Discovery of novel hedgehog antagonists from cell-based screening: Isosteric modification of p38 bisamides as potent inhibitors of SMO

Article abstract of DOI:10.1016/j.bmcl.2012.04.104

Cell-based subset screening of compounds using a Gli transcription factor reporter cell assay and shh stimulated cell differentiation assay identified a series of bisamide compounds as hedgehog pathway inhibitors with good potency. Using a ligand-based optimization strategy, heteroaryl groups were utilized as conformationally restricted amide isosteres replacing one of the amides which significantly increased their potency against SMO and the hedgehog pathway while decreasing activity against p38α kinase. We report herein the identification of advanced lead compounds such as imidazole 11c and 11f encompassing good p38α selectivity, low nanomolar potency in both cell assays, excellent physiochemical properties and in vivo pharmacokinetics.

Full text of DOI:10.1016/j.bmcl.2012.04.104

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4907–4911
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel hedgehog antagonists from cell-based screening:
Isosteric modification of p38 bisamides as potent inhibitors of SMO
⇑
Bin Yang , Alexander W. Hird, Daniel John Russell, Benjamin P. Fauber, Les A. Dakin, Xiaolan Zheng,
Qibin Su, Robert Godin, Patrick Brassil, Erik Devereaux, James W. Janetka
AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham, MA 02451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cell-based subset screening of compounds using a Gli transcription factor reporter cell assay and shh
stimulated cell differentiation assay identified a series of bisamide compounds as hedgehog pathway
inhibitors with good potency. Using a ligand-based optimization strategy, heteroaryl groups were utilized
as conformationally restricted amide isosteres replacing one of the amides which significantly increased
Received 11 February 2012
Revised 19 April 2012
Accepted 23 April 2012
Available online 30 April 2012
their potency against SMO and the hedgehog pathway while decreasing activity against p38
report herein the identification of advanced lead compounds such as imidazole 11c and 11f encompass-
ing good p38 selectivity, low nanomolar potency in both cell assays, excellent physiochemical proper-
ties and in vivo pharmacokinetics.
a kinase. We
Keywords:
a
Hedgehog pathway
Smoothened
Patched
Ó 2012 Elsevier Ltd. All rights reserved.
Amide isosteres
p38
Heterocycles
The Hedgehog pathway affects numerous biological processes,
such as cell differentiation and proliferation, and particularly
embryogenesis, where the pathway regulates embryo patterning.
During embryogenesis, Hedgehog pathway typically is activated by
the secreted Hedgehog ligands, which directly bind to their receptor
protein, 12-pass transmembrane protein Patched1 (Ptch1),1 and
inhibit its repression of the G protein-coupled receptor-like protein
Smoothened (SMO).2 Recently it has been reported that primary cilia
plays a pivotal role in Hedgehog pathway.3 Under basal conditions,
Ptch1 is localized to the primary cilium and SMO is sequestered in
endosomes;4 Hedgehog ligands induce Ptch1 movement out of and
consequently SMO trafficking into primary cilium. The pathway
activation ultimately leads to the modulation of Gli zinc-finger
transcription factors, permitting transcription activation of Hedge-
hog-responsive genes whose expression is crucial for tissue pattern-
ing, growth and differentiation and tissue homeostasis.
carcinomas, medulloblastomas, and rhabdomyosarcomas.6 Onco-
genic mutations in SMO and Su(fu) have also been identified.7 Phar-
macological inhibitors of the Hedgehog pathway should have great
therapeutic value. Particularly, the SMO antagonist cyclopamine is
known to block tumor progression in a variety of mouse cancer
models.8 More recently, known SMO antagonist Vismodegib (GDC-
0449) demonstrated good response rate in basal cell carcinoma pa-
tients and has since been approved by FDA to treat metastatic basal
cell carcinoma and locally advanced basal cell carcinoma.9
To identify Hedgehog pathway inhibitors, we screened a subset
of over 40,000 compounds against a cell-based Gli-Luciferase as-
say10 and a selection of active compounds from the Gli-Luciferase
assay in a C3H10T1/2 Hedgehog-dependent differentiation assay.11
A series of bisamide compounds, including compound 1a (Fig. 1),
were among the initial hits identified. In both Hedgehog pathway
assays, this series of compounds offered attractive potency and
The linkage of the Hedgehog pathway to diseases, such as cancer,
is established, and a number of different oncogenetic mechanisms
related to the Hedgehog pathway have been identified. Autocrine
or paracrine Hedgehog signaling was found in certain neoplasms,
such as small-cell lung cancers and pancreatic adenocarcinomas.5
Ligand-independent Hedgehog target gene expression can also lead
to tumorigenesis, exemplified by Gorlin’s syndrome patients who
are heterozygous for Ptch1mutations and susceptible to basal cell
2
O
O
5
N
N
N
1
H
H
O
N
F
1a
⇑
Corresponding author. Tel.: +1 781 839 4135; fax: +1 781 839 4230.
