Effect of the piperazine unit and metal-binding site position on the solubility and anti-proliferative activity of ruthenium(II)- and osmium(II)-arene complexes of isomeric indolo[3,2-c ]quinoline - Piperazine hybrids

Article abstract of DOI:10.1021/ic500825j

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 [(η⁶-p-cymene)M(L^1-3)Cl]Cl, where L¹ = 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), L² = 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), L³ = 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.

Full text of DOI:10.1021/ic500825j

Article
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Effect of the Piperazine Unit and Metal-Binding Site Position on the
Solubility and Anti-Proliferative Activity of Ruthenium(II)- and
Osmium(II)- Arene Complexes of Isomeric
Indolo[3,2‑c]quinolinePiperazine Hybrids
Lukas K. Filak,†,‡ Danuta S. Kalinowski,*,§ Theresa J. Bauer,† Des R. Richardson,*,§
and Vladimir B. Arion*,†
†Institute of Inorganic Chemistry, University of Vienna, Wahringer Strasse 42, A-1090 Vienna, Austria
̈
‡School of Chemical Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
§Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney,
New South Wales 2006, Australia
S
* Supporting Information
ABSTRACT: 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 L1−3 were
prepared in situ and isolated as six ruthenium and osmium
complexes [(η6-p-cymene)M(L1−3)Cl]Cl, where L1 = 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), L2 = 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), L3 = 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.
mitochondrial malate dehydrogenase.14,15 However, their
clinical use was hampered by their limited aqueous solubility.
INTRODUCTION
■
The search for more effective ruthenium complexes and
One way to potentially overcome this problem was the
organoruthenium compounds as chemotherapeutic agents
coordination of these organic compounds to metal ions.
with fewer side effects than currently clinically applied platinum
Complexes with gallium(III),16,17 ruthenium,18 copper(II),19 as
compounds continues to attract attention.1−8 In fact, the use of
well as organoruthenium(II) and organoosmium(II) com-
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
pounds have been synthesized, and due to their improved
aqueous solubility, their antiproliferative activity has been
investigated.20−24 In fact, interesting structure−activity relation-
ships 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
Received: April 8, 2014
© XXXX American Chemical Society
A
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Inorganic Chemistry
Article
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 indoloqui-
noline 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 indoloquino-
line 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 moi-
eties.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
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).
active compounds, namely, indolo[3,2-c]quinolines (Figure 1,
top right and bottom).
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 indoloquino-
line 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 counterparts23 (Figure 1, top right). However,
the complexes with indoloquinolines were approximately 1
order of magnitude more cytotoxic.23
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]+
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
Chart 1. Ruthenium- and Osmium-Arene Complexes with Indoloquinoline−Piperazine Hybrid Ligands Modified at Position 2,
4 or 8
B
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Article
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-2-κN-amine]-
chloridoosmium(II) Chloride, [1b]Cl. The compound was synthe-
sized by following the general synthetic procedure (A) starting from 2-
amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline (0.15 g, 0.45
mmol), [OsCl(μ-Cl)(η6-p-cymene)]2 (0.16 g, 0.20 mmol) and 2-
pyridinecarboxaldehyde (39 μL, 0.41 mmol) in dry ethanol (3 mL).
Yield: 178 mg, 52%. Anal. Calcd for C36H38Cl2N6Os·1.5H2O (Mr =
842.89), %: C, 51.30; H, 4.90; N, 9.97. Found, %: C, 51.43; H, 4.97; N,
9.87. Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥ 8.4
(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.
