Design, Palladium-Catalyzed Synthesis, and Biological Investigation of 2-Substituted 3-Aroylquinolin-4(1H)-ones as Inhibitors of the Hedgehog Signaling Pathway

Article abstract of DOI:10.1021/acs.jmedchem.6b01135

2-Substituted 3-aroylquinolin-4(1H)-ones, prepared through a palladium-catalyzed carbonylative cyclization of N-(2-iodoaryl)enaminones, proved to inhibit efficiently the Hedgehog pathway through direct antagonism of the wild-type and drug-resistant form o

Full text of DOI:10.1021/acs.jmedchem.6b01135

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Article
Design, palladium-catalyzed synthesis, and biological
investigation of 2-substituted 3-aroylquinolin-4(1H)-
ones as inhibitors of the Hedgehog signaling pathway.
Romina Alfonsi, Bruno Botta, Sandro Cacchi, Lucia Di Marcotullio, Giancarlo
Fabrizi, Roberta Faedda, Antonella Goggiamani, Antonia Iazzetti, and Mattia Mori
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01135 • Publication Date (Web): 25 Jan 2017
Downloaded from http://pubs.acs.org on January 26, 2017
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Design, palladium-catalyzed synthesis, and
biological investigation of 2-substituted 3-
aroylquinolin-4(1H)-ones as inhibitors of the
Hedgehog signaling pathway
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Romina Alfonsi,† Bruno Botta,‡ Sandro Cacchi,‡ Lucia Di Marcotullio,†# Giancarlo Fabrizi,‡
Roberta Faedda,† Antonella Goggiamani,‡* Antonia Iazzetti,‡ and Mattia Mori ∫
† Department of Molecular Medicine, Sapienza University, Viale Regina Elena 291, 00161
Rome, Italy
‡ Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza University, Piazzale A. Moro 5,
00185 Rome, Italy
∫ Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Viale Regina Elena
291, 00161 Rome, Italy.
# Istituto Pasteur, Fondazione Cenci-Bolognetti; Sapienza University, Viale Regina Elena 291,
00161 Rome, Italy.
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ABSTRACT. 2-Substituted 3-aroylquinolin-4(1H)-ones, prepared through a palladium-catalyzed
carbonylative cyclization of N-(2-iodoaryl) enaminones, proved to inhibit efficiently the
Hedgehog pathway through direct antagonism of the wild-type and drug-resistant form of the
Smoothened receptor. Notably, these compounds repressed the Hh-dependent growth events and
the proliferation of tumor cells with aberrant activation of the Hh pathway, which plays a crucial
role in development and tumorigenesis.
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INTRODUCTION
Hedgehog (Hh) signaling is crucial for tissue development and stemness and its deregulation is
found in many tumors.1 In recent years, the Smoothened (Smo) receptor has emerged as the most
promising target for the development of anticancer drugs targeting the Hh pathway, as
underlined by the FDA-approved drug Vismodegib. Moreover, a number of Smo antagonists
have been developed up to advanced clinical trials.2 However, the clinical development of these
molecules has failed due to different issues, including pharmacokinetics, noncanonical Hh
signaling activation and, particularly, the emergence of drug resistant Smo mutations, thus
raising the need for novel Hh inhibitors. As part of a program devoted to the modulation of the
Hh signaling pathway by small molecules,3-7 we designed a number of new chemotypes for Smo
antagonists by coupling synthetic feasibility criteria with pharmacophoric and steric features. In
particular, new potential synthetic scaffolds were preliminary tested in silico by means of
molecular docking towards the crystallographic structure of the Smo receptor.8 Among tested
molecules, 2-substituted 3-aroylquinolin-4(1H)-ones 2 proved to fit within the antagonists
binding site of Smo, which is located within its heptahelical bundle, with the highest affinity as
well as with the best pharmacophoric overlapping with respect to crystallographic Smo
antagonists (Supporting Information (SI), Figure S1).
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For these reasons, this class of compounds appeared to us as a valuable target for the inhibition
of the Hh signaling pathway. Therefore, a number of derivatives have been designed in silico,
synthesized, and tested in vitro and ex vivo.
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RESULTS AND DISCUSSION
CHEMISTRY. For the synthesis of 2-substituted 3-aroylquinolin-4(1H)-ones, we envisioned
that the palladium-catalyzed carbonylative cyclization of readily available N-(2-iodoaryl)
enaminones 19 might be a convenient approach (Scheme 1).
Scheme 1. Palladium-catalyzed carbonylative cyclization of N-(2-iodoaryl) enaminones 1 to 2-
substituted 3-aroylquinolin-4(1H)-ones 2.
On the basis of the carbonylative cyclization of related 2-substituted N-(2-haloaryl)-2-
propenoates described by Torii and co.,10 we initially used the same reaction conditions
[Pd(OAc)2, PPh3, K2CO3, DMF, CO (20 atm), 120 °C] for the conversion of 1a, our model
system, into the corresponding 2a. However, 2a was isolated only in 5% yield after 24 h along
with a 76% yield of the recovered starting material and a 7% yield of indole, derived from the
direct palladium-catalyzed cyclization of 1a.11-14 Slightly better results, but still unsatisfactory
from a synthetic point of view, were obtained prolonging the reaction time to 72 h (2a: 30%;
recovered 1a: 53%; indole derivative: 13%).
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Apparently, the presence of a ketonic group (our work) instead of an ester group (Torii work)
in the β-amino-α,β-unsaturated carbonyl fragment of the substrate makes it more difficult the
carbonylative cyclization reaction. Thus, we started a study to explore the influence of bases,
solvents, ligands, the source of Pd(0) species, the pressure of carbon monoxide, and the reaction
time on the reaction outcome. Some of the results of this study are listed in Table 1 and show
that 2a could be isolated in satisfactory yields by using Pd2(dba)3, Cs2CO3 in MeCN at 100 °C
under 20 atm of carbon monoxide in the presence of bulky monodentate ligands such as SPhos
(Table 1, entry 6) and XPhos, (Table 1, entry 9), the best result being obtained with the latter.