E-mail address: bin.yang@astrazeneca.com (B. Yang).
Figure 1. Structure of a bisamide hit identified from the cell-based Gli-Luciferase
assay.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.104

4908
B. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4907–4911
Table 1
preliminary SAR. Herein we report our lead optimization of this
series to improve its potency and selectivity against p38-type
kinases.
The synthesis of the bisamide compounds (e.g., 1a) was re-
ported previously.12 Compound 1a offered considerable potency
in the firefly reporter assay against the Hh pathway with
Activities of bisamide compounds against Hedgehog pathway and p38
a
R2
H
R1
N
O
IC50 = 0.45
l
M, and it appeared to be selective over the renilla fire-
M). The biophysical properties of this series
R1
R2
Firefly
EC50
(lM)
p38
Enz
IC50
a
Solubility
Hu
PPB
(%
fly assay13 (IC50 >30
l
(l
M)
are poor in general; aqueous solubility of compound 1a is ꢀ1
lM
and its strong protein binding in plasma gave smaller than 1% free
fraction. In addition, one of the main potential concerns as a
Hedgehog pathway inhibitor is that the bisamide series are known
(lM)
free)
O
N
O
1a
1b
0.45
0.45
0.025
0.03
<1
<1
N
N
N
as potent p38 MAP kinases inhibitors (p38
a IC50 = 0.025 lM for
compound 1a). The initial screening results in the firefly reporter
assay for this series indicated a wide tolerance of various function-
alities on the phenyl ether side of the molecule, but those structural
variations did not meaningfully attenuate p38
for a selection of compounds and their activities in Hh pathway
firefly reporter and p38 kinase assays are summarized in Table 1.
It is noteworthy that the 2-pyridyl group on the right side of the
molecule appeared to be optimal for Hh pathway inhibition, and
other heteroaryls or alkyl groups reduced potency. Further instal-
lation of solubilizing basic amine groups on the right hand side
of the molecule failed to attenuate their activities on p38 kinase,
and it further reduced their potency on Hh pathway inhibition.
Some improvement on solubility was observed, although with lim-
ited significance (data not shown).
Structural optimization efforts focused on the modifications of
C-5 substitution on the middle phenyl group. It was reasoned that
reducing the molecular weight from the left hand side of molecule
could potentially improve both potency and the physical
properties of this series of compounds.
O
N
O
1.4
3.6
a activity. The data
N
N
a
O
O
O
N
N
O
S
1c
>3
0.017
<1
2
O
O
O
1d
1e
3.2
1.1
0.166
0.051
<1
<1
<1
<1
N
N
N
O
N
N
OH
O
N
O
b
N
a
MeO
HO
MeO
O
O
O
2
3
4
O
N
O
O
H
N
O
H
N
N
d
c
HO
MeO
O
O
6
5
O
N
N
O
f
H
N
O
e
H
N
N
N
H
N
O
H
NH2
O
8
7
Scheme 1. Synthesis of benzimidazole amide compound 8. Reagents and conditions: (a) 2-(bromomethyl)pyridine, K2CO3, acetonitrile/water (80%); (b) NaOH (1 N), MeOH,
reflux (90%); (c) 3-amino-4-methylbenzoic acid methyl ester, HATU, DIPEA, DMF (99%); (d) NaOH (1 N), MeOH, reflux (91%); (e) 1,2-diaminobenzene, HATU, DIPEA, DMF
(93%); (f) AcOH, reflux (99%).

B. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4907–4911
4909
Table 2
Nitrogen-containing heterocycles as amides isosteres increased Hh pathway potency and reduced p38
a potency
O
N
H
N
R
O
11
R
Firefly IC50
0.005
(lM)
shh EC50
(l
M)
BST p38
30
a
IC50
(
lM)
Solubility (
lM)
Hu PPB (% free)
8
0.03
<1
<1
N
HN
N
11a
<0.04
0.0392
1.6
90
5.1
N
N
11b
11c
<0.016
0.018
0.011
15
190
5.1
2.2
N
<0.006
0.0024
100
12
350
<1
N
CF3
11d
11e
11f
11g
11h
11i
0.022
0.015
0.013
0.089
0.007
0.021
0.027
1.2
ND
5.6
9.5
3.2
6.3
5.6
N
H
NH
N
0.0026
<0.012
0.005
0.007
0.009
0.021
5.9
2.7
19
19
25
80
15
120
46
N
N
H
N
N
N
36
HN
N
NH
100
100
N
11j
N
N
N
11k
11l
0.012
0.009
0.008
0.029
100
100
5
1.8
<1
F3C
N
<1
N
H
O
N
11m
0.042
>0.145
30
22
3.3
S
N
S
11n
11o
11p
11q
0.044
>0.45
0.021
>0.40
0.028
ND
14
NV
15
22
<1
<1
<1
2
<1
N
N
<1
N
0.017
0.04
1.1
1.8
N
N
H2N
N
11r
11s
<0.019
0.061
0.008
0.115
15
7
5.2
ND
N
NH
N
3.5
ND
N

4910
B. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4907–4911
O
O
OH
B
OH
B
N
N
a
NH2
N
HO
HO
HO
+
O
O
4
9
10
O
N
b
R
N
O
11
Scheme 2. Syntheses of heterocyclic amides using Suzuki coupling reactions. Reagents and conditions: (a) HATU, DIPEA, DMF, rt (50%); (b) heterocyclic halides, Pd(PPh3)4,
dioxane/water, 100–150 °C.
Nitrogen-containing heterocycles are regularly used as isosteres
Table 3
for amide groups in medicinal chemistry. Interestingly, using hetero-
cycles as amide replacements to improve SMO antagonist activity is
recently reported.14 Particularly, some heteroaryls groups, such as
benzimidazoles, widely appear in numerous SMO inhibitors.15 To
explore the structure–activity relations of C-5 substitution on com-
pound 1a, it was reasoned that heterocyclic moieties could be poten-
tial surrogates for the C-5 amide group in compound 1a.16 In addition,
X-ray crystal structure revealed hydrogen bondings between the C-5
In vivo CD-1 murine DMPK properties for selected heteroaryls
Compound Dosea (mg/kg)
Cl
PO AUC Vss
IV Half %F
IV
PO
(mL/min/kg)
(lg⁄h/L) (L/kg) life (h)
11c
11f
3.0
3.0
10.0
7.5
210
8.3
100
12535
12
0.5
0.9
1.0
22
83
a
The compounds were formulated using DMA/TEG/saline 40/40/20%.
amide group of the bisamide compounds and p38
a
protein residues
As illustrated in Table 2, the majority of the heterocyclic analogs
Glu71 and Asp168. We envisioned that amide isosteres could poten-
tially disrupt these hydrogen bondings and hence attenuate their
p38 activity. Aiming to improve Hh pathway inhibition potency and
to limit p38 inhibition activity associated with both bisamide series,
benzimidazole group was first introduced as an amide isostere, and
its synthesis is shown in Scheme 1.
Phenol 2 was directly alkylated under mild basic conditions in
acetonitrile. Notably, it was found that a small fraction of water
presence in the solvent significantly facilitated the reaction rate.
