Synthesis and biological evaluation of SANT-2 and analogues as inhibitors of the hedgehog signaling pathway

Article abstract of DOI:10.1016/j.bmc.2009.06.008

Hedgehog (Hh) signaling plays an important role in cell signaling of embryonic development and adult tissue homeostasis. In vertebrates, the hh gene encodes three different unique proteins: sonic hedgehog (Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh). Disruption of the Hh signaling pathway leads to severe disorders in the development of vertebrates whereas aberrant activation of the Hh pathway has been associated with several malignancies including Gorlin syndrome (a disorder predisposing to basal cell carcinoma, medulloblastoma and rhabdomyosarcoma), prostate, pancreatic and breast cancers. In vivo evidence suggests the antagonism of excessive Hh signaling provides a route to unique mechanism-based anti-cancer therapies. Recently the small molecule SANT-2 was identified as a potent antagonist of Hh-signaling pathway. Here, we describe the synthesis, SAR studies as well as biological evaluation of SANT-2 and its analogues. Fifteen SANT-2 derivatives were synthesized and analyzed for their interference with the expression of the Hh target gene Gli1 in a reporter gene assay. By comparison of structure and activity important molecular descriptors for Gli inhibition could be identified. Furthermore we identified derivative TC-132 that was slightly more potent than the parent compound SANT-2. Selected compounds were tested for Hh related teratogenic effects in the small teleost model medaka. Albeit Gli expression has indicated a 16-fold higher Hh-inhibiting activity than observed for the plant alkaloid cyclopamine, none of the tested compounds were able to induce the cyclopamine-specific phenotype in the medaka assay.

Full text of DOI:10.1016/j.bmc.2009.06.008

Bioorganic & Medicinal Chemistry 17 (2009) 4943–4954
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of SANT-2 and analogues as inhibitors
of the hedgehog signaling pathway
Anita Büttner a, , Katrin Seifert a, , Thomas Cottin a, Vasiliki Sarli a, Lito Tzagkaroulaki a,
Stefan Scholz b, Athanassios Giannis a,
*
a University of Leipzig, Institute for Organic Chemistry, Johannisallee 29, D-04103 Leipzig, Germany
b UFZ—Helmholz Centre for Environmental Research, Department of Cell Toxicology, Permoserstraße 15, 04318 Leipzig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Hedgehog (Hh) signaling plays an important role in cell signaling of embryonic development and adult
tissue homeostasis. In vertebrates, the hh gene encodes three different unique proteins: sonic hedgehog
(Shh), desert hedgehog (Dhh) and indian hedgehog (Ihh). Disruption of the Hh signaling pathway leads to
severe disorders in the development of vertebrates whereas aberrant activation of the Hh pathway has
been associated with several malignancies including Gorlin syndrome (a disorder predisposing to basal
cell carcinoma, medulloblastoma and rhabdomyosarcoma), prostate, pancreatic and breast cancers. In
vivo evidence suggests the antagonism of excessive Hh signaling provides a route to unique mecha-
nism-based anti-cancer therapies. Recently the small molecule SANT-2 was identified as a potent antag-
onist of Hh-signaling pathway.
Here, we describe the synthesis, SAR studies as well as biological evaluation of SANT-2 and its
analogues. Fifteen SANT-2 derivatives were synthesized and analyzed for their interference with the
expression of the Hh target gene Gli1 in a reporter gene assay. By comparison of structure and activity
important molecular descriptors for Gli inhibition could be identified. Furthermore we identified deriva-
tive TC-132 that was slightly more potent than the parent compound SANT-2. Selected compounds were
tested for Hh related teratogenic effects in the small teleost model medaka. Albeit Gli expression has indi-
cated a 16-fold higher Hh-inhibiting activity than observed for the plant alkaloid cyclopamine, none of
the tested compounds were able to induce the cyclopamine-specific phenotype in the medaka assay.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 3 April 2009
Revised 29 May 2009
Accepted 2 June 2009
Available online 10 June 2009
Keywords:
SANT-2
Cyclopamine
Hedgehog
Medaka
Cyclopia
1. Introduction
from endosomes to the tip of the cilium resulting in the inhibi-
tion of processing of transcription factors Gli1/2.4 Inappropriate
The Hedgehog (Hh) signaling pathway regulates numerous
processes involved in patterning, proliferation, survival and
growth in embryonic development as well as in adult tissue
homeostasis.1 In mammals, three Hedgehog genes, Sonic (Shh)
Indian (Ihh) and Desert (Dhh) expressed specifically in different
cell types are known. After their biosynthesis they are
post-translationally modified with a palmitoyl group at their
N-terminal cystein residue and with a cholesterol moiety at their
C-terminal glycin residue.2 Hh proteins are secreted lipidated
proteins that bind 12 pass transmembrane receptors Patched1
(Ptch1) and Ptch2. They are binding to these receptors induces
internalisation of Patch and derepression of the activity of
Shh signaling has been associated with several malignancies
including Gorlin syndrome (an autosomal dominant inherited
disorder predisposing to basal cell carcinoma, medulloblastoma
and rhabdomyosarcoma) as well as tumors of the gastrointesti-
nal tract, small cell lung cancers, pancreatic carcinomas and
prostate cancers.5–8
Antagonism of excessive Hh signaling may provide a route to
unique mechanism-based anti-cancer therapies. Therefore small
molecules inhibitors of Hh signaling are of great therapeutic poten-
tial.9 Recently, a number of small molecules that act as Shh antag-
onists have been reported both in scientific and patent literature.
They include the steroidal alkaloid cyclopamine,10 biarylcarboxa-
mides,11 CUR61414,12 SANT1-4,13,14 JK184,15 and GANT61.16 Fur-
thermore, important insights into the mechanism of action of
some of these inhibitors were published.17 For example it was
shown that cyclopamine drives Smo to the primary cilium were
it is inhibited by this alkaloid. Conversely, other antagonists
including SANT-2 and GANT61 abrogate the Shh-dependent trans-
location of Smo to the primary cilium.
Smoothened (Smo),
a 7 pass transmembrane receptor. After
binding of Hh to Ptch, Smo is phosphorylated3 and translocated
* Corresponding author. Fax: +49 341 9736599.
E-mail address: giannis@uni-leipzig.de (A. Giannis).
These authors contributed equally to this work.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.008

