Design, synthesis, and biological study of 6,7-dihydro-5H-pyrano[2,3-d] pyrimidine derivatives as novel hedgehog signaling pathway inhibitors

Article abstract of DOI:10.1007/s00044-014-0959-3

A novel series of hedgehog signaling pathway inhibitors were designed by replacing the pyrimidine nucleus of our earlier reported compounds with 6,7-dihydro-5H-pyrano[2,3-d]pyrimidine scaffold. Among this new class of hedgehog signaling pathway inhibitors, compounds 14 and 18 exhibited promising potency in vitro compared to GDC-0449. Compound 18 was advanced to profile its pharmacokinetic characteristics, and showed moderate pharmacokinetic properties in vivo, indicating that the 6,7-dihydro-5H-pyrano[2,3-d]pyrimidine skeleton is a promising scaffold for further exploration as hedgehog signaling pathway inhibitors. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0959-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0959-3
ORIGINAL RESEARCH
Design, synthesis, and biological study of 6,7-dihydro-5H-
pyrano[2,3-d]pyrimidine derivatives as novel hedgehog
signaling pathway inhibitors
•
Minhang Xin Liandi Zhang Han Shen Jun Wen Chongxing Tu
•
•
•
•
•
Zhaoyu Liu Lingfei Cheng Xinge Zhao
•
Received: 28 October 2013 / Accepted: 13 February 2014
Ó Springer Science+Business Media New York 2014
Abstract A novel series of hedgehog signaling pathway
inhibitors were designed by replacing the pyrimidine
nucleus of our earlier reported compounds with 6,7-dihy-
dro-5H-pyrano[2,3-d]pyrimidine scaffold. Among this new
class of hedgehog signaling pathway inhibitors, com-
pounds 14 and 18 exhibited promising potency in vitro
compared to GDC-0449. Compound 18 was advanced to
profile its pharmacokinetic characteristics, and showed
moderate pharmacokinetic properties in vivo, indicating
that the 6,7-dihydro-5H-pyrano[2,3-d]pyrimidine skeleton
is a promising scaffold for further exploration as hedgehog
signaling pathway inhibitors.
Introduction
The Hedgehog (Hh) signaling pathway plays very key roles
in several normal biological processes including cell pro-
liferation, survival signals control, embryological devel-
opment, tissue patterning, and stem cells maintenance (Ng
and Curran, 2011). However, when this signaling pathway
becomes abnormally disrupted or hyperactivated, they
express hallmarks of many cancer types. Mechanistic
researches indicate that genetic mutations (mainly in
receptor patched, GPCR-like receptor smoothened, or
Suppressor of fused) or Hh ligand overexpression in this
pathway are implicated in a variety of solid and hemato-
logic tumors such as basal cell carcinoma (BCC), medul-
loblastoma, rhabdomyosarcoma, melanoma, glioblastoma,
leukemia, lymphoma, hepatocellular, pancreatic, gastric,
colorectal, esophageal, lung, ovarian, and prostate cancers
(Onishi and Katano, 2011). Hence, inhibition of Hh sig-
naling pathway is thought to have considerable therapeutic
potential for addressing numerous unmet clinical needs.
To date, several small molecules which are capable of
inhibiting the Hh signaling pathway have been advanced
into clinical trials (Ruch and Kim, 2013). Among these,
vismodegib was approved by FDA in 2012 for treatment of
metastatic BCC (Dlugosz et al., 2012). Moreover, it is still
undergoing other clinical trials to profile its therapeutic
potential against cancers, such as medulloblastoma, mul-
tiple myeloma, leukemia, chondrosarcoma, pancreatic,
gastric, gastroesophageal, prostate, and small-cell lung
cancers, presently. Likewise, other candidates including
sonidegib (NVP-LDE225, Phase III), LY-2940680 (Phase
II), BMS-833923 (XL-139, Phase II), NVP-LEQ506
(Phase I), PF-04449913(Phase I), and TAK-441 (Phase I),
have recently been validated in clinical stages for com-
bating some cancer types by a single or combinational
Keywords Hedgehog signaling pathway Á Inhibitors Á
6,7-Dihydro-5H-pyrano[2,3-d]pyrimidine Á Novel
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-0959-3) contains supplementary
material, which is available to authorized users.
M. Xin (&)
Department of Medicinal Chemistry, School of Pharmacy,
Health Science Center, Xi’an Jiaotong University, No. 76, Yanta
West Road, Xi’an 710061, People’s Republic of China
e-mail: xmhcpu@163.com
M. Xin Á L. Zhang Á H. Shen Á J. Wen Á Z. Liu Á L. Cheng Á
X. Zhao
Jiangsu Simcere Pharmaceutical Co. Ltd., Jiangsu Key
Laboratory of Molecular Targeted Antitumor Drug Research,
No. 699-18, Xuan Wu District, Nanjing 210042, People’s
Republic of China
H. Shen Á J. Wen Á C. Tu Á L. Cheng Á X. Zhao
Nanjing BioSciKin Co. Ltd., Small Molecule Drug Discovery
Platform, No. 699-18, Xuan Wu District, Nanjing 210042,
People’s Republic of China
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Med Chem Res
Fig. 1 Representative Hh signaling pathway inhibitors
Fig. 2 Design of the 6,7-dihydro-5H-pyrano[2,3-d]pyrinidine derivatives
therapies (Fig. 1) (Hadden, 2013). Considering the prom-
ising therapeutic potential of Hh signaling pathway inhib-
itors in many solid and hematologic tumors, therefore, the
novel Hh signaling pathway inhibitors are still needed in
terms of exploring new anti-cancer therapeutic benefits for
solving unmet clinical needs.
derivatives bearing simple alkyl substituents at C5- and/or
C6-position with suitable size showed similar potency,
demonstrating that a sizable hydrophobic unit adjacent to the
pyrimidine ring was allowed (Huang et al., 2012). This result
inspired us to attempt some scaffold modifications by con-
ceptual cyclization of the 5-position and the 6-position of
pyrimidine, thereby being capable of developing new scaf-
fold as novel Hh signaling pathway inhibitors (Fig. 2).
