4-(3-Halo/amino-4,5-dimethoxyphenyl)-5-aryloxazoles and-N-methylimidazoles That Are cytotoxic against combretastatin a resistant tumor cells and vascular disrupting in a cisplatin resistant germ cell tumor model

Article abstract of DOI:10.1021/jm100345r

New combretastatin A analogues featuring oxazole or N-methylimidazole bridged Z-alkenes and halo-or amino-substituted A-rings were tested against various cancer cell lines and in testicular germ cell tumor xenografts in mice. Imidazoles with 3-halo-4,5-dimethoxy substituted A-rings and 3-amino-4-methoxy substituted B-rings (7b and 8b) were efficacious at nanomolar concentrations against cells of combretastatin A refractory HT-29 colon carcinoma, multidrug-resistant MCF-7/Topo breast carcinoma, and cisplatin-resistant 1411HP testicular germ cell tumor. They induced apoptosis and inhibited tubulin polymerization. While well tolerated by mice at high doses, these imidazoles initiated extensive intratumoral hemorrhage and regressions of highly vascularized 1411HP xenografts.

Full text of DOI:10.1021/jm100345r

J. Med. Chem. 2010, 53, 6595–6602 6595
DOI: 10.1021/jm100345r
4-(3-Halo/amino-4,5-dimethoxyphenyl)-5-aryloxazoles and -N-methylimidazoles That Are Cytotoxic
against Combretastatin A Resistant Tumor Cells and Vascular Disrupting in a Cisplatin Resistant Germ
Cell Tumor Model
Rainer Schobert,*,‡ Bernhard Biersack,‡ Andrea Dietrich,† Katharina Effenberger,‡ Sebastian Knauer,‡ and
Thomas Mueller*,†
†Department of Internal Medicine IV, Oncology/Hematology, Martin Luther University Halle-Wittenberg, Ernst-Grube-Strasse 40,
D-06120 Halle, Germany, and ‡Organic Chemistry Laboratory, University Bayreuth, Universita€tsstrasse 30, D-95440 Bayreuth, Germany
Received March 16, 2010
New combretastatin A analogues featuring oxazole or N-methylimidazole bridged Z-alkenes and halo-
or amino-substituted A-rings were tested against various cancer cell lines and in testicular germ cell
tumor xenografts in mice. Imidazoles with 3-halo-4,5-dimethoxy substituted A-rings and 3-amino-4-
methoxy substituted B-rings (7b and 8b) were efficacious at nanomolar concentrations against cells of
combretastatin A refractory HT-29 colon carcinoma, multidrug-resistant MCF-7/Topo breast carci-
noma, and cisplatin-resistant 1411HP testicular germ cell tumor. They induced apoptosis and inhibited
tubulin polymerization. While well tolerated by mice at high doses, these imidazoles initiated extensive
intratumoral hemorrhage and regressions of highly vascularized 1411HP xenografts.
Introduction
unsatisfactorypharmacologicalproperties.11 In another study
(3-halo-4,5-dimethoxyphenyl)stilbenes 2, close analogues of
combretastatin A-3, showed increased affinities for tubulin
and a more selective activity profile against tumor cells
including resistant ones. However, because of their insuffi-
cient water solubility, they require formulations as phosphate
prodrugs in order to reach optimum uptake rates.15 Fluori-
nated combretastatin derivatives were also synthesized by
various other groups.16-18 Herein we report new water-solu-
ble, stable, and nontoxic oxazole and imidazole-bridged
combretastatin derivatives with halo- or amino-substituted
A-rings that show selective antitumoral activity in vitro and in
vivo.
