Ru(η6-arene) complexes of combretastatin-analogous oxazoles with enhanced anti-tumoral impact

Article abstract of DOI:10.1016/j.ejmech.2010.07.061

Oxazole-bridged combretastatin A-4 analogues bind to tubulin and exert anti-vascular and anti-angiogenic effects. When linked to Ru(η⁶- arene) complex fragments, conjugates with additional cytotoxic activity result which can ruthenate bionucleophiles such as DNA and proteins. For instance, the Ru(II)(p-cymene)(isonicotinate)Cl₂ complex 6a of the known 4-(3,4,5-trimethoxyphenyl)-5-(3-hydroxy-4-methoxyphenyl)-oxazole 4a was far more active than the latter against cells of the p53-competent wild-type form of HCT-116 colon carcinoma at low 0.01 μM concentrations. A fast reaction of 6a with nucleophilic N-acetyl-l-cysteine was observed in NMR studies. The Ru(arene) complexes 6a-c were also more efficacious against combretastatin-refractory p53(+) cells of human HT-29 colon carcinoma when compared to their parent 4-(3,4-dimethoxy-5-methoxy/halo-phenyl)-5-(3-hydroxy-4-methoxyphenyl)-oxazoles 4a-c. These cells are rich in ABC-transporters which are responsible for their multi-drug resistance and for which conjugates 6 are less good substrates than the phenols 4. Unlike 4a, its complex 6a also diminished the motility of human 518A2 melanoma cells in a wound-healing assay which is indicative of anti-metastatic activity in solid tumors. Overall, the Ru(arene) complex conjugates 6 broaden the anti-tumoral spectrum of the combretastatin A-4 analogues 4 considerably.

Full text of DOI:10.1016/j.ejmech.2010.07.061

European Journal of Medicinal Chemistry 45 (2010) 4890e4896
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Ru(h
6-arene) complexes of combretastatin-analogous oxazoles with enhanced
anti-tumoral impact
,
Bernhard Biersack a, Katharina Effenberger a, Sebastian Knauer a, Matthias Ocker b, Rainer Schobert a
*
a Organic Chemistry Laboratory, University of Bayreuth, 95440 Bayreuth, Germany
b Institute for Surgical Research, Philipps-University of Marburg, Baldingerstrasse, 35043 Marburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxazole-bridged combretastatin A-4 analogues bind to tubulin and exert anti-vascular and anti-angio-
genic effects. When linked to Ru(
6-arene) complex fragments, conjugates with additional cytotoxic
activity result which can ruthenate bionucleophiles such as DNA and proteins. For instance, the Ru(II)
(p-cymene)(isonicotinate)Cl2 complex 6a of the known 4-(3,4,5-trimethoxyphenyl)-5-(3-hydroxy-
4-methoxyphenyl)-oxazole 4a was far more active than the latter against cells of the p53-competent
Received 30 March 2010
Received in revised form
28 July 2010
Accepted 28 July 2010
Available online 6 August 2010
h
wild-type form of HCT-116 colon carcinoma at low 0.01
mM concentrations. A fast reaction of 6a with
nucleophilic N-acetyl- -cysteine was observed in NMR studies. The Ru(arene) complexes 6aec were also
L
Keywords:
more efficacious against combretastatin-refractory p53(þ) cells of human HT-29 colon carcinoma when
compared to their parent 4-(3,4-dimethoxy-5-methoxy/halo-phenyl)-5-(3-hydroxy-4-methoxyphenyl)-
oxazoles 4aec. These cells are rich in ABC-transporters which are responsible for their multi-drug
resistance and for which conjugates 6 are less good substrates than the phenols 4. Unlike 4a, its complex
6a also diminished the motility of human 518A2 melanoma cells in a wound-healing assay which is
indicative of anti-metastatic activity in solid tumors. Overall, the Ru(arene) complex conjugates 6
broaden the anti-tumoral spectrum of the combretastatin A-4 analogues 4 considerably.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Ruthenium complexes
Combretastatin
Anticancer drugs
Multi-drug resistance
Oxazoles
1. Introduction
and hence to tumor relapses [5]. Another strategy is the adjuvant
administration of stable Ru(arene) complexes which were
Tubulin polymerisation inhibitors such as combretastatin A-4
phosphate (CA-4-P) or colchicine-derived ZD 6126 have been
attracting attention since the discovery of their vascular disrupting
properties [1]. CA-4-P is currently on phase II and III trials against
platinum-resistant ovarian cancer and anaplastic thyroid carci-
noma [2]. Its pronounced tumor vascular damaging activity orig-
frequently found to have anti-tumor and anti-metastatic activity
based upon mechanisms different from those of the common Pt(II)
drugs [6]. Chemically stable derivatives of CA-4-P, which itself is
prone to isomerise in vitro to a biologically inactive E-alkene, were
obtained by integration of the Z-alkene in five-membered
heterocycles such as imidazoles or oxazoles [1]. It has also been
shown that attachment of halogen atoms (Cl, Br) in the meta
position of ring A of CA-4 increased the tubulin affinity as well as
the cytotoxic effects on certain cancer cells [7]. Previously, we have
reported beneficial synergistic effects of combretastatin-analogous
chalcones upon covalent attachment to platinum complex frag-
ments [8]. In continuation of this approach we now investigated
similar ruthenium complex conjugates of known and new
halogen-substituted oxazole-bridged combretastatin analogues.
inates from a functional inhibition of the VE-cadherin/b-catenin
complex which is required for endothelial cellecell adhesion
during neovessel assembly [3]. The activation of Rho signalling
which in turn causes a reorganisation of the actin skeleton is also
believed to play a role [4]. A drawback of CA-4 is its insufficient
cytotoxicity which necessitates combination regimens with
cisplatin or taxol in the therapy of solid tumors. Treatment with
CA-4 alone often led to the persistence of peripheral cancer cells
2. Results and discussion
Abbreviations: ABC, ATP-binding cassette; CAM, chorioallantoic membrane;
MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PBS, phos-
phate buffered saline; PPTS, pyridinium p-toluenesulfonate; SRB, sulforhodamine-
B; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
* Corresponding author. Tel.: þ49 0 921 552679; fax: þ49 0 921 552671.
