A SAR study of novel antiproliferative ruthenium and osmium complexes with quinoxalinone ligands in human cancer cell lines

Article

pubs.acs.org/jmc

A SAR Study of Novel Antiproliferative Ruthenium and Osmium

Complexes with Quinoxalinone Ligands in Human Cancer Cell Lines

Werner Ginzinger,^†Gerhard Muhlgassner,^†Vladimir B. Arion,*^,†Michael A. Jakupec,^†Alexander Roller,^†

̈

Markus Galanski,^†Michael Reithofer,^†Walter Berger,^‡and Bernhard K. Keppler*^,†

^†University of Vienna, Institute of Inorganic Chemistry, Wahringer Strasse 42, A-1090 Vienna, Austria

̈

^‡Medical University of Vienna, Department of Medicine I, Institute of Cancer Research, Borschkeg. 8a, A-1090 Vienna, Austria

S

* Supporting Information

ABSTRACT: A series of ruthenium(II) arene complexes with

3-(1H-benzimidazol-2-yl)-1H-quinoxalin-2-one, bearing phar-

macophoric groups of known protein kinase inhibitors, and

related benzoxazole and benzothiazole derivatives have been

synthesized. In addition, the corresponding osmium complexes

of the unsubstituted ligands have also been prepared. The

compounds have been characterized by NMR, UV−vis, and IR

spectroscopy, ESI mass spectrometry, elemental analysis, and

by X-ray crystallography. Antiproliferative activity in three

human cancer cell lines (A549, CH1, SW480) was determined

by MTT assays, yielding IC₅₀values of 6−60 μM for three

unsubstituted metal-free ligands, whereas values for the metal

complexes vary in a broad range from 0.3 to 140 μM.

Complexation with osmium of quinoxalinone derivatives with benzimidazole or benzothiazole results in a more consistent

increase in cytotoxicity than complexation with ruthenium. For selected compounds, the capacity to induce apoptosis was

confirmed by fluorescence microscopy and flow-cytometric analysis, whereas cell cycle effects are only moderate.

intervene therapeutically with kinase inhibitors to block either

the kinase−substrate interaction or the ATP binding site.^18,19

Recently, Meggers and co-workers developed a new class of

metal-based kinase inhibitors inspired by the ATP competitive

staurosporine molecule (Figure 1).²⁰⁻²³The metal within these

rather inert complexes serves as a scaffold for shaping the

molecule. Some new inhibitors show high selectivity and

overcome resistance by irreversible covalent binding to the

target.²⁴In this context, coordination might provide a new

mode of action so that keeping at least one moderately reactive

site could be advantageous in some cases.

In China, chronic myelocytic leukemia has commonly been

treated with the traditional recipe Danggui Longhui Wan, a

mixture of 11 herbal medicines. The ingredient Indigo naturalis

was suggested to be responsible for the antileukemic effects.

Finally, the activity was attributed to a minor constituent of the

blue powder, namely the red-colored isomer of indigo,

indirubin (Figure 2a), which was found to inhibit cyclin

dependent kinases.^25,26In the meantime not only a large

number of derivatives has been synthesized, but also other

targets have been identified.²⁷⁻³²The crystal structure of Thr-

160 phospho-CDK2-cyclin A with indirubin-5-sulfonate

revealed that the inhibitor binds to the ATP binding site of

the protein through the lactam moiety and the N¹′−H group by

INTRODUCTION

■

Since the approval of cisplatin for the treatment of cancer, a

number of other metal complexes have been investigated for

their therapeutic potential.¹⁻³Among them, ruthenium

compounds have received respectable attention because of

interesting features such as iron mimicking behavior or the

availability of several oxidation states under physiological

conditions.⁴Two ruthenium compounds, [ImH] trans-

[RuCl₄(Im)(dmso-S)] (NAMI-A, Im = imidazole) and

[IndH] trans-[RuCl₄(Ind)₂)] (KP1019, Ind = 1H-indazole),

have successfully completed phase I clinical trials.⁵⁻⁷NAMI-A

has already entered a phase II study.⁸Recently, half-sandwich

ruthenium(II) and osmium(II) arene compounds were

discovered as a new class of potential anticancer drugs.^9,10

The arene ligand stabilizes the oxidation state +II at the metal

center. Furthermore, the metal−arene moiety is believed to

facilitate uptake by diffusion through the cell membrane. In

some cases, the arene ligand may also play a crucial role in the

interaction with intracellular targets (e.g., intercalation with

DNA).¹¹The remaining three coordination sites are occupied

by monodentate or chelating ligands, which control the

reactivity toward biomolecules and thereby the mode of action

of the drug.¹²⁻¹⁶

Protein kinases are involved in the regulation of most cellular

processes, such as metabolism, proliferation, damage repair, and

apoptosis. Perturbations of these signaling pathways are quite

common in cancer cells.¹⁷Therefore efforts have been made to

Received: January 20, 2012

Published: March 15, 2012

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RESULTS AND DISCUSSION

■

Syntheses of Ligands and Complexes. Because the

reported pathways to the ligands studied in this work turned

out to be not very reliable in terms of reproducibility and yields,

new synthetic strategies were developed. 3-(1H-Benzimidazol-

2-yl)-1H-quinoxalin-2-one, its derivatives, and oxazole ana-

logues were synthesized in four steps. By reaction of ethyl

2‑(ethoxycarbonyl)acetimidate hydrochloride (1) with suitable

1,2-phenylenediamines or 2-aminophenols cyclization products,

2a−f have been obtained. Bromination of the α-methylene

group in acetic acid resulted in 3a−f. This reaction requires a

high quality of acetic acid and sodium acetate. The dibromo

derivatives are quite unstable and could not be purified. The

dimethyl compound 2c, being a stronger base than the other

intermediates, partly formed a salt with acetic acid, which may

account for higher contaminations of 3c. The following

cyclization with suitable 1,2-phenylenediamines led to the

ligands 4a−k (Scheme 1a). Alternatively, 4a was synthesized by

condensation of ethyl 3-oxo-3,4-dihydro-quinoxaline-2-carbox-

ylate and 1,2-phenylenediamine in ethylene glycol under argon

at 170 °C. However, this pathway could only be applied

successfully to the unsubstituted compound, although with a

much lower yield. The corresponding thiazole analogues were

synthesized by another approach because bromination of ethyl

2-(benzothiazol-2-yl)acetate led to prevalent formation of side

products. First, diethyl sodium oxalacetate was condensed with

1,2-phenylenediamines to give 5a−b. Then ester hydrolysis and

nitrosation followed by decarboxylation⁴¹afforded 6a and 6b

(Scheme 1b). Finally, the oximes were converted into the

corresponding benzothiazoles 4l−o using 2-mercaptoanilines.

Figure 1. Straurosporine (a) and new metal-based kinase inhibitors

developed by Meggers et al.²⁰⁻²³(b,c).

formation of three hydrogen bonds.³³A similar pharmaco-

phoric group, which is a basic structural element of promising

tyrosine kinase inhibitors, is present in 4-amino-3-(1H-

benzimidazol-2-yl)-1H-quinolin-2-ones (Figure 2b)³⁴⁻³⁶and

in 3-(3-amino-1Hg f-indol-2-yl)-1H-quinoxalin-2-ones (Figure

2c).^37,38

Combining the structures of the benzimidazolylquinolinone

and the indolylquinoxalinone in Figure 2 to the motif of 3-(1H-

benzimidazol-2-yl)-1H-quinoxalin-2-one results in a compound

that incorporates the indirubin pharmacophore together with a

vicinal diimine, which should be well suited for coordination to

metal ions. Using Krohnke intermediates, this compound type

̈

has already been synthesized by Westphal et al.^39,40Besides the

benzimidazole derivative, they prepared the corresponding

benzoxazole and benzothiazole analogues in the same way.

Herein we present new synthetic approaches for the family of

3-(benzazol-2-yl)-1H-quinoxalin-2-ones and the first evaluation

of their antiproliferative potency. The benzimidazole derivative

was compared with the corresponding azoles containing oxygen

and sulfur. In addition, chloro and methyl substituents with

inverse inductive effects on the electron density of the aromatic

system were introduced at the benzene moieties in order to

explore some structure−activity relationships. However, the

solubility of the heterocyclic substances is extremely poor.

Coordination to an organometallic ruthenium(II) and osmium

(II) arene scaffold should not only diminish this problem but

may also help to enhance anticancer activity. To elucidate the

influence of the metal, the structures of corresponding

ruthenium and osmium complexes were examined by X-ray

diffraction and compared with the metal-free ligands. Moreover,

the impact of the metal on the reactivity of the monodentate

chlorido ligand was studied by monitoring aquation and

backward anation. Structure−activity relationships were

elucidated by means of the MTT assay in three human cancer

cell lines, and effects of selected compounds on the cell cycle

were investigated by flow cytometry. Furthermore, induction of

apoptosis was proved by fluorescence microscopy.

1

The H NMR spectrum of the crude product 4l displayed a

1:1:1 triplet of ammonium, which indicated that the expected

intermediate 2,3-dihydrobenzothiazole is oxidized to benzo-

thiazole by the initially released hydroxylamine. Only in the

case of 4o oxygen atmosphere for completion of the oxidation

reaction was necessary.

The low solubility of the ligands in suitable organic solvents

precluded the use of chromatographic methods or straightfor-

ward recrystallization procedures for their further purification.

4a was dissolved in acetic acid and reprecipitated by addition of

diethylether, followed by dissolution in orthophosphoric acid

and reprecipitation with NaOH. To remove inorganic salts, the

suspended compound was boiled in water. It should be noted

that hydrochlorides of all benzimidazole derivatives are rather

poorly soluble too. 4h was recrystallized from ethylene glycol.

However, to remove traces of this solvent, treatment in boiling

methanol was necessary. 4l was dissolved in a mixture of

ethanol and THF and precipitated by pouring the solution into

a huge amount of water. Afterward, the compound was

recrystallized from o-xylene. Finally, 4l was dissolved in THF

and precipitated with water. Because the compound possesses

poor solubilities, all steps required large volumes of solvents.

Figure 2. ATP competitive protein kinase inhibitors with the same pharmacophore: (a) indirubin, (b) 4-amino-3-(1H-benzimidazol-2-yl)-1H-

quinolin-2-one, (c) 3-(3-amino-1H-indol-2-yl)-1H-quinoxalin-2-one.