Combretastatin A-4 (Figure 1) was first isolated from the
bark of the South African Cape Bushwillow (Combretum
caffrum).1 Fosbretabulin, its water-soluble phosphate pro-
drug, selectively damaged tumoral vasculature in clinical
trials.2 A drawback of combretastatin A-4 is its insufficient
cytotoxicity which necessitates combination regimens with
other drugs. A recent phase Ib trial revealed that combina-
tions of fosbretabulin with carboplatin and paclitaxel were
well tolerated and had antitumor activity in heavily pretreated
patients with advanced cancer.3 Treatment with combretast-
atin A-4 alone often led to the persistence of peripheral, quic-
kly revascularizing cancer cells and hence to tumor relapses.4
The related catechol combretastatin A-1 and its bisphosphate
prodrug (OXi4503) are also strongly vascular disrupting5,6
and led to tumor regressions especially when combined with
other anticancer drugs such as bevacizumab7 or doxorubicin.8
Catechols are known to undergo redox cycling via quinoid
intermediates, thus mediating the generation of reactive oxy-
gen species and the alkylation of bionucleophiles.6,9,10 Che-
mically stable derivatives of combretastatin A-4, which itself
tends to isomerize to a biologically inactive E-alkene,11-13
were obtained by incorporation of the Z-alkene in hetero-
cycles such as imidazoles, oxazoles,14 isoxazolines, pyridines,
or triazoles.4 For instance, imidazole 1 retains the tubulin
affinity of the parent combretastatins while showing im-
proved water solubility and pharmacokinetics. However, it
is inferior to combretastatin A-4 with respect to cytotoxicity.
The analogous highly cytotoxic oxazoles are hampered by
Results and Discussion
Chemistry. The new imidazoles and oxazoles were pre-
pared from the corresponding halo- and nitro-substituted
p-toluenesulfonylmethyl isocyanides (TosMICsa ). Commer-
cially available 5-chloro- and 5-bromovanillin 3a/b were
reacted with iodomethane to give the veratraldehydes
4a/b,15 which were converted to the tosylmethylformamides
5a/b by reaction with p-toluenesulfinic acid and formamide in
the presence of camphorsulfonic acid (CSA). In the same
way, 5-nitrovanillin as obtained by reaction of vanillin with
fuming nitric acid in acetic acid was methylated to give
veratraldehyde 4c,19,20 which was converted to formamide
5c. Subsequently, the formamides 5 were dehydrated to the
a Abbreviations: CAM, chorioallantoic membrane; CSA, camphor-
sulfonic acid; DME, 1,2-dimethoxyethane; DMF, N,N-dimethylforma-
mide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide;
NBT, nitro blue tetrazolium; ROS, reactive oxygen species; SRB,
sulforhodamine B; TBAI, tetrabutylammonium iodide; TosMIC,
p-toluenesulfonylmethyl isocyanide; TUNEL, terminal deoxynucleotidyl
transferase mediated dUTP nick end labeling.
*To whom correspondence should be addressed. For R.S.: phone/
fax, þ49 921 552679/1; e-mail, Rainer.Schobert@uni-bayreuth.de.
For T.M.: phone/fax, þ49 345 5577278/9; e-mail, thomas.mueller@
medizin.uni-halle.de.
r
2010 American Chemical Society
Published on Web 08/23/2010
pubs.acs.org/jmc

6596 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Schobert et al.
TosMIC derivatives 6 by treatment with phosphoroxychlor-
ide (Scheme 1).
imidazoles 7b-d and 8b-d with 3 M HCl/dioxane afforded
the respective water-soluble mono- or bis-hydrochlorides.
The nitro-substituted compounds 9a-e were prepared
likewise from the nitro-TosMIC derivative 6c and the corre-
sponding aldehydes and imines (Scheme 3). The amines
10a-d were obtained by Pd-catalyzed transfer hydrogena-
tion. The 3-chloroindole 9e was reduced with Zn/HCl to
amine 10e. Finally, the compounds 10 were converted to
water-soluble hydrochlorides.
The halo-substituted TosMIC derivatives 6a/b were con-
verted to N-methylimidazoles 7/8 by reaction with N-methyl-
imines generated from the corresponding arylaldehydes
(Scheme 2). Selective reduction of the nitro group of 7a/8a
with Zn/HCl gave the amines 7b/8b. Treatment of the
Biological Evaluation. Figure 2 shows the growth inhibi-
tion in MTT assays of compounds 1, 7b, 8b, 10a, and 10e in
cells of the human tumor cell lines 518A2 melanoma, HL-60
leukemia, HT-29 colon carcinoma, KB-V1/Vbl cervix car-
cinoma, and MCF-7/Topo breast carcinoma. Table 1 sum-
marizes the IC50 (72 h) values as calculated from the
Scheme 3. Synthesis of 4-(3-Aminoaryl)-Substituted Oxazoles
and N-Methylimidazoles 10a
Figure 1. Combretastatins A-1, A-3, and A-4 and known analo-
gues 1 and 2: potent inhibitors of tubulin polymerization.