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Table 1. Palladium-catalyzed carbonylative cyclization of 1a. Optimization studies.a
Entry Pd/ligand
CO
(atm)
Solvent Time Yield %
(h)
24
72
72
39
48
72
48
72
72
68
24
72
48
of 2ab,c
d
1
Pd(PPh3)4
1
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
MeCN
THF
‒
2
Pd(PPh3)4
5
tracese
tracesf
53
3
Pd(PPh3)4
10
20
20
20
4
Pd(PPh3)4
5
Pd2(dba)3/SPhos
Pd2(dba)3/SPhos
56
6
64
7
Pd2(dba)3/Xantphos 20
59
8
Pd2(dba)3/dppf
20
20
20
20
20
30
36g
68
9
Pd2(dba)3/XPhos
Pd2(dba)3/XPhos
Pd2(dba)3/XPhos
Pd2(dba)3/XPhos
Pd2(dba)3/XPhos
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11
12
13
41h
54i
DMF
MeCN
MeCN
42j
44k
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a Unless otherwise stated, reactions were carried out on a 0.30 mmol scale using 0.05 equiv of
[Pd], 0.05 equiv of ligand, 2 equiv of Cs2CO3 in 5 mL of solvent at 100 °C under an atmosphere
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b
of carbon monoxide (see Table). Yields are given for isolated products. Unless otherwise
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d
stated, no evidence of 1a was obtained after the allotted reaction time. The corresponding
indole was isolated in 78% yield. e 1a was recovered in 93% yield. f 1a was recovered in 96%
g
h
i
yield. 1a was recovered in 35% yield. 1a was recovered in 50% yield. The corresponding
indole was isolated in 14% yield. j The reaction was carried out using 2 equiv of K2CO3. 1a was
recovered in 33% yield. k 1a was recovered in 4% yield.
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Table 2. Synthesis of 3-aroylquinolin-4(1H)-ones 2 via palladium-catalyzed carbonylative
cyclization of N-(2-iodoaryl) enaminones 1.a
Entry
1
2a
(yield %)b,c
R
Ar1
Ar2
1
2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
2a (68)
2b (65)d
2c (73)e
2d (82)
2e (62)
2f (82)
2g (62)
2h (57)
2i (74)
3-CF3-C6H4
3-F-C6H4
3
4
3-MeO-C6H4
3-Me-C6H4
4-Me-C6H4
4-Cl-C6H4
4-MeO-C6H4
4-CN-C6H4
Ph
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7
8
9
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14
15
16
4-MeCO-C6H4
4-MeCO2-C6H4 Ph
2j (91)
2k (83)
2l (60)f
2m (58)g
2n (78)
2o (47)
2p (60)
4-Me-C6H4
Ph
4-MeO-C6H4
4-HO-C6H4
4-MeCO-C6H4
Ph
Ph
4-Cl-C6H4
Ph
4-MeO Ph
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4-Me
4-F
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
2q (88)
2r (70)h
2s (71)
2t (37)
4-Cl
4-Br
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a Unless otherwise stated, reactions were carried out on a 0.30 mmol scale using 0.025 equiv of
Pd2(dba)3, 0.05 equiv of XPhos, 2 equiv of Cs2CO3 in 5 mL of MeCN at 100 °C for 72 h under
b
20 atm of carbon monoxide. Yields are given for isolated products. Unless otherwise stated,
c
d
no evidence of the starting material was obtained after the allotted reaction time. The starting
e
f
material was recovered in 15% yield. The starting material was recovered in 10% yield. The
starting material was recovered in 14% yield. g The starting material was recovered in 12% yield.
h The starting material was recovered in 8% yield.
Using the optimized conditions, we next explored the scope and generality of the process. As
shown in Table 2, good to high yields are usually obtained with enaminones bearing both
electron-rich and electron-poor as well as neutral aromatic rings (Table 2, entries 1-7). 3-
Aroylquinolin-4(1H)-ones containing chlorine substituents, which may be key intermediates for
increasing the molecular complexity via transition metal-catalyzed coupling reactions, can be
successfully prepared (Table 2, entries 7, 15, and 19), although the yield is only moderate when
Ar1 bears a strongly electron-withdrawing substituent (Table 2, entry 15). A moderate yield of
the desired 3-aroylquinolin-4(1H)-one derivative is obtained with an N-(2-iodoaryl) β-enaminone
containing a bromine substituent in the aniline fragment (Table 2, entry 20).
This synthesis of 2-substituted 3-aroylquinolin-4(1H)-ones can also be carried out through a
process that omits the isolation of enaminone intermediates. In practice, satisfactory results can
be obtained by adding Pd2(dba)3, Xphos, Cs2CO3, MeCN, and CO (20 atm) to the crude mixture
derived from the reaction of 2-iodoanilines with α,β-ynones after evaporation of the volatile
materials. As an example, under these conditions 2a was isolated in 61% overall yield (Scheme
2).
Scheme 2. Sequential synthesis of 2a from 2-iodoaniline and 1,3-diphenylprop-2-yn-1-one.
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BIOLOGICAL EVALUATION. The Hh inhibitory activity of synthesized molecules was
investigated in a luciferase reporter assay, which is widely used for characterizing Hh inhibitors.
