Cl, this procedure led to the coprecipitation of small
amounts of side products. Pure compounds, obtained when condensation
reactions in the presence of [RuCl(μ-Cl)(η6-p-cymene)]2 were carried out in water,
were isolated from concentrated aqueous solutions. All complexes were
characterized by elemental analysis, ESI mass spectrometry, and spectroscopic
methods. The compounds are moderately hygroscopic; water, which had
to be included in the elemental formulas, is a property common to
nearly all metal complexes with modified indoloquinolines reported
so far.[23,27−29] The in situ formation of the ligands during the complex syntheses of [1a]Cl–[3a]Cl and [1b]Cl–[3b]Cl was confirmed by the presence of strong peaks at m/z 691 and m/z 781, respectively, attributed to the [M–Cl]+ ion.The 1D and 2D NMR spectra showed chemical shifts
typical for this class of compounds,[23,27−29] in particular, the rather upfield-shifted resonances for carbon
atom C6a (for the atom numbering scheme used for the assignment
of resonances see Supporting Information, Scheme
S1) at approximately 106–107 ppm. Although a weak resonance
for quaternary carbon atom C6 could be seen in complexes
[1a]Cl and [1b]Cl, this was not detected
in the remaining four metal complexes. Note that such isolated quaternary
carbons are generally hard to detect, as they do not display in standard
2D 1H–13C NMR experiments like HSQC or
HMBC. They also have much longer relaxation times compared to those
with protons in close proximity, leading to weakening or even suppression
of the signal in standard 13C NMR experiments. However,
the shift of approximately 158.5 ppm for C6 in the 13C NMR spectra of [1a]Cl and [1b]Cl is in good agreement with that featured by related indoloquinolines,
in which the piperazine ring is substituted by an ethylenediamine
moiety.[23] Due to the presence of the stereogenic
metal center, the adjacent cymene protons showed diastereotopic splitting,
leading to four distinct signals for the aromatic protons. In addition,
two resonances were observed for the protons of the isopropyl groups.
This pattern is typical for racemic ruthenium-arene complexes, in
which bulky ligand(s) hinder the free rotation of the arene.[59,60] Furthermore, because of the flexibility of the piperazine ring,
the unequivocal assignment of the resonances observed was not possible.
Therefore, the CH2 resonances present
as broad singlets were assigned as CH2mp.
Stability in DMSO, Solubility in Phosphate Buffer, and Hydrolytic
Behavior
Complexes [3a]Cl and [3b]Cl remain intact when dissolved in DMSO. ESI mass spectra of solutions
of [3a]Cl and [3b]Cl in DMSO diluted with
methanol 1:100 showed intense peaks with m/z 691
and 781, respectively, attributed to [M–Cl]+ ions
(Supporting Information, Figures S1 and S2). Basically, the same mass spectra were obtained for 19 h old DMSO
solutions of [3a]Cl and [3b]Cl after dilution
with methanol. Hence, the dissolution of these complexes in DMSO does
not lead to an appreciable replacement of Cl– with
DMSO.The complexes presented herein are readily soluble in
aqueous media. The solubility of all six complexes [1a,b]Cl–[3a,b]Cl in phosphate
buffer (20 mM NaH2PO4/Na2HPO4 buffer at pH 7.40) exceeded 7 mM, regardless of the position
of the coordinating moiety.The stability of [2a]Cl and [2b]Cl in aqueous media over 24 h was further
investigated by HPLC (Figure 2). Their stability
in deionized water (Supporting Information, Figure
S3) is very similar to that in buffered solution at physiological
pH (Supporting Information, Figure S4;
20 mM NaH2PO4/Na2HPO4 buffer
at pH 7.40). As can be seen from Supporting Information,
Figure S4, greater than 90% of both the ruthenium complex [2a]Cl as well as its osmium counterpart [2b]Cl
were hydrolyzed after 24 h, without any noticeable difference in the
hydrolysis rate. A closer assessment revealed that [2b]Cl gave almost exclusively one hydrolysis product. However, in the
case of its ruthenium analogue [2a]Cl, a second, minor
species appeared between the parent peak and the less-retained hydrolysis
peak (see Supporting Information, Figures S3 and
S4 for chromatograms). Attempts to establish the nature of
the predominant hydrolysis reactions were then performed. The fractions
containing the two peaks from the hydrolysis of [2a]Cl
were collected and subjected to high-resolution ESI mass spectrometry.
