Synthesis, Modification, and Biological Evaluation of a Library of Novel Water-Soluble Thiopyridone-Based Organometallic Complexes and Their Unexpected (Biological) Behavior

A Journal of

Accepted Article

Title: Synthesis, Modification, and Biological Evaluation of a Library

of Novel Water-Soluble Thiopyridone-Based Organometallic

Complexes, and Their Unexpected (Biological) Behavior

Authors: Sophia Harringer, Barbara Happl, Marius Ozenil, Caroline

Kast, Michaela Hejl, Debora Wernitznig, Anton Legin,

Andreas Schweikert, Natalie Gajic, Alexander Roller, Gunda

Koellensperger, Michael A Jakupec, Wolfgang Kandioller, and

Bernhard K Keppler

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content of this Accepted Article.

To be cited as: Chem. Eur. J. 10.1002/chem.201905546

Link to VoR: http://dx.doi.org/10.1002/chem.201905546

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Synthesis, Modification, and Biological Evaluation of a Library of

Novel Water-Soluble Thiopyridone-Based Organometallic

Complexes, and Their Unexpected (Biological) Behavior

Sophia Harringer,^[a]Barbara Happl,^{[a, b, c]}Marius Ozenil,^[c]Caroline Kast,^[a]Michaela Hejl,^[a]Debora

Wernitznig,^[a]Anton Legin,^[a]Andreas Schweikert,^[d]Natalie Gajic,^[a]Alexander Roller,^[a]Gunda

Koellensperger,^[d]Michael A. Jakupec,^{[a, e]}Wolfgang Kandioller,^{*[a, e]}and Bernhard K. Keppler^{[a, e]}

Abstract:

A

series of 16 dinuclear thiopyridone-based

6% weight loss.^[3]In later studies this lead compound was

replaced by its sodium analogue BOLD-100 (formerly IT-139 and

NKP-1339; Chart 1 ) due to superior solubility of the latter, which then

showed activity in patients with advanced solid tumors, such as non-

small cell lung cancer, colorectal carcinoma and gastrointestinal

neuroendocrine tumors, in a clinical phase I/II study.^[7]Reversible

adduct formation with blood proteins is thought to foster

accumulation in tumor tissue. Therefore, the proposed mode of

action is based on binding to non-classic targets and the

exploitation of the enhanced permeability and retention (EPR)

effect.^[4,8]Due to its fast growth rate, tumor tissue lacks proper

vascular architecture with enhanced permeability allowing for

sufficient nutrient supply, whereas a lack of lymphatic drainage

leads to enhanced retention of (macro-)molecules inside the

tumor cells.^[9]Furthermore, BOLD-100 was suggested to interact

with the cell’s protein machinery, and the endoplasmic

organometallics with excellent water-solubility, increased stability and

remarkable cytotoxicity were synthesized and characterized. The

complexes of this work formed dimeric species featuring a double

positive charge in polar protic solvents, accounting for their

outstanding solubility in aqueous solution. Most of them displayed

higher antiproliferative activity than their parental thiomaltol complex,

with unexpected cytotoxicity trends depending on the employed metal

center, ligand modification, and cell line. Insights into their behavior in

biological systems were gathered by means of amino-acid interaction

studies, cytotoxicity tests in 3D spheroid models, laser ablation,

cellular accumulation measurements, as well as cell cycle

experiments.

Introduction

reticulum’s (ER) chaperone GRP78,

a

glucose-regulated

protein.^[3]This protein is highly expressed in cancer cells to

regulate responses to ER stress, which is constitutively elevated

in cancer cells.^[10]

Much effort has been expended to develop alternatives to classic

platinum-based single agent or combination therapy due to

reoccurring clinical limitations, such as severe side effects or

ineffectiveness caused by drug resistance.^[1–3]Ruthenium-

containing compounds are amongst the most promising

candidates, as several ruthenium complexes show redox

behavior under physiological conditions, accumulate preferably in

tumor tissue and feature more coordination sites allowing

additional modifications compared to platinum(II) coordination

compounds.^[4]This field of research was tremendously stimulated

when the exceptional anticancer properties of trans-[tetrachlorido-

bis(1H-indazole)ruthenate(III)] (KP1019) were discovered.^[3,5,6]

This compound showed preclinical activity against autochthonous

colon cancer in a rat model with an efficacy of up to 95% reduction

of tumor volume at a well tolerable dose causing no more than

[a]

S. Harringer, B. Happl, C. Kast, M. Hejl, D. Wernitznig, A. Legin, N.

Gajic, A. Roller, Dr. M. A. Jakupec, Dr. W. Kandioller, Prof. Dr. Dr.

B. K. Keppler

Chart 1. Representatives of clinically studied ruthenium(III)-based

compounds, and most extensively investigated ruthenium(II) piano-stool

complexes.

Institute of Inorganic Chemistry, University of Vienna

Waehringer Strasse 42, 1090 Vienna (Austria)

E-mail: wolfgang.kandioller@univie.ac.at

B. Happl

[b]

[c]

The development of organometallic complexes brought to light

another class of ruthenium compounds, which contain the metal

in its +II oxidation state, stabilized via the introduction of a facially

coordinated arene ligand.^[7,11]These so-called “piano-stool”

complexes feature an arene ligand, resembling the stool’s seat

Ludwig Boltzmann Institute Applied Diagnostics, General Hospital of

Vienna

Waehringer Guertel 18–20, 1090 Vienna (Austria)

B. Happl, M. Ozenil

Department of Biomedical Imaging and Image-guided Therapy,

Division of Nuclear Medicine, Medical University of Vienna

Spitalgasse 23, 1090 Vienna (Austria)

[d]

[e]

A. Schweikert, G. Koellensperger

Department of Analytical Chemistry, University of Vienna

Waehringer Strasse 38, 1090 Vienna (Austria)

Research Cluster “Translational Cancer Therapy Research”

Waehringer Strasse 42, 1090 Vienna (Austria)

Supporting information for this article is given via a link at the end of

the document.

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and three coordination sites available for mono-, bi- or tridentate

ligands, mimicking the legs.^[12]Two representatives of this class

showed promising results in preclinical investigations, namely

RAPTA-C and RM175 (Chart 1 ).^[13]While RAPTA-C’s antiangiogenic

and antimetastatic activity is supposed to be pH-dependent, RM175

reduces tumor growth in vivo based on DNA interactions.^[14,15]

These findings specifically highlight the importance of the

coordination sphere for pharmacokinetic and pharmacodynamic

properties, which can be fine-tuned via proper ligand

modification.^[16]As complex stability is another fundamental

aspect for sufficient anticancer activity or lack thereof, a stable

coordination site might prevent premature reaction with

biomolecules and subsequent inactivation. This has been

demonstrated by the replacement of O,O-chelating pyrone

complexes by their S,O-derivatives yielding IC₅₀values in the low

to sub µM range.^[17,18]On the other hand, ruthenium(II) pyridone-

complexes showed increased stability in aqueous solution, which

can be explained by their stronger pseudo aromatic character

respective primary amines. Synthesis was performed according

to literature for 3-hydroxy-1,2-dimethyl-pyridin-4(1H)-one, 3-

hydroxy-2-methyl-1-phenylpyridin-4(1H)-one, and 1-benzyl-3-

hydroxy-2-methylpyridin-4(1H)-one.^[20]However,

a twentyfold

reaction time was applied in order to improve yields. These

optimized reaction conditions also enabled a one-step synthesis

of

3-hydroxy-2-methyl-1-(naphthalene-1-yl)pyridine-4(1H)-one

with yields of up to 74%. These results are superior to previously

published literature, where a three step reaction protocol with an

overall yield of 43% has been described.^[21]Subsequently the

synthesized pyridones were thionated by use of Lawesson’s

reagent under inert conditions yielding the desired S,O-ligands in

moderate to good yields (33-83%). Complexation was carried out

according to established literature procedures under inert

conditions by means of Schlenk technique to avoid undesired side

reactions.^[22,23]The respective thiopyridone was deprotonated

with sodium methoxide in dry methanol and the dimeric metal

precursor of choice was added to the solution. The applied

enhancing complex stability by at least 1.5 log units.^[19]In this work, reaction time is strongly dependent on the coordination motif.

we prove that the combination of thiomaltol and pyridone motifs

in a new class of organometallic thiopyridone complexes leads to

increased bioactivity as well as enhanced stability in a biologically

relevant pH range of 5.8–7.9. Furthermore, the impact of metal

center and ligand variation on the physicochemical and biological

properties was investigated. Apart from MTT assays in monolayer

cultures of human cancer cells, additional studies have been

carried out in order to gather insights into the behavior of these

compounds in biological systems (e.g. incubation with possible

target molecules, cellular accumulation, cell cycle analysis, and

cytotoxicity in multicellular tumor spheroid models).

While standard procedures for pyrone-complexes with an

O,O-coordination motif require at least several hours or even

overnight syntheses,^[24]thiopyrone-complexes featuring an

S,O-coordination motif react much faster with reported reaction

times of 3.5 h at most. Thus, the mixtures were stirred at room

temperature for 1–3.5 h, depending on the utilized ligand. Pure

complexes were obtained after work up and purification by

crystallization/precipitation from dichloromethane/diethyl ether, or

dichloromethane/n-hexane in moderate to excellent yields (33–

83%). In total a library consisting of 16 new thiopyridone-based

organometallics has been synthesized bearing 3-hydroxy-1,2-

dimethylpyridine-4(1H)-thione (a),

phenylpyridine-4(1H)-thione

3-hydroxy-2-methyl-1-

(b),

benzylpyridine-4(1H)-thione (c), or 3-hydroxy-2-methyl-1-

(naphthalene-1-yl)pyridine-4(1H)-thione (d) as chelating ligands.

The obtained complexes were characterized by ¹H, ¹³C and

2D NMR, ESI-MS measurements, elemental analysis, and single

crystal X-ray crystallography, where possible. ¹H, ¹³C and

2D NMR of all complexes recorded in deuterated dimethyl

sulfoxide d₆-DMSO, showed peaks according to the expected

monomeric species (Figures S7-38). Successful complexation

could be confirmed by lowfield field shifts of the ligand’s proton

signals (-0.03 ppm) and vanishing of the hydroxygroup’s signal.

Four signals could be expected for the aromatic protons of the

p-cymene arene due to the presence of a chiral metal center.

However, in accordance with literature, only two doublets could

be observed for the aromatic η⁶-p-cymene protons.^[25–27]None of

Results and Discussion

Scheme 1. Overview of synthesized thiopyridone-based piano-stool

complexes. (i) methylamine, water and reflux overnight (1a), or aniline,

benzylamine,

or

1-naphthylamine,

0.38 M

HCl

and

microwave

irradiation (t=30 min, T=165 °C) (1b-d); (ii) Lawesson’s reagent, toluene abs.,

Schlenk technique, reflux 4-16h; (iii) [(p-cym)/(Cp*)MCl₂]₂, NaOMe, MeOH abs.,

40 °C, 1-3.5h.

the

phenylic

protons

of

the

3-hydroxy-2-methyl-1-phenylpyridine-4(1H)-thione (1b)

complexes become chemically equivalent due to the proximity

and resulting sterical hindrance caused by the neighboring methyl

group. The spectrum changes drastically when employing a

The compound library was designed in order to optimize

pharmacokinetic and pharmacodynamic properties via variation

of the metal center, and substitution of the thiopyridone

scaffold (Scheme 1 ). Generally, the ligands were chosen to cover

a broad range of characteristics, such as lipophilicity and steric

volume. In this synthetic route, commercially available maltol was

converted to four different pyridones by treatment with the

3-hydroxy-2-methyl-1-benzylpyridine-4(1H)-thione (1c)

ligand

instead, where benzylic protons become chemically equivalent

and form a doublet and a triplet signal. This can be explained by

the bridging CH₂group which enables more degrees of freedom

for this ligand system compared to its aniline congener. The

complexes prepared in this work showed exceptionally good

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aqueous solubility (up to 10 mg/mL), thus ¹H-, ¹³C-, and 2D-NMR

sets of ruthenium 3a–c and osmium compounds 4a–c were

recorded in deuterium oxide (Figures S39–50). However, Rh and

Ir complexes exhibited limited water solubility. Therefore, only the

respective NMRs of Rh methyl and benzyl complexes (5a, 5c)

and Ir methyl compound 6a were recorded in D₂O (Figures S51–

56). ¹H-NMR spectra of organoruthenium(II) and -osmium(II)

complexes show two η⁶-p-cymene methylene signals and four

aromatic signals compared to one methylene signal and two

aromatic signals in d₆-DMSO. Herein, we propose the formation

of a dimeric complex under polar protic conditions (denoted with

* at the respective compound abbreviation henceforth). In

¹H-NMR spectra the protons of the methylene groups Hg form two

doublets with an integral of 6H and a geminal coupling constant

of ²J(H,H) = 15 Hz confirms their chemical inequality. Additionally,

a DACH effect can be observed for the methylene signals as well

as the aromatic signals, accounting for the deviation from the

expected 1:1 signal intensity. Furthermore, the four sharply

resolved aromatic signals in polar protic solvents compared to two

doublets in polar aprotic solvents can be explained by the

presence of a dimeric species. As the dimeric, thiolato-bridged

complex is expected to be more rigid it can prevent signal

broadening, which is usually caused by dynamic effects according

to literature.^[26]In this case, the proximity of the two p-cymene

arenes hinders their rotation, reducing symmetry and leading to

signal separation. Theoretically, 2²=4 stereoisomers should be

observed for these dimers, as a result of the two stereogenic

metal centers. However, the total number is reduced to three,

because the (R,S) and (S,R) configurations are identical due to

the molecular symmetry. The thiopyridone ligands face into the

same direction in the (R,R) and (S,S) conformation, enabling π-

stacking. Consequently, these diastereomers should be

thermodynamically more stable compared to the (R,S)≡(S,R) form,

which was also supported by means of crystal structure analysis

(see X-ray discussion). NMR spectra show only one set of signals

for complexes 3 a–d and 4 a–d in d₆-DMSO, but at least two

different configurations for 3d* and 4d* can be observed for

spectra measured in D₂O (Figure 2 ). There are several possible

explanations for this phenomenon. First of all, it is possible that

the formation of the dimeric complex is not complete and some

complex is still present in its monomeric form. Secondly, it is

possible that the bulky naphthyl ligand favors the (R,S)≡(S,R)

configuration. Finally, the proximity of the sterically demanding

ligands could lead to the formation of rotamers in the (R,R) and

(S,S) conformation, due to the naphthyl moieties limited rotation.

numerous publications based on pyrone-, pyridone-, and

thiopyridone ruthenium and osmium piano-stool complexes

dimerization as observed in this work has not been described

yet.^{[17–19,24,26,32]}Therefore, the bridging effect may be attributed to

the N-substituted backbone of the new thiopyridone ligands.