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a

Scheme 1. Novel Synthetic Pathways to Ligands 4a−k (a) and 4l−o (b)

a

Reagents and conditions: (i) EtOH/H₂O, SOCl₂, Ar, 0 °C−rt. (ii) MeOH_abs, Ar, Δ. (iii) AcOH, NaOAc, Br₂, Ar, rt. (iv) EtOH_abs, NaOAc, Ar, Δ.

(v) AcOH. (vi) NaOH, Δ, NaNO₂, H₂SO₄. (vii) EtOH, HCl. Compounds with underlined numbers were characterized by X-ray diffraction.

Therefore all ligands with exception of the three biologically

tested derivatives were used for complexation without extensive

purification.

with the exception of 8b and 8d. [RuCl(arene)L − HCl]⁺and

[RuCl(arene)L − H + Na]⁺ions are also abundant (Scheme 2).

For osmium-containing complexes, similar mass spectra have

been obtained. Peaks with higher m/z values were also

The quinoxalinone ligands were reacted with 0.5 equiv of the

dimeric bis[chlorido(μ-chlorido)(η⁶-p-cymene)ruthenium(II)]

(7a) or bis[chlorido(μ-chlorido)(η⁶-p-cymene)osmium(II)]

(7b) in absolute ethanol at 70 °C. Normally, the presence of

small amounts of concentrated hydrochloric acid improved the

quality and the yield of the products. Some complexes were

isolated by filtration from the cooled reaction mixture, while the

others were precipitated by addition of diethyl ether or other

solvents. For purification, the organometallic compounds were

dissolved in highly diluted hydrochloric acid, or in some cases

just in water, filtered, and the filtrates immediately frozen and

lyophilized afterward. The ruthenium η⁶-benzene complex 10a

(an analogue of 8a) was prepared similarly to the p-cymene

derivatives.

a

Scheme 2. Synthesis of Complexes 8a−o, 9a, 9h, and 9l

Characterization of Compounds. Positive ion ESI mass

spectra of the ligands 4a, 4h, and 4l displayed strong peaks of

the ions [M + Na]⁺and [2M + Na]⁺. While the spectrum of 4a

contains a peak with m/z 263 attributed to [M + H]⁺, the

formation of such ions was not observed for 4h and 4l.

However, the mass spectra of the latter measured in the

negative ion mode showed peaks which have been assigned to

the ions [M − H]⁻and [2M − 2H + Na]⁻. The peak attributed

to [RuCl(arene)L]⁺was found for all ruthenium complexes

a

Reagents and conditions: EtOH_abs, Ar, 70 °C. Compounds with

underlined numbers were characterized by X-ray diffraction.

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registered but could not be unequivocally identified. UV−vis

spectra of metal-free and metal-based compounds in methanol

(Figures S1−S4, Supporting Information) display several

structured bands, which were characterized by their first

derivatives. NMR spectra were recorded in DMSO-d₆with

exception of complexes with oxazole derivatives and 10a (with

η⁶-bound benzene), which decompose in this medium very fast.

Therefore, in those cases, MeOH-d₄was used as a solvent.

Sometimes even for complexes with benzimidazole and

benzothiazole derivatives a very slow dissociation in DMSO-

d₆with liberation of the metal-free quinoxalinone ligand and

formation of [RuCl₂(p-cymene)(dmso)] was observed. Fre-

quently, directly after dissolution the signals in ¹H NMR

spectra appeared as quite broad lines and their resolution into

multiplets was found to be time-dependent. Consequently, the

¹H NMR spectra were usually recorded once more after all

other experiments. ¹H,¹H-ROESY spectra facilitated the

assignment of resonances in the case of metal-based

compounds by showing NOE interactions between p-cymene

and quinoxalinone ligands. These were particularly helpful for

discerning C⁵and C⁸or C⁴′ and C⁷′ because cross-peaks for

long-range coupling between N¹H and C⁸or C^4awere not

Figure 3. Time-dependent changes of NMR spectra of 8a in D₂O.

similarly. The batches were still slightly contaminated but used

for investigations of the reactions with chloride and the pH

dependent behavior without further purification. Their ¹H

resonances agree well with those for the species generated upon

aquation of the corresponding chlorido complexes.

The reaction of 11a with sodium chloride in D₂O monitored

by integration of the doublets at 8.56 and 8.47 ppm

demonstrated the reversibility of the aquation. The hydrolysis

of the compounds occurred markedly slower than that of

recently reported maltol-derived organometallic complexes,

where a complete aquation within seconds was observed.⁴³This

is in agreement with earlier observations, that ruthenium arene

complexes containing N-donor ligands normally display a

slower hydrolysis, than those with O- and/or S-donor

atoms.^44,45The aquation of the osmium analogue 9a and the

reverse anation reaction of the aqua complex 12a with sodium

chloride are even slower, in accord with results published

elsewhere.⁴⁶

The quality of the data did not allow a reliable calculation of

the rate constants because fitting with a model including

pseudo-first-order kinetics for aquation and second-order

kinetics for backward anation did not give adequate results.

Especially, for the chloride concentration in the backward

reaction, a fractional order is quite conceivable. Nonetheless,

the rate of the conversion of the chlorido-species into the aqua-

species complies with the following order: benzoxazole

derivative > benzimidazole derivative > benzothiazole deriva-

tive. The rate is markedly higher for ruthenium complexes than

that for the corresponding osmium analogues (Figures 4−5,

S10−S14 of Supporting Information). It should be also stressed

that in isotonic saline the amount of the aqua complex in

equilibrium is negligible.

1

always detectable. In the case of 8a, a H,¹³C-HSQC-TOCSY

was performed for unambiguous distinction of carbons to

which four protons with δ at 7.50−7.21 ppm (Figure S5,

Supporting Information) were attached. The ¹H,¹³C-HMBC of

8e showed cross-peaks from N¹′H to C³and C^3a′, while in the

¹H,¹⁵N-HMBC, besides the cross-peak belonging to C⁵H and

N⁴with a ¹⁵N shift of 248.8 ppm, another, actually very weak

peak at 135.1 ppm was found, pointing to C⁸H. This was

confusing because ¹H,¹⁵N-HSQC spectrum, measured with 128

scans, showed only one signal at about 135 ppm for N¹′. A

repeated measurement with 768 scans, however, delivered two

cross-peaks with ¹⁵N shift of 134.6 ppm (N¹′) and 135.1 ppm

(N¹) (Figures S6−S9, Supporting Information). Often,

1

compounds did not give any peak in H,¹⁵N-HSQC due to

very broad NH-signals. In many cases, comparison of the

spectra for differently substituted derivatives was required to

1

ensure a correct assignment. Generally, a downfield shift of H

resonances of the coordinated ligands, compared to those of

the uncoordinated ones, has been observed. Thereby

ruthenium shows a more pronounced effect than osmium.

For recently described maltol derived ruthenium p-cymene

complexes, the diastereotopic signal pattern of the four

aromatic p-cymene protons is lost on going from aprotic to

protic solvents, indicating a fast inversion at the metal center

there.⁴²However, this was not the case for the compounds

presented here.

Interconversion of Chlorido- and Aqua-Species. The

behavior of 8a in aqueous solution was monitored by ¹H NMR

spectroscopy. Figure 3 shows the time dependence of the NMR

spectra. The result suggests a hydrolysis of the Ru−Cl bond

and the formation of the monoaqua species in aqueous

solution.

Attempts to prepare the monoaqua species by treatment of

the initial chlorido complex with AgNO₃in D₂O failed because

of abundant formation of side products. Therefore, [Ru(p-

cymene)(4a)(H₂O)](NO₃)₂(11a) was prepared by an

alternative route. First, the chlorido-bridged dimer 7a was

treated with silver nitrate in water. After removal of silver

chloride, the intermediate was allowed to react in situ with 4a

to form 11a. The corresponding aqua complexes (11h, 11l,

12a, 12h, and 12l) of 8h, 8l, 9a, 9h, and 9l were prepared

Figure 4. NMR monitored reaction of 11a with NaCl in D₂O at 298

□

K. ( ) Aqua species in 4.2 mM NaCl. (Δ) Formed chlorido species in

■

▲

4.2 mM NaCl. ( ) Aqua species in 1.4 mM NaCl. ( ) Formed

chlorido species in 1.4 mM NaCl.

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in accord with the NMR data. Even for the chlorido species 8a,

a deprotonation step at pH 4−8 is plausible, which is

responsible for the dramatic decrease of the solubility from

20 mg mL⁻¹in H₂O to 0.15 mg mL⁻¹in phosphate buffered

saline (pH 7.2) due to formation of a nonelectrolyte complex.

Comparison with the corresponding oxazole- and thiazole-

derivatives (solubilities: 8h, 7.7 mg mL⁻¹in H₂O, 1.1 mg mL⁻¹

in PBS; 8l, 7.0 mg mL⁻¹in H₂O, 0.48 mg mL⁻¹in PBS; see

titration curves in the Figures S15−S20, Supporting Informa-

tion) indicates that between pH 4 and 8 both the aqua ligand

and the amide of the quinoxalinone are deprotonated, although

for the unsubstituted 2-quinoxalinone, a pK_aof 9.2 was

reported.⁴⁷The third deprotonation step observed for the

benzimidazole derivatives was tentatively assigned to the N¹′H

group.

Only the NMR titration curves of the osmium complex 12l

(aquation product of 9l) showed a sigmoidal form expected for

one deprotonation step. The solubility of 9l decreased only by a

factor of 5.5 from 1.1 mg mL⁻¹in H₂O to 0.20 mg mL⁻¹in

phosphate buffered saline (pH 7.2). UV−vis titrations of 12l

were not suitable for pK_adetermination because changes of the

absorption above pH 6 were quite small and depended strongly

on experimental conditions (Figures S21, S22, Supporting

Information). For [Ru(p-cymene)(bpy)(H₂O)]²⁺(bpy = 2,2′-

bipyridine), a pK_avalue for coordinated H₂O of 7.2 was

reported,⁴⁸for the osmium analogue [Os(p-cymene)(bpy)-

(H₂O)]²⁺, pK_a= 5.8 was found.⁴⁹Therefore an overlapping of

the deprotonation steps at the quinoxalinone and the aqua

ligands is at least conceivable for 12l.

□

Figure 5. NMR monitored aquation of 9a in D₂O at 298 K. ( )

Formed aqua species. (Δ) Chlorido species. Reaction of 12a with

■

▲

NaCl in D₂O: ( ) aqua species in 4.3 mM NaCl. ( ) Formed

chlorido species in 4.3 mM NaCl.

pK_aValues. Attempts to estimate pK_avalues by titrations of

the aqua complexes with a base monitored by NMR

spectroscopy revealed a complex dependence of the chemical

shift on pD as shown for 11a (aquation product of 8a, Figure

6).

