Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N'-chelate ligands to rhenium(i) complexes and the anticancer activity of newly synthesized oximine rhenium(i) complexes against human melanoma and leukemia cells in vitro

Journal of Inorganic Biochemistry 104 (2010) 774–789

Contents lists available at ScienceDirect

Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N′-chelate ligands

to rhenium(^I) complexes and the anticancer activity of newly synthesized oximine

☆

rhenium(^I) complexes against human melanoma and leukemia cells in vitro

Stefan Wirth ^a, Andreas U. Wallek ^a, Anna Zernickel ^a, Florian Feil ^a, M. Sztiller-Sikorska ^b,

b,

K. Lesiak-Mieczkowska ^b, Christoph Bräuchle ^a, Ingo-Peter Lorenz ^a,, M. Czyz

⁎

a

Ludwig-Maximilians University Munich, Department of Chemistry and Biochemistry, Butenandtstr. 5-13 (House D), D-81377 Munich, Germany

Medical University of Lodz, Department of Molecular Biology of Cancer, 6/8 Mazowiecka, 92-215 Lodz, Poland

b

a r t i c l e i n f o

a b s t r a c t

Article history:

The synthesis, structural characterization and biological activity of eight ortho-quinone(N-aryl)-oximine

rhenium(^I) complexes are described. The reaction of the halogenido complexes (CO)₅ReX (X=Cl (4), Br (5))

with 2-nitroso-N-arylanilines {(C₆H₃ClNO)NH(C₆H₄R)} (R=p-Cl, p-Me, o-Cl, H) (3a–d) in tetrahydrofurane

(THF) yields the complexes fac-(CO)₃XRe{(C₆H₃ClNO)NH(C₆H₄R)} (6a–d, 7a–d) with the tautomerized

ligand acting as a N,N′-chelate. The substitution of two carbonyl ligands leads to the formation of a nearly

planar 5-membered metallacycle. During coordination the amino-proton is shifted to the oxygen of the

nitroso group which can be observed in solution for 6 and 7 by ¹H NMR spectroscopy and in solid state by

Received 6 November 2009

Received in revised form 24 March 2010

Accepted 26 March 2010

Available online 1 April 2010

Keywords:

Rhenium

Oximine

crystal structure analysis. After puriﬁcation, all compounds have been fully characterized by their ¹H and ¹³

C

NMR, IR, UV/visible (UV/Vis) and mass spectra. The X-ray structure analyses revealed a distorted octahedral

coordination of the CO, X and N,N′-chelating ligands for all Re(^I) complexes. Biological activity of four

oximine rhenium(^I) complexes was assessed in vitro in two highly aggressive cancer cell lines: human

metastatic melanoma A375 and human chronic myelogenous leukemia K562. Chlorido complexes (6a and

6c) were more efﬁcient than bromido compounds (7d and 7b) in inducing apoptotic cell death of both types

of cancer cells. Melanoma cells were more susceptible to tested rhenium(^I) complexes than leukemia cells.

None of the ligands (3a–d) showed any signiﬁcant anticancer activity.

N,N′-chelates

In vitro anticancer activity

Melanoma

Leukemia

1. Introduction

various biological metabolic processes [13–21] has also generated a

renewed interest in this class of compounds.

The chemistry of C-nitroso compounds started in 1874 with the

synthesis of 4-nitroso-N,N′-dimethylaniline [1] and nitrosobenzene

[2] by A. von Baeyer. Their ﬁrst coordination to metals (Cd(^II) and

Zn(^II)) was reported by Pickard and Kenyon in 1907 [3]. Since then a

considerable variety of synthetic routes to high-yield preparations of

C-nitroso compounds has been developed. The most up to date

reviews on this topic have been recently published [4,5]. Not only due

to its rich coordination chemistry [6] the family of C-nitroso

compounds has been extensively investigated during the last decades.

Its relevance in organic chemistry [7] was ﬁrst proved 1899 by the

Ehrlich–Sachs reaction [8]. Examples published in recent years are

application in ene reactions [9] or hetero Diels–Alder reactions [10–

12]. The discovery of the important roles of C-nitroso compounds in

In this context the convenient availability of 2-nitroso-N-arylani-

lines [22] has drawn our attention from N,O-bridging [23–25] and -

chelating [26] to N,N′-chelating ligands. Before 2007 this class of

compounds was mostly reported as a by-product [27–29]. Examples

are the photochemical cyclization of N-acyl-2-nitroarylanilines

[30,31] or the Fischer–Hepp rearrangement [32]. Only two compara-

ble compounds with additional functional groups (methyl 6-hydroxy-

4-methyl-3-nitroso-2-(phenylamino)benzoate [33] and 2-nitroso-

1,3,5-tris(phenylamino)benzene [34]) were obtainable in good yields

earlier. In coordination chemistry this ligand system is mentioned in

few binuclear Pd(^II) complexes [35–38]. There, it is formed by the

reaction of a tetranuclear Pd(^I) cluster with nitrosoarenes. Contrary to

these results, the reaction of 2-nitroso-N-arylanilines with Re(^I)

halogenido complexes of the type Re(CO)₅X (X=Cl, Br) leads to a

metal-induced tautomerization. An o-quinoid system is formed and

the amino-proton is shifted to the oxygen of the nitroso group. In this

report we describe the synthesis and characterization of eight Re(^I)

complexes showing this tautomeric behaviour and the results of

testing for biological activity of four of these complexes. We have

☆

Dedicated to Prof. Dr. Hubert Schmidbaur on the Occasion of his 75th Birthday.

Corresponding authors. Fax: +49 89 2180 77867.

⁎

E-mail addresses: ipl@cup.uni-muenchen.de (I.-P. Lorenz),

malgorzata.czyz@umed.lodz.pl (M. Czyz).

doi:10.1016/j.jinorgbio.2010.03.014

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

775

complexes presented here are the ﬁrst combining the imine and the

oxime function in one and the same o-quinoid system.

2. Results and discussion

2.1. Synthesis and characterization of ligands 3a–d

Scheme 1. Synthesis of C-nitroso compounds 3a–d.

The 2-nitroso-N-arylaniline ligands 3a–d were synthesized in a

one-pot reaction from anilines 1a–d and 1-chloro-4-nitrobenzene (2)

with potassium-tert-butoxide and acetic acid in dimethylformamide

(DMF) (Scheme 1). Modiﬁcations made on this literature method [22]

are described in the experimental section.

selected 6a, 6c, 7d, and 7b for biological studies. The cytotoxicity of

the drugs was evaluated in A375, a human melanoma cell line

exhibiting high metastatic potential, and K562, a Bcr–Abl-positive

human chronic myelogenous leukemia (CML) cell line derived from a

patient in blast crisis.

Metal-induced tautomerization reactions have been examined

especially in relation with Pt(^II) pyrimindine [39] and adenine [40]

model nucleobase interactions. In these examples a shift of the

equilibrium to the “wrong” tautomer could lead to base-mispairing in

nucleic acids. Moreover, metal-induced proton migration in com-

plexes is an important attribute in connection with the design of

molecular electronic devices [41]. The capacity of intercalation into

DNA [42,43] and in general the strong metal–ligand π-interaction

have also attracted great interest into o-quinone ligand systems. Much

literature on this topic is concerned with o-quinoid diimines [44–48],

less with dioximes [49–52], but to the best of our knowledge the Re(^I)

The synthesis yields ligands 3a–d as air stable, dark green or brown

powders, soluble for example in dichloromethane, tetrahydrofurane

or acetone and nearly insoluble in pentane or n-hexane. Mass

spectrometric investigation in the direct electron-impact ionization

mode with detection of positive ions (DEI⁺) shows the expected [M⁺

]

peak and an assignable fragmentation pattern for all ligands. In the ¹H

NMR of 3a–d spectra a broad singlet in the range of 11.60 to

12.06 ppm can be identiﬁed as the amino-proton signal. Another

broad singlet at δ=8.64–8.66 ppm can be assigned to the proton in

ortho-position of the NO-group. The remaining signals of the protons

in meta-position and the second aromatic ring are observed at

δ=6.93–7.52 ppm. Exemplarily the ¹³C NMR spectrum of 3c is

depicted in Fig. 1.

Fig. 1. ¹³C NMR spectra of 3c at A) 25 °C and B) −60 °C.

776

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

For each ligand one C_qand one C_Hpeak was “missing” and a very

broad signal around 140 ppm could be detected at room temperature

(Fig. 1). Measurement at −60 °C shows two “new” peaks at 130.8

(C_q1) and 142.2 ppm (C_H3) which can be assigned to the carbons in

ortho-position of the nitroso group. “Freezing” rotation along the C–N

bond leads to

a signiﬁcant splitting of the broad signal

(Δ=11.4 ppm). This is induced of course by the different substituents

but in fact more due to the large magnetic anisotropy [53] of the NO-

group. This effect of an asymmetric substituent upon the relative

chemical shifts of the ortho-carbons is a known phenomenon and

established for aromatic nitroso compounds [54,55].

The IR spectra of ligand 3a–d (KBr pellets) show ν(C–H)

absorptions between 3100 and 2900 cm⁻¹but lack a pronounced

ν(N–H) band, apparently as a consequence of the strong intramolec-

ular hydrogen bonding [32]. Therefore, a broad and weak absorption

between 2900–2650 cm⁻¹is observed due to N…H…O bonding.

Surprisingly a very weak absorption for ν(N–H) is detected in liquid

phase IR spectra at higher wave numbers (3a: 3372 cm⁻¹; 3b:

3377 cm⁻¹; 3c: 3373 cm⁻¹; 3d: 3378 cm⁻¹in CH₂Cl₂). Nitroso

stretching absorptions are assigned according to the reports of

Gowenlock et al. in the range of 1488–1513 cm⁻¹for monomeric

aromatic ArNO compounds [21,55,56]. The allocation of these bands is

supported due to the fact that they disappear after complexation. This

demonstrates the fundamental change in chemical character of the

N―O bond when tautomerization from nitroso to oxime happens.

Measurement of UV/Vis spectra of 3a–d in dichloromethane

revealed four intense absorptions for each ligand (Table 1). Three

are located in the UV area and are originated from π–π* transitions of

the aromatic rings. The fourth is situated in the visible range and is

identiﬁed as π–π* NO transition.

Fig. 2. Molecular structure of 5-Chloro-2-nitroso-N-p-tolylaniline (3b) with π–π

stacking (open bond) and 3-centered intermolecular hydrogen bond (dashed lines).

The thermal ellipsoids are drawn at the 50% probability level [57]. Aromatic and

aliphatic hydrogen atoms as well as parts of further molecules of 3b are omitted for

clarity.

Since no crystallographic information was available for this com-

pound class in the literature, molecular structures of 3b (Fig. 2) and 3d

have been determined by X-ray diffraction analysis. Single crystals were

obtained by slow sublimation at 55 °C and 1.0×10⁻³mbar. The

structure analysis revealed two planar aromatic rings (C(1)–C(6) and

C(7)–C(12)) whereas the ﬁrst ring shows some quinoid contribution.

C―C bond lengths for C(3)−C(4) and C(5)–C(6) are noticeable

shortened in comparison to the remaining aromatic ring. Furthermore

C―N bond lengths of N(1)–C(1) and N(2)–C(2) are always a bit shorter

than N(1)–C(7). In both cases the nitroso group, located in the plane of

the C(1)–C(6) ring and the amine, is stabilized by an intramolecular

hydrogen bond. N―O bond lengths are in the expected range (1.13–

1.29 Å) [21] for nitrosoarenes.

In both structures a secondary intermolecular interaction of the

amine proton forms a 3-centered hydrogen bond. In 3b the

intermolecular acceptor is chlorine (Cl(1)) (Fig. 2). For 3d the

additional acceptor is a nitroso oxygen whereas the interacting

molecule forms a second “back bonding” H-bridge. As further

intermolecular interaction a π–π stacking [58,59] of the C(1)–C(6)

ring occurs (Fig. 2). In the unit cell of 3b for example, two of these

rings show absolutely coplanar arrangement with a plane-to-plane

distance of 3.50 Å. A parallel displacement of only 1.00 Å is calculated

for the centroids. For 3d a slight deviation from coplanarity is

observed.