Subsequent hydrolyses on methyl esters and amides coupling
steps using HATU generated bisamide 7, which was cyclized upon
refluxing in acetic acid to produce the benzimidazole, 8.17 Satisfac-
torily, compound 8 significantly improved the potency in both fire-
fly reporter assay (4.7 nM) and the differentiation assay
(EC50 = 30 nM). Even more encouragingly, compound 8 diminished
the activity against p38 kinases, given that its observed IC50 for
either retained or improved the potency to inhibit Hh pathway in
both cell assays, with some of the compounds in Table 2 reaching
close to the lowest testing concentration of both the firefly reporter
assay and the differentiation assay. Substitution of C-5 amide
group by imidazoles was systematically explored (compound
11a–11j). Between the unsubstituted 2-imidazole (11a)18 and 5-
imidazole (11f),19 similar inhibition activities against Hh pathway
were observed, and both showed diminished activities against p38
kinase. While the monosubstituted analogs on both imidazoles
(11b, 11d, 11e, 11g, 11h, and 11i) significantly further reduced
p38 inhibition potency, the disubstituted imidazole analogs
(11c,20 11j) showed no p38
concentration (100 M). Pyrazole and thiazole analogs (11k, 11l,
a inhibition at the maximum testing
l
11n) inhibited Hh pathway at comparable potency to imidazols,
but these analogs also gave less than desirable solubility and high-
er plasma protein binding. Notably, installation of a morpholine
group as a solubilizing group on a thiazole ring (11m) was detri-
mental to the potency in the cell differentiation assay. All three
pyridine (11o, 11p, 11q) analogs were synthesized. Compared to
3 and 4 substituted pridines (11o, 11q), 2-substituted pyridine
analog (11p) demonstrated superior potency in both cell assays,
consistent with what others have previously reported.14,21 The
amino-pyridazine analogs offered superior potency, but at the ex-
pense of solubility. Purine as a heterocycle with more structural
complexity was also installed, only to be proved with inferior po-
tency, particularly in the differentiation assay (11s).
p38
a
kinase is greater than 100
l
M (Table 2). However, the solu-
M, and it also suffers
bility of compound 8 at pH 7.4 is less than 1
l
from high plasma protein binding, with free fraction in human
plasma less than 1%.
Given the improved potency and reduced p38 activity of the
benzimidazole compound 8, and to further address its poor physi-
cal properties, a variety of heterocycles were next explored as
amide isosteres at the C5 position of compound 1a. Suzuki cou-
pling reaction was used as the key step to introduce a large set
of structurally diverse heterocycles, and their syntheses are out-
lined in Scheme 2.
Benzoic acid 4 was directly coupled with anilino boronic acid 9
using HATU to produce the boronic acid intermediate 10, setting
the stage for the installation of a variety of heterocycles by Suzuki
coupling. As shown in Table 3, a large array of nitrogen-containing
heterocycles was successfully introduced as C-5 substituents,
including imidazole, pyrazole, thiazole, pyridine, and purine. The
yields of the coupling reactions ranged from 30% to 70%. The ana-
logs shown in Table 3 were characterized by LCMS and 1H NMR.
To further confirm the diminished p38 activity of this new ser-
ies of hetercyclic analogs, compound 11f was tested in a whole
blood cell assay for its activity against p38. Satisfactorily, no inhib-
itory activity against p38 was observed at the maximum concen-
tration tested (50 lM). Moreover, to determine whether the
inhibitors acted specifically on SMO, we used a SMO binding assay
and this series of compounds were found to competitively displace
BODIPY labeled cyclopamine in HeLa cells engineered to express
either human or mouse SMO.22

B. Yang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4907–4911
cells per well in DMEM supplemented with 10% FBS and 1%
4911
L
-glutamine
Based on their overall profile, compounds 11c and 11f were
chosen for in vivo pharmacokinetic measurements in mouse. As
illustrated in Table 3, the dimethyl substitution on imidazole ring
in compound 11c demonstrated enormous impact on in vivo
pharmacokinetics. Compared to its analog 11c, compound 11f
has significantly lower clearance and higher overall exposure,
and it also demonstrates excellent oral bioavailability.
overnight at 37 °C. The following day, the media was exchanged for a 50%
CM + shh (DMEM with 0.5% FBS, 1% -glutamine + shh ligand). Compounds
were solubilized in 100% DMSO to a concentration of 10 mM and then serially
diluted threefold also in a 100% DMSO solution. The highest concentration on
the cell plate was 33 lM and the lowest was 0.00045 lM. The compounds
were dosed onto the cells and the plates were incubated for 20 h. The cell
plates were then assayed for luciferase and renilla activity by using the
Promega Dual-Glo Luciferase Assay System (Cat #2590) per manufacturer’s
instructions. Plates were read on the Tecan Ultra at 50 ms integration time per
well.