E-mail address: Rainer.Schobert@uni-bayreuth.de (R. Schobert).
2.1. Chemistry
Wang et al. reacted p-toluenesulfonyl 3,4,5-trimethox-
ybenzylisocyanide 1a with O-benzylisovanillin 2a to obtain oxazole
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.061

B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
4891
Scheme 2. Reagents and conditions: (i) Isonicotinic acid, Et3N, C6H2Cl3COCl, DMAP,
DMF/toluene (1:5), r.t., 16 h; (ii) [Ru(arene)Cl2]2, CH2Cl2, r.t., 3 h.
518A2 melanoma cells. However, in the HL-60 cells the complexes
6b and c exceeded their parent halo-oxazoles 4b and c. The toluene
complex 6d was less efficacious when compared to the p-cymene
complex 6a but reached the activity of the halo compounds.
In order to elucidate how HL-60 cells perish once treated with
compounds 4a or 6a we carried out TUNEL-assays that detect DNA
Scheme 1. Reagents and conditions: (i) K2CO3, DME/methanol, 2 h, reflux (for 3a) or r.t.
(for 3b and c); (ii) HCO2NH4, 5% Pd/C, MeOH, 2 h, reflux; (iii) PPTS, ethanol, 1 h, reflux.
4a after deprotection of benzyl ether 3a by Pd-catalysed transfer
hydrogenation [9]. The halo substituted isocyanides 1b (Br) and 1c
(Cl) were prepared according to the method reported for 1a (cf.
Supporting Information) and were treated with THP-protected
isovanillin 2b to generate the oxazoles 3b and c. These gave the
oxazoles 4b and c after deprotection with PPTS in ethanol
(Scheme 1, Table 1). The use of the THP-group is recommended in
these cases since Pd-catalysed hydrogenation of benzyl ethers has
occasionally led to unwanted dehalogenation affording 3,4-dime-
thoxyphenyl derivatives [10].
The ligands 5 were prepared by esterification of isonicotinic acid
with oxazoles 4aec under Yamaguchi conditions using 2,4,6-tri-
chlorobenzoyl chloride and 4-(N,N-dimethylamino)pyridine
(DMAP) (Scheme 2). Subsequently, the ligands 5 and half an
equivalent of [Ru(arene)Cl2]2 were mixed in CH2Cl2 to leave the
product complexes 6 as ochre-coloured powders in high yields
(>80%). They are soluble in organic solvents and well-resolved
NMR spectra were obtained of solutions in CDCl3. These NMR
spectra revealed distinct downfield shifts for the pyridine signals at
positions 2 and 6 indicating the coordination of the ligand to Ru(II).
fragmentation,
Supporting Information). Complex 6a gave rise to 73.7% ꢀ 5.1
apoptotic cells when applied at 10 M for 16 h. In contrast, oxazole
a late event in the course of apoptosis (cf.
m
4a merely led to 6.1% ꢀ 5.8 apoptotic cells under identical condi-
tions. This discrepancy between 4a and 6a is most likely originating
from the propensity of the latter to metallate bionucleophiles such
as DNA in a way reminiscent of cisplatin. The apoptosis of HL-60
cells induced by compounds 4a and 6a was not associated with
mitochondrial damage according to an assay carried out with the
fluorescence dye JC-1 that detects changes in the mitochondrial
membrane potential (cf. Supporting Information) [11]. In contrast,
both compounds 4a and 6a reduced the number of intact mito-
chondria in 518A2 cells treated for 72 h significantly to ca. 50% of an
untreated control.
The boost in efficacy of Ru(p-cymene) complex 6a over oxazole
4a became more apparent in tests against two congenerous chemo-
sensitive human cancer cell lines, a p53-competent wild-type and
a p53(ꢁ/ꢁ)-mutant form of HCT-116 colon carcinoma. The cells
were incubated for up to 120 h with 4a or 6a at concentrations
The signals of the
h
6-bound arene ligand were shifted towards
higher field (between 5 and 6 ppm in the 1H NMR spectra)
compared to normal benzene derivatives. The NMR spectra of the
Ru(arene) complexes 6aed featured supernumerary highfield
shifted arene signals which we assign to isomers or rotamers of the
respective complexes (ratio ca. 1:10).
ranging from 0.01 mM to 10 mM before being stained with trypan
blue. This visualisation method is generally more accurate than
MTT or SRB assays since it renders cell proliferation numbers rather
than surrogate parameters. Compound 4a was quite active in HCT-
116 cells, especially in the p53(ꢁ/ꢁ) mutants (Fig. 1). The complex
2.2. Biological evaluation
MTT tests with the oxazoles 4 and their ruthenium complexes
6aed revealed extraordinary efficacies against cells of human
518A2 melanoma and HL-60 leukaemia with IC50(72 h) reaching
the picomolar range (cf. Supporting Information). These cancer cell
lines are sensitive to most anti-tumor compounds including met-
allodrugs such as cisplatin and combretastatins. Complex 6a and its
parent oxazole 4a showed the highest activity and virtually iden-
tical cytotoxicity profiles in both cell lines. The compounds 4b/6b
and 4c/6c also showed pairwise equal albeit lesser activities against
Table 1
Oxazoles 3 and 4.