Table 1

UV/Vis absorption data of 3a–3d and 6a–7d: in CH₂Cl₂

ﬂuorescence data of 3c and 6a–7d in CH₂Cl₂λ_max[nm].

λ

_max[nm] (ε [M⁻¹cm⁻¹]) and

UV

Visible

3a

253(13300), 278(17100), 312(14900) 461(8200)

3b 249(12900), 269(12100), 312(12700) 467(6900)

3c

253(12000), 275^a(15500), 312^a

(13400)

457(7200)

λ_em523^b523^b

3d 250(12000), 274(12400), 312(12400) 464(6800)

471(4500), 548^a(9700)

2.2. Synthesis and characterization of Re(^I) complexes 6a–7d

6a

λ_em

321(4500)

607^b

6b 320(4500)

474(5200), 541^a(10500)

The novel oximine rhenium(^I) complexes (6a–7d) are obtained as

illustrated in Scheme 2. Reﬂuxing Re(CO)₅X (X=Cl (4), Br (5)) in dry

THF leads to the substitution of two CO ligands. Completeness of the

replacement can be monitored with liquid phase IR spectroscopy by a

shift of the ν(CO) bands.

λ_em

573^b

6c

322^a(6400)

438(4500), 470(4800), 555^a

(12600)

λ_em510^b

6d 321(4900)

λ_em

606^b

470(4800), 544^a(11200)

612^b

Addition of one equivalent of 3a–d in dry THF and workup as

described in the experimental section yields the complexes 6a–7d

quantitatively. Complexes 6a–7d are obtained as dark green (6a–7b)

or dark purple (7c, 7d) powders. They are slowly decomposing when

exposed to moist air, soluble in dichloromethane or chloroform and

insoluble in pentane. Mass spectrometric investigations of complexes

6a–7d (FAB⁺mode) exhibit the parent peak as well as a comparable

fragmentation pattern resulting from successive loss of CO and

halogenido ligands.

7a

358(6200)

475(5400), 549^a(11000)

λ_em

600^c

7b 357(5400)

λ_em

481(5500), 544^a(9900)

586^c

7c

362(6400)

441(3800), 474(4400), 557^a

(11200)

λ_em

601^c

7d 358(5600)

λ_em

473(4700), 546^a(9800)

608^c

a

Excitation at the marked absorption peak.

Accuracy of the measurement: 3c, 6a–d: 3 nm.

Accuracy of the measurement: 7a–d: 10 nm.

Comparison of the ¹H NMR spectra of 6a–7d with the

corresponding ligand spectra (3a–d) shows similar tendencies. In all

b

c

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

777

Scheme 2. Synthesis of the N,N′-chelate complexes 6a–7d.

cases the broad singlet of the acidic proton shows a large shift to

higher ﬁeld due to migration from the amino group (3a–d: 11.60–

12.06 ppm) to the nitroso oxygen (6a–7d: 8.31–9.50 ppm). All three

signals of the ortho-quinoid system are shifted to higher ﬁeld after

coordination. The protons of the second, aromatic ring of complexes

6a–7d show no consistent tendencies compared to the ligand spectra.

In the ¹³C NMR spectra of 6a–7d all signals for the CO ligands, the

quaternary aromatic carbons and the aromatic C_Hcarbons are

detected in the expected areas. Since rotation of the nitroso group

along C(2)–N(2) is inhibited after coordination, all signals are

observable separately at room temperature. In comparison to the

starting materials 3a–d no general direction of the shifts can be

identiﬁed in products 6a–7d.

strength. The stronger absorptions in the visible region arise from

π–π* transitions of the ligand, mostly with no distinct maxima, shifted

there by its tautomerization into an o-quinoid form. All spectra show

this bathochromic shift in comparison to the corresponding electronic

transitions of 3a–d (Table 1). Compounds 6a–7d showed weak

ﬂuorescence in the visible range with a ﬂuorescence quantum yield of

about 10⁻⁵. Excitation at the absorption maxima of 6a–7d at around

550 nm resulted in ﬂuorescence maxima at around 610 nm (Table 1).

For bromido complexes 7a–7d the signal to noise ratio (SNR) was at

the detection limit and about one order of magnitude lower than the

SNR of 6a–6d with chlorido ligand. Therefore the error of the

ﬂuorescence maxima position is signiﬁcantly increased. That in

consideration, the ﬂuorescence maxima position of the chlorido

complex compared with the corresponding bromido complex seems

to be similar, while the different ligands (3a–d) have a small inﬂuence

on the maxima. It is noteworthy that the ligand in its aromatic form

(only tested for 3c) exhibited also very weak ﬂuorescence at around

523 nm when exciting at 275 nm or 312 nm, but not around 610 nm.

This indicates that the ﬂuorescence at 510 nm in 6c is only caused by

the rhenium carbonyl part and that around 610 nm results from the

ligand which has been changed into its o-quinoid form showing lower

frequencies. Selected ﬂuorescence spectra are available as supporting

information.

The molecular structures of complexes 6a–7d were determined by

X-ray diffraction analysis. Single crystals were obtained by isothermic

diffusion of n-pentane into a solution of 6a–7d in CH₂Cl₂or CHCl₃. The

X-ray structure analysis revealed a distorted octahedral coordination

for all Re(^I) complexes, consisting of the CO, halogenido and N,N′-

chelating ligands 3a–d.

Crystal data and details of structure reﬁnement for compounds 3b,

3d and 6a–7d are summarized in Table 5. Selected bond lengths and

angles are listed in Table 2. Exemplarily, only 6a is depicted in Fig. 3,

ORTEP-plots of all other structures and hydrogen bond data are

IR spectra of 6a–7d in liquid phase show three intense ν(CO)

absorptions. This is in accordance with C_S–symmetry of their facial

arrangement. Surprisingly measurement of 7a–7d in KBr pellets

exhibit up to ﬁve ν(CO) bands what apparently can be caused by

packing effects in solid state. In the area around 3000 cm⁻¹ν(C–H)

absorptions are detected overlapping with a broad band between 3050

and 3200 cm⁻¹attributed to ν(O―H). Comparison of IR data of

different transition metal complexes containing an oxime or oximine

function [60–64] with spectra of 6a–7d leads to the following

assignment: absorptions at 1597–1606 cm⁻¹to ν(C=N) of the

imine moiety, bands from 1543–1553 cm⁻¹to ν(C=N) of the oxime

moiety. The interval associated with ν(N―O) (1040–1060 cm⁻¹

)

shows two strong absorptions very close together for each complex,

again caused by packing effects.

The UV/Vis spectroscopic investigations of 6a–7d in CH₂Cl₂

revealed typically three and in two cases (6c and 7c) four absorptions.

One is located in the UV area and is caused by a d⁶-metal-to-ligand

charge-transfer transition, the rest appears in the visible range. The

difference of about 30 m between chlorido (6a–6d) and bromido

complexes (7a–7d) may be caused by their different ligandﬁeld

Table 2

Selected bond lengths (Å) and angles (°) of compounds 3b, 3d and 6a–7d.

Compound

3b

3d

6a

1.948(4)

6b

1.943(4)

6c

6d

1.904(12)

7a

7b

1.936(5)

7c

7d

Re(1)–C(13)/C(14) 6b

Re(1)–C(14)/C(15) 6b

Re(1)–C(15)/C(16) 6b

Re(1)–Cl 6/Br 7

Re(1)–N(1)

Re(1)–N(2)

O(1)–N(2)

N(2)–C(2)

C(1)–C(2)

N(1)–C(1)

–

1.947(7)

1.937(7)

1.913(8)

2.480(2)

2.129(5)

2.136(5)

1.369(7)

1.308(8)

1.456(9)

1.313(8)

1.439(8)

73.8(2)

1.951(4)

1.937(4)

1.905(5)

2.621(1)

2.133(3)

2.120(3)

1.370(3)

1.310(4)

1.456(5)

1.315(4)

1.433(4)

73.95(11)

99.88(13)

96.57(14)

−4.7(2)

1.962(8)

1.912(8)

1.913(10)

2.614(1)

2.128(6)

2.137(5)

1.379(7)

1.303(8)

1.471(9)

1.314(9)

1.448(9)

73.7(2)

1.927(7)

1.932(7)

1.901(8)

2.636(1)

2.129(4)

2.112(5)

1.309(6)

1.345(7)

1.447(7)

1.314(7)

1.431(7)

74.99(17)

96.4(2)

–

1.921(4)

1.911(4)

2.481(1)

2.120(3)

2.141(3)

1.384(3)

1.298(4)

1.460(5)

1.310(4)

1.451(4)

73.27(10)

104.13(13)

94.83(13)

4.3(2)

1.937(3)

1.897(4)

2.486(1)

2.134(3)

2.139(3)

1.374(3)

1.312(4)

1.461(4)

1.314(4)

1.439(4)

73.90(9)

103.04(11)

95.15(11)

8.9(2)

1.927(10)

1.904(12)

2.478(2)

2.134(7)

2.136(7)

1.379(8)

1.304(11)

1.468(12)

1.333(10)

1.441(11)

73.8(3)

1.936(5)

1.899(6)

2.623(1)

2.133(3)

2.127(3)

1.375(4)

1.312(5)

1.456(6)

1.319(5)

1.437(5)

73.71(13)

100.67(16)

97.54(16)

5.1(3)

–

1.258(2)

1.388(2)

1.434(3)

1.350(2)

1.431(2)

–

1.259(2)

1.382(2)

1.430(2)

1.357(2)

1.420(2)

–

N(1)–C(7)

N(1)–Re(1)–N(2)

C(13)/C(14) 6b–Re(1)–N(2)

C(14)/C(15) 6b–Re(1)–N(1)

N(2)–Re(1)–N(1)–C(1)

C(1)–N(1)–C(7)–C(12)

–

103.1(2)

95.1(2)

103.1(3)

96.5(3)

−7.1(6)

−110.2(10)

103.5(2)

95.4(3)

–

96.5(2)

1.4(4)

69.1(7)

–

1.8(5)

1.4(5)

74.4(3)

62.2(3)

69.8(4)

76.1(4)

96.4(8)

−103.9(4)

117.7(5)

99.7(8)

778

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

Scheme 3. Average bond lengths of 6a–7d in comparison with average values in 3b and

3d (in brackets).

dependence of this angle on the ligands was not identiﬁable. This

may be an effect of π–π-interaction between the aromatic or quinoid

rings since in every structure an absolute coplanar arrangement of

these moieties is observed. An exception is given in 7d where only

some rings of the same type are coplanar. For complexes 6a–7c a

triclinic crystal system in the space group P-1 is observed, containing

two complex molecules in the unit cell. An intermolecular hydrogen

bond connects the oxime function of one molecule and the halogenido

ligand of another molecule in the next unit cell and vice versa. Thus

each of these pairs is connected through two intermolecular hydrogen

bonds. In complex 7d a monoclinic crystal system in the space group

C2/c with eight molecules in one unit cell is observed. The only

difference to 6a–7c is the lack of intermolecular hydrogen bonds

between complex molecules of 7d. Instead of these, a hydrogen bond

to the CH₂Cl₂enclosed in the cell is observed. This may be the reason

for the discrepancy. Apparently intermolecular hydrogen bonds

between the complex molecules are an integral factor in the

arrangement within the unit cell of these compounds.

Fig. 3. Molecular structure of Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-4-chlor-

ophenyl)-oximine-N,N′}rhenium(I) (6a). The thermal ellipsoids are drawn at the 50%

probability level [57]. Aromatic hydrogen atoms are omitted for clarity.

available as supporting information. Bond lengths Re–CO are in the

expected range for fac-Re(CO)₃X-complexes with quinoid ligands

[46,65], just as well as the Re–X (X=Cl, Br) bonds. The Re―CO_axbond

in trans-position to the halogenido ligand is considerably shorter than

the Re―CO_eqbonds. This indicates a π-accepting character of the N,N′-

chelates. Re–N distances in 6a–7d show no recognizable preference

for a shorter imine or oxime bonding and are close to bond lengths

recently reported for an ortho-quinoid diimine Re(^I) complex [46] or a

Re(^I) nitroso complex [66]. Only few structurally characterized

rhenium oximato complexes suitable for comparison are known.