1
mg/mL. H NMR (500 MHz, DMSO-d6): 13.15 (s, 1H, H11), 9.62−
3
9.59 (m, 2H, H13 + H15), 8.73 (s, 1H, H1), 8.52 (d, 1H, J = 7.6 Hz,
EXPERIMENTAL SECTION
H18), 8.36−8.31 (m, 1H, H17), 8.05 (d, 1H, 3J = 8.8 Hz, H4), 8.01 (d,
1H, 3J = 8.1 Hz, H7), 7.97 (dd, 1H, 3J = 8.8 Hz, 4J = 2.4 Hz H3), 7.92−
7.88 (m, 1H, H16), 7.79 (d, 1H, 3J = 8.0 Hz, H10), 7.55−7.51 (m, 1H,
■
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
3
H9), 7.45−7.40 (m, 1H, H8), 6.48 (d, 1H, J = 5.8 Hz, Hcy2/Hcy2′),
3
3
6.11 (d, 1H, J = 5.8 Hz, Hcy1/Hcy1′), 5.92 (d, 1H, J = 5.8 Hz, Hcy2
/
Hcy2′), 5.84 (d, 1H, J = 5.8 Hz, Hcy1/Hcy1′), 3.58 (br s, 4H, CH2mp),
2.76 (s, 4H, CH2mp), 2.44−2.36 (m, 4H, Hcy3 + CH3mp), 2.33 (br s,
3H, Hcy5), 0.94−0.92 (m, 6H, Hcy4 + Hcy4′). 13C NMR (125 MHz,
DMSO-d6): 168.23 (CH, C13), 158.59 (Cq, C6), 156.49 (Cq, C13a),
156.42 (CH, C15), 147.52 (Cq, C2), 145.52 (Cq, C4a), 142.58 (Cq, C11a),
140.77 (CH, C17), 139.12 (Cq, C10a), 130.29 (CH, C16), 130.20 (CH,
C18), 129.30 (CH, C4), 125.52 (CH, C9), 124.54 (CH, C3), 122.20
(CH, C7), 121.47 (CH, C8), 121.44 (Cq, C6b), 115.72 (CH, C1), 115.63
(Cq, C11b), 112.51 (CH, C10), 107.01 (Cq, C6a), 98.55 (Cq, Ccy1a), 97.19
(Cq, Ccy2a), 79.66 (CH, Ccy2/Ccy2′), 79.24 (CH, Ccy2/Ccy2′), 76.40 (CH,
3
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.
C
cy1/Ccy1′), 75.75 (CH, Ccy1/Ccy1′), 55.12 (CH2, CH2mp), 49.49 (CH2,
CH2mp), 31.25 (CH, Ccy3), 22.60 (CH3, Ccy4/Ccy4′), 22.33 (CH3, Ccy4
/
Ccy4′), 18.89 (CH3, Ccy5) ppm, CH3 not observed.
mp
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-4-κN-amine]-
chloridoruthenium(II) Chloride, [2a]Cl. 4-Amino-6-(4-methylpi-
perazin-1-yl)-indolo[3,2-c]quinoline (0.14 g, 0.43 mmol) and [RuCl-
(μ-Cl)(η6-p-cymene)]2 (0.12 g, 0.19 mmol) were suspended in
deionized water (2.5 mL) in a Schlenk tube. 2-Pyridinecarboxaldehyde
(37 μL, 0.39 mmol) was added, and the resulting suspension was
stirred under an argon atmosphere at room temperature for 24 h. The
resulting precipitate was filtered off and dried in vacuo. Yield: 45 mg,
41%. Anal. Calcd for C36H38Cl2N6Ru·2.5H2O (Mr = 771.74), %: C,
56.03; H, 5.62; N, 10.89. Found, %: C, 56.12; H, 5.61; N, 10.87.
Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥ 6.4 mg/
mL. 1H NMR (500 MHz, DMSO-d6): 13.27 (s, 1H, H11), 9.70 (d, 1H,
3J = 5.5 Hz, H15), 9.34 (s, 1H, H13), 8.66 (d, 1H, 3J = 8.6 Hz, H1), 8.42
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-2-κN-amine]-
chloridoruthenium(II) Chloride, [1a]Cl. 2-Amino-6-(4-methyl-
piperazin-1-yl)-indolo[3,2-c]quinoline (50 mg, 0.15 mmol) and
[RuCl(μ-Cl)(η6-p-cymene)]2 (0.04 g, 0.07 mmol) were suspended
in deionized water (1 mL) in a Schlenk tube. 2-Pyridinecarbox-
aldehyde (13 μL, 0.14 mmol) was added, and the resulting suspension
was stirred under an argon atmosphere at room temperature for 24 h.
The resulting precipitate was filtered off and dried in vacuo at 45 °C.