4944
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
SANT-2, a member of the benzimidazole class of compounds, is
exhibit significantly different IC50 values. According to the IC50-val-
ues and their confidence intervals the derivatives could be classi-
fied into four different groups (Fig. 4B–E). Group 1 (Fig. 4B) had
similar IC50-values as the parent compound SANT-2. Group 2
(Fig. 4C) had approximately twofold higher IC50 values. About
10-fold higher IC50-values were observed for group 3 (Fig. 4D).
No activity at all could be detected for compounds summarised
in group 4 (Fig. 4E). Analysis of the molecular structure revealed
striking similarities for most of the compounds within one of the
groups. Group 1 comprised chemicals with modifications exclu-
sively at the western aryl moiety. With the exception of compound
KS-B all members of this group had at least one ethoxy or methoxy
substitution at position 3 of the phenyl ring. Compounds summa-
rised in group 2 were characterised by the absence of ethoxy and
methoxy substitution in positions 3, 4 and 5 of the phenyl ring
and/or the presence of a cyano, dimethylamino, chloride or fluoride
substitution in position 3 of the N-phenyl ring. Compounds with an
additional methoxy group at position 2 of the phenyl ring or re-
moval of the Cl-substitution at the benzimidazole formed group
3 of SANT-derivatives. Group 4 comprised compounds with substi-
tutions within the benzimidazole ring. The data clearly show that
the benzimidazole core is essential for inhibitory properties. Minor
effects are obtained by changes at the benzoic acid moiety. One
derivative (TC132) was shown to be slightly more potent than
the parent compound SANT-2. Similar observations were made
for a structurally related group of compounds, that is, JRK184
and its derivatives.15 In JRK184 derivatives the imidazopyridine
core—the analogous moiety to the benzimidazole core in SANT-
derivatives—is essential for inhibition of Gli1. Substitutions of the
phenyl ring were of minor relevance. TC132, the compound with
the highest efficacy in the present study, exhibited an IC50 of
80 nM. Some JRK derivates showed a slightly higher activity with
IC50 values of about 30 nM. Both studies show that the identifica-
tion of molecular descriptors important for Shh inactivation—mea-
sured by Gli1-inhibition—provides a possibility for the generation
of further derivatives with higher bioactivity.
a very potent inhibitor of hedgehog signaling effective in nanomo-
lar concentrations by inhibiting both wild type and oncogenic
Smo.13
The aim of this study was to establish a synthetic route to SANT-
2 and its analogues in order to evaluate their potential inhibitory
effects on the hedgehog pathway. Furthermore we wanted to per-
form some SAR studies using these analogues. We modified SANT-
2 at its benzimidazole moiety and altered the pattern of substitu-
tion particularly at the phenyl ring. Shh inhibition was measured
by analyzing the expression of the hedgehog target transcription
factor gene Gli1 in a reporter gene assay. Furthermore, teratogenic
effects were analyzed in the small model teleost medaka (Oryzias
latipes) for selected compounds. Shh-antagonists such as the plant
alkaloid cyclopamine are known to induce the cyclopia phenotype
in vertebrates. For teleosts this phenotype has been described in
zebrafish and medaka18 with more severe manifestation in the me-
daka. Cyclopia19 or hosoprosencephaly is a disorder that is charac-
terized by failure of the embryo’s forebrain to divide and to form a
bilateral hemisphere during development. Exposure of medaka
embryos was performed in order to identify Shh-associated or
other potential non-Shh related teratogenic effects. These disor-
ders may be important indicators for side effects in preclinical
studies.
2. Results and discussion
2.1. Synthesis of SANT-2 and analogues
Commercially available benzene-1,2-diamine reacted with 2-
chloro-5-nitrobenzoyl chloride and yielded amide 3 which was
subsequently cyclised to benzimidazole 4 under acidic condi-
tions.20 Reduction of the nitro group in the presence of iron pow-
der, HCl and EtOH afforded aniline 5, which was further used for
N-acylation reactions, Figure 1. Additionally, we synthesized benz-
oxazole, benzothiazole, imidazole, and indole derivatives as SANT-
2 analogues as depicted in Figures 2 and 3.
2.3. Induction of cyclopia phenotype in the medaka
2.2. Inhibition of Gli1 reporter gene expression
Compounds interfering with the Shh signaling pathway, such as
the Smo binding alkaloid cyclopamine can induce the cyclopia phe-
notype in vertebrates. This phenotype, which is characterized by
one fusion eye, has been observed in mammals and birds21 and
in a less severe form also for the small teleost medaka. The medaka
offers the opportunity to screen for Shh-related teratogenic effects
by screening for the cyclopia phenotype in a small-scale system.
The different derivatives were tested for their interference with
the Shh signaling pathway by an established reporter gene assay
based on the inhibition of the target gene Gli1. The IC50-values
and their corresponding confidence intervals for inhibition of the
Gli1 reporter gene were determined (Table 1, Fig. 4A). Compounds
with non-overlapping confidence intervals were considered to
Cl
Cl
NH2
NH2
H
i
ii
+
N
O
O2N
O2N
NH2
O
OH
1
2
3
Cl
H
N
Cl
Cl
H
H
N
N
N
iii
iv
HN
N
N
O2N
H2N
Ar
O
6-15
5
4
6 Ar=3,4,5-triethoxyphenyl
8 Ar=4-dimethylaminophenyl 9 Ar=4-cyanophenyl
11 Ar=4-flurophenyl
7 Ar=3,4,5-trimethoxy phenyl
12 Ar=2-methoxyphenyl
13 Ar=3-trifluromethylphenyl 14 Ar=4-chlorophenyl
15 Ar=4-methoxyphenyl
Figure 1. Reagents and conditions: (i) (a) (COCl)2, DMF cat., CH2Cl2, rt 2 h; (b) Et3N, THF, rt, 12 h; (ii) CH3COOH, reflux, 2 h; (iii) Fe, HCl, EtOH, reflux, 2 h; (iv) (a) ArCOOH,
(COCl)2, DMF cat., CH2Cl2, rt 2.5 h; (b) Et3N, THF, rt, 2.5 h.

A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
4945
O
O
NO2
NO2
NO2
NH2
NH2
EtO
EtO
N
N
N
Cl
16
iii
NH2
N
H
ii
NO2
i
HN
Cl
N
H
N
H
19
18
Cl
OEt
Cl
2
17
Cl
Cl
O
O
O
N
NH2
SH
N
21
iii
NH2
EtO
EtO
H
ii
N
N
N
H
S
S
iv
S
NO2
23
24
22
20
Cl
Cl
Cl
OEt
OEt
Cl
O
NH2
OH
EtO
EtO
N
N
N
H
ii
iii
26
O
v
O
O
30
NH2
NO2
29
25
28
Figure 2. Reagents and conditions: (i) (a) Et3N, THF, rt, 12 h; (b) CH3COOH, reflux, 2 h; (ii) Fe, HCl, EtOH, reflux, 2–3 h; (iii) 3,4,5-triethoxybenzoyl chloride, Et3N, DMAP, THF,
rt 15–36 h; (iv) I2, DMF, rt 15 h; (v) (a) Et3N, THF, rt, 15 h; (b) p-TsOH, xylole, reflux, 4 h.
Cl
Cl
O
Cl
Cl
O
NO2
NH2
N
EtO
EtO
N
N
21
H
ii
iii
N
H
i
HN
N
H
N
H
NH2
34
NO2
Cl
NH2
Cl
32
33
OEt
31
Cl
O
Cl
N
EtO
EtO
N
N
N
iv
ii
iii
NH2
N
H
HN
N
H
N
N
H
H
37
NO2
35
NO2
36
OEt
32
Cl
Cl
Cl
O
Cl
O
EtO
EtO
NO2
iii
NH2
N
H
ii
NO2
NH3Cl
39
Me
N
H
HN
N
H
N
H
v
42
OEt
38
41
40
Figure 3. Reagents and conditions: (i) (a) I2, K2CO3, t-BuOH, 70 °C, 3 h; (ii) Fe, HCl, EtOH, reflux, 2–3 h; (iii) (a) 3,4,5-triethoxybenzoic acid, (COCl)2, DMF cat.; (b) Et3N, CH2Cl2,
rt, 12 h; (iv) DIB, K2CO3, DMSO, rt 3d; (v) (a) AcOH, EtOH, 80 °C, 12 h; (b) PPA, 120 °C, 15 min.
Table 1
IC50-values, slope (p) of non-linear regression curves (Hill-slope equation), corresponding standard errors (SE) and confidence intervals for the inhibition of Gli1 reporter gene
expression in Shh-Light II cells by SANT-2 and its derivatives (see also Fig. 4 for graphical representation and compound structures)
Compound
No.
IC50 (nM)
SE
Confidence interval
Slope (p)
SE
Lower level
Upper level
TC132
SANT-2
KSA
7
6
15
13
10
9
79.8
97.9
117
119
163
185
190
224
251
876
1325
1312
n.d.
7.9
8.9
9.7
65
81
99
98
118
140
145
201
239
259
287
301
1207
3653
1593
—
1.3
1.1
1.3
1.1
1.1
1.6
0.8
1.2
1.3
1.4
0.64
2.11
—
0.18
0.10
0.12
0.12
0.10
0.21
0.09
0.13
0.11
0.23
0.19
0.37
—
KSB
13
96
TC135
TC134
TC133
KSC
TC136
TC137
TC145
Cyclopamine
TC146
TC147
KS79
16
23
29
27
23
94
603
95
—
133
144
140
174
209
702
647
1246
—
8
14
11
12
18
23
28
32
35
40
n.d.
n.d.
n.d.
n.d.
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
KS80
KS83
Cyclopamine was included for comparison and as well-known Shh inhibiting reference compound. Gli1 expression was measured in shh light II cells co-treated with the
hedgehog activator SAG. IC50 values were calculated from 4 replicated independent experiments except TC137 (N = 3), TC145 (N = 2) and cyclopamine (N = 2). n.d. = no effect
detected within the tested range of concentrations. Highest tested concentration of each compound = 5000 nM. Numbers in column 2 refer to numbers given in the
description for the chemical synthesis.

4946
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
A
B
D
O
O
O
O
O
NH
NH
NH
NH
N
N
N
N
O
O
O
N
H
N
H
N
H
N
H
O
O
Cl
SANT-2 (6)
NH
O
O
O
O
Cl
TC132 (7)
Cl
TC145 (19)
TC137 (12)
O
O
NH
E
O
N
O
N
NH
NH
F3C
N
N
N
N
H
H
O
O
Cl
Cl
O
S
O
KSA (15)
KSB (13)
O
O
Cl
O
O
Cl
O
O
C
NH
NH
TC146 (23)
N
TC147 (28)
N
N
O
O
N
H
H
NH
NH
Cl
Cl
N
N
N
N
TC134 (9)
N
TC135 (10)
O
O
O
O
N
H
NH
H
NH
NH
O
O
Cl
O
O
Cl
N
N
N
H
KS79 (32)
KS80 (35)
H
Cl
Cl
N
Cl
O
O
NH
TC133 (8)
KSC (14)
O
N
N
N
H
O
O
Cl
H
F
Cl
KS83 (40)
TC136 (11)
Figure 4. (A) IC50-values (in nM) for the inhibition of Gli1 reporter gene expression in Shh- Light II cell exposed to 100 nM SAG and various concentrations of SANT-2 and its
derivatives. Cyclopamine was included for comparison and as well-known Shh inhibiting reference compound. Data points represent IC50-values upper and lower
confidence interval. Each compound was tested in four independent experiments, except TC137 (N = 3), TC145 (N = 2) and cyclopamine (N = 2). Numbers in brackets refer to
numbers given in the description for the chemical synthesis. (B–E) Structures of SANT-2 derivatives grouped according to their efficacy in inhibiting Gli1 reporter gene
expression (see Section 2 for further details).
Other teratogenic effects or unknown bioactivities of the test com-
pound may be detected as well. In contrast to the cellular screening
system based on Gli1 expression, it provides the advantage to rep-
resent a complex system based on a complete organism. In the
present study, SANT-2 and two of the most potent Gli1-inhibiting
derivatives (TC132, KSB) identified from the Gli1-reporter gene as-
say, were also analyzed for their effects on medaka development.
Cyclopamine was used as reference compound. It provokes the
cyclopia phenotype in concentrations of about 10-fold (12.5
lM)
above the IC50-values (1.3 M) of Gli1-reporter gene expression.
l