Re–N bond lengths observed in 6a–7d are slightly longer than

reported for an oximato ligand chelating Re(^V) (2.099(3) Å) [67] or a

dioxime ligand system chelating Re(^III) (2.028(9)–2.108(9) [68] /

2.03(1)–2.17(1) [69]). Crystallographic information for Re(^I) is only

available for a monodentating, nonaromatic oximato ligand. This

shows a somewhat longer Re―N bond length (2.183(6) Å) [70] than

6a–7d.

After coordination the former aromatic system of 3a–d with only

some quinoid contribution shows clearly quinoid topology. A

signiﬁcant elongation of the N―O bond (Scheme 3) to an expected

scale (1.319(4) Å [67], 1.368 Å [69], 1.396(9) Å [70]) conﬁrms the

oxime nature of the former nitroso group. Shortening of N(1)–(C1)

and N(2)–C(2) and explicitly alternating C―C bond lengths indicate

the o-quinoid form of the C(1)–C(6) ring. This is also in accordance to

a recently published bond length pattern for o-quinoid ligands in

different oxidation states[71]. The effect of coordination to rhenium

on the average bond lengths in the second aromatic ring is negligible.

The bidentate ligands 3a–d bind to rhenium(^I) via formation of a

nearly planar metallacycle. This is conﬁrmed by torsion angels close to

0° within the 5-membered ring. Distortion of the octahedral

coordination sphere becomes evident by the small ligand bite angles

N(1)–Re–N(2) around 74° and the angles between the chelating

nitrogen atoms and the equatorial carbonyls. Complexes 6a–7d

exhibit on the oxime-side (N(2)–Re(1)–CO_eq) an average angle of

101.7°. The imine-side (N(1)–Re(1)–CO_eq) shows values close to 95°.

The bridging nitrogen atom (N1) is trigonal planar in geometry

consistent with its sp²nature, whereas the sum of surrounding angles

is always very close to 360°.

2.3. Biological evaluation

2.3.1. Oximine rhenium(^I) complexes inhibit melanoma adherent cell

proliferation more efﬁciently than proliferation of leukemia cells

Concentration–response and time course analyses were per-

formed using four rhenium(^I) complexes (6a, 6c, 7d, and 7b). In

some experiments, the 2-nitroso-N-arylaniline ligands (3a–3d) were

included. Tetrazolium derivative reduction (MTT) assay was used to

assess the inﬂuence of the drugs on the metabolic activity of adherent

melanoma cells (A375) in relation to untreated control cells. Cell

proliferation of leukemic K562 cells cultured in suspension was

determined using Trypan blue dye exclusion assay. First, IC₅₀values

were estimated for the cancer cell inhibition of proliferation as shown

in Fig. 4. IC₅₀values obtained for melanoma A375 cells treated with

6a, 6c, 7d, and 7b for 2 days were 0.9, 0.7, 1.3, and 1.8 μM, respectively

(Fig. 4A).

Higher concentrations of oximine rhenium(^I) complexes were

necessary to reduce proliferation of K562 cells to 50% compared to the

control cells (Fig. 4B). But similarly to the results obtained for

melanoma cells, 6a and 6c were more efﬁcient in induction of growth

arrest (IC₅₀of 3.4 and 3 μM, respectively) than 7d and 7b with IC₅₀of

7.5 μM and 7.8 μM, respectively. Ligand 3c reduced cell proliferation

to 96% and 90% compared to the control cells when it was used at the

concentrations of 4 μM and 8 μM, respectively. The other ligands were

also much less efﬁcient than their respective rhenium(^I) complexes

(not shown).

The second aromatic ring connected to N(1) is highly turned out of

the plane deﬁned by the metallacycle (69.1(7)°–117.7(5)°). A

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

779

Fig. 5. Oximine rhenium(I) complexes inhibit melanoma (A) and leukemia (B) cell

proliferation. A time course. A375 cells were treated with 1.4 μM and K562 cells with

8 μM oximine rhenium(^I) complexes for up to 3 days. Viable, adherent A375 melanoma

cells were quantiﬁed daily by MTT assay and viable K562 leukemia cells by Trypan blue

exclusion test. The data are the mean SD of three independent experiments done in

triplicates (Pb0.05 except for 7b on day 1).

Fig. 4. Concentration–response analyses of the inﬂuence of oximine rhenium(I)

complexes on melanoma and leukemia cell proliferation. (A) MTT assay was used to

study cytostatic effects of oximine rhenium(^I) complexes in human melanoma cell line

A375. The mean of the absolute absorbance values given by drug-treated cells was

divided by the mean of the absolute absorbance of DMSO-treated control sample and

expressed as relative number of viable adherent cells. (B) Trypan blue exclusion test

was applied to assess cytostatic effects of the tested drugs in human CML cell line K562

as described in Materials and Methods. Cell proliferation is expressed as the percentage

of viable cell number in the control culture. IC50 values for each compound in each cell

line were calculated (see text for the results). The data are the mean SD of three

independent experiments done in triplicates.

concentrations of drugs in the range from 1 μM to 100 μM, 6c and

6a reduced viability to 50% of the control at the concentrations of 7 μM

and 8 μM, respectively. The same was observed for 7d and 7b at

concentrations as high as 33 μM and 29 μM, respectively (Fig. 7B).

Ligands 3a–d did not signiﬁcantly affect K562 cell viability even at the

concentration of 20 μM (not shown).

Next, time course analyses were performed. Viable melanoma cells

were quantiﬁed daily by MTT assay, leukemia cells were evaluated by

Trypan blue staining (Fig. 5).To get similar reduction of proliferation

in both cell lines, concentrations of drugs used in these experiments

were 1.4 μM in A375 cell cultures (Fig. 5A) and 8 μM in K562 cell

cultures (Fig. 5B). As expected 6c and 6a were more efﬁcient than 7d

and 7b in both cell types.

2.3.3. Induction of apoptosis in A375 melanoma cells and K562 leukemic

cells by oximine rhenium(^I) complexes

Cell death was assessed in melanoma cells treated with oximine

rhenium(^I) complexes by morphological characteristics of apoptotic

and necrotic cells in ﬂuorescence microscopy after staining with

acridine orange and ethidium bromide (AO/EB) (Fig. 8). More than

300 cells were analyzed and then the percentages of early/late

apoptotic or necrotic cells were calculated. Compound 6c, which most

efﬁciently reduced proliferation of adherent melanoma cells, induced

apoptosis in more than 80% of the cells when applied at the

concentrations of 1 μM and 2 μM (Table 3). For 6a and 7d,

concentration of 2 μM was required to stimulate apoptosis to this

extent. Compound 7b, the least efﬁcient in inhibiting proliferation of

adherent melanoma cells, was also the least effective in inducing cell

death. None of the tested drugs, including ligands, stimulated necrosis

in melanoma cells. Therefore, we could assume that in the applied

conditions apoptotic cell death was the major cause of reduced

proliferation of melanoma cells.

Bcr–Abl-expressing leukemic K562 cells are highly resistant to

apoptosis induced by chemotherapeutic agents. In the current study,

they were assessed on day 3 for the evidence of apoptotic cell death by

double staining with AO/EB (Fig. 9A). In addition, DAPI staining was

used to show the condensation and fragmentation of the nuclei

(Fig. 9B). Neither DAPI staining nor double staining with AO/EB for

early and late apoptosis showed an induction of apoptosis after 3 days

2.3.2. Effects of oximine rhenium(^I) complexes on cancer cell viability

Changes in A375 melanoma cell viability in response to oximine

rhenium(^I) complexes were assessed by propidium iodide staining

and FACS analysis (Fig. 6). On the second day of treatment, only 6c at

the concentration of 1 μM signiﬁcantly reduced the viability of

melanoma cells to 53% (47% 5 of cells were PI-positive; Pb0.05).

The other oximine rhenium(^I) complexes (6a, 7b, and 7d) applied at

the concentration of 1 μM or ligands (3a–d) used at the concentration

of 2 μM did not induce the appearance of PI-permeable cells. When

concentration of rhenium(^I) complexes was raised to 2 μM viability of

melanoma cells was decreased to the level below 30%.

Trypan blue exclusion test was used to assess viability of K562 cells

treated with oximine rhenium(^I) complexes. In this assay, the number

of dead cells taking up Trypan blue was expressed as percentage of the

total cell number (viable and dead) in each experimental condition.

First, K562 cells were treated continuously for 3 days with 8 μM of the

oximine rhenium(^I) complexes. On day 3, similar effectiveness was

observed for 6c and 6a, whereas 7d and 7b were still ineffective

(Fig. 7A). When the viability of K562 cells was tested at the

780

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

Fig. 6. Effects of the oximine rhenium(I) complexes on melanoma cell viability. A375 melanoma cells were treated for 2 days with 6c, 6a, 7d, and 7b at concentrations of 1 μM and

2 μM, or with ligands 3a–d at the concentration of 2 μM. The percentages of cells with PI-permeable membrane were assessed by FACS analysis in combined populations of adherent

and ﬂoating cells. Selected histograms from a representative experiment are shown. The data are the mean SD of two independent experiments (Pb0.05) except from 2 μM

oximine rhenium(^I) complexes when only one experiment was done.

of treatment with 8 μM 7d and 7b. At the same concentration, 6c and

6a induced apoptotic cell death in the majority of K562 cells. More

than 90% of cells were either in early or late stage of apoptosis after

3 days of treatment with these drugs (Table 4). Compound 6c was the

most efﬁcient in induction of apoptosis. This was clearly visible in the

experiment showing fragmented nuclei which appeared in the

presence of 6c at a concentration as low as 4 μM (Fig. 9B). As

expected, ligands (3a–d) did not induce apoptotic cell death when

used at concentrations of 8 μM and 20 μM. Necrosis was also not

induced in these conditions.

In summary, current studies of anticancer activity of four oximine

rhenium(^I) complexes performed in melanoma and leukemia cell

lines have revealed that in two series of halogenido Re(^I) complexes

(X=Cl, Br), chlorido complexes were more efﬁcient as anticancer

drugs in vitro. Chlorido complexes 6c and 6a possessed signiﬁcant

cytostatic activity against leukemia K562 cells and melanoma A375

cells. This activity was much higher than for bromido compounds 7d

and 7b. Lethal effects of chlorido Re(^I) complexes in melanoma and

leukemia cells were obtained at different concentrations of com-

pounds. Chlorido Re(^I) complexes at the concentration of 1 μM were

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

781

part of the cytotoxic activity of these compounds might be connected

with Bcr–Abl pathway.

Melanoma is the most aggressive type of skin cancer and is highly

resistant to all currently used chemotherapeutics [74]. The alkylating

drug dacarbazine (DTIC), which is approved for treatment of

metastatic melanoma, results in clinical responses of 5–10% of

patients [75]. Human A375 melanoma cell line used in our study, is

considered as having high metastatic potential and is highly resistant

to anticancer drugs. Therefore, the obtained results with oximine

rhenium(^I) complexes, especially those chlorido compounds 6c and

6a, are encouraging and need further investigation. One reason for

higher susceptibility of melanoma cells than leukemia cells to Re(^I)

complexes could be the inﬂuence of the drugs on the adhesive

potential of melanoma cells. In addition to clearly visible apoptosis

induced by oximine rhenium(^I) complexes, they might affect cell

adhesion. By reducing the number of adherent cells in culture they

could inﬂuence the relative number of cells able to proliferate. It

should also be taken into account, that any single-agent chemother-

apy tested in clinical setting for melanoma patients has resulted in

response rates below 20%.[76,77] Therefore, instead of oximine

rhenium(^I) complexes alone, combination with other drugs, including

those highly speciﬁc for deregulated pathways in melanoma cells,

should be considered for future evaluations.