Results and discussion
Pyrano[2,3-d]pyrimidines
and
dihydro-5H-pyrano
[2,3-d]pyrimidines ring systems are regarded as highly
valuable heterocyclic scaffolds in drug discovery (EI-
Agrody et al., 2001). Numerous pyrano[2,3-d]pyrimidine
derivatives have been abundantly described to have various
interesting pharmocologic activities (EI-Agrody et al.,
2001; Huang et al., 2012). Whereas 6,7-dihydro-5H-pyrano
[2,3-d]pyrimidine nucleus is considered as a bioisoster of
pyrano[2,3-d]pyrimidine. However, it was found in organic
synthesis and medicinal chemistry that only few reports
validated its pharmaceutical application (Herrera et al.,
2006). Considering that, in connection with our pursuit of
developing novel Hh signaling pathway inhibitors con-
taining new scaffold, we are interested in the development
Design of the 6,7-dihydro-5H-pyrano[2,3-d]pyrinidine
derivatives
In previous studies, we detailed the discovery of a novel
series of highly potent Hh signaling pathway inhibitors
structurally featured by 4-(2-pyrimidinylamino)benzamides
(Xin et al., 2013; Xin et al., 2014a). SAR studies of this series
revealed that substituents outside the B-ring were limited to
alter the substitutions at C5- and/or C6-position of the
pyrimidine core, derivatizing some significantly potent
analogs, such as 5-methyl (1), 5-ethyl (2), 6-methyl (3),
6-propyl (4), and 5,6-dimethyl (5) (Fig. 2). All of these
123

Med Chem Res
Scheme 1 Reagents and conditions: a CH3SCN, Tf2O, anhydrous
CH2Cl2, -78–0 °C, 40 h, 23 %, b MCPBA, CH2Cl2, rt, 5 h, 98 %,
c 10 % NaOH/MeOH, reflux, 2 h, 91 %, d POCl3, DIPEA, reflux,
18 h, 52 %, e 4-Trifluoromethoxyphenylboronic acid, Pd(PPh3)2Cl2,
2 M Na2CO3, dioxane, reflux, 2 h, 32 %, f methyl 4-aminobenzoate,
Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux, 13 h, 86 %, g 2 M
NaOH, THF/MeOH, reflux, 5 h, 100 %, h 2,6-Dimethylaniline,
HATU, TEA, DMF, rt for 16 h, 80 %, i 3-Amino-4-methylbenzyl
alcohol, HATU, DMF, DIPEA, 80 °C, 17 h, 79 %, j SOCl2, CH2Cl2,
rt, 17 h, 93 %, k DMF, K2CO3, rt for 6 h, N-methylpiperazine for 17,
71 %, morpholine for 18, 80 %, pyrrolidine for 19, 100 %, diethyl-
amine for 20, 94 %, octahydrocyclopenta[c]pyrrole for 21, 81 %,
2-oxa-6-azaspiro[3.3]heptane for 22, 90 %
of new scaffold containing this dihydro-5H-pyrano[2,3-d]
pyrimidine heterocyclic system. Particularly, 6,7-dihydro-
5H-pyrano[2,3-d]pyrimidine core could be considered as
pyrimidine derivatizing nucleus by an ethoxy linker con-
nected the 5-position of pyrimidine to the 6-position.
Accordingly, herein we report the synthesis of this new
6,7-dihydro-5H-pyrano[2,3-d]pyrimidine series as well as
the evaluation of their potencies toward the Hh signaling
pathway (Fig. 2). Furthermore, the pharmacokinetic (PK)
property of compound 18 is also profiled. To the best of our
knowledge, this is the first report that 6,7-dihydro-5H-
pyrano[2,3-d]pyrimidine is capable of a surrogate as
pyrimidine derivatizing skeleton and its systemic synthesis
in drug discovery.
delta-valerolactone 6 and methyl thiocyanate (CH3SCN),
the bicyclic pyrano[2,3-d]pyrimidinyl 7 was constructed in
the presence of trifluoromethanesulfonic anhydride (Tf2O)
with 23 % yield. The dimethylthio group of 7 was oxidized
by 3-chloroperoxybenzoic acid (MCPBA) to provide bis-
methylsulfonyl 8. Nucleophilic displacement of both
methylsulfonyl groups using reflux sodium hydroxide
(NaOH) solution gave diketone 9, followed by chlorination
using phosphoryl chloride (POCl3) to afford the key
building block 10. Then, the readily prepared 10 was
reacted with p-trifluoromethoxylphenylboronic acid under
palladium-catalyzed Suzuki–Miyaura coupling condition to
yield 4-phenyl-substituted 11, and then followed by
Buchwald–Hartwig cross-coupling reaction of 11 with
methyl 4-aminobenzoate under palladium acetate
[Pd(OAc)2] catalysis, to afford the intermediate 12 which
was easily hydrolyzed to the free acid 13. 6,7-Dihydro-5H-
pyrano[2,3-d]pyrimidine derivative 14 was achieved by
condensation of 13 with 2,6-dimethylaniline. Derivatives
17–22 were synthesized from carboxylic acid 13 following
three steps, which were condensation with 3-amino-4-
methylbenzyl alcohol to give 15, chlorination of 15 with
thionyl chloride (SOCl2) to provide 16, and nucleophilic
Synthesis of the 6,7-dihydro-5H-pyrano[2,3-
d]pyrimidine derivatives (14 and 17–22)
The 6,7-dihydro-5H-pyrano[2,3-d]pyrimidine derivatives
(14 and 17–22) were prepared as shown in Scheme 1. At
first, we constructed the key intermediate 2,4-dichloro-6,7-
dihydro-5H-pyrano[2,3-d]pyrimidine 10 following a four-
step synthetic route. Starting from commercially available
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Med Chem Res
MRT(0-t) T1/2
Table 1 Hh signaling pathway inhibitions of 6,7-dihydro-5H-pyrano
[2,3-d]pyrimidine derivatives
Table 2 PK profile of compound 18
Compounds Cmax AUC Vz
Cl
(ng/mL) (hr*ng/ (mL/kg) (mL/hr/ (h)
mL) kg)
(h)
GDC-0449 1,249.82 2,455.65 954.15 406.23 2.23
18 441.74 1,568.39 4,551.44 621.01 5.02
1.64
5.14
Compound 18 (as well as GDC-0449) was formulated using 5 %
DMA ? 5 % Tween-80, iv 1 mg/kg
based on the previous SAR study. It was found that only the
morpholine 18 (6.9 nM) generated satisfactorily equipotent
potency compared to GDC-0449; while other amine deriva-
tives, such as N-methylpiperazine 17 (16.7 nM), pyrrolidine
19 (16.1 nM), diethylamine 20 (15.4 nM), and bicyclic
amines 21 (17.6 nM) and 22 (15.7 nM), exhibited decreased
activity compared to GDC-0449 (Table 1).