A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
4947
The tested compounds were 11–16-fold more potent inhibitors of
Gli1 expression if compared to cyclopamine. However, none of
them induced the cyclopia phenotype in the tested range of con-
mechanism of action of cyclopamine and SANT-2 (vide supra)
should be taken into consideration.
centrations (0.01–100
lM). At concentrations close to lethality
2.4. Conclusions
(10–50 M), in the range of concentrations for cyclopamine-in-
l
duced cyclopia, a strong delay in development was observed indi-
cating toxic effects (Fig. 5). The failure to induce the cyclopia
phenotype in the embryo is conflicting, as strong inhibitors of
Gli1 expression should be expected to induce this phenotype as
well. The relatively weak sensitivity of the medaka embryo assay
(ca. 10-fold less if compared to the IC50-values of cyclopamine)
does not explain this failure. Based on the IC50-values of the Gli1 re-
porter gene assay, induction of the cyclopia phenotype by SANT-2
derivatives should have been expected in the range of 100–
1000 nM. Differences in the uptake and distribution of cyclop-
amine and SANT-2 derivatives or reduced bioavailability due to
storage in the lipophilic yolk may have caused the failure to induce
cyclopia. However, injection of SANT-2 at the 1-cell stage into the
cytoplasm, the yolk or the perivitellin space did not cause cyclopia
as well (data not shown). Alternatively, additional unknown bioac-
tivities of SANT-2 and its derivatives that are not detected by the
reporter gene assay may overlap Shh inhibition and prevent the
manifestation of the cyclopia phenotype. Finally the different
We have shown that modifications of aryl moieties and partic-
ularly the benzimidazole core in SANT-2 reduce the ability of
Figure 5. Development of medaka embryos exposed to potential Shh inhibiting
compounds. Exposure to the Shh reference inhibitor cyclopamine induced the
cyclopia phenotype (indicated by a reduced distance of the eyes, cp). SANT-2 and its
derivatives did not induce cyclopia but provoked developmental delays shown by
smaller and less pigmented eyes. Blood hemorrhagia (bh) was also observed in
some cases. All images were recorded at 5 days post fertilization and are
representative examples of 15 embryos exposed per compound and concentrations.
Figure 6. Examples of concentration–response curves for cyclopamine (N = 2),
SANT-2 (N = 4) and the SANT-2 derivative TC132 (N = 4). Numbers refer to
independent experiments.
The scale bar represents 100 lm.

4948
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
Data were first normalised so that individual dose–response curves
converged to 0 and 100. Subsequently, these normalised data were
used to calculate the IC50 and corresponding SE and confidence
intervals based on 2–4 replicates. Examples for the calculation of
concentration–response curves are shown for cyclopamine, SANT-
2 and TC132 in Figure 6. Calculations were performed using the
software jmp (SAS Institute Inc., Cary, NC). IC50 with non-overlap-
ping confidence intervals (p < 0.05) were considered as significantly
different.
derivatives to inhibit the Shh signaling pathway. The chlorine sub-
stituent in SANT-2 is also important for activity. Exposure of meda-
ka embryos to SANT-2 and derivatives did not provoke the cyclopia
phenotype, although this effect could have been expected based on
the inhibition of Gli1 expression relative to the inhibition by
cyclopamine. The analysis of Gli1-inhibition and phenotypic effects
represent a powerful combination to identify potential Shh-inhib-
itors and the molecular descriptors of their biological activity. Both
assays provide an efficient way to identify potential drug candidate
compounds for the treatment of Shh-associated cancers. While
screening of Gli1-expression is principally able to identify Shh
antagonists, the embryo assay can be used to identify teratogenic,
toxic and unwanted side effects in a complex system and limit the
number of compounds subjected to further analysis.
4. Chemistry
4.1. General
All materials were obtained from commercial suppliers and
used without further purification. All anhydrous reactions were
performed under argon atmosphere. All reactions were monitored
by TLC (silica gel, GF254) with UV light and ninhydrin visualization.
Flash chromatography was performed on Merck Silica Gel 60. Melt-
ing points were uncorrected and were determined on a Büchi Melt-
ing Point B-540 apparatus. 1H and 13C NMR spectra were recorded
on Varian Gemini 2000, Varian Mercury plus 300 and Varian Mer-
cury plus 400 NMR spectrometers at room temperature. High-res-
olution (HR) mass spectra were obtained with a 7 T APEX II mass
spectrometer. Yields are not optimised.
3. Experimental
3.1. Biological tests
Cell assay inhibition of Gli1-expression was measured in Shh-
Light II cells (ATCC CRL-2795, LGC, Wesel, Germany) co-exposed
to 100 nM of the Hh agonist SAG as described by Beachy et al.9
Stock solutions of the potential inhibitors of Hh were prepared in
ethanol and introduced in different concentration into the culture
medium to reach a final solvent concentration of 0.1%. After treat-
ment for 48 h cells were washed with PBS. Harvest and lysis of the
cells, measurement of reporter gene and constitutive Renilla lumi-
nescence were performed with the Dual LuciferaseÒ reporter gene
system according to the manufacturer’s instructions (Promega,
Mannheim, Germany). Luminescence was recorded by using a
GENios reader (TECAN, Crailsheim, Germany). For determination
of IC50 values, the relative luminescence units per second RLU/s
(quotient of firefly and renilla luminescence) were plotted against
the inhibitor concentrations. Each assay was replicated at two to
four times (independent experiments) with three technical repli-
cates each.
Cl
O
N
H
NH2
NO2
4.1.1. N-(2-Aminophenyl)-2-chloro-5-nitrobenzamide (3)
To solution of 2-chloro-5-nitrobenzoic acid (4.03 g,
a
1
19.99 mmol) in dry CH2Cl2 (20 ml) was added oxalylchloride
(1.80 ml, 20.99 mmol), followed by two drops of DMF at 0 °C under
argon atmosphere. The resulting mixture was stirred at 0 °C for
30 min and then 1.5 h at room temperature. The solvent was evap-
orated in vacuo to give a yellow solid. The crude acyl chloride
(1.1 equiv) was added into an ice-cooled solution of 1,2-diamino-
benzene 2 (2.12 g, 19.60 mmol) and Et3N (4.13 ml, 29.40 mmol) in
anhydrous THF (80 ml). After stirring over night at room tempera-
ture the volatiles were removed under reduced pressure, and the
Analysis of constitutive Renilla luminescence was used to nor-
malise for any potential unspecific inhibition of Gli1-reporter gene
luminescence. Furthermore, microscopic observations of cells did
not indicate any toxic effects up to a concentration of 5
highest concentration tested for all compounds.
lM, the
Medaka cyclopia assay Medaka of the CAB strain (kindly pro-
vided by Professor J. Wittbrodt, EMBL, Heidelberg, Germany) were
cultured at 26 °C and a light cycle of 14:10 h light/dark. For breed-
ing, one female and one male were placed in 3 L tanks in a circula-
tion system. Eggs were collected within 1–2 h after fertilization by
removing them from the female’s belly. Fertilized eggs were ex-
posed to different inhibitor concentrations in local tap water (pH
8–8.2, water hardness 1.2–2.4 mM bivalent ions, conductivity
520e560 mS/cm) supplemented with 0.1% methylene blue. Com-
pounds were added by stock solutions in ethanol with final solvent
concentrations of 1%. Controls were treated with 1% ethanol only.
Exposure started at 4–5 hpf and was continued until 5 days post
fertilisation at 26 °C. Embryos were visually inspected for the
cyclopia phenotype (reduced eye distance). Images were recorded
with a digital consumer camera attached to a dissection micro-
scope (MZ 16F, Leica, Wetzlar, Germany).
residue was purified by silica gel FC (EtOAc/hexane, 3:2, v/v) to ob-
tain 3 as a yellow solid (3.89 g, 68%); mp = 170–171 °C; 1H NMR
(300 MHz, (CD3)2SO): d = 5.00 (s, 2H), 6.58 (dt, J = 7.5 Hz, 1.5 Hz,
1H), 6.76 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 6.98 (dt, J = 7.8 Hz, 1.5 Hz,
1H), 7.26 (dd, J = 7.8 Hz, 1.5 Hz, 1H), 7.85 (d, J = 8.7 Hz), 8.30 (dd,
J = 8.7 Hz, 2.7 Hz, 1H), 8.59 (d, J = 2.7 Hz, 1H), 9.89 (s, 1H); 13C
NMR (75 MHz, (CD3)2SO): d = 115.8, 116.0, 121.9, 124.1, 125.4,
126.2, 126.9, 131.1, 137.2, 138.0, 142.9, 146.1, 163.2; ESI-HRMS
m/z calcd for C13H10ClN3O3: 314.03029 [M+Na+], found: 314.02021.
Cl
H
N
N
3.2. Statistical analysis
O2N
Concentration–response curves of Gli1-reporter gene lumines-
cence were fitted with the Hill-slope equation:
4.1.2. 2-(2-Chloro-5-nitrophenyl)-1H-benzo[d]imidazole (4)
N-(2-Aminophenyl)-2-chloro-5-nitrobenzamide
3
(3.58 g,
12.26 mmol) was dissolved in glacial AcOH (130 ml) and refluxed
for 2 h. After treatment with ice-water (190 ml) the obtained
precipitate was washed with a small among of ice water. Again,
Max ꢀ Min
y ¼ Min þ
ꢀ
ꢁꢀp
x
IC50
1 þ