Scheme 1. Synthesis of TosMIC Derivatives 6a
a Reagents and conditions: (i) ArCHO, K2CO3, DME/MeOH, 2 h,
reflux, 42-86% (for X = O); ArCHO, MeNH2 (33% in EtOH), AcOH,
EtOH, 2 h, reflux (for X = NMe); then 6c, K2CO3, DME/EtOH, 3 h,
reflux, 64-74%; (ii) HCO2NH4, Pd/C (5%), MeOH, 2 h, reflux,
67-84% (for 10a-d); Zn, HCl, THF, 10 min, room temp, 64% (for 10e).
a Reagents and conditions: (i) MeI, K2CO3, TBAI, DMF, 24 h, room
temp, 80-90%; (ii) HCONH2, CSA, p-toluenesulfinic acid, 16 h, 60 °C,
51-58%; (iii) POCl3, Et3N, DME, 3 h, -5 °C, 57-74%.
Scheme 2. Synthesis of 4-(3-Haloaryl)-Substituted N-Methylimidazoles 7 and 8a
a Reagents and conditions: (i) ArCHO, MeNH2 (33% in EtOH), AcOH, EtOH, 2 h, reflux; then 6a/b, K2CO3, DME/EtOH, 3 h, reflux, 52-99%.
(ii) For 7a or 8a: Zn, HCl, THF, 15 min, room temp, yield 40/91% (7b 2HCl/8b 2HCl). (iii) For 7c,d or 8c,d: 3 M HCl/dioxane, 15 min, room temp,
3
3
46-66%.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6597
Figure 2. Cell growth inhibition by compounds 1, 7b, 8b, 10a, and 10e at various concentrations (9, 100 μM; 2, 1 μM; 1, 0.01 μM; [, 0.001
μM) in cells of human 518A2 melanoma, HL-60 leukemia, HT-29 colon carcinoma, KB-V1/Vbl cervix carcinoma, and MCF-7/Topo breast
carcinoma upon incubation for 24-72 h (x-axis). Y-Axis shows number of viable cells relative to untreated controls as ascertained by the MTT
assay (RCN = relative cell numbers).
Table 1. Inhibitory Concentrationsa (IC50 [nM]) of Imidazoles 1, 7, 8, 10c-e and oxazoles 10a,b, and Combretastatin A-4 (CA4) When Applied to
Various Human Cancer Cell Lines
IC50 (nM) for cell line
compd
518A2
HL-60
HT-29
KB-V1/Vbl
MCF-7/Topo
H12.1b
1411HPb
1
7b
>100000
62 ( 11
400 ( 100
2300 ( 200
6.4 ( 1.4
66 ( 16
860 ( 140
2.0 ( 0.9
110 ( 40
2800 ( 300
>10000
>100000
0.2 ( 0.05
>10 000
64 ( 14
300 ( 200
400 ( 170
>10000
320 ( 180
c
c
0.06 ( 0.01
530 ( 30
0.2 ( 0.1
0.02 ( 0.01
510 ( 10
4.3 ( 0.8
57 ( 7
>15 000
>100 000
>50 000
14 ( 4
∼ 30
∼ 60
7c
7d
c
c
c
c
0.02 ( 0.01
0.1 ( 0.02
9700 ( 300
0.2 ( 0.01
3.8 ( 1.2
0.9 ( 0.1
4600 ( 400
>10 000
c
c
c
c
8b
8c
200 ( 60
c
340 ( 160
c
∼ 22
∼ 54
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
c
8d
c
c
10a
10b
10c
10d
10e
CA4
0.01 ( 0.01
c
1.23 ( 0.48
c
c
c
c
c
3100 ( 600
18 ( 7
0.04 ( 0.02
0.1 ( 0.0
160 ( 60
0.4 ( 0.1
3.8 ( 1.8
500 ( 200
3600 ( 140
a Values are derived from concentration-response curves (MTT, 72 h; Figure 2, S18) by applying the four-parameter Hill model. b SRB tests, 96 h
(S19). IC50 (96 h; cisplatin): ∼400 nM (H12.1), ∼3.5 μM (1411HP). c Not measured.
dose-response curves of Figure 2 according to the “four para-
meter Hill model”. It also includes the IC50 values for other
analogous compounds and cell lines whose dose-response curves
are depicted in the Supporting Information. The haloamino-
substituted imidazoles 7b and 8b were distinctly more cyto-
toxic than the known reference compound 1 with IC50 con-
centrations in the lower nanomolar range, even in the com-
bretastatin A-4-resistant HT-29 and the multidrug-resistant

6598 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Schobert et al.