Oxazole
R1
R2
Yield (%)
3a
4a
3b
4b
3c
4c
OMe
OMe
Br
Br
Cl
Bn
H
THP
H
THP
H
89 [9]
90 [9]
45
93
70
Fig. 1. Relative numbers as to trypan blue staining of viable cells of colon carcinomas
HCT-116 and HCT-116 (p53ꢁ/ꢁ) when treated with 10
mM (A), 1 mM (-), 0.1 mM (:),
Cl
84
0.05 M ( ), or 0.01 M (>) of oxazoles 4a and 6a for the indicated times.
m
6
m

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B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
Fig. 2. Relative numbers of viable cells (y-axis) of HT-29 colon carcinoma treated with 10
4 or complexes 6 for the indicated times (in h, x-axis) as to MTT-assays.
mM (A), 1 mM (-), 0.1 mM (:), 0.01 mM (>), 0.001 (,) and 0.0001 mM (6) of compounds
6a showed the same activity profile in the HCT-116 (p53ꢁ/ꢁ)
mutant cells, but was more active than oxazole 4a in the wild-type
(cf. Supporting Information). Probably, p53-competent but not p53-
null cells of HCT-116 are able to repair the cellular damage inflicted
by low concentrations of 4a while 6a causes additional DNA lesions
via ruthenation thwarting efficient repair in p53(þ) cells.
cells of HCT-116 at the lowest concentration tested (0.01
mM).
Similar results were obtained from the apoptosis induction assay
Fig. 3. Chicken embryos with surrounding blood vessels immediately after adding the test compounds 4a or 6a (left), after one day (middle) and after three days (right). The top
row shows a negative control (PBS). Pictures are representative of several independent tests.

B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
4893
MTT-assays were also carried out with cells of p53(þ) com-
bretastatin-refractory human HT-29 colon carcinoma in order to
evaluate the influence of the arene ligand and of halogen substitu-
tion of the A-ring (Fig. 2). Generally, the complexes 6 were active at
lower concentrations than their respective parent oxazoles 4. This is
further evidence for the Ru(arene) fragment contributing to the
overall cytotoxic effect and also for the chemical stability of the
conjugates. As a further control we proved that mixtures of 4a and
Ru(p-cymene)(pyridine)Cl2, a surrogate for the complex Ru(p-
cymene)(isonicotinic acid)Cl2 that would result from hydrolysis of
conjugate 6a, showed an activity profile identical to that of 4a alone.
The oxazoles 4aec also feature a phenolic OH-group which makes
them amenable to glucuronidation and thus potential substrates for
the multi-drug resistance relevant ABC-transporters of types MRP-1
and MRP-3. These transporters are overexpressed in HT-29 cells and
are responsible for their resistance to colchicine or camptothecin
derivatives bearing phenolic OH-groups [12,13]. HL-60 cells lack
such drug transporters which fact might explain the greater activi-
ties of compounds 4 in these cells. No difference in activity was
observed between the p-cymene complex 6a and the corresponding
toluene analogue 6d (not shown).
observed for the aryl protons of the p-cymene ligand. The methyl
and isopropyl protons of the latter experienced a slight highfield
shift and the signals also became smaller and broader (cf.
Supporting Information). This experiment showed that ruthenation
of thiols is quite effective even under water-free conditions. In
a biological context this means that complexes like 6a are not only
capable of modifying cysteine and methionine containing peptides
and proteins, but also tucked-away components of membranes and
of active sites of enzymes.
The effects of the compounds 4a and 6a on the development of
embryonal blood vessels compared to a negative control (PBS) were
documented by a CAM assay (Fig. 3) [14]. Within the first 24 h of
incubation with 4a a slight shrinking of the vessels was visible
which ended up with a complete degradation after three days.
Complex 6a had little visible effect during the first 24 h. As with the
negative control there was a continuous growth of the blood
vessels. Only in the following two days this compound exhibited an
anti-angiogenic effect. It is unclear whether this long induction
period is a trait of complex 6a or is due to its slow degradation to
oxazole 4a, which is the causative agent proper.
The wound-healing assay is a useful method for gauging the
anti-migrative activity of drug candidates [15]. It was carried out
with the compounds 4a and 6a and with 518A2 melanoma cells.
Complex 6a diminished the motility of these cells to a greater
extent than the parent oxazole 4a even at concentrations as low as
Metallodrugs usually also react with intracellular S-nucleophiles
such as glutathion. 1H NMR spectra of complex 6a dissolved in
DMF-d7 and treated with N-acetyl-L-cysteine revealed a fast reac-
tion within minutes. A new highfield signal for the pyridine protons
2-H and 6-H appeared at 8.9 ppm and a downfield shift was
0.01
mM (Fig. 4). While little difference was visible after 6 h, the
Fig. 4. Temporal progress of wound-healing in colonies of 518A2 melanoma cells treated with compounds 4a and 6a (0.01 mM).

