Synthesis and biological evaluation of novel benzamide derivatives as potent smoothened antagonists

Article abstract of DOI:10.1016/j.bmcl.2014.01.006

A series of novel benzamide derivatives were prepared and evaluated using cell-based measurements. Among these compounds, 10f significantly inhibited Hedgehog signaling and showed equivalent or more potency than GDC-0449 in different tests. Furthermore, compound 10f potently inhibited the proliferation of Daoy, a medulloblastoma cell line that is reported to be resistant to GDC-0449, which indicated a promising prospect in the treatment of Hedgehog signaling pathway related cancer in clinical trial.

Full text of DOI:10.1016/j.bmcl.2014.01.006

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1426–1431
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of novel benzamide derivatives
as potent smoothened antagonists
Tian-Ming Wu a, , Dao-Cai Wang b, , Pu Xiang a, Jian-Nan Zhang a, Ya-Xiong Sang a, Hong-Jun Lin a,
Jie Chen a, Gang Xie a, Hang Song b, Ying-Lan Zhao a, , Yong-Mei Xie a,
⇑
⇑
a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China
b Department of Pharmaceutical and Biological Engineering, College of Chemical Engineering, Sichuan University, Chengdu 610065, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzamide derivatives were prepared and evaluated using cell-based measurements.
Among these compounds, 10f significantly inhibited Hedgehog signaling and showed equivalent or more
potency than GDC-0449 in different tests. Furthermore, compound 10f potently inhibited the prolifera-
tion of Daoy, a medulloblastoma cell line that is reported to be resistant to GDC-0449, which indicated
a promising prospect in the treatment of Hedgehog signaling pathway related cancer in clinical trial.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 15 October 2013
Revised 30 December 2013
Accepted 6 January 2014
Available online 15 January 2014
Keywords:
Amide derivatives
Hedgehog
Smoothened antagonist
In addition to the important role in the regulation of stem cell
and progenitor cell differentiation during multiple developmental
processes, Hedgehog (Hh) signaling pathway has also been impli-
cated in various type of cancers, such as skin cancer,1 breast can-
cer,2 prostate cancer3 and brain cancer.4 Typically, Hedgehog
pathway is activated when the secreted Hedgehog ligands directly
bind to 12-pass transmembrane protein Patched1 (PTCH1),5 reliev-
ing its repression of the G protein-coupled receptor-like protein
Smoothened (SMO),6 which subsequently signals to the glioma-
associated (Gli) family of transcription factors and induces a
change in the transcriptional profile of the cell.
Antagonists of SMO showed effectively anticancer activity, and
a number of these antagonists were under investigation in clinical
trials, such as CUR614147 and IPI-926.8 More recently, one known
SMO antagonist Vismodegib (GDC-0449) has achieved promising
results for the treatment of patients with advanced BCC, high-
lighted by a 55% overall response rate in early-stage clinical stud-
ies,9 and has been approved by FDA for the treatment of metastatic
basal cell carcinoma and locally advanced basal cell carcinoma.10
These data suggests that SMO is a promising therapeutic target
for Hh signaling pathway related diseases.
the C-3 substitution on the middle phenyl group of GDC-0449 with
urea or amide spacer, and expected that the terminal NH group of
the urea or amide moiety may form additional hydrogen bond(s) at
the receptor site to improve SMO antagonist activity. The SMO
antagonist activity was identified by dual luciferase assay on
Shh-light
2 cells (NIH3T3 cell line stably transfected with
8⁄Gli-binding site luciferase and Renilla reporters).11 The following
initial hit offered considerable potency in Light 2 assay with
IC50 = 300 nM (Fig. 1).
Encouraged by this result, a series of compounds based on the
lead structure was synthesized. To begin with, the target amide
derivatives 6a–d were prepared according to the route described
Aiming to design, synthesize and obtain novel SMO inhibitor for
the treatment of Hh dependent malignancies, we initially replaced
⇑
Corresponding authors. Tel.: +86 28 85164063; fax: +86 28 85164060.
E-mail addresses: zhaoyinglan@scu.edu.cn (Y.-L. Zhao), xieym@scu.edu.cn (Y.-M.
Xie).
Figure 1. Structures of GDC-0449 and the initial hit identified from the cell-based
These two authors contributed equally as first authors.
Gli-Luciferase assay.
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2014.01.006