Taken together, our data demonstrated that albeit to different

extent, newly synthesized oximine rhenium(^I) complexes investigat-

ed in this study could induce apoptotic cell death in leukemia and

melanoma cells. To our knowledge, this is the ﬁrst study showing the

anticancer therapeutic potential of rhenium complexes against

melanoma and leukemia cells. Further studies are necessary to

unravel the exact mechanism(s) of the cellular responses evoked by

these compounds as well to verify their effectiveness in in vivo

models.

Fig. 7. Effects of the oximine rhenium(I) complexes on leukemia cell viability. (A)

Aliquots of K562 cell cultures treated with 8 μM oximine rhenium(^I) complexes 6c, 6a,

7d, and 7b were removed daily for a determination of the number of viable and dead

cells by Trypan blue exclusion test. The number of viable cells is expressed as

percentage of total cell number. (B) K562 cells were treated with oximine rhenium(^I)

complexes at different concentrations and the number of viable and dead cells was

assessed on day 3 by Trypan blue exclusion test. The data are the mean SD of two

independent experiments done in triplicates (Pb0.05).

3. Experimental

3.1. General

apparently effective in stimulating cell death only in melanoma cells,

6c was the most efﬁcient in this respect. In leukemic K562 cells, higher

concentrations were necessary to achieve a similar level of inhibition,

but again compound 6c was the most effective. Comparison of

cytotoxic and cytostatic effects revealed that apoptotic cell death,

deﬁned as loss of cell membrane integrity, was the major cause of

reduced proliferation in melanoma and leukemia cells. This was

assessed in A375 melanoma cells as PI- and AO/EB-membrane

permeability and in K562 leukemia cells as Trypan blue- and AO/EB-

membrane permeability, and shown as nuclei fragmentation. Bro-

mido compounds 7d and 7b were much less effective against

melanoma and leukemia cells, however, 7d exerted some effect on

proliferation of adherent melanoma cells. It is also interesting that

among chlorido and bromido complexes, those having ligand with no

substituent in para position (6c and 7d) showed higher cytotoxic

activity than those with either methyl (7b) or chlorine (6a)

substituents. It is of note that ligands themselves (3a–d) did not

stimulate cellular death in melanoma and leukemia cells. The

relationship between chemical structure and the vulnerability of

cancer cells to drug-induced apoptosis needs to be further explored.

K562 cell line derived from a patient in blast crisis is commonly

accepted as a cellular model of advanced phase of chronic myelog-

enous leukemia (CML) [72,73]. It was reported by many groups that

commonly used anticancer drugs do not efﬁciently induce apoptosis

in K562 cells, mainly due to the constitutive Bcr–Abl activity and a

negligible level of p53. Only imatinib, by directly targeting activity of

fusion kinase Bcr–Abl, induces mitochondria-dependent apoptosis in

K562 cell line [73]. Therefore, the results obtained for the newly

synthesized oximine rhenium(^I) complexes suggested that at least a

All experiments and manipulations were performed under dry

argon atmosphere using Schlenk and vacuum-line techniques. Re

(CO)₅X (X=Cl (4), Br (5)) [78] were prepared according to a literature

procedure. The published synthesis of 2-nitroso-N-arylanilines [22]

(3a–d) has to be modiﬁed to achieve good yields. Solvents were

puriﬁed by standard procedures; dichloromethane was distilled from

calcium hydride, n-pentane and n-heptane were distilled from lithium

aluminium hydride and tetrahydrofurane was distilled from sodium.

All solvents were stored under a dry argon atmosphere with 3 Å

molecular sieves (dichloromethane) respectively sodium pieces (n-

pentane, n-heptane, THF). NMR spectra were recorded with a Jeol

Eclipse 270, Jeol Eclipse 400 or Jeol EX 400 spectrometer at ambient

temperature unless stated otherwise. All chemical shifts are given in

ppm relative to TMS. The splitting of proton resonances in the reported

¹H NMR spectra is deﬁned as s = singlet, d = doublet, dd = doublet of

doublets, ddd = doublet of doublets of doublets, dddd = doublet of

doublets of doublets of doublets, m = multiplet and br s = broad

singlet. IR spectra were measured in the range of 4000–400 cm⁻¹

using a PerkinElmer Spectrum One FT-IR spectrometer. The intensity

of reported IR signals is deﬁned as vs = very strong, s = strong, m =

medium and w = weak. UV/visible (UV/Vis) data was recorded with a

Varian Cary 50 UV/Vis spectrophotometer. The emission spectra were

recorded on a FS900 ﬂuorescence spectrometer (Edinburgh Analytical

Instruments). Mass spectra were obtained by a Jeol MStation JMS-700

in direct electron-impact ionization mode (DEI) or positive ion fast

atom bombardment ionization mode (FAB⁺) (3-nitrobenzyl alcohol

(NBA) matrix). Multi-isotope containing fragments refer to the isotope

with the highest abundance. Elemental analyses were performed with

782

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

Fig. 8. The oximine rhenium(I) complexes induce apoptosis but not necrosis in melanoma cells. Double staining of A375 melanoma cells following 2 day exposure to oximine

rhenium(^I) complexes at the concentrations of 1 μM and 2 μM. Cells were stained with nucleic acid selective ﬂuorochromes: membrane-permeable acridine orange and impermeable

ethidium bromide. Representative microscopic ﬁelds are shown. Viable cells had bright green chromatin with organized structure. In early apoptotic cells, the chromatin was

condensed or fragmented but still stained green. In late apoptotic cells, it was condensed or fragmented and stained orange. Necrotic cells had bright orange chromatin with

organized structure. Ten different ﬁelds were randomly selected for counting 300 cells and the percentages of early and late apoptotic cells, and necrotic cells were calculated.

Quantitative data are presented in Table 3.

a Heraeus elementar varioEL by the Micoanalytical Laboratory of the

Department of Chemistry and Biochemistry, LMU.

cooled mixture of conc. AcOH (6 mL) in DMF (6 mL) was added

(change of colour to brown). The solution was allowed to reach room

temperature, poured into water and extracted three times with EtOAc.

The combined organic layers were washed three times with water,

one time with brine, then a solution of NaHCO₃and water again, then

dried with Na₂SO₄. After evaporation the crude product was puriﬁed

by column chromatography on silica gel (dichloromethane–pentane).

3.2. Synthesis of ligands 3a–d

A solution of tBuOK (24 mmol, 2.69 g) in 8 mL DMF was cooled

(acetone/dry ice/−78 °C) till it was nearly freezing. First a cooled

solution of aniline 1a–d (8 mmol) in 4 mL DMF (change of colour to

light yellow or green), then a cooled solution of 1-chloro-4-

nitrobenzene (2) (8 mmol) in 4 mL DMF was added dropwise (change

of colour to purple). After stirring at this temperature for 5–10 min a

3.2.1. 5-Chloro-N-(4-chlorophenyl)-2-nitrosoaniline (3a)

Reagents: 1.02 g (8.00 mmol) 1a, 1.26 g (8.00 mmol) 2. Yield:

1.01 g (3.78 mmol, 47%), brown powder. — ¹H NMR (400 MHz,

3

4

CDCl₃): δ=6.99 (dd, J_H,H=8.7 Hz, J_H,H=1.7 Hz, 1 H, H4), 7.05 (d,

⁴J_H,H=1.9 Hz, 1 H, H6), 7.18–7.23 (m, 2H, H8+H12), 7.39–7.44 (m,

2H, H9+H11), 8.65 (br s, 1H, H3), 11.81 (br s, 1H, NH) ppm. ¹³C NMR

(100 MHz, CDCl₃): δ=114.3 (C6), 119.3 (C4), 126.3 (C8+C12), 130.2

(C9+C11), 132.5 (C10), 135.2 (C7), 141.2 (br, C1+C3), 145.0 (C5),

Table 3

Dual staining with acridine orange and ethidium bromide indicates an induction of

apoptosis but not necrosis after 2 days of treatment of melanoma A375 cells with

indicated concentrations of tested compounds.

155.2 (C2) ppm. IR (KBr, cm⁻¹): ν

̃=2963 (w), 2925 (w), 2854 (w),

[µM]

Early apoptosis

Late apoptosis

Necrosis

1612 (m), 1592 (s), 1560 (s), 1504 ν(N=O) (m), 1489 (m), 1462 (m),

1338 (m), 1262 (s), 1154 (vs), 1106 (vs), 1092 (vs), 1012 (m), 942

(m), 809 (s), 798 (vs), 560 (m). UV/Vis (CH₂Cl₂): λ_max(ε)=253

(13,300), 278 (17,100), 312 (14,900), 461 nm (8200 M⁻¹cm⁻¹). MS

(DEI): m/z (%)=266.2 (13) [M⁺], 249.2 (100) [M⁺–H–O], 235.2 (29)

[M⁺–H–NO], 231.2 (16) [M⁺–Cl], 214.2 (4) [M⁺–Cl–OH], 201.2 (28)

[M⁺–Cl–NO], 166.2 (16) [M⁺–2Cl–NO]. C₁₂H₈Cl₂N₂O (267.11 g mol⁻¹):

calcd. C 53.96, H 3.02, N 10.49; found C 53.88, H 3.13, N 10.38.

Control

6a

–

1

2

1

2

1

2

1

2

1

11

17

29

14

3

26

3

15

2

6

23

73

50

70

10

54

11

43

2

1

9

1

4

1

3

5

6c

7d

7b

3c

3.2.2. 5-Chloro-2-nitroso-N-p-tolylaniline (3b)

Reagents: 857 mg (8.00 mmol) 1b, 1.26 g (8.00 mmol) 2. Yield:

873 mg (3.54 mmol, 44%), dark green powder. Green crystals were

A minimum of 300 cells was counted and all four cellular states were recorded. Then,

the percentages of early or late apoptotic or necrotic cells were calculated. Bold data

points indicate signiﬁcant differences (Pb0.05) from the control data points.

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

783

Fig. 9. The oximine rhenium(I) complexes induce apoptosis but not necrosis in leukemia cells. (A) Double staining of K562 leukemia cells following 3 day exposure to oximine

rhenium(^I) complexes at the indicated concentrations. The microscopic ﬁelds obtained for K562 cells exposed to ligands 3a–c are included. Quantitative data are presented in

Table 4. (B) K562 cells were stained with DAPI for nuclear fragmentation and analyzed by ﬂuorescence microscopy. Arrows indicate some examples of cells with fragmented nuclei.

obtained by slow sublimation of 3b at 55 °C and 1.0×10⁻³mbar. — ¹H

NMR (270 MHz, CDCl₃): δ=2.39 (s, 3H, CH₃), 6.93 (dd,³J_H,H=8.8 Hz,

⁴J_H,H=1.9 Hz, 1H, H4), 7.05 (d, ⁴J_H,H=2.0 Hz, 1H, H6), 7.10–7.16 (m,

2H, H8+H12), 7.21–7.27 (m, 2H, H9+H11), 8.65 (br s, 1H, H3), 12.06

(br s, 1H, NH) ppm. ¹³C NMR (100 MHz, CDCl₃): δ=21.2 (CH₃), 114.6

(C6), 118.7 (C4), 125.1 (C8+C12), 130.6 (C9+C11), 133.7 (C7),

784

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

(267.11 gmol⁻¹): calcd. C 53.96, H 3.02, N 10.49; found C 53.93, H

2.90, N 10.36.

Table 4

Dual staining with acridine orange and ethidium bromide indicates an induction of

apoptosis but not necrosis after 3 days of treatment of K562 cells with indicated

concentrations of tested drugs.