X-ray Crystallography. The results of the X-ray diffraction

studies of three ligands and six metal complexes are shown in

Figures 8−10 and S22−S36 of the Supporting Information. A

Figure 6. pD dependence of a ¹H NMR chemical shift (most probably

C⁵H) of 11a in D₂O, titrated with DNO₃and NaOD.

The shape of the curve suggests three pK_avalues, the first at

pD 5−6, the second at pD 7−8, and the third at pD 10−11. To

verify these findings, titrations monitored by UV−vis spectros-

copy were performed. The form of the UV−vis titration curves

is strongly affected by other species present in solution (e.g.,

perchlorate, triflate, or chloride, sodium or potassium, Figure

7). In the case of chloride-free medium, the results are generally

Figure 8. Associates of 4l formed by hydrogen bonds; thermal

ellipsoids are drawn at 40% probability level. Dashed lines represent

hydrogen bonds.

more detailed discussion of the crystal structures is given in the

Supporting Information.

The unsubstituted benzazolylquinoxalinone ligands crystal-

lized in the monoclinic space groups C2/c (4a) or P2₁/c (4h,

4l). In each case, the asymmetric unit consists of two

molecules, which mainly differ by the twist angle between the

quinoxalinone and the benzazole moiety ranging from 1.7° to

4.6° for the first and from 7.3° to 12.2° for the second

molecule. Thereby 4h shows the smallest and 4a the largest

distortion from planarity. A three-dimensional network of

hydrogen bonds in 4a is presumably responsible for its poor

solubility in many solvents (Figure S23, Supporting Informa-

tion). H-Bond stabilized chains and tetrameric assemblies are

observed in the crystal structures of 4h and 4l, correspondingly

(Figures S25 and 8, Supporting Information).

▲

Figure 7. pH dependence of the absorbance at 490 nm. ( ) 2.3 ×

10⁻⁵mM 11a in H₂O acidified with HClO₄to pH 3, titrated with

KOH. ( ) 2.2 × 10⁻⁵mM 8a in aqueous 1 M KCl acidified with HCl

□

to pH 3, titrated with KOH.

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The organometallic compounds show the expected pseudo-

octahedral “piano-stool” configuration (see Figure 9 for 8a and

Figure 10 for 9l). The complexes are chiral due to the presence

of the stereogenic metal center crystallizing as a racemic

mixture of both enantiomers.

The ligands are always coordinated to the metal via the N4 of

the quinoxalinone moiety and the atom N13 of the benzazole

group.

Some average structural data concerning the coordination

sphere are summarized in Table 1.

Table 1. Selected Average Bond Lengths and Bond Angles in

8a, 8h, 8l, 9a, 9h, and 9l (M = Ru or Os; C^cy= Carbon of the

Aromatic p-Cymene Ring; Cent^cy= Centroid of the Six C^cy)

average bond length [Å]

average bond angle [deg]

M−Cl1

M−N4

2.399(12)

2.126(13)

2.084(15)

1.695(4)

2.21(3)

Cl1−M−N4

86(1)

Cl1−M−N13

N4−M−N13

Cent^cy−M−Cl1

Cent^cy−M−N4

Cent^cy−M−N13

86(1)

M−N13

76.0(4)

128(1)

132(2)

130.8(7)

M−Cent^cy

M−C^cy

Figure 9. ORTEP plot of the complex cation of 8a with atom

numbering scheme. The thermal ellipsoids are drawn at 50%

probability level. Selected bond lengths (Å) and bond angles (deg)

for 8a: Ru1−Cent^cy1.7005(11), av Ru1−C^cy2.21(3), Ru1−N4

2.145(2), Ru1−N13 2.074(2), Ru1−Cl1 2.4137(8), av C^cy−C^cy

1.416(5), C10−N1 1.389(3), N1−C2 1.358(3), C2−O2 1.235(3),

C2−C3 1.473(4), C3−N4 1.327(3), N4−C5 1.397(3), C3−C12

1.431(4), C19−N11 1.380(3), N11−C12 1.362(3), C12−N13

1.326(3), N13−C14 1.395(3) Å. Cent^cy−Ru1−Cl1 128.99(5)°,

Cent^cy−Ru1−N4 133.39(7)°, Cent^cy−Ru1−N13 129.74(7)°, N4−

Ru1−Cl1 85.13(7)°, N13−Ru1−Cl1 84.77(7)°, N13−Ru1−N4

76.00(8)°, C2−N1−C10 122.8(2)°, C3−N4−C5 117.6(2)°, N13−

C12−N11 112.5(2)°, C12−N11−C19 106.5(2)°, Θ_{(N4−C3−C12−N13)}

−2.5(4)°.

a

M−C22/C26

2.18(7)

b

M−C24

2.251(12)

C^cy−C^cy

1.419(7)

a

b

Average of the shortest M−C^cybond. Average of the longest M−C^cy

bond.

A comparison of bond lengths in metal-free and coordinated

quinoxalinone ligands revealed mere insignificant (within 3 σ)

or just minor changes. The twist between the quinoxalinone

and the benzazole moieties in the coordinated ligand is mainly

dependent on the packing (see Supporting Information, pages

33−44), which is strongly affected by the content of

cocrystallized water and correlates with the space group (C2/

c: water free, 20.3(2)°; P1: 2H O, 10.5(7)°). In general, no

̅

2

marked structural differences between the corresponding

ruthenium and osmium complexes were observed. Structural

similarity with the osmium analogues has already been noted

for other organo-ruthenium compounds.⁴⁶

Cytotoxicity in Cancer Cells. The cytotoxic potency of the

quinoxalinone derivatives 4a, 4h, and 4l, the ruthenium(II)

(8a−o, 10a) and osmium(II) complexes (9a, 9h, 9l) was

determined in the human tumor cell lines A549 (nonsmall cell

lung cancer), CH1 (ovarian carcinoma), and SW480 (colon

carcinoma) by means of the colorimetric MTT assay.

In general, the CH1 cells were more sensitive to the

compounds investigated in this study than the other cell lines.

The IC₅₀values of the three unsubstituted metal-free ligands

(the substituted derivatives could not be tested for solubility

reasons) vary within small ranges of micromolar concentrations

in each cell line, whereas the potencies of the metal complexes

vary within 2 orders of magnitude, with IC₅₀values ranging

from high nanomolar to medium micromolar concentrations,

depending on the ligand substitutions and the metal ion

(Table 2).

The following structure−activity relationships can be

deduced from the cytotoxicity data: The quinoxalinone

derivatives 4a (with a benzimidazole moiety), 4h (with

benzoxazole), and 4l (with benzothiazole) hardly differ in

their cytotoxicity in CH1 and SW480 cells (IC₅₀: 6.7−8.6 μM

and 14−23 μM, respectively). In A549 cells, 4a (IC₅₀: 17 μM)

is the most potent of these three compounds, while 4l (IC₅₀: 61

μM) is the least potent in this cell line. Complexation to

ruthenium or osmium bound to cymene results in more clear-

cut differences, with a rank order of cytotoxicity in CH1 and

Figure 10. ORTEP plot of the complex cation of 9l with atom

numbering scheme. The thermal ellipsoids are drawn at 50%

probability level. Selected bond lengths (Å) and bond angles (deg)

for 9l: Os1−Cent^cy1.6959(14), av Os1−C^cy2.21(3), Os1−N4

2.118(2), Os1−N13 2.101(2), Os1−Cl1 2.3951(7), av C^cy−C^cy

1.420(4), C10−N1 1.389(4), N1−C2 1.331(4), C2−O2 1.232(4),

C2−C3 1.469(4), C3−N4 1.329(4), N4−C5 1.408(4), C3−C12

1.427(4), C19−S11 1.730(3), S11−C12 1.719(3), C12−N13

1.330(4), N13−C14 1.401(4) Å. Cent^cy−Os1−Cl1 129.47(5)°,

Cent^cy−Os1−N4 130.87(8)°, Cent^cy−Os1−N13 130.70(7)°, N4−

Os1−Cl1 84.90(7)°, N13−Os1−Cl1 86.35(7)°, N13−Os1−N4

76.20(9)°, C2−N1−C10 123.6(3)°, C3−N4−C5 117.0(3)°, N13−

C12−S11 116.6(2)°, C12−S11−C19 88.69(14)°, Θ_{(N4−C3−C12−N13)}

1.3(4)°.

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cytotoxicity are mostly small and there is no consistent

tendency with regard to the effects of complexation. The

ruthenium complex 10a (IC₅₀: 34−140 μM), where cymene is

replaced by η⁶-bound benzene, is by an order of magnitude less

active than the cymene-containing analogue 8a (IC₅₀: 4.3−17

μM) and still considerably less active than noncoordinated 4a

(IC₅₀: 8.6−23 μM). In contrast to 8a, which is stable in water

for at least several weeks, 10a decomposes within a few days in

aqueous solution, forming an amorphous unidentified insoluble

product. This precipitation may account for the observed

reduction of biological activity.

Furthermore, the consequences of chloro and methyl

substitutions at the quinoxalinone derivatives for the cytotoxic

potency of the ruthenium complexes were studied. A

comparison of the ruthenium complexes of benzimidazole

and benzothiazole derivatives reveals a favorable effect of chloro

substitutions, as chloro-only (8b, 8d, 8n) and mixed chloro/

methyl substituted species (8f, 8g, 8o) are at least as potent as

or more potent than the corresponding methyl-only substituted

species (8c, 8e, 8m), which in turn are at least as potent as

complexes 8a and 8l with unsubstituted ligands. For the

complexes with benzoxazole ligands, the effects of methyl

substituents are not consistent; only substitution in positions 6

and 7 (see atom numbering Scheme 1a) of the quinoxalinone

moiety in complex 8k is more likely to be advantageous.

Complexes with chloro substituents at benzoxazole ligands

could not be obtained in sufficient purity because these

substituents further destabilize these generally not very robust

compounds. Anyway, the partially somewhat higher cytotox-

icities resulting from the substituents hardly outweigh the

pronounced decrease of solubility with all its disadvantages for

drug formulation and bioavailability.