T.-M. Wu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1426–1431
1427
Scheme 1. Reagents and conditions: (a) Boc2O, Et3N, DMAP, THF, 25 °C, 98.2%; (b) Fe, NH4Cl in H2O, ethanol, 90 °C, 52%; (c) 2-chloro-4-(methylsulfonyl)benzoyl chloride,
Et3N, DMAC, 78.6%, (d) TFA/CH2Cl2 = 10%, 25 °C, 87.1%; (e) substituted phenyl isocyanates, pyridine, 25 °C.
Table 2
Table 1
Activities of compounds against the Hh pathway at a concentration of 2 lM
Activities of compounds against the Hh pathway at a concentration of 2 lM
O
S
O
O
S
H
N
H
N
O
H
N
H
N
H
N
R
R
O
O
Cl
Cl
O
O
Cl
Cl
Entry
R
Gli-LUC Shh-light2 inhibition (%)
28.4 10
Entry
R
Gli-LUC Shh-light 2 inhibition (%)
CF3
7a
6a
73.8
66.1
3
2
O
O
OMe
7b
7c
3.6 10
6b
6c
6d
OMe
N
0
53.2
5
Cl
Br
N
S
7d
7e
30.5
51.4
6
7
55.4
5
Cl
Cl
F
Scheme 2. Reagents and conditions: (a) aromatic acyl chlorides, THF, Et3N, 25 °C.
in Scheme 1. The amino group of 2-chloro-5-nitroaniline (1) was
protected by Boc, and then the nitro group was reduced to amino
group to give compound 3. Compound 3 was reacted with 2-
chloro-4-(methylsulfonyl) benzoyl chloride in the presence of tri-
ethylamine to obtain compound 4. After deprotection of the Boc
group of compound 4 with TFA in dichloromethane, the resulting
amine (5) is finally condensed with appropriate aryl isocyanate
to gain the target urea derivatives 6a–d.
Scheme 3. Reagents and conditions: (a) (i) SOCl2, reflux; (ii) 5-amino-2-chloro-
benzoic acid, Et3N, DMAC, 25 °C, 79.8%, (b) (iii) HOBT, DCC, DMF, 0 °C; (iv) aromatic
amines, 0 °C; (v) 25 °C.
Dual luciferase assay was performed to measure the inhibition
activities of each compound at concentration of 2 lM. Except com-
pound 6c, compounds 6a, 6b and 6d exhibited good Hh signaling
inhibition activities (Table 1). However, when the concentrations
of 6a–d decreased to 0.5 lM, none of the above compounds
showed satisfying potency (data not shown).
Subsequently, we preceded our investigation by modifying the
urea moiety to two different amide-based derivatives with
opposite directions. One group of amide-based derivative was pre-
pared according to the route described in Scheme 2. Compound 5
was reacted with aromatic acyl chlorides in the presence of trieth-
ylamine to yield 7a–e. Nevertheless, the inhibition activities of
compounds 7a–e in the Light 2 assay (Table 2) were all far behind
the reported potency of GDC-0449 (IC50 = 20 nM).12