3.2.4. 5-Chloro-2-nitroso-N-phenylaniline (3d)

[µM]

Early apoptosis

Late apoptosis

Necrosis

Reagents: 745 mg (8.00 mmol) 1d, 1.26 g (8.00 mmol) 2. Yield:

775 mg (3.33 mmol, 42%), dark green powder. Green crystals were

obtained by slow sublimation of 3d at 55 °C and 1.0×10⁻³mbar. — ¹H

Control

DMSO

6a

−

–

0.2

0.3

2.4

45.0

0.0

30.1

42.5

0.2

2.4

51.2

0.2

3.0

58.3

0.2

0.6

0.0

0.2

0.1

0.0

0.1

0.8

0.7

1.8

51.6

1.5

12.9

49.5

0.7

2.5

45.1

2.2

0.9

39.1

0.2

0.9

1.1

0.9

0.2

0.8

1.3

0.3

0.9

1.3

1.0

0.5

1.2

1.0

0.7

0.5

0.9

0.7

1.1

0.2

1.7

1.6

1.4

2.1

1.6

1.0

2.0

0.9

1.5

2.3

4

8

2

4

8

4

8

20

8

10

20

4

8

4

8

4

8

20

4

3

4

NMR (270 MHz, CDCl₃): δ=6.96 (dd, J_H,H=8.8 Hz, J_H,H=2.0 Hz,

1H, H4), 7.11 (d, ⁴J_H,H=2.0 Hz, 1H, H6), 7.23–7.28 (m, 2H, H8+H12),

7.28–7.34 (m, 1H, H10), 7.40–7.49 (m, 2H, H9+H11), 8.66 (br s, 1H,

H3), 12.02 (br s, 1H, NH) ppm. ¹³C NMR (100 MHz, CDCl₃): δ=114.5

(C6), 119.0 (C4), 125.1 (C8+C12), 127.0 (C10), 130.0 (C9+C11),

136.5 (C7), 141.4 (br, C1+C3), 144.9 (C5), 155.2 (C2) ppm. IR (KBr,

6c

7d

7b

cm⁻¹): ν

̃=3084 (w), 3021 (w), 2924 (w), 1608 (m), 1587 (s), 1559

(vs), 1498 ν(N=O) (s), 1457 (m), 1354 (m), 1333 (m), 1160 (m),

1153 (m), 1098 (vs), 1081 (m), 1073 (m), 941 (m), 796 (m), 534 (s).

UV/Vis (CH₂Cl₂): λ_max(ε)=250 (12000), 274 (12400), 312 (12400),

464 nm (6800 M⁻¹cm⁻¹). MS (DEI): m/z (%)=231.3 (9) [M⁺–H],

215.3 (100) [M⁺–H–O], 201.3 (25) [M⁺–H–NO], 167.3 (23) [M⁺–H–

NO–Cl]. C₁₂H₉ClN₂O (232.67 gmol⁻¹): calcd. C 61.95, H 3.90, N 12.04;

found C 62.06, H 3.88, N 11.82.

3a

3c

3d

3b

8

20

0.0

1.7

3.3. Synthesis of complexes 6a–d and 7a–d

A minimum of 300 cells was counted and all four cellular states were recorded. Then,

the percentages of early or late apoptotic or necrotic cells were calculated. Bold data

points indicate signiﬁcant differences (Pb0.05) from the control data points.

Re(CO)₅X (X=Cl, Br) (4 and 5) was dissolved in 20 mL dry THF and

reﬂuxed for 20 h. The elimination of two CO ligands leading to the

intermediates 4′ and 5′ could be observed by liquid phase IR

spectroscopy in THF. The resulting pale-yellow mixture was added

to a solution of one equivalent of ligand 3a–d in 10 mL dry THF.

Stirring at room temperature resulted in a change of colour from red

brown to dark violet. Full conversion to the desired complexes 6a–d

and 7a–d was again monitored by liquid phase IR spectroscopy after

stated reaction time. After evaporation of nearly all THF (rest 2–3 mL)

30–40 mL of dry n-heptane was added and subsequently the solvent

was again removed till complexes 6a–d and 7a–d precipitate as dark

green or purple solids. After ﬁltration the solids were washed four

times with 5 mL dry n-heptane and dried in vacuo.

137.1 (C10), 141.7 (br, C1+C3), 144.8 (C5), 155.2 (C2) ppm. IR (KBr,

cm⁻¹): ν

̃=3084 (w), 3034 (w), 2913 (w), 1605 (m), 1554 (s), 1509

ν(N=O) (s), 1488 (s), 1436 (m), 1350 (m), 1333 (s), 1143 (s), 1100

(vs), 1175 (s), 1018 (m), 945 (m), 938 (m), 813 (m), 800 (s), 509 (m).

UV/Vis (CH₂Cl₂): λ_max(ε)=249 (12900), 269 (12100), 312 (12700),

467 nm (6900 M⁻¹cm⁻¹). MS (DEI): m/z (%)=245.3 (6) [M⁺–H],

229.3 (100) [M⁺–H–O], 214.3 (19) [M⁺–H–NO], 180.3 (17) [M⁺–H–

NO–Cl], 166.3 (5) [M⁺–H–NO–Cl–Me]. C₁₃H₁₁ClN₂O (246.69 g mol⁻¹):

calcd. C 63.29, H 4.49, N 11.36; found C 63.39, H 4.27, N 11.32.

3.2.3. 5-Chloro-N-(2-chlorophenyl)-2-nitrosoaniline (3c)

Reagents: 1.02 g (8.00 mmol) 1c, 1.26 g (8.00 mmol) 2. Yield:

812 mg (3.04 mmol, 38%), brown powder. — ¹H NMR (270 MHz,

CDCl₃): δ=7.02 (dd, J_H,H=9.2 Hz, J_H,H=2.0 Hz, 1 H, H4), 7.02

3.3.1. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-4-chlorophenyl)-

oximine-N,N′}rhenium(I) (6a)

3

4

3

(d, J_H,H=1.9 Hz, 1 H, H6), 7.24 (ddd, J_H,H=7.9 Hz, J_H,H=7.4 Hz,

Reagents: 135 mg (0.373 mmol) 4, 100 mg (0.373 mmol) 3a,

reaction time: 20 h. Yield: 196 mg (0.342 mmol, 92%), dark green

powder. Dark red crystals were obtained by slow isothermic diffusion

of n-pentane into a solution of 6a in dichloromethane. — ¹H NMR

(270 MHz, CDCl₃): δ=6.70 (dd, ⁴J_H,H=1.8 Hz, ⁵J_H,H=0.7 Hz, 1H, H6),

6.86 (dd, ³J_H,H=10.0 Hz, ⁴J_H,H=1.8 Hz, 1H, H4), 6.99–7.10 (m, 1H, H8

⁴J_H,H=1.7 Hz, 1H, H10), 7.34 (ddd, J_H,H=7.9 Hz, J_H,H=7.4 Hz,

3

⁴J_H,H=1.6 Hz, 1H, H11), 7.46 (ddd, J_H,H=8.0 Hz, J_H,H=1.7 Hz,

3

4

⁵J_H,H=0.3 Hz, 1H, H12), 7.52 (ddd, J_H,H=7.9 Hz, J_H,H=1.6 Hz,

3

4

⁵J_H,H=0.4 Hz, 1 H, H9), 8.64 (br s, 1H, H3), 11.60 (br s, 1H, NH)

ppm. ¹H NMR (400 MHz, CDCl₃, −60 °C): δ=7.02 (d, J_H,H

=

4

3

4

3

5

1.8 Hz, 1H, H6), 7.06 (dd, J_H,H=8.7 Hz, J_H,H=1.8 Hz, 1H, H4),

or 12), 7.32–7.42 (m, 1H, H8 or 12), 7.52 (dd, J_H,H=9.9 Hz, J_H,H=

7.25 (dd, J_H,H=7.6 Hz,³J_H,H=7.6 Hz, 1H, H10), 7.34 (dd, J_H,H

=

0.7 Hz, 1H, H3), 7.52 (dd, ³J_H,H=7.6 Hz, ⁴J_H,H=1.5 Hz, 2H, H9+H11),

9.43 (br s, 1H, NOH) ppm. ¹³C NMR (100 MHz, CDCl₃): δ=117.4 (C6),

118.2 (C3), 123.6 (C8 or 12), 124.7 (C8 or 12), 130.1 (C9 or 11), 130.4

(C9 or 11), 131.1 (C4), 134.4 (C10), 143.6 (C5), 147.8 (C7), 155.3 (C2),

163.7 (C1), 176.8 (CO), 193.7 (CO), 194.5 (CO) ppm. IR (KBr, cm⁻¹):

3

7.6 Hz, J_H,H=7.6 Hz, 1H, H11), 7.45 (d, J_H,H=7.8 Hz, 1H, H12),

3

7.51 (d, J_H,H=7.9 Hz, 1H, H9), 8.88 (d, J_H,H=8.7 Hz, 1H, H3),

12.03 (br s, 1H, NH) ppm. ¹³C NMR (68 MHz, CDCl₃): δ=114.5

(C6), 119.5 (C4), 126.2 (C12), 127.7 (C10), 127.9 (C11), 129.4

(C8), 130.9 (C9), 134.4 (C7), 138.9 (br, C1+C3), 144.8 (C5), 155.4

(C2) ppm. ¹³C NMR (100 MHz, CD₂Cl₂, −60 °C): δ=113.6 (C6),

118.9 (C4), 125.8 (C12), 127.4 (C10), 127.5 (C11), 128.1 (C8),

130.0 (C9), 130.8 (C1), 133.1 (C7), 142.2 (C3), 144.0 (C5), 154.6

ν̃=3098 (w), 3084 (w), 2033 ν(CO) (vs), 1950 ν(CO) (vs), 1930

ν(CO) (vs), 1604 ν(C=N) (w), 1553 ν(C=N) (w), 1483 (m), 1421

(m), 1398 (w), 1303 (m), 1189 (m), 1161 (w), 1093 (w), 1054 ν(N–

O) (m), 1045 ν(N–O) (m), 1013 (w), 931 (w), 829 (w), 803 (w), 586

(C2) ppm. IR (KBr, cm⁻¹): ν

̃

=3086 (w), 2963 (w), 2923 (w),

(w) 543 (w), 518 (w), 444 (w). IR (THF, cm⁻¹): ν

̃

=2029 ν(CO) (vs),

1605 (s), 1589 (s), 1581 (s), 1565 (vs), 1506 ν(N=O) (m), 1476

(m), 1349 (m), 1339 (m), 1158 (s), 1105 (vs), 1079 (s), 946 (s),

804 (m), 750 (vs), 549 (m). UV/Vis (CH₂Cl₂): λ_max(ε)=253

(12000), 275 (15500), 312 (13400), 457 nm (7200 M⁻¹cm⁻¹).

MS (DEI): m/z (%)=266.1 (6) [M⁺], 249.1 (26) [M⁺–H–O], 235.1

(14) [M⁺–H–NO], 231.2 (100) [M⁺−Cl], 216.2 (10) [M⁺–Cl–O],

201.2 (13) [M⁺–Cl–NO], 166.2 (14) [M⁺–2Cl–NO]. C₁₂H₈Cl₂N₂O

1952 ν(CO) (m), 1918 ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2033 ν(CO)

(vs), 1954 ν(CO) m), 1926 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max(ε)=

321 (4500), 471 (4500), 548 nm (9700 M⁻¹cm⁻¹). MS (FAB⁺): m/z

(%)=572.0 (76) [M⁺], 544.1 (50) [M⁺–CO], 537.1 (100) [M⁺–Cl],

488.1 (78) [M⁺–3CO], 453.1 (78) [M⁺–3CO–Cl]. C₁₅H₈Cl₃N₂O₄Re

(572.80 gmol⁻¹): calcd. C 31.45, H 1.41, N 4.89; found C 31.28, H 1.39,

N 4.72.