KB-V1 and MCF-7/Topo cells. The fluoro and N,N-di-
methylaminoimidazoles 7c/8c and 7d/8d were generally less
active than their amino congeners 7b/8b (cf. Supporting
Information).
The diamino substituted oxazole 10a and imidazole 10e
were more efficacious at low concentrations than 1 and 7b/8b
against both multidrug-resistant cells, i.e., KB-V1/Vbl and
MCF-7/Topo. The reduced activity of compounds 10b-d
when compared to that of 10a emphasizes the importance of
the 3-amino group at the B-ring. The imidazoles 7b and 8b
were also tested in vitro in a testicular germ cell tumor model
comprising the highly chemosensitive cell line H12.1 and the
cell line 1411HP, which is intrinsically resistant to cisplatin
and other conventional chemotherapeutic drugs. These cell
lines were chosen because they maintain their individual in
vitro chemosensitivities to cisplatin when grown as xenograft
tumors in mice.21 Unlike cisplatin, imidazole 7b/8b showed
similar activity against the two germ cell tumor cell lines with
IC50(96 h) ≈ 20-60 nM (cf. Supporting Information). They
also inhibited the polymerization of tubulin to a comparable
extent when tested with the tubulin polymerization assay kit
by Cytoskeleton. Figure 3 shows the time dependency of this
process in samples containing 7b, 8b, or the reference com-
pounds combretastatin A-4, colchicine, or vincristine.
In TUNEL assays, which allow the detection of apoptosis by
labeling the 30-OH ends of DNA fragments with fluorescein-
tagged nucleotides, compounds 7b, 8b, and 10a were
found to induce death in HL-60 cells predominantly in an
apoptotic way (∼60% after 16 h of incubation with 10 μM
drug). The extent of apoptosis-related mitochondrial dam-
age in HL-60 cells was ascertained by means of the fluo-
rescence dye JC-1 that detects changes in the mitochondrial
Figure 3. Effects of 3 μM each of compounds 7b, 8b, vincristine,
colchicine, or combretastatin A-4 on the polymerization of tubulin
as ascertained with a fluorescence-based assay kit from Cytoskele-
ton. Data are representative of four to five independent experi-
ments. RFU = relative fluorescence units.
Figure 4. Chicken embryos with surrounding blood vessels immediately after adding combretastatin A-4 (CA-4) or 7b (left), after 1 day
(middle), and after 3 days (right). The top row shows a negative control. Pictures are representative of at least two independent runs.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6599
Figure 5. In vivo effects of 7b and 8b in mouse xenografts of germ cell tumor cell line 1411HP: intratumoral hemorrhage 24 h after treatment
(A); tumor response following single-dose (B) and dual-dose (C) applications; tumor response to repeated applications (D). Arrows indicate
administration of compounds.
membrane potential.22 Only ∼60% of the mitochondria
were intact after incubation with the more cytotoxic com-
pounds 7b-d, 8b-d, and 10a/e, while treatment with 1 left
74% of the mitochondria unaffected. The involvement of
reactive oxygen species in the apoptosis of treated HL-60
cells was assessed with the colorimetric nitro blue tetrazo-
lium (NBT) assay but found to be insignificant (cf. Support-
ing Information).
double doses of 7b on days 16/17 and 35/36. As shown in
Figure 5D, regressions and prolonged periods of stabiliza-
tion following each application were achieved. Notably, even
the third course of treatment was tolerated well and the
mouse had regained its original body weight by this time
(cf. Supporting Information). These preliminary data demo-
nstrate the potential of 7b and 8b for the treatment of
resistant tumors.