1428
T.-M. Wu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1426–1431
Table 3
The other group of amide-based derivatives was prepared as
shown in Scheme 3. To begin with, the 2-chloro-4-(methylsulfo-
nyl) benzoic acid compound 8, was converted to corresponding
acyl chloride and then reacted with 5-amino-2-chlorobenzoic acid
to obtain intermediate compound 9. Coupling of compound 9 with
amines in the presence of DCC and HOBT gave target compounds
10a–k. The Hh signaling inhibition activities of this group of com-
pounds are shown in Table 3. The preliminary results were supe-
rior to the previous compounds. The logical explanation may be
that the amide moiety, in this particular orientation, geometrically
permit appropriate fitting of the molecule at the receptor site.
The effects of substituents on the terminal phenyl ring were
also studied. Compounds 10a–h showed higher activity than com-
pounds 10i–j. This may be attributed to that methyl, ethyl, hydro-
xyl, methoxyl or ethoxy substituents on the terminal phenyl ring of
this kind of compounds enhance the Hh signaling inhibition activ-
ity. Compound 10k also possessed good Hh signaling inhibition
activity with heteroaromatic ring.
Activities of compounds against the Hh pathway at a concentration of 2 lM
O
S
O
O
H
N
R
N
H
Cl
O
Cl
Entry
R
Gli-LUC Shh-light2 inhibition (%)
10a
73
2
1
10b
10c
67.5
65.9
76.3
56.8
7
2
2
The compounds whose Hh signaling inhibition activities over
70% at concentration of 2 lM, that is, compounds 10a, d, f, h, k,
were selected for further investigation. Consequently, we obtained
two high potent Hh signaling inhibitors, compound 10d
(IC50 = 100 nM) and compound 10f (IC50 = 40 nM) (Fig. 2). Given
the optimized potencies, our top priority is to confirm the
anti-SMO target specificity. We constructed a HEK293T cell line
SMO-293T,13 that stably over expressed human SMO, for SMO-
binding competition assay.14 Both compounds 10d and 10f showed
significant competition of Cyclopamine-binding site against
BODIPY-Cyclopamine (Fig. 3), which sufficiently indicated 10d
and 10f are specific SMO antagonists. Unfortunately, the Hh signal-
ing inhibition activity of compound 10d in vivo was not very good
due to the poor water-solubility.
We then turned our effort to understand the resulting conse-
quences of compound 10f under the condition of Hh ligand in-
duced activation. Down-regulation of alkaline phosphatase (AP)15
in mouse multipotent mesenchymal stem cells and Gli target gene
mPTCH1 in a mouse fibroblast cell NIH3T3 were measured (Fig. 4A
and B). In both detections, compound 10f was found to be equiva-
lent or more potent than GDC-0449. Given the fact that GDC-0449
failed to kill medulloblastoma cells in patient, we expected that
compound 10f could lead a positive respond in vitro. Proliferation
inhibiting potency was determined by MTT assay16 on four human
cancer cell lines, one of which was human medulloblastoma cell
line Daoy, which is reported to be resistant to GDC-0449 in vitro.17
During the MTT assay, a common chemotherapeutics drug adria-
mycin (ADM) was utilized as positive control. Surprisingly,
10d
10e
OH
10f
72.2
69.8
5
4
10g
O
O
10h
10i
70.4
3
Cl
0
Cl
Cl
10j
46.6 10
Cl
N
10k
72.9
1
Figure 2. Hh signaling inhibition activities were determined by Light 2 assay. Shh-light2 cells were incubated with Shh-N-conditioned medium and various concentrations of
GDC-0449 (A), compounds 10d (B) and 10f (C), respectively. Compound 10f (IC50 = 40 nM) showed equivalent potency to that of GDC0449 (IC50 = 33 nM).

T.-M. Wu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1426–1431
1429
Figure 3. Compounds 10d or 10f can block the binding of BODIPY-cyclopamine to SMO. SMO-293T cells were incubated with 5 nM BODIPY-cyclopamine (green) (A) and then
exposed to different concentrations of GDC-0449 (B and C), compound 10d (D–F) or 10f (G–I). All cells were counterstained with Hochest 33342 (blue). The concentrations at
which compound 10d or 10f effectively competed for SMO-binding site correlate with the IC50 in Light 2 assay, respectively.
Figure 4. Compound 10f can block down-stream signaling of Hh pathway and inhibit cancer cell proliferation. Down-regulated expression of alkaline phophatase (AP) in
mMSC (A) and mPTCH1mRNA in NIH3T3 (B) were measured. Notably, compound 10f exhibited better proliferation inhibition activity in cancer cells than GDC-0449, as
measured by MTT assay (C). In the MTT assay, ADM and the two compounds were added to the medium at a concentration of 10
human sHH, SMO, and Gli1 was normalized to the human housekeeping gene actin.
lM, respectively. (D) The expression level of
compound 10f demonstrated relatively better activity than GDC-
0449 in inhibiting cell proliferation in these cancer cell lines, espe-
cially in Daoy (Fig. 4C). The differences in cell growth inhibition
were attributed to correlation between Hh signaling pathway
and carcinogenesis of different kinds of cancers, which were
demonstrated by quantitative detection of mRNA expression level
of Hedgehog signaling18 (Fig. 4D).
Genetic analysis in mice, Drosophila and zebrafish indicated a sin-
gle homologue of SMO is responsible for suppression of Gli.19 That
made it available to utilize the Gli-dependent-RFP transgenic