4894
B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
scratch on the glass bottom representing the “wound” was almost
grown over or “healed” after 24 h post-incubation with 4a but still
gaping in the case of 6a. This immobilising effect of the ruthenium
fragment is reminiscent of the well-documented anti-metastatic
activity of other Ru complexes, e.g., NAMI-A and RAPTA-C [6].
column chromatography (silica gel 60) to leave oxazole 3b (80 mg,
45%) as a colourless oil; Rf 0.47 (ethyl acetate/n-hexane, 1:2, v/v);
nmax(ATR)/cmꢁ1 2938, 1590, 1557, 1509, 1484, 1463, 1411, 1357,
1260, 1228, 1200, 1173, 1036, 1021, 999, 854, 697; 1H NMR
(300 MHz, CDCl3)
d 1.5e1.7 (4H, m), 1.8e2.0 (4H, m), 3.4e3.5 (1H,
m), 3.78 (3H, s), 3.8e3.9 (7H, m), 5.2e5.3 (1H, m), 6.89 (1H, d,
J ¼ 8.5 Hz), 7.16 (1H, d, J ¼ 1.9 Hz), 7.23 (1H, dd, J 8.5, J ¼ 2.1 Hz), 7.34
(1H, d, J ¼ 1.9 Hz), 7.45 (1H, d, J ¼ 2.1 Hz), 7.86 (1H, s); 13C NMR
3. Conclusion
Combretastatin A-4 analogues were prepared that feature
A-rings modified by bromine and chlorine at position 3 and/or B-
rings modified by attachment of dichlorido(arene)(isonicotinate)
ruthenium(II) groups as well as an alkene bond fixed in the
bioactive Z-configuration by being part of an oxazole ring. While all
compounds inhibited the growth of human HL-60 leukaemia and
518A2 melanoma cells at picomolar concentrations, their activities
in cells of human colon carcinomas HT-29, which is CA-4-refrac-
tory, and HCT-116 as wild-type and p53(ꢁ/ꢁ) mutant forms, were
more discriminative and structure-dependent. Unlike the known
anisole 4a, its Ru(II) complex 6a was quite active at the lowest
(75.5 MHz, CDCl3) d 19.0, 25.1, 30.3, 56.1, 60.6, 62.4, 97.9,111.3,112.2,
116.3, 117.7, 121.0, 121.4, 123.9, 129.4, 132.1, 146.1, 146.2, 146.5, 149.3,
151.0, 153.6; MS (EI) m/z 491 (7%), 489 (7), 408 (82), 407 (93), 406
(73), 405 (100), 392 (33), 390 (37), 361 (80), 282 (20), 152 (21), 85
(88). Anal C23H24BrNO6 (C, H, N).
4.1.2. 4-(30-Chloro-40,50-dimethoxyphenyl)-5-(400-methoxy-300-
tetrahydropyranyloxyphenyl)-oxazole 3c
Analogously to oxazole 3b, compound 3c (110 mg, 70%) was
obtained from TosMIC reagent 1c (R1 ¼ Cl) (128 mg, 0.35 mmol),
THP-protected isovanillin 2b (83 mg 0.35 mmol) [17] and K2CO3
(320 mg, 2.3 mmol) as a colourless oil; Rf 0.54 (ethyl acetate/n-
hexane,1:1, v/v); nmax(ATR)/cmꢁ1 2939,1562, 1510,1488,1463,1411,
1358, 1254, 1227, 1200, 1173, 1047, 1021, 1000, 859, 658; 1H NMR
concentration tested (0.01 mM) in p53-competent wild-type HCT-
116 cells. The ruthenium complexes 6aec were generally more
active in combretastatin A-4-refractory HT-29 cells than the cor-
responding oxazoles 4aec. Halogenation alone as in the oxazoles
4b and c led only to a slight improvement of efficacy against HT-29
cells when compared to 4a. The activity enhancing effect of the Ru
(II) moiety is presumably due to an auxiliary DNA- or protein-
modifying mode of action since complex 6a also reacted rapidly
(300 MHz, CDCl3)
d 1.5e1.7 (4H, m), 1.8e2.0 (4H, m), 3.4e3.5 (1H,
m), 3.77 (3H, s), 3.8e3.9 (7H, m), 5.25 (1H, t, J ¼ 3.4 Hz), 6.88 (1H, d,
J ¼ 8.5 Hz), 7.12 (1H, d, J ¼ 2.0), 7.21 (1H, dd, J ¼ 8.5 Hz), 7.28 (1H, d,
J ¼ 2.0 Hz), 7.33 (1H, d, J ¼ 2.1 Hz), 7.85 (1H, s); 13C NMR (75.5 MHz,
CDCl3)
d 18.9, 25.0, 30.2, 55.9, 56.0, 60.6, 62.2, 97.8, 110.4, 112.1,
with common bionucleophiles such as N-acetyl-
L
-cysteine in cell-
116.2, 120.9, 121.0, 121.3, 128.2, 128.7, 132.2, 145.9, 146.3, 149.2,
150.9, 153.6; MS (EI) m/z 445 [Mþ] (6%), 364 (27), 363 (83), 362 (72),
361 (100), 348 (17), 346 (42), 318 (36), 151 (30), 85 (77). Anal
C23H24ClNO6 (C, H, N).
free 1H NMR experiments. In addition, complex 6a lowered the
motility of 518A2 melanoma cells more efficiently than the parent
oxazole 4a and thus might have a beneficial effect on aggressively
metastasising tumors. So, by attaching a Ru(arene) fragment to
modified combretastatin-mimicking oxazoles a significant broad-
ening of their anti-cancer spectrum can be achieved.