To this end, NIH3T3 Shh Light II cells, stably incorporating a Gli-responsive firefly luciferase
reporter (Gli-RE),15 were treated with the synthetic Smo agonist SAG16 alone or in combination
with the tested molecules to evaluate their ability to suppress Hh signaling. At the maximum
concentration of 20 ꢀM, molecules 2b, 2j, 2h, 2n, and 2s showed mild activity as Hh inhibitor
(SI, Figure S2A), while 2d and 2t showed high activity in this assay (Figure 1A). The remaining
15 compounds proved to be inactive (2c, 2g, 2m, SI, Figure S2B) or cytotoxic (data not shown),
as observed by a significant modulation of the internal control Renilla. In particular, 2d and 2t
proved to be the most potent Hh inhibitors of the test set, having an IC50 of 0.9 and 0.85 µM,
respectively (Figure 1A).
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Figure 1. Inhibition of Hh signaling and predicted binding mode of 2d and 2t. A) Dose–response
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curve of 2d and 2t in NIH T3 Shh-Light II cells. Treatment time was 48 h, and normalization
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was against Renilla luciferase. Data show the mean SD of three independent experiments.
*P<0.01 vs CTR. B-C) Predicted binding mode of 2d (B) and 2t (C) within the crystallographic
heptahelical bundle of the Smo receptor (PDB ID: 4JKV).17 Smo is shown as green transparent
surface. Residues interacting with small molecules are showed as green sticks, and are labeled.
2d and 2t are showed as cyan sticks. Polar interactions are highlighted by black dashed lines.
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Moreover, compounds 2d and 2t suppressed Hh genes signature in genetically defined
Ptch1-/- mouse embryonic fibroblasts (Ptch1-/- MEFs) (SI, Figure S3), in which deletion of
the inhibitory Ptch1 receptor releases Smo function and leads to a constitutive activation
of Gli transcription factors.18 According with molecular modeling, these molecules bind
the antagonists site of Smo, which is located within the heptahelical bundle, and establish
H-bond interactions with Arg400, a key residue for ligand binding highlighted in
crystallographic studies.8,19,20 In addition, 2d and 2t establish hydrophobic interactions with a
cluster of aromatic and hydrophobic residues including Leu221, Phe222, Met230, Ile234,
Trp281, Met301, Val386, Tyr394, Trp480, Phe484, Pro513, and Leu522 (Figure 1B-C).
Notably, these two Smo antagonists bind in a highly similar manner to each other within
the heptahelical bundle, with the aroylquinolin-4(1H)-one core being H-bonded to
Arg400, and Ar2 ring (Scheme 2 and Table 2) stacked with Trp480 or Tyr394. The Ar1
ring is docked in a hydrophobic sub-pocket bounded by Phe222 and Val386. The peculiar
meta-methoxy group of 2d is projected towards the solvent area at the upper part of Smo,
whereas the bromine atom of 2t is projected towards the lower intracellular portion of
Smo.
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These chemical moieties interact in hydrophobic sub-pockets of the Smo antagonist site.
Moreover, comparison between 2t and its corresponding chlorine derivative 2s, which proved
mild inhibition of the Hh signaling, emphasizes the relevance of hydrophobic halogen
substitutions within the inner part of the Smo heptahelical bundle. All inactive molecules bear
substitutions to the Ar1 ring, which are probably disadvantaged by the steric hindrance due to the
presence of Phe222 side chain, whereas substitutions to the Ar2 ring are favored only in meta
position. Finally, both 2d and 2t nicely overlap with crystallographic Smo antagonists and may
represent valuable starting points for further optimization based on molecular elongation (SI,
Figure S1).
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To test the ability of compounds 2d and 2t to interfere directly with the Smo receptor, we
carried out a displacement assay by using the Bodipy-Cyclopamine (BC), a fluorescent
derivative of cyclopamine that interacts with Smo at the level of its heptahelical bundle.8 We
used this assay to verify the capability of compounds 2d and 2t to bind both Smo WT or mutant
D477G, the mouse orthologous of the first described human Smo point mutation (D473H), which
confers resistance to Smo antagonist Vismodegib treatment.21,22 Notably, 2d and 2t proved to be
not sensitive to the D477G drug−resistant Smo mutation, showing dose−dependent effects and
equal binding affinity corresponding to similar IC50 values against both wild-type and drug-
resistant Smo (2d on mSmo-WT IC50 = 13.76 ꢀM, on mSmo–D477G IC50 = 12.41 ꢀM; 2t on
mSmo-WT IC50 = 23.96 ꢀM, on mSmo–D477G IC50 = 29.35ꢀM) (SI, Figure S4).
Hh signaling is crucial for the proliferation and differentiation of granule cell progenitors
(GCPs) that populate the external germinal layer (EGL) of the cerebellar cortex during the first
week after birth in mice.23 This process is triggered by Purkinje cell-derived Shh and is required
for intense GCPs proliferation and proper development of the granule neuron lineage. After the
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first week of age, a physiological withdrawal of Hh signal is responsible for the GCPs
proliferation arrest, leading to terminal differentiation of these cells. Aberrant regulation or
inappropriate reactivation of Hh pathway is responsible of the tumorigenic conversion of GCPs,
considered as the cell of origin of medulloblastoma (MB), an aggressive and the most common
pediatric brain tumor.24,25 To assess the efficacy of compound 2d and 2t to interfere with Hh-
dependent growth events, we tested their ability to suppress Hh signaling in SAG-activated
GCPs isolated from 4–day–old mice. As shown in SI, Figure S5, these compounds significantly
inhibited both mRNA (SI, Figure S5A) and protein level (SI, Figure S5B) of several Hh target
genes and marker related to cell proliferation.
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Figure 2. Ex vivo cell cultures from Ptch1+/- mice MBs. MB cells were freshly isolated from
Ptch1+/- mice tumor, cultured and treated with compound 2d or 2t (10 µM) or DMSO only, as
control. (A) After the indicated times, a trypan blue count was performed to determine the
growth rate of viable cells. (B) Hh target genes mRNA expression levels were determined by
qRT-PCR after 24h of treatment and normalized to endogenous control (β2-microglobulin and
HPRT). In all experiments data show the mean SD of three independent experiments. *P< 0.05
vs CTR (DMSO).