1430
T.-M. Wu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1426–1431
Figure 5. Compound 10f exhibited stronger potency than GDC-0449 in abrogating Gli-dependent RFP in zebrafish model. Generally, Gli-dependent RFP (red) (B) expressed in
dorsal cord (A). (C) In blank group, the embryos at 2hpf were treated with DMSO. (D–F) Ablation of Gli-dependent RFP by 20, 40 and 80 M GDC-0449 in 36-hpf embryos. (G–
I) Ablation of Gli-dependent RFP by 20, 40 and 80 M compound 10f in 36-hpf embryos.
l
l
zebrafish model to investigate SMO antagonist in vivo.20 The red
florescence protein (RFP) expressed in somite tissues adjacent to
the midline structure reflected Hh signaling inhibition activity. In
this test, the 2 h post-fertilization (hpf) embryos were exposed
to compound 10f and GDC-0449 and incubated until 36 hpf.
The inhibitory effect was analyzed at concentrations of 20, 40
006. These data include MOL files and InChiKeys of the most
important compounds described in this article.
References and notes
1. Bale, A. E.; Yu, K. P. Hum. Mol. Genet. 2001, 10, 757.
2. Mukherjee, S.; Frolova, N.; Sadlonova, A.; Novak, Z.; Steg, A.; Page, G. P.; Welch,
D. R.; Lobo-Ruppert, S. M.; Ruppert, J. M.; Johnson, M. R.; Frost, A. R. Cancer Biol.
Ther. 2006, 5, 674.
3. 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.
4. Rudin, C. M.; Hann, C. L.; Laterra, J.; Yauch, R. L.; Callahan, C. A.; Fu, L.; Holcomb,
T.; Stinson, J.; Gould, S. E.; Coleman, B.; LoRusso, P. M.; Von Hoff, D. D.; de
Sauvage, F. J.; Low, J. A. N. Eng. J. Med. 2009, 361, 1173.
and 80
GDC-0449, treatment with compound 10f at 20
reduced RFP expression in RFP-positive embryos at 36 hpf. With
the concentration increasing to 40 and 80 M, compound 10f
lM, respectively. Similar to the positive control
lM significantly
l
showed stronger inhibitory effect in lessening the Gli-dependent
RFP (Fig. 5).
5. Rohatgi, R.; Scott, M. P. Nat. Cell Biol. 2007, 9, 1005.
In conclusion, we have described the design, synthesis and bio-
logical evaluation of novel benzamide derivatives as potent
smoothened antagonists. Our work focused on the obtainment of
candidate smoothened antagonists with higher Hh signaling inhi-
bition activities, that is, compound 10d21 and 10f.21 Both in vitro
and in vivo tests indicated that compound 10f was a specific
SMO antagonist, which significantly down-regulated Hh-induced
cellular factors. Especially, compound 10f not only effectively
inhibited the GDC-0449-resistant cell line, but also exhibited supe-
rior potency in zebrafish. These results offered a very promising
prospect for the treatment of Hh signaling pathway related cancer
in clinical trial which is resistant to GDC-0449. Further report on
the detection of pharmacokinetic properties and validation of
tumor inhibition in mouse model are forthcoming.
6. Kalderon, D. Cell 2000, 103, 371.
7. 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.
8. Tremblay, M. R.; Lescarbeau, A.; Grogan, M. J.; Tan, E.; Lin, G.; Austad, B. C.; Yu,
L. C.; Behnke, M. L.; Nair, S. J.; Hagel, M.; White, K.; Conley, J.; Manna, J. D.;
Alvarez-Diez, T. M.; Hoyt, J.; Woodward, C. N.; Sydor, J. R.; Pink, M.;
MacDougall, J.; Campbell, M. J.; Cushing, J.; Ferguson, J.; Curtis, M. S.;
McGovern, K.; Read, M. A.; Palombella, V. J.; Adams, J.; Castro, A. C. J. Med.
Chem. 2009, 52, 4400.
9. Von Hoff, D. D.; LoRusso, P. M.; Rudin, C. M.; Reddy, J. C.; Yauch, R. L.; Tibes, R.;
Weiss, G. J.; Borad, M. J.; Hann, C. L.; Brahmer, J. R.; Mackey, H. M.; Lum, B. L.;
Darbonne, W. C.; Marsters, J. C.; de Sauvage, F. J.; Low, J. A. N. Eng. J. Med. 2009,
361, 1164.
10. Dlugosz, A.; Agrawal, S.; Kirkpatrick, P. Nat. Rev. Drug Disc. 2012, 11, 437.
11. Taipale, J.; Chen, J. K.; Cooper, M. K.; Wang, B. L.; Mann, R. K.; Milenkovic, L.;
Scott, M. P.; Beachy, P. A. Nature 2000, 406, 1005.
12. Yauch, R. L.; Dijkgraaf, G. J. P.; Alicke, B.; Januario, T.; Ahn, C. P.; Holcomb, T.;
Pujara, K.; Stinson, J.; Callahan, C. A.; Tang, T.; Bazan, J. F.; Kan, Z.; Seshagiri, S.;
Hann, C. L.; Gould, S. E.; Low, J. A.; Rudin, C. M.; de Sauvage, F. J. Science 2009,
326, 572.
Acknowledgment
13. Construction of SMO-293T cell line. SMO human SMO cDNA (NM_005631.3)
was purchased from Origene and was cloned into cytomegalovirus promoter-
driven pCDH lentivirus plasmid to obtain SMO-pCDH. HEK 293T cells were
This work was supported by China Postdoctoral Science Special
Foundation (201104690).
cultured in DMEM containing 10% FBS, 100 units/ml penicillin and 100 lg/ml
streptomycin to 50% confluency in 6-well plate and then transfected with
pCHD-SMO, pspax2, pMD2.0G (2:2:1) and lipofectamine LTX (Life Technology)
according to the manufactures’ protocol. After 48 h, the medium containing
virus was collected and transferred to another HEK 293T cultured in 12-well
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.01.
plate with 60% confluency. 2
lg/ml puromycin was added to select smo-
overexpression stable clone, named SMO-293T.