The most abundant mass at m/z 373.1397
could be attributed to the doubly charged fragment [M–2Cl]2+, whereas the second peak at m/z 736.2786 was assigned to the hydroxido species [M–2Cl+OH]+, clearly stemming from hydrolysis of the chlorido ligand.
The fraction containing the peak with the longer retention time showed
mainly the parent ion [M–Cl]+ at m/z 781.2442, and, to a lesser extent, the doubly
charged [M–2Cl]2+ ion. These data suggest that the ,-bound
ligand remained coordinated to the metal center, showing no signs
of ligand cleavage.
Figure 2
Hydrolysis curves of 75 μM solutions of complex
[2a]Cl (top) and [2b]Cl (bottom) in 20 mM
phosphate buffer at pH 7.40. For both complexes, the chlorido species
concentration (■) steadily decreases with an estimated half-lifetime
of approximately 6 h. In the case of [2b]Cl, only one
hydrolysis product is formed (◆), whereas in the case of [2a]Cl, a third species (▲) is formed to a minor extent,
exhibiting an area under curve (AUC) of approximately 12% compared
to the staring material after 24 h. Data were recorded every hour
over 24 h using HPLC with UV detection at 300 nm (see Experimental Section for details). Quantification was done
by normalizing the AUC to the AUC at 0 h.
Hydrolysis curves of 75 μM solutions of complex
[2a]Cl (top) and [2b]Cl (bottom) in 20 mM
phosphate buffer at pH 7.40. For both complexes, the chlorido species
concentration (■) steadily decreases with an estimated half-lifetime
of approximately 6 h. In the case of [2b]Cl, only one
hydrolysis product is formed (◆), whereas in the case of [2a]Cl, a third species (▲) is formed to a minor extent,
exhibiting an area under curve (AUC) of approximately 12% compared
to the staring material after 24 h. Data were recorded every hour
over 24 h using HPLC with UV detection at 300 nm (see Experimental Section for details). Quantification was done
by normalizing the AUC to the AUC at 0 h.
X-ray Crystallography
The results of the X-ray diffraction
studies of [(η6-p-cymene)Ru(HL1)Cl]Cl2·EtOH·2H2O ([1aH]Cl2·EtOH·2H2O), [(η6-p-cymene)Ru(HL2)Cl]Cl2·1.5PrOH·0.5H2O ([2aH]Cl2·1.5i-PrOH·0.5H2O), and [(η6-p-cymene)Ru(L2)Cl]Cl·0.5EtOH·H2O ([2a]Cl·0.5EtOH·H2O) are
shown in Figures 3–5, respectively. The complexes [1aH]Cl2·EtOH·2H2O, [2aH]Cl2·1.5PrOH·0.5H2O, and [2a]Cl·0.5EtOH·H2O crystallized
in the monoclinic centrosymmetric space groups P21/n, P21/c and noncentrosymmetric Pc, respectively.
All complexes studied contain a stereogenic ruthenium center and crystallize
as racemates. The asymmetric units of the first two compounds consist
of a complex dication, two chlorides as counteranions, and cocrystallized
solvent, while that of the third complex consists of a complex monocation,
one chloride as counteranion, and cocrystallized solvent molecules.
Figure 3
ORTEP
view of the cation [(η6-p-cymene)Ru(HL1)Cl]2+ in [1aH]Cl2·EtOH·2H2O with thermal displacement parameters drawn at the 50% probability
level. Selected bond distances (Å) and angles (deg): Ru–N19
2.091(3), Ru–N26 2.081(4), Ru–Cl1 2.387(1), Ru–Carene(av) 2.20(1), N5–C6 1.318(5), C6–N12 1.402(6);
N19–Ru–N26 77.18, ΘC1–C2–N19–C20 −33.6(6), ΘN19–C20–C21–N26 0.6(6).
