Cytotoxic and antivascular 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5- (halophenyl)-imidazoles

Article abstract of DOI:10.1016/j.bmcl.2011.09.005

A series of 1-methyl-4,5-diphenylimidazoles 6 with various patterns of m-halogen substitution at the 5-phenyl ring were tested for cytotoxicity in cancer and nonmalignant cell lines and for their capacity to prevent tube formation in HUVEC cultures. Unlik

Full text of DOI:10.1016/j.bmcl.2011.09.005

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6270–6273
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Cytotoxic and antivascular 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5-(hal-
ophenyl)-imidazoles
Bernhard Biersack a, Yazh Muthukumar b, Rainer Schobert a, , Florenz Sasse b
⇑
a Organic Chemistry Laboratory, University Bayreuth, Universitaetsstrasse 30, 95440 Bayreuth, Germany
b Helmholtz Center for Infection Research (HZI), Inhoffenstrasse 7, 38124 Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1-methyl-4,5-diphenylimidazoles 6 with various patterns of m-halogen substitution at the
5-phenyl ring were tested for cytotoxicity in cancer and nonmalignant cell lines and for their capacity
to prevent tube formation in HUVEC cultures. Unlike the monofluoro and difluoro derivatives 6a and
6e, the monobromo and diiodo analogs 6c and 6h were strongly cytotoxic and inhibited the polymeriza-
tion of tubulin and the tube formation by HUVEC. The dibromo derivative 6g displayed a unique selec-
tivity for KB-3-1 cervix and PC-3 prostate cancer cells. It also inhibited the tube formation by HUVEC
and the polymerization of tubulin which is indicative of its potential antiangiogenic activity in solid
tumors.
Received 22 June 2011
Revised 31 August 2011
Accepted 1 September 2011
Available online 7 September 2011
Keywords:
Combretastatin A-4
HUVEC
Angiogenesis
Anticancer agents
Cytotoxicity
Ó 2011 Elsevier Ltd. All rights reserved.
Combretastatin A-4 (CA-4, 1a, Fig. 1) was first isolated from the
bark of the South African Cape Bushwillow (Combretum caffrum)
and was later shown to have pronounced antivascular properties.1
Prodrugs of 1a with improved bioavailability such as the phos-
phate fosbretabulin have been investigated in a number of clinical
trials which proved their selective impact on tumor vasculature.2
Other CA-4 analogs are also being studied, for example, the serine
amide ombrabulin which is currently in phase III trials for the
treatment of NSCLC.3 Vascular disrupting agents (VDA) are an
intriguing alternative for the therapy of highly vascularized tumors
or such no longer responding to conventional chemotherapy.4 The
mechanism of action of VDA is typically associated with destabili-
zation of microtubules, activation of Rho signaling and reorganiza-
tion of the cellular actin cytoskeleton. Morphologically, endothelial
cells exposed to VDA get rounded and blebby which eventually
leads to the collapse of the tumor blood vessels and thus to tumor
necrosis.5 However, due to their insufficient cytotoxicity, 1a and its
prodrugs have to be administered as part of combination regimens
with other anticancer drugs such as carboplatin or bevacizumab to
prevent tumor relapse.6–8 In addition, they tend to isomerize to the
biologically inactive trans-form.9,10 Halogenated derivatives of 1a
seem to be less susceptible to biooxidative deactivation, yet retain
their antivascularity only in certain cases. Several vascular disrupt-
ing fluoro derivatives, for example, 1b11 have been recently re-
ported.12–15 The nature of the halogen substituent was found to
be particularly decisive for the activity of analogs with 3,
5-dihalo-substituted phenyl rings. The dibromo (1c) and diiodo
(1d) derivatives showed a much higher activity in human umbilical
vein endothelial cells (HUVEC) than the difluoro congener and
when compared with 1a.15 The 3,5-dibromo-4-methoxyphenyl
O
R1
R
Br
N
A
MeO
N
MeO
B
R2
R3
Br
N
HN
OMe
1a: R1, R2 = OMe, R3 = OH
1b: R1, R2 = OMe, R3 = F
1c: R1, R2 = Br, R3 = OH
1d: R1, R2 = I, R3 = OH
2a: R = Me
2b: R = H
MeO
OMe
R
N
N
NH2
OMe
3a: R = OMe
3b: R = Cl
3c: R = Br
x 2HCl
⇑
Corresponding author.
E-mail address: rainer.schobert@uni-bayreuth.de (R. Schobert).
Figure 1. Structures of combretastatin A derivatives 1, ceratamines 2, and N-
methyl-imidazoles 3.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.09.005