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In order to verify the effect of our selected compounds to impair the Hh-dependent tumor
growth, we turned to a MB model. To this end, primary MB cells were freshly isolated from
Ptch+/- mice tumors and tested in short-term cultures to keep Hh sensitivity in vitro.26-28
Compound 2d and 2t significantly arrested the proliferation of Ptch+/- MB cells (Figure 2A) and
inhibited Hh target gene mRNA levels (Figure 2B).
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Figure 3. 2d and 2t inhibit Hh–dependent BCC tumor cell growth. ASZ001 BCC cells were
treated with compound 2d or 2t or DMSO only, as control. (A) After the indicated times, a
trypan blue count was performed to determine the growth rate. (B) Hh target genes mRNA
expression levels in ASZ001 BCC cells were determined by qRT–PCR after 48h treatment with
compound 2d or 2t or DMSO only, as control. Results were normalized to endogenous control
(β2-microglobulin and HPRT). All data show the mean SD of three independent experiments.
*P < 0.05, **P < 0.01 versus CTR (DMSO).
Furthermore, we tested the efficacy of 2d and 2t to suppress the proliferation of a different Hh-
driven tumor cells, by using ASZ001 basal cell carcinoma cells (BCC), which have been
previously characterized as an Hh-dependent tumor cell line harboring Ptch1 deletion.29 At 10
ꢀM concentration, both molecules impaired ASZ001 BCC cell proliferation in a dose-dependent
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manner (Figure 3A), also in agreement with the significant decrease of Hh signature genes
observed after drug–treatment, as showed in Figure 3B.
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CONCLUSIONS
In conclusion, 2-substituted 3-aroylquinolin-4(1H)-ones were prioritized in silico in a search
for efficient small molecule inhibitors of the Hh signaling pathway. To access these compounds,
an efficient palladium-catalyzed carbonylative cyclization of N-(2-iodoaryl) enaminones was
developed. The reaction tolerates a variety of useful functionalities including ether, keto, cyano,
ester, and chlorine substituents. 2-Substituted 3-aroylquinolin-4(1H)-ones can also be prepared
via a sequential process from 2-iodoanilines and α,β-ynones omitting the isolation of N-(2-
iodoaryl) enaminone intermediates. Notably, a number of synthesized 2-substituted 3-
aroylquinolin-4(1H)-ones proved to inhibit the Hh pathway in vitro by binding to the Smo
receptor, the major upstream transducer of the pathway. The two most active compounds 2d and
2t were profiled for their mechanism of action, and proved to be not sensitive to the D477G drug-
resistant mutant of the Smo receptor. Finally, these 2-substituted 3-aroylquinolin-4(1H)-ones
were able to repress the growth of MB cells from Ptch+/- mice and ASZ001 BCC cells in a dose
dependent manner, thus opening new venues for the discovery of anticancer lead compounds
targeting the Hh pathway and overcoming drug resistance.
EXPERIMENTAL SECTION
General procedures. All of the reagents, catalysts, bases and solvents are commercially
1 13
available and were used as purchased, without further purification. H NMR (400.13 MHz), C
NMR (100.6 MHz), and 19F spectra (376.5 MHz) were recorded with a Bruker Avance 400
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spectrometer. Infrared spectra were recorded on a Jasco FT/IR-430 spectrophotometer. Melting
points were determined with a Büchi B-545 apparatus and are uncorrected.
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Mass spectra were determined with a QP2010 Gas Chromatograph Mass spectrometer (EI ion
source). Compounds 2a-t were converted into the corresponding N-methyl derivatives to obtain
suitable mass data according to the following typical procedure.
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To a stirred solution of 3-benzoyl-2-phenylquinolin-4(1H)-one (32.5 mg, 0.10 mmol) in 3 mL
of DMF, NaH (2.4 mg, 0.12 mmol) was added and the mixture was stirred at room temperature
for 0.5 h. After this time, MeI (7.4 ꢀl, 0.12 mmol) was added and the reaction was stirred until
the disappearance of the starting material. Then, the reaction mixture was quenched by water
(5mL) and extracted by diethyl ether (3×15 mL). The combined organic phases were dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The crude 3-benzoyl-1-methyl-2-
phenylquinolin-4(1H)-one was dissolved in diethyl ether and injected to the mass spectrometer.
HRMS was performed on a Bruker BioApex Fourier transform ion cyclotron resonance (FT-
ICR) mass spectrometer operated in ESI-HRMS (positive) mode, equipped with an Apollo I ESI
source, a 4.7 T superconducting magnet, and a cylindrical infinity cell (Bruker Daltonics,
Billerica, MA, USA).
Purity of 2a-t (found to be ≥95%) was determined by HPLC on a Jasco HPLC system using a
Macherey-Nagel NUCLEODUR Sphinx RP, 5 µm column and was determined to be ≥95%.
HPLC conditions: eluent, acetonitrile/water 80:20; flow rate, 1.0 mL/min; UV detection, 254 nm;
room temperature; injection volume: 10 µL.
Materials. N-(2-Iodoaryl)enaminones were prepared through conjugate addition of 2-
iodoanilines with the α,β-ynones, in turn prepared via Sonogashira cross-coupling of terminal
alkynes with aroyl chlorides.9
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Molecular Modeling. The crystallographic structure of the Smoothened receptor in complex
with an antitumor agent (PDB ID: 4JKV)17 was used as receptor in molecular modeling studies.