Compared to maltol and thiomaltol analogues the electron density

in the ligand system is enhanced, due to the stronger +M effect of

the introduced amine functionality. Additionally, in accordance

with the HSAB principle the transition metals’ affinity is expected

to be higher towards sulfur than oxygen, because of its softer

character. The combination of these two effects may be a crucial

factor for the formation of the discovered thiolato bridges. The

dimeric species is connected via two thiolato-bridges, resulting in

a double positive charge which would furthermore account for the

exceptional solubility in water (Figure 1 ).

ESI-MS investigations showed peaks according to [M-Cl]⁺

fragments for all synthesized complexes. As mass spectra show

m/z peaks it is impossible to distinguish the monomeric and

dimeric species by their mass peaks only. Nevertheless, clear

differences should be visible in their metal patterns, which clearly

show a monomeric species. These findings are counterintuitive,

as

samples

are

prepared

in

aqueous

solution

(ACN/MeOH+1% H₂O), which should lead to formation of the

dimeric complex. However, it is likely that the dimer is cleaved

upon electrospray ionization and disintegrates into two

monomeric fragments.

Figure 1. General structure of the proposed dimers featuring a double positive

charge under polar protic conditions.

X-ray diffraction analysis

Single crystals of 3a–d, 4a–d, 5b, 6b, and 6d suitable for X-ray

diffraction analysis were obtained by slow diffusion method from

dichloromethane/diethyl

ether (3b,

3c,

4a,

4b),

dichloromethane/n-hexane (3d, 4c, 4d, 5b*, 6b*, 6d),

methanol/diethyl ether (3b*, 3c*, 4a*) or dichloromethane/ethyl

acetate (3a*). Five monomeric (3d, 4b–d, 6d), three dimeric (3a*,

5b*, 6b*) and three structures for both configurations (3b–c, 3b*–

c*, 4a, 4a*) could be collected. Monomeric crystal structures

obtained from polar aprotic solvents confirm the adaption of a

‘piano-stool’ configuration, with the arene (p-cymene or Cp*)

constituting the stool’s seat, and the bidentate chelate

(thiopyridone) and the chloride-leaving group resembling its legs.

Crystals grown in polar protic solvents confirm the findings of our

NMR measurements that thiopyridone-based organometallic

complexes form dimers under these conditions. The most

important parameters for complexes 3b (ruthenium featuring a

thioaniline ligand), and 4a (osmium coupled to thiodeferiprone) as

Generally, thiolato-bridged dimerization has already been

reported for organoruthenium complexes.^[28–31]However, despite

Figure 2. ¹H-NMR aromatic p-cymene signal comparison of Ru(II) complexes

3c*, 3d* and Os(II) derivative 4d* in D₂O

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well as the respective dimers 3b* and 4b* are summarized in

Table 1 , and their structures are shown in Figure 3 . While

Ru-complex 3b crystallizes in monoclinic space groups in the

monomeric (P2₁/n) and 3b* (C2/c) form, monomeric

Os-compound 4a crystallizes in the monoclinic space group C2/c

and the respective dimer 4a* in the triclinic space group P1.

According to expectations, C=S bond lengths are increased upon

Table 1. Selected bond lengths of Ru^IIand Os^IIcompounds 3b, and 4a as well

as their dimeric counterparts 3b* and 4a*.

3b

3b*

4a

4a*

M-S [Å]

2.366

-

2.074

2.439

1.719

1.309

2.372

2.414

2.091

-

1.755

1.313

1.678

3.566

12.5

2.366

-

2.398

2.437

2.085

-

1.773

1.321

1.680

3.550

23.5

M-O [Å]

M-Cl [Å]

C=S [Å]

C-O [Å]

π-plane centroid [Å] 1.654

M-M

2.082

2.448

1.736

1.320

1.649

-

dimerization

(3b/3b*: 1.719Å/1.755

Å,

and

4a/4a*: 1.736 Å/1.773 Å). This indicates a higher stability of the

four-membered dimeric scaffold, featuring two sulfur atoms and

two metal centers. On the other hand, M-O, and C-O distances

showed only a slight increase of around 0.011 Å in 3b* and

0.017 Å in 4a*. Additionally, the arene π-plane distance increases

approximately the same, with 0.024 Å (3b*), and 0.026 Å (4a*),

respectively. Compared to reported values for the respective

Os-thiomaltol parent compound, the M-S bond (4a: 2.366,

Os-TM: 2.378) and π-plane centroid distance (4a: 1.649, Os-TM:

1.662) are slightly shorter, while the M-Cl bond is slightly longer

in compound 4a, indicating higher stability of the thiopyridone

counterparts (4a: 2.488 Å, Os-TM: 2.436 Å).^[18]On the other hand,

the distances between M-O, and C-O are approximately the same.

-

Torsion M-S-M [°]

-

Stability in aqueous solution

In order to investigate complex stabilities, UV/Vis

spectrophotometry was conducted for all complexes. All

ruthenium (3a*-d*), rhodium (5a*-d*), and iridium (6a*-d*)

compounds were stable over 24 h in water at 25 °C. UV/Vis

spectra showed a slight decrease in complex concentration,

which can be attributed to slow precipitation of the product from

the UV/Vis cuvette over time (Figures S57-72). However,

osmium complexes 4a*-d* showed decomposition over time, with

UV/Vis spectra showing isosbestic points at 220 nm, 270 nm,

300 nm, and 370 nm respectively (Figures S61-64). This process

is fastest for complex 4b* followed by complexes 4a* and finally

4c* in PBS. Decomposition of the osmium compounds was

slightly slower in pure water (Figure 4 ).

Figure 4. UV/Vis spectra of complex 4b* in PBS (A) and H2O (B)

measured over 24h.

In order to investigate a possible pH influence on complex-stability

and to gather further insights into the instability of the osmium

complexes, UHPLC kinetic measurements were carried out at pH

values of 5.8, 6.2, 6.7, 7.2 and 7.9 in phosphate buffered solution

via UHPLC. Due to its biological relevance this pH area is of particular

interest, as the reported blood pH value is 7.4 and the acidic

extracellular milieu of solid tumors is often 0.5-1.0 units lower than in

healthy tissue.^[27,33]Solutions of all complexes (500 µM in 20 mM

phosphate buffer, pH 7.2) and of complexes 3a*-d*, 4c*, 5b*, and

6c* (500 µM in 20 mM phosphate buffer, pH 5.8, 6.2, 6.7, and 7.9)

were incubated at 25 °C over 24 h and periodically analyzed via

gradient UHPLC runs on a C18-RP column. Due to pump pressure

fluctuations the retention times tended to shift ±0.18 min. The

investigated ruthenium, rhodium and iridium complexes (3a*-d*, 5b*,

and 6c*) proved to be stable over a wide range of biologically relevant

pH values without any degradation or side-product formation (Figure

5 , Figures S73-101). This is in accordance to expectations as the

Figure 3. ORTEP views of monomeric Ru complex 3b (top left), and Os

complex 4a (top right) as well as their dimeric congeners 3b* (bottom left) and

4a* (bottom right).

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experimental conditions in aqueous solution should induce

formation of the thiolato-bridged dimers.

(N-Ac-Met, N-Ac-Cys, and N-Ac-His) at 37 °C over 168 h and

measured in 24 h intervals via HPLC runs (Figures S102-105).

ESI-MS analysis revealed N-Ac-Cys adduct formation for all

analyzed complexes, and a N-Ac-Met adduct with ruthenium

complex 3c to a lesser extent. All adduct peaks could already be

found at 0 h, and HPLC chromatograms and ESI-MS analyses

showed no additional adduct formation over time. In addition

ESI-MS spectra of 3c showed peaks with a dimeric metal pattern,

according

to

[((p-cym)Ru)₂L(N-Ac-Cys)₂]²⁺

(m_exp513.5,

m_calc= 513.7), and [((p-cym)Ru)₂L(N-Ac-Met)]⁺(m_exp891.1,

m_calc= 892.1) fragments, while the spectra for osmium complex

Figure 5. UHPLC stability investigation of complex 3c* after 0 h (A; 5.15

min) and 48 h (B; 4.93 min) at pH 7.2 in phosphate buffer, proving stability

of the complex under these conditions. Minor shifts in retention time are

due to fluctuating pump pressure.

4c

revealed

the

preferential

formation

of

a

[((p-cym)Os)₂L(N-Ac-Cys)₂]²⁺(m_exp601.6, m_calc= 601.8) adduct

(Figure 6 ). Furthermore, [((Cp*)Rh)₂L(N-Ac-Cys)₂]²⁺(m_exp515.6,

m_calc= 514.9)

and

[((Cp*)Rh)₂L(N-Ac-Cys)]⁺

(m_exp867.1,

m_calc= 869.8) fragments could be observed for rhodium complex

However, in aqueous solution decomposition of the osmium

compound 4c* was observed. At pH 7.2 and 7.9 formation of a

second species was observed after 24 h. On the other hand, at

pH 5.8, 6.2 and 6.7 complex 4c* was stable over 24 h, which

5c, and iridium complex 6c revealed formation of

[((Cp*)Ir)₂L(N-Ac-Cys)]²⁺

(m_exp523.1,

m_calc= 523.2)

and

[((Cp*)Ir)₂L(N-Ac-Cys)₂]²⁺(m_exp604.7, m_calc= 604.8) adducts

respectively. Overall the [((Ar)M)₂L(N-Ac-Cys)₂]²⁺species was the

most abundant species for all analyzed complexes. On the other

hand, no adduct formation with N-Ac-His was observed. All

adducts are based on preceding dissociation of one thiopyridone

ligand, which seems to be a crucial step enabling amino acid

interaction. These findings indicate a very high affinity of

thiopyridone complexes towards sulfur containing amino acids

rather than nitrogen containing amino acids such as L-histidine

under biologically relevant conditions. Furthermore, they are in

accordance with previously published results where preferential

reactivity of pyrone- and pyridone-based ruthenium(II) complexes

towards L-cysteine and L-methionine was reported.^[34]Generally,

the dimeric complexes of this work showed unexpectedly high

affinity towards sulfur containing amino acids as adduct formation

was observed immediately, and adduct ratios showed only minor

changes over time. This behavior strongly deviates from the

reactivity of monomeric thiomaltol derivatives, where adduct

formation was only observed after 3–24 h respectively.^[18]These

findings prove once more the importance of the attached ligand

scaffolds for biological activity and reactivity of piano stool

complexes, as minor variations thereof may cause major changes

in biological studies. Furthermore, it is impossible to do

extrapolations between related ligand systems, and in-depth

examination is crucial in order to elucidate a possible mode of

action or activation.

indicates

a

pH dependent stability in the case of

osmium-thiopyridone complexes. In order to elucidate the fate of

complex 4c* and a possible concentration effect on its stability,

additional NMR and MS experiments were carried out. NMR

experiments at a concentration of 6 mg/mL in either d₆-DMSO or

1

D₂O indicated no decomposition over 24 h. Therefore, H-NMR

kinetics of 4c* in D₂O/1% d₆-DMSO (starting concentration

6 mg/mL) were measured with no visible changes over 24 h.

Consequently, the sample was diluted 1:1 (3 mg/mL) and kinetics

were measured over 24 h a second time. This step was repeated

six times in total (after 24 h, 48 h, 72 h, 96 h, 168 h and, 192 h;

resulting in a final concentration of 176 µM). At a dilution of

375 µg/mL formation of a second species became apparent,

indicated by an additional septet at 2.77 ppm with a 1:4 intensity.