Figure 5
ORTEP view of the cation [(η6-p-cymene)Ru(L2)Cl]+ in [2a]Cl·0.5EtOH·H2O with thermal displacement
parameters drawn at the 50% probability level. Selected bond distances
(Å) and angles (deg): Ru–N19 2.096(2), Ru–N26 2.073(2),
Ru–Cl1 2.3838(8), Ru–Carene(av) 2.20(2),
N5–C6 1.311(4), C6–N12 1.393(4); N19–Ru–N26
76.4(1), ΘC4a–C4–N19–C20 −51.2(4),
ΘN19–C20–C21–N26 1.7(4).
ORTEP
view of the cation [(η6-p-cymene)Ru(HL1)Cl]2+ in [1aH]Cl2·EtOH·2H2O with thermal displacement parameters drawn at the 50% probability
level. Selected bond distances (Å) and angles (deg): Ru–N19
2.091(3), Ru–N26 2.081(4), Ru–Cl1 2.387(1), Ru–Carene(av) 2.20(1), N5–C6 1.318(5), C6–N12 1.402(6);
N19–Ru–N26 77.18, ΘC1–C2–N19–C20 −33.6(6), ΘN19–C20–C21–N26 0.6(6).ORTEP view of the cation [(η6-p-cymene)Ru(HL2)Cl]2+ in
[2aH]Cl2·1.5PrOH·0.5H2O with thermal displacement parameters
drawn at the 40% probability level. Selected bond distances (Å)
and angles (deg): Ru–N19 2.073(4), Ru–N26 2.085(4),
Ru–Cl1 2.382(1), Ru–Carene(av) 2.194(7),
N5–C6 1.314(5), C6–N12 1.400(6); N19–Ru–N26
77.1(1), ΘC4a–C4–N19–C20 −60.4(5),
ΘN19–C20–C21–N26 1.0(5).ORTEP view of the cation [(η6-p-cymene)Ru(L2)Cl]+ in [2a]Cl·0.5EtOH·H2O with thermal displacement
parameters drawn at the 50% probability level. Selected bond distances
(Å) and angles (deg): Ru–N19 2.096(2), Ru–N26 2.073(2),
Ru–Cl1 2.3838(8), Ru–Carene(av) 2.20(2),
N5–C6 1.311(4), C6–N12 1.393(4); N19–Ru–N26
76.4(1), ΘC4a–C4–N19–C20 −51.2(4),
ΘN19–C20–C21–N26 1.7(4).All three complexes have a typical
“three-leg piano-stool” geometry of ruthenium(II)-arene
complexes, with a η6 π-bound p-cymene ring forming the seat and three other donor atoms (two nitrogens,
N19 and N26, of indolo[3,2-c]quinoline and one chloride
ligand) as the legs of the stool. In the first two complexes, the
ligand is protonated at N15, while in the last complex the ligand
is neutral. The protonation is corroborated by the presence of hydrogen-bonding
interactions of the type N15–H···Cl2ii in [1aH]Cl2 and N15–H···Cl3ii and N15–H···Cl3iii in [2aH]Cl2. Hydrogen-bonding interactions of the type
N–H···Cl, in which N11 acts as a proton donor
and Cl– as proton acceptor, are evident in all three
crystal structures (for hydrogen bond geometries see Supporting Information, Table S1).The piperazine ring
in all three compounds adopts a chair conformation. The conformations
of the neutral ligand L2 in the cation [(η6-p-cymene)Ru(L2)Cl]+ and the
protonated ligand (HL2)+ in [(η6-p-cymene)Ru(HL2)Cl]2+ are
similar, as can be seen in Supporting Information,
Figure S5 showing a model fitting plot for the two cations.