Compounds
R
–
IC50 (nM)
14
17
5.2
16.7
N
N
N
N
N
N
18
19
20
21
6.9
16.1
15.4
17.6
O
PK profile of compound 18
Analog 18 was chosen as a representative of this new scaffold
to profile the PK properties in vivo, and the result is illustrated
in Table 2. After intravenous injection with 1 mg/kg in SD
rats, positive control GDC-0449 displayed satisfactory PK
profiles, with high exposure (AUC = 2,455.65 h 9 ng/mL)
and lower clearance (Cl = 406.23 mL/hr/kg). Compared to
GDC-0449, compound 18 exhibited moderate PK properties,
with an acceptable exposure (AUC = 1,568.39 h 9 ng/mL)
and relatively low clearance (Cl = 621.01 mL/hr/kg), and a
large volume of distribution (Vz = 4,551.44 mL/kg). How-
ever, 18 showed lower peak plasma concentrations
(Cmax = 441.74 ng/mL), meaning that it should be adminis-
trated in a high dose for generating in vivo antitumor efficacy
in Hh signaling dependent or operative solid tumor models.
Thus, some more efforts should be made for optimizing the
PK characteristics as well as in vitro biological activities for
the next structural modification.
22
15.7
N
O
1
–
–
1.3
7.2
GDC-0449
substitution of 16 with the corresponding amines to afford
17–22 (Scheme 1).
Hh signaling pathway inhibitions of 6,7-dihydro-5H-
pyrano[2,3-d]pyrimidine derivatives
The Hh signaling pathway inhibitory activity of our series of
6,7-dihydro-5H-pyrano[2,3-d]pyrimidine derivatives (14
and 17–22) were assessed using a luciferase reporter in
NIH3T3 cell carrying a stably transfected Gli reporter con-
struct (Gli-luciferase reporter cell lines) (Ohashi et al., 2012;
Xin et al., 2014b). GDC-0449 was used as positive control.
The vitro IC50 for this seriesare illustrated in Table 1. It was
found that all the analogs of 6,7-dihydro-5H-pyrano[2,3-d]
pyrimidine derivatives showed good Hh signaling inhibitory
activity with IC50 range from 5.2 to 17.6 nM. The B-ring of
6,7-dihydro-5H-pyrano[2,3-d]pyrimidine scaffold was
proved to be an appropriate surrogate for the parent pyrimi-
dine core; whereas the 6,7-dihydro-5H-pyrano[2,3-d]pyrim-
idine analog 14 displayed Hh inhibitory activity with an IC50
of 5.2 nM, despite showing weaker potency than the mother
compound 1 (1.3 nM), showing slightly more potency than
GDC-0449 (7.2 nM). Some aliphatic amine substitutions
were incorporated on C-5 of D-ring, derivatizing compounds
17–22, in order to improve the physicochemical properties
Conclusion
In this report, a novel series of 6,7-dihydro-5H-pyrano[2,3-d]
pyrimidine derivatives as Hh signaling pathway inhibitors
have been designed and synthesized, and their Hh signaling
pathway inhibitory activities were evaluated by in vitro
Gli-luciferase reporter assay. Among this new class of Hh
signaling pathway inhibitors, compound 18 exhibited
promising potency in vitro and moderate pharmacokinetic
properties in vivo compared to GDC-0449. Based on this
study, more extensive investigation is deserved to expand
so as to understand the structure–activity relationship in
depth and to further optimize the biological activities and
PK properties.
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Med Chem Res
Materials and methods
Chemistry
CH3), 2.53 (s, 3H, CH3), 2.52 (t, 2H, ArCH2), 2.04 (m, 2H,
CH2) ppm; MS (ESI) m/z: [M ? H]? = 229.0.
2,4-Bis(methylsulfonyl)-6,7-dihydro-5H-pyrano[2,3-
Melting points were measured with a Melt-Temp II appa-
ratus and uncorrected. The Nuclear Magnetic Resonance of
1H-NMR spectra (400 MHz) and 13C-NMR (100 MHz)
spectra were recorded on a Bruker BioSpin AG (Ultra-
shield Plus AV 400) spectrometer as deuterochloroform
(CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6) solutions.
The chemicals shift values were expressed in parts per
million (ppm) relative to tetramethylsilane as an internal
standard (d = 0) and signals were reported as s (singlet), d
(doublet), d (double doublet), t (triplet), and m (multiplet).
Mass spectra were recorded on an Agilent technologies
6120 quadrupole LC/MS spectrometer Electrospray Ioni-
zation (ESI) method. High-resolution mass spectra
(HRMS) were measured on a Water Q-T of micro mass
spectrometer. The purity of the compounds was verified by
the HPLC study performed on an Agilent Eclipse C18
(4.6 9 150 mm, 5 lm, Agilent) column using a mixture of
solvent methanol/water or CH3CN/H2O at the flow rate of
2 mL/min and peak detection at 254 nm under UV. The
solvents (such as CH2Cl2, EtOAc, MeOH, EtOH, and
others) were C.P. grade purchased from Nanjing Chemical
Co., Ltd. and used without further purification. Column
chromatography was carried out on silica gel (200–300
mesh, Qindao Ocean Chemical Company, China). Thin-
layer chromatography (TLC) analyses were carried out on
silica gel GF254 (Qindao Ocean Chemical Company,
China). GDC-0449 was synthesized according to the lit-
erature (Robarge et al., 2009). Concentration and evapo-
ration of the solvent after reaction or extraction was carried
out on rotary evaporator operated at reduced pressure.
d]pyrimidine (8) (Herrera et al., 2006)
In a 250-mL reaction vial, MCPBA (14 g, 6.85 eq) was
added fractionally to a solution of 7 (2.66 g, 1 eq) in
CH2Cl2 (70 mL). After 5 h of stirring at room temperature,
the reaction was quenched by 5 % Na2S2O3 solution
(150 mL). The organic phase was separated and washed by
saturated NaHCO3 solution. After dried by anhydrous
Na2SO4, the organic layer was concentrated in vacuo, and
the residue was crystallized from CH3OH, providing the
intermediate 8 (2.54 g, 98 %) as a white solid. Rf = 0.28
1
(PE : EtOAc = 5:1). H-NMR (400 MHz, DMSO-d6) d
4.59 (t, 2H, OCH2), 3.45 (s, 3H, CH3), 3.34 (s, 3H, CH3),
3.33 (t, 2H, ArCH2), 2.16 (m, 2H, CH2) ppm; MS (ESI)
m/z: [M ? H]? = 293.1.