L
In conclusion, structural modifications on a series of bisamide
compounds led to the discovery of a novel class of heterocyclic
amide compounds as potent Hh pathway inhibitors. Using nitro-
gen-containing heterocycles as amide isosteres, the heterocylic
amide compounds demonstrated superior potency against Hh
pathway in both the Gli reporter cell assay and shh stimulated cell
differentiation assay, and significantly reduced p38 inhibition
activity associated with the bisamide compounds. While a large
set of structurally diverse heterocycles can be tolerated for their
potency against Hh pathway, they also offer distinctly different
profiles in physical properties and in vivo pharmacokinetics.
Imidazole compounds were selected as the front runners for more
advanced studies due to their excellent potency and physical prop-
erties. A balance of Hh pathway potency, physical properties, and
in vivo pharmacokinetic properties make 11f a suitable tool for
in vivo pharmacodynamic and efficacy studies of Hh pathway inhi-
bition. Further reports on the optimization of the heterocyclic
amides series are forthcoming.
11. Differentiation assay: C3H10T1/2 cells are plated into 384-well plates at a
concentration of 5000 cells/well in DMEM/10% FBS. The following day the
media is changed to 20% CM (low serum media DMEM/2% FBS + Shh ligand).
Compounds are solubilized in 100% DMSO to a concentration of 10 mM and
then serially diluted three fold in 100% DMSO. The highest concentration in the
cell plate is 30 lM and the lowest is 3 nM. The compounds are then added to
the cells. Cell plates are incubated with compound for 72 h and then assayed
for alkaline phosphatase production using pNp as a substrate. Briefly, after 72 h
of incubation the media is aspirated from the cells and washed with 30
lL of
PBS. PBS is aspirated off the cells and 15
l
L of 1 Â RIPA cell lysis buffer is added
on to the cells. The cell plates are then incubated at À80 °C for 30 min to insure
proper cell lysis. The plates are then thawed back to room temperature. The
substrate solution containing pNp at 1 mg/mL in diethanolamine buffer pH 9.8
is then added onto the lysed cells. The plates are incubated at 30 °C overnight
for color development and read at absorbance of 405 nm.
12. Brown, D. S.; Belfield, A. J.; Brown, G. R.; Campbell, D.; Foubister, A.; Masters, D.
J.; Pike, K. G.; Snelson, W. L.; Wells, S. L. Bioorg. Med. Chem. Lett. 2004, 14, 5383.
13. A constitutively active Renilla luciferase construct was transfected into NIH-
3T3 cells. The Renilla luciferase assay was used as control for the Gli1 reproter
assay. Compounds that inhibited Renilla luciferase were filtered out.
14. Brown, M. L.; Aaron, W.; Austin, R. J.; Chong, A.; Huang, T.; Jiang, B.; Kaizerman,
J. A.; Lee, G.; Lucas, B. S.; McMinn, D. L.; Orf, J.; Rong, M.; Toteva, M. M.; Xu, G.;
Ye, Q.; Zhong, W.; DeGraffenreid, M. R.; Wickramasinghe, D.; Powers, J. P.;
Hungate, R.; Johnson, M. G. Bioorg. Med. Chem. Lett. 2011, 21, 5206.
15. Romer, J. T.; Kimura, H.; Magdaleno, S.; Sasai, K.; Fuller, C.; Baines, H.; Connelly,
M.; Stewart, C. F.; Gould, S.; Rubin, L. L.; Curra, T. Cancer Cell 2004, 6, 229; (b)
Peukert, S.; Miller-Moslin, K. ChemMedChem 2010, 5, 500.
Acknowledgements
The authors thank Mutka Bagul for performing the shh stimu-
lated cell differentiation assay. Camil Joubran, Tatyana Friedman,
and Nancy DeGrace provided analytical support. We also thank
Michael H. Block for helpful discussions.