Table 2. Cytotoxicity of Ligands (4a, 4h, 4l), Ruthenium(II)

Complexes (8a−o, 10a), and Osmium(II) Complexes (9a,

9h, 9l) in Three Human Cancer Cell Lines

a

IC₅₀(μM)

compd

A549

CH1

SW480

Ligands

4a

4h

4l

17

38

2

7

8.6 1.9

7.5 1.0

23

14

20

4

3

61 15

6.7 1.1

Ru(II) Complexes

4.3 1.2

8a

8b

8c

8d

8e

8f

17

3.9 0.6

13

5.4 0.7

11

3

5.3 0.7

2.3 0.7

4.5 0.6

1.8 0.3

2.6 0.8

3.4 0.6

1.6 0.2

9.0 0.8

0.34 0.11

1.8 0.4

1

0.54 0.18

1.6 0.7

2

4.1 0.7

3.8 0.8

1.7 0.2

8g

8h

8i

0.93 0.12

6.2 1.8

28

56

14

22

12

7

6

4

3

2

1

4.3 0.7

14

19

1

3

8j

5.7 1.1

8k

8l

3.8 0.5

8.0 0.7

2.2 0.3

1.7 0.3

1.0 0.2

1.0 0.1

1.2 0.4

8m

8n

8o

10a

0.52 0.10

0.40 0.03

0.36 0.06

34 11

7.8 0.4

3.9 0.2

140 17

40

5

Os(II) Complexes

0.92 0.29

2.8 0.7

9a

9h

9l

3.0 0.4

4.7 0.6

12

0.91 0.36

37

6

1

2.3 0.5

0.36 0.02

a

50% inhibitory concentrations in A549, CH1, and SW480 cells after

exposure for 96 h in the MTT assay. Values are means standard

Cell Cycle Perturbations. The impact of the lead structure

indirubin-3′-monoxime as well as compounds 4a, 8a, 8h, 8l, 9a,

9h, and 9l on cell cycle progression of exponentially growing

A549 cells was studied by flow cytometry of propidium iodide-

stained cells (Figure 11). Of these compounds, indirubin-3′-

monoxime and the uncomplexed, unsubstituted quinoxalinone

derivative 4a have the most pronounced effects. In accordance

with literature data,^25,50indirubin-3′-monoxime induces a cell

cycle arrest in G2/M phases. Similarly, 4a induces a marked

increase of the G2/M fraction, accompanied by a decrease of

the G0/G1 and S fractions. The maximum effects of indirubin-

3′-monoxime and 4a were observed at concentrations of 40 and

160 μM, respectively, inducing increases of the G2/M fraction

from 10% (in untreated controls) to 49% and 35%, respectively.

Exposure to 40 μM 4a for 24 h results in an increase of the G2/

M fraction to 29%, which is about half the effect of indirubin-3′-

monoxime, corresponding to their different cytotoxic potencies

(IC₅₀values after 96 h: 17 and 7.9 μM,⁵¹respectively).

In contrast, the complexes 8a and 9a exert only marginal

effects on the cell cycle despite their equal or even higher

cytotoxicity. In particular, the apparent G2/M-arresting effect

of 4a is nearly completely abolished by complexation to

ruthenium or osmium. The consequences of replacing the

benzimidazole moiety of these complexes with benzoxazole or

benzothiazole are moderate, with an increase of the G0/G1

fraction from 49 to 72% and a concomitant decrease of the S

fraction from 37 to a minimum of 12% induced by 100 μM 8h

being the most conspicuous effect.

deviations obtained from at least three independent experiments.

SW480 cells depending on the variable heterocyclic moiety as

follows: benzothiazole > benzimidazole > benzoxazole; or 8l

(IC₅₀: 1.2 and 2.2 μM) > 8a (IC₅₀: 4.3 and 5.3 μM) > 8h (IC₅₀:

6.2 and 9.0 μM, respectively), and 9l (IC₅₀: 0.36 and 0.91 μM)

> 9a (IC₅₀: 0.92 and 4.7 μM) > 9h (IC₅₀: 2.8 and 12 μM,

respectively). In A549 cells, the ruthenium complexes are rather

similar in activity (IC₅₀: 17−28 μM), whereas the osmium

compound 9h (IC₅₀: 37 μM) is considerably less cytotoxic than

9a (IC₅₀: 3.0 μM) and 9l (IC₅₀: 2.3 μM). The generally lower

cytotoxicity of the complexes containing benzoxazole deriva-

tives may be explained by their lower thermodynamic and

kinetic stability. As already mentioned, it was not possible to

measure 2D-NMR spectra in d₆-DMSO due to fast

decomposition, while those of the analogue complexes with

benzothiazole and benzimidazole ligands did not pose such

severe problems. Still, the presence of the indirubin

pharmacophore, as in the benzimidazole derivatives, is not an

essential prerequisite for cytotoxicity, and exchange of nitrogen

for sulfur may even be advantageous from the mere aspect of in

vitro potency.

A comparison of the uncomplexed quinoxalinone derivatives

with benzimidazole (4a) or benzothiazole (4l), their ruthenium

cymene complexes (8a, 8l), and the osmium analogues (9a, 9l)

reveals that complexation with osmium results in a more

pronounced and more consistent increase in cytotoxicity (5−27

times lower IC₅₀values) than complexation with ruthenium

(equal to 9 times lower IC₅₀values). For the analogue

benzoxazole-containing compounds (4h, 8h, 9h), differences in

Induction of Apoptosis. The capacity of 8a, 8l, 9a, and 9l

to induce apoptotic cell death was verified in a qualitative

manner by fluorescence microscopy of SW480 cells treated for

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Figure 11. Concentration-dependent impact of indirubine-3′-monoxime, 4a, 8a, 8h, 8l, 9a, 9h, and 9l on the cell cycle distribution of A549 cells after

exposure for 24 h. Cells were analyzed for DNA content by flow cytometry after staining with propidium iodide.

24 h with low micromolar concentrations (1.25−10 μM) of the

compounds and subsequently stained with DAPI. The

microscopic images depicted in Figure 12 give clear evidence

of the formation of apoptotic bodies, recognizable by the

characteristic fragmentation of nuclei with bright condensed

chromatin, for treatment with each of the compounds.

According to the flow-cytometric annexin V/propidium

iodide assay, the ruthenium and osmium complexes with

unsubstituted ligands derived from benzimidazole or benzo-

thiazole are all capable of inducing apoptosis in SW480 colon

cancer cells in a concentration-dependent manner, but with

different potencies corresponding to their different cytotox-

icities in the MTT assay (Figure 13). Variation of the

heteroatom in the ligand has a more pronounced impact on

the potencies of the compounds than the change of the central

metal. At a concentration of 10 μM and within 48 h, complexes

8l and 9l with benzothiazole-derived ligands induce apoptosis

in totals of about 67 and 43%, respectively, whereas the

benzimidazole-based analogues 8a and 9a exert comparatively

modest effects with totals of about 13 and 8%, respectively

(Figure 14). However, especially at higher concentrations of

the latter two compounds, apoptosis is accompanied by

noteworthy fractions of necrotic cells (about 28 and 22% at

20 μM, respectively). Comparison of apoptosis-to-necrosis

ratios reveals marked differences depending on the variable

heteroatom in the ligand. Benzothiazole-derived compounds

yield much higher ratios (8l: 10 μM, 4.7; 20 μM, 4.6. 9l: 10

μM, 4.0; 20 μM, 6.3) than benzimidazole analogues (8a: 10

μM, 1.3; 20 μM, 0.64. 9a: 10 μM, 2.9; 20 μM, 1.0). For a

complete set of percentage values averaged from three

independent experiment series, see Table S2 in the Supporting

Information.

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differences between the two metals. The coordination of

quinoxalinone derivatives to the metal results in a shift of the

pK_avalue for the lactam N−H from ca. 9 to 7. Thereby the

solubility is impaired, but it is still much higher than that of the

metal-free ligand. Complex 10a containing a η⁶-bound benzene

seems to decompose with formation of an insoluble product,

leading to IC₅₀values higher than those of the metal-free

ligand. The lower activity of complexes with benzoxazole

derivatives may also be due to diminished complex stability.

The chlorido ligand is a potential leaving group that could

provide a site for coordinative interaction with targets, with

reactivity depending on the central ion. For the compounds

presented herein, the higher inertness of osmium tends to be

advantageous in terms of cytotoxicity in cancer cells. This might

indicate that either coordination does not play any role in the

mode of action or that the higher reactivity of ruthenium

compounds results in deactivating side reactions. Evidently,

effects on the cell cycle seem to be diminished by coordination

to ruthenium or osmium, although cytotoxicity is enhanced or

at least maintained and selected complexes all induce apoptosis

in the low micromolar concentration range, suggesting that

other effects not affecting the cell cycle contribute to their

activity in vitro. All these findings should be taken into

consideration for the future design of new enzyme-targeted

metal-based drugs, which certainly open up new possibilities

beyond the limitations of purely organic compounds.

Figure 12. Formation of apoptotic bodies upon treatment of SW480

cells with quinoxalinone complexes: (A) untreated control; (B)

untreated control with late mitotic nuclei; (C) 8a (2.5 μM); (D) 8l (5

μM); (E) 9a (5 μM); (F) 9l (1.25 μM).

EXPERIMENTAL SECTION

■

All solvents and reagents, if available in analytical grade, were

purchased from commercial suppliers and used without further

purification. Water from a reverse osmotic demineralization facility

was distilled before use. Indirubin-3′-monoxime²⁵and the precursors

[(η⁶-p-cymene)RuCl₂]₂(7a),^52,53[(η⁶-benzene)RuCl₂]₂,⁵²and [(η⁶-p-

cymene)OsCl₂]₂(7b)⁵⁴were prepared according to literature

protocols. H₂[OsCl₆] was prepared by using hydrazine dihydro-

chloride for the reduction of osmium tetroxide in HCl.⁵⁵In such a

way, heating of the hazardous volatile OsO₄was avoided. Elemental

analyses of all biologically tested compounds were carried out on Carlo

Erba (EA 1108 CHNS), Perkin-Elmer (2400 CHN), and Mettler-

CONCLUSIONS

■

A series of new ruthenium and osmium arene complexes

containing quinoxalinone ligands with structural features that

may contribute to specific interactions with biological targets

has been prepared and tested for cytotoxicity in the three

human cancer cell lines CH1 (ovarian carcinoma), SW480

(colon carcinoma), and A549 (nonsmall cell lung carcinoma).