6,7-Dihydro-5H-pyrano[2,3-d]pyrimidine-2,4-dione (9)
(Herrera et al., 2006)
In a 250-mL reaction vial, intermediate 8 (3.34 g, 1 eq)
was suspended in a 10 % NaOH methanolic solution
(115 mL), and the mixture was refluxed for 2 h. After
cooled to room temperature, the reaction was poured into
ice water, and adjusted to a pH 1–2 by 3 N HCl in an ice
bath. The precipitated solid was collected by filtration and
dried in vacuo to give 9 (1.5 g, 91 %) as a white solid.
Rf = 0.55 (CH2Cl2
:
CH3OH = 20:1). 1H-NMR
(400 MHz, CDCl3) d 11.19 (s, 1H, OH), 10.69 (s, 1H, OH),
4.24 (t, 2H, OCH2), 2.17 (t, 2H, ArCH2), 1.84 (m, 2H,
CH2) ppm; MS (ESI) m/z: [M ? H]? = 169.0.
2,4-Dimethylthio-6,7-dihydro-5H-pyrano[2,3-d]pyrimidine
(7) (Herrera et al., 2006)
2,4-Dichloro-6,7-dihydro-5H-pyrano[2,3-d]pyrimidine
(10) (Bergeron et al., 2010)
In a 250-mL reaction vial, a solution of Tf2O (30 mL,
1.5 eq) in anhydrous CH2Cl2 (30 mL) was slowly added to
a solution of delta-valerolactone (5 g, 1 eq) and CH3SCN
(13.5 mL, 4 eq) in anhydrous CH2Cl2 (50 mL) under
-78 °C. The mixture was stirred for 2 h under -70 °C,
then warmed to 0 °C, and stirred for 40 h at 0 °C. After the
resulting mixture was adjusted to a pH 8–9 with saturated
NaHCO3 solution, the organic layer was separated and the
aqueous layer was extracted by CH2Cl2. The organic layer
was combined and dried by anhydrous Na2SO4, The sol-
vent was removed in vacuo and the residue was crystallized
from n-hexane to give the intermediate 7 (3.4 g, 23 %) as a
white solid. Rf = 0.64 (PE : EtOAc = 5:1). 1H-NMR
(400 MHz, DMSO-d6) d 4.32 (t, 2H, OCH2), 2.57 (s, 3H,
In a 25-mL reaction vial, DIPEA (8 mL) was slowly added to
a mixture of 9 (1.0 g, 1 eq) and POCl3 (8 mL); the reaction
mixture was refluxed for 18 h. After cooled to room tem-
perature, the reaction was poured slowly into ice and stirred
vigorously for 30 min, followed by extraction with CH2Cl2.
The organic layer was washed with water and saturated
brine, and dried by anhydrous Na2SO4. The mother liquor
was concentrated and the residue was purified by chroma-
tography (PE : EtOAc = 5:1) to generate the intermediate 10
(636 mg, 52 %) as a white solid. Rf = 0.25 (PE :
1
EtOAc = 5:1). H-NMR (400 MHz, DMSO-d6) d 4.43 (t,
2H, OCH2), 2.78 (t, 2H, ArCH2), 2.10 (m, 2H, CH2) ppm; MS
(ESI) m/z: [M ? H]? = 205.0.
123

Med Chem Res
2-Chloro-4-(4-(trifluoromethoxy)phenyl)-6,7-dihydro-5H-
N-(2,6-Dimethylphenyl)-4-((4-(4-
pyrano[2,3-d]pyrimidine (11)
(trifluoromethoxy)phenyl)-6,7-dihydro-5H-pyrano[2,3-d]
pyrimidin-2-yl)amino)benzamide (14)
In a 25-mL reaction vial, a 2 M Na2CO3 solution (2 mL) was
added to a suspension of Pd(PPh3)2Cl2 (140 mg, 0.1 eq) and
4-trifluoromethoxylphenylboronic acid (416 mg, 1 eq) and
intermediate 10 (412 mg, 1 eq) in dioxane (10 mL). The
mixture was reflux for 2 h under N2 atmosphere, then cooled
to room temperature and filtrated. The filtrate was diluted
with H2O (100 mL) and extracted with EtOAc. After dried
by anhydrous Na2SO4, the liquor was evaporated and puri-
fied by chromatography (PE : EtOAc = 5:1) to provide 11
(210 mg, 32 %) as a white solid. Rf = 0.32 (PE :
In a 25-mL reaction vial, HATU (114 mg, 1.25 eq) and TEA
(56 mg, 2 eq) were added to a solution of 13 (51 mg, 1 eq)
and 2,6-dimethylaniline (15 mg, 1 eq) in DMF (10 mL). The
mixture was stirred at room temperature for 16 h under N2
atmosphere and then poured into ice water (30 mL), fol-
lowing extraction with EtOAc. The liquor was concentrated
in vacuo and the residue was purified by chromatography (PE
: EtOAc = 2:1) to acquire the product 14 (42 mg, 80 %) as a
white solid, mp 246–248 °C. Rf = 0.18 (PE : EtOAc = 2:1).