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

785

3

3.3.2. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-p-tolyl)-oximine-N,

N′}rhenium(I) (6b)

1H, H8 or 12), 7.34–7.40 (m, 1H, H8 or 12), 7.43 (dddd, J_H,H=

3

4

7.5 Hz, J_H,H=7.5 Hz, J_H,H=1.2 Hz, J_H,H=1.2 Hz, 1H, H10), 7.50

3

5

3

Reagents: 133 mg (0.368 mmol) 4, 91 mg (0.368 mmol) 3b,

reaction time: 22 h. Yield: 170 mg (0.308 mmol, 84%), dark green

powder. Black crystals were obtained by slow isothermic diffusion of

n-pentane into a solution of 6b in chloroform. — ¹H NMR (270 MHz,

(dd, J_H,H=10.0 Hz, J_H,H=0.5 Hz, 1H, H3), 7.56 (ddd, J_H,H=

7.5 Hz,³J_H,H=7.5 Hz, J_H,H=1.3 Hz, 2H, H9+H11), 8.94 (br s, 1H,

NOH) ppm. ¹³C NMR (100 MHz, CD₂Cl₂): δ=117.8 (C3), 117.9 (C6),

122.2 (C8 or 12), 123.1 (C8 or 12), 128.4 (C10), 129.8 (C9), 129.8

(C11), 131.1 (C4), 143.1 (C5), 149.5 (C7), 155.5 (C2), 163.4 (C1), 177.6

4

5

CDCl₃): δ=2.45 (s, 3 H, CH₃), 6.75 (dd, J_H,H=1.8 Hz, J_H,H=0.7 Hz,

3

4

1H, H6), 6.84 (dd, J_H,H=9.9 Hz, J_H,H=1.8 Hz, 1H, H4), 6.93–7.05

(m, 1H, H8 or 12), 7.29–7.35 (m, 3H, H9+H11+H8 or 12), 7.49 (dd,

(CO), 194.6 (CO), 195.6 (CO) ppm. IR (KBr, cm⁻¹): ν

̃=3054 (w), 3032

(w), 2935 (w), 2032 ν(CO) (vs), 1950 ν(CO) (vs), 1938 ν(CO) (vs),

1606 ν(C=N) (m), 1590 (w), 1548 ν(C=N) (w), 1451 (w), 1421 (m),

1398 (w), 1294 (s), 1187 (m), 1056 ν(N–O) (s), 1047 ν(N–O) (s),

940 (w), 856 (w), 802 (m), 703 (m), 626 (w), 587 (w) 544 (w). IR (THF,

³J_H,H=9.9 Hz, J_H,H=0.7 Hz, 1H, H3), 9.09 (br s, 1H, NOH) ppm. ¹³C

5

NMR (100 MHz, CDCl₃): δ=21.4 (CH₃), 117.9 (C3), 118.0 (C6), 122.0

(C8 or 12), 123.2 (C8 or 12), 130.3 (C9 or 11), 130.6 (C9 or 11), 131.3

(C4), 138.8 (C10), 142.7 (C5), 147.1 (C7), 155.3 (C2), 163.2 (C1),

cm⁻¹): ν

̃

=2029 ν(CO) (vs), 1951 ν(CO) (m), 1917 ν(CO) (m). IR

=2032 ν(CO) (vs), 1952 ν(CO) (s), 1925 ν(CO) (s).

177.2 (CO), 193.9 (CO), 195.0 (CO) ppm. IR (KBr, cm⁻¹): ν

̃

=3049

(CH₂Cl₂, cm⁻¹): ν

̃

(w), 3025 (w), 2927 (w), 2032 ν(CO) (vs), 1955 ν(CO) (vs), 1937

ν(CO) (vs), 1605 ν(C=N) (m), 1599 (m), 1548 ν(C=N) (m), 1501

(m), 1421 (m), 1397 (m), 1296 (s), 1189 (m), 1161 (m), 1054 ν(N–O)

(s), 1048 ν(N–O) (s), 1017 (w), 931 (w), 803 (m), 588 (w), 543 (w),

UV/Vis (CH₂Cl₂): λ_max(ε)=321 (4900), 470 (4800), 544 nm

(11200 M⁻¹cm⁻¹). MS (FAB⁺): m/z (%)=538.0 (72) [M⁺], 510.0

(45) [M⁺–CO], 503.0 (100) [M⁺–Cl], 454.0 (72) [M⁺–3CO], 419.0 (68)

[M⁺–Cl–3CO]. C₁₅H₉Cl₂N₂O₄Re (538.36 gmol⁻¹): calcd. C 33.46, H 1.69,

N 5.20; found C 33.69, H 1.76, N 5.02.

456 (w). IR (THF, cm⁻¹): ν

ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2028 ν(CO) (vs), 1950 ν(CO) (m), 1916

=2031 ν(CO) (vs), 1951 ν(CO) (s),

̃

1924 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max(ε)=320 (4500), 474 (5200),

541 nm (10500 M⁻¹cm⁻¹). MS (FAB⁺): m/z (%)=552.1 (71) [M⁺],

524.1 (49) [M⁺–CO], 517.1 (100) [M⁺–Cl], 568.1.0 (84) [M⁺–3CO],

433.1 (78) [M⁺–Cl–3CO]. C₁₆H₁₁Cl₂N₂O₄Re (552.38 gmol⁻¹): calcd. C

34.79, H 2.01, N 5.07; found C 35.05, H 2.12, N 5.15.

3.3.5. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-4-chlorophenyl)-

oximine-N,N′}rhenium(I) (7a)

Reagents: 128 mg (0.315 mmol) 5, 84 mg (0.315 mmol) 3a,

reaction time: 22 h. Yield: 169 mg (0.274 mmol, 87%), dark green

powder. Brown crystals were obtained by slow isothermic diffusion of

n-pentane into a solution of 7a in dichloromethane. — ¹H NMR

(400 MHz, CDCl₃): δ=6.74 (dd, ⁴J_H,H=1.8 Hz, ⁵J_H,H=0.7 Hz, 1H, H6),

3.3.3. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-2-chlorophenyl)-

oximine-N,N′}rhenium(I) (6c)

Reagents: 136 mg (0.376 mmol) 4, 100 mg (0.376 mmol) 3c,

reaction time: 28 h. Yield: 194 mg (0.339 mmol, 90%), dark green

powder. Brown crystals were obtained by slow isothermic diffu-

sion of n-pentane into a solution of 6c in dichloromethane. — ¹H

3

4

3

6.89 (dd, J_H,H=9.9 Hz, J_H,H=1.8 Hz, 1H, H4), 7.04 (br d, J_H,H

=

7.6 Hz, 1H, H8 or 12), 7.42 (br d, ³J_H,H=7.6 Hz, 1H, H8 or 12), 7.52 (dd,

³J_H,H=7.6 Hz, J_H,H=1.3 Hz, 2H, H9+H11), 7.55 (dd, J_H,H=9.9 Hz,

⁵J_H,H=0.7 Hz, 1H, H3), 8.94 (br s, 1H, NOH) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ=117.3 (C6), 117.7 (C3), 123.5 (C8 or 12), 125.0 (C8 or 12),

130.1 (C9 or 11), 130.4 (C9 or 11), 130.9 (C4), 134.4 (C10), 143.1 (C5),

147.9 (C7), 154.8 (C2), 163.0 (C1), 176.3 (CO), 192.9 (CO), 194.2 (CO)

4

3

4

5

NMR (400 MHz, CD₂Cl₂): δ=6.57 (dd, J_H,H=1.8 Hz, J_H,H=0.7 Hz,

3

4

1H, H6), 6.91 (dd, J_H,H=10.0 Hz, J_H,H=1.9 Hz, 1H, H4), 7.39

(ddd, J_H,H=8.0 Hz, J_H,H=7.3 Hz,⁴J_H,H=1.9 Hz, 1H, H10), 7.48

ppm. IR (KBr, cm⁻¹): ν

̃=3101 (w), 3084 (w), 2037 ν(CO) (vs), 1953

3

(ddd, J_H,H=7.9 Hz, J_H,H=7.2 Hz,⁴J_H,H=1.4 Hz, 1H, H11), 7.53

ν(CO) (vs), 1940 ν(CO) (vs), 1930 ν(CO) (vs), 1604 ν(C=N) (m),

1544 ν(C=N) (w), 1483 (m), 1423 (m), 1264 (s), 1193 (m), 1153 (w),

1056 ν(N―O) (s), 1047 ν(N―O) (m), 1014 (m), 932 (m), 824 (w),

3

(ddd, J_H,H=7.9 Hz, J_H,H=1.9 Hz,⁵J_H,H=0.4 Hz, 1H, H12), 7.54

3

4

3

5

3

(dd, J_H,H=9.9 Hz, J_H,H=0.7 Hz, 1 H, H3), 7.60 (ddd, J_H,H

=

8.1 Hz, J_H,H=1.4 Hz,⁵J_H,H=0.4 Hz, 1H, H9), 9.05 (br s, 1H, NOH)

ppm. ¹³C NMR (100 MHz, CD₂Cl₂): δ=117.6 (C6), 117.7 (C3),

125.2 (C12), 126.1 (C8), 128.3 (C11), 129.4 (C10), 130.5 (C9),

131.1 (C4), 143.9 (C5), 146.0 (C7), 154.7 (C2), 164.5 (C1), 177.2

798 (w), 585 (w) 516 (m), 443 (w). IR (THF, cm⁻¹): ν

̃

=2030 ν(CO)

4

(vs), 1954 ν(CO) (m), 1921 ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2034

ν(CO) (vs), 1955 ν(CO) (m), 1928 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max

(ε)=358 (6200), 475 (5400), 549 nm (11000 M⁻¹cm⁻¹). MS

(FAB⁺): m/z (%)=617.8 (86) [M⁺], 589.8 (47) [M⁺–CO], 537.0

(100) [M⁺–Br], 533.9 (81) [M⁺–3CO], 453.0 (73) [M⁺–3CO–Br].

C₁₅H₈BrCl₂N₂O₄Re (617.25 gmol⁻¹): calcd. C 29.19, H 1.31, N 4.54;

found C 29.03, H 1.29, N 4.39.

(CO), 194.2 (CO), 195.1 (CO) ppm. IR (KBr, cm⁻¹): ν

̃=3110 (w),

3070 (w), 2031 ν(CO) (vs), 1960 ν(CO) (vs), 1936 ν(CO) (vs),

1605 ν(C=N) (m), 1548 ν(C=N) (w), 1469 (m), 1421 (m), 1401

(w), 1297 (m), 1191 (w), 1161 (w), 1055 ν(N―O) (m), 1046

ν(N―O) (m), 935 (w), 800 (w), 763 (w), 740 (w) 623 (w), 588

(w), 547 (w), 456 (w). IR (THF, cm^{− 1}): ν

̃

=2029 ν(CO) (vs), 1953

3.3.6. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-p-tolyl)-oximine-N,

N′}rhenium(I) (7b)

ν(CO) (m), 1924 ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2033 ν(CO)

(vs), 1953 ν(CO) (m), 1932 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max(ε)=

322 (6400), 438 (4500), 470 (4800), 555 nm (12600 M^{− 1}cm⁻¹).

MS (FAB⁺): m/z (%)=571.9 (75) [M⁺], 544.0 (32) [M⁺–CO], 537.1

(100) [M⁺–Cl], 509.1 (13) [M⁺–Cl–CO], 488.0 (26) [M⁺–3CO],

Reagents: 129 mg (0.318 mmol) 5, 78 mg (0.318 mmol) 3b, reaction

time: 23 h. Yield: 167 mg (0.280 mmol, 88%), dark green powder. Dark

red crystals were obtained by slow isothermic diffusion of n-pentane

into a solution of 7b in dichloromethane. — ¹H NMR (270 MHz, CDCl₃):

4

5

481.1 (10) [M⁺–Cl–2CO], 453.1 (54) [M⁺–Cl–3CO], 452.1 (54) [M⁺

–

δ=2.45 (s, 3H, CH₃), 6.79 (dd, J_H,H=1.8 Hz, J_H,H=0.7 Hz, 1H, H6),

Cl–3CO–H], 416.1 (18) [M⁺–2Cl–3CO–H]. C₁₅H₈Cl₃N₂O₄Re

(572.80 g mol⁻¹): calcd. C 31.45, H 1.41, N 4.89; found C 31.52, H

1.51, N 4.81.