Additionally, the formation of a third methylene signal with a 1:1.5

intensity at 1.08 ppm could be observed. It was impossible to

measure 2D-NMR sets throughout the experiment, due to the low

sample concentration. Instead HR ESI-MS analysis of the 176 µM

sample after 168 h was carried out in order to gather further

insights into the nature of the newly formed species. Based on the

combined findings, we propose that the decomposition of 4c* is

initiated by cleavage of the arene moiety, indicated by the

formation of [OsL(CH₃OH)]⁺(m_exp= 454.0133, m_calc= 454.0517),

[OsLCl]^-(m_exp= 454.0176,

[OsL(Cl)(CH₃O)]^-(m_exp= 484.0283,

m_calc= 453.9910),

m_calc= 484.0070), and

[OsLCl₂(CH₃OH)]^-(m_exp= 520.0036, m_calc= 520.9860) fragments

respectively. These findings suggest a completely different

activation mode for osmium(II) thiopyridone complexes where the

p-cymene moiety seems to act as the leaving group.

[((p-cym)Os)₂L(N-Ac-Cys)₂]²⁺

Investigation of amino acid interaction by HPLC-MS

An important factor for side-effects and drug inactivation is the

premature interaction of administered anticancer agents with

biomolecules on the way to the target site. In order to acquire

details about the reactivity of thiopyridone organometallics

towards possible binding partners, complexes 3c, 4c, 5c, and 6c

were incubated in phosphate buffered solutions at pH 7.2 with N-

acetyl-L-methionine, N-acetyl-L-cysteine, and N-acetyl-L-histidine

Figure 6. MS spectrum of osmium complex 4c after amino acid incubation

for 0 h. The formation of a [((p-cym)Os)₂L(N-Ac-Cys)2]²⁺(m_exp601.6,

m_calc= 601.8) adduct is clearly visible, and metal patterns indicate the

presence of a dimeric species.

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Cytotoxicity

Table 2. Cytotoxicity of organometallic thiopyridone complexes 3a-6d, their

Previously, closely related maltol organometallics had exhibited

only moderate activity against the three human cancer cell lines

CH1/PA-1 (ovarian teratocarcinoma), SW480 (colon carcinoma),

and A549 (non-small cell lung carcinoma),^[35]which was

subsequently improved via introduction of the S,O-coordination

motif, e.g., by thiomaltol, resulting in IC₅₀values in the low to sub-

micromolar range.^[18]To expand our knowledge on the cytotoxic

behaviour of compounds featuring a thiopyridone motif, we now

tested ligands 2a-d and the respective thiopyridone complexes

3a–6d in the above mentioned cancer cell lines (Table 2 ; Figures

S108–S111).

thiomaltol analogues and well-studied drug (candidates).

IC50 [µM]^[a]

Compound

A549

SW480

CH1/PA-1

2a

66 ± 6

1.6 ± 1.2^[b]

0.79 ± 0.12

1.6 ± 0.1

>200

3.2 ± 0.3

0.52 ± 0.03

0.28 ± 0.04

0.35 ± 0.02

55 ± 18

15 ± 1

0.52 ± 0.05

n/a

2b

2c

2d

0.62 ± 0.08

>200

3a

3b

2.4 ± 0.8

1.3 ± 0.1

2.1 ± 0.3

>200

2.8 ± 0.1

3.1 ± 0.2

4.0 ± 0.3

>200

0.94 ± 0.08

1.04 ± 0.03

1.6 ± 0.3

112 ± 27

1.13 ± 0.03

1.3 ± 0.2

1.3 ± 0.3

11 ± 2

3c

3d

4a

In general, variation of the thiopyridone ligand had a pronounced

impact on the cytotoxic activity of these compounds in human

cancer cell lines. Complexes featuring the methylamine ligand 2a

were less active than their respective thiomaltol parent

compounds in all tested cell lines by factors of 5 to >100, with IC₅₀

values ranging from 4.2 to >200 µM, respectively. Furthermore,

the more lipophilic compounds featuring the bulky naphthylamine

ligand 2d were generally more active (except for osmium

compound 4d in SW480 cells, which showed four times higher

IC₅₀values than the osmium thiomaltol complex). Overall,

complexes featuring phenyl and benzyl ligands (2b and 2c) are

by far the most active of the established compound library, with

IC₅₀values in the low to sub-micromolar range. Overall, the lowest

IC₅₀values were achieved with the Rh-benzyl compound 5c.

Apart from the different ligands, variation of the metal center

seemed to have a marked impact on cytotoxicity in the tested

cancer cell lines. In each ligand series, organo-osmium

derivatives were the least active, followed by the respective Ru

analogues, while Rh and Ir complexes showed distinctly higher

cytotoxic potency. The most active compound series featured the

benzyl ligand 2c, where Ru (3c) and Os (4c) analogues had IC₅₀

values in the low micromolar range, and their Rh (5c) and Ir (6c)

counterparts were found to be even more active with IC₅₀values

in the high nanomolar range. Therefore, the cytotoxicity of 5c and

6c is up to 20 times higher than that of 3c and 4c, depending on

the cell line. Overall, Ru and Os organometallics were most active

in the broadly chemosensitive CH1/PA-1 cells, followed by the

markedly multidrug-resistant A549 cells and least active in

SW480 cells, which show a certain extent of chemoresistance due

to P-gp expression. Interestingly, the IC₅₀of the complexes

featuring either of these two metals was higher by a factor of up

to 3 in SW480 cells than in the cell line A549. These findings are

in accordance with well-established Pt^IIdrugs (e.g., cisplatin),

which are up to 22 times more active in CH1/PA-1 cells than in

SW480 cells.^[36]Contrary to this, the Rh and Ir complexes were

revealed to be highly active in SW480 cells, followed by CH1/PA-1

and least active in A549 cells, where IC₅₀values were up to 21

times higher. This pattern reveals an interesting cell line sensitivity,

with a strong dependency on the employed metal center.

Summarizing, it seems that the ligand, as well as the metal center,

impact the anticancer activity in this library of organometallics.

Additionally, different trends in cell line sensitivities could be

observed depending on the employed metal center.

4b

2.2 ± 1.1

2.2 ± 1.2

2.4 ± 0.3

117 ± 19

2.0 ± 0.8

0.72 ± 0.11

1.3 ± 0.2

63 ± 6

5.8 ± 0.3

5.6 ± 0.7

7.7 ± 0.3

5.5 ± 1.1

0.67 ± 0.04

0.28 ± 0.02

1.0 ± 0.1

4.2 ± 0.2

0.59 ± 0.08

0.54 ± 0.07

0.80 ± 0.15

4.3 ± 0.2

2.0 ± 0.2

1.0 ± 0.1

0.73 ± 0.10

3.5 ± 0.3

62 ± 9

4c

4d

5a

5b

0.66 ± 0.08

0.37 ± 0.04

0.48 ± 0.06

7.0 ± 1.6

0.82 ± 0.05

0.57 ± 0.05

0.68 ± 0.02

3.3 ± 0.5

2.0 ± 0.2

1.0 ± 0.1

0.57 ± 0.03

0.16 ± 0.03

50 ± 6

5c

5d

6a

6b

2.2 ± 0.8

1.1 ± 0.2

2.0 ± 0.4

7.7 ± 1.8

4.1 ± 0.3

5.9 ± 0.8

5.8 ± 1.7

1.3 ± 0.4

156 ± 11

6c

6d

Ru-TM

Os-TM^[18]

Rh-TM^[18]

Ir-TM^[18]

Cisplatin^[36]

BOLD-100^[18]

[a] 50% inhibitory concentrations in human carcinoma cell lines A549, SW480,

and CH1/PA-1. Values are means ± SDs obtained by the MTT assay (exposure

time: 96 h). [b] High SD due to a broad shoulder at about 50% in the

concentration-effect curves.

Cellular accumulation and lipophilicity

In order to examine possible determinants for the strong

differences in cytotoxicity between Ru and Rh congeners, the

cellular accumulation of both methyl (3a, 5a) and benzyl

analogues (3c, 5c) was measured in SW480 cells (Table 3 ).

These experiments revealed strong deviations in intracellular

metal levels, depending on the ligand, as well as the metal center.

Intracellular accumulation was highest for the Rh-benzyl

compound 5c, followed by its Ru counterpart 3c, while complexes

featuring a methyl ligand showed lower cellular accumulation

overall. These findings are in good accordance with the measured

IC₅₀values for these complexes 3a, 3c, 5a and 5c in SW480 cells,

where benzyl complexes 3c and 5c exhibited highest cytotoxicity.

Additional experiments to determine the chromatographic

lipophilicity indices ϕ₀were carried out in order to further support

these results. Therefore, a well-established literature-known

procedure was applied, where the retention times of all complexes

was compared to a dead time marker in isocratic RP-UHPLC runs

with different mobile phase compositions.^[7],[37]In accordance with

the commonly employed logP value, a higher ϕ₀value correlates

to a higher lipophilicity of the respective compound.^[37]The ϕ₀

values for all Ru, Rh, and Ir complexes can be found in the

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supporting information (Table S36). However, due to stability

issues no ϕ₀values could be obtained for Os compounds (4a–d).

According to expectations, complex lipophilicity increases when

employing carbon enriched ligands; however, the impact of the

applied metal center was marginal. Consequently the general

trend for each transition metal series was, that the naphthyl

complexes featuring ligand d revealed highest ϕ₀values, followed

by benzyl complexes c and aniline complexes b, which showed

similar lipophilicity, and the lowest indices were obtained for

methyl-compounds featuring ligand a. The findings for methyl (a)

and benzyl (c) complexes establish a good correlation between

lipophilicity and cellular uptake, as well as lipophilicity and

cytotoxicity for each metal series (Figure 7 , Figure S106–107).

While the Ru-methyl organometallic 3a revealed the lowest

lipophilicity index, the lowest cytotoxicity and low cellular

accumulation levels, Ru-benzyl compound 3c showed

a

Figure 7. Scatter plot of cellular uptake vs. log(IC₅₀) of complexes 3a, 3c, 5a,

5c, 6a, and 6c in SW480 cells.

comparably higher ϕ₀value, together with distinctly higher IC₅₀

values and intracellular accumulation levels which were 7 times

as high. The same correlations could be observed for Rh

complexes 5a and 5c, where the cellular accumulation of the

benzyl complex was manifold higher than that of the methyl

counterpart even though the applied concentration was lower (5a:

50 µM; 5c: 20 µM), associated with IC₅₀values in the nanomolar

range and the highest ϕ₀value. This is counterintuitive as the

applied concentration was lower for Rh-benzyl (6c, 20 µM)

compared to Rh-methyl (5a, 50 µM). Finally, Ir-methyl 6a and

benzyl 6c organometallics revealed the same pattern. Although,

lipophilicity indices of both Ir compounds were between their

respective Ru and Rh counterparts, the cellular accumulation

levels were drastically higher. Ir-methyl compound 6a revealed

cellular concentrations about 47 times as high as the observed

values for Ru-methyl complex 3a, and 82 times as high as

Rh-methyl compound 5a.On the other hand, Ir-benzyl 6c

concentrations were 11 times as high as Ru-benzyl 3c, and

2 times as high as for Rh-benzyl 5c. While the deviations for

benzyl compounds c can be explained by the molecular weight of

the Ir central ion, which is two times as high as Ru and Rh, further

studies have to be conducted to completely elucidate the reason

for the higher values of methyl compound 6a.

Cell cycle studies

The influence of two ligands, as well as seven complexes on cell

cycle distribution was tested in two of the cancer cell lines (SW480,

CH1/PA-1) by means of flow cytometry. The cell cycle distribution

(G1, S, G2/M phase in %; Table S37) was investigated upon DNA

staining with propidium iodide. Based on their cytotoxic potency,

rhodium compounds 5b and 5d as well as benzyl ligand 2c and

the corresponding complexes 3c (Ru), 5c (Rh), and 6c (Ir) were

chosen and, in order to check for correspondence with cytotoxicity,

the least active methyl ligand 2a and the respective complexes 5a

(Rh) and 6a (Ir) were included for comparison.

In CH1/PA-1 cells the benzyl-featuring 2c ligand has little effect

on cell cycle distribution, while the corresponding complexes 3c

(Ru), 5c (Rh) and 6c (Ir) show increasing activity in the following

order: Rh<Ru<Ir (Table S38; 6%, 10%, 13% decrease in G1

phase at 2.5 µM, respectively). The most pronounced effect on

CH1/PA-1 cells was observed for Ru-benzyl complex 3c at a

concentration of 10 µM, which led to about 16% decrease in G1

phase in favor of S and G2/M phases. The ligand alone at the

same concentration decreased the G1 phase by 9%. Interestingly,

in SW480 cells ligand 2c showed an effect starting at 2.5 µM (18%

decrease in G1), while complexes 3c, 5c, and 6c exhibited scarce

effects at applicable concentrations (<9% decrease in G1 phase),

despite their partially high cytotoxicity.

In the methyl subgroup, featuring ligand 2a as well as compounds

5a (Rh) and 6a (Ir), the metal complexes showed comparable

effect on CH1/PA-1 cells by shifting the cell cycle distribution

towards S and G2/M phases by up to 14% in total (5a at 40 µM)

and both were more effective than 2a. SW480 cells that were

more sensitive than CH1/PA-1 to higher drug concentrations (cell

detachment at concentrations > 20 µM was observed) showed

minor cell cycle perturbations upon drug exposure. In this case

only Rh complex 5a caused a noteworthy effect with 10%

decrease in G1 phase at a concentration of 10 µM.

Table 3. Cellular accumulation in SW480 cells and chromatographic lipophilicity

indices of complexes 3a, 3c, 5a, 5c, 6a and 6c in comparison to their IC₅₀values.