1H-NMR (400 MHz, DMSO-d6) d 9.84 (s, 1H, CONH), 9.55
(s, 1H, NH), 7.93 (m, 4H, ArH), 7.84 (d, 2H, J = 8.4 Hz,
ArH), 7.54 (d, 2H, J = 8.0 Hz, ArH), 7.12 (s, 3H, ArH), 4.41
(t, 2H, OCH2), 2.69 (t, 2H, ArCH2), 2.18 (s, 6H, 2 9 CH3),
1.91 (m, 2H, CH2) ppm; 13C-NMR (100 MHz, DMSO-d6) d
167.67, 164.56, 164.20, 157.66, 148.74, 143.83, 136.97,
135.70, 135.60, 130.81, 129.09, 128.21, 127.61, 127.52,
126.46, 126.23, 120.62, 118.24, 117.37, 104.20, 67.50,
22.29, 22.05, 18.06; MS (ESI) m/z: [M ? H]? = 535.3;
HRMS(ESI) m/z calcd for C29H25N4O3F3Na [M ? Na]?
557.1784, found 557.1776.
1
EtOAc = 5:1). H-NMR (400 MHz, DMSO-d6) d 7.64 (d,
2H, J = 8.4 Hz, ArH), 7.31 (d, 2H, J = 8.4 Hz, ArH), 4.48
(t, 2H, OCH2), 2.82 (t, 2H, ArCH2), 2.01 (m, 2H, CH2) ppm;
MS (ESI) m/z: [M ? H]? = 331.0.
Methyl 4-((4-(4-(trifluoromethoxy)phenyl)-6,7-dihydro-5H-
pyrano[2,3-d]pyrimidin-2-yl)amino)benzoate (12)
In a 25-mL reaction vial, Pd(OAc)2 (19 mg, 0.1 eq), BINAP
(108 mg, 0.2 eq), and Cs2CO3 (850 mg, 3 eq) were added to
a solution of 11 (301 mg, 1 eq) and methyl 4-aminobenzoate
(131 mg, 0.95 eq) in dioxane (5 mL). The mixture was
refluxed for 13 h under N2 atmosphere. After cooled to room
temperature and filtrated, the filtrate was evaporated and
purified by chromatography (PE : EtOAc = 5:1) to provide
the 12 (334 mg, 86 %) as a white solid. Rf = 0.15 (PE :
N-(5-(Hydroxymethyl)-2-methylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)-6,7-dihydro-5H -pyrano[2,3-
d]pyrimidin-2-yl)amino)benzamide (15)
In a 25-mL reaction vial, HATU (356 mg, 1.2 eq) and DIPEA
(201 mg, 2 eq) were added to a solution of 13 (337 mg, 1 eq)
and 3-amino-4-methylbenzyl alcohol (107 mg, 1 eq) in DMF
(5 mL). The reaction mixture was stirred at 80 °C for 17 h
under N2 atmosphere. The resulting mixture was cooled to
room temperature, poured into ice water (50 mL), and
extracted by EtOAc. After dried over anhydrous Na2SO4, the
organic layer was concentrated and purified by chromatog-
raphy (CH2Cl2 : CH3OH = 50:1) to provide 15 (340 mg,
79 %)asa white solid. Rf = 0.27(CH2Cl2 : CH3OH = 50:1).
1H-NMR (400 MHz, DMSO-d6) d 9.85 (s, 1H, CONH), 9.63
(s, 1H, NH), 7.88(s, 4H, ArH), 7.83(d, 2H, J = 8.4 Hz, ArH),
7.51 (d, 2H, J = 8.4 Hz, ArH), 7.29 (s, 1H, ArH), 7.21 (d, 1H,
J = 8.0 Hz, ArH), 7.10(d, 1H, J = 7.6 Hz, ArH), 5.17(t, 1H,
OH), 4.46 (d, 2H, J = 5.6 Hz, CH2OH), 4.40 (t, 2H, OCH2),
2.68 (t, 2H, ArCH2), 2.20 (s, 3H, CH3), 1.90 (m, 2H, CH2)
ppm; MS (ESI) m/z: [M ? H]? = 551.0.
1
EtOAc = 2:1). H-NMR (400 MHz, DMSO-d6) d 7.98 (d,
2H, J = 8.4 Hz, ArH), 7.70 (m, 4H, ArH), 7.32 (d, 2H,
J = 8.8 Hz, ArH), 7.22 (s, 1H, NH), 4.46 (t, 2H, OCH2), 3.86
(s, 3H, CH3), 2.75 (t, 2H, ArCH2), 1.99 (m, 2H, CH2) ppm;
MS (ESI) m/z: [M ? H]? = 446.0.
4-((4-(4-(Trifluoromethoxy)phenyl)-6,7-dihydro-5H-
pyrano[2,3-d]pyrimidin-2-yl)amino)benzoic acid (13)
In a 25-mL reaction vial, 2 M NaOH (1.5 mL) was added
to a solution of 12 (334 mg, 1 eq) in THF (5 mL) and
MeOH (5 mL). The reaction mixture was refluxed for 5 h,
then cooled to room temperature, poured into ice water
(30 mL), and stirred vigorously. After acidification with
1 N HCl, the precipitated solid was collected by filtration
and washed with ice water, dried in vacuo to provide 13
(337 mg, 100 %) as a white solid. Rf = 0.05 (PE :
1
EtOAc = 2:1). H-NMR (400 MHz, DMSO-d6) d 9.47 (s,
N-(5-(Chloromethyl)-2-methylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)-6,7-dihydro-5H -pyrano[2,3-
d]pyrimidin-2-yl)amino)benzamide (16)
1H, NH), 7.81 (d, 2H, J = 8.4 Hz, ArH), 7.75 (d, 2H,
J = 8.0 Hz, ArH), 7.64 (d, 2H, J = 8.4 Hz, ArH), 7.52 (d,
2H, J = 8.4 Hz, ArH), 4.37 (t, 2H, OCH2), 2.66 (t, 2H,
ArCH2), 1.88 (m, 2H, CH2) ppm; MS (ESI) m/z:
[M ? H]? = 432.0.