6.87 (dd, J_H,H=9.9 Hz, J_H,H=1.8 Hz, 1H, H4), 6.93–7.03 (m, 1H, H8

or 12), 7.29–7.35 (m, 3H, H9+H11+H8 or 12), 7.51 (dd, ³J_H,H=9.9 Hz,

⁵J_H,H=0.7 Hz, 1H, H3), 8.31 (br s, 1H, NOH) ppm. ¹³C NMR (100 MHz,

CDCl₃): δ=21.3 (CH₃), 117.5 (C3), 117.7 (C6), 121.9 (C8 or 12), 123.4

(C8 or 12), 130.3 (C9 or 11), 130.6 (C9 or 11), 131.1 (C4), 138.8 (C10),

142.2 (C5), 147.2 (C7), 154.8 (C2), 162.5 (C1), 176.6 (CO), 193.1 (CO),

3

4

3.3.4. Tricarbonyl-chlorido-{4-chloro-o-quinone-(N-phenyl)-oximine-N,

N′}rhenium(I) (6d)

Reagents Reagents: 147 mg (0.406 mmol) 4, 94 mg (0.406 mmol)

3d, reaction time: 22 h. Yield: 192 mg (0.357 mmol, 88%), dark green

powder. Red crystals were obtained by slow isothermic diffusion of n-

pentane into a solution of 6d in dichloromethane. — ¹H NMR

194.5 (CO) ppm. IR (KBr, cm⁻¹): ν

̃=3097 (w), 3089 (w), 3024 (w),

2919 (w), 2034 ν(CO) (vs), 1957 ν(CO) (vs), 1951 ν(CO) (vs), 1939

ν(CO) (vs), 1925 ν(CO) (vs), 1604 ν(C=N) (m), 1544 ν(C=N) (w),

1502 (m), 1454 (w), 1423 (m), 1267 (s), 1240 (m), 1193 (m), 1153 (m),

1055 ν(N–O) (s), 932 (w), 795 (m), 623 (w) 586 (w), 514 (w). IR (THF,

4

5

(400 MHz, CD₂Cl₂): δ=6.75 (dd, J_H,H=1.7 Hz, J_H,H=0.5 Hz, 1H,

3

4

H6), 6.89 (dd, J_H,H=10.0 Hz, J_H,H=1.8 Hz, 1H, H4), 7.10–7.16 (m,

cm⁻¹): ν

̃=2030 ν(CO) (vs), 1952 ν(CO) (m), 1919 ν(CO) (m). IR

786

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

(CH₂Cl₂, cm⁻¹): ν

̃

=2032 ν(CO) (vs), 1953 ν(CO) (s), 1926 ν(CO) (s).

3.4. Cell culture conditions and drug treatment

UV/Vis (CH₂Cl₂): λ_max(ε) =357 (5400), 481 (5500), 544 nm

(9900 M⁻¹cm^{− 1}). MS (FAB⁺): m/z (%)=596.0 (83) [M⁺], 568.0

(44) [M⁺–CO], 517.2 (100) [M⁺–Br], 512.1.0 (84) [M⁺–3CO], 433.1

(75) [M⁺–Br–3CO]. C₁₆H₁₁BrClN₂O₄Re (596.83 gmol^{− 1}): calcd. C

32.20, H 1.86, N 4.69; found C 32.53, H 1.89, N 4.57.

Tested oximine rhenium(^I) complexes and ligands were diluted

in DMSO and stored at −20 ºC. Working solutions were prepared in

RPMI 1640 medium immediately before use. The human K562 cell

line was a gift of Prof. Jean Claude D'Halluin (INSERM 125, Lille,

France). This Bcr–Abl-positive cell line was maintained as described

previously [79]. For experiments, K562 cells were seeded at the

density 3.5×10⁴mL^{− 1}, 22 h later the tested rhenium(I) complexes

or ligands at indicated concentrations were added to cells cultured

in suspension. A375, a human melanoma adherent cell line with

high metastatic potential, derived from a 54 year old female with

malignant melanoma (a gift of Prof. Piotr Laidler, Jagiellonian

University, Poland) was maintained in RPMI 1640 medium supple-

mented with 10% FBS and antibiotics. For drug exposure experi-

ments, culture medium was substituted with fresh medium

containing 0.5% FBS and tested compounds at indicated concentra-

tions. Equivalent ﬁnal concentration of DMSO was used in the

control cultures.

3.3.7. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-2-chlorophenyl)-

oximine-N,N′}rhenium(I) (7c)

Reagents: 118 mg (0.291 mmol) 5, 78 mg (0.291 mmol) 3c,

reaction time: 22 h. Yield: 154 mg (0.250 mmol, 86%), dark pur-

ple powder. Black crystals were obtained by slow isothermic diffusion

of n-pentane into a solution of 7c in dichloromethane. — ¹H NMR

4

5

(270 MHz, CDCl₃): δ=6.54 (dd, J_H,H=1.8 Hz, J_H,H=0.7 Hz, 1H, H6),

6.89 (dd, ³J_H,H=9.9 Hz, ⁴J_H,H=1.8 Hz, 1H, H4), 7.37 (ddd, ³J_H,H=7.9 Hz,

³J_H,H=7.5 Hz,⁴J_H,H=1.8 Hz, 1H, H10), 7.46 (ddd, ³J_H,H=7.8 Hz, ³J_H,H

=

7.5 Hz,⁴J_H,H=1.5 Hz, 1H, H11), 7.58 (dd, J_H,H=9.9 Hz, J_H,H=0.7 Hz,

3

5

1H, H3), 7.58 (ddd, ³J_H,H=7.9 Hz, ⁴J_H,H=1.5 Hz,⁵J_H,H=0.4 Hz, 1H, H9),

7.64 (ddd, J_H,H=7.8 Hz, J_H,H=1.8 Hz,⁵J_H,H=0.4 Hz, 1H, H12), 9.50

(br s, 1H, NOH) ppm. ¹³C NMR (100 MHz, CDCl₃): δ=117.3 (C6), 117.6

(C3), 125.7 (C12), 126.1 (C8), 128.4 (C11), 129.5 (C10), 130.6 (C4),

130.6 (C9), 143.2 (C5), 146.2 (C7), 154.3 (C2), 163.8 (C1), 176.3 (CO),

3

4

3.5. Proliferation and viability of cancer cells

3.5.1. Leukemia cell viability and proliferation assay

193.0 (CO), 194.2 (CO) ppm. IR (KBr, cm⁻¹): ν

̃=3097 (w), 3086 (w),

Cell proliferation and viability were determined by using Trypan

blue dye exclusion assay (Sigma-Aldrich, St. Louis, MO, USA). K562

cells were seeded at the density 4×10⁴mL⁻¹, 22 h later the oximine

rhenium(^I) complexes or ligands were added at indicated concentra-

tions. Non-treated cells were cultivated as control. Proliferation rate

or cell viability was evaluated as described previously [79]. Brieﬂy,

treatment with drugs was carried out up to 3 days and aliquots were

removed daily for determination of dead and viable cell number using

Trypan blue dye exclusion test. In proliferation assay, only viable cells

were counted that did not take up Trypan blue. Comparison was

made relative to values obtained for the untreated control and

expressed as percentage of the control. In viability assay, the number

of viable cells not taking up Trypan blue is expressed as percentage

of the total cell number (viable and dead) in each experimental

condition. The medium was not changed during the induction period.

Each experiment was conducted in triplicate and repeated three

times.

2025 ν(CO) (vs), 1956 ν(CO) (vs), 1947 ν(CO) (vs), 1934 ν(CO) (vs),

1926 ν(CO) (vs), 1602 ν(C=N) (m), 1544 ν(C=N) (m), 1468 (m), 1453

(w), 1425 (m), 1391 (w), 1278 (s), 1198 (w), 1149 (w), 1060 ν(N–O)

(s), 1047 ν(N–O) (m), 948 (w), 935 (w), 806 (m), 625 (w), 586 (w), 518

(w), 452 (w). IR (THF, cm⁻¹): ν

1926 ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2031 ν(CO) (vs), 1954 ν(CO) (m),

=2034 ν(CO) (vs), 1955 ν(CO)

̃

(m), 1935 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max(ε)=362 (6400), 441

(3800), 474 (4400), 557 nm (11200 M⁻¹cm⁻¹). MS (FAB⁺): m/z (%)=

617.9 (92) [M⁺], 589.8 (33) [M⁺–CO], 537.0 (100) [M⁺–Br], 534.0

(21) [M⁺–3CO], 509.0 (13) [M⁺–Br–CO], 496.0 (37) [M⁺–3CO–H–Cl],

481.0 (12) [M⁺–Br–2CO], 453.0 (58) [M⁺–Br–3CO], 452.0 (54) [M⁺–Br–

3CO–H], 417.1 (16) [M⁺–Br–3CO–H–Cl]. C₁₅H₈BrCl₂N₂O₄Re

(617.25 gmol⁻¹): calcd. C 29.19, H 1.31, N 4.54; found C 29.26, H 1.45,

N 4.55.

3.3.8. Bromido-tricarbonyl-{4-chloro-o-quinone-(N-phenyl)-oximine-N,

N′}rhenium(I) (7d)

Reagents: 141 mg (0.347 mmol) 5, 81 mg (0.347 mmol) 3d,

reaction time: 20 h. Yield: 178 mg (0.305 mmol, 88%), dark purple

powder. Black crystals were obtained by slow isothermic diffusion of

n-pentane into a solution of 7d in dichloromethane. — ¹H NMR

(270 MHz, CDCl₃): δ=6.75 (dd, ⁴J_H,H=1.8 Hz, ⁵J_H,H=0.7 Hz, 1H, H6),

3.5.2. Melanoma cell proliferation assay

Cell proliferation was measured by a colorimetric MTT (3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.

Brieﬂy, cells were seeded in 24-well plates and allowed to adhere

for 6 h in medium containing 10% fetal bovine serum (FBS). Next, they

were treated for 2 days with tested compounds at indicated

concentrations in the culture medium containing 0.5% FBS. MTT

reagent (thiazolyl blue tetrazolium bromide; Sigma-Aldrich;

0.84 mg mL⁻¹in PBS) was added and left in the cultures for 3 h at

37 ºC prior to addition of 800 μL solubilization reagent (DMSO in

optical grade). The reduction of a tetrazolium component into DMSO-

soluble formazan product was monitored at a wavelength of 540 nm

using a spectrophotometer. The mean of the absolute absorbance

values given by drug-treated cells was divided by the mean of the

absolute absorbance of DMSO-treated control sample and expressed

as relative number of viable adherent cells. Data show the mean of at

least three independent experiments SD. IC₅₀was calculated. For

time course, the number of viable, adherent melanoma cells was

estimated each day by MTT assay after incubation with tested

compounds at the concentration of 1.4 µM.

3

4

3

6.88 (dd, J_H,H=9.9 Hz, J_H,H=1.8 Hz, 1H, H4), 7.08 (br d, J_H,

3

_H=7.2 Hz, 1H, H8 or 12), 7.42 (dddd, J_H,H=7.3 Hz, J_H,H=7.3 Hz,

⁴J_H,H=1.4 Hz, J_H,H=1.4 Hz, 1H, H10), 7.43–7.48 (m, 1H, H8 or 12),

4

3

5

7.50–7.58 (m, 2H, H9+H11), 7.53 (dd, J_H,H=9.9 Hz, J_H,H=0.7 Hz,

1H, H3), 8.53 (br s, 1H, NOH) ppm. ¹³C NMR (100 MHz, CDCl₃):

δ=117.6 (C3), 117.6 (C6), 122.0 (C8 or 12), 123.4 (C8 or 12), 128.6

(C10), 129.8 (C9 or 11), 130.1 (C9 or 11), 131.0 (C4), 142.5 (C5), 149.6

(C7), 154.8 (C2), 162.7 (C1), 176.4 (CO), 193.0 (CO), 194.4 (CO) ppm.