Small molecules 2a-t were sketched and built with the VIDA suite of OpenEye,30 while
conformational analysis was performed with Omega2 using default parameters and allowing the
storage of up to 400 conformers for each molecule.31,32 Molecular docking simulations were
performed with the Hybrid docking program,33-35 using settings previously described.3
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Typical procedure for the preparation of α,β-ynones: synthesis of 1,3-diphenylprop-2-yn-
1-one. Benzoyl chloride (843.5 mg, 697 ꢀL, 6.0 mmol), PdCl2(PPh3)2 (70 mg, 0.1 mmol), and
Et3N (607.1 mg, 834 ꢀL, 6 mmol) in anhydrous THF (20 mL) were stirred for 10 min under an
argon atmosphere at room temperature. CuI (38 mg, 0.2 mmol) was added and the resulting
reaction mixture was stirred for 10 min before adding ethynylbenzene (511.0 mg, 549 ꢀL, 5.0
mmol). After 2 h at room temperature, the reaction mixture was diluted with ethyl acetate,
washed with 2 N HCl, with a saturated NH4Cl solution, and with brine. The organic phase was
separated, dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was
purified by chromatography on SiO2 (25-40 ꢀm), eluting with a 95/5 (v/v) n-hexane/AcOEt
mixture to afford 958 mg (93% of yield) of 1,3-diphenylprop-2-yn-1-one: yellow solid; mp 43-
45°C; 1H NMR (400 MHz, CDCl3): δ 8.26 (d, J = 7.3 Hz, 2 H), 7.71 (d, J = 7.2 Hz, 2 H), 7.66 (t,
J = 7.2 Hz, 1 H), 7.57-7.43 (m, 5 H); 13C NMR (100 MHz, CDCl3): δ 178.1, 137.1, 134.2, 133.1,
130.8, 129.6, 128.8, 128.7, 120.3, 93.1, 87.0; IR (KBr): 2200, 1633, 1315, 1290, 690 (cm-1); MS
m/z (relative intensity) 206 (M+, 76), 178 (100), 129 (98), 75 (39), 51 (39).
Typical procedure for the preparation of N-(2-iodoaryl)enaminones (1): synthesis of 3-(2-
iodophenylamino)-1,3-diphenylprop-2-en-1-one (1a). An oven-dried Schlenk tube was
charged with 824 mg of 1,3-diphenylprop-2-yn-1-one (4 mmol), 876 mg of o-iodoaniline (4
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mmol), and anhydrous MeOH (1.5 mL). The reaction tube was sealed and stirred at 120 °C until
the disappearance of the starting materials. Then, the reaction mixture was cooled to room
temperature, the solvent was evaporated, and the residue was purified by chromatography on
SiO2 (25-40 ꢀm), eluting with a 95/5 (v/v) n-hexane/AcOEt mixture to afford 1.44 g (85% yield)
of 1a.
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3-(2-Iodophenylamino)-1,3-diphenylprop-2-en-1-one (1a). Yellow solid; mp 123-125°C; 1H
NMR (400 MHz, CDCl3) : δ 12.63 (bs, 1 H), 8.02 (d, J = 6.8 Hz, 2 H), 7.84 (d, J = 8.0 Hz, 1 H),
7.53-7.44 (m, 3 H), 7.39-7.30 (m, 4 H), 6.94 (t, J = 6.8 Hz, 1 H), 6.71 (t, J = 6.4 Hz, 1 H), 6.49
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(d, J = 8.0 Hz, 1 H), 6.21 (s, 1 H); C NMR (100 MHz, CDCl3): δ 190.1, 160.7, 141.7, 139.6,
139.2, 135.6, 131.5, 129.7, 128.5, 128.3, 128.0, 127.4, 125.7, 98.0, 93.9; IR (KBr): 3330, 2923,
1590, 1567, 1321, 1214, 750 ( cm-1); MS m/z (relative intensity) 425 (M+, 10), 298 (75), 193
(34), 105 (100), 77 (79).
Typical procedure for the cyclization of N-(2-iodoaryl)enaminones: synthesis of 3-
benzoyl-2-phenylquinolin-4(1H)-one (2a). A stainless steel reaction vessel was charged with
Pd2(dba)3 (0.0075 mmol, 6.9 mg), XPhos (0.015 mmol, 7.2 mg), Cs2CO3 (0.6 mmol, 195.6 mg),
3-(2-iodophenylamino)-1,3-diphenylprop-2-en-1-one (0.30 mmol, 127.5 mg), and MeCN (5
mL). The reactor was sealed, pressured to 20 atm with CO, warmed to 100°C, and stirred for 72
h. After cooling and eliminating CO, the volatile materials were evaporated at reduced pressure
and the residue was purified by chromatography on SiO2 (25-40 ꢀm) eluting with a n-
hexane/ethyl acetate/methanol mixture (57:38:5) to afford 66.4 mg (68% yield) of 2a: pale
yellow solid; 66.4 mg, yield: 68%; mp: 280-282°C. IR (KBr): 3255, 3060, 1731, 1668, 1508
(cm-1). 1H NMR (400.13 MHz, DMSO d6): δ 12.14 (bs, 1 H), 8.11(d, J = 8.0 Hz, 1 H), 7.79-7.73
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(m, 4 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.48-7.41 (m, 8 H); C NMR (100.6 MHz, DMSO d6): δ
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196.2, 175.6, 149.9, 140.3, 138.4, 134.0, 133.5, 132.9, 130.5, 129.4, 129.1, 129.0, 128.9, 125.3,
125.2, 124.5, 120.7, 119.3. MS m/z (%) N-methyl derivative: 339 (M+, 30), 324 (38), 207 (98),
133 (20), 105 (61), 77 (100); HRMS [M+H]+: calcd for C22H16NO2 326.11756, found 326.11740.
HPLC purity: 99.22%; tR = 3.40 min.