The most promising of the new oxazoles and imidazoles
were tested for antiangiogenic and vasculature disrupting
properties using the CAM assay. In this test the vascular
system of a fertilized chicken embryo is used as a model.23
Figure 4 depicts the effects of combretastatin A-4 and 7b on
the development of embryonal blood vessels compared to a
negative control (PBS). Both led to a dramatic vessel shrink-
age within 24 h after treatment and to a complete degrada-
tion of the vascular system within 3 days. Interestingly, the
bromo analogue 8b displayed virtually no effect on this
regular embryonal vasculature, which highlights the pivotal
role of the halogen substituent. The halogen-free oxazole 10a
also showed no significant antiangiogenic effect.
We then evaluated 7b and 8b in mouse xenografts of germ
cell tumor cell line 1411HP. Two mice bearing tumors of ∼1
cm3 volume were treated with a single dose of 30 mg/kg body
weight of 7b or 8b which caused transient swelling and strong
hemorrhages in the tumors visible after 24 h as a red-blue to
brown coloring (Figure 5A). Eventual regression and slow
regrowth was observed (Figure 5B). Both mice tolerated this
treatment very well. Two mice with xenografts of ∼2.5 cm3
volume were administered 20 mg/kg body weight of 7b on 2
consecutive days. The resulting dramatic regressions, leading
even to a stabilization in one case, are shown in Figure 5C,D.
The other xenograft regrew and was given two further
Conclusions
The new N-methyl-4-(3-halo-4,5-dimethoxyphenyl)-5-(3-
amino-4-methoxyphenyl)imidazoles 7b and 8b combine che-
mical stability, good water solubility, and low general toxicity
in mice with excellent antitumoral efficacy against various
cancer cell lines in vitro, including multidrug-resistant ones,
and against certain chemoresistant tumor xenografts in mice.
The nature of the halogen substituent is crucial for the
selectivity and the magnitude of their bioactivity. Unlike its
chloro congener 7b, the bromoimidazole 8b was vascular
disrupting in tumor xenografts while leaving regular vascu-
lature in chicken embryos (CAM assay) alone. The new
imidazoles are also strong inducers of cancer cell apoptosis.
Their properties warrant further in vivo evaluations in order
to fully elucidate their mode of action and to establish
optimized treatment schedules including combinations with
other chemotherapeutic agents.
Experimental Section
Chemistry. All starting compounds and combretastatin A-4
were purchased from Aldrich. Compound 1 was prepared
according to literature.11 For chromatography Merck silica
gel 60 (230-400 mesh) was used. The following instruments

6600 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18
Schobert et al.
were used: melting points (uncorrected), Gallenkamp; IR spec-
tra, Perkin-Elmer Spectrum One FT-IR spectrophotometer
with ATR sampling unit; nuclear magnetic resonance spectra,
BRUKER Avance 300 spectrometer; chemical shifts are given in
parts per million (δ) downfield from tetramethylsilane as inter-
nal standard; mass spectra, Varian MAT 311A (EI); microana-
lyses, Perkin-Elmer 2400 CHN elemental analyzer. All tested
compounds are >95% pure by elemental analysis.
406 (46), 405 (87) [Mþ], 404 (77), 403 (100) [Mþ], 391 (23), 390
(71), 389 (62), 388 (87), 341 (58), 313 (58), 206 (33), 164 (26).