Of note is the slightly different orientation of the metal-binding
site in both cations, which can be described by the torsional angle
ΘC4a–C4–N19–C20 that differs
by 9.2° and by the more divergent orientation of the six-membered
piperazine heterocycle in two compounds. In addition, protonation
of the piperazine ring led to counterclockwise rotation of the p-cymene ring by approximately 39° around p-cymene ring centroid-Ru vector in [(η6-p-cymene)Ru(L2)Cl]+ (see Supporting Information, Figure S5).
Anti-Proliferative
Activity Against Tumor Cells
The ability of the isomeric
ruthenium- and osmium-arene complexes of the indoloquinoline–piperazine
hybrid ligands to inhibit cellular proliferation was examined using
human SK-N-MC neuroepithelioma cells. These cells were utilized since
the ability of other metal complexes to affect their growth is well-characterized.[52−54] The novel complexes [1a,b]Cl–[3a,b]Cl were compared to a number of relevant
positive controls that form metal complexes and have been extensively
examined in this cell type. These included: (1) DFO, which is used
clinically for the treatment of iron overload disease;[61] (2) Dp44mT, an iron chelator that demonstrates
potent antitumor activity in vitro(53) and in vivo;[62] and (3) doxorubicin (DOX), which is a clinically used cytotoxic
agent.[63,64]As previously observed,[53] the control chelator Dp44mT demonstrated the
most potent antiproliferative effects of all the agents examined (IC50: 0.004 ± 0.003 μM; Table 2). In contrast, the other positive controls, DOX (IC50: 0.15 ± 0.05 μM; Table 2) and
DFO (IC50: 9.83 ± 0.39 μM; Table 2), demonstrated moderate and poor ability to inhibit the growth
of SK-N-MC cells, respectively.
Table 2
IC50 (μM)
Values of [1a,b]Cl–[3a,b]Cl at Inhibiting the Growth of SK-N-MC Neuroepithelioma
Cells As Determined by the MTT Assay
compound
IC50 (μM)a
DFO
9.83 ± 0.39
Dp44mT
0.004 ± 0.003
DOX
0.15 ± 0.05
[1a]Cl
39.81 ± 2.07
[1b]Cl
>50
[2a]Cl
18.41 ± 2.22
[2b]Cl
19.40 ± 1.28
[3a]Cl
>50
[3b]Cl
41.24 ± 2.37
Assays conducted
for 72 h. Results are mean ± SD (three experiments).
Assays conducted
for 72 h. Results are mean ± SD (three experiments).Of the ruthenium and osmium complexes
that contain the metal-binding site at the position 4 of the indoloquinoline
scaffold, complexes [2a]Cl (IC50: 18.41 ±
2.22 μM; Table 2) and [2b]Cl (IC50: 19.40 ± 1.28 μM; Table 2) demonstrated significantly (p < 0.001) greater antiproliferative effects than the other ruthenium
and osmium complexes examined. However, the anticancer activity of
[2a]Cl and [2b]Cl was significantly (p < 0.001) less potent than that of the positive controls,
Dp44mT, DFO, or DOX. Although [1a]Cl and [3b]Cl demonstrated comparable IC50 values (39.81 ±
2.07 μM cf. 41.24 ± 2.37 μM, respectively), both
[1b]Cl and [3a]Cl did not exhibit marked
antiproliferative effects with IC50 values > 50 μM.Interestingly, these data illustrated the important role of the
position of the metal-binding site on the anticancer activity of these
isomeric Ru- and Os-arene complexes with indoloquinoline–piperazine
conjugates. Those complexes containing the metal-binding site located
at the 4 position of the indoloquinoline scaffold, namely, ([2a]Cl and [2b]Cl) demonstrated the most potent
antiproliferative effects. In contrast, the Ru and Os complexes that
contained the metal-binding site located at the 2 or 8 positions exhibited
significantly (p < 0.001) decreased anticancer
effects. Importantly, the position of the metal-binding site played
a more critical role than the identity of the complexed metal in the
antiproliferative activity of these agents.