B. Biersack et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6270–6273
6271
motif is also represented in natural anticancer compounds, for
example, in the marine ceratamines 2.16 N-Methyl-4,5-diaryl imid-
azole analogs of 1a are stable to cis–trans isomerization and are or-
ally applicable in most cases.17 Recently, we published their
preparation by van Leusen reaction of p-toluenesulfonylmethyl
isocyanide (TosMIC) with aryl aldehydes as an alternative to the
palladium-mediated aryl coupling developed by Bellina et al.18
We had also identified imidazoles 3 as examples of this type with
R1
6a: R1 = F, R2 = OMe
6b: R1 = Cl, R2 = OMe
6c: R1 = Br, R2 = OMe
6d: R1 = I, R2 = OMe
6e: R1, R2 = F
O
O
OMe
C
CHO
N
S
R2
F
(i), (ii)
(iii), (iv)
N
F
N
6f: R1, R2 = Cl
F
OMe
6g: R1, R2 = Br
x HCl
OMe
OMe
6h: R1, R2 = I
4
5
Scheme 1. Synthesis of 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5-(halophenyl)-imidazoles 6. Reagents and conditions: (i) HCONH2, CSA, p-toluenesulfinic acid, 16 h, 60 °C;
(ii) POCl3, Et3N, DME, 3 h, À5 °C, 31% (two steps); (iii) ArCHO, MeNH2 (33% in EtOH), AcOH, EtOH, 2 h, reflux; then 5, K2CO3, DME/EtOH, 6 h, reflux; (iv) 3 M HCl/dioxane,
CH2Cl2, 10 min, rt, 25–91% (two steps).
Table 1
Inhibitory concentrations IC50 [nM] of 3 and 6 for the growth of cancer and nontumor derived cellsa and for the formation of tubes by HUVECb
Cell line/compound
L929
220
135
10,000
220
25
40
31,000
2400
4400
18
KB-3-1
PC-3
PtK2
450
70
5000
480
440
180
23,000
4300
1200
330
NHDF
HUVEC
3a
3b
6a
6b
6c
6d
6e
6f
70
16
2000
190
5
90
140
970
7600
240
175
40
52,000
7180
400
290
70
n/mc
1800
n/mc
70
10,000
n/mc
110
90
n/mc
16,000
n/mc
10
n/mc
27,000
n/mc
270
21,000
1150
14
6g
6h
3
3
3300
80
a
Values are derived from dose–response curves obtained by measuring the percentual absorbance of viable cells relative to untreated
controls (100%) after 5 days exposure of the cells to the test compounds in the MTT assay.
b
Minimal inhibitory concentrations [nM] for HUVEC tube formation.
Not measured.
c
Figure 2. Micrographs showing the degree of tube formation by HUVEC after exposure for 20 h to various concentrations of compounds 6e, 6g, and 6h which inhibited tube
formation at minimal inhibitory concentrations of 11 lg/mL, 135 ng/mL, or 50 ng/mL, respectively. Black arrows indicate tubes formed by HUVEC.