A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
4949
the filtrate was treated with ice (200 g) and after filtration the
combined precipitates were washed with a small among of ether
and dried over night. The crude material was purified by FC (hex-
CD3)2SO): d = 1.24 (t, J = 7.1 Hz, 3H), 1.35 (t, J = 6.9 Hz, 6H), 4.01
(q, J = 7.0 Hz, 2H), 4.10 (q, J = 7.1 Hz, 4H), 7.22–7.29 (m, 4H),
7.61–7.64 (m, 3H), 8.02 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 8.38 (d,
J = 2.7 Hz, 1H), 10.33 (br, 1H), 12.69 (br, 1H); 13C NMR (50 MHz,
CD3)2SO): d = 15.4, 16.2, 65.0, 68.8, 107.1, 112.6, 122.5 123.4,
124.3, 126.1, 129.8, 130.6, 131.2, 139.1, 140.9, 143.8, 149.6,
152.9, 165.7; ESI-HRMS m/z calcd for C26H27ClN3O4: 480.16901
[M+H+], found: 480.16825.
ane/EtOAc, 2:1 v/v, then gradient of hexane/acetone, 4:1–0:1, v/v)
and gave 4 as a yellow solid (2.71 g, 81%); mp = 198–199 °C; 1H
NMR (300 MHz, (CD3)2SO): d = 7.23 (dd, J = 6.0 Hz, 3.3 Hz, 2H),
7.65 (dd, J = 6.0 Hz, 3.3 Hz, 2H), 7.90 (d, J = 8.7 Hz, 1H), 8.28 (dd,
J = 8.7 Hz, 3.0 Hz, 1H), 8.74 (d, J = 2.7 Hz, 1H); 13C NMR (100 MHz,
(CD3)2SO): d = 115.8, 122.4, 125.0, 126.4, 131.2, 132.1, 138.1,
139.4, 146.3, 147,6; ESI-HRMS m/z calcd for C13H8ClN3O2:
569.05023 [2M+Na+], found: 569.05028.
O
NH
N
O
Cl
H
N
H
N
O
O
Cl
N
H2N
4.2.2. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-3,4,5-
trimethoxybenzamide (7, TC-132)
4.1.3. 3-(1H-Benzo[d]imidazol-2-yl)-4-chloroaniline (5)
White solid, yield 41%. Mp = 257–258 °C; 1H NMR (300 MHz,
(CD3)2SO): d = 3.73 (s, 3H), 3.87 (s, 6H), 7.22–7.25 (m, 2H), 7.32
(s, 2H), 7.62–7.65 (m, 3H), 8.03 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 8.38
(d, J = 2.7 Hz, 1H), 10.38 (s,1H), 12.69 (s, 1H); 13C NMR
(75 MHz, (CD3)2SO): d = 56.1, 60.1, 105.4, 122.7, 123.6, 125.4,
129.4, 129.8, 130.6, 138.3, 140.6, 148.9, 152.7, 165.0; ESI-HRMS
m/z calcd for C23H20ClN3O4: 438.12151 [M+H+], found:
438.12149.
To a solution of 2-(2-chloro-5-nitrophenyl)-1H-benzimidazole
4 (2.40 g, 8.77 mmol) in ethanol (31 ml) was added iron powder
(1.47 g, 26.28 mmol) and concd HCl (3.7 ml). The mixture was
heated to reflux under argon for 2 h. The cooled solution was di-
luted with EtOAc (120 ml) and water (120 ml), and the layers were
separated. The aqueous layer was washed with EtOAc (120 ml)
three times. The combined organic layers were filtered through
Celite, washing with water (100 ml) and satd K2CO3 solution
(100 ml) and dried over Na2SO4. The solvent was removed under
reduced pressure and the residue was purified by column chroma-
tography using EtOAc as eluent to furnish 5 as a brown solid
(1.62 g, 76%); mp = 209–210 °C; 1H NMR (300 MHz, (CD3)2SO):
d = 5.47 (s, 2H), 6.69 (dd, J = 8.7 Hz, 3.0 Hz, 1H); 7.11 (d,
J = 3.0 Hz, 1H), 7.19–7.22 (m, 3H), 7.54–7.63 (m, 2H), 12.50 (s,
1H); 13C NMR (50 MHz, (CD3)2SO): d = 115.4, 116.4, 116.6, 116.9,
122.0, 129.9, 130.5, 147.9, 149.9; ESI-HRMS m/z calcd for
C13H10ClN3: 244.06360 [M+H+], found: 244.06357.
O
NH
N
N
H
N
Cl
4.2.3. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-4-
(dimethylamino)benzamide (8)
4.2. General procedure for preparing benzimidazoles 6–15
Orange solid, yield 90%. Mp = 208–215 °C; 1H NMR (200 MHz,
(CD3)2SO): d = 2.96 (br, 6H), 6.73 (d, J = 9.2 Hz, 2H), 7.19–7.24 (m,
2H), 7.54–7.62 (m, 2H), 7.79–7.90 (m, 3H), 7.99 (dd, J = 9.2 Hz,
2.6 Hz, 1H), 8.40 (d, J = 2.6 Hz, 1H), 10.14 (s, 1H); 13C NMR
(50 MHz, (CD3)2SO): d = 40.3, 111.4, 111.8, 121.1, 123.0, 123.8,
125.4, 129.9, 130.5, 131.1, 132.7, 139.7, 149.8, 153.2, 154.8,
166.0; ESI-HRMS m/z calcd for C22H19ClN4O: 391.13202 [M+H+],
found: 391.13209.
Substituted benzoic acids (4.01 mmol) were dissolved in dry
CH2Cl2 (4 ml) and cooled to 0 °C under argon. Oxalylchloride
(0.4 ml, 4.21 mmol) and two drops of DMF were added and the
resulting mixture was stirred at 0 °C for 1.5 h and then 1 h at room
temperature. The solvent was evaporated in vacuo to give a solid
that was dried in vacuo. Part of the obtained crude acyl chloride
(1.73 mmol, 1.05 equiv) was dissolved in THF (5 ml), and added drop
wise into an ice-cooled solution of the corresponding aniline of type
5 (1.64 mmol) and Et3N (2.46 mmol) in anhydrous THF (5 ml). After
stirring an additional 1 h at 0 °C and 2.5 h at room temperature the
solvent was removed under reduced pressure, and the residue was
chromatographed on silica gel to obtain the corresponding anilides.
O
NH
N
N
H
Cl
O
N
NH
N
O
4.2.4. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-4-
cyanobenzamide (9)
N
H
Red solid, yield 61%. Mp = 225–227 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 7.25 (d, J = 4.8 Hz, 2H), 7.61–7.71 (m, 3H), 8.00 (dd,
J = 8.4 Hz, 2.8 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 8.15 (d, J = 8.4 Hz,
2H), 8.45 (d, J = 2.8 Hz, 1H), 10.77 (s, 1H), 12.72 (s, 1H); 13C NMR
(100 MHz, (CD3)2SO): d = 111.9, 114.1, 118.3, 119.0, 121.8, 122.7,
122.8, 123.5, 125.9, 128.6, 130.0, 130.7, 132.5, 134.8, 138.0,
138.4, 143.1 148.9, 164.3; ESI-HRMS m/z calcd for C21H13ClN4O:
373.08507 [M+H+], found: 373.08512.
O
O
Cl
4.2.1. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-3,4,5-
triethoxybenzamide (6, SANT-2)
SANT-2 was obtained as a white solid in 80% yield from 3,4,5-
triethoxybenzoic acid. Mp = 210–211 °C; 1H NMR (300 MHz,