Conclusions
In this study, the two bioactive substructures, namely, that of
the indoloquinoline backbone with a piperazine heterocycle, were combined
to improve the aqueous solubility of the resulting products, which
can act as ligands to form ruthenium and osmium complexes. In addition
to incorporating the piperazine heterocycle, these studies also examined
the possibility of varying the position of the same metal-binding
site on the indoloquinoline backbone. This resulted in the generation
of three different structural isomers of the indoloquinoline–piperazine
hybrid ligands. The effect of this structural combination on the antiproliferative
activity of the resulting ruthenium- and osmium-arene complexes [1a,b]Cl–[3a,b]Cl was studied.The data obtained illustrate the important
role of the position of the metal-binding site on the anticancer activity
of these isomeric ruthenium- and osmium-arene complexes with indoloquinoline–piperazine
hybrids. Whereas the position of the metal-binding site does not have
a marked effect on the solubility of [1a,b]Cl–[3a,b]Cl in phosphate buffer,
the complexes containing the metal-binding site located at the position
4 of the indoloquinoline scaffold ([2a]Cl and [2b]Cl) demonstrated the most potent antiproliferative effects.
In contrast, the ruthenium and osmium complexes that contain the metal-binding
site located at the 2 or 8 position exhibited decreased antiproliferative
activity. Importantly, the position of the metal-binding site played
a more critical role than the identity of the complexed metal in the
antiproliferative efficacy of these agents. These data highlight important
structure–activity relationships that can be further utilized
in the design of more potent anticancer ruthenium and osmium complexes
based on indoloquinoline–piperazine hybrids.
Authors: Aleksandar Savić; Marija Dulović; Jelena M Poljarević; Sonja Misirlić-Denčić; Maja Jovanović; Andrija Bogdanović; Vladimir Trajković; Tibor J Sabo; Sanja Grgurić-Šipka; Ivanka Marković Journal: ChemMedChem Date: 2011-07-29 Impact factor: 3.466
Authors: Johanna Faist; Werner Seebacher; Robert Saf; Reto Brun; Marcel Kaiser; Robert Weis Journal: Eur J Med Chem Date: 2011-11-18 Impact factor: 6.514
Authors: C Schultz; A Link; M Leost; D W Zaharevitz; R Gussio; E A Sausville; L Meijer; C Kunick Journal: J Med Chem Date: 1999-07-29 Impact factor: 7.446
Authors: Paul V Bernhardt; Philip C Sharpe; Mohammad Islam; David B Lovejoy; Danuta S Kalinowski; Des R Richardson Journal: J Med Chem Date: 2009-01-22 Impact factor: 7.446
Authors: Wolfgang F Schmid; Stefanie Zorbas-Seifried; Roland O John; Vladimir B Arion; Michael A Jakupec; Alexander Roller; Mathea Sophia Galanski; Ion Chiorescu; Haralabos Zorbas; Bernhard K Keppler Journal: Inorg Chem Date: 2007-04-03 Impact factor: 5.165
Authors: Lukas K Filak; Simone Göschl; Petra Heffeter; Katia Ghannadzadeh Samper; Alexander E Egger; Michael A Jakupec; Bernhard K Keppler; Walter Berger; Vladimir B Arion Journal: Organometallics Date: 2013-01-17 Impact factor: 3.876
Authors: Anatoly Dobrov; Vladimir B Arion; Norbert Kandler; Werner Ginzinger; Michael A Jakupec; Anna Rufińska; Nikolai Graf von Keyserlingk; Mathea Sophia Galanski; Christian Kowol; Bernhard K Keppler Journal: Inorg Chem Date: 2006-03-06 Impact factor: 5.165
Authors: Christopher Wittmann; Felix Bacher; Eva A Enyedy; Orsolya Dömötör; Gabriella Spengler; Christian Madejski; Jóhannes Reynisson; Vladimir B Arion Journal: J Med Chem Date: 2022-02-01 Impact factor: 7.446
Figure 1
Chart 1
Scheme 1
Figure 2
Figure 3
Figure 5