6272
B. Biersack et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6270–6273
6g was selectively efficacious against the cervix and prostate can-
cer cells for yet unknown reason. The anticancer active compounds
6c, 6g, and 6h were in almost all cases less harmful to the non-
tumor derived L929, PtK2, and NHDF cells than to the cancer cells.
Figure 2 shows the inhibitory effects of selected test compounds
on the tube formation propensity of HUVEC. While tube formation
was observed even in the presence of ca. 4 lg/mL (=12 lM) of the
tris-fluoro derivative 6e, the dibromo analog 6g was required at
concentrations only slightly greater than 130 ng/mL (=270 nM) to
prevent tube formation. The most effective compound, the diiodo
derivative 6h, suppressed tube formation even at this low concen-
tration (cf. Supplementary data file for other compounds).
The capacities of compounds 3a, 6a, 6c, 6e, 6g, and 6h to inhibit
the polymerization of tubulin as assessed by a standard turbidi-
metric method employing freshly isolated porcine microtubule
protein21 correlated well with their inhibitory effects on HUVEC
tube formation and their cytotoxicities. The highly cytotoxic and
HUVEC tube inhibiting compounds 6c, 6g, and 6h were also strong
inhibitors of tubulin polymerization (Fig. 3). The least cytotoxic
and least effective HUVEC tube suppressor 6e did not interfere
with tubulin polymerization. Interestingly, the known trimethoxy-
phenyl compound 3a while strongly cytotoxic, had but a minute
effect on tubulin polymerization. Except for 6e, a destruction of
microtubules was observed for all test compounds when applied
Figure 3. Tubulin polymerization in vitro at 37 °C in the presence of 25
compounds 3a and 6a, 6c, 6e, 6g, and 6h (control MeOH).
lg/mL of
exceptional in vivo activity and tolerance.19 Now we sought to fur-
ther optimize the drug profiles of similar imidazoles by attaching
halogen substituents to the tetrasubstituted A-ring.
The new 1-methyl-4-(3-fluoro-4-methoxyphenyl)-5-(halophe-
nyl)imidazoles 6 feature A- and B-rings transposed with respect
to 3 and were prepared from TosMIC 5. 3-Fluoro-4-methoxybenz-
aldehyde 4 was reacted with p-toluenesulfinic acid and formamide
in the presence of camphorsulfonic acid (CSA) to give a tosylmeth-
ylformamide intermediate, which was dehydrated to 5 by POCl3.
The halogenated benzaldehydes used for the van Leusen reaction
at 10 lg/mL to PtK2 potoroo (Potorous tridactylis) kidney epithelial
cells which are easy to monitor due to their flat shape (Fig. 4). The
greatest damage was caused by compounds 6h and 6c (not shown
since similar to that of 6h), which is in line with their high cytotox-
icity in these cells.
In conclusion, we prepared eight new N-methylimidazole-
bridged analogs 6a–h of combretastatin A-4 (1a) featuring a
3-fluoro-4-methoxyphenyl B-ring and various m-halogenated
A-rings. Some structure–activity relations already emerged in our
tests for cytotoxicity and antiangiogenic activity. In both series of
imidazoles 6 with mono- and dihalogenated A-rings the antiprolif-
erative activity was lowest for the fluoro (6a and 6e) and the chloro
(6b and 6f) derivatives. Substitution of one m-methoxy group of
the A-ring by bromine or replacement of both m-methoxy groups
by two iodine atoms led to strongly cytotoxic and antiangiogenic
agents 6c and 6h. Their activities exceeded even that of the known
trimethoxy derivative 3a. This confirms recent findings by Myers et
al.15 who discovered that bigger bromo (1c) or iodo (1d), but not
small fluoro substituents at both meta-positions of ring A of stilb-
enoid analogs of 1a led to enhanced potency against HUVEC cells.
The dibromophenyl substituted imidazole 6g was a potent sup-
pressor of HUVEC tube formation, of tubulin polymerization, and
with
5 were prepared according to known literature meth-
ods12,15,20 and converted to their N-methylimines. Reaction of the
latter with 5 and addition of HCl/dioxane finally afforded the imi-
dazolium salts 6 (Scheme 1).
Growth inhibition of cells of L929 mouse fibroblasts, human KB-
3-1 cervix carcinoma, PC-3 prostate cancer, PtK2 potoroo kidney
cells and primary human dermal fibroblasts (NHDF) by compounds
3 and 6 was assessed by MTT assays (Table 1). The monobromo
derivative 6c and the diiodo compound 6h proved most active.
They were also distinctly more cytotoxic to cancer cells than the
known trimethoxy derivative 3a and also than 3b, the best per-
former of our previously published imidazole series.19 In contrast,
the monofluoro (6a), difluoro (6e) and dichloro (6f) analogs were of
moderate cytotoxicity. Compounds 6c and 6h were also most ac-
tive with respect to suppressing tube formation in HUVEC cultures
at minimal inhibitory concentrations of ca. 100 nM. The dibromide
Figure 4. Effects on the microtubules network in PtK2 potoroo cells exposed for 16 h to 10 lg/mL of compounds 3a, 6a, 6e, 6g, or 6h then fixed and stained with antitubulin
antibodies.

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