4950
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
O
4.2.8. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-3-
(trifluoromethyl)benzamide (13)
NH
White solid, yield 57%. Mp = 223–224 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 7.22–7.26 (m, 2H), 7.59 (d, J = 7.2 Hz, 1H), 7.68 (d,
J = 9.2 Hz, 1H), 7.70 (d, J = 7.2 Hz, 1H), 7.79 (t, J = 7.6 Hz, 1H),
7.97–8.03 (m, 2H), 8.29 (d, J = 7.6 Hz, 1H), 8.34 (s, 1H), 8.42 (d,
J = 2.4 Hz, 1H), 10.74 (s, 1H); 12.71 (s, 1H); 13C NMR (100 MHz,
(CD3)2SO): d = 111.9, 119.0, 121.8, 122.8, 123.6, 124.3, 125.8,
128.5, 129.4, 129.9, 130.0, 130.7, 132.0, 134.8, 135.3, 138.1,
143.1, 148.9, 164.2; 19F NMR (376.4 MHz, CDCl3): d = ꢀ56.7 (s,
3F); ESI-HRMS m/z calcd for C21H13ClF3N3O: 416.07720 [M+H+],
found: 416.07756.
N
N
H
Cl
4.2.5. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-
benzamide (10)
White solid, yield 58%. Mp = 272 °C; 1H NMR (200 MHz,
(CD3)2SO): d = 7.20–7.24 (m, 2H), 7.48–7.63 (m, 6H), 8.0 (dd,
J = 7.8 Hz, 1.6 Hz, 3H), 8.44 (d, J = 2.8 Hz, 1H), 10.53 (s, 1H), 12.70
(s, 1H); 13C NMR (75 MHz, (CD3)2SO): d = 111.8, 119.0, 121.9,
122.6, 123.4, 125.5, 127.7, 128.4, 129.9, 130.5, 131.8, 134.4,
138.4, 143.0, 149.0, 165.7; ESI-HRMS m/z calcd for C20H14ClN3O:
348.08982 [M+H+], found: 348.08987.
O
NH
N
N
H
O
Cl
Cl
NH
N
4.2.9. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-4-
chlorobenzamide (14)
N
H
F
Cl
Yellow solid, yield 95%. Mp = 201–204 °C; 1H NMR (200 MHz,
(CD3)2SO): d = 7.22–7.28 (m, 2H), 7.57–7.73 (m, 5H), 8.00–8.05
(m, 3H), 8.42 (d, J = 3.0 Hz, 1H), 10.59 (s, 1H), 12.70 (br, 1H); 13C
NMR (100 MHz, (CD3)2SO): d = 111.8, 119.0, 121.8, 122.6, 122.8,
123.4, 125.6, 128.5, 129.7, 130.6, 133.1, 134.7, 136.7, 138.2,
143.1, 148.9, 164.6; ESI-HRMS m/z calcd for C20H13Cl2N3O:
382.05084 [M+H+], found: 382.05058.
4.2.6. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-4-
fluorobenzamide (11)
White solid, yield 94%. Mp = 241 °C; 1H NMR (300 MHz,
(CD3)2SO): d = 7.23–7.24 (m, 2H), 7.37 (t, J = 8.7 Hz, 2H), 7.58–
7.63 (m, 2H), 7.70 (d, J = 6.6 Hz, 1H), 7.99 (dd, J = 8.7 Hz, 2.4 Hz,
1H), 8.08 (dd, J = 8.7 Hz, 5.7 Hz, 2H), 8.44 (d, J = 2.4 Hz, 1H), 10.55
(s, 1H), 12.70 (s, 1H); 13C NMR (100 MHz, (CD3)2SO): d = 115.3,
115.5, 111.8/119.0 (d, J = 720 Hz), 121.8, 122.6, 122.8, 123.4,
125.5, 129.9, 130.4, 130.5/130.6 (d, J = 1.5 Hz), 130.8/130.9 (d,
J = 3.0 Hz), 134.8, 138.3, 143.1, 149.0, 164.6, 163.0/165.5 (d,
J = 247.7 Hz); ESI-HRMS m/z calcd for C20H13ClFN3O: 366.08039
[M+H+], found: 366.08042.
O
NH
N
N
H
H3CO
Cl
O
4.2.10. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-4-
methoxybenzamide (15)
O
NH
N
Orange solid, yield 88%. Mp = 188–191 °C; 1H NMR (300 MHz,
(CD3)2SO): d = 3.99 (s, 3H), 7.06 (d, J = 8.7 Hz, 2H), 7.21–7.24 (m,
2H), 7.56–7.72 (m, 4H), 7.96–8.00 (m, 2H), 8.41 (d, J = 2.7 Hz,
1H), 10.37 (s, 1H), 12.67 (br, 1H); 13C NMR (100 MHz, (CD3)2SO):
d = 55.5, 113.7, 114.1, 113.7, 122.6, 123.3, 125.2, 126.2, 129.7,
129.9, 130.5, 138.6, 142.2, 149.1, 162.2, 165.1; ESI-HRMS m/z calcd
for C21H16ClN3O2: 378.10038 [M+H+], found: 378.10033.
N
H
Cl
4.2.7. N-(3-(1H-Benzo[d]imidazol-2-yl)-4-chlorophenyl)-2-
methoxybenzamide (12)
White solid, yield 84%. Mp = 181–182 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 3.89 (s, 3H), 7.06 (t, J = 7.6 Hz, 1H), 7.17 (d,
J = 8 Hz, 1H), 7.23–7.24 (m, 2H), 7.48–7.52 (m, 1H), 7.59–7.64 (m,
4H), 7.90 (dd, J = 8.8 Hz, 2.4 Hz, 1H), 8.36 (d, J = 2.4 Hz, 1H), 10.39
(s, 1H), 12.72 (s, 1H); 13C NMR (100 MHz, (CD3)2SO): d = 55.9,
111.7, 112.0, 119.1, 120.4, 121.7, 122.1, 122.7, 124.7, 125.4,
129.6, 130.1, 130.5, 132.2, 138.2, 149.0, 156.5, 164.9, 170.3; ESI-
HRMS m/z calcd for C21H16ClN3O2: 378.10038 [M+Na+], found:
400.09233.
N
N
H
NO2
4.2.11. 2-(3-Nitrophenyl)-1H-benzo[d]imidazole (17)
To a stirred solution of diaminobenzene (1.26 g, 11.7 mmol) in
THF (50 ml) triethyl amine (2.50 ml, 17.5 mmol) and 3-nitro-
benzoyl chloride (2.27 g, 12.2 mmol) were added at 0 °C. The solu-
tion was stirred for 15 h at rt. The solvent was removed under
reduced pressure and the crude intermediate product was purified
O
NH
by column chromatography (n-hexane/ethyl acetate 1:1 v/v) yield-
N
ing the intermediate amide (1.29 g, 5.0 mmol) as a yellow solid. It
was dissolved in acetic acid (45 ml) and refluxed for 2 h. After cool-
ing down to rt the solution was poured on ice and extracted with
dichloromethane (3 ꢁ 50 ml). The organic phases were dried over
F3C
N
H
Cl