Comparison of structural aspects did not reveal significant

Figure 13. Percentages (means standard derivations) of viable, apoptotic, and necrotic SW480 cells after exposure to Ru and Os arene complexes

with quinoxalinone ligands, 8a (A), 8l (B), 9a (C), and 9l (D) for 48 h, as obtained by flow cytometric quantification.

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Figure 14. Flow cytometric quantification of viable, apoptotic, and necrotic SW480 cells after treatment with Ru and Os arene complexes with

quinoxalinone ligands. Evaluation by annexin V-FITC versus propidium iodide staining of SW480 cells after 48 h incubation of 10 μM of compounds

8a (A), 8l (B), 9a (C), and 9l (D) compared to untreated control (E). Each bottom left quadrant contains viable, the bottom right early apoptotic,

the top right late apoptotic and the top left necrotic cells (numbers indicate the respective percentages of total cell populations).

Toledo (DL 21) microanalyzers at the Microanalytical Laboratory of

the University of Vienna and are within 0.4%, confirming ≥95%

purity, with exception of 10a. An Esquire 3000 ion trap mass

spectrometer (Bruker Daltonics, Bremen, Germany) with an

orthogonal ESI ion source was used for MS measurements. The

solutions in methanol were introduced via flow injection using a Cole-

Parmer 74900 single-syringe infusion pump (Vernon Hills, IL).

Expected and experimental isotope distributions were compared.

Infrared spectra were obtained using a Bruker Vertex 70 instrument

equipped with a Specac Golden Gate single reflection diamond ATR

unit (4000−550 cm⁻¹). UV−vis spectra were recorded on a Perkin-

Elmer Lambda 650 UV−vis spectrophotometer using samples

dissolved in methanol (800−210 nm). For the description of

shoulders, the wavelength of the appropriate local minimum of the

first derivative absolute value was quoted. ¹H, ¹³C, ¹H,¹H−COSY,

¹H,¹H-TOCSY, ¹H,¹H-ROESY, ¹H,¹³C-HSQC, ¹H,¹³C-HMBC,

¹H,¹³C-HSQC-TOCSY, ¹H,¹⁵N-HSQC, and ¹H,¹⁵N-HMBC NMR

spectra were recorded in DMSO-d₆, MeOH-d₄, or D₂O with a Bruker

Avance III 500 MHz FT-NMR spectrometer. The 2D NMR spectra

curves, created with Excel software by plotting the absorbance at a

selected wavelength against the pH.

Crystallographic Structure Determinations. X-ray diffraction

quality single crystals of the ligands 4a, 4h, and 4l were obtained by

slow diffusion of diethyl ether into their solutions in DMF. Single

crystals of the corresponding ruthenium(II) complexes (8h, 8l) and

osmium(II) complexes (9a, 9h, 9l) were obtained by slow diffusion of

sodium chloride into solutions of the compounds in water. Crystals of

8a grew from D₂O solution during NMR measurements after addition

of sodium chloride. Measurements were performed on a Bruker X8

APEXII CCD diffractometer at 100 K. Single crystals were positioned

at 40 mm from the detector and measured under the following

conditions: (4a) 1044 frames for 100 s over 1°, (4h) 2405 frames for

20 s over 1°, (4l) 1234 frames for 100 s over 1°, (8a) 931 frames for

20 s over 1°, (8h) 782 frames for 60 s over 1°, (8l) 1626 frames for 80

s over 1°, (9a) 2548 frames for 20 s over 1°, (9h) 1277 frames for 50 s

over 1°, and (9l) 1395 frames for 50 s over 1°. The data were

processed using the SAINT software package.⁵⁶Crystal data, data

collection parameters, and structure refinement details are given in

Table S1, Supporting Information. 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 at calculated positions and refined

with a riding model. The following computational resources were

used: structure solution, SHELXS-97;⁵⁷refinement, WinGX,⁵⁸

SHELXL-97;⁵⁹calculations, PLATON;⁶⁰molecular diagrams, XP 5.1

(Bruker, 1998);⁶¹computer, Pentium Dual Core; scattering factors.⁶²

Crystallographic data have been deposited with the Cambridge

Crystallographic Data Center with the numbers CCDC 863692−

863700. Copy of data can be obtained, free of charge, on application to

The Cambridge Crystallographic Data Center, 12 Union Road,

Cambridge CB2 1EZ, U.K. (deposit@ccdc.com.ac.uk).

were measured in a gradient-enhanced mode. The residual ¹H and ¹³

C

present in the solvents were used as internal references. ¹⁵N shifts are

quoted relative to external NH₄Cl. For the monitoring of the time-

dependent interconversion between chlorido- and aqua-species,

tetramethylammonium nitrate (0.13 mM) was used as internal

reference. The automatically generated series of proton spectra were

baseline corrected and integrated using AU programs included in

Topspin 2.0 (Bruker Biospin Inc., Fremont, CA). The normalized

peak areas were plotted against the time with Excel software

(Microsoft Office Excel 2003, SP3, Microsoft Corporation).

Abbreviation for NMR data: cy = p-cymene.

NMR Titrations. Complexes 11a, 11h, 11l, 12a, 12h, or 12l (3 mg)

were dissolved in D₂O (0.6 mL) at ∼298 K. The pD value was

measured directly in the NMR tubes with an Eco Scan pH6 pH meter

equipped with a glass-micro combination pH electrode (Orion

9826BN), which was calibrated with standard buffer solutions of pH

4.00, 7.00, and 10.00. The pH titration was accomplished by addition

of NaOD (0.4−0.0004% in D₂O) and DNO₃(0.4−0.0004% in D₂O).

The observed shifts of quinoxalinone ligand or cymene protons in the

¹H NMR spectra were plotted against the pD value with Excel

software.

Cell Lines and Culture Conditions.⁴²CH1 cells originate from

an ascites sample of a patient with a papillary cystadenocarcinoma of

the ovary and were a generous gift from Lloyd R. Kelland, CRC

Centre for Cancer Therapeutics, Institute of Cancer Research, Sutton,

UK. SW480 (adenocarcinoma of the colon) and A549 (nonsmall cell

lung cancer) cells were kindly provided by Brigitte Marian (Institute of

Cancer Research, Department of Medicine I, Medical University of

Vienna, Austria). All cell culture reagents were obtained from Sigma-

Aldrich Austria. Cells were grown in 75 cm²culture flasks (Iwaki) as

adherent monolayer cultures in Minimal Essential Medium (MEM)

supplemented with 10% heat-inactivated fetal calf serum, 1 mM

UV Titrations. The titrations were performed in a quartz cell,

whereby the pH was measured with the pH electrode used for NMR

titrations. Details are specified in the legends of the corresponding

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sodium pyruvate, and 2 mM L-glutamine. Cultures were maintained at

37 °C in a humidified atmosphere containing 5% CO₂and 95% air.

Cytotoxicity in Cancer Cell Lines. Cytotoxicity in the cell lines

mentioned above was determined by the colorimetric MTT (3-(4,5-

dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, purchased

from Sigma-Aldrich) microculture assay,⁶³with modifications as

described previously.⁴²For this purpose, cells were harvested from

culture flasks by trypsinization and seeded in 100 μL aliquots into 96-

well microculture plates (Iwaki/Asahi Technoglass). Cell densities of

1.5 × 10³cells/well (CH1), 2.5 × 10³cells/well (SW480), and 4 × 10³

cells/well (A549) were chosen in order to ensure exponential growth

of untreated controls throughout drug exposure. Cells were allowed to

settle and resume proliferation in drug-free complete culture medium

for 24 h. The not sufficiently water-soluble test compounds were

dissolved in DMSO and diluted in complete culture medium such that

the maximum DMSO content did not exceed 0.5% (especially in the

case of 4a, 4h, and 4l, this procedure yielded opaque but colloidal

solutions from which no precipitates could be separated by

centrifugation). Only 8h−k, 9h, and 10a were directly dissolved in

medium under sonication. Appropriate dilutions were added in 100 μL

aliquots to the microcultures, and cells were exposed to the test

compounds for 96 h. At the end of exposure, all media were replaced

by 100 μL/well RPMI1640 culture medium (supplemented with 10%

heat-inactivated fetal bovine serum and 4 mM ^L-glutamine) plus 20

μL/well MTT (5 mg/mL) dissolved in phosphate-buffered saline

(PBS). After incubation for 4 h, the supernatants were removed and

the formazan crystals formed by viable cells were dissolved in 150 μL

of DMSO per well. Optical densities at 550 nm were measured with a

microplate reader (Tecan Spectra Classic), using a reference

wavelength of 690 nm to correct for unspecific absorption. The

quantity of viable cells was expressed as percentage of untreated

controls, and 50% inhibitory concentrations (IC₅₀) were calculated

from concentration−effect curves by interpolation. Evaluation is based

on means from at least three independent experiments, each

comprising three replicates per concentration level.

advanced apoptotic stage or in necrotic cells. For this purpose, 2 × 10⁵

SW480 cells per well were seeded out in 6-well plates and allowed to

recover for 24 h. Cells were then exposed to different concentrations

of the test compounds for 48 h. The supernatant and cells detached by

trypsinization were collected and transferred into FACS tubes,

centrifuged at 1200 rpm for 3 min, and the supernatant was discarded.

After resuspension in 0.5 mL of binding buffer, cells were incubated

for 5 min with 1 μL of annexin V-FITC from Bio Vision. PI was added

shortly before measurement in an effective concentration of 1 μg/mL.

Fluorescence of 10000 cells was measured by flow cytometry with a

FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ)

using FL1 channel for annexin V-FITC and FL2 channel for PI

staining. Resulting dot blots were quantified by Cell Quest Pro

software (Becton Dickinson and Company, Franklin Lakes, NJ).

Synthesis of Ligands and Metal Complexes. Microanalytical

1

data, mass spectrometry, IR- and UV−vis-, H and ¹³C NMR spectra

for all biologically tested compounds can be found in Supporting

Information, along with the syntheses of precursors.

General Procedure A. A mixture of ethyl 2-(1H-benzimidazol-2-yl)-

2,2-dibromoacetate or a substituted derivative (1 equiv), 1,2-

phenylenediamine or a substituted derivative (1.1−1.4 equiv), and

sodium acetate (3 equiv) in ethanol (96%, 7−8 mL/mmol) was

sonicated until the starting compounds were dissolved at room

temperature. In some cases, a yellow product started to precipitate

prior the complete dissolution of starting material so that there was

never a clear (red) solution. Subsequently, the reaction mixture was

refluxed under argon (to avoid formation of oxidation side products

from excess phenylendiamine) for 60 min. Afterward, the formed

suspension was cooled and stored in a freezer (−20 °C) for 2−3 h.