Yield: 45 mg, 41%. Anal. Calcd for C36H38Cl2N6Ru·3.75H2O (Mr =
794.26), %: C, 54.44; H, 5.77; N, 10.58. Found, %: C, 54.35; H, 5.54;
N, 10.57. Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥
6.4 mg/mL. 1H NMR (500 MHz, deuterated dimethyl sulfoxide
(DMSO-d6)): 13.18 (s, 1H, H11), 9.66 (d, 1H, 3J = 5.4 Hz, H15), 9.20
(s, 1H, H13), 8.80 (s, 1H, H1), 8.41−8.34 (m, 2H, H17 + H18), 8.09−
3
3
(d, 1H, J = 7.5 Hz, H18), 8.40−8.36 (m, 1H, H17), 8.29 (d, 1H, J =
7.6 Hz, H3), 7.99 (d, 1H, J = 8.0 Hz, H7), 7.97−7.93 (m, 1H, H16),
3
7.81 (d, 1H, 3J = 8.1 Hz, H10), 7.76−7.71 (m, 1H, H2), 7.57−7.52 (m,
1H, H9), 7.45−7.41 (m, 1H, H8), 6.15 (d, 1H, 3J = 6.1 Hz, Hcy2), 5.74
3
(d, 1H, J = 6.1 Hz, Hcy1), 5.50−5.47 (m, 2H, Hcy1′ + Hcy2′), 3.55−
3.30 (br s, 4H, CH2mp, in part overlapped with H2O signal), 2.80−2.60
(br s, 4H, CH2mp), 2.50−2.42 (m, 1H, Hcy3), 2.45−2.25 (br s, 3H,
CH3mp), 2.21 (s, 3H, Hcy5), 0.98 (d, 3H, Hcy4), 0.93 (d, 3H, Hcy4′). 13
C
3
8.05 (m, 2H, H3 + H4), 8.01 (d, 1H, J = 8.0 Hz, H7), 7.96−7.92 (m,
NMR (125 MHz, DMSO-d6): 172.34 (CH, C13), 156.74 (CH, C15),
155.39 (Cq, C13a), 147.00 (Cq, C4), 142.72 (Cq, C11a), 140.78 (CH,
C17), 139.35 (Cq, C10a), 135.30 (Cq, C4a), 130.17 (CH, C18), 129.46
(CH, C16), 125.67 (CH, C9), 124.45 (CH, C3), 123.48 (CH, C1),
123.28 (CH, C2), 122.05 (CH, C7), 121.60 (CH, C8), 121.26 (Cq, C6b),
117.20 (Cq, C11b), 112.64 (CH, C10), 106.84 (Cq, C6a), 105.38 (Cq,
3
1H, H16), 7.80 (d, 1H, J = 8.0 Hz, H10), 7.55−7.51 (m, 1H, H9),
7.45−7.40 (m, 1H, H8), 6.20 (d, 1H, 3J = 6.2 Hz, Hcy2/Hcy2′), 5.90 (d,
1H, 3J = 6.2 Hz, Hcy1/Hcy1′), 5.72 (d, 1H, 3J = 6.2 Hz, Hcy2/Hcy2′), 5.69
(d, 1H, J = 6.2 Hz, Hcy1/Hcy1′), 3.60 (br s, 4H, CH2mp), 2.81 (br s,
3
4H, CH2mp), ∼2.50 (m, 1H, Hcy3, overlapped with DMSO signal), 2.42
(br s, 3H, CH3mp), 2.25 (s, 3H, Hcy5), 1.01−0.96 (m, 6H, Hcy4). 13C
NMR (125 MHz, DMSO-d6): 167.40 (CH, C13), 158.46 (Cq, C6),
156.73 (CH, C15), 155.03 (Cq, C13a), 147.53 (Cq, C2), 145.39 (Cq, C4a),
142.64 (Cq, C11a), 140.62 (CH, C17), 139.14 (Cq, C10a), 130.39 (CH,
C18), 129.45 (CH, C16), 129.32 (CH, C4), 125.53 (CH, C9), 124.16
(CH, C3), 122.19 (CH, C7), 121.46 (CH, C8), 121.44 (Cq, C6b), 115.71
(Cq, C11b), 115.20 (CH, C1), 112.52 (CH, C10), 107.02 (Cq, C6a),
105.42 (Cq, Ccy2a), 104.56 (Cq, Ccy1a), 87.43 (CH, Ccy2/Ccy2′), 87.10
(CH, Ccy2/Ccy2′), 85.74 (CH, Ccy1/Ccy1′), 85.25 (CH, Ccy1/Ccy1′), 55.0
(CH2, CH2mp), 49.3 (CH2, CH2mp), 46.2 (CH3, CH3mp), 31.01 (CH,
C
cy2a), 104.5 (Cq, Ccy1a), 87.10 (CH, Ccy2), 86.4 (CH, Ccy2′), 85.7 (CH,
Ccy1′), 85.01 (CH, Ccy1), 48.9 (CH2, CH2mp), 30.99 (CH, CH2cy3),
22.42 (CH3, Ccy4/Ccy4′), 22.35 (CH3, Ccy4/Ccy4′), 19.10 (CH3, Ccy5
)
mp
mp
ppm, C6, one CH2 and CH3 not observed.