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Sequential synthesis of 2-substituted 3-aroylquinolin-4(1H)-ones from 2-iodoanilines and
α,β-ynones: synthesis of 3-benzoyl-2-phenylquinolin-4(1H)-one (2a) from 2-iodoaniline and
1,3-diphenylprop-2-yn-1-one. An oven-dried Schlenk tube was charged with 103 mg of 1,3-
diphenylprop-2-yn-1-one (0.5 mmol), 109.5 mg of o-iodoaniline (0.5 mmol), and anhydrous
MeOH (1.0 mL). The tube was sealed and stirred at 120 °C for 72 h. Then, the reaction mixture
was cooled to room temperature and the solvent was evaporated at reduced pressure. The residue
was dissolved in 5mL of MeCN and the resulting solution was poured into a stainless steel
reaction vessel along with Pd2(dba)3 (0.0125 mmol, 11.2 mg), XPhos (0.025 mmol, 12.0 mg),
and Cs2CO3 (1.0 mmol, 326 mg). The reactor was sealed, pressured to 20 atm with CO, warmed
to 100°C and stirred for 72h. After cooling and eliminating CO, the volatile materials were
evaporated at reduced pressure and the residue was purified by chromatography on SiO2 (25-40
ꢀm), eluting with a n-hexane/ethyl acetate/methanol mixture (57:38:5) to afford 99.0 mg (61%
yield) of 2a.
2-Phenyl-3-(3-(trifluoromethyl)benzoyl)quinolin-4(1H)-one (2b). White solid; 76.7 mg,
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yield: 65%; mp: 238-240 °C; IR (KBr): 3255, 1683, 1670, 1508 (cm-1); H NMR (400.13 MHz,
DMSO d6): δ 12.28 (bs, 1 H), 8.12-8.11(m, 2 H), 8.02 (s, 1 H), 7.94 (d, J = 7.2 Hz, 1 H), 7.78 (s,
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2 H), 7.69 (t, J = 7.6 Hz, 1 H), 7.47-7.45 (m, 6 H); C NMR (100.6 MHz, DMSO d6): δ 195.0,
175.6, 151.3, 140.3, 139.3, 134.0, 133.6, 133.1, 130.54, 130.50, 130.0, 129.7 (q, J= 28.4
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Hz),129.6, 125.4, 125.3, 125.1 (q, J= 4.2 Hz), 124.3 (q, J= 272 Hz), 119.7, 119.5; F (376.5
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MHz, DMSO d6): δ - 61.2; MS m/z (%) N-methyl derivative: 407 (M+, 46), 392 (67), 378 (86),
207 (59), 173 (33), 145 (100), 95 (32), 77 (35); HRMS [M+H]+: calcd for C23H15F3NO2
394.10494, found 394.10524. HPLC purity: 99.65%; tR = 3.63 min.
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3-(3-Fluorobenzoyl)-2-phenylquinolin-4(1H)-one (2c). White solid; 75.2 mg, yield: 73%;
mp: 260-262°C. IR (KBr): 3260, 1681, 1506 (cm-1). 1H NMR (400.13 MHz, DMSO d6): δ 12.19
(bs, 1 H), 8.11(d, J = 8.0 Hz, 1 H), 7.77 (bs, 2 H), 7.65 (d, J = 7.6 Hz, 1 H), 7.56-7.42 (m, 9 H);
13C NMR (100.6 MHz, DMSO d6): δ 195.1, 175.6, 162.7 (d, J = 238 Hz), 150.6, 140.87 (d, J =
6.1 Hz), 140.3, 134.0, 133.0, 131.2 (d, J = 8.1 Hz), 130.5, 129.1, 129.0, 125.7 (d, J = 4.1 Hz),
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125.36, 125.33, 124.5, 120.3 (d, J = 20.2 Hz), 120.2, 119.4, 115.4 (d, J = 22.1 Hz); F (376.5
MHz, DMSO d6) δ - 112.8; MS m/z (%) N-methyl derivative: 357 (M+, 38), 342 (46), 207 (54),
123 (52), 95 (100), 77 (19); HRMS [M+H]+: calcd for C22H15F3NO2 344.10813, found
344.10811. HPLC purity: 98.42%; tR = 3.42 min.
3-(3-Methoxybenzoyl)-2-phenylquinolin-4(1H)-one (2d). Pale yellow solid; 87.4 mg, yield:
82%; mp: 248-250 °C; IR (KBr): 3260, 1675, 1571, 1504 (cm-1); 1H NMR (400.13 MHz, DMSO
d6): δ 12.19 (bs, 1 H), 8.10 (d, J = 8.0 Hz, 1 H), 7.78-7.75 (m, 2 H), 7.50-7.32 (m, 8 H), 7.27 ( s,
1 H), 7.15-7.32 (m, 1 H), 3.76 (s, 3 H); 13C NMR (100.6 MHz, DMSO d6): δ 195.9, 175.5, 159.8,
149.9, 140.4, 139.9, 134.1, 132.89, 132.87, 130.5, 130.2, 129.1, 128.9, 125.3, 125.2, 124.4,
122.4, 120.7, 119.4, 113.5, 55.7; MS m/z (%) N-methyl derivative: 384 (M+, 10), 369 (57), 207
(39), 151 (11), 135 (83), 95 (28), 77 (100); HRMS [M+H]+: calcd for C23H18NO3 356.12812,
found 356.12836. HPLC purity: 99.31%; tR = 3.38 min.
3-(3-Methylbenzoyl)-2-phenylquinolin-4(1H)-one (2e). Yellow solid; 63.1 mg, yield: 62%;
mp: 257-259 °C; IR (KBr): 3255, 1666, 1571, 1508 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.13 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.79-7.73 (m, 2 H), 7.59 (d, J = 10.0 Hz, 2 H), 7.49-
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7.29 (m, 8 H), 2.31 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 196.2, 175.5, 149.8, 140.3,
138.5, 138.3, 134.14, 134.10, 132.8, 130.4, 129.6, 129.1, 128.9, 126.8, 125.3, 125.2, 124.4,
120.9, 119.3, 21.3; MS m/z (%) N-methyl derivative: 353 (M+, 37), 338 (31), 262 ( 42), 207 (11),
119 (67), 91 (100); HRMS [M+H]+: calcd for C23H18NO2 340.13321, found 362.11554. HPLC
purity: 99.81%; tR = 3.55 min.
