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

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.


■ INTRODUCTION
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 cyclindependent 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 sixmembered pyridine ring has led to another class of biologically active compounds, namely, indolo [3,2- 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 sp 3 -hybridized nitrogen donor atoms with sp 2 -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 NH 2 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 metalfree 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 [(η 6 -p-cymene)M(L 1−3 )Cl] + Figure 1. Original paullone, also named indolo [3,2-d]benzazepin-6one (top left), 11 modified paullone (top middle), 23  (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 osmiumarene complexes with indoloquinoline−piperazine hybrids and can be utilized further for the design and development of more effective chemotherapeutic agents.
(   Physical Measurements. One-dimensional (1D) 1 H and 13 C NMR and two-dimensional (2D) 1 where n is the number of reflections and p is the total number of parameters refined. spectrometers at 500.32 or 500.10 ( 1 H), and 125.82 or 125.76 ( 13 C) MHz, respectively, by using DMSO-d 6 as a solvent at room temperature and standard pulse programs. 1 H and 13 C 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 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. 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 4,4dimethyl-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 (IC 50 ) 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. 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 2nitroindolo[3,2-c]quinoline-6-one 4a was chlorinated using POCl 3 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[3,2c]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 H 2 and Pd/C as shown in Scheme 1.

■ RESULTS AND DISCUSSION
Further condensation reactions of the indoloquinoline− piperazine hybrids 7a−7c with 2-pyridinecarboxaldehyde in the presence of [RuCl(μ-Cl)(η 6 -p-cymene)] 2 or [OsCl(μ-Cl)(η 6 -pcymene)] 2  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 Na 2 SO 4 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)(η 6 -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 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 C 6a (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 C 6 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 1 H− 13 C 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 13 C NMR experiments. However, the shift of approximately 158.5 ppm for C 6 in the 13 C 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 CH 2 resonances present as broad singlets were assigned as CH 2 mp .  (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 NaH 2 PO 4 / Na 2 HPO 4 buffer at pH 7.40). As can be seen from Supporting Information, Figure S4,  O crystallized in the monoclinic centrosymmetric space groups P2 1 /n, P2 1 /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.

Stability in DMSO, Solubility in
All three complexes have a typical "three-leg piano-stool" geometry of ruthenium(II)-arene complexes, with a η 6 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···Cl2 ii in [1aH]Cl 2 and N15−H···Cl3 ii and N15−H··· Cl3 iii in [2aH]Cl 2 . 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 L 2 in the cation [(η 6 -p-cymene)Ru(L 2 )Cl] + and the protonated ligand (HL 2 ) + in [(η 6 -p-cymene)Ru(HL 2 )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 Θ C4a−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°a round p-cymene ring centroid-Ru vector in [(η 6 -p-cymene)-Ru(L 2 )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 wellcharacterized. 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 (IC 50 : 0.004 ± 0.003 μM; Table 2). In contrast, the other positive controls, DOX (IC 50 : 0.15 ± 0.05 μM; Table 2) and DFO (IC 50 : 9.83 ± 0.39 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 metalbinding 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.