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-4-κN-amine]-
chloridoosmium(II) Chloride, [2b]Cl. The compound was synthe-
sized by following the general synthetic procedure (A) starting from 4-
amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline (0.15 g, 0.45
mmol), [OsCl(μ-Cl)(η6-p-cymene)]2 (0.16 g, 0.20 mmol), and 2-
pyridinecarboxaldehyde (39 μL, 0.41 mmol) in dry ethanol (3 mL).
Yield: 156 mg, 45%. Anal. Calcd for C36H38Cl2N6Os·1.75H2O (Mr =
C
C
cy3), 22.24 (CH3, Ccy4/Ccy4′), 22.16 (CH3, Ccy4/Ccy4′), 18.93 (CH3,
cy5) ppm.
C
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Inorganic Chemistry
Article
Table 1. Crystal Data and Details of Data Collection for [1aH]Cl2·EtOH·2H2O, [2aH]Cl2·1.5iPrOH·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
fw
C38H49Cl3N6O3Ru
845.25
C40.5H52Cl3N6O2Ru
862.30
C37H43Cl2N6O1.5Ru
767.74
space group
α, Å
P21/n
P21/c
Pc
19.2712(14)
7.8687(5)
25.5459(19)
91.724(4)
3872.0(5)
4
15.2896(5)
19.5424(6)
15.3917(5)
113.677(2)
4211.8(2)
4
10.0291(4)
9.4460(4)
19.2958(7)
93.479(2)
1824.62(13)
2
b, Å
c, Å
β, deg
V [Å3]
Z
λ [Å]
0.710 73
1.450
0.710 73
1.360
0.710 73
1.397
ρcalcd, g cm−3
cryst size, mm3
T [K]
0.12 × 0.08 × 0.02
150(2)
0.25 × 0.13 × 0.03
100(2)
0.15 × 0.10 × 0.04
100(2)
μ, mm−1
0.657
0.604
0.615
a
R1
0.0519
0.0480
0.0362
b
wR2
0.1137
0.1422
0.0922
c
GOF
1.084
1.058
1.036
a
b
c
2
R1 = Σ||F0| − |Fc||/Σ|F0|. wR2 = {Σ[w(F02 − Fc2)2]/Σ[w(F0 )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.
847.39), %: C, 51.03; H, 4.94; N, 9.92. Found, %: C, 50.98; H, 4.94; N,
9.88. Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥ 7.0
mg/mL. 1H NMR (500 MHz, DMSO-d6): 13.23 (s, 1H, H11), 9.76 (s,
1H, H13), 9.64 (d, 1H, 3J = 5.6 Hz, H15), 8.64 (d, 1H, 3J = 7.4 Hz, H1),
8.56 (d, 1H, 3J = 7.6 Hz, H18), 8.38−8.33 (m, 1H, H17), 8.15 (d, 1H, 3J
= 7.5 Hz, H3), 7.99 (d, 1H, 3J = 8.0 Hz, H7), 7.93−7.89 (m, 1H, H16),
7.81 (d, 1H, 3J = 8.1 Hz, H10), 7.75−7.70 (m, 1H, H2), 7.58−7.52 (m,
1H, H9), 7.45−7.41 (m, 1H, H8), 6.45 (d, 1H, 3J = 5.6 Hz, Hcy2), 5.95
(d, 1H, 3J = 5.6 Hz, Hcy1), 5.69 (d, 1H, 3J = 5.6 Hz, Hcy2′), 5.64 (d, 1H,
3J = 5.6 Hz, Hcy1′), 3.60−3.20 (br s, 4H, CH2mp, in part overlapped
with H2O signal), 2.80−2.40 (br s, 4H, CH2mp, in part overlapped with
DMSO signal), 2.38−2.20 (m, 7H, Hcy3 + CH3mp + Hcy5), 0.91 (d, 3H,
NMR (125 MHz, DMSO-d6): 167.25 (CH, C13), 156.51 (CH, C15),
155.31 (Cq, C13a), 146.40 (Cq, C8), 145.41 (Cq, C4a), 144.21 (Cq, C11a),
140.47 (CH, C17), 139.39 (Cq, C10a), 130.16 (CH, C18), 129.38 (CH,
C3), 129.15 (CH, C16), 128.26 (CH, C4), 124.20 (CH, C2), 122.49
(CH, C1), 121.72 (Cq, C6b), 120.14 (CH, C9), 115.97 (Cq, C11b),
115.58 (CH, C7), 112.86 (CH, C10), 106.68 (Cq, C6a), 104.86 (2 Cq,
Ccy1a + Ccy2a), 87.58 (CH, Ccy2), 87.11 (CH, Ccy2′), 85.19 (CH, Ccy1),
84.99 (CH, Ccy1′), 54.8 (CH2, CH2mp), 49.8 (CH2, CH2mp), 46.0 (CH3,
CH3mp), 30.98 (CH, Ccy3), 22.37 (CH3, Ccy4), 21.93 (CH3, Ccy4′),
19.04 (CH3, Ccy5) ppm, C6 not observed.