Cellular accumulation

Compound

Φ₀

IC₅₀[µM]

[fg/cell]^[c]

3a

3c

5a

5c

6a

6c

20.72 ± 5.1

147.05 ± 54.28

11.82 ± 34.9

5.51

6.04

5.91

6.28

5.75

6.22

54.65 ± 18.42

3.06 ± 0.2

5.54 ± 1.07

0.28 ± 0.02

4.18 ± 0.19

0.54 ± 0.07

854.59 ± 66.47

985.82 ± 152.14

1679.68 ± 332.72

[c] Cellular accumulation in SW480 cells, determined by inductively coupled

plasma mass spectrometry (ICP-MS) after 2 h exposure at concentrations of

20 µM (5c), or 50 µM (3a, 3c, 5a, 6a, 6c).

Of the remaining two Rh complexes with an aniline (5b) or

naphthyl (5d) ligand, only 5b showed a pronounced impact on cell

cycle distribution. Moreover, 5b is the only compound to cause a

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G1 phase decrease by more than 20% in both cell lines (at

concentrations of 10 µM in CH1/PA-1 and 5 µM in SW480).

In conclusion, the compounds show pronounced cytotoxic activity

in cancer cells at low to sub-micromolar concentrations (Table 2 ),

in some cases reversing the activity towards the intrinsically more

chemoresistant SW480 cell line, but only some of them (in

particular Rh aniline complex 5b and benzyl ligand 2c) provoked

a pronounced cell cycle perturbation upon 24 h exposure.

However, even their effects are markedly smaller than those of

etoposide, an established drug known to cause cell cycle arrest

and hence used as a positive control.

Figure 8. Representative images of HCT 116 multicellular spheroids after

treatment with the indicated compounds (at about their respective IC₅₀) after 96 h

Cytotoxicity in 3D spheroid tumor models

compared to an untreated control.

Thiopyridone complexes 3a (Ru), 5a (Rh) and 6a (Ir) featuring

methyl ligand 2a and benzyl derivatives 3a (Ru), 5c (Rh) and 6c

(Ir) were also tested in three different human cancer cell lines

(A549, HCT-116, CH1/PA-1), from which multicellular spheroid

models were grown and treated with the desired organometallics

for 96 h (Figure 8 ). As these models mimic solid tumors more

closely, it is possible to gather more insights into the cytotoxic

behavior of organometallics.^[38]However, the 50% inhibitory

concentrations were higher in 3D models than in 2D monolayer

cultures (up to 200 times; values listed in Table 4 ). This effect has

also been reported for other metallodrugs in literature.^[39]Still,

these results confirmed the general cytotoxicity trends of 2D

models, where benzyl complexes (c) were more active than the

respective methyl (a) congeners. Additionally, Rh and Ir

complexes were least active in the more resistant A549 cell line

and showed approximately the same cytotoxicity in CH1/PA-1

and HCT-116 cells. The only exception to this trend was Ir benzyl

complex 6c where a lower concentration was required in HCT-116

cells for 50% inhibition compared to both A549 and CH1/PA-1 cell

lines. Interestingly, Ru compounds 3a and 3c show a different

cytotoxicity pattern. While methyl complex 3a is distinctly more

active in CH1/PA-1 cells, benzyl-based complex 3c showed

similar activity in all three cell lines.

Laser Ablation-ICP-MS studies of 3D tumor models

Laser

ablation-inductively

coupled

plasma-mass

spectrometry (LA-ICP-MS) is a powerful tool to determine the

distribution of (metal-)drugs in tissue and small organisms (e.g.

platinum and ruthenium drugs in vivo).^[40,41],[42]This is of special

interest as bio distribution in solid tumors is a crucial factor in

cancer treatment and thus drug development.^[43]In this work, we

report the distribution of ruthenium compounds featuring a methyl

and benzyl ligand (3a and 3c), as well as the respective

rhodium (5a, 5c) and iridium (6a, 6c) compounds in 3D colon

cancer HCT-116 tumor spheroids. The metal content is reported

in ions per extraction. Interestingly, the distribution patterns for

methyl-based compounds generally indicate metal disposition

throughout the spheroid tissue. This is in accordance with

published findings for bioreductive Pt prodrugs, which

accumulated in the necrotic core as well.^[43,44]Accordingly, Ru

levels of compound 3a are highest in the center of the tumor

spheroid, while rhodium complex 5a accumulated throughout the

spheroid sections with higher concentrations at the outer cell

layers (proliferating cells), as well as in isolated compartments in

the center. On the other hand, iridium organometallic 6a revealed

an even distribution throughout the tumor spheroid section ().

Contrary, complexes featuring a benzyl ligand exhibited highest

accumulation levels at the outer cell layers according to

literature.^[45]This indicates different disposition of methyl- and

benzyl-compounds in biological systems. This is in accordance

with uptake studies (see section above).

Table 4. IC₅₀values of selected compounds in multicellular spheroids of three

cell lines after 96 h drug exposure.

IC50 [µM]

Compound

A549

CH1/PA-1

HCT-116

3a

3c

5a

5c

6a

6c

>400

267 ± 14

242 ± 11

144 ± 7

59 ± 10

131 ± 14

114 ± 3

>400

211 ± 28

140 ± 20

62 ± 1

287 ± 3

367 ± 24

79 ± 5

270 ± 17

136 ± 5

138 ± 6

78 ± 2

Figure 9. LA-ICP-TOF-MS images of HTC 116 tumor spheroid sections

incubated with the respective organometallic ruthenium (3a, 3c), rhodium

(5a, 5c), or iridium (6a, 6c) complexes at the respective IC₅₀concentration

for 96 h; scale bar 100 µM.

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Conclusions

DMSO) or deuterated water (D₂O). CHNS elemental analyses

were carried out on a Eurovector EA3000 elemental analyzer in

the Microanalytical Laboratory of the University of Vienna. High

resolution electronspray ionization mass spectra were recorded

on a Bruker Maxis UHR qTOF Mass Spectrometer at the Core

Facility for Mass Spectrometry of the University of Vienna (Faculty

of Chemistry).

A library consisting of 16 new thiopyridone-based piano stool

complexes was synthesized and characterized by standard

analytical methods. These compounds showed increased stability

in aqueous solution, and higher cytotoxicity compared to their

pyridone and thiomaltol parental compounds.^[18,19]These findings

could partly be explained by the formation of dimeric species

featuring a double positive charge under physiological conditions.

It is noteworthy that despite their structural similarity, the results

for osmium compounds deviated in all analytical studies, and

were not investigated further due to stability issues under

physiologically relevant conditions. In order to gather first insights

into a possible mode of action as well as SARs, these complexes

were investigated by HPLC incubations with N-protected amino

The X-ray intensity data were measured on Bruker D8 Venture

and X8 APEX2 diffractometer equipped with multilayer

monochromator’s, Mo K/α INCOATEC micro focus sealed tube

and Oxford and Cryoflex2 cooling systems. The structure was

solved by direct methods, Patterson or charge flipping and refined

by full-matrix least-squares techniques. Non-hydrogen atoms

were refined with anisotropic displacement parameters. Hydrogen

atoms were inserted at calculated positions and refined with riding

model. The following software was used: Bruker SAINT software

package^[52]using a narrow-frame algorithm for frame integration,

SADABS^[53]for absorption correction, OLEX2^[54]for structure

solution, refinement, molecular diagrams and graphical user-

interface, Shelxle^[55]for refinement and graphical user-interface

SHELXS-2015^[56]for structure solution, SHELXL-2015^[57]for

refinement, Platon^[58]for symmetry check and π-π interactions

proof. Experimental data and CCDC-Code (Available online:

http://www.ccdc.cam.ac.uk/conts/retrieving.html ) can be found in

Table S1.

acids, revealing

a

distinct affinity for sulfur containing

biomolecules (e.g. l-cysteine). Furthermore, clear trends could be

observed in inhibitory concentrations, where benzyl complexes of

Rh and Ir proved to be most active, with IC₅₀values in the high

nanomolar range. These patterns were also observed in cell

uptake experiments, where the most toxic compounds showed

the highest cellular accumulation. However, no distinct effect on

any cell cycle phase could be observed. Consequently, the mode

of action of thiopyridone complexes may significantly differ from

that of their thiomaltol congeners, which proved to cause profound

S-phase accumulation.^[18]In order to completely elucidate

possible SARs as well as a mode of action further experiments

need to be conducted and the thiopyridone library may be

expanded.

UV/Vis spectra were recorded using a Hewlett Packard 8452A

diode-array spectrophotometer between 200-800 nm. The path

length (l) was either 1 or 2 cm. UV/Vis spectra were recorded to

confirm complex stability in aqueous systems and over a pH

range 5.8–7.9.

Analytical UHPLC was carried out on a Thermo Scientific^TM

UltiMate 3000 UHPLC equipped with a silica-based XBridge

C18 (4.6 × 150 nm, 5 μm) column.

Amino acid incubation studies were performed on an Agilent 1260

Infinity HPLC equipped with a silica-based Waters Atlantis T3 (4.6

× 150 nm) column, which was coupled to a Bruker amazon SL

ESI-IT mass spectrometer.

Experimental Section

Materials and methods

All dimeric metal precursors [Ru(p-cym)Cl₂]₂,^[46][Os(p-

cym)Cl₂]₂,^[47][Rh(Cp*)Cl₂]₂,^[48]and [Ir(Cp*)Cl₂]₂,^[49]and ligands

1a,^[50]and 2a,^[51]were prepared according to literature. The

solvents used were purchased from commercial sources and

dried before use if needed. Methanol (HPLC grade, Fisher) and

DCM (HPLC grade, Fisher) were used for column

chromatography. 3-Hydroxy-2-methyl-4H-pyran-4-one (≥99.0%;

Stability in aqueous solution

Stock solutions containing the desired complex in DMSO (5 mM)

were diluted with phosphate buffer to a final concentration of

50 µM compound in 1% DMSO/20 mM buffer and directly

analyzed via gradient UHPLC runs over 24 h. Phosphate buffers

at pH 5.8, 6.2, 6.7, 7.2 and 7.9 were employed to ensure a

constant pH value. The instrument was set to 0.6 mL/min flow rate,

37 °C sampler temperature, 25 °C column compartment, 225 nm

detection wavelength, 595% MeOH in MiliQ water + 0.1% formic

acid over 7.0 min.

maltol),

aniline (≥99.0%),

1-phenylmethanamine (99%;

benzylamine), hydrazine dihydrochloride (>98.0%), HCl, and

HNO₃were purchased from Sigma-Aldrich. Iridium(III) chloride,

osmium(VIII)

tetroxide,

rhodium(III)

chloride·H₂O,

and

ruthenium(III) chloride·x H₂O were purchased from Johnson

Matthey. Lawesson’s reagent (99%), and hydrochloric acid (37%),

were purchased from Acros-Fisher α-terpinene (90%) was

Lipophilicity indices

Stock solutions containing the desired complex in DMSO (5 mM)

purchased

from

Alfa

Aesar.

1,2,3,4,5-

Pentamethylcyclopentadiene (>93%, TCI Europe), sodium

methoxide (ca. 95%, Fluka), and naphthalene-1-amine (>99%,

Merck-Schuchardt) were purchased and used as received. ¹H,

¹³C (APT) and 2D-NMR spectra were recorded on a Bruker

Avance III^TM500 MHz FT-NMR spectrometer. H-NMR spectra

were measured at 500.10 MHz and ¹³C-NMR spectra at

125.75 MHz from solutions in deuterated dimethyl sulfoxide (d₆-

were diluted with phosphate buffer (pH 7.2) to

a final

concentration of 500 µM compound in 1% DMSO/20 mM buffer

and directly analyzed by four different isocratic UHPLC

runs (Δ = 5%). The lipophilicity parameters logk_wand lipophilicity

indices Φ₀were calculated according to literature. ^[7],[37]

1

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HPLC-MS incubation studies

spheroids had been grown. After incubation for 96 h, 20 µl of a

440 µM resazurin sodium salt solution in PBS were added to each

well, and the plates were incubated for further 16 h. Fluorescence

was measured with a Synergy HT reader (BioTek) with an

excitation wavelength of 530 nm and emission wavelength of

590 nm. For both assays, at least three independent experiments

(or two in case of poor activity) were performed, each with

triplicates per concentration level, and the 50% inhibitory

concentrations (IC₅₀) relative to untreated controls were

interpolated from concentration–effect curves.

For amino acid incubation studies, buffered solutions as

described for stability investigations (buffer at pH 7.2) were

prepared and additionally contained N-Ac-Met, N-Ac-His, and N-

Ac-Cys (500 μM each). The instrument was set to 0.5 mL/min flow

rate, 37 °C sampler temperature, 25 °C column compartment,

225 nm detection wavelength, 595% MeOH in MiliQ water + 0.1%

formic acid over 30.0 min.

Cell Lines and Culture Conditions

A549 (non-small cell lung carcinoma), SW480 and HCT-116 (both

colon carcinoma) were kindly provided by Brigitte Marian, Institute

of Cancer Research, Department of Medicine I, Medical

University of Vienna, Austria. The cell line CH1/PA-1 (ovarian

teratocarcinoma) was kindly provided by Lloyd R. Kelland (CRC

Centre for Cancer Therapeutics, Institute of Cancer Research,

Sutton, UK). All cell culture media (including supplements) and

reagents were obtained from Sigma-Aldrich, and all plasticware

from StarLab, unless indicated otherwise.