The solid was filtered off by suction and washed with cold ethanol (1−

2 mL/mmol), water (4−5 × 2−4 mL/mmol), methanol (2 × 1−2

mL/mmol), and diethyl ether (3 × 3−4 mL/mmol). Finally, the crude

product was dried in vacuo.

3-(1H-Benzimidazol-2-yl)-1H-quinoxalin-2-one (4a). The reaction

was performed according to procedure A using 3a (5.47 g, 15.0 mmol)

and 1,2-phenylenediamine (2.22 g, 20.0 mmol). The crude product

was dissolved in acetic acid (45 mL) and precipitated by addition of

diethyl ether (100 mL). The solid was filtered off, washed with diethyl

ether (3 × 25 mL), and dried in vacuo. Then the material was

suspended in water (100 mL) and dissolved by addition of

orthophosphoric acid (85%, 5.1 mL, 75 mmol). The solution was

filtered and the filtrate poured into a solution of sodium hydroxide

(4.59 g, 113 mmol) in water (100 mL). After cooling (+2 °C) for 1 h,

the precipitate was filtered off and washed with water (3 × 70 mL).

The solid was suspended in water (100 mL), heated to 100 °C for 30

min, filtered off again, and washed with water (2 × 70 mL). Finally, the

product was dried in vacuo. Yield: 3.18 g (80%). Anal. (C₁₅H₁₀N₄O):

C, H, N. Alternatively, the compound was synthesized by reaction of

ethyl 3-oxo-3,4-dihydro-quinoxaline-2-carboxylate⁶⁵(0.22 g, 1.0

mmol) and 1,2-phenylenediamine (0.13 g, 1.2 mmol) in ethylene

glycol (4.0 mL) under argon at 170 °C for 3 h. After cooling to room

temperature, the mixture was allowed to stand in a freezer (−20 °C)

for 3−4 h. Then the crude product was filtered off by suction, washed

with methanol (3 × 3 mL), and dried in vacuo. The purification was

performed as described above using acetic acid (3.5 mL), diethyl ether

(8 mL, 3 × 5 mL), 1 M aqueous orthophosphoric acid (4 mL), 1 M

aqueous sodium hydroxide (4.5 mL), and water (5 × 10 mL). Yield:

0.09 g (34%).

Cell Cycle Analyses. A549 cells (1 × 10⁶cells) were seeded into

Petri dishes and allowed to recover for 24 h. Cells were then exposed

for 24 h to the test compounds, dissolved as described for the MTT

assays. Control and treated cells were collected, washed with PBS,

fixed in 70% ice-cold ethanol, and stored at −20 °C. To determine cell

cycle distributions, cells were transferred in physiological NaCl

solution into PBS, incubated with 10 μg/mL RNase A for 30 min at

37 °C, and treated with 5 μg/mL propidium iodide for 30 min, and

their fluorescence was measured by flow cytometry, using a FACS

Calibur instrument (Becton Dickinson, Palo Alto, CA). The resulting

DNA histograms were analyzed by Cell Quest Pro software (Becton

Dickinson and Company, New York, USA).

Fluorescence Microscopy. Induction of apoptosis in SW480 was

verified by fluorescence microscopy after DAPI staining. For this

purpose, 2.5 × 10⁵cells/well were seeded in culture medium onto

cover glasses in 6-well plates and allowed to attach for 24 h at 37 °C.

Cells were then exposed to various concentrations of the test

compounds for 24 h. The supernatant was removed, and cells were

treated for 15 min with 1 μg/mL DAPI (4′,6-diamidino-2-phenyl-

indole) in methanol. After washing the cells with methanol, the cover

glasses were mounted on microscopy slides with mounting medium.

Fluorescence images were taken on an Olympus BX-40 microscope

using an excitation wavelength of 345 nm.

Annexin V/Propidium Iodide Assay. This assay was applied to

determine the capacity of inducing cell death of the compounds under

investigation. The method was described by Aubry et al.⁶⁴and allows

differentiation of early and late apoptosis as well as necrosis. In early

apoptosis, phosphatidylserine flips from the inner side of the plasma

membrane to the cell surface. The Ca²⁺-dependent affinity of

phosphatidylserine to an annexin V-FITC conjugate enables the

fluorometric detection of this process by stimulation at a wavelength of

488 nm, resulting in emission at a wavelength of 530 nm. Staining with

propidium iodide (PI), having an absorption wavelength of 990 nm

and an emission wavelength of 612 nm, on the other hand, requires a

loss of cell membrane integrity and therefore only works in the

3-(5,6-Dichloro-1H-benzimidazol-2-yl)-1H-quinoxalin-2-one (4b).

The reaction was performed by following procedure A, but reducing

the reaction time to 10 min, starting from 3b (1.51 g, 3.5 mmol) and

1,2-phenylenediamine (0.52 g, 4.7 mmol). Yield: 1.03 g (88%). The

crude product was used for complexation reactions without further

purification.

3-(5,6-Dimethyl-1H-benzimidazol-2-yl)-1H-quinoxalin-2-one

(4c). 3c (0.82 g, 2.1 mmol) and 1,2-phenylenediamine (0.32 g, 2.8

mmol) were refluxed in absolute methanol (20 mL) under argon for

30 min. Then 4-methylmorpholine (0.53 mL, 4.7 mmol) was added.

Subsequently, the reaction mixture was refluxed for further 3 h. After

cooling to 50 °C, the precipitate was filtered off by suction and washed

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with methanol (2 × 7 mL), water (5 × 10 mL), methanol (2 × 7 mL),

and diethyl ether (3 × 10 mL). The crude product was recrystallized

from ethylene glycol (60 mL), filtered off, washed with methanol (3 ×

8 mL), and dried in vacuo. Yield: 0.27 g (43%).

3-(1H-Benzimidazol-2-yl)-6,7-dichloro-1H-quinoxalin-2-one (4d).

The reaction was performed according to procedure A starting from 3a

(1.09 g, 3.0 mmol) and 4,5-dichloro-1,2-phenylenediamine (0.73 g, 4.0

mmol). Yield: 0.94 g (94%). The crude product was used for

complexation without further purification.

3-(1H-Benzimidazol-2-yl)-6,7-dimethyl-1H-quinoxalin-2-one (4e).

4,5-Dimethyl-1,2-phenylenediamine (0.46 g, 3.3 mmol) and sodium

acetate (0.83 g, 10 mmol) were dissolved in a mixture of ethanol (96%,

20 mL) and water (1 mL). Activated charcoal was added, and the

resulting suspension was stirred at room temperature for 40 min.

Subsequently, the charcoal was removed by filtration through a small

column with silica (3 g). To the filtrate, 3a (1.09 g, 3.0 mmol) was

added as a solid and the reaction mixture immediately sonicated until

the starting compound had dissolved. Subsequently, the flask was

closed under argon and allowed to stand at room temperature for 2 h.

Then the formed solid was filtered off by suction and washed with

ethanol (10 mL), water (4 × 15 mL), ethanol (2 × 10 mL), and

diethyl ether (3 × 15 mL). Finally, the product was dried in vacuo.

Yield: 0.84 g (96%).

3-(5,6-Dichloro-1H-benzimidazol-2-yl)-6,7-dimethyl-1H-quinoxa-

lin-2-one (4f). The reaction was performed according to procedure A,

applying a longer refluxing time (1.5 h), starting from 3b (2.16 g, 5.0

mmol) and 4,5-dimethyl-1,2-phenylenediamine (0.97 g, 7.0 mmol).

The crude product was suspended in water (100 mL), heated to 100

°C for 1 h, filtered off, and washed with water (3 × 15 mL). Finally,

the product was dried in vacuo. Yield: 1.68 g (70%).

three times from ethanol (3 × 320 mL). The product was dried in

vacuo. Yield: 1.40 g (37%).

3-(Benzoxazol-2-yl)-6,7-dimethyl-1H-quinoxalin-2-one (4k). A

suspension of 3d (2.65 g, 7.3 mmol), 4,5-dimethyl-1,2-phenylendi-

amine (1.33 g, 9.7 mmol), and sodium acetate (1.84 g, 22 mmol) in

ethanol (96%, 25 mL) was sonicated at 30 °C for 5 min before water

(7.0 mL) was added and the sonication continued for additional 5 min.

Subsequently, the reaction mixture was allowed to stand at 30 °C for

further 3.5 h. The precipitate formed was filtered off by suction and

washed with ethanol (2 × 15 mL), water (4 × 30 mL), ethanol (15

mL), and diethyl ether (4 × 30 mL). The product was dried in vacuo

and used without further purification in the next step. Yield: 0.93 g

(44%).

3-Benzothiazol-2-yl-1H-quinoxalin-2-one (4l). To a suspension of

6a (4.74 g, 25 mmol) in ethanol (96%, 150 mL) was added 2-

aminothiophenol (3.0 mL, 28 mmol). The mixture was refluxed under

argon for 30 min. After the addition of hydrochloric acid (37%, 2.9

mL, 35 mmol), refluxing was continued for 2.5 h. Subsequently, the

reaction mixture was cooled with an ice bath for 1 h. The yellow solid

was filtered off and washed with cold ethanol (10 mL), water (5 × 12

mL), cold ethanol (5 mL), and diethyl ether (3 × 12 mL). The crude

material was dissolved in a mixture of ethanol (750 mL) and

tetrahydrofuran (150 mL) at 70 °C, filtered, and the filtrate poured

into water (3500 mL). The precipitate was filtered off and washed with

water (3 × 12 mL), cold ethanol (5 mL), and diethyl ether (2 × 12

mL). The dried substance was recrystallized from o-xylene (1000 mL).

Thereby insoluble material was removed by hot filtration. After drying

in vacuo at 80 °C, the yellow powder was dissolved in THF (2500

mL), filtered, and poured into water (10000 mL). The precipitate was

filtered off by suction, washed with water (3 × 50 mL), and dried in

vacuo at 70 °C. Yield: 1.90 g (27%). Anal. (C₁₅H₉N₃OS): C, H, N, S.

3-(Benzothiazol-2-yl)-6,7-dimethyl-1H-quinoxalin-2-one (4m).