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-8-κN-amine]-
chloridoosmium(II) Chloride, [3b]Cl. The compound was synthe-
sized by following the general synthetic procedure (A) starting from 8-
amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline (0.15 g, 0.45
mmol), [OsCl(μ-Cl)(η6-p-cymene)]2 (0.16 g, 0.20 mmol), and 2-
pyridinecarboxaldehyde (39 μL, 0.41 mmol) in dry ethanol (3 mL).
Yield: 204 mg, 58%. Anal. Calcd for C36H38Cl2N6Os·2.25H2O (Mr =
856.40), %: C, 50.49; H, 5.00; N, 9.81. Found, %: C, 50.49; H, 4.93; N,
9.99. Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥ 6.8
mg/mL. 1H NMR (500 MHz, DMSO-d6): 13.44 (s, 1H, H11), 9.57 (d,
1H, 3J = 5.6 Hz, H15), 9.51 (s, 1H, H13), 8.54 (d, 1H, 3J = 7.9 Hz, H1),
3
3J = 6.7 Hz, Hcy4), 0.87 (d, 3H, J = 6.7 Hz, Hcy4′). 13C NMR (125
MHz, DMSO-d6): 172.90 (CH, C13), 156.87 (Cq, C13a), 156.40 (CH,
C15), 147.01 (Cq, C4), 142.69 (Cq, C11a), 140.93 (CH, C17), 139.35 (Cq,
C
10a), 135.34 (Cq, C4a), 130.32 (CH, C16), 130.01 (CH, C18), 125.69
(CH, C9), 124.86 (CH, C3), 123.67 (CH, C1), 123.11 (CH, C2), 122.05
(CH, C7), 121.62 (CH, C8), 121.26 (Cq, C6b), 117.16 (Cq, C11b),
112.64 (CH, C10), 106.86 (Cq, C6a), 98.4 (Cq, Ccy1a), 97.25 (Cq, Ccy2a),
79.1 (CH, Ccy2), 78.4 (CH, Ccy2′), 76.04 (CH, Ccy1′), 75.3 (CH, Ccy1),
55.2 (CH2, CH2mp), 49.0 (CH2, CH2mp), 45.0 (CH3, CH3mp), 31.26
(CH, Ccy3), 22.85 (CH3, Ccy4), 22.52 (CH3, Ccy4′), 19.07 (CH3, Ccy5
ppm, C6 not observed.
)
3
8.48 (d, 1H, J = 7.7 Hz, H18), 8.34−8.29 (m, 1H, H17), 8.21 (s, 1H,
H7), 7.94−7.89 (m, 2H, H10 + H4), 7.89−7.83 (m, 2H, H9 + H16),
7.72−7.67 (m, 1H, H3), 7.56−7.53 (m, 1H, H2), 6.44 (d, 1H, 3J = 5.7
(η6-p-Cymene)[6-(4-methylpiperazin-1-yl)-κN-(pyridin-2-yl-
methylene)-11H-indolo[3,2-c]quinolin-8-κN-amine]-
chloridoruthenium(II) Chloride, [3a]Cl. The compound was
synthesized by following the general synthetic procedure (A) starting
from 8-amino-6-(4-methylpiperazin-1-yl)-indolo[3,2-c]quinoline (0.15
g, 0.45 mmol), [RuCl(μ-Cl)(η6-p-cymene)]2 (0.13 g, 0.20 mmol), and
2-pyridinecarboxaldehyde (39 μL, 0.41 mmol) in dry ethanol (3 mL).