A549, CH1/PA-1 and SW480 cells were grown in MEM

supplemented with 10% fetal calf serum (FCS; from Biowest), 1

mM sodium pyruvate, 4 mM L-glutamine and 1% v/v nonessential

amino acids (from 100× solution) and L-glutamine. HCT-116 cells

were maintained in McCoy’s 5a medium supplemented with 10%

FCS and L-glutamine. Adherent monolayer cultures were grown

in 75 cm²flasks. For spheroid culture, A549, CH1/PA-1 and HCT-

116 cells were harvested from culture flasks by trypsinization,

resuspended in their respective supplemented medium and

seeded in ultra-low attachment round-bottom 96-well plates

(Corning Costar^TM) at a density of 500 viable cells per well. Plates

were incubated for 96 h to allow spheroid formation prior to use

for the tests. All cultures were incubated in a humidified incubator

at 37 °C with 5% CO₂.

Cell cycle studies

CH1/PA-1 and SW480 cells were harvested by trypsinization,

8×10⁴and 1.2×10⁵cells per well were seeded into 12-well plates,

respectively. The cells were allowed to attach and resume

proliferation for 24 h after the seeding. Etoposide was used as a

positive control.^[59]Dimethyl sulfoxide (DMSO) or MEM stock

solutions of the tested compounds were diluted with MEM and

added onto the cells so that the final DMSO content (where

applied) did not exceed 0.5% (controls were treated accordingly).

After continuous exposure for 24 h at 37 °C and under 5% CO₂,

the cells were harvested by trypsinization and centrifuged at 300

g for 3 min. The cells were washed with PBS (1 mL) and

resuspended in propidium iodine (PI) containing HSF buffer (600

µL; 0.1% Triton X-100, 0.1% sodium citrate, 50 µg/mL PI in PBS).

After incubation overnight at 4 °C in the dark, 5×10³cells were

measured by flow cytometry with a Millipore Guava easyCyte 8HT

instrument (Luminex, USA). Data were evaluated by means of

FlowJo software (Tree Star) using Watson Pragmatic

algorithms.^[60]The fitting of the curve was chosen to keep root

mean square error between 1.0 and 2.0, alongside with good

fitting visual model.

LA-ICP-TOF-MS imaging

Cytotoxicity tests

Cytotoxicity of the compounds in monolayer cultures was

determined by the colorimetric MTT assay (MTT = 3-(4,5-

An Analyte Excite Excimer 193nm laser ablation system

(Teledyne Photon Machines, Bozeman, MT, U.S.A.), equipped

with a prototype Cobalt ablation cell and the aerosol rapid

introduction system (ARIS), was coupled to an icpTOF 2R ICP-

TOF-Ms instrument(TOFWERK AG, Thun, Switzerland). The

optimised He carrier gas flow was 0.5 L min^-1, to which Ar was

added as a makeup gas (~0.9 L min^-1) through the low dispersion

mixing bulb of the ARIS before entering the plasma. The

performance and settings of the instruments were optimised on a

daily basis using the standard reference material NIST SRM612

(National Institute for Standards and Technology, Gaithersburg,

MD, U.S.A.). Optimisation criteria are the signal intensities of the

isotopes ⁵⁹Co⁺, ¹¹⁵In⁺and ²³⁸U⁺as well as the ratios of ²³⁸U¹⁶O⁺to

²³⁸U⁺(oxide formation, < 2%) and ²³⁸U⁺to ²³²Th⁺(elemental

fractionation, ~1). The specified mass resolving power of the ICP-

TOF-MS is m/Δm = 6000 (FWHM definition) and the analysable

mass range in the used operation mode is m/z = 14 – 256. The

plasma Ar gas flow was 14 L min^-1, the auxiliary Ar gas flow was

~0.8 L min^-1and the radio frequency power was 1440 W. Laser

ablation was performed with a square spot with 5 µm in diameter

and a repetition rate of 200 Hz. The fixed dosage mode was used

with dosage set to 2 and the shift between lines was 2.5 µm

resulting in an effective ablation area of 2.5 x 2.5 µm. Tumor

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

bromide).

1×10³CH1/PA-1, 2×10³SW480 and 3×10³A549 cells were

seeded in 100 μL supplemented MEM per well into 96-well flat-

bottom microculture plates. After 24 h, most test compounds were

dissolved in DMSO (Fisher Scientific) first, except for 5a, 5b, 6a

and 6b, which were dissolved in MEM; all were serially diluted in

MEM (to final DMSO contents not exceeding 0.5% v/v) and added

in 100 μL per well. After 96 h, the drug-containing medium was

replaced with 100 µL of RPMI 1640/MTT mixture [6 parts of RPMI

1640 medium (supplemented with 10% heat-inactivated fetal

bovine serum and 4 mM L-glutamine), 1 part of MTT solution in

phosphate-buffered saline (5 mg/mL)]. After incubation for 4 h,

the mixture was replaced with 150 μL DMSO per well to dissolve

the formazan product formed by viable cells. Optical densities at

550 nm (and at a reference wavelength of 690 nm) were

measured with a microplate reader (ELx808, Bio-Tek).

Cytotoxicity in multicellular spheroids was determined by the

fluorimetric resazurin assay. For this purpose, 100 µl of the

compound dilutions in the appropriate medium (MEM or McCoy’s

5a medium) were added to each well of the plates where

10.1002/chem.201905546

Chemistry - A European Journal

FULL PAPER

spheroid sections were removed quantitatively with a laser

fluence of 0.75 J cm^-2. Data was recorded using TofPilot 1.3.4.0

(TOFWERK AG, Thun, Switzerland). LA-ICP-TOF-MS data was

further processed with HDIP version 1.2.5.beta4 (Teledyne

Photon Machines, Bozeman, MT, U.S.A.).

performed according to the general complexation protocol using

ligand 2b (142 mg, 0.653 mmol), sodium methoxide (39 mg,

0.719 mmol)

and

bis[dichlorido(η⁶-p-

cymene)ruthenium(II)] (200 mg, 0.327 mmol) and a reaction time

of 1.5 h. The product was crystallized from DCM/Et₂O and

isolated as red crystals. Yield: 205 mg (65%).

General Procedures

Monomer

3b:

¹H-NMR (500.10 MHz,

d₆-DMSO,

General protocol for Os^II/Ru^IIarene and Ir^III/Rh^IIICp* halide

complex’ syntheses: Syntheses of all complexes were

performed by dissolving the respective ligand (1 eq.) and sodium

methoxide (1.1 eq.) in absolute methanol (10 mL) and the

solution was stirred under Ar atmosphere at RT for 20 min. The

respective dimeric metal precursor (0.45 eq.) was added and the

resulting dark colored mixture was stirred at RT or 40 °C for

several hours (0.5-3.5 h depending on the complex). Afterwards,

the solvent was concentrated under reduced pressure and the

crude product was dissolved in dichloromethane. In order to

remove insoluble by-products, the solution was filtrated and the

filtrate was concentrated in vacuo. Precipitation or crystallization

from DCM/Et₂O, or DCM/n-hexane afforded the desired products

in moderate to good yields (33-83%).

25 °C): δ = 1.22 (d, ³J (H,H) = 7 Hz, 6H, Hg), 2.11 (s, 3H, H1),

2.14 (s, 3H, Ha), 2.73 (hept, ³J (H,H) = 7 Hz, 1H, Hf), 5.84 (d,

³J (H,H) = 6 Hz, 2H, Hd), 6.01 (d, ³J (H,H) = 6 Hz, 2H, Hc),

7.36 (d, ³J (H,H) = 6 Hz, 1H, H5), 7.48–7.54 (m, 3H, H6, H8, H12),

7.59–7.64 (m, 3H, H9, H10, H11) ppm; ¹³C-NMR (125.75 MHz,

d₆-DMSO, 25 °C): δ = 13.9 (C1), 17.5 (Ca), 22.2 (Cg), 30.4 (Cf),

85.4 (Cd), 88.9 (Cc), 102.4 (Ce), 105.8 (Cb), 120.8 (C5),

126.0 (C8, C12), 128.8 (C6), 129.9 (C9, C11), 130.1 (C10),

134.1 (C2), 141.3 (C7), 162.1 (C4), 166.7 (C3) ppm.

Dimer 3b*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 1.03 (d,

³J (H,H) = 7 Hz, 6H, Hg), 1.18 (d, ³J (H,H) = 7 Hz, 6H, Hg),

2.05 (s, 6H, H1), 2.33 (s, 6H, Ha), 2.68–2.78 (m, ³J (H,H) = 7 Hz,

2H, Hf), 5.56 (d, ³J (H,H) = 6 Hz, 2H, Hd), 5.61 (d, ³J (H,H) = 6 Hz,

2H, Hc), 5.76 (d, ³J (H,H) = 6 Hz, 2H, Hc), 5.98 (d,

³J (H,H) = 6 Hz, 2H, Hd), 7.06 (d, ³J (H,H) = 8 Hz, 2H, H8, H12),

7.32 (d, ³J (H,H) = 8 Hz, 2H, H8, H12), 7.50–7.57 (m, 6H, H5, H6,

H9, H11), 7.59 (dd, ³J (H,H) = 8 Hz, ³J (H,H) = 8 Hz, 2H, H9,

H11), 7.68–2.78 (m, 2H, H10) ppm; ¹³C-NMR (125.75 MHz, D₂O,

25 °C): δ = 14.8 (C1), 17.6 (Ca), 20.8 (Cg), 21.8 (Cg), 30.1 (Cf),

80.7 (Cd), 84.0 (Cc), 85.2 (Cd), 86.0 (Cc), 103.2 (Ce), 107.1 (Cb)

124.3 (C8, C12), 125.6 (C8, C12), 126.4 (C5), 129.3 (C6),

130.2 (C9, C11), 130.3 (C9, C11), 131.0 (C10), 140.5 (C7),

140.9 (C2), 148.7 (C4), 171.6 (C3) ppm.

Chlorido[1,2-dimethyl-3-oxo-κO-pyridine-4(1H)-thionato-κS](η⁶-

p-cymene)-ruthenium(II) (3a): The synthesis was performed

according to the general complexation protocol using ligand

2a (101 mg,

0.719 mmol,)

0.653 mmol,),

and

sodium

methoxide (39 mg,

bis[dichlorido(η⁶-p-

cymene)ruthenium(II)] (200 mg, 0.327 mmol) and a reaction time

of 1.5 h. The product was precipitated from DCM/Et₂O and

isolated as a red powder. Yield: 176 mg (63%).

Monomer

3a:

¹H-NMR (500.10 MHz,

d₆-DMSO,

ESI-HR-MS^-m/z found (calculated): [M]^-538.0158 (538.0158).

Elemental analysis calcd (%) for C₂₂H₂₄ClNORuS: C 54.26,

H 4.97, N 2.88, S 6.58; found: C 54.26, H 4.98, N 2.90, S 5.60.

25 °C): δ = 1.21 (d, ³J (H,H) = 7 Hz, 6H, Hg), 2.14 (s, 3H, H1),

2.44 (s, 3H, Ha), 2.67–2.77 (m, ³J (H,H) = 7 Hz, 1H, Hf), 3.86 (s,

3H, H7), 5.85 (d, ³J (H,H) = 6 Hz, 2H, Hd), 6.01 (d,

³J (H,H) = 6 Hz, 2H, Hc), 7.27 (d, ³J (H,H) = 6 Hz, 1H, H5),

7.54 (d, ³J (H,H) = 6 Hz, 1H, H6) ppm. ¹³C-NMR (125.75 MHz,

d₆-DMSO, 25 °C): δ = 11.9 (C1), 17.5 (Ca), 22.1 (Cg), 30.4 (Cf),

43.3 (C7), 87.2 (Cd), 89.0 (Cc), 102.6 (Ce), 106.6 (Cb),

120.8 (C5), 126.1 (C6), 137.7 (C2), 159.1 (C4), 166.8 (C3) ppm.

Dimer 3a*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 0.87 (d,

Chlorido[1-benzyl-2-methyl-3-(oxo-κO)-pyridine-4(1H)-thionato-

κS](η⁶-p-cymene)ruthenium(II) (3c):

The

synthesis

was

performed according to the general complexation protocol using

ligand 2c (227 mg, 0.980 mmol), sodium methoxide (58 mg,

1.078 mmol)

and

bis[dichlorido(η⁶-p-

cymene)ruthenium(II)] (300 mg, 0.490 mmol) and a reaction time

³J (H,H) = 7 Hz, 6H, Hg), 1.04 (d, ³J(H,H) = 7 Hz, 6H, Hg), 2.05 (s, of 1 h. The product was crystallized from DCM/Et₂O and isolated

6H, H1), 2.21 (s, 6H, Ha), 2.53–2.62 (m, ³J (H,H) = 7 Hz, 2H, Hf),

3.81 (s, 6H, H7), 5.38 (d, ³J (H,H) = 6 Hz, 2H, Hd), 5.42 (d,

³J (H,H) = 6 Hz, 2H, Hc), 5.60 (d, ³J (H,H) = 6 Hz, 2H, Hd),

5.82 (d, ³J (H,H) = 6 Hz, 2H, Hc), 7.26 (d, ³J (H,H) = 6 Hz, 2H,

as red crystals. Yield: 163 mg (33%).