16. While the manuscript of this report was in preparation, others have published
SMO antagonists containing benzimidazoles and other heterocycles (a)
Buttner, A.; Seifert, K.; Cottin, T.; Sarli, V.; Tzagkaroulaki, L.; Scholz, S.;
Giannis, A. Bioorg. Med. Chem. 2009, 17, 4943; (b) Robarge, K. D.; Brunton, S. A.;
Castanedo, G. M.; Cui, Y.; Dina, M. S.; Goldsmith, R.; Gould, S. E.; Guichert, O.;
Gunzner, J. L.; Halladay, J.; Jia, W.; Khojasteh, C.; Koehler, M. F. T.; Kotkow, K.;
La, H., et al J. Med. Chem. 2009, 52, 3829; (c) Stanton, B. Z.; Peng, L. F. Mol.
BioSyst. 2010, 6, 44; (d) Mahindroo, N.; Punchihewa, C.; Fujii, N. J. Med. Chem.
2009, 52, 3839; (e) Ontoria, J. M.; Bufi, L. L.; Torrisi, C.; Bresciani, A.; Giomini, C.;
Rowley, M.; Serafini, S.; Bin, H.; Hao, W.; Steinkuehler, C., et al Bioorg. Med.
Chem. Lett. 2011, 21, 5274; (f) Dessole, G.; Branca, D.; Ferrigno, F.; Kinzel, O.;
Muraglia, E.; Palumbi, M. C.; Rowley, M.; Serafini, S.; Steinkuehler, C.; Jones, P.
Bioorg. Med. Chem. Lett. 2009, 19, 4191; (g) Miller-Moslin, K.; Peukert, S.; Jain, R.
K.; McEwan, M. A.; Karki, R.; Llamas, L.; Yusuff, N.; He, F.; Li, Y.; Sun, Y., et al J.
Med. Chem. 2009, 52, 3954; (h) Remsberg, J. R.; Lou, H.; Tarasov, S. G.; Dean, M.;
Tarasova, N. I. J. Med. Chem. 2007, 50, 4534; (i) Peukert, S.; Jain, R. K.; Geisser,
A.; Sun, Y.; Zhang, R.; Bourret, A.; Carlson, A.; Dasilva, J.; Ramamurthy, A.;
Kelleher, J. F. Bioorg. Med. Chem. Lett. 2009, 19, 328.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.04.104.
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d6) d ppm 12.54 (s, 1H), 9.83 (s, 1H), 8.59 (d, J = 4.6 Hz, 1H), 7.98 (d, J = 8.8 Hz,
2H), 7.90 (d, J = 1.5 Hz, 1H), 7.85 (td, J = 7.7, 1.8 Hz, 1H), 7.72 (dd, J = 8.0, 1.6 Hz,
1H), 7.54 (d, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.1, 5.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H),
7.16 (d, J = 8.8 Hz, 2H), 7.11 (s, 2H), 5.28 (s, 2H), 2.23 (s, 3H); LCMS
(M+H) = 385.
19. Experimental characterization of compound 11f: 1H NMR (DMSO-d6) d ppm
12.54 (s, 1H), 9.83 (s, 1H), 8.59 (d, J = 4.6 Hz, 1H), 7.98 (d, J = 8.8 Hz, 2H), 7.90
(d, J = 1.5 Hz, 1H), 7.85 (td, J = 7.7, 1.8 Hz, 1H), 7.72 (dd, J = 8.0, 1.6 Hz, 1H), 7.54
(d, J = 7.8 Hz, 1H), 7.36 (dd, J = 7.1, 5.1 Hz, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.16 (d,
J = 8.8 Hz, 2H), 7.11 (s, 2H), 5.28 (s, 2H), 2.23 (s, 3H). LCMS (M+H) = 385.
20. Experimental characterization of compound 11c: 1H NMR (300 MHz, DMSO-d6)
d ppm 2.33 (s, 3 H), 2.65 (s, 3 H), 3.68 (s, 3 H), 5.37 (s, 2 H), 7.19 (d, J = 8.85 Hz, 2
H), 7.32 (m, 1 H), 7.49 (m, 2 H), 7.57 (s, 1 H), 7.68 (d, J = 7.91 Hz, 1 H), 7.74 (s, 1
H), 8.01 (d, J = 8.85 Hz, 3 H), 8.67 (br s, 1 H), 9.94 (s, 1 H), 14.47 (br s, 1 H); LCMS
(M+H) = 413.
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10. Gli1 reporter assay: NIH-3T3 cells stably expressing an 8X GBS-Luciferase
reporter were plated into 384-well Matrix plates at a concentration of 10,000