In a 25-mL reaction vial, SOCl2 (730 mg, 10 eq) was
added to a solution of 15 (340 mg, 1 eq) in CH2Cl2
123

Med Chem Res
(10 mL). After stirred at room temperature for 17 h, the
reaction mixture was washed with saturated NaHCO3
solution and water, and dried over anhydrous Na2SO4. The
solvent was removed in vacuo and the residue was purified
by chromatography (CH2Cl2 : CH3OH = 100:1) to give
the chloride 16 (329 mg, 93 %) as a white solid. Rf = 0.52
7.09 (d, 1H, J = 8.0 Hz, ArH), 4.46 (t, 2H, OCH2), 3.71 (m,
4H, (CH2)2O), 3.51 (s, 2H, PhCH2N), 2.74 (t, 2H, ArCH2),
2.47 (s, 4H, N(CH2)2), 2.31 (s, 3H, PhCH3), 2.00 (m, 2H,
CH2) ppm; 13C-NMR (100 MHz, CDCl3) d 168.10, 165.38,
165.16, 157.69, 149.93, 143.26, 136.36, 135.95, 130.38,
128.21, 127.78, 125.90, 123.76, 120.73, 118.12, 104.19,
68.05, 66.94, 63.08, 53.52, 29.70, 17.57; MS (ESI) m/z:
[M ? H]? = 620.3; HRMS(ESI) m/z calcd for
C33H33N5O4F3 [M ? H]? 620.2440, found 620.2434.
1
(CH2Cl2 : CH3OH = 50:1). H-NMR (400 MHz, DMSO-
d6) d 9.87 (s, 1H, CONH), 9.69 (s, 1H, NH), 7.91 (s, 4H,
ArH), 7.83 (d, 2H, J = 8.4 Hz, ArH), 7.53 (d, 2H,
J = 8.0 Hz, ArH), 7.44 (s, 1H, ArH), 7.28 (d, 1H,
J = 8.0 Hz, ArH), 7.23 (d, 1H, J = 8.0 Hz, ArH), 4.76 (s,
2H, CH2Cl), 4.40 (t, 2H, OCH2), 2.68 (t, 2H, ArCH2), 2.23
(s, 3H, CH3), 1.91 (m, 2H, CH2) ppm; MS (ESI) m/z:
[M ? H]? = 569.0.
N-(2-Methyl-5-(pyrrolidin-1-ylmethyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)-6,7-dihydro-5H-pyrano[2,3-
d]pyrimidin-2-yl)amino)benzamide (19)
Similar procedure of 17 was employed, and the compound 19
(56 mg, 100 %) was obtained as a white solid. mp
116–118 °C. Rf = 0.16 (CH2Cl2 : CH3OH = 20:1). 1H-
NMR (400 MHz, DMSO-d6) d 9.86 (s, 1H, CONH), 9.64 (s,
1H, NH), 7.91 (s, 4H, ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH),
7.54 (d, 2H, J = 8.0 Hz, ArH), 7.30 (s, 1H, ArH), 7.21 (d,
1H, J = 8.0 Hz, ArH), 7.10 (d, 1H, J = 7.6 Hz, ArH), 4.41
(t, 2H, OCH2), 3.58 (s, 2H, PhCH2N), 2.69 (t, 2H, ArCH2),
2.47 (s, 4H, N(CH2)2), 2.21 (s, 3H, CH3), 1.90 (m, 2H, CH2),
1.70 (s, 4H, CH2CH2) ppm; 13C-NMR (100 MHz, DMSO-
d6) d 167.65, 164.75, 164.19, 157.64, 148.75, 143.89,
136.95, 136.43, 131.99, 130.82, 129.95, 128.34, 126.56,
126.30, 120.65, 117.27, 104.21, 59.01, 53.39, 23.05, 22.06,
21.54, 17.63; MS (ESI) m/z: [M ? H]? = 604.3;
HRMS(ESI) m/z calcd for C33H33N5O3F3 [M ? H]?
604.2543, found 604.2536.
N-(2-Methyl-5-((4-methylpiperazin-1-yl)methyl)phenyl)-4-
((4-(4-(trifluoromethoxy)phenyl)-6,7- dihydro-5H-
pyrano[2,3-d]pyrimidin-2-yl)amino)benzamide (17)
In a 10-mL reaction vial, N-methylpiperazine (44 mg, 5 eq)
and K2CO3 (24 mg, 3 eq) were added to a solution of 16
(50 mg, 1 eq) in DMF (2 mL). The mixture was stirred at
room temperature for 6 h and poured into ice water (20 mL),
extracted by EtOAc. After dried over anhydrous Na2SO4, the
organic layer was concentrated and purified by chromatog-
raphy (CH2Cl2 : CH3OH = 20:1) to give the product 17
(40 mg, 71 %) as a white solid, mp 184–186 °C. Rf = 0.15
1
(CH2Cl2 : CH3OH = 20:1). H-NMR (400 MHz, DMSO-
d6) d 9.88 (s, 1H, CONH), 9.67 (s, 1H, NH), 7.92 (s, 4H,
ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 2H,
J = 8.0 Hz, ArH), 7.34 (s, 1H, ArH), 7.25 (d, 1H,
J = 7.6 Hz, ArH), 7.12 (d, 1H, J = 6.4 Hz, ArH), 4.41 (t,
2H, OCH2), 3.55 (s, 2H, PhCH2N), 2.69 (t, 2H, ArCH2), 2.68
(m, 8H, N(CH2CH2)2N), 2.23 (s, 3H, CH3), 1.91 (m, 2H,
CH2) ppm; 13C-NMR (100 MHz, DMSO-d6) d 167.65,
164.78, 164.20, 157.63, 148.74, 143.94, 136.95, 136.60,
130.82, 130.11, 128.37, 126.24, 120.65, 117.27, 104.24,
67.52, 60.58, 52.75, 49.63, 45.38, 21.54, 17.64; MS (ESI)
m/z: [M ? H]? = 633.3; HRMS(ESI) m/z calcd for
C34H36N6O3F3 [M ? H]? 633.2809, found 633.2801.