Monomer 3c: (500.10 MHz,

¹H-NMR

d₆-DMSO,

25 °C): δ = 1.18 (d, ³J (H,H) = 7 Hz, 6H, Hg), 2.12 (s, 3H, Ha),

2.33 (s, 3H, H1), 2.68 (hept, ³J (H,H) = 7 Hz, 1H, Hf), 5.50 (s, 2H,

3

H5),

7.35 (d,

³J (H,H) = 6 Hz,

2H,

H6) ppm;

¹³C-

H7), 5.83 (d, J (H,H) = 6 Hz, 2H, Hd), 5.98 (d, J (H,H) = 6 Hz,

2H, Hc), 7.10 (d, ³J (H,H) = 7 Hz, 2H, H9, H13), 7.31–7.36 (m, 1H,

NMR (125.75 MHz, D₂O, 25 °C): δ = 12.3 (C1), 17.6 (Ca),

20.7 (Cg), 21.7 (Cg), 30.1 (Cf), 44.7 (C7), 80.3 (Cc), 83.9 (Cc),

84.9 (Cd), 86.2 (Cd), 103.2 (Ce), 106.9 (Cb), 126.6 (C5),

129.4 (C6), 141.6 (C2), 146.1 (C4), 171.3 (C3) ppm.

ESI-HR-MS⁺m/z found (calculated): [M]⁺390.0460 (390.0464).

Elemental analysis calcd (%) for C₁₇H₂₂ClNORuS·1.25 H₂O:

C 45.63, H 5.52, N 3.13, S 7.17; found: C 45.60, H 5.55, N 3.09,

S 7.07.

3

H5), 7.36–7.42 (m, 2H, H10, H12), 7.46 (d, J (H,H) = 6 Hz, 1H,

H11),

7.73 (d,

³J (H,H) = 6 Hz,

1H,

H6) ppm;

¹³C-

NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 12.1 (C1), 17.5 (Ca),

22.4 (Cg), 30.4 (Cf), 58.1 (C7), 78.9 (Cd), 81.1 (Cc), 94.5 (Ce),

98.7 (Cb), 121.0 (C5), 126.7 (C9, C13), 128.3 (C11), 129.1 (C10,

C12),

130.4 (C6),

135.0 (C8),

136.5 (C2),

161.2 (C4),

169.0 (C3) ppm.

Dimer 3c*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 0.88 (d,

³J (H,H) = 7 Hz, 6H, Hg), 0.98 (d, ³J (H,H) = 7 Hz, 6H, Hg),

1.57 (s, 6H, H1), 2.16 (s, 6H, Ha), 2.48–2.56 (m, ³J (H,H) = 7 Hz,

Chlorido[2-methyl-3-(oxo-κO)-1-phenylpyridine-4(1H)-thionato-

κS](η⁶-p-cymene)ruthenium(II) (3b):

The

synthesis

was

10.1002/chem.201905546

Chemistry - A European Journal

FULL PAPER

2H, Hf), 5.05 (d, ²J (H,H) = 15 Hz, 2H, H7a), 5.13 (d,

²J (H,H) = 15 Hz, 2H, H7b), 5.40 (d, ³J (H,H) = 6 Hz, 2H, Hd),

5.45 (d, ³J (H,H) = 7 Hz, 2H, Hc), 5.50 (d, ³J (H,H) = 7 Hz, 2H,

78.8 (Cc), 81.3 (Cd), 95.1 (Cb), 97.1 (Ce), 120.6 (C5), 130.2 (C6),

137.4 (C2), 159.6 (C4), 168.3 (C3) ppm.

Dimer 4a*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 0.94 (d,

³J (H,H) = 6 Hz, 6H, Hg), 1.15 (d, ³J (H,H) = 6 Hz, 6H, Hg),

2.24 (s, 6H, H1), 2.38 (s, 6H, Ha), 2.55–2.64 (m, ³J (H,H) = 7 Hz,

2H, Hf), 3.92 (s, 6H, H7), 5.76 (dd, ³J (H,H) = 6 Hz,

3

Hd), 5.66 (d, J (H,H) = 7 Hz, 2H, Hc), 7.09 (dd, J (H,H) = 8 Hz,

⁴J (H,H) = 2 Hz, 4H, H9, H13), 7.33–7.40 (m, 6H, H10, H11, H12),

7.56 (d, ³J (H,H) = 6 Hz, 2H, H5), 7.60 (d, ³J (H,H) = 6 Hz, 2H,

H6) ppm; ¹³C-NMR (125.75 MHz, D₂O, 25 °C): δ = 12.3 (C1),

17.5 (Ca), 21.1 (Cg), 21.9 (Cg), 30.2 (Cf), 60.1 (C7), 73.0 (Cc),

76.5 (Cc), 76.9 (Cd), 79.0 (Cd), 95.1 (Ce), 99.0 (Cb), 125.1 (C5),

127.7 (C9, C13), 129.3 (C6), 129.5 (C10, C12), 130.3 (C11),

132.7 (C8), 141.4 (C2), 146.4 (C4), 172.7 (C3) ppm.

ESI-HR-MS⁺m/z found (calculated): [M]⁺452.0626 (452.0622).

Elemental anaylsis calcd (%) for C₂₃H₂₆ClNORuS·0.5 H₂O:

C 54.16, H 5.34, N 2.75, S 6.29; found: C 54.04, H 5.36, N 2.78,

S 6.39.

3

³J (H,H) = 6 Hz, 4H, Hc, Hd), 5.96 (d, J (H,H) = 6 Hz, 2H, Hd),

6.16 (d, ³J (H,H) = 6 Hz, 2H, Hc), 7.47 (dd, ³J (H,H) = 6 Hz,

³J (H,H) = 6 Hz, 4H, H5, H6) ppm; ¹³C-NMR (125.75 MHz,

298.2 K, D₂O): δ = 12.5 (C1), 17.6 (Ca), 21.1 (Cg), 22.1 (Cg),

30.2 (Cf), 44.8 (C7), 72.3 (Cc), 75.5 (Cd), 76.3 (Cc), 78.8 (Cd),

96.5 (Cb), 99.2 (Ce), 126.5 (C5), 130.2 (C6), 142.3 (C2),

145.3 (C4), 172.5 (C3) ppm.

ESI-HR-MS^-m/z found (calculated): [M]^-550.0398 (550.0398).

Elemental anaylsis calcd (%) for C₁₇H₂₂ClNOOsS: C 39.72,

H 4.31, N 2.72, S 6.24; found: C 39.62, H 4.20, N 2.80, S 6.19.

Chlorido[2-methyl-1-(naphthalene-1-yl)-3-(oxo-κO)-pyridine-

4(1H)-thionato-κS](η⁶-p-cymene)ruthenium(II) (3d):

The

synthesis was performed according to the general complexation

Chlorido[2-methyl-3-(oxo-κO)-1-phenylpyridine-4(1H)-thionato-

κS](η⁶-p-cymene)osmium(II) (4b): The synthesis was performed

according to the general complexation protocol using 2b (137 mg,

0.632 mmol), sodium methoxide (38 mg, 0.696 mmol) and

protocol

methoxide (39 mg,

using

2d (174 mg,

0.719 mmol)

0.653 mmol),

and

sodium

bis[dichlorido(η⁶-p-

cymene)ruthenium(II)] (200 mg, 0.327 mmol) and a reaction time

of 1 h. The product was crystallized from DCM/Et₂O and isolated

as red crystals. Yield: 248 mg (63%).

bis[dichlorido(η⁶-p-cymene)osmium(II)] (250 mg,

0.316 mmol)

and a reaction time of 1.5 h. The product was crystallized from

DCM/Et₂O and isolated as orange crystals. Yield: 213 mg (58%).

Monomer 4b: ¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =

¹H-NMR

(500.10 MHz,

d₆-DMSO,

25 °C): δ = 1.24

(d,

³J (H,H) = 7 Hz, 6H, Hg), 1.98 (s, 3H, H1), 2.17 (s, 3H, Ha),

2.75 (sept., ³J (H,H) = 7 Hz, 1H, Hf), 5.84-5.95 (m, 2H, Hd),

6.05 (s, 2H, Hc), 7.01 (d, ³J (H,H) = 6 Hz, 1H, H5), 7.47 (d,

³J (H,H) = 8 Hz, 1H, H8), 7.60-7.66 (m, 2H, H9, H14), 7.71–

1.24 (d, J(H,H) = 7 Hz, 6H; Hg), 2.16 (s, 3H, H1), 2.22 (s, 3H,

3

Ha), 2.69 (sept., ³J (H,H) = 7 Hz, 1H, Hf), 5.96 (d, ³J (H,H) = 5 Hz,

2H, Hd), 6.15 (d, ³J (H,H) = 7 Hz, 2H, Hc), 7.46 (d,

³J (H,H) = 6 Hz, 1H, H5), 7.51–7.56 (m, 2H, H8, H12), 7.59 (d,

³J (H,H) = 7 Hz, 1H, H6), 7.60–7.66 (m, 3H, H9, H10, H11) ppm;

3

7.66 (m, 3H, H6, H10, H13), 8.17 (d, J (H,H) = 8 Hz, 1H, H15),

3

8.24 (d, J (H,H) = 8 Hz, 1H, H12) ppm; ¹³C-NMR (125.75 MHz,

¹³C-NMR (125.75 MHz,

d₆-DMSO,

25 °C): δ =

13.9 (C1),

d₆-DMSO, 25 °C): δ = 13.1 (C1), 17.5 (Ca), 22.3 (Cg), 22.6 (Cg),

30.5 (Cf), 79.0 (Cc), 81.4 (Cd), 103.7 (Ce), 106.1 (Cb),

17.4 (Ca), 22.5 (Cg), 30.5 (Cf), 78.5 (Cd), 81.3 (Cc), 95.7 (Ce),

98.2 (Cb), 120.7 (C5), 126.1 (C8, C12), 129.9 (C9, C11),

130.0 (C10), 130.2 (C6), 136.3 (C2), 141.1 (C7), 162.4 (C4),

168.3 (C3) ppm.

120.7 (C5),

121.2 (C15),

124.7 (C8),

125.8 (C9/C13),

126.1 (C9/C13), 127.5 (C6), 127.7 (C11, C16), 128.8 (C12, C14),

129.8 (C5), 130.7 (C10), 133.7 (C16), 137.0 (C2, C7), 167.0 (C3,

C4) ppm.

ESI-HR-MS⁺m/z found (calculated): [M]⁺572.0163 (572.0162).

Elemental analysis calcd (%) for C₂₆H₂₆ClNORuS·0.5 CH₂Cl₂:

C 54.92, H 4.70, N 2.42, S 5.53; found: C 54.88, H 4.59, N 2.54,

S 5.44.

Dimer 4b*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 1.01 (d,

³J (H,H) = 7 Hz, 6H, Hg), 1.19 (d, ³J (H,H) = 7 Hz, 6H, Hg),

2.15 (s, 6H, H1), 2.42 (s, 6H, Ha), 2.66 (hept, ³J (H,H) = 7 Hz, 2H,

Hf), 5.86 (d, ³J (H,H) = 6 Hz, 2H, Hd), 5.91 (d, ³J (H,H) = 6 Hz, 2H,

3

Hc), 6.05 (d, J (H,H) = 6 Hz, 2H, Hc), 6.25 (d, J (H,H) = 6 Hz,

2H, Hd), 7.06 (d, ³J (H,H) = 8 Hz, 2H, H8/H12), 7.33 (d,

³J (H,H) = 8 Hz, 2H, H8/H12), 7.51–7.64 (m, 6H, H6, H9, H11),

7.64–7.71 (m, 4H, H5, H10) ppm; ¹³C-NMR (125.75 MHz, D₂O,

25 °C): δ = 14.9 (C1), 17.6 (Ca), 21.1 (Cg), 22.1 (Cg), 30.2 (Cf),

72.6 (Cc), 75.6 (Cd), 76.8 (Cc), 78.9 (Cd), 96.5 (Ce), 99.6 (Cb),

124.3 (C8, C12), 125.6 (C8, C12), 126.2 (C5), 130.1 (C9, C11),

130.3 (C6), 131.1 (C10), 140.4 (C7), 141.8 (C2), 147.8 (C4),

172.7 (C3) ppm.

[Chlorido[1,2-dimethyl-3-(oxo-κO)-pyridine-4(1H)-thionato-

κS](η⁶-p-cymene)osmium(II)] (4a): The synthesis was performed

according to the general complexation protocol using 2a (70 mg,

0.448 mmol), sodium methoxide (27 mg, 0.493 mmol) and

bis[dichlorido(η⁶-p-cymene)osmium(II)] (177 mg,

0.224 mmol)

and a reaction time of 1.5 h. The product was crystallized from

DCM/Et₂O and isolated as orange crystals. Yield: 130 mg (56%).

Monomer 4a: ¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =

ESI-HR-MS^-m/z found (calculated): [M]^-612.0555 (612.0555).

Elemental anaylsis calcd (%) for C₂₂H₂₄ClNOOsS: C 45.86,

H 4.20, N 2.43, S 5.56; found: C 45.93, H 4.19, N 2.59, S 5.54.