To a suspension of 6b (0.87 g, 4.0 mmol) in ethanol (96%, 50 mL)

was added 2-aminothiophenol (0.48 mL, 4.4 mmol). The mixture was

refluxed under argon for 45 min. After the addition of hydrochloric

acid (37%, 0.42 mL, 5 mmol), refluxing was continued for 3 h. The

reaction mixture was allowed to cool to ca. 40 °C. The precipitate was

filtered off by suction and washed with ethanol (2 × 5 mL). The crude

material was refluxed in ethanol (50 mL) for 2.5 h. Subsequently,

water (30 mL) was added and the boiling was continued for 1 h. The

suspension was cooled to ca. 40 °C. The solid was filtered off and

washed with water (3 × 15 mL), ethanol (5 mL), and diethyl ether (3

× 15 mL). Finally, the product was dried in vacuo and used for

complexation without further purification. Yield: 0.74 g (60%).

3-(5-Chlorobenzothiazol-2-yl)-1H-quinoxalin-2-one (4n). To a

suspension of 6a (0.57 g, 3.0 mmol) in ethanol (96%, 30 mL) was

added 2-amino-4-chloro-thiophenol (0.55 g, 3.3 mmol). The mixture

was refluxed under argon for 45 min. After the addition of

hydrochloric acid (37%, 0.42 mL, 5 mmol), refluxing was continued

for 5.5 h. Subsequently, the reaction mixture was cooled with an ice

bath for 15 min. The yellow solid was filtered off and washed quickly

with cold ethanol (3 × 5 mL), water (5 × 15 mL), cold ethanol (5

mL), and diethyl ether (3 × 10 mL). Finally, the product was dried in

vacuo and used for complexation without further purification. Yield:

0.72 g (76%).

6,7-Dichloro-3-(5,6-dimethyl-1H-benzimidazol-2-yl)-1H-quinoxa-

lin-2-one (4g). The compound was prepared as described above for 4e

starting from 4,5-dichloro-1,2-phenylenediamine (0.37 g, 2.0 mmol),

sodium acetate (0.39 g, 4.7 mmol), and 3c (0.57 g, 1.46 mmol). The

amounts of all solvents used were reduced to the half compared to

those used for the synthesis of 4e. Yield: 0.43 g (82%).

3-(Benzoxazol-2-yl)-1H-quinoxalin-2-one (4h). 3d (2.55 g, 7.0

mmol) and 1,2-phenylenediamine (1.03 g, 9.3 mmol) were stirred in

absolute methanol (50 mL) under argon at 50 °C for 30 min. Then 4-

methylmorpholine (0.53 mL, 4.7 mmol) was added and the reaction

mixture was stirred for further 5.5 h at 50 °C. After cooling to 5 °C,

the precipitate was filtered off by suction, washed with cold methanol

(2 × 7 mL), diethyl ether (4 × 15 mL), water (5 × 15 mL), methanol

(5 mL), and diethyl ether (3 × 15 mL). The crude product was

recrystallized from ethylene glycol (40 mL), filtered off, washed with

methanol (3 × 8 mL), and dried in vacuo. To remove all ethylene

glycol, the substance was dissolved in boiling methanol (450 mL).

After cooling to room temperature, the solvent was evaporated at

1

reduced pressure to /₃of the initial volume. The suspension was

chilled in a freezer (−20 °C) for 4 h before the precipitate was filtered

off by suction, washed with cold methanol (10 mL), and dried in

vacuo at 120 °C. Yield: 0.55 g (30%). Anal. (C₁₅H₉N₃O₂): C, H, N.

3-(5-Methylbenzoxazol-2-yl)-1H-quinoxalin-2-one (4i). To a

solution of 3e (10.3 g 27.3 mmol) in ethanol (96%, 50 mL) was

added a solution of 1,2-phenylendiamine (3.98 g, 36 mmol) and

sodium acetate (6.76 g, 81 mmol) in a mixture of ethanol (45 mL) and

water (25 mL). The red solution formed was allowed to stand under

argon and light protection at room temperature for 20 h. The

precipitate was filtered off by suction and washed with ethanol (2 × 10

mL), water (4 × 25 mL), ethanol (10 mL), and diethyl ether (3 × 25

mL). The product was dried in vacuo and used without further

purification in the next step. Yield: 4.78 g (63%).

3-(6-Methylbenzoxazol-2-yl)-1H-quinoxalin-2-one (4j). 3f (4.53 g,

12 mmol), 1,2-phenylenediamine (1.77 g, 16 mmol), and sodium

acetate (2.09 g, 25 mmol) were dissolved in a mixture of ethanol (96%,

40 mL) and water (10 mL). After 30 min sonication, the reaction

mixture was stirred under argon and light protection at room

temperature for 70 h. The solid was filtered off by suction and washed

with ethanol (2 × 12 mL), water (3 × 15 mL), ethanol (10 mL), and

diethyl ether (3 × 12 mL). The dried crude product was recrystallized

3-(5-Chlorobenzothiazol-2-yl)-6,7-dimethyl-1H-quinoxalin-2-one

(4o). 6b (0.87 g, 4.0 mmol) and 2-amino-4-chloro-thiophenol (0.73 g,

4.4 mmol) were suspended in ethanol (96%, 40 mL) and refluxed for

75 min. Then hydrochloric acid (37%, 0.59 mL, 7 mmol) was added,

and refluxing continued for 18 h. After cooling to ca. 50 °C, the crude

product was filtered off by suction and washed with ethanol (2 × 10

mL) and water (4 × 15 mL). Subsequently, the orange material was

suspended in water (40 mL) and refluxed under oxygen atmosphere

for 2.5 h. Then the yellow solid was filtered off from the hot

suspension and washed with boiling water (3 × 15 mL). Finally, the

product was dried in vacuo over phosphorus pentoxide and used for

complexation without further purification. Yield: 1.06 g (77%).

General Procedure B. A mixture of the quinoxalinone-ligand (1

equiv) and 7a or 7b (0.5 equiv) in absolute ethanol (7 mL/mmol

ligand) was stirred at 70 °C for 45 min. Then hydrochloric acid (37%,

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0.25 equiv) was added and stirring at 70 °C continued for further 7 h.

Subsequently, the mixture was cooled and allowed to stand in a freezer

(−20 °C) overnight. The precipitate was filtered off by suction and

washed with diethyl ether (4 × 15 mL/mmol ligand). The crude

product was dried in vacuo, dissolved in hydrochloric acid (10 mM,

varying amounts), the solution filtered to remove solid impurities, the

filtrate immediately frozen, and then lyophilized.

mL) was added. The precipitate was filtered off and washed with

diethyl ether (3 × 7 mL). The crude material was treated with

dichloromethane (60 mL), the solution was separated from

undissolved material by filtration, and the filtrate was poured into

diethyl ether (170 mL). The solid formed was filtered off, washed with

diethyl ether (10 mL), and dried in vacuo. The substance was

dissolved in hydrochloric acid (10 mM, 120 mL) and the solution

filtered. The filtrate was immediately frozen, and lyophilized. Yield:

0.26 g (38%). Anal. (C₂₅H₂₂Cl₄N₄ORu·2.5H₂O): C, H, N, Cl.

T-4-R/S-[3-(1H-Benzimidazol-2-yl-κN³′)-6,7-dimethyl-1H-quinox-

alin-2-one-κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-

ruthenium(II) Chloride (8e). A mixture of 4e (0.12 g, 0.4 mmol) and

7a (0.12 g, 0.2 mmol) in absolute ethanol (4 mL) was stirred at 70 °C

for 1 h, allowed to cool to room temperature, and stirred for a further

1.5 h. Then dry diethyl ether (10 mL) was added under vigorous

stirring. Then 20 min later, the precipitate was filtered off and washed

with diethyl ether (3 × 10 mL). The crude product was dried in vacuo,

dissolved in hydrochloric acid (10 mM, 80 mL), and the solution

filtered. The filtrate was immediately frozen and lyophilized. Yield:

0.18 g (70%). Anal. (C₂₇H₂₈Cl₂N₄ORu·2.5H₂O): C, H, N, Cl.

T-4-R/S-Chlorido-[3-(5,6-dichloro-1H-benzimidazol-2-yl-κN³′)-

6,7-dimethyl-1H-quinoxalin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methyl-

benzene)-ruthenium(II) Chloride (8f). The complex was prepared

according to general procedure B starting from 4f (0.29 g, 0.8 mmol)

and 7a (0.25 g, 0.4 mmol) and by using hydrochloric acid (10 mM,

150 mL) for lyophilization. Yield: 0.31 g (54%). Anal.

(C₂₇H₂₆Cl₄N₄ORu·2.5H₂O): C, H, N, Cl.

T-4-R/S-Chlorido-[6,7-dichloro-3-(5,6-dimethyl-1H-benzimidazol-

2-yl-κN³′)-1H-quinoxalin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylben-

zene)-ruthenium(II) Chloride (8g). A mixture of 4g (0.15 g, 0.4

mmol) and 7a (0.12 g, 0.2 mmol) in absolute ethanol (5 mL) was

stirred under argon at 70 °C for 2.5 h. After cooling to room

temperature, acetone (12 mL) was added. The solid was filtered off

and washed with acetone (2 × 7 mL) and diethyl ether (3 × 10 mL).

The dried crude material was dissolved in hydrochloric acid (10 mM,

120 mL) at 70 °C and insoluble impurities removed by filtration. The

filtrate was frozen and lyophilized. Yield: 0.14 g (51%). Anal.

(C₂₇H₂₆Cl₄N₄ORu·H₂O): C, H, N, Cl.

General Procedure C. A mixture of the quinoxalinone-ligand (1

equiv) and 7a or 7b (0.5 equiv) in absolute ethanol (8 mL/mmol

ligand) was stirred at 70 °C for 45 min. After the addition of

hydrochloric acid (37%, 0.25 equiv), stirring at 70 °C was continued

for a further 5.5 h. The mixture was cooled to −20 °C, and absolute

diethyl ether (varying amounts) was added under vigorous stirring.

The suspension was allowed to stand at −20 °C for 1 h before the

precipitate was filtered off by suction and washed with diethyl ether (4

× 15 mL/mmol ligand). The crude product was dried in vacuo,

dissolved in hydrochloric acid (10 mM, varying amounts), and the

solution filtered. The filtrate was immediately frozen and lyophilized.