N-(5-((Diethylamino)methyl)-2-methylphenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)-6,7-dihydro- 5H-pyrano[2,3-
d]pyrimidin-2-yl)amino)benzamide (20)
Similar procedure of 17 was employed, and the title com-
pound 20 (50 mg, 94 %) was obtained as a white solid. mp
168–170 °C. Rf = 0.23 (CH2Cl2 : CH3OH = 20:1). 1H-
NMR (400 MHz, DMSO-d6) d 9.87 (s, 1H, CONH), 9.65 (s,
1H, NH), 7.91 (s, 4H, ArH), 7.84 (d, 2H, J = 8.8 Hz, ArH),
7.54 (d, 2H, J = 8.4 Hz, ArH), 7.31(s, 1H, ArH), 7.21 (d, 1H,
J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 7.6 Hz, ArH), 4.41 (t,
2H, OCH2), 3.54 (s, 2H, PhCH2N), 2.69 (t, 2H, ArCH2), 2.51
(m, 4H, N(CH2)2), 2.21 (s, 3H, PhCH3), 1.91 (m, 2H, CH2),
1.00 (m, 6H, 2 9 CH3) ppm; 13C-NMR (100 MHz, DMSO-
d6) d 168.15, 165.24, 164.69, 158.14, 149.25, 144.39, 137.44,
136.94, 131.31, 130.41, 128.84, 127.21, 126.80, 121.13,
117.77, 104.70, 68.00, 56.82, 46.49, 29.48, 22.79, 22.04,
18.13, 11.89; MS (ESI) m/z: [M ? H]? = 606.3;
HRMS(ESI) m/z calcd for C33H35N5O3F3 [M ? H]?
606.2701, found 606.2692.
N-(2-Methyl-5-(morpholinomethyl)phenyl)-4-((4-(4-
(trifluoromethoxy)phenyl)-6,7-dihydro-5H-pyrano[2,3-
d]pyrimidin-2-yl)amino)benzamide (18)
Similar procedure of 17 was employed to provide compound
18 (42 mg, 90 %) as a white solid. mp 237–239 °C.
Rf = 0.18 (CH2Cl2 : CH3OH = 20:1). 1H-NMR (400 MHz,
CDCl3) d 7.95 (s, 1H, CONH), 7.86 (d, 2H, J = 8.4 Hz,
ArH), 7.79 (d, 2H, J = 8.4 Hz, ArH), 7.68 (d, 2H,
J = 8.0 Hz, ArH), 7.62 (s, 1H, NH), 7.44 (s, 1H, ArH), 7.35
(d, 2H, J = 8.0 Hz, ArH), 7.18 (d, 1H, J = 7.6 Hz, ArH),
123

Med Chem Res
N-(5-((Hexahydrocyclopenta[c]pyrrol-2(1H)-yl)methyl)-2-
methylphenyl)-4-((4-(4-(trifluoro methoxy)phenyl)-6,7-
dihydro-5H-pyrano[2,3-d]pyrimidin-2-
cells were treated with DMEM and 10 % FBS and 1 lg/
mL Puromycin. The cells were seeded onto 96-well plates
at 2 9 104cells/well and cultured in the condition of 5 %
CO2 and 37 °C overnight. After incubation, all the pre-
pared compounds (including GDC-0449, as an internal
yl)amino)benzamide (21)
Similar to the preparation of 17, the title compound 21
(46 mg, 81 %) was obtained as a white solid. mp
194–196 °C. Rf = 0.18 (CH2Cl2 : CH3OH = 20:1). 1H-
NMR (400 MHz, DMSO-d6) d 9.87 (s, 1H, CONH), 9.65 (s,
1H, NH), 7.92 (s, 4H, ArH), 7.84 (d, 2H, J = 8.4 Hz, ArH),
7.54 (d, 2H, J = 8.0 Hz, ArH), 7.31 (s, 1H, ArH), 7.22 (d,
1H, J = 7.6 Hz, ArH), 7.11 (d, 1H, J = 7.2 Hz, ArH), 4.41
(t, 2H, OCH2), 3.57 (s, 2H, PhCH2N), 2.69 (t, 2H, ArCH2),
2.67 (m, 2H, N(CH2)2), 2.22 (s, 3H, CH3), 2.21 (m, 2H,
N(CH2)2), 1.90 (m, 2H, CH2), 1.61 (m, 2H, 2 9 CH), 1.26-
1.60 (m, 6H, octahydrocyclopentane-3 9 CH2) ppm; 13C-
NMR (100 MHz, DMSO-d6) d, 167.65, 164.77, 164.18,
157.64, 148.74, 143.92, 136.94, 136.46, 130.81, 130.02,
128.35, 126.65, 126.28, 125.93, 120.63, 118.78, 117.28,
104.20, 67.50, 60.57, 58.40, 41.67, 32.39, 22.30, 22.06,
17.63; MS (ESI) m/z: [M ? H]? = 644.3; HRMS(ESI) m/z
calcd for C36H37N5O3F3 [M ? H]? 644.2857, found
644.2849.
standard control) diluted in
a serial 8 9 solution
(0.05–300 nM) containing 0.5 % FBS and 0.7 lg/mL
Sonic Hh agonist were added to each well (n = 4 wells per
concentration). The cells were incubated for an additional
48 h. To determine the assay window, cells were incubated
in media containing 0.1 % DMSO with or without Sonic
Hh (0 % or 100 % inhibition control), respectively. Cells
were then harvested and lysed in reporter lysis buffer,
and luciferase activities were measured using a Dual-
LuciferaseÒ Reporter Assay System (Promega E1910). The
activity of the Gli reporter was defined as the ratio of
Firefly/Renilla luciferase activities.
In vivo pharmacokinetic Study in SD rats
Six healthy male SD rats (weight ranging from 180 to
240 g, three for GDC-0449 and three for compound 18)
were administered with GDC-0449 or compound 18 at a
dose of 1 mg/kg body weight for iv administration. The
dosing volume was 5 mL/kg. After administration, blood
samples collected at each time point (2 min, 5 min,
15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h) for
PK analyses (n = 3) were centrifuged at 4,000 rpm for
5 min at 4 °C. Plasma samples were analyzed after protein
precipitation with CH3CN acidified with 1 % HCOOH.
LC/MS/MS (Agilent 6418B) analysis of compound 18 was
performed under optimized conditions to obtain the best
sensitivity and selectivity of the analyte in the selected
reaction monitoring mode containing an internal standard.
Plasma concentration–time data were measured by a non-
compartmental approach using the software WinNonlin
Enterprise, version 5.2 (Pharsight Co.,Mountain View, CA,
USA) (Xin et al., 2014b).