IR (KBr, cm⁻¹): ν

̃=3106 (w), 3083 (w), 2038 ν(CO) (vs), 1959 ν(CO)

(vs), 1925 ν(CO) (vs), 1908 ν(CO) (vs), 1597 ν(C=N) (m), 1545

ν(C=N) (m), 1451 (m), 1418 (w), 1392 (m), 1285 (s), 1184 (w), 1151

(w), 1051 ν(N–O) (s), 1042 ν(N–O) (m), 938 (w), 803 (m), 763 (w),

703 (m), 623 (w), 579 (w) 540 (w). IR (THF, cm⁻¹): ν

̃

=2030 ν(CO)

(vs), 1953 ν(CO) (m), 1920 ν(CO) (m). IR (CH₂Cl₂, cm⁻¹): ν

̃

=2033

ν(CO) (vs), 1954 ν(CO) (m), 1927 ν(CO) (m). UV/Vis (CH₂Cl₂): λ_max

(ε)=358 (5600), 473 (4700), 546 nm (9800 M⁻¹cm⁻¹). MS (FAB⁺):

m/z (%)=582.0 (87) [M⁺], 554.0 (46) [M⁺–CO], 503.1 (100) [M⁺

–

3.5.3. Flow cytometric analysis of melanoma cell viability

Br], 498.1 (74) [M⁺–3CO], 419.2 (67) [M⁺–Br–3CO]. C₁₅H₉BrClN₂O_4-

Re (582.81 gmol⁻¹): calcd. C 30.91, H 1.56, N 4.81; found C 31.24, H

1.64, N 4.74.

The cytotoxicity of tested compounds on cultured melanoma cells

was detected by propidium iodide (PI; Sigma) staining and FACS

analysis. Cells were treated with tested drugs at indicated

Table 5

Crystal data and details of structural reﬁnement on 3b, 3d and 6a–7d.

3b

3d

6a

6b

6c

6d

7a

7b

7c

7d

Formula

C₁₃H₁₁ClN₂O

246.692

200(2)

0.71073

Monoclinic

P21/c

9.957(2)

15.364(3)

8.390(2)

90

112.38(3)

90

1186.9(5)

4

1.3806

0.305

512

C₁₂H₉ClN₂O

232.665

200(2)

0.71073

Monoclinic

P21/c

10.599(2)

6.658(2)

15.413(3)

90

99.59(3)

90

1072.4(4)

4

1.4411

0.333

480

C₁₅H₈Cl₃N₂O₄Re

572.800

200(2)

0.71073

Triclinic

P-1

8.862(2)

10.524(2)

11.074(2)

71.76(3)

66.85(3)

80.71(3)

901.0(4)

2

C₁₆H₁₁Cl₂N₂O₄Re

552.382

200(2)

0.71073

Triclinic

P-1

8.754(2)

10.732(2)

11.525(2)

116.22(3)

101.44(3)

101.02(3)

902.9(5)

2

C₁₆H₁₀Cl₅N₂O₄Re

657.732

200(2)

0.71073

Triclinic

P-1

9.273(2)

10.129(2)

11.110(2)

86.19(3)

85.36(3)

79.82(3)

1022.2(4)

2

C₁₅H₉Cl₂N₂O₄Re

538.355

200(2)

0.71073

Triclinic

P-1

9.027(2)

9.796(2)

10.499(2)

109.38(3)

101.12(3)

99.30(3)

833.5(4)

2

C₁₅H₈BrCl₂N₂O₄Re C₂₁H₂₃BrClN₂O₄Re C₁₆H₁₀BrCl₄N₂O₄Re C₁₆H₁₁BrCl₃N₂O₄Re

FW [g mol⁻¹

]

617.251

200(2)

668.982

200(2)

702.183

200(2)

667.739

200(2)

Temperature [K]

Wavelength [Å]

Crystal system

Space group

a [Å]

b [Å]

c [Å]

α (°)

β (°)

0.71073

Triclinic

P-1

0.71073

Triclinic

P-1

0.71073

Triclinic

P-1

0.71073

Monoclinic

C2/c

6.769(2)

11.821(2)

12.065(2)

93.42(3)

105.14(3)

102.05(3)

904.7(3)

2

6.777(2)

11.949(2)

15.573(3)

69.53(3)

88.26(3)

77.92(3)

1153.9(5)

2

9.344(2)

10.198(2)

11.318(2)

86.08(3)

85.79(3)

80.49(3)

1059.1(4)

2

25.708(5)

10.095(2)

17.501(3)

90

102.45(3)

90

γ (°)

V [Å³]

4435.1(15)

8

Z

ρ_calc.[g cm⁻³

]

2.1114

7.210

540

2.0318

7.048

524

2.1370

6.623

624

2.1451

7.631

508

2.2659

1.9254

2.2019

2.0001

µ [mm⁻¹

F(000)

]

9.242

7.141

8.152

7.665

576

644

660

2512

Crystal size [mm]

θ range [°]

Index ranges

0.16×0.13×0.02

3.45–25.37

−11≤h ≤ 11

−18≤k≤17

−10≤l≤10

7202

0.33×0.18×0.16

3.90–26.34

−13≤h ≤ 10

−8≤k≤6

−19≤l≤19

4750

0.30×0.17×0.12

3.88–26.35

−11≤h≤11

−13≤k≤13

−13≤l≤13

10164

0.11×0.10×0.09

3.28–27.52

−11≤h≤11

−13≤k≤13

−14≤l≤14

19819

0.23×0.22×0.14

3.17–27.56

−12≤h≤12

−13≤k≤12

−14≤l≤14

22333

0.17×0.08×0.02

3.70–25.50

−10≤h≤10

−10≤k≤11

−12≤l≤12

5763

0.21×0.12×0.10

3.20–26.41

−8≤h≤8

−14≤k≤14

−15≤l≤15

17978

0.19×0.16×0.13

3.73–26.35

−8≤h≤8

−14≤k≤14

−19≤l≤19

9553

0.24×0.18×0.15

3.70–26.33

−10≤h≤11

−12≤k≤12

−13≤l≤14

10588

0.16×0.08×0.03

3.16–27.00

−32≤h≤32

−12≤k≤12

−21≤l≤22

40519

Reﬂns collected

Independent reﬂns

R_int

2151

0.0420

2166

0.0284

3644

0.0304

4122

0.0389

4681

0.0610

3068

0.0587

3700

0.0328

4655

0.0282

4281

0.0249

4832

0.0474

Completeness to

[%]

θ

99.0

99.3

99.4

99.2

99.0

99.5

98.9

99.2

99.6

Reﬁnement

method

Full-matrix

least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²least-squares on F²

Data/restraints/

parameters

Goodness-of-ﬁt

on F²

2151/0/159

1.039

2166/0/149

0.876

3644/0/227

0.964

4122/0/228

1.036

4681/2⁎/239

3068/0/219

0.909

3700/0/227

1.153

4655/0/226

1.023

4281/2⁎/239

4832/10⁎/264

1.046

1.056

1.060

Final R indices

[IN2σ(I)]

R indices

R₁=0.0374

wR₂=0.0872

R₁=0.0580

wR₂=0.0979

R₁=0.0355

wR₂=0.0766

R₁=0.0631

wR₂=0.0813

R₁=0.0207

wR₂=0.0389

R₁=0.0270

wR₂=0.0395

R₁=0.0216

wR₂=0.0447

R₁=0.0257

wR₂=0.0458

R₁=0.0399

wR₂=0.1021

R₁=0.0471

wR₂=0.1065

R₁=0.0442

wR₂=0.0818

R₁=0.0707

wR₂=0.0876

R₁=0.0213

wR₂=0.0478

R₁=0.0236

wR₂=0.0487

R₁=0.0239

wR₂=0.0535

R₁=0.0331

wR₂=0.0567

R₁=0.0359

wR₂=0.1001

R₁=0.0459

wR₂=0.1033

R₁=0.0371

wR₂=0.0906

R₁=0.0505

wR₂=0.0993

(all data)

Largest diff. peak/ 0.208 and −0.218 0.176 and −0.205 1.291 and −0.710 0.805 and −0.897 2.051 and −2.413 2.348 and −1.723 0.586 and −0.851 1.328 and −0.740 2.548 and −1.803 1.672 and −1.518

hole [e Å⁻³

CCDC number

]

730545

730546

730547

730548

730549

730550

730551

730552

730553

730554

*Restraints only used for reﬁnement of solvent molecules.

788

S. Wirth et al. / Journal of Inorganic Biochemistry 104 (2010) 774–789

concentrations for 2 days. After treatment, both attached (harvested

by trypsinization) and ﬂoating cells were collected, centrifuged at

400×g for 5 min and stained with PI (8 μg mL⁻¹) for 10 min at room

temperature in the dark. PI-positive cells were identiﬁed on a

FACSCalibur (Becton Dickinson). 10,000 events were analyzed for

each sample and results were processed by using CellQuest software

(Becton Dickinson).

contain the supplementary crystallographic data for this paper. These

data can be obtained free of charge from The Cambridge Crystallo-

graphic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.

4. Conclusions

The present paper has reported the synthesis of the ﬁrst o-quinoid

oximine complexes. Two series of halogenido Re(^I) compounds

(X=Cl, Br) have been synthesized and subjected to full spectroscopic

characterisation to ensure a systematic approach to this new

conﬁguration. Two ligands known from literature and two new

ligands have been employed for this study. Structures from all novel

complexes (6a–7d) and from two of the ligands (3b and 3d) have

been conﬁrmed by single crystal X-ray crystallography, so there is no

doubt about the reported oximine character and the proton shift.

Biological studies have revealed that the newly synthesized oximine

rhenium(^I) complexes could induce apoptotic cell death in leukemia

and melanoma cells, thus reducing proliferation of drug-treated

cancer cells. Chlorido complexes (6a and 6c) were more efﬁcient than

bromido compounds (7d and 7b) in stimulating apoptosis. None of

the tested ligands (3a–d) showed any signiﬁcant anticancer activity.

As systematic investigations in this ligand system just started, work

on a larger variety of functional groups and transition metals is in

progress at the moment. First experiments with different metal

centers indicate that the proton shift cannot be anticipated in general.

3.6. Fluorescence microscopy

3.6.1. Acridine orange/ethidium bromide staining

Cell death was studied morphologically using ﬂuorescent dyes:

acridine orange (AO) and ethidium bromide (EB). Brieﬂy, the

melanoma cells were cultured for 2 days and leukemia cells for

3 days with or without tested agents at indicated concentrations. Cells

(1×10⁵) were collected by centrifugation and resuspended in 20 μL of

staining solution mixture of 100 µg mL⁻¹of EB and 100 µg mL⁻¹of

AO (1:1) (Sigma Chemical Co.). Then, they were examined by

ultraviolet ﬂuorescence microscopy (Olympus BX 41). In each

experiment, more than 300 cells were analyzed and then percentages

of early/late apoptotic or necrotic cells were calculated. The cells with

bright green chromatin with organized structure were counted as

viable. The cells with green, condensed and fragmented chromatin

were counted as early apoptotic cells. In late apoptotic cells,

chromatin was condensed or fragmented and stained orange. Necrotic

cells had bright orange chromatin with organized structure.

Acknowledgments

3.6.2. DAPI staining

Apoptosis was also evaluated by DAPI (4′,6′-diamidino-2-pheny-

lindole) staining. K562 cells were cultured for 3 days with or without

tested compounds at indicated concentrations. 5×10⁵cells were

collected by centrifugation, washed with PBS and ﬁxed with ice-cold

70% ethanol overnight at −20 °C. After washings in PBS, cells were

incubated for 10 min with 20 μM DAPI (0.5 μg mL⁻¹, Molecular

Probes, Eugene, Oregon, USA) at room temperature in the darkness.

Finally, the cells were examined by ultraviolet ﬂuorescence micros-

copy (Olympus BX 41). Apoptotic cells were identiﬁed qualitatively as

cells with condensed chromatin and fragmented nuclei.

Financial support by the Center for Integrated Protein Science

Munich (CIPS, LMU Excellent) is gratefully acknowledged. We are

grateful to Dr. Marta Stasiak for her help in FACS and Mrs. Grazyna Kus

for technical work. This research was supported by Grant 503-1099-2

from the Medical University of Lodz.

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Data represent the mean SD from at least three separate

experiments. The signiﬁcance of an apparent difference in mean

values for any tested parameter was validated by a Student's paired t

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3.8. X-ray structure determinations

Single crystal X-ray diffraction data were collected on a Nonius

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Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N'-chelate ligands to rhenium(i) complexes and the anticancer activity of newly synthesized oximine rhenium(i) complexes against human melanoma and leukemia cells in vitro

DOI: 10.1016/j.jinorgbio.2010.03.014

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