4.1.3. 4-(30-Bromo-40,50-dimethoxyphenyl)-5-(300-hydroxy-400-
methoxyphenyl)-oxazole 4b
A mixture of compound 3b (78 mg, 0.16 mmol), ethanol (30 mL)
and PPTS (100 mg, 0.4 mmol) was heated under reflux for 1 h and
then concentrated in vacuum. The residue was purified by column
chromatography (silica gel 60) affording oxazole 4b (60 mg, 93%) as
a colourless oil; Rf 0.31 (ethyl acetate/n-hexane, 2:5, v/v); nmax
(ATR)/cmꢁ1 3500, 2937, 2837, 1589, 1557, 1509, 1485, 1461, 1441,
1401, 1367, 1270, 1260, 1229, 1130, 1105, 1040, 1024, 997, 899, 857,
4. Experimental protocols
Melting points were recorded using a GALLENKAMP apparatus
and are uncorrected. IR: PERKINeELMER Spectrum One FT-IR
spectrophotometer equipped with an ATR sampling unit. NMR:
BRUKER Avance 300 spectrometer; chemical shifts are given in
parts per million (
d) downfield from Me4Si as internal standard;
804, 760, 697, 657; 1H NMR (300 MHz, CDCl3)
d 3.77 (3H, s), 3.86
coupling constants (J) are given in Hz. MS: VARIAN MAT 311A (EI).
Microanalyses: PERKINeELMER 2400 CHN elemental analyser.
Microanalyses indicated by the symbols of the elements were
within ꢀ0.2% of the theoretical values for all new compounds. The
starting compounds and pure solvents were purchased from
the usual sources and were used without further purification. The
solvents were dried by distillation over CaH2 (DMF) or Na (toluene).
TosMIC reagent 1a and oxazoles 3a and 4a were prepared according
to a literature procedure [9]. [Ru(toluene)Cl2]2 was prepared anal-
ogously to [Ru(benzene)Cl2]2 [16]. Merck silica gel 60 (230e400
mesh) was used for column chromatography.
(3H, s), 3.90 (3H, s), 6.84 (1H, d, J ¼ 8.4 Hz), 7.10 (1H, dd, J ¼ 8.4 Hz,
J ¼ 2.1 Hz), 7.1e7.2 (2H, m), 7.45 (1H, d, J ¼ 1.9 Hz), 7.87 (1H, s); 13C
NMR (75.5 MHz, CDCl3)
d 56.0, 60.6, 110.7, 111.2, 113.3, 117.7, 119.4,
121.5, 124.0, 129.2, 132.2, 145.8, 145.9, 147.4, 149.5, 153.5; MS (EI) m/
z 407 [Mþ] (81%), 405 (Mþ] (81), 361 (17), 151 (100), 81 (28). Anal
C18H16BrNO5 (C, H, N).
4.1.4. 4-(30-Chloro-40,50-dimethoxyphenyl)-5-(300-hydroxy-400-
methoxyphenyl)-oxazole 4c
Analogously to oxazole 4b, compound 4c (76 mg, 84%) was
obtained from THP-ether 3c (111 mg, 0.25 mmol) and PPTS (100 mg,
0.4 mmol) as a colourless solid of m.p. 124 ꢂC; Rf 0.32 (ethyl acetate/
4.1. Chemistry
n-hexane, 2:3, v/v); lmax(MeOH)/nm 302 (3
/dm3 molꢁ1 cmꢁ1
11400); nmax(ATR)/cmꢁ1 3500, 3004, 2970, 2939, 2838, 1582, 1562,
1509,1489,1440,1401,1365,1272,1258,1235,1217,1131,1105,1045,
1024, 997, 900, 869, 806, 759, 728, 709, 657; 1H NMR (300 MHz,
4.1.1. 4-(30-Bromo-40,50-dimethoxyphenyl)-5-(400-methoxyphenyl-
300-tetrahydropyranyloxy)-oxazole 3b
A mixture of TosMIC reagent 1b (R1 ¼ Br) (144 mg, 0.35 mmol),
THP-protected isovanillin 2b (83 mg 0.35 mmol) [17], K2CO3
(320 mg, 2.3 mmol) and CH2Cl2/MeOH (1:3, 20 mL) was stirred at
room temperature for 2 h. The volatiles were removed in vacuum
and the remainder was taken up in ethyl acetate. The resulting
solution was washed with water and brine, dried over Na2SO4,
filtered and concentrated in vacuum. The residue was purified by
CDCl3) d 3.78 (3H, s), 3.87 (3H, s), 3.89 (3H, s), 5.90 (1H, s), 6.84 (1H, d,
J ¼ 8.4 Hz), 7.09 (1H, dd, J ¼ 8.4 Hz, J ¼ 2.2 Hz), 7.14 (1H, d, J ¼ 2.2 Hz),
7.17 (1H, d, J ¼ 2.1 Hz), 7.28 (1H, d, J ¼ 2.1 Hz), 7.87 (1H, s); 13C NMR
(75.5 MHz, CDCl3)
d 56.0, 56.1, 60.7, 110.5, 110.7, 113.3, 119.4, 121.1,
121.5,128.4,128.6,132.4,145.8,145.9,147.4,149.4,153.7; MS (EI) m/z
364 (34%), 363 (89), 362 (81), 361 (100), 348 (22), 346 (76), 318 (45),
300 (23). Anal C18H16ClNO6 (C, H, N).