3

1.22 (d, J(H,H) = 7 Hz, 6H; Hg), 2.22 (s, 3H; Ha), 2.48 (s, 3H,

H1), 2.62–2.70 (m, ³J (H,H) = 7 Hz, 1H, Hf), 3.89 (s, 3H, H7),

5.93 (d, ³J (H,H) = 5 Hz, 2H, Hd), 6.10 (d, ³J (H,H) = 5 Hz, 2H,

Chlorido[1-benzyl-2-methyl-3-(oxo-κO)-pyridine-4(1H)-thionato-

κS](η⁶-p-cymene)osmium(II) (4c): The synthesis was performed

according to the general complexation protocol using 2c (146 mg,

0.632 mmol), sodium methoxide (38 mg, 0.696 mmol) and

3

Hc), 7.34 (d, J (H,H) = 6 Hz, 1H, H6), 7.61 (d, J (H,H) = 6 Hz,

1H, H5) ppm; d₆-DMSO,

¹³C-NMR (125.75 MHz,

25 °C): δ = 12.0 (C1), 17.4 (Ca), 22.4 (Cg), 30.5 (Cf), 43.4 (C7),

bis[dichlorido(η⁶-p-cymene)osmium(II)] (250 mg,

0.316 mmol)

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and a reaction time of 1.5 h. The product was crystallized from

DCM/Et₂O and isolated as orange crystals. Yield: 171 mg (46%).

Monomer 4c: ¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =

1.19 (d, J (H,H) = 7 Hz, 6H, Hg), 2.20 (s, 3H, Ha), 2.37 (s, 3H,

H1), 2.60–2.69 (m, ³J (H,H) = 7 Hz, 1H, Hf), 5.55 (s, 2H, H7),

5.94 (d, ³J (H,H) = 6 Hz, 2H, Hc), 6.10 (d, ³J(H,H) = 6 Hz, 2H, Hd), synthesis was performed according to the general complexation

7.11 (d, ³J (H,H) = 7 Hz, 2H, H9, H13), 7.34 (dd, ³J (H,H) = 7 Hz,

³J (H,H) = 7 Hz, ³J (H,H) = 7 Hz,

1H, H11), 7.40 (dd,

C 47.60, H 4.07, N 2.09, S 4.80; found: C 47.65, H 4.05, N 2.18,

S 4.76.

Chlorido[1,2-dimethyl-3-oxo-κO-pyridine-4(1H)-thionato-κS](η⁵-

3

1,2,3,4,5-pentamethylcyclopentadienyl)rhodium(III) (5a):

The

protocol using ligand 2a (50 mg, 0.320 mmol), sodium

methoxide (21 mg, 0.384 mmol) and bis[dichlorido(η⁵-1,2,3,4,5-

pentamethyl-cyclopentadienyl)rhodium (III)] (89 mg, 0.144 mmol)

and a reaction time of 2.0 h. The product was crystallized from

DCM/n-hexane and isolated as red crystals. Yield: 86 mg (70%).

³J(H,H) = 7 Hz, 2H, H10, H12), 7.46 (d, ³J (H,H) = 6 Hz, 1H, H5),

7.79 (d, ³J (H,H) = 6 Hz, 1H, H6) ppm; ¹³C-NMR (125.75 MHz,

d₆-DMSO, 25 °C): δ = 12.1 (C1), 17.5 (Ca), 22.4 (Cg), 30.4 (Cf),

58.1 (C7), 78.9 (Cc), 81.1 (Cd), 94.5 (Cb), 98.7 (Ce), 121.0 (C5),

126.7 (C9, C13), 128.3 (C11), 129.1 (C10, C12), 130.4 (C6),

135.0 (C8), 136.5 (C2), 161.2 (C4), 169.0 (C3) ppm.

Monomer

5a:

¹H-NMR

(500.10 MHz,

d₆-DMSO,

25 °C): δ =1.64 (s, 15H, CH₃, Cp*), 2.45 (s, 3H, H1), 3.86 (s, 3H,

3

H7), 7.22 (d, J (H,H) = 6 Hz, 1H, H5), 7.54 (d, J (H,H) = 6 Hz,

1H, H6) ppm; ¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ =

8.4 (CH_3,Cp*), 12.3 (C1), 43.5 (C7), 98.9 (Cq, Cp*), 121.5 (C5),

Dimer 4c*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 0.95 (d,

³J (H,H) = 7 Hz, 6H, Hg), 1.09 (d, ³J (H,H) = 7 Hz, 6H, Hg),

1.81 (s, 6H, H1), 2.32 (s, 6H, Ha), 2.54 (hept., ³J (H,H) = 7 Hz, 2H, 129.4 (C6), 142.0 (C2), 148.0 (C4), 171.0 (C3) ppm.

Hf), 5.09 (d, ²J (H,H) = 15 Hz, 2H, H7), 5.23 (d, ²J (H,H) = 15 Hz,

2H, H7), 5.82 (d, ³J (H,H) = 6 Hz, 2H, Hd), 5.86 (d,

Dimer 5a*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 1.19 (s, 15H,

CH₃, Cp*), 1.60 (s, 15H, Cp*), 2.14 (s, 3H, H1), 2.71 (s, 3H, H1),

3

³J (H,H) = 6 Hz, 2H, Hc), 5.91 (d, J (H,H) = 6 Hz, 2H, Hc), 6.08

3.94 (s, 3H, H7), 4.08 (s, 3H, H7), 7.31 (d, J (H,H) = 6 Hz, 1H,

3

(d, J (H,H) = 6 Hz, 2H, Hd), 7.17–7.22 (m, 4H, H9, H13), 7.45–

H5), 7.41 (d, ³J (H,H) = 6 Hz, 1H, H6), 7.70 (s, 2H, H5, H6) ppm;

3

7.49 (m, 6H, H10, H11, H12), 7.56 (d, J (H,H) = 6 Hz, 2H, H5),

¹³C-NMR (125.75 MHz,

D₂O,

25 °C): δ = 7.1 (CH₃,

Cp*),

7.74 (d, ³J (H,H) = 6 Hz, 2H, H6) ppm; ¹³C-NMR (125.75 MHz,

D₂O, 25 °C): δ = 12.3 (C1), 17.5 (Ca), 21.1 (Cg), 21.9 (Cg),

30.2 (Cf), 60.1 (C7), 73.0 (Cc), 76.5 (Cd), 76.9 (Cc), 79.0 (Cd),

95.1 (Cb), 99.0 (Ce), 125.1 (C5), 127.7 (C9, C13), 129.3 (C11),

7.9 (CH3, Cp*), 11.9 (C1), 12.9(C1), 44.5 (C7), 45.2 (C7),

96.5 (Cq, Cp*), 97.0 (Cq, Cp*), 125.2 (C5), 126.0 (C5),

128.7 (C6), 130.2 (C6), 141.0 (C2), 143.5 (C2), 149.6 (2 C4),

169.5 (C3), 170.7 (C3) ppm. 1:1 ratio

129.5 (C10, C12), 130.3 (C6), 132.6 (C8), 141.4 (C2), 146.4 (C4), ESI-HR-MS⁺m/z found (calculated): [M]⁺392.0552 (392.0550).

172.7 (C3) ppm.

Elemental anaylsis calcd (%) for C₁₇H₂₃ClNORhS·H₂O: C 45.80,

H 5.65, N 3.14, S 7.19; found: C 45.73, H 5.38, N 3.15, S 7.46.

ESI-HR-MS^-m/z found (calculated): [M]^-626.0714 (626.0711).

Elemental anaylsis calcd (%) for C₂₃H₂₆ClNOOsS·0.5 CH₂Cl₂:

C 44.61, H 4.30, N 2.21, S 5.07; found: C 44.54, H 4.25, N 2.25,

S 4.92.

Chlorido[1-phenyl-2-methyl-3-oxo-κO-pyridine-4(1H)-thionato-

κS](η⁵-1,2,3,4,5-pentamethylcyclopentadienyl)rhodium(III) (5b):

The synthesis was performed according to the general

complexation protocol using ligand 2b (75 mg, 0.345 mmol),

sodium methoxide (22 mg, 0.414 mmol) and bis[dichlorido(η⁵-

Chlorido[2-methyl-1-(naphthalene-1-yl)-3-(oxo-κO)-pyridine-

4(1H)-thionato-κS](η⁶-p-cymene)osmium(II) (4d): The synthesis

was performed according to the general complexation protocol

using 2d (168 mg, 0.628 mmol), sodium methoxide (37 mg,

1,2,3,4,5-pentamethyl-cyclopentadienyl)rhodium

(III)] (96 mg,

0.155 mmol) and a reaction time of 2.0 h. The product was

crystallized from DCM/n-hexane and isolated as red crystals.

Yield: 63 mg (42%).

¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =1.68 (s, 15H,

CH_3,Cp*), 2.08 (s, 3H, H1), 7.30 (d, ³J (H,H) = 6 Hz, 1H, H5),

7.45-7.55 (m, 3H, H9, H10, H11), 7.57–7.67 (m, 3H, H6, H8, H12),

7.58–7.67 (m, 1H, H6) ppm; ¹³C-NMR (125.75 MHz, d₆-DMSO,

25 °C): δ = 8.5 (CH_3,Cp*), 14.2 (C1), 97.8 (Cq, Cp*), 121.3 (C5),

126.0 (C8, C12), 128.7 (C6), 129.9 (C9, C11), 130.0 (C10),

0.691 mmol)

and

bis[dichlorido(η⁶-p-

cymene)osmium(II)] (248 mg, 0.314 mmol) and a reaction time of

1 h. The product was crystallized from DCM/Et₂O and isolated as

orange crystals. Yield: 248 mg (63%).

Monomer 4d: ¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =

3

1.24 (d, J (H,H) = 7 Hz, 6H, Hg), 2.02 (s, 3H, H1), 2.24 (s, 3H,

Ha), 2.66–2.74 (m, ³J (H,H) = 7 Hz, 1H, Hf), 5.98 (d,

³J (H,H) = 5 Hz, 1H, Hd), 6.02 (d, ³J (H,H) = 5 Hz, 1H, Hc),

6.16 (d, ³J (H,H) = 5 Hz, 2H, Hc, Hd), 7.04 (d, ³J (H,H) = 6 Hz, 1H, 135.8 (C2), 141.4 (C7), 161.3 (C4), 166.2 (C3) ppm.

H5), 7.55 (d, ³J (H,H) = 8 Hz, 1H, H8), 7.60–7.66 (m, 1H, H9),

7.66–7.71 (m, 2H, H10, H14), 7.71–7.78 (m, 2H, H6, H13),

ESI-HR-MS⁺m/z found (calculated): [M]⁺454.0708 (454.0706).

Elemental anaylsis calcd (%) for C₂₂H₂₅ClNORhS·1.1 H₂O:

C 51.84, H 5.38, N 2.75, S 6.29; found: C 51.71, H 5.19, N 2.86,

S 6.23.

3

8.17 (d, J (H,H) = 8 Hz, 1H, H15), 8.24 (d, J (H,H) = 8 Hz, 1H,

H12) ppm; ¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ =

13.1 (C1), 17.5 (Ca), 22.3 (Cg), 22.6 (Cg), 30.5 (Cf), 79.0 (Cc),

79.4 (Cd), 81.4 (Cb), 95.3 (Ce), 120.7 (C5), 121.1 (C15),

124.8 (C8), 125.8 (C9, C13), 127.5 (C6), 127.7 (C11, C16),

128.8 (C12, C14), 130.5 (C5), 130.7 (C10), 133.7 (C16),

137.0 (C2, C7), 167.8 (C4), 168.5 (C3) ppm.

Chlorido[1-benzyl-2-methyl-3-oxo-κO-pyridine-4(1H)-thionato-

κS](η⁵-1,2,3,4,5-pentamethylcyclopentadienyl)rhodium(III) (5c):

The synthesis was performed according to the general

complexation protocol using ligand 2c (50 mg, 0.216 mmol),

sodium methoxide (14 mg, 0.259 mmol) and bis[dichlorido(η⁵-

ESI-HR-MS^-m/z found (calculated): [M]^-662.0711 (662.0712).

Elemental anaylsis calcd (%) for C₂₆H₂₆ClNOOsS·0.5 CH₂Cl₂:

1,2,3,4,5-pentamethyl-cyclopentadienyl)rhodium

(III)] (60 mg,

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Chemistry - A European Journal

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0.0973 mmol) and a reaction time of 2.0 h. The product was

crystallized from DCM/n-hexane and isolated as red crystals.

Yield: 72 mg (74%).

protocol using ligand 2a (50 mg, 0.322 mmol), sodium

methoxide (26 mg, 0.483 mmol) and bis[dichlorido(η⁵-1,2,3,4,5-

pentamethyl-cyclopentadienyl)iridium(III)] (128 mg, 0.161 mmol)

and a reaction time of 2.0 h. The product was precipitated from

DCM/n-hexane and isolated as an orange solid. Yield: 124 mg

(74%).

Monomer

5c:

¹H-NMR

(500.10 MHz,

d₆-DMSO,

25 °C): δ =1.65 (s, 15H, CH_3,Cp*), 2.36 (s, 3H, H1), 5.51 (s, 2H,

H7), 7.10 (d, ³J (H,H) = 7 Hz, 2H, H9, H13), 7.34 (d,

³J (H,H) = 6 Hz, 2H, H10, H12), 7.40 (dd, ³J (H,H) = 7 Hz,

³J (H,H) = 7 Hz, 2H, H5, H11), 7.58–7.70 (m, ³J (H,H) = 6 Hz, 1H,

H6) ppm; ¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 8.4 (CH_3,

Cp*), 12.3 (C1), 58.3 (C7), 98.7 (Cq, Cp*), 121.8 (C5), 126.6 (C9,

C13), 128.2 (C10, C12), 129.1 (C11). 129.5 (C6), 135.1 (C8),

136.9 (C2), 159.7 (C4), 166.6 (C3) ppm.