Yield: 189 mg, 60%. Anal. Calcd for C36H38Cl2N6Ru·2.5H2O (Mr =
771.74), %: C, 56.03; H, 5.62; N, 10.89. Found, %: C, 56.09; H, 5.49;
N, 11.04. Solubility in sodium phosphate buffer (20 mM, pH 7.40): ≥
5.6 mg/mL. 1H NMR (500 MHz, DMSO-d6): 13.37 (s, 1H, H11), 9.62
(d, 1H, 3J = 5.6 Hz, H15), 9.10 (s, 1H, H13), 8.52 (d, 1H, 3J = 7.9 Hz,
H1), 8.37−8.34 (m, 2H, H17 + H18), 8.29 (s, 1H, H7), 7.95−7.89 (m,
4H, H4 + H9 + H10 + H16), 7.71−7.67 (m, 1H, H3), 7.57−7.53 (m, 1H,
3
3
Hz, Hcy2), 6.03 (d, 1H, J = 5.7 Hz, Hcy1), 5.81 (d, 1H, J = 5.6 Hz,
Hcy2′), 5.60 (d, 1H, 3J = 5.6 Hz, Hcy1′), 3.70 (br s, 2H, CH2mp), 3.48 (s,
2H, CH2mp2), 2.85 (br s, 2H, CH2mp), 2.64 (br s, 2H, CH2mp), 2.44−
2.25 (m, 7H, Hcy3 + Hcy5 + CH3mp), 0.94 (d, 3H, J = 6.9 Hz, Hcy4),
3
0.91 (d, 3H, J = 6.9 Hz, Hcy4′). 13C NMR (125 MHz, DMSO-d6):
3
168.05 (CH, C13), 156.74 (Cq, C13a), 156.20 (CH, C15), 146.36 (Cq,
C8), 145.38 (Cq, C4a), 144.24 (Cq, C11a), 140.63 (CH, C17), 139.49 (Cq,
C
10a), 130.04 (CH, C16 or C18), 129.98 (CH, C16 or C18), 129.40 (CH,
C3), 128.26 (CH, C4), 124.24 (CH, C2), 122.54 (CH, C1), 121.63 (Cq,
C6b), 120.46 (CH, C9), 116.05 (CH, C7), 115.99 (Cq, C11b), 112.86
(CH, C10), 106.64 (Cq, C6a), 98.79 (Cq, Ccy1a), 96.63 (Cq, Ccy2a), 79.66
(CH, Ccy2), 79.10 (CH, Ccy2′), 75.67 (CH, Ccy1), 75.45 (CH, Ccy1′),
54.8 (CH2, CH2mp), 49.5 (CH2, CH2mp), 46.0 (CH3, CH3mp), 31.25
3
3
H2), 6.15 (d, 1H, J = 6.2 Hz, Hcy2), 5.81 (d, 1H, J = 6.2 Hz, Hcy1),
(Cq, Ccy3), 22.57 (CH3, Ccy4), 22.27 (CH3, Ccy4′), 18.99 (CH3, Ccy5
ppm, C6 not observed.
)
5.62 (d, 1H, 3J = 6.1 Hz, Hcy2′), 5.44 (d, 1H, J = 6.1 Hz, Hcy1′), 3.71
3
Physical Measurements. 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
(br s, 2H, CH2mp), 3.49 (br s, 2H, CH2mp), 2.86 (br s, 4H, CH2mp),
2.64 (br s, 2H, CH2mp), 2.56−2.48 (m, 1H, Hcy3, overlapped in part
with DMSO signal), 2.43−2.27 (br s, 3H, CH3mp), 2.26 (s, 3H, Hcy5),
1.00 (d, 3H, J = 6.9 Hz, Hcy4), 0.97 (d, 3H, J = 6.9 Hz, Hcy4′). 13C
3
3
D
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a
Scheme 1. Synthetic Pathways to Prepare the Isomeric Indoloquinoline-Piperazine Hybrids 7a, 7b, and 7c
a
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.
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.
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium (MTT) assay by stand-
ard 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.
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]Cl2·1.5iPrOH·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
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-
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-pyridinecarbox-
aldehyde.
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
E
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osmium complexes [1a,b]Cl−[3a,b]Cl were formed in a single
reaction mixture in situ. This suggests that the indoloquino-
line−piperazine hybrids were stabilized through their coordi-
nation 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.