B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
4895
4.1.5. 5-(300-Isonicotinoyl-400-methoxyphenyl)-4-(30,40,50-
trimethoxyphenyl)-oxazole 5a
temperature for 3 h. A mixture of ethyl acetate and n-hexane
(50 mL, 1:4) was added and the resulting suspension was stirred for
Isonicotinic acid (22 mg, 0.18 mmol) was suspended in dry DMF
another 5 min. The precipitate was collected, washed with
(1 mL). Et3N (29
m
L, 0.20 mmol) and 2,4,6-trichlorobenzoyl chloride
n-hexane and dried in vacuum to give complex 6a (63 mg, 75%) as
a yellow solid of m.p. 145 ꢂC (dec.); nmax(ATR)/cmꢁ1 2962, 2938,
2842, 1752, 1582, 1515, 1464, 1414, 1375, 1272, 1235, 1168, 1125,
1084, 1057, 1006, 838, 762, 692, 654; 1H NMR (300 MHz, CDCl3)
(33 L, 0.20 mmol) were added and the mixture was stirred for
m
20 min at room temperature. Compound 4a (65 mg, 0.18 mmol) and
DMAP (45 mg, 0.36 mmol) dissolved in a mixture of dry DMF/
toluene (5 mL, 1:4) were added and the reaction mixture was
stirred for 16 h at room temperature. After dilution with ethyl
acetate and washing with water the organic phase was dried over
Na2SO4, filtered and concentrated in vacuum. The residue was
purified by column chromatography (silica gel 60) giving iso-
nicotinate 5a (51 mg, 61%) as a colourless gum; Rf 0.32 (ethyl
acetate/n-hexane, 2:1, v/v); nmax(ATR)/cmꢁ1 2934, 1750, 1582, 1514,
1498, 1463, 1414, 1373, 1298, 1125, 1062, 1003, 837; 1H NMR
d
1.19 (6H, d, J ¼ 6.9 Hz), 1.32 (6H, d, J ¼ 6.9 Hz), 2.04 (3H, s), 2.13
(3H, s), 2.8e2.9 (1H, m), 2.9e3.1 (1H, m), 3.8e4.0 (12H, m), 4.61
(2H, d, J ¼ 6.1 Hz), 5.17 (2H, d, J ¼ 6.1 Hz), 5.25 (2H, d, J ¼ 6.1 Hz),
5.47 (2H, d, J ¼ 6.1 Hz), 6.89 (2H, s), 7.0e7.1 (1H, m), 7.43 (1H, s),
7.5e7.6 (1H, m), 7.91 (1H, s), 7.94 (2H, d, J ¼ 6.7 Hz), 9.29 (2H, d,
J ¼ 6.7 Hz); 13C NMR (75.5 MHz, CDCl3)
d 18.3, 22.3, 30.7, 56.1, 61.0,
82.4, 83.0, 97.4, 104.0, 104.8, 112.6, 121.1, 123.9, 126.3, 127.4, 134.2,
137.6, 139.3, 144.4, 149.5, 151.2, 153.5, 156.0, 161.9; MS (EI) m/z 462
(16%), 393 (42), 351 (23), 270 (19), 228 (24), 185 (10), 147 (25), 124
(45), 106 (100), 78 (47). Anal C35H36Cl2N2O7Ru (C, H, N).
(300 MHz, CDCl3) d 3.78 (6H, s), 3.83 (6H, s), 6.89 (2H, s), 7.02 (1H, d,
J ¼ 8.6 Hz), 7.45 (1H, s), 7.56 (1H, d, J ¼ 8.6 Hz), 7.89 (1H, s), 7.95 (2H,
d, J ¼ 6.0 Hz), 8.82 (2H, d, J ¼ 6.0 Hz); 13C NMR (75.5 MHz, CDCl3)
d
56.0, 60.9, 104.8, 112.5, 121.4, 121.6, 123.2, 126.1, 127.3, 134.1, 136.2,
4.1.9. Dichlorido(h6-p-cymene)[5-(300-isonicotinoyl-400-
methoxyphenyl)-4-(30-bromo-40,50-dimethoxyphenyl)-oxazole]
ruthenium(II) 6b
Analogously to the synthesis of 6a, complex 6b (73 mg 84%) was
obtained from ligand 5b (54 mg, 0.11 mmol) and [Ru(p-cymene)
Cl2]2 (32 mg, 0.052 mmol) as a yellow solid of m.p. 147e148 ꢂC
(dec.); nmax(ATR)/cmꢁ1 2964, 2940, 1753, 1555, 1514, 1486, 1462,
1414, 1269, 1232, 1129, 1084, 1058, 1041, 1000, 859, 762, 693; 1H
138.0, 139.5, 144.4, 149.4, 150.8, 151.4, 153.4, 163.1; MS (EI) m/z 462
[Mþ] (100%), 447 (29), 419 (8), 278 (7), 106 (68). Anal C25H22N2O7
(C, H, N).
4.1.6. 5-(300-Isonicotinoyl-400-methoxyphenyl)-4-(30-bromo-40,50-
dimethoxyphenyl)-oxazole 5b
Analogously to 5a, compound 5b (204 mg, 64%) was obtained
from isonicotinic acid (68 mg, 0.63 mmol), Et3N (100
m
L,
NMR (300 MHz, CDCl3)
d
1.31 (6H, d, J ¼ 6.9 Hz), 2.12 (3H, s), 2.9e3.1
0.72 mmol), 2,4,6-trichlorobenzoyl chloride (110 L, 0.72 mmol),
m
(1H, m), 3.80 (3H, s), 3.83 (3H, s), 3.87 (3H, s), 5.24 (2H, d,
J ¼ 6.0 Hz), 5.47 (2H, d, J ¼ 6.0 Hz), 7.03 (1H, d, J ¼ 8.5 Hz), 7.15 (1H,
s), 7.41 (1H, s), 7.47 (1H, s), 7.5e7.6 (1H, m), 7.90 (1H, s), 7.96 (2H, d,
J ¼ 6.6 Hz), 9.29 (2H, d, J ¼ 6.6 Hz); 13C NMR (75.5 MHz, CDCl3)
oxazole 4b (320 mg, 0.79 mmol) and DMAP (155 mg, 1.26 mmol) as
a colourless solid of m.p. 191e192 ꢂC; Rf 0.16 (ethyl acetate/n-
hexane, 1:1, v/v); nmax(ATR)/cmꢁ1 3079, 2936, 1750, 1558, 1509,
1483, 1458, 1411, 1363, 1300, 1272, 1258, 1165, 1092, 988, 858, 751,
d
18.3, 22.3, 30.7, 56.1, 60.7, 82.4, 83.0, 97.4, 104.0, 111.1, 112.2, 112.7,
685; 1H NMR (300 MHz, CDCl3)
d
3.78 (3H, s), 3.83 (6H, s), 7.02 (1H,
117.9, 121.1, 121.2, 123.9, 126.2, 129.0, 133.0, 137.7, 139.4, 144.8, 146.4,
149.6, 151.5, 153.8, 156.0, 161.9; MS (EI) m/z 512 (68%), 510 (70), 495
(5), 271 (5), 234 (5), 119 (28), 106 (100), 78 (42). Anal
C34H33BrCl2N2O6Ru (C, H, N).