N-(5-(2-Oxa-6-azaspiro[3.3]heptan-6-ylmethyl)-2-
methylphenyl)-4-((4-(4-(trifluoromethoxy)phenyl)-6,7-
dihydro-5H-pyrano[2,3-d]pyrimidin-2-
yl)amino)benzamide (22)
Similar to the preparation of 17, the title compound 22
(30 mg, 90 %) was acquired as a white solid. mp
175–177 °C. Rf = 0.18 (CH2Cl2 : CH3OH = 20:1). 1H-
NMR (400 MHz, DMSO-d6) d 9.86 (s, 1H, CONH), 9.62 (s,
1H, NH), 7.90 (s, 4H, ArH), 7.83 (d, 2H, J = 8.4 Hz, ArH),
7.54 (d, 2H, J = 8.4 Hz, ArH), 7.22 (s, 1H, ArH), 7.19 (d,
1H, J = 7.6 Hz, ArH), 7.03 (d, 1H, J = 7.6 Hz, ArH), 4.60
(s, 4H, N(CH2)2), 4.40 (t, 2H, OCH2), 3.49 (s, 2H, PhCH2N),
3.32 (s, 4H, (CH2)2O), 2.68 (t, 2H, ArCH2), 2.19 (s, 3H,
PhCH3), 1.89 (m, 2H, CH2) ppm; 13C-NMR (100 MHz,
DMSO-d6) d 167.65, 164.72, 164.18, 157.64, 148.75,
143.91, 136.95, 136.51, 132.11, 130.82, 130.04, 128.35,
117.27, 126.32, 126.29, 125.63, 120.65, 118.78, 104.22,
79.91, 67.51, 62.73, 61.72, 28.98, 22.30, 22.06, 17.63; MS
(ESI) m/z: [M ? H]? = 632.3; HRMS(ESI) m/z calcd for
C34H33N5O4F3 [M ? H]? 632.2493, found 632.2485.
Acknowledgments This work was supported by the National Major
Science and Technology Project of China (Innovation and Develop-
ment of New Drugs, No. 2011ZX09401-008).
Conflict of interest The authors have reported no conflict of
interest.
In vitro Gli-luciferase reporter assay
References
Bergeron P, Cohen F, Estrada A, Koehler MF, Ly C, Lyssikatos JP,
Pei Z, Lee W, Zhao X (2010) Oxo-heterocycle fused pyrimidine
compounds, compositions and methods of use. Patent Applica-
tion US 2010/0331305
Dlugosz A, Agrawal S, Kirkpatrick P (2012) Vismodegib. Nat Rev
Drug Disc 11:437–438
The Hh signaling inhibition of the 6,7-dihydro-5H-pyr-
ano[2,3-d]pyrimidine derivatives were assessed through a
luciferase reporter assay in NIH3T3 cells carrying a stably
transfected Gli reporter construct (Gli-luc reporter cell line)
(Ohashi et al., 2012; Xin et al., 2013). NIH3T3/Gli-luc
123

Med Chem Res
EI-Agrody AM, EI-Latif MS Abd, EI-Hady NA, Fakery AH, Bedair
AH (2001) Heteroaromatization with 4-hydroxycoumarin part II:
synthesis of some new pyrano[2,3-d]pyrimidines, [1,2,4]triazolo
[1,5-c]pyrimidines and pyrimido[1,6-b][1,2,4]triazine deriva-
tives. Molecules 6:519–527
Robarge KD, Brunton SA, Castanedo GE, Cui MS, Goldsmith R,
Gould SE, Guichert O, Gunzner JL, Halladay J, Jia W, Khojasteh
C, Koehler MF, Kotkow K, La H, LaLonde RL, Lau K, Lee L,
Marshall D, Marsters JC, Murray LJ, Qian C, Rubin LL, Salphati
L, Stanley MS, Stibbard JH, Sutherlin DP, Ubhayaker S, Wang
S, Wong S, Xie M (2009) GDC-0449-A potent inhibitor of the
hedgehog pathway. Bioorg Med Chem Lett 19:5576–5581
Ruch JM, Kim EJ (2013) Hedgehog signaling pathway and cancer
therapeutics: progress to date. Drugs 73:1–11
Xin M, Wen J, Tang F, Tu C, Shen H, Zhao X (2013) The discovery
of novel of N-(2-pyrimidinylamino) benzamide derivatives as
potent hedgehog signaling pathway inhibitors. Bioorg Med
Chem Lett 23:6777–6783
Xin M, Wen J, Tang F, Tu C, Huang W, Shen H, Zhao X, Cheng L,
Wang M, Zhang L (2014a) Synthesis and evaluation of novel of
4-(2-pyrimidinylamino) benzamides inhibitors of hedgehog
signaling pathway. Bioorg Med Chem Lett 24:983–988
Xin M, Zhang L, Tang F, Tu C, Wen J, Zhao X, Liu Z, Cheng L, Shen
H (2014b) Design, synthesis and evaluation of pyrrolo[2,1-f]
[1,2,4]triazine derivatives as novel hedgehog signaling pathway
inhibitors. Bioorg Med Chem 22:1429–1440
Hadden MK (2013) Hedgehog pathway inhibitors: a patent review
(2009-present). Exp Opin Ther Pat 23:345–361
Herrera A, Martinez-Alvarez R, Ramiro P, Almy J (2006) An easy
synthesis of pyranopyrimidines. Monatsh Chem 137:1421–1430
Huang X, Su J, Rao AU, Tang H, Zhou W, Zhu X, Chen X, Liu Z,
Huang Y, Degrado S, Xiao D, Qin J, Aslanian R, McKittrick BA,
Greenfeder S, Heek MV, Chintala M, Palani A (2012) SAR
studies of C2 ether of 2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,
3H) –triones as nicotinic acid receptor (NAR) agonist. Bioorg
Med Chem 22:854–858
Ng JM, Curran T (2011) The Hedgehog’s tale: developing strategies
for targeting cancer. Nat Rev Cancer 11:493–501
Ohashi T, Oguro Y, Tanaka T, Shiokawa Z, Shibata S, Sato Y,
Yamakawa H, Hattori H, Yamamoto Y, Kondo S, Miyamoto M,
Tojo H, Baba A, Sasaki S (2012) Discovery of pyrrolo[3,2-c]
quinoline-4-one derivatives as novel hedgehog signaling inhib-
itors. Bioorg Med Chem 20:5496–5506
Onishi H, Katano M (2011) Hedgehog signaling pathway as a
therapeutic target in various types of cancer. Cancer Sci
102:1756–1760
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