1-Methyl-5-(300-amino-400-methoxyphenyl)-4-(30-chloro-40,50-
dimethoxyphenyl)imidazole Bis(hydrochloride) (7b 2HCl): Typi-
3
cal Procedure. Compound 7a (109 mg, 0.27 mmol) was dissolved
in THF (7.5 mL). Zn powder (107 mg, 1.36 mmol) was added
followed by a mixture of concentrated HCl (230 μL) in THF
(1 mL). After being stirred for 15 min at room temperature, the
reaction mixture was poured into water and treated with aqu-
eous NaHCO3 to adopt pH 8. The water phase was extracted
with ethyl acetate, and the organic phase was dried over Na2SO4,
filtered. The filtrate was concentrated in vacuum. The residue
was purified by column chromatography (silica gel 60, 5%
methanol/ethyl acetate, Rf = 0.66), giving crude 7b. This crude
product was dissolved in DCM (5 mL) and treated with 3 M
HCl/dioxane (1 mL). After the mixture was stirred for 15 min,
the solvent was removed and the oily residue was recrystallized
from an ethanol/n-hexane mixture giving the bis(hydrochloride)
salt of 7b. Yield: 42 mg (0.095 mmol, 40%); colorless solid of mp
180-183 °C; UV (MeOH) λmax (ε) 255 (14940). Anal. (C19H22-
Cl3N3O3) C, H, N. νmax (ATR)/cm-1 3009, 2781, 2578, 1635,
1552, 1517, 1497, 1445, 1409, 1304, 1271, 1147, 1113, 1047, 1025,
998, 830, 762, 739, 721; 1H NMR (300 MHz, DMSO-d6) δ 3.63
(3 H, s), 3.69 (3 H, s), 3.75 (3 H, s), 3.92 (3 H, s), 7.08 (1 H, d, J =
2.1 Hz), 7.16 (1 H, d, J = 2.1 Hz), 9.36 (1 H, s), 7.2-7.4 (3 H, m);
13C NMR (75.5 MHz, DMSO-d6) δ 33.9, 56.1, 56.2, 60.4, 110.7,
112.4, 117.6, 119.6, 123.6, 127.3, 127.6, 129.7, 135.7, 145.0,
153.5; m/z (EI) 375 (20) [Mþ], 374 (15), 373 (54) [Mþ], 358
(25), 296 (15), 252 (25), 237 (16), 70 (14), 61 (23), 43 (100).
Biological Studies. 1. Cell Lines and Culture Conditions. The
HL-60 cells were obtained from the German Collection of
Biological Material (DSMZ), Braunschweig, Germany. The
human 518A2 melanoma cells and the testicular germ cell tumor
cell lines H12.1 and 1411HP were cultured in the Department of
Oncology and Hematology, Medical Faculty of the Martin
Luther University, Halle, Germany. The KB-V1/Vbl and the
MCF-7/Topo cells were obtained from the Institute of Phar-
macy of the University Regensburg, Germany, and the colon
HT-29 cells from the University Hospital Erlangen, Germany.
The HL-60 and the HT-29 cells were grown in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 100 IU/
mL penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/mL
amphotericin B, and 250 μg/mL gentamycine (all from Gibco,
Egenstein, Germany). The 518A2 and the KB-V1/Vbl cells were
cultured in Dulbecco’s modified Eagle medium (D-MEM,
Gibco) containing 10% FCS, 100 IU/mL penicillin G, 100
μg/mL streptomycin sulfate, 0,25 μg/mL amphotericin B, and
250 μg/mL gentamycine. The MCF-7/Topo cells were grown in
E-MEM medium (Sigma) supplemented with 2.2 g/L NaHCO3,
110 mg/L sodium pyruvate, and 5% FCS. The cells were
maintained in a moisture-saturated atmosphere (5% CO2) at
37 °C in 75-mL culture flasks (Nunc, Wiesbaden, Germany).
They were serially passaged following trypsinization by 0.05%
N-[(Toluene-4-sulfonyl)-(3-chloro-4,5-dimethoxyphenyl)methyl]-
formamide (5a): Typical Procedure. 5-Chloroveratraldehyde
(5.67 g, 23.44 mmol), p-toluenesulfinic acid (3.01 g, 19.29 mmol),
and camphorsulfonic acid (110 mg, 0.47 mmol) were treated
with formamide (10 mL). Upon heating to 65 °C, the reaction
mixture turned into a solution, and after 2 h the product began
to precipitate. After the mixture was stirred for 16 h, the
precipitate was filtered, washed with methanol, and dried in
vacuum. Yield: 4.57 g (11.92 mmol, 51%); colorless solid of mp
157-158 °C; νmax (ATR)/cm-1 3190, 3107, 2947, 1690, 1593,
1576, 1484, 1470, 1423, 1403, 1308, 1283, 1250, 1215, 1143, 1121,
1
1078, 1053, 999, 860, 822, 788, 769, 659, 689; H NMR (300
MHz, DMSO-d6) δ 2.41 (3 H, s), 3.76 (3 H, s), 3.79 (3 H, s), 6.45
(1 H, d, J = 10.7 Hz), 7.24 (1 H, d, J = 1.9 Hz), 7.30 (1 H, d, J =
1.9 Hz), 7.43 (1 H, d, J = 8.4 Hz), 7.72 (2 H, d, J = 8.4 Hz), 7.98
(1 H, s), 9.74 (1 H, d, J = 10.7 Hz); 13C NMR (75.5 MHz,
CDCl3) δ 21.1, 56.3, 60.3, 69.5, 113.5, 122.4, 126.7, 127.1, 129.2,
129.6, 133.2, 144.9, 145.4, 153.1, 160.2; m/z (EI) 382 (4), 278 (6),
227 (89), 192 (76), 156 (57), 113 (55), 91 (100), 77 (67), 63 (92).