d, J ¼ 8.7 Hz), 7.14 (1H, d, J ¼ 2.0 Hz), 7.42 (1H, d, J ¼ 2.0 Hz), 7.50
(1H, d, J ¼ 2.2 Hz), 7.52 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.2 Hz), 7.88 (1H, s),
7.96 (2H, d, J ¼ 6.0 Hz), 8.8e8.9 (2H, m); 13C NMR (75.5 MHz, CDCl3)
d
56.0, 60.6, 111.0, 112.6, 117.9, 121.2, 121.4, 123.3, 123.9, 126.0, 128.9,
132.7, 136.3, 139.6, 144.9, 146.3, 149.5, 150.7, 151.6, 153.7, 163.0; MS
(EI) m/z (EI) 512 [Mþ] (75%), 510 [Mþ] (64), 106 (100). Anal
C24H19BrN2O6 (C, H, N).
4.1.10. Dichlorido(h6-p-cymene)[5-(300-isonicotinoyl-400-
methoxyphenyl)-4-(30-chloro-40,50-dimethoxyphenyl)-oxazole]
ruthenium(II) 6c
Analogously to the synthesis of 6a, complex 6c (82 mg, 88%) was
obtained from 5c (58 mg, 0.12 mmol) and [Ru(p-cymene)Cl2]2
(38 mg, 0.06 mmol) as a yellow solid of m.p. 167e168 ꢂC (dec.);
nmax(ATR)/cmꢁ1 2965, 2932,1753,1561,1514,1490,1463,1415,1369,
1269, 1232, 1167, 1129, 1084, 1047, 1002, 896, 869, 821, 762, 692; 1H
4.1.7. 5-(300-Isonicotinoyl-400-methoxyphenyl)-4-(30-chloro-40,50-
dimethoxyphenyl)-oxazole 5c
Analogously to the synthesis of 5a, compound 5c (58 mg, 81%)
was obtained from isonicotinic acid (20 mg, 0.16 mmol), Et3N
(24
m
L, 0.17 mmol), 2,4,6-trichlorobenzoyl chloride (26
m
L,
NMR (300 MHz, CDCl3)
d
1.31 (6H, d, J ¼ 6.9 Hz), 2.12 (3H, s), 2.9e3.1
0.17 mmol), oxazole 4c (51 mg, 0.15 mmol) and DMAP (37 mg,
0.30 mmol) as a colourless oil; Rf 0.40 (ethyl acetate/n-hexane,
2:1, v/v; nmax(ATR)/cmꢁ1 2938, 2841, 1748, 1595, 1562, 1488, 1408,
1365, 1296, 1265, 1168, 1130, 1062, 998, 896, 752, 702; 1H NMR
(1H, m), 3.80 (3H, s), 3.82 (3H, s), 3.88 (3H, s), 5.25 (2H, d,
J ¼ 6.0 Hz), 5.47 (2H, d, J ¼ 6.0 Hz), 7.03 (1H, d, J ¼ 8.6 Hz), 7.11 (1H,
s), 7.30 (1H, s), 7.40 (1H, s), 7.5e7.6 (1H, m), 7.90 (1H, s), 7.95 (2H, d,
J ¼ 6.7 Hz), 9.29 (2H, d, J ¼ 6.7 Hz); 13C NMR (75.5 MHz, CDCl3)
(300 MHz, CDCl3)
d
3.78 (3H, s), 3.82 (3H, s), 3.85 (3H, s), 7.02 (1H, d,
d 18.3, 22.3, 30.7, 56.1, 60.8, 82.4, 83.0, 97.4, 104.0, 110.4, 111.8, 112.7,
J ¼ 8.7 Hz), 7.11 (1H, d, J ¼ 2.0 Hz), 7.30 (1H, d, J ¼ 2.0 Hz), 7.42 (1H,
d, J ¼ 2.0 Hz), 7.51 (1H, dd, J ¼ 8.7 Hz, J ¼ 2.0 Hz), 7.88 (1H, s), 7.96
(2H, d, J ¼ 6.0 Hz), 8.8e8.9 (2H, m); 13C NMR (75.5 MHz, CDCl3)
121.1, 121.8, 123.9, 126.3, 128.3, 128.6, 133.0, 137.7, 139.4, 144.8,
146.2, 149.6, 151.4, 153.9, 156, 161.9; MS (EI) m/z 568 (31%), 468 (36),
466 (77), 284 (5), 232 (4), 109 (23), 106 (100), 78 (39). Anal
C34H33Cl3N2O6Ru (C, H, N).
d
56.0, 60.7, 110.3, 112.6, 121.1, 121.2, 121.4, 123.2, 126.0, 128.3, 128.6,
132.9, 136.2, 139.6, 144.8, 145.3, 149.5, 150.7, 151.6, 153.8, 163.0; MS
(EI) m/z 469 (59%), 468 [Mþ] (50), 467 (42), 466 [Mþ] (98), 453 (33),
451 (75), 332 (42), 293 (43), 282 (23), 106 (100), 78 (86). Anal
C24H19ClN2O6 (C, H, N).