T.-M. Wu et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1426–1431
1431
14. For binding assay, SMO-293T cells were incubated with DMEM containing 0.5%
FBS, Hochest 33342, 10 nM BODIPY-cyclopamine and various concentration of
candidate compounds for 4 h. Then cells were washed with PBS, and then
images were acquired by Array Scan VTI (Thermo).
15. Stappenbeck, F.; Xiao, W.; Epperson, M.; Riley, M.; Priest, A.; Huang, D. W.;
Nguyen, K.; Jung, M. E.; Thies, R. S.; Farouz, F. Bioorg. Med. Chem. Lett. 2012, 22,
5893.
human Gli1, forward, 5GTGCAAGTCAAGCCAGAACA3, and reversed, 5ATAGGG
GCCTGACTGGAGAT3; human actin, forward, 5GCACTCTTCCAGCCTTCCTTCC3,
and reversed, 5TCACCTTCACCGTTCCAGTTTTT3; mouse PTCHED, forward,
5ATGCTCCTTTCCTCCTGAAACC3, and reversed, 5TGAACTGGGCAGCTATGAA
GTC3; mouse GAPDH, forward, 5CGTCCCGTAGACAAAATGGTGAA3, and
reversed, 5GCCGTGAGTGGAGTCATACTGGAACA3.
19. Chen, J. K.; Taipale, J.; Young, K. E.; Maiti, T.; Beachy, P. A. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 14071.
16. MTT assay: In brief, cells were incubated with 10
lM ADM, 10 lM GDC-0449 or
10 M compound 10f in 96-well plate for 48 h. After removing the medium, a
freshly prepared MTT (0.5 mg/ml) was added to the plate. And the absorbance
was measured at 570 nm.
l
20. Yang, H. B.; Xiang, J.; Wang, N. D.; Zhao, Y.; Hyman, J.; Li, S.; Jiang, J.; Chen, J. K.;
Yang, Z.; Lin, S. J. Biol. Chem. 2009, 284, 20876.
21. Selected data: Compound 10d: 1H NMR (DMSO, 400 MHz) d 10.98 (s, 1H), 10.06
(s, 1H), 8.15 (s, 1H), 7.91–8.04 (m, 3H), 7.80–7.82 (m, 1H), 7.57 (d, 1H,
J = 8.4 Hz), 7.18 (d, 1H, J = 6.8 Hz), 7.07–7.13 (m, 2H), 3.35 (s, 3H), 2.28 (s, 3H),
2.18 (s, 3H); MS m/z 513.2 (M+Na)+. Compound 10f: 1H NMR (DMSO, 400 MHz)
d 10.98 (s, 1H), 10.42 (s, 1H), 9.44 (s, 1H), 8.15 (d, 1H, J = 1.6 Hz), 8.01–8.03 (m,
1H), 7.88–7.92 (m, 2H), 7.78–7.80 (m, 1H), 7.57 (d, 1H, J = 8.8 Hz), 7.31–7.32
(m, 1H), 7.06–7.14 (m, 2H), 6.50–6.53 (m, 1H), 3.35 (s, 3H); MS m/z 501.1
(M+Na)+.
17. Ohshima-Hosoyama, S.; Davare, M. A.; Hosoyama, T.; Nelon, L. D.; Keller, C. J.
Neurooncol. 2011, 105, 475.
18. Quantitative real-time PCR: Total RNA was extracted from cancer cells using
RNA purification kit (Axygen). Single strand cDNA was synthesized by iScript
Kit (Bio-Rad), and was assayed using SYBRGreen qPCR kit (Bio-Rad). The primer
sequences used were as follows: human sHH, forward, 5ACCGAGGGCTGGGA
CGAAGA3, and reversed, 5ATTTGGCCGCCACCGAGTT3; human SMO, forward,
5GGGAGGCTACTTCCTCATCC3, and reversed, 5GGCAGCTGAAGGTAATGAGC3;