Dimer 5c*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 1.15 (s, 15H,

CH_3,Cp*), 1.55 (s, 9H, CH1), 1.57 (s, 45H, CH₃, Cp*), 2.46 (s, 3H,

H1), 5.27 (s, 6H, H7), 5.66 (s, 2H, H7), 7.11-7.15 (m, 6H, H9,

H13), 7.23-7.26 (m, 2H, H9, H13), 7.38 (m, 3H, H10, H11, H12),

7.40-7.46 (m, 12H, H5, H10, H11, H12), 7.64 (d, ³J (H,H) = 6 Hz,

2H, H6), 7.81 (s, 1H, H5), 7.95 (s, 1H, H6) ppm; ¹³C-

NMR (125.75 MHz, D₂O, 25 °C): δ = 7.1 (CH_3,Cp*), 7.8 (CH_3,

Cp*), 12.1 (C1), 13.3 (C1), 60.4 (C7), 61.0 (C7), 96.5 (Cq, Cp*),

97.1 (Cq, Cp*), 124.6 (C5), 126.4 (C5), 127.1 (C9, C13),

127.4 (C9, C13), 128.8 (C10, C12), 128.9 (C6, C11), 129.3 (C10,

C11, C12), 130.3 (C6), 133.1 (C8), 133.5 (C8), 140.9 (C2),

143.2 (C2), 145.3 (C4), 150.6 (C4), 170.2 (C3), 171.3 (C4) ppm.

1:3 ratio

Monomer

6a:

¹H-NMR

(500.10 MHz,

d₆-DMSO,

25 °C): δ = 1.71 (s, 15H, CH₃, Cp*), 3.90 (s, 3H, H1), 7.33 (d,

3

³J (H,H) = 6 Hz, 1H, H5), 7.63 (d, J (H,H) = 6 Hz, 1H, H6) ppm;

¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 8.1 (CH₃, Cp*),

12.2 (C1), 43.4 (C7), 93.7 (Cq, Cp*), 121.3 (C5), 130.4 (C6),

138.3 (C2), 156.8 (C4), 167.5 (C3) ppm, H7 signal under DMSO

peak.

Dimer 6a*: ¹H-NMR (500.10 MHz, D₂O, 25 °C): δ = 1.10 (s, 15H,

CH₃, Cp*), 1.55 (s, 120H, CH3, Cp*), 2. 09(s, 24H, H1), 2.69 (s,

3H, H1), 3.90 (s, 24H, H7), 4.03 (s, 3H, H7), 7.28 (d,

³J (H,H) = 6 Hz, 8H, H5), 7.41 (d, ³J (H,H) = 6 Hz, 8H, H6),

7.60 (d, ³J (H,H) = 6 Hz, 1H, H5), 7.69 (d, ³J (H,H) = 6 Hz, 1H,

H6) ppm; ¹³C-NMR (125.75 MHz, D₂O, 25 °C): δ = 7.0 (CH₃, Cp*),

7.7 (CH3, Cp*), 11.7 (C1), 13.0 (C1), 44.5 (C7), 45.3 (C7),

89.9 (Cq, Cp*), 90.8 (Cq, Cp*), 125.0 (2 C5), 129.7 (C6),

131.3 (C6),

143.8 (2

C2),

148.5 (2 C4),

169.3 (C3),

170.6 (C3) ppm. 1:8 ratio.

ESI-HR-MS⁺m/z found (calculated): [M]⁺482.1104 (482.1123).

Elemental anaylsis calcd (%) for C₁₇H₂₃ClIrNOS·1.2 H₂O: C 37.90,

H 4.75, N 2.60, S 5.95; found: C 37.98, H 4.82, N 2.58, S 5.87.

ESI-HR-MS⁺m/z found (calculated): [M]⁺468.0854 (468.0863).

Elemental anaylsis calcd (%) for C₂₃H₂₇ClNORhS·0.75 H₂O:

C 53.39, H 5.55, N 2.71, S 6.20; found: C 53.21, H 5.47, N 2.62,

S 5.99.

Chlorido[1-phenyl-2-methyl-3-oxo-κO-pyridine-4(1H)-thionato-

κS](η⁵-1,2,3,4,5-pentamethylcyclopentadienyl)iridium(III) (6b):

The synthesis was performed according to the general

complexation protocol using ligand 2b (54 mg, 0.250 mmol),

sodium methoxide (20 mg, 0.380 mmol) and bis[dichlorido(η⁵-

1,2,3,4,5-pentamethyl-cyclopentadienyl)iridium(III)] (100 mg,

0.130 mmol) and a reaction time of 2.0 h. The product was

precipitated from DCM/n-hexane and isolated as a red powder.

Yield: 120 mg (83%).

Chlorido[1-(naphthalene-1-yl)-2-methyl-3-oxo-κO-pyridine-4(1H)-

thionato-κS](η⁵-1,2,3,4,5-

pentamethylcyclopentadienyl)rhodium(III) (5d): The synthesis

was performed according to the general complexation protocol

using ligand 2d (85 mg, 0.320 mmol,), sodium methoxide (26 mg,

0.480 mmol)

and

bis[dichlorido(η⁵-1,2,3,4,5-pentamethyl-

cyclopentadienyl)rhodium (III)] (100 mg, 0.160 mmol) and

a

¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =1.74 (s, 15H, CH₃,

3

reaction time of 2.0 h. The product was precipitated from DCM/n-

Cp*), 2.19 (s, 3H, H1), 7.45 (d, J (H,H) = 6 Hz, 1H, H5), 7.54–

1

hexane and isolated as a red powder. Yield: 136 mg (79%). H-

7.59 (m, 2H, H8, H12), 7.60–7.66 (m, 4H, H6, H9, H10,

H11) ppm; ¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ =

8.1 (CH₃, Cp*), 14.2 (C1), 93.8 (Cq, Cp*), 121.4 (C5), 126.0 (C8,

C12), 129.9 (C6), 130.1 (C9, C11), 130.2 (C10), 137.2 (C2),

141.1 (C7), 159.4 (C4), 167.5 (C3) ppm.

ESI-HR-MS⁺m/z found (calculated): [M]⁺544.1265 (544.1280).

Elemental anaylsis calcd (%) for C₂₂H₂₅ClIrNOS·0.75 H₂O:

C 44.58, H 4.51, N 2.36, S 5.41; found: C 44.25, H 4.52, N 2.35,

NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =1.71 (s, 15H, CH_3,Cp*),

1.99 (s, 3H, H1), 7.04 (d, ³J(H,H) = 8 Hz, 1H, H5), 7.42 (d,

³J (H,H) = 7 Hz, 1H, H8), 7.62–7.77 (m, 5H, H6, H9, H10, H13,

H14), 8.16 (d, ³J (H,H) = 8 Hz, 1H, H15), 8.23 (d, ³J(H,H) = 8 Hz,

1H, H12) ppm.

¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 8.5 (CH₃, Cp*),

13.4 (C1), 98.6 (Cq, Cp*), 120.7 (C5), 121.8 (C15), 124.6 (C8),

125.7 (C9, C13), 127.4 (C6), 127.7 (C11, C16), 128.7 (C12, C14), S 5.43.

129.6 (C5), 130.6 (C10), 133.7 (C16), 136.8 (C7), 137.2 (C2),

162.0 (C4), 166.2 (C3) ppm.

Chlorido[1-benzyl-2-methyl-3-oxo-κO-pyridine-4(1H)-thionato-

ESI-HR-MS⁺m/z found (calculated): [M]⁺504.0856 (504.0863).

Elemental anaylsis calcd (%) for C₂₆H₂₇ClNORhS·0.25 H₂O:

C 57.36, H 5.09, N 2.57, S 5.89; found: C 57.24, H 5.24, N 2.50,

S 5.56.

κS](η⁵-1,2,3,4,5-pentamethylcyclopentadienyl)iridium(III) (6c):

The synthesis was performed according to the general

complexation protocol using ligand 2c (58 mg, 0.250 mmol),

sodium methoxide (20 mg, 0.380 mmol) and bis[dichlorido(η⁵-

1,2,3,4,5-pentamethyl-cyclopentadienyl)iridium(III)] (100 mg,

0.130 mmol) and a reaction time of 2.0 h. The product was

precipitated from DCM/n-hexane and isolated as an orange

powder. Yield: 115 mg (78%).

Chlorido[1,2-dimethyl-3-oxo-κO-pyridine-4(1H)-thionato-κS](η⁵-

1,2,3,4,5-pentamethylcyclopentadienyl)iridium(III) (6a):

The

synthesis was performed according to the general complexation

10.1002/chem.201905546

Chemistry - A European Journal

FULL PAPER

¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =1.71 (s, 15H, CH₃,

Cp*), 2.42 (s, 3H, H1), 5.56 (s, 2H, H7), 7.13 (d, ³J (H,H) = 6 Hz,

2H, H9, H13), 7.40 (dd, ³J (H,H) = 7 Hz, ³J (H,H) = 7 Hz, 1H,

H11), 7.46 (d, ³J (H,H) = 6 Hz, 2H, H10, H12), 7.46 (d,

[6]

[7]

F. Lentz, A. Drescher, A. Lindauer, M. Henke, R. A. Hilger,

C. G. Hartinger, M. E. Scheulen, C. Dittrich, B. K. Keppler,

U. Jaehde, Anticancer. Drugs 2009, 20, 97–103.

C. A. Riedl, M. Hejl, M. H. M. Klose, A. Roller, M. A.

Jakupec, W. Kandioller, B. K. Keppler, Dalt. Trans. 2018,

47, 4625–4638.

3

³J (H,H) = 6 Hz, 1H, H5), 7.80 (d, J (H,H) = 6 Hz, 1H, H6) ppm;

¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 8.1 (CH₃, Cp*),

12.3 (C1), 58.2 (C7), 93.8 (Cq, Cp*), 121.7 (C5), 126.7 (C9, C13),

128.3 (C11), 129.1 (C10, C12), 130.5 (C6), 134.8 (C8),

137.5 (C2), 158.3 (C4), 168.0 (C3) ppm.

ESI-HR-MS⁺m/z found (calculated): [M]⁺558.1422 (558.1436).

Elemental anaylsis calcd (%) for C₂₃H₂₇ClIrNOS: C 46.57, H 4.59,

N 2.36, S 5.41; found: C 46.46, H 4.75, N 2.44, S 5.42.

[8]

P. C. Bruijnincx, P. J. Sadler, Curr. Opin. Chem. Biol. 2008,

12, 197–206.

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H. Maeda, J. Wu, T. Sawa, Y. Matsumura, K. Hori, J.

Control. Release 2000, 65, 271–284.

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[11]

[12]

[13]

[14]

M. Ni, Y. Zhang, A. S. Lee, Biochem. J. 2011, 434, 181–

188.

M. A. Bennett, M. J. Byrnes, I. Kováčik, J. Organomet.

Chem. 2004, 689, 4463–4474.

Chlorido[1-(naphthalene-1-yl)-2-methyl-3-oxo-κO-pyridine-4(1H)-

thionato-κS](η⁵-1,2,3,4,5-

pentamethylcyclopentadienyl)iridium(III) (6d): The synthesis was

performed according to the general complexation protocol using

ligand 2d (67 mg, 0.250 mmol), sodium methoxide (20 mg,

Y. K. Yan, M. Melchart, A. Habtemariam, P. J. Sadler,

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54, 3–25.

0.34 mmol)

and

bis[dichlorido(η⁵-1,2,3,4,5-pentamethyl-

cyclopentadienyl)iridium(III)] (100 mg, 0.13 mmol) and a reaction

time of 2.0 h. The product was precipitated from DCM/n-hexane

and isolated as an orange powder. Yield: 114 mg (72%).

¹H-NMR (500.10 MHz, d₆-DMSO, 25 °C): δ =1.71 (s, 15H, CH₃,

Cp*), 1.99 (s, 3H, H1), 7.07 (d, ³J(H,H) = 8 Hz, 1H, H5), 7.55 (d,

³J (H,H) = 7 Hz, 1H, H8), 7.61–7.82 (m, 5H, H6, H9, H10, H13,

H14), 8.17 (d, ³J (H,H) = 8 Hz, 1H, H15), 8.25 (d, ³J(H,H) = 8 Hz,

1H, H12) ppm.

¹³C-NMR (125.75 MHz, d₆-DMSO, 25 °C): δ = 8.1 (CH₃, Cp*),

13.4 (C1), 93.9 (Cq, Cp*), 120.8 (C5), 121.8 (C15), 124.8 (C8),

125.7 (C9, C13), 127.5 (C6), 127.6 (C11, C16), 128.7 (C12, C14),

130.8 (C5, C10), 133.7 (C16), 136.9 (C7), 137.7 (C2), 160.5 (C4),

167.6 (C3) ppm.

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ESI-HR-MS⁺m/z found (calculated): [M]⁺594.1424 (594.1436).

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Article Doi

Synthesis, Modification, and Biological Evaluation of a Library of Novel Water-Soluble Thiopyridone-Based Organometallic Complexes and Their Unexpected (Biological) Behavior

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