3-Chloro-4,5-dimethoxyphenyl(tosyl)methyl Isocyanide (6a):
Typical Procedure. Formamide 5a (4.57 g, 11.92 mmol) was
suspended in dry DME (100 mL) and cooled to -10 °C. POCl3
(3.4 mL, 36.1 mmol) was added, and a mixture of Et3N (8.3 mL,
59.5 mmol) in DME (10 mL) was dropped slowly to the reaction
mixture. After being stirred for 2 h at -5 °C, the reaction
mixture was poured into ice-water. The water phase was
extracted with ethyl acetate, and the organic phase was washed
with saturated aqueous NaHCO3 and brine, dried over Na2SO4,
filtered, and concentrated in vacuum. By refrigeration overnight
a yellow solid crystallized from the residue, which was collected
and dried in vacuum. Yield: 2.48 g (6.79 mmol, 57%); yellow
solid of mp 115 °C; νmax (ATR)/cm-1 2920, 2136, 1593, 1577,
1492, 1452, 1423, 1325, 1294, 1276, 1238, 1199, 1137, 1082, 1053,
1
1002, 862, 826, 759, 705, 683; H NMR (300 MHz, CDCl3) δ
2.45 (3 H, s), 3.79 (3 H, s), 3.87 (3 H, s), 5.49 (1 H, s), 6.76 (1 H, d,
J = 2.1 Hz), 6.88 (1 H, d, J = 2.1 Hz), 7.34 (2 H, d, J = 8.5 Hz),
7.64 (2 H, d, J 8.5 = Hz); 13C NMR (75.5 MHz, CDCl3) δ 21.7,
56.2, 60.8, 75.6, 110.8, 122.2, 122.6, 128.6, 129.9, 130.0, 130.4,
146.9, 147.4, 153.8, 166.6; m/z (EI) 365 (2) [Mþ], 278 (7), 246
(10), 210 (100), 155 (23), 91 (54), 66 (20).
1-Methyl-4-(30-chloro-40,50-dimethoxyphenyl)-5-(400-methoxy-
300-nitrophenyl)imidazole (7a): Typical Procedure. A mixture of
4-methoxy-3-nitrobenzaldehyde (76 mg, 0.42 mmol) and 33%
MeNH2/ethanol (260 μL, 2.10 mmol) in ethanol (15 mL) was
treated with AcOH (150 μL) and refluxed for 2 h. After the
mixture was cooled down to room temperature, compound 6a
(153 mg, 0.42 mmol) dissolved in DME (10 mL) and K2CO3 (500
mg, 3.62 mmol) were added. The reaction mixture was refluxed
for 3 h. The solvent was evaporated and the residue diluted with
ethyl acetate, washed with water and brine, dried over Na2SO4,
filtered, and concentrated in vacuum. The residue was purified
by column chromatography (silica gel 60). Yield: 110 mg (0.26
mmol, 62%); yellow oil; Rf = 0.24 (ethyl acetate/methanol
95:5); νmax (ATR)/cm-1 2939, 1623, 1600, 1566, 1524, 1505,
1483, 1461, 1396, 1342, 1325, 1263, 1230, 1189, 1167, 1112, 1086,
1047, 994, 889, 873, 863, 828, 816, 762, 737, 698, 675; 1H NMR
(300 MHz, CDCl3) δ 3.44 (3 H, s), 3.64 (3 H, s), 3.75 (3 H, s), 3.95
(3 H, s), 6.9-7.0 (2 H, m), 7.15 (1 H, d, J = 8.7 Hz), 7.45 (1 H,
dd, J = 8.7 Hz, J = 2.2 Hz), 7.50 (1 H, s), 7.78 (1 H, d, J = 2.2
Hz); 13C NMR (75.5 MHz, CDCl3) δ 32.1, 55.7, 56.6, 60.5,
109.2, 114.2, 119.7, 122.2, 126.0, 127.3, 127.9, 130.6, 136.4,
137.5, 137.8, 139.7, 144.0, 152.9, 153.4; m/z (EI) 407 (42),
€
trypsin/0.02% EDTA (PAA Laboratories, Colbe, Germany).