A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
4951
sodium sulphate and 17 was obtained as a orange solid (1.21 g,
43%); mp = 184–187 °C; 1H NMR (400 MHz, (CD3)2SO): d = 7.15
(d, J = 7.2 Hz, 1H), 7.31–7.33 (m, 2H), 7.67–7.70 (m, 2H) 7.78–
7.82 (m, 1H), 8.37–8.40 (m, 2H), 10.35 (br, 1H); 13C NMR
(75 MHz, (CD3)2SO): d = 116.0, 116.1, 122.6, 125.9, 126.2, 130.0,
130.2, 134.3, 136.1, 147.7, 163.6; ESI-HRMS m/z calcd for
C13H9N3O2: 240,07675 [M+H+], found: 240.07677.
and dried over sodium sulfate. The solvent was removed under
reduced pressure. The crude product was purified by column chro-
matography (hexane/EtOAc 5:1 v/v) yielding 28 (2.06 g, 99%) as a
yellow solid; mp = 170–172 °C; 1H NMR (400 MHz, (CD3)2SO):
d = 7.45–7.54 (m, 2H), 7.85–7.91 (m, 2H), 7.99 (d, J = 8.8 Hz, 1H),
8.39 (dd, J = 8.8 Hz, 2.8 Hz, 1H), 8.87 (d, J = 2.8 Hz, 1H); 13C NMR
(100 MHz, (CD3)2SO): d = 111.3, 120.5, 125.3, 126.3, 126.6, 126.8,
133.1, 138.7, 138.7, 140.9, 146.4, 150.0, 158.2; ESI-HRMS m/z calcd
for C13H8ClN2O3: 275.02234 [M+H+], found: 275.02180.
Cl
N
4.3. General procedure for preparing benzamines 18, 23, 29
S
NO2
The procedure is described for 18 (3-(1H-benzo[d]imidazol-2-
yl)benzenamine). A suspension containing 2-(3-nitrophenyl)-1H-
benzo[d]imidazole (1.15 g, 4.81 mmol), iron (0.81 g, 14.4 mmol)
and concd HCl (2.0 ml) in ethanol (17 ml) was refluxed for 3 h.
After cooling down to rt the mixture was diluted with EtOAc
(30 ml) and washed with water (20 ml) and brine (20 ml). The or-
ganic phase was dried over Na2SO4. After removing the solvent un-
der reduced pressure the crude product was chromatographed on
silica gel to obtain 18 (758 mg, 75%) as a grey solid.
4.2.12. 2-(2-Chloro-5-nitrophenyl)benzo[d]thiazole (22)
A solution of 2-aminothiophenol 20 (1.0 ml, 9.27 mmol), 2-
chloro-5-nitrobenzaldehyde 21 (1.72 g, 9.27 mmol) and iodine
(1.18 g, 4.63 mmol) in DMF (2.5 ml) was stirred at rt for 15 h.
The solvent was removed under reduced pressure. The crude prod-
uct was purified by column chromatography (hexane/EtOAc 3:1 v/
v
) yielding 22 (2.0 g, 74%) as a yellow solid; mp = 106 °C; 1H NMR
(400 MHz, (CD3)2SO): d = 6.83–6.85 (m, 1H), 6.94–6.98 (m, 1H),
7.01–7.03 (m, 1H), 7.16 (d, J = 3.2 Hz, 1H), 7.80 (d, J = 8.8 Hz, 1H),
8.16 (dd, J = 8.4 Hz, 2.8 Hz, 1H,), 8.31 (d, J = 3.2 Hz, 1H); 13C NMR
(100 MHz, (CD3)2SO): d = 119.9, 121.4, 121.7, 123.5, 124.3, 126.0,
131.4, 137.3, 137.9, 143.1, 147.0, 151.6, 161.3; ESI-HRMS m/z calcd
for C13H8ClN2O2S: 290.99950 [M+H+], found: 290.99895.
N
N
H
NH2
4.3.1. 3-(1H-Benzo[d]imidazol-2-yl)benzenamine (18)
Grey solid, yield 75%. Mp = 247–249 °C; 1H NMR (200 MHz,
(CD3)2SO): d = 5.27 (br, 2H), 6.65 (d, J = 7.8 Hz, 1H), 7.10–7.28 (m,
4H), 7.40–7.46 (m, 2H), 7.59 (d, J = 8.2 Hz, 1H), 12.68 (br, 1H);
13C NMR (50 MHz, (CD3)2SO): d = 111.1, 111.9, 114.0, 115.5,
118.7, 121.4, 122.2, 129.3, 130.7, 134.9, 143.8, 149.1, 152.1; ESI-
HRMS m/z calcd for C13H12N3: 210.10312 [M+H+], found:
210.10257.
Cl
O
NH
NO2
OH
4.2.13. 2-Chloro-N-(2-hydroxyphenyl)-5-nitrobenzamide (27)
A solution of 2-aminophenol 25 (4.0 g, 19.8 mmol), 2-chloro-5-
nitrobenzoyl chloride 26 (4.4 g, 19.8 mmol), and triethyl amine
(3.98 ml, 28.4 mmol) in THF (8.0 ml) was stirred at rt for 15 h.
The solvent was removed under reduced pressure. The crude
product was purified by column chromatography (hexane/EtOAc
Cl
N
S
NH2
1:1, v/v) yielding 27 (5.09 g, 88%) as a yellow solid; mp = 190–
192 °C; 1H NMR (300 MHz, (CD3)2SO): d = 6.80–6.85 (m, 1H),
6.90 (m, 1H), 7.00–7.06 (m, 1H), 7.79–7.84 (m, 2H), 8.29 (dd,
J = 8.9 Hz, 2.6 Hz, 1H), 8.42 (d, J = 2.7 Hz, 1H), 9.78 (br, 1H), 9.93
(br, 1H); 13C NMR (75 MHz, (CD3)2SO): d = 115.8, 119.0, 123.8,
124.2, 125.2, 125.5, 125.9, 131.2, 137.3, 137.7, 146.0, 149.1,
163.2; ESI-HRMS m/z calcd for C13H10ClN2O4: 293.03291 [M+H+],
found: 293.03236.
4.3.2. 3-(Benzo[d]thiazol-2-yl)-4-chlorobenzenamine (23)
Yellow solid, yield 57%. Mp = 118–120 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 5.55 (br, 2H), 6.75 (dd, J = 8.6 Hz, 2.6 Hz, 1H), 7.26
(d, J = 8.8 Hz, 1H), 7.45–7.49 (m, 2H), 7.53–7.57 (m, 1H), 8.07 (d,
J = 8.4 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H); 13C NMR (100 MHz,
(CD3)2SO): d = 115.6, 117.0, 117.5, 122.0, 122.9, 125.5, 126.5,
131.1, 131.3, 135.3, 148.2, 152.0, 164.2; ESI-HRMS m/z calcd for
C13H10ClN2S: 261.02532 [M+H+], found: 261.02477.
Cl
N
O
Cl
N
NO2
O
4.2.14. 2-(2-Chloro-5-nitrophenyl)benzo[d]oxazole (28)
NH2
A solution of 2-chloro-N-(2-hydroxyphenyl)-5-nitrobenzamide
27 (2.20 g, 7.52 mmol, see above) and p-toluenesulfonic acid
monohydrate (3.06 g, 16.1 mmol) in o-xylole (75 ml) was refluxed
for 4 h. After cooling down to rt the solution was diluted with
EtOAc (70 ml), washed with NaHCO3 (2 ꢁ 60 ml, half-concen-
trated) and the aqueous phase reextracted with EtOAc (30 ml).
The combined organic phases were washed with brine (70 ml)
4.3.3. 3-(Benzo[d]oxazol-2-yl)-4-chlorobenzenamine (29)
Yellow solid, yield 96%. Mp = 138–139 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 5.62 (br, 2H), 6.78 (dd, J = 8.4 Hz, 2.8 Hz, 1H), 7.27
(d, J = 8.8 Hz, 1H), 7.35 (d, J = 2.8 Hz, 1H), 7.35–7.46 (m, 2H), 7.77
(dd, J = 7.0 Hz, 2.2 Hz, 1H), 7.83 (dd, J = 7.0 Hz, 2.5 Hz, 1H); 13C
NMR (100 MHz, (CD3)2SO): d = 110.9, 115.9, 117.5, 118.0, 120.0,

4952
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
124.8, 125.3, 125.7, 131.5, 141.1, 148.1, 149.9, 160.9; ESI-HRMS m/
z calcd for C13H10ClN2O: 245.04817 [M+H+], found: 245.04762.
(d, J = 2.4 Hz, 1H), 8.69 (s, 1H), 10.43 (br, 1H); 13C NMR
(100 MHz, CD3)2SO): d = 14.7, 15.4, 64.3, 68.1, 106.4, 111.0, 120.2,
122.9, 124.2, 125.1, 125.2, 126.1, 129.0, 131.7, 138.6, 140.2,
141.0, 150.0, 152.2, 160.0, 165.1; ESI-HRMS m/z calcd for
C26H26ClN2O5: 481.15302 [M+H+], found: 481.15248.
4.4. General procedure for preparing benzimidazoles 19, 24, 30
The procedure is described for N-(3-(1H-benzo[d]imidazol-2-
Cl
N
yl)phenyl)-3,4,5-triethoxybenzamide 19.
A solution of 3-(1H-
benzo[d]imidazol-2-yl)benzenamine 18 (280 mg, 1.34 mmol),
3,4,5-triethoxybenzoyl chloride (383 mg, 1.41 mmol, 1.05 equiv),
DMAP (17 mg, 0.14 mmol) and triethyl amine (0.28 ml, 2.01 mmol)
in dry THF (8.0 ml) was stirred at rt for 15 h. The solvent was re-
moved under reduced pressure. The crude product was chromato-
graphed on silica gel yielding 18 (474 mg, 79%) as a white solid.
N
H
NO2
4.4.4. 2-(2-Chloro-5-nitrophenyl)-4,5-dihydro-1H-imidazole
O
(32)
NH
H
To
a solution of 2-Chloro-5-nitrobenzaldehyde 21 (2.56 g,
N
13.78 mmol) in t-BuOH (138 ml) was added ethylendiamine
(0.91 g, 15.16 mmol). The obtained mixture was stirred at rt under
an argon atmosphere for 30 min, and then K2CO3 (5.71 g,
41.33 mmol) and iodine (4.37 g, 17.22 mmol) were added to the mix-
ture and stirred at 70 °C. After 3 h, the mixture was quenched with
satd Na2SO3 solution until iodine colour almost disappeared, and
was extracted with CHCl3 (3 ꢁ 150 ml). The organic layer was
washed with satd NaHCO3 solution and brine, and dried over Na2SO4.
After filtration the mixture was evaporated in vacuo and purified by
silica gel FC (MeOH/EtOAc 1:1 v/v) to give 32 (3.0 g, 99%) as an orange
solid; mp = 123–124 °C; 1H NMR (300 MHz, CDCl3): d = 3.80 (s, 4H),
5.10 (br, 1H), 7.55 (d, J = 9 Hz, 1H), 8.15 (dd, J = 8.7 Hz, 2.7 Hz, 1H),
8.56 (d, J = 2.7 Hz, 1H); 13C NMR (50 MHz, CDCl3): d = 50.6, 125.5,
126.6, 131.6, 132.1, 139.0, 146.5, 161.7; ESI-HRMS m/z calcd for
C9H8ClN3O2: 226.03778 [M+H+], found: 226.03757.
O
N
O
O
4.4.1. N-(3-(1H-Benzo[d]imidazol-2-yl)phenyl)-3,4,5-
triethoxybenzamide (19)
White solid, yield 79%. Mp = 201–202 °C; 1H NMR (200 MHz,
(CD3)2SO): d = 1.24 (t, J = 7.0 Hz, 3H), 1.35 (t, J = 6.9 Hz, 6H), 3.94–
4.16 (m, 6H), 7.15–7.18 (m, 2H), 7.31 (s, 2H), 7.45–7.65 (m, 3H),
7.83–7.93 (m, 2H), 8.61 (s, 1H), 10.23 (br, 1H), 12.89 (br, 1H); 13C
NMR (50 MHz, (CD3)2SO): d = 14.7, 15.4, 64.2, 68.0, 106.3, 111.3,
118.8, 119.0, 121.4, 121.6, 121.9, 122.4, 129.1, 129.3, 130.5,
135.0, 139.7, 140.0, 143.8, 151.1, 152.2, 164.8; ESI-HRMS m/z calcd
for C26H28N3O4: 446.20798 [M+H+], found: 446.20743.
O
Cl
N
NH
N
H
S
O
N
O
O
Cl
NO2
4.4.5. 2-(2-Chloro-5-nitrophenyl)-1H-imidazole (35)
4.4.2. N-(3-(Benzo[d]thiazol-2-yl)-4-chlorophenyl)-3,4,5-
triethoxybenzamide (24)
To a mixture of 2-(2-Chloro-5-nitrophenyl)-4,5-dihydro-1H-
imidazole 33 (0.82 g, 3.64 mmol) and K2CO3 (0.55 g, 4.0 mmol) in
DMSO (36 ml) was added DIB (1.29 g, 4.0 mmol). Then the mixture
was stirred for 3 days at rt under argon. Then, the reaction mixture
was diluted with satd NaHCO3 solution (20 ml) and EtOAc (15 ml),
and was stirred for 5 min. After extraction with EtOAc (3 ꢁ 80 ml)
the organic layer was dried over Na2SO4, concentrated and purified
Yellow solid, yield 77%. Mp = 171–174 °C; 1H NMR (300 MHz,
(CD3)2SO): d = 1.24 (t, J = 7.1 Hz, 3H), 1.35 (t, J = 6.9 Hz, 6H), 4.01
(q, J = 7.0 Hz, 2H), 4.11 (q, J = 7.0 Hz, 4H), 7.30 (s, 2H), 7.48–7.54
(m, 1H), 7.56–7.62 (m, 1H), 7.67 (d, J = 9.0 Hz, 1H), 8.06 (dd,
J = 9.0 Hz, 2.7 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.18–8.21 (m, 1H),
8.68 (d, J = 2.4 Hz, 1H), 10.40 (br, 1H); 13C NMR (75 MHz,
(CD3)2SO): d = 14.7, 15.4, 64.3, 68.0, 106.4, 122.2, 122.7, 123.0,
123.7, 125.4, 125.8, 126.8, 129.0, 131.2, 135.4, 138.7, 140.2,
151.8, 152.2, 163.2, 165.1; ESI-HRMS m/z calcd for C26H26ClN2O4S:
497.13018 [M+H+], found: 497.12963.
by FC (hexane/EtOAc 4:1–2:1 + 1% amine, v/v) to give 35 (0.54 g,
66%) as a yellow solid; mp = 170–172 °C; 1H NMR (400 MHz,
CDCl3): d = 7.27 (d, J = 10.8 Hz, 2H), 7.61 (d, J = 8.8 Hz, 1H), 8.11
(dd, J = 9.2 Hz, 2.8 Hz, 1H), 9.19 (d, J = 2.8 Hz, 1H), 10.37 (br, 1H);
13C NMR (100 MHz, CDCl3): d = 123.5, 125.9, 127.3, 130.0, 131.8,
135.5, 141.9, 147.3; ESI-HRMS m/z calcd for C9H6ClN3O2:
224.022013 [M+H+], found: 224.02201.
O
NH
O
O
N
Cl
O
O
Cl
N
H
4.4.3. N-(3-(Benzo[d]oxazol-2-yl)-4-chlorophenyl)-3,4,5-
triethoxybenzamide (30)
NO2
White solid, yield 82%. Mp = 191–193 °C; 1H NMR (400 MHz,
CD3)2SO): d = 1.28 (t, J = 7.2 Hz, 3H), 1.39 (t, J = 6.8 Hz, 6H), 4.03
(q, J = 6.8 Hz, 2H), 4.14 (q, J = 6.9 Hz, 4H), 7.32 (s, 2H), 7.48–7.52
(m, 2H), 7.71 (d, J = 8.8 Hz, 1H), 8.08 (d, J = 2.8 Hz, 1H), 8.10
4.4.6. 2-(2-Chloro-5-nitrophenyl)-1H-indole (40)
2-Chloro-5-nitroacetophenone 39 (0.50 g, 2.51 mmol) and
phenylhydrazine hydrochloride 38 (0.54 g, 3.758 mmol) were