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3-(4-Methylbenzoyl)-2-phenylquinolin-4(1H)-one (2f). Brown solid; 83.5 mg, yield: 82%;
mp 329-331 °C; IR (KBr): 3254, 1664, 1571, 1500 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.12 (bs, 1 H), 8.10 (d, J = 8.0 Hz, 1 H), 7.78-7.75 (m, 2 H), 7.68 (d, J = 8.0 Hz, 2 H), 7.48-7.40
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(m, 6 H), 7.23 (d, J = 8.0 Hz, 2 H), 2.34 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 195.7,
175.5, 149.6, 143.8, 140.4, 136.0, 134.2, 132.8, 130.4, 129.60, 129.57, 129.1, 128.9, 125.3,
125.2, 124.3, 120.9, 119.3, 21.6; MS m/z (%) N-methyl derivative: 353 (M+, 28), 338 (23), 262
(29), 207 (25), 119 (84), 91 (100); HRMS [M+H]+: calcd for C23H18NO2 340.13321, found
340.13270. HPLC purity: 95.09%; tR = 3.52 min.
3-(4-Chlorobenzoyl)-2-phenylquinolin-4(1H)-one (2g). Pale yellow solid; 66.9 mg, yield:
62%; mp: 269-270 °C; IR (KBr): 3250, 1675, 1508 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
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12.20 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.82-7.76 (m, 4 H) 7.51-7.41 (m, 8 H); C NMR
(100.6 MHz, DMSO d6) δ 195.1, 175.5, 150.4, 140.3, 138.3, 137.2, 134.0, 133.0, 131.3, 130.5,
129.2, 129.1, 129.0, 125.3, 124.5, 120.25, 120.24 119.4; MS m/z (%) N-methyl derivative: 373
(M+, 20), 344 (100), 262 (13), 207 (40), 111 (30), 75 (36); HRMS [M+H]+: calcd for
C22H15ClNO2 360.07858, found 360.07850. HPLC purity: 99.43%; tR = 3.68 min.
3-(4-Methoxybenzoyl)-2-phenylquinolin-4(1H)-one (2h). Pale yellow solid; 60.8 mg, yield:
57%; mp: 266-268 °C; IR (KBr): 3250, 1662, 1500 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.14 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.77-7.75 (m, 4 H) 7.48-7.39 (m, 6 H), 6.94 (d, J = 7.6
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Hz, 2 H), 3.80 (s, 1H); C NMR (100.6 MHz, DMSO d6) δ 194.6, 175.5, 163.5, 149.4, 140.4,
134.2, 132.8, 131.8, 131.5, 130.4, 129.1, 129.0, 125.3, 125.2, 124.3, 121.0, 119.3, 114.3, 55.9;
MS m/z (%) N-methyl derivative: 369 (M+, 27), 354 (15), 262 (21), 207 (32), 135 (100), 77 (51);
HRMS [M+H]+: calcd for C23H18NO3 356.12812, found 356.12835. HPLC purity: 99.23%; tR =
3.35 min.
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4-(4-Oxo-2-phenyl-1,4-dihydroquinoline-3-carbonyl)benzonitrile (2i). Pale yellow solid;
77.8 mg, yield: 74%; mp: 251-252 °C; IR (KBr): 3251, 2226, 1671, 1515, 1152 (cm-1); 1H NMR
(400.13 MHz, DMSO d6): δ 12.29 (bs, 1 H), 8.10 (d, J = 8.0 Hz, 1 H), 7.96-7.90 (m, 4 H) 7.78
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(s, 2 H), 7.46-7.44 (m, 6H); C NMR (100.6 MHz, DMSO d6) δ 195.5, 175.6, 151.4, 141.8,
140.3, 134.0, 133.2, 133.1, 130.5, 129.8, 129.1, 120.0, 125.5, 125.3, 124.7, 119.7, 119.5, 118.7,
115.2; MS m/z (%) N-methyl derivative: 364 (M+ 7), 324 (100), 262 (21), 207 (70), 77 (72);
HRMS [M+H]+: calcd for C23H14N2O2 351.11280, found 373.09466. HPLC purity: 95.44%; tR =
3.30 min.
2-(4-Acetylphenyl)-3-benzoylquinolin-4(1H)-one (2j). Yellow solid; 101.3 mg, yield: 91%;
mp: 250-252 °C; IR (KBr): 3251, 1685, 1550, 1513 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.26 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.99 (d, J = 8.4 Hz, 2 H), 7.82 (d, J = 7.2 Hz, 2 H),
7.77 (d, J = 3.6 Hz, 2 H), 7.63 (d, J = 8.0 Hz, 2 H), 7.58 (t, J = 7.2 Hz, 1 H), 7.47-7.43 (m, 3 H),
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2.58 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 197.9, 196.1, 175.5, 149.2, 140.3, 138.4,
138.3, 138.1, 133.5, 133.0, 129.6, 129.4, 129.1, 128.6, 125.37, 125.36, 124.6, 120.9, 119.4, 27.3;
MS m/z (%) N-methyl derivative: 381 (M+ 8), 366 (100), 281 (33), 207 (66), 105 (21), 77 (44);
HRMS [M+H]+: calcd for C24H18NO3 368.12812, found 368.12850. HPLC purity: 99.65%; tR =
3.25 min.