T-4-R/S-[3-(1H-Benzimidazol-2-yl-κN³′)-1H-quinoxalin-2-one-

κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II)

Chloride (8a). To a suspension of 4a (0.26 g, 1.0 mmol) in absolute

ethanol (10 mL) was added 7a (0.25 g, 0.5 mmol). The mixture was

stirred at room temperature for 15 min. Then the turbid dark-red

solution was filtered. After the addition of absolute ethanol (5 mL)

and diisopropyl ether (20 mL), the mixture was refluxed for 15 min

and allowed to cool to room temperature. Subsequently, the reaction

mixture was placed in a freezer overnight. The solid formed was

filtered off by suction and washed with diethyl ether (3 × 12 mL).

After drying, the crude product was dissolved in water (60 mL) and

the solution filtered. The filtrate was immediately frozen and

lyophilized. Yield: 0.28 g (45%). Anal. (C₂₅H₂₄Cl₂N₄ORu·2H₂O): C,

H, N, Cl.

T-4-R/S-Chlorido-[3-(5,6-dichloro-1H-benzimidazol-2-yl-κN³′)-

1H-quinoxalin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylbenzene)-

ruthenium(II) Chloride (8b). A mixture of 4b (0.34 g, 1.0 mmol) and

7a (0.31 g, 0.5 mmol) in absolute ethanol (10 mL) was allowed to stir

at room temperature for 30 min. Then the mixture was heated to

boiling and immediately filtered. Addition of diethyl ether to the

cooled filtrate resulted in precipitation of the product, which was

filtered off and washed with diethyl ether (3 × 12 mL). The crude

solid was dissolved in dichloromethane (80 mL), the solution filtered,

and the product precipitated again by addition of diethyl ether (150

mL). After washing with diethyl ether (25 mL) and drying in vacuo,

the substance was dissolved in hydrochloric acid (10 mM, 80 mL) and

the solution filtered. The filtrate was immediately frozen, and

lyophilized. Yield: 0.25 g (57%). Anal. (C₂₅H₂₂Cl₄N₄ORu·2H₂O): C,

H, N, Cl.

T-4-R/S-Chlorido-[3-(5,6-dimethyl-1H-benzimidazol-2-yl-

κN³′)1H-quinoxalin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylbenzene)-

ruthenium(II) Chloride (8c). A mixture of the 4c (0.20 g, 0.70 mmol)

and 7a (0.21 g, 0.35 mmol) in absolute ethanol (5 mL) was stirred at

70 °C for 30 min, forming a pulp. After the addition hydrochloric acid

(37%, 0.009 mL, 0.1 mmol), stirring at 70 °C was continued for 8.5 h.

To reduce the viscosity, the pulp was sonicated and shaken from time

to time. Subsequently, the thick suspension was cooled and allowed to

stand in the refrigerator at 2 °C for 4 h. The solid was filtered off and

washed with absolute ethanol (3 mL) and diethyl ether (4 × 10 mL).

The crude product was dried in vacuo, dissolved in water (220 mL),

and the solution filtered. The filtrate was immediately frozen, and

lyophilized. This purification step was repeated using water (100 mL).

Yield: 0.25 g (57%). Anal. (C₂₇H₂₈Cl₂N₄ORu·0.7H₂O): C, H, N, Cl.

T-4-R/S-[3-(1H-Benzimidazol-2-yl-κN³′)-6,7-dichloro-1H-quinoxa-

lin-2-one-κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-

ruthenium(II) Chloride (8d). A mixture of 4d (0.33 g, 1.0 mmol) and

7a (0.31 g, 0.5 mmol) in absolute ethanol (10 mL) was allowed to stir

at room temperature for 30 min and then heated to 70 °C for 10 min.

When cooled to room temperature, activated charcoal (0.05 g) was

added, whereupon the mixture was stirred at room temperature for 90

min. Then the charcoal was removed by filtration through a small

column with cellulose powder (1.5 g). The filtrate was concentrated to

2.5 mL under reduced pressure, and under sonication diethyl ether (25

T-4-R/S-[3-(Benzoxazol-2-yl-κN³′)-1H-quinoxalin-2-one-κN⁴]-

chlorido-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II) Chloride

(8h). The complex was prepared according to general procedure B

starting from 4h (0.11 g, 0.4 mmol) and 7a (0.12 g, 0.2 mmol) and by

using hydrochloric acid (10 mM, 20 mL) for lyophilization. The

complex was dissolved in water (20 mL), filtered, the filtrate

immediately frozen, and lyophilized again. Yield: 0.15 g (62%). Anal.

(C₂₅H₂₃Cl₂N₃O₂Ru·2H₂O): C, H, N, Cl.

T-4-R/S-Chlorido-[3-(5-methylbenzoxazol-2-yl-κN³′)-1H-quinoxa-

lin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II)

Chloride (8i). The complex was prepared according to procedure C

starting from 4i (0.17 g, 0.6 mmol) and 7a (0.19 g, 0.3 mmol). Diethyl

ether (10 mL) was used for precipitation and hydrochloric acid (10

mM, 50 mL) for lyophilization. Yield: 0.30 g (80%). Anal.

(C₂₆H₂₅Cl₂N₃O₂Ru·2H₂O): C, H, N, Cl.

T-4-R/S-Chlorido-[3-(6-methylbenzoxazol-2-yl-κN³′)-1H-quinoxa-

lin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II)

Chloride (8j). The complex was prepared according to general

procedure B starting from 4j (0.22 g, 0.70 mmol) and 7a (0.22 g, 0.35

mmol) and by using hydrochloric acid for lyophilization (10 mM, 40

mL). Yield: 0.37 g (84%). Anal. (C₂₆H₂₅Cl₂N₃O₂Ru·2H₂O): C, H, N,

Cl.

T-4-R/S-[3-(Benzoxazol-2-yl-κN³′)-6,7-dimethyl-1H-quinoxalin-2-

one-κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II)

Chloride (8k). The complex was prepared according to general

procedure B starting from 4k (0.18 g, 0.6 mmol) and 7a (0.19 g, 0.3

mmol) and by using hydrochloric acid for lyophilization (10 mM, 100

mL). Yield: 0.32 g (77%). Anal. (C₂₇H₂₇Cl₂N₃O₂Ru·3H₂O): C, H, N,

Cl.

T-4-R/S-[3-(Benzothiazol-2-yl-κN³′)-1H-quinoxalin-2-one-κN⁴]-

chlorido-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II) Chloride

(8l). The complex was prepared according to general procedure B

starting from 4l (0.11 g, 0.4 mmol) and 7a (0.12 g, 0.2 mmol) and by

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using hydrochloric acid for lyophilization (10 mM, 25 mL). Yield: 0.18

g (66%). Anal. (C₂₅H₂₃Cl₂N₃ORuS·1.6H₂O): C, H, N, S, Cl.

T-4-R/S-[3-(Benzothiazol-2-yl-κN³′)-6,7-dimethyl-1H-quinoxalin-

2-one-κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium-

(II) Chloride (8m). The complex was prepared according to general

procedure B starting from 4m (0.12 g, 0.4 mmol) and 7a (0.12 g, 0.2

mmol) and by using hydrochloric acid for lyophilization (10 mM, 400

mL). The lyophilizate was dissolved in dichloromethane (130 mL),

insoluble impurities removed by filtration, and the filtrate evaporated

to dryness under reduced pressure. The residue was dissolved in water

(120 mL), filtered, the filtrate immediately frozen, and lyophilized

again. Yield: 0.12 g (46%). Anal. (C₂₇H₂₇Cl₂N₃ORuS·2H₂O): C, H, N,

S, Cl.

AUTHOR INFORMATION

■

Corresponding Author

*Phone: +431427752600. Fax: +431427752680. E-mail:

vladimir.arion@univie.ac.at (V.B.A.); bernhard.keppler@

univie.ac.at (B.K.K.).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS

■

The authors are indebted to the FFG (Austrian Research

Promotion Agency, project no. 811591 FA526003) and to the

Austrian Council for Research and Technology Development

and COST (European Cooperation in the Field of Scientific

and Technical Research) for financial support. We also thank

Anatoly Dobrov for ESI mass spectra measurements, Wolfgang

Kandioller for NMR measurements, and Alexander Egger for

carrying out some of the MTT assays. Irene Herbacek

(Institute of Cancer Research, Department of Medicine I,

Medical University of Vienna) is gratefully acknowledged for

flow cytometry analyses of cell cycle effects, and Prof. Verena

Dirsch and Daniel Schachner (both Department of Pharma-

cognosy, University of Vienna) for providing FACS equipment

and technical assistance for flow cytometric cell death analyses.

T-4-R/S-Chlorido-[3-(5-chlorobenzothiazol-2-yl-κN³′)-1H-quinox-

alin-2-one-κN⁴]-(η⁶-1-isopropyl-4-methylbenzene)-ruthenium(II)

Chloride (8n). The complex was prepared according to general

procedure B starting from 4n (0.13 g, 0.4 mmol) and 7a (0.12 g, 0.2

mmol) and by using hydrochloric acid for lyophilization (10 mM, 200

mL). Yield: 0.21 g (82%). Anal. (C₂₅H₂₂Cl₃N₃ORuS·H₂O): C, H, N,

S, Cl.

T-4-R/S-Chlorido-[3-(5-chlorobenzothiazol-2-yl-κN³′)-6,7-dimeth-

yl-1H-quinoxalin-2-one-κN⁴]-(η6−1-isopropyl-4-methylbenzene)-

ruthenium(II) Chloride (8o). The complex was prepared according to

general procedure B starting from 4o (0.14 g, 0.4 mmol) and 7a (0.12

g, 0.2 mmol) and by using hydrochloric acid for lyophilization (10

m M , 7 0 m L ) . Y i e l d : 0 . 1 9

g

( 7 0 % ) . A n a l .

(C₂₇H₂₆Cl₃N₃ORuS·1.5H₂O): C, H, N, S, Cl.

T-4-R/S-[3-(1H-Benzimidazol-2-yl-κN³′)-1H-quinoxalin-2-one-

κN⁴]-chlorido-(η⁶-1-isopropyl-4-methylbenzene)-osmium(II) Chlor-

ide (9a). The complex was prepared according to procedure C

starting from 4a (0.11 g, 0.4 mmol) and 7b (0.16 g, 0.2 mmol).

Diethyl ether (70 mL) was used for precipitation and hydrochloric

acid (10 mM, 50 mL) for lyophilization. Yield: 0.13 g (46%). Anal.

(C₂₅H₂₄Cl₂N₄OOs·2H₂O): C, H, N.

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A SAR study of novel antiproliferative ruthenium and osmium complexes with quinoxalinone ligands in human cancer cell lines

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