A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
4953
mixed with EtOH (2.5 ml) and two drops of glacial AcOH and
4.6. General procedure for preparing benzamides 34, 37, 42
heated slowly to 80 °C and the mixture was stirred over night at
this temperature. The solvent was evaporated to give a yellow so-
lid. PPA (1.5 ml) was added and the mixture was slowly heated to
120 °C, stirred for 15 min and poured on ice and neutralised with
2 M NaOH (5 ml). The obtained precipitate was filtrated to give
40 (0.68 g, 100%) as a brown solid in an almost pure state; 1H
NMR (300 MHz, (CD3)2SO): d = 7.05 (t, J = 7.4 Hz, 1H), 7.11 (d,
J = 2.1 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.62
(d, J = 8.1 Hz, 1H), 7.89 (d, J = 8.7 Hz, 1H), 8.17 (dd, J = 8.7 Hz,
2.7 Hz, 1H), 8.56 (d, J = 2.4 Hz, 1H), 11.74 (s, 1H); 13C NMR
(75 MHz, (CD3)2SO): d = 104.7, 11.6, 119.7, 120.8, 122.8, 123.0,
124.5, 127.8, 131.9, 132.2, 136.7, 137.4, 146.6; ESI-HRMS m/z calcd
for C14H9ClN2O2: 273.04253 [M+H+], found: 273.04256.
The procedure is described for 37 (KS79). 3,4,5-triethoxybenzoic
acid (139 mg, 0.55 mmol) was dissolved in dry CH2Cl2 (1 ml) and
cooled to 0 °C under argon. Oxalylchloride (0.05 ml, 0.57 mmol) and
two drops of DMF were added and the resulting mixture was stirred
at 0 °C for 1.5 h and then 2 h at room temperature. The solvent was
evaporated in vacuo to give a solid that was dried in vacuo. The crude
acyl chloride (129 mg, 0.47 mmol, 0.95 equiv) was dissolved in CH2Cl2
(2 ml), and added drop wise into an ice-cooled solution of 4-chloro-3-
(1H-imidazol-2-yl)benzamine (36) (96 mg, 0.50 mmol) and Et3N
(0.1 ml, 0.74 mmol) in anhydrous CH2Cl2 (2 ml). After stirring addi-
tional 15 min at 0 °C and at room temperature over night the solvent
was removed under reduced pressure, and the residue was chromato-
graphed on silica gel to obtain 37 as a yellow oil (36 mg, 18%).
4.5. General procedure for preparing benzamines 33, 36, 41
O
The procedure is described for 36. A suspension containing 2-(2-
chloro-5-nitrophenyl)-1H-imidazole 35 (142 mg, 0.64 mmol), iron
(106 mg, 1.89 mmol) and concd HCl (0.27 ml) in ethanol (3 ml) was
refluxedfor 3 h. Aftercoolingdowntortthe mixturewasdilutedwith
EtOAc (9 ml) and washed with water (9 ml) and satd NaHCO3 solu-
tion (9 ml). The aqueous layer was washed with EtOAc (3 ꢁ 9 ml)
and the organic phase was dried over Na2SO4. After removing the sol-
vent under reduced pressure the crude product was chromato-
graphed on silica gel to obtain 36 (110 mg, 89%) as a yellow oil.
NH
H
N
O
N
O
O
Cl
4.6.1. N-(4-Chloro-3-(1H-imidazol-2-yl)phenyl)-3,4,5-
triethoxybenzamide (37)
Compound 37 was obtained as a yellow oil in 20% yield from 4-
chloro-3-(1H-imidazol-2-yl)benzamine 36. 1H NMR (300 MHz,
(CD3)2SO): d = 1.24 (t, J = 7.2 Hz, 3H), 1.35 (t, J = 6.9 Hz, 6H), 4.00
(q, J = 7.2 Hz, 2H), 4.10 (q, J = 6.6 Hz, 4H), 7.06–7.25 (m, 4H), 7.51
(d, J = 8.7 Hz, 1H), 7.87 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 8.18 (d,
J = 2.7 Hz, 1H), 10.23 (s, 1H), 12.24 (br, 1H); 13C NMR (50 MHz,
(CD3)2SO): d = 14.8, 15.5, 64.3, 68.1, 106.4, 107.5, 121.6, 122.8,
129.2, 130.3, 138.2, 142.8, 152.2, 165.0; ESI-HRMS m/z calcd for
C22H24ClN3O4: 430.15281 [M+H+], found: 430.15286.
Cl
N
N
H
NH2
4.5.1. 4-Chloro-3-(1H-imidazol-2-yl)benzamine (36)
Yellow oil, yield 89%. 1H NMR (300 MHz, (CD3)2SO): d = 5.33 (s,
2H), 6.56 (dd, J = 8.4 Hz, 2.7 Hz, 1H), 6.96–7.17 (m, 4H), 12.03 (br,
1H); 13C NMR (75 MHz, (CD3)2SO): d = 115.3, 115.8, 116.4, 117.4,
123.5, 130.2, 143.6, 145.7, 147.7; ESI-HRMS m/z calcd for C9H8ClN3:
194.04795 [M+H+], found: 194.04806.
Cl
O
O
N
N
H
Cl
N
HN
O
N
O
H
NH2
4.6.2. N-(4-Chloro-3-(4,5-dihydro-1H-imidazol-2-yl)phenyl)-
3,4,5-triethoxybenzamide (34)
4.5.2. 4-Chloro-3-(4,5-dihydro-1H-imidazol-2-yl)benzamine (33)
Colourless oil, yield 50%. 1H NMR (200 MHz, (CD3)2SO): d = 3.13 (s,
4H), 5.30 (s, 2H), 6.54 (dd, J = 8.8 Hz, 2.6 Hz, 1H), 6.70 (d, J = 2.4 Hz,
1H), 7.02 (d, J = 8.8 Hz, 1H); 13C NMR (100 MHz, (CD3)2SO): d = 48.6,
115.4, 115.8, 116.8, 129.9, 131.7, 147.4, 163.4; ESI-HRMS m/z calcd
for C9H10ClN3: 196.06360 [M+H+], found: 196.06353.
Yellow oil, yield 81%. 1H NMR (400 MHz, CD3)2SO): d = 1.15 (t,
J = 7.1 Hz, 3H), 1.29 (t, J = 6.9 Hz, 6H), 3.80–3.98 (m, 10H), 6.33
(dd, J = 8.7 Hz, 2.7 Hz, 1H), 6.55 (d, J = 2.7 Hz, 1H), 6.57 (s, 2H),
6.69 (d, J = 8.7 Hz, 1H); 13C NMR (75 MHz, (CD3)2SO): d = 14.8,
15.4, 48.5, 52.9, 64.0, 67.6, 106.8, 115.7, 115.9, 116.5, 128.9,
129.7, 131.2, 138.7, 147.2, 151.3, 156.8, 167.3; ESI-HRMS m/z calcd
for C22H26ClN3O4: 432.16846 [M+H+], found: 432.16861.
Cl
N
H
O
NH
NH2
O
4.5.3. 4-Chloro-3-(1H-indol-2-yl)benzamine (41)
N
H
Yellow oil, yield 80%. 1H NMR (300 MHz, (CD3)2SO): d = 5.36 (s,
2H), 6.59 (dd, J = 8.7 Hz, 2.7 Hz, 1H), 6.70 (d, J = 2.1 Hz, 1H), 6.86 (d,
J = 2.7 Hz, 1H), 7.00 (t, J = 7.5 Hz, 1H), 7.10 (t, J = 8.1 Hz, 1H), 7.18
(d, J = 8.7 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H),
11.23 (s, 1H); 13C NMR (100 MHz, (CD3)2SO): d = 102.2, 11.3,
114.7, 115.5, 117.1, 119.1, 120.0, 121.4, 127.9, 130.6, 131.5,
135.4, 136.3, 147.8; ESI-HRMS m/z calcd for C14H11ClN2:
243.06835 [M+H+], found: 243.06815.
O
O
Cl
4.6.3. N-(4-Chloro-3-(1H-indol-2-yl)phenyl)-3,4,5-
triethoxybenzamide (42)
Colourless oil, yield 90%. 1H NMR (300 MHz, (CD3)2SO): d = 1.24
(t, J = 7.2 Hz, 3H), 1.35 (t, J = 6.9 Hz, 6H), 4.01 (q, J = 7.2 Hz, 2H),