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
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
adaption29 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 salt46 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. Micro-
crystalline 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.
1
detect, as they do not display in standard 2D H−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
mp
singlets were assigned as CH2
.
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
F
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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 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,
Figure 4. ORTEP view of the cation [(η6-p-cymene)Ru(HL2)Cl]2+ in
[2aH]Cl2·1.5iPrOH·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).
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.
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
N,N-bound ligand remained coordinated to the metal center,
showing no signs of ligand cleavage.
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·
1.5iPrOH·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]Cl2·1.5iPrOH·0.5H2O, and
[2a]Cl·0.5EtOH·H2O crystallized in the monoclinic centro-
symmetric 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 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
G
dx.doi.org/10.1021/ic500825j | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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).
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.
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 counter-
clockwise 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).
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 indoloquino-
line−piperazine conjugates. Those complexes containing the
metal-binding site located at the 4 position of the indoloquino-
line 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.
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
vitro53 and in vivo;62 and (3) doxorubicin (DOX), which is a
clinically used cytotoxic agent.63,64
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.
As previously observed,53 the control chelator Dp44mT
demonstrated the most potent antiproliferative effects of all the
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.
agents examined (IC50: 0.004
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.
0.003 μM; Table 2). In
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
a
compound
IC50 (μM)
DFO
9.83 0.39
0.004 0.003
0.15 0.05
39.81 2.07
>50
Dp44mT
DOX
[1a]Cl
[1b]Cl
[2a]Cl
[2b]Cl
[3a]Cl
[3b]Cl
18.41 2.22
19.40 1.28
>50
ASSOCIATED CONTENT
* Supporting Information
Synthesis of 7-nitroisatin, 2-amino-6-(4-methylpiperazin-1-yl)-
■
41.24 2.37
S
a
Assays conducted for 72 h. Results are mean
experiments).
SD (three
indolo[3,2-c]quinoline, 4-nitroindolo[3,2-c]quinolin-6-one, 6-
H
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Inorganic Chemistry
Article
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geometries and crystallographic data in CIF format. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
(18) Schmid, W. F.; Zorbas-Seifried, S.; John, R. O.; Arion, V. B.;
Jakupec, M. A.; Roller, A.; Galanski, M.; Chiorescu, I.; Zorbas, H.;
Keppler, B. K. Inorg. Chem. 2007, 46, 3645−3656.
■
Corresponding Authors
*E-mail: vladimir.arion@univie.ac.at. (V.B.A.)
*E-mail: danutak@med.usyd.edu.au. (D.S.K.)
*E-mail: d.richardson@med.usyd.edu.au. (D.R.R.)
(19) Primik, M. F.; Muhlgassner, G.; Jakupec, M. A.; Zava, O.;
̈
Dyson, P. J.; Arion, V. B.; Keppler, B. K. Inorg. Chem. 2010, 49, 302−
311.
(20) Schmid, W. F.; John, R. O.; Muhlgassner, G.; Heffeter, P.;
̈
Notes
Jakupec, M. A.; Galanski, M.; Berger, W.; Arion, V. B.; Keppler, B. K. J.
Med. Chem. 2007, 50, 6343−6355.
The authors declare no competing financial interest.
(21) Schmid, W. F.; John, R. O.; Arion, V. B.; Jakupec, M. A.;
Keppler, B. K. Organometallics 2007, 26, 6643−6652.
ACKNOWLEDGMENTS
■
(22) Muhlgassner, G.; Bartel, C.; Schmid, W. F.; Jakupec, M. A.;
̈
We thank Prof. Dr. M. Galanski for recording the 2D NMR
spectra of complexes [1a,b]Cl−[3a,b]Cl, A. Dobrov and Dr. N.
Lloyd for ESI mass spectra measurements, and A. Roller for
collection of the X-ray data. We are also indebted to the
Austrian Science Fund (FWF) for financial support of Project
No. P22339−N19. D.R.R. thanks the National Health and
Medical Research Council of Australia for a Senior Principal
Research Fellowship (1062607) and Project Grants (1060482
and 1021607). D.S.K. acknowledges the NHMRC for Project
Grant 1048972 and also the Sydney Medical School
Foundation of The University of Sydney for a Helen and
Robert Ellis Fellowship.
Arion, V. B.; Keppler, B. K. J. Inorg. Biochem. 2012, 116, 180−187.
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