4.1.11. Dichlorido(h6-toluene)[5-(300-isonicotinoyl-400-
methoxyphenyl)-4-(30,40,50-trimethoxyphenyl)-oxazole](toluene)
ruthenium(II) 6d
Analogously to the synthesis of 6a, complex 6d (85 mg, 71%) was
obtained from ligand 5a (77 mg, 0.17 mmol) and [Ru(toluene)Cl2]2
(44 mg, 0.083 mmol) as a yellow solid of m.p. 145 ꢂC (dec); nmax(-
ATR)/cmꢁ1 3061, 2939, 2842,1750,1582,1515,1497,1452,1415,1374,
1299, 1272,1263,1125, 1086,1057,1005, 839, 761, 692, 654; 1H NMR
4.1.8. Dichlorido(h6-p-cymene)[5-(300-isonicotinoyl-400-
methoxyphenyl)-4-(30,40,50-trimethoxyphenyl)-oxazole]ruthenium
(II) 6a
A solution of ligand 5a (51 mg, 0.11 mmol) and [Ru(p-cymene)
Cl2]2 (34 mg, 0.06 mmol) in CH2Cl2 (5 mL) was stirred at room
(300 MHz, CDCl3)
d 2.14 (3H, s), 2.18 (3H, s), 3.8e4.0 (12H, m), 4.83

4896
B. Biersack et al. / European Journal of Medicinal Chemistry 45 (2010) 4890e4896
(2H, d, J ¼ 5.7 Hz), 5.32 (2H, d, J ¼ 5.7 Hz), 5.5e5.6 (1H, m), 5.6e5.8
(2H, m), 6.88 (2H, s), 7.0e7.1 (1H, m), 7.43 (1H, s), 7.5e7.6 (1H, m),
7.90 (1H, s), 7.94 (2H, d, J ¼ 6.6 Hz), 9.31 (2H, d, J ¼ 6.6 Hz); 13C NMR
created by fixing a thin plastic foil on top of a cup and covered. This
was done by opening the shell at the flat end where the air sac
resides and by letting the content slip out. After another 2e4 days
the first blood vessels became visible. Then 10 nmol of the
(75.5 MHz, CDCl3) d 19.0, 56.0, 60.9, 79.8, 79.9, 81.5, 86.0, 87.3,100.6,
104.8,112.6,121.0,123.9,126.3,127.3,134.2,137.7,139.2,144.3,149.5,
151.2, 153.4, 156.1, 161.8. Anal C32H30Cl2N2O7Ru (C, H, N).
substance to be tested (in 10 mL PBS with 1% DMSO) was applied
directly onto the embryonal vessels. PBS was used as a negative
control. Finally, incubation was continued for upto three days and
the results were documented by photography [14].
4.2. Biological studies
4.2.1. Cell lines and culture conditions
4.2.4. Wound-healing assay
All cell lines were obtained from the German Centre of Biolog-
ical Materials (DSMZ), Braunschweig, Germany, apart from the
human 518A2 melanoma cells which were a gift from the Depart-
ment of Oncology and Hematology of the Martin-Luther University,
Halle, Germany. The HL-60 and colon cancer cells were grown in
RPMI-1640 medium (Gibco) supplemented with 10% fetal calf
518A2 cells (1 ꢃ 105 cells/mL) were seeded out in 6-well plates
and cultured for 24 h to obtain a subconfluent monolayer. Then an
artificial wound was created by scraping off a narrow strip of cells
using a 10
mL plastic tip. The medium was changed and the cells
were treated with 0.01
mM of the test compounds for up to 24 h and
microscopically analyzed following 6 h and 24 h of incubation [15].
serum (FCS, Gibco), 100 IU/mL penicillin G, 100
mg/mL streptomycin
sulfate, 0.25 g/mL amphotericin B and 250 g/mL Gentamycin. The
518A2 melanoma cells were cultured in Dulbecco’s Modified Eagle
m
m
Acknowledgments
Medium (D-MEM, Gibco) containing 10% FCS, 100 IU/mL penicillin
We thank the Deutsche Forschungsgemeinschaft for financial
support (grant Scho 402/8-3).
G, 100
mg/mL streptomycin sulfate, 0.25 mg/mL amphotericin B and
250 g/mL gentamycine. Mycoplasm contamination was routinely
m
monitored, and only mycoplasm-free cultures were used.
Appendix. Supporting information
4.2.2. Cytotoxicity assays
Supplementary data associated with this article can be found in
online version at doi:10.1016/j.ejmech.2010.07.061.
MTT assay: MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide] (ABCR) was used to identify viable cells which
reduce it to a violet formazan [18]. HL-60 cells (5 ꢃ 105 cells/mL)
were seeded and cultured for 24 h, the adherent 518A2 and HT-29
cells (5 ꢃ104 cells/mL) and HF cells (5 ꢃ104 cells/mL) were cultured
for 24 h. Incubation (5% CO2, 95% humidity, 37 ꢂC) following
treatment with the test compounds (dilution series ranging from
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4.2.3. CAM assay
Fertilised chicken eggs received from a nearby farm directly
after laying were incubated at 36e38 ꢂC and a relative humidity of
60%. During the growth the eggs were held in an inclined position
and turned from time to time in order to avoid an adherence to the
shell. After four days each embryo was transferred into a cavity