4954
A. Büttner et al. / Bioorg. Med. Chem. 17 (2009) 4943–4954
9. Di Magliano, M. P.; Hebrok, M. Nat. Rev. Cancer 2003, 3, 903; Rubin, L. L.; de
Sauvage, F. J. Nat. Rev. Cancer 2006, 5, 1026.
10. Cooper, M. K.; Porter, J. A.; Young, K. E.; Beachy, P. A. Science 1998, 280, 1603.
11. Jain Rishi, K.; Kelleher, J., III; Peukert, S.; Sun, Y. PCT Int. Appl., 2007; WO
2007120827 A2 20071025.
12. Williams, J. A.; Guicherit, O. M.; Zaharian, B. I.; Xu, Y.; Chai, L.; Wichterle, H.;
Kon, C.; Gatchalian, C.; Porter, J. A.; Rubin, L. L.; Wang, F. Y. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 4616.
13. Chen, J. K.; Taipale, J.; Young, K. E.; Maiti, T.; Beachy, P. A. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 14071.
4.10 (q, J = 6.6 Hz, 4H), 6.81 (d, J = 1.5 Hz, 1H), 7.02 (t, J = 7.4 Hz,
1H), 7.12 (t, J = 7.4 Hz, 1H), 7.26 (s, 1H), 7.42 (d, J = 7.8 Hz, 1H),
7.55–7.59 (m, 2H), 7.76 (dd, J = 8.7 Hz, 2.4 H, 1H), 8.12 (d,
J = 2.4 Hz, 1H); 13C NMR (75 MHz, (CD3)2SO): d = 14.8, 15.5, 64.3,
68.1, 102.9, 106.4, 111.5, 119.4, 120.3, 121.1, 121.9, 122.6, 125.4,
127.9, 129.3, 130.6, 131.5, 134.5, 136.6, 138.3, 140.1, 152.2,
165.0; ESI-HRMS m/z calcd for C27H27ClN2O4: 479.17321 [M+H+],
found: 479.17287.
14. (a) Guicherit, O. M.; Boyd, E. A.; Brunton, S .A.; Price, S.; Stibbard, J. H. A.;
MacKinnon, C. H. PCT Int. Appl., 2006; WO 2006050506 A1 20060511.; (b)
Beachy, P. A.; Chen, J. K.; Taipale, A. J. N. PCT Int. Appl., 2003; WO 2003088970
A2 20031030.
References and notes
15. Lee, J.; Wu, X.; Di Magliano, M. P.; Peters, E. C.; Wang, Y.; Hong, J.; Hebrok, M.;
Ding, S.; Cho, C. Y.; Schultz, P. G. ChemBioChem 2007, 8, 1916.
16. Lauth, M.; Berström, A.; Shimokawa, T.; Toftgard, R. Proc. Natl. Acad. Sci. U.S.A.
2007, 104, 8455.
17. Wang, Y.; Zhouc, Z.; Walsh, C. T.; McMahon, A. P. Proc. Natl. Acad. Sci. U.S.A.
2009, 106, 2623.
1. (a) Taipale, J.; Beachy, P. A. Nature 2001, 411, 349; (b) Ingham, P. W.; McMahon,
A. P. Gene Dev. 2001, 15, 3059.
2. (a) Porter, J. A.; Young, K. E.; Beachy, P. A. Science 1996, 274, 255; (b) Porter, J.
A.; Ekker, S. C.; Park, W. J.; von Kessler, D. P.; Young, K. E.; Chen, C. H.; Ma, Y.;
Woods, A. S.; Cotter, R. J.; Koonin, E. V.; Beachy, P. A. Cell 1996, 86, 21; Mann, R.
K.; Beachy, P. A. Annu. Rev. Biochem. 2004, 73, 891.
18. (a) Ferrari, S.; Yanega, R. Effect of Cyclopamine on Medaka (Oryzias latipes)
embryos. Course Experiment, Developmental Biology, Franklin&Marshall-
College. 2000. http://www.swarthmore.edu/NatSci/sgilber1/DB_lab/Student/
Medaka_cyclo.html.; (b) Loucks, E.; Schwend, T.; Ahlgren, S. Birth Defects
Research Part A: Clinical and Molecular Teratology, 2007.
19. Chiang, C.; Litingtung, Y.; Lee, E.; Young, K. E.; Corden, J. L.; Westphal, H.;
Beachy, P. A. Nature 1996, 383, 407.
20. Gong, B.; Hong, F.; Kohm, C.; Bonham, L.; Klein, P. Bioorg. Med. Chem. Lett. 2004,
14, 1455.
3. Aikin, R. A.; Ayers, K. L.; Therond, P. P. EMBO Rep. 2008, 9, 330.
4. (a) Huangfu, D.; Anderson, K. V. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 11325; (b)
Rohatgi, R.; Milenkovic, L.; Scott, M. P. Science 2007, 317, 372.
5. Fukushima, N.; Walter, K. M.; Ueko, T.; Sato, N.; Matsubayashi, H.; Cameron, J.
L.; Hruban, R. H.; Canto, M.; Yeo, C. J.; Goggins, M. Cancer Biol. Ther. 2003, 2, 78.
6. Karhadkar, S. S.; Bova, G. S.; Abdallah, N.; Dhara, S.; Gardner, D.; Maitra, A.;
Isaacs, J. T.; Berman, D. M.; Beachy, P. A. Nature 2004, 431, 707.
7. Berman, D. M.; Karhadkar, S. S.; Maitra, A.; Montes De Oca, R.; Gerstenblith, M.
R.; Briggs, K.; Parker, A. R.; Shimada, Y.; Eshleman, J. R.; Watkins, D. N.; Beachy,
P. A. Nature 2003, 425, 846.
21. (a) Keeler, R. F.; Binns, W. Teratology 1968, 1, 5; (b) Incardona, J.; Gaffield, W.;
Kapur, R.; Roelink, H. Development 1998, 125, 3553.
8. Corcoran, R. B.; Scott, M. P. J. Neurooncol. 2001, 53, 307.