Mycoplasma contamination was routinely monitored, and only
mycoplasma-free cultures were used.
2. Determination of Tumor Cell Growth (MTT Assay). MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
(ABCR) was used to identify viable cells that reduce it to a violet
formazan.24 HL-60 leukemia cells (5 ꢀ 105/mL) and cells (5 ꢀ
104/mL) of 518A2 melanoma, HT-29 colon, KB-V1/Vbl cervix,
and MCF-7/Topo breast carcinoma were seeded out in 96-well
tissue culture plates and cultured for 24 h. Incubation (5% CO2,
95% humidity, 37 °C) of the cells following treatment with the
test compounds was continued for 24, 48, or 72 h. Blank and
solvent controls were treated identically. MTT in phosphate
buffered saline (5 mg/mL) was added to a final concentration of
0.05% (HL-60, 518A2) or 0.1% (HT-29, KB-V1/Vbl, MCF-7/
Topo). After 2 h the formazan precipitate was dissolved in 10%
sodium dodecyl sulfate in DMSO containing 0.6% acetic acid in

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 18 6601
the case of the HL-60 cells. For the adherent 518A2, KB-V1/
Vbl, MCF-7/Topo, and HT-29 cells the microplates were swiftly
turned to discard the medium before adding the solvent mixture.
The microplates were gently shaken in the dark for 30 min, and
absorbance at 570 and 630 nm (background) was measured with
an ELISA plate reader. All experiments were carried out in
quadruplicate. The percentage of viable cells was calculated as
the mean ( SD with controls set to 100%.
3. CAM Assay. Fertilized chicken eggs received from a nearby
farm directly after laying have been incubated at 36-38 °C and a
relative humidity of 60%. During the growth the eggs have been
held in an inclined position and turned from time to time in
order to avoid an adherence to the shell. After 4 days each
embryo was transferred into a cavity 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
letting the content slip out. There the growth continued for
another 2-4 days until the first blood vessels became visible.
Then an amount of 10 nmol of the substance to be tested (in 10
μL PBS with 1% DMF) was applied directly on the embryonal
vessels. As a reference, PBS was used. Finally incubation con-
tinued for up to another 3 days.25,26
4. SRB Cytotoxicity Assay. Dose-response curves of the
testicular germ cell tumor cell lines exposed to drug concentra-
tions of 0.001-10 μM were established using the sulforhoda-
mine B (SRB) microculture colorimetric assay27 and performed
as previously described.21 Briefly, cells were seeded into 96-well
plates on day 0 at cell densities previously determined to ensure
exponential cell growth during the period of the experiment. On
day 1, cells were treated with the drugs dissolved in medium to
give the appropriate concentrations for indicated times, and the
percentage of surviving cells relative to untreated controls was
determined on day 5.
5. Tubulin Polymerization Assay. Analysis of tubulin polym-
erization was performed using the tubulin polymerization assay
kit (Cytoskeleton) according to manufactures instructions. The
assay is fluorescence-based, and tubulin polymerization was
followed by measuring RFU (relative fluorescence units) on
the SpectraFluorPlus (Tecan, Switzerland) using the following
filters: excitation 360 nm, emission 465 nm.
effects on mitochondria; in vivo body weight-time curves;
maximal slopes of tubulin polymerization curves. This material
is available free of charge via the Internet at http://pubs.acs.org.
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Acknowledgment. This work was supported by a grant
from the Deutsche Forschungsgemeinschaft (Grant Scho
402/8-3). We thank Franziska Reipsch for her excellent
technical assistance.
Supporting Information Available: Syntheses and character-
ization of derivatives 4-5b/c, 7c/d, 8a-d, 9, 10; MTT and SRB
assays; TUNEL assays with HL-60 cells; ROS generation and

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