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Methyl 4-(3-benzoyl-4-oxo-1,4-dihydroquinolin-2-yl)benzoate (2k). Pale yellow solid; 95.5
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mg, yield: 83%; mp: 253-254 °C; IR (KBr): 3254, 1727, 1629, 1508 (cm-1); H NMR (400.13
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MHz, DMSO d6): δ 12.26 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.98 (d, J = 8.4 Hz, 2 H), 7.79-
7.76 (m, 4 H), 7.63 (d, J = 8.0 Hz, 2 H), 7.57 (t, J = 7.2 Hz, 1 H), 7.46-7.41 (m, 3 H), 3.85 (s, 3
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H); C NMR (100.6 MHz, DMSO d6): δ 195.9, 175.5,166.0, 149.0, 140.3, 138.5, 138.3, 133.5,
133.1, 131.3, 129.7, 129.6, 129.4, 129.0, 125.4, 125.2, 124.6, 120.9, 119.4, 52.8; MS m/z (%) N-
methyl derivative: 397 (M+, 11), 367 (60), 278 (63), 214 (93), 133 (85), 55 (100). HRMS
[M+H]+: calcd for C24H18NO4 384.12303, found 384.12320. HPLC purity: 98.71%; tR = 3.33
min.
3-Benzoyl-2-p-tolylquinolin-4(1H)-one (2l). Pale yellow solid; 61.1 mg, yield: 60%; mp:
276-278 °C; IR (KBr): 3252, 2896,1669, 1541, 1509 (cm-1); 1H NMR (400.13 MHz, DMSO d6):
δ 12.09 (bs, 1 H), 8.10 (d, J = 7.6 Hz, 1 H), 7.81-7.72 (m, 4 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.45-
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7.35 (m, 5 H), 7.22 (d, J= 8.0 Hz, 2 H), 2.29 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ
196.3, 175.5, 149.9, 140.4, 140.3, 138.4, 133.4, 132.8, 131.2, 129.5, 129.4, 129.1, 129.0, 125.3,
125.2, 124.3, 120.6, 119.3, 21.3; MS m/z (%) N-methyl derivative: 353 (M+, 47), 338 (46), 324
(76), 276 (36), 262 (16), 207 (29), 105 (62), 77 (100); HRMS [M+H]+: calcd for C23H18NO2
340.13321, found 340.13308. HPLC purity: 99.19%; tR = 3.52 min.
3-Benzoyl-2-(4-methoxyphenyl)quinolin-4(1H)-one (2m). Yellow solid; 61.8 mg, yield:
58%; mp: 250-252 °C; IR (KBr): 3255, 1627, 1509 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.07 (bs, 1 H), 8.08 (d, J = 7.6 Hz, 1 H), 7.80-7.74 (m, 4 H), 7.56 (t, J = 7.2 Hz, 1 H), 7.45-7.37
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(m, 5 H), 6.98 (d, J = 8.4 Hz, 2 H), 3.77 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 196.4,
175.5, 161.0, 149.6, 140.4, 138.4, 133.4, 132.8, 130.7, 129.4, 129.0, 126.2, 125.3, 125.1, 124.3,
120.4, 119.3,114.4, 55.8; MS m/z (%) N-methyl derivative: 369 (M+, 37), 354 (40), 292 ( 26),
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207 (51), 105 (56), 77 (100); HRMS [M+H]+: calcd for C23H18NO3 356.12812, found 356.12788.
HPLC purity: 98.84%; tR = 3.37 min.
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3-Benzoyl-2-(4-hydroxyphenyl)quinolin-4(1H)-one (2n). Yellow solid; 79.8 mg, yield: 78%;
mp: 289-291 °C; IR (KBr): 3453, 3255, 1644, 1508 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
11.95 (bs, 1 H), 9.95 (bs, 1 H), 8.08 (d, J = 7.6 Hz, 1 H), 7.78-7.71 (m, 4 H), 7.55 (t, J = 7.2 Hz,
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1 H), 7.44-7.40 (m, 3 H), 7.29 (d, J = 8.4 Hz, 2 H), 6.77 (d, J = 8.4 Hz, 2 H); C NMR (100.6
MHz, DMSO d6): δ 196.4, 175.5, 159.5, 150.0, 140.4, 138.4, 133.4, 132.7, 130.7, 129.3, 129.0,
125.3, 125.0, 124.6, 124.2, 120.2, 119.2, 115.7; MS m/z (%) N-methyl derivative: 355 (M+, 33),
340 (63), 292 ( 31), 207 (15), 105 (53), 77 (100); HRMS [M+H]+: calcd for C22H16NO3
342.11247, found 342.11219. HPLC purity: 98.76%; tR = 3.02 min.
2-(4-Acetylphenyl)-3-(4-chlorobenzoyl)quinolin-4(1H)-one (2o). Yellow solid; 56.5 mg,
yield: 47%; mp: 294-296 °C; IR (KBr): 3254, 1687, 1509 (cm-1); 1H NMR (400.13 MHz, DMSO
d6): δ 12.32 (bs, 1 H), 8.10 (d, J = 7.6 Hz, 1 H), 8.00 (d, J = 8.4 Hz, 2 H), 7.84-7.74 (m, 4 H),
7.62 (d, J = 8.4 Hz 2 H), 7.53-7.50 (m, 2 H), 7.46-7.42(m, 1 H), 2.59 (s, 3 H); 13C NMR (100.6
MHz, DMSO d6): δ 197.9, 195.0, 175.5, 149.7, 140.3, 138.4, 138.2, 137.2, 133.7, 133.1, 131.3,
129.6, 129.2, 128.6, 125.3, 124.7, 120.4, 119.4, 114.7, 27.3; MS m/z (%) N-methyl derivative:
415 (M+, 12), 401 (18), 367 (59), 278 (65), 214 (100), 207 (20), 133 (94), 55 (100); HRMS
[M+H]+: calcd for C24H17ClNO3 402.08915, found 402.08884. HPLC purity: 97.79%; tR = 3.48
min.
3-Benzoyl-6-methoxy-2-phenylquinolin-4(1H)-one (2p). Pale yellow solid; 63.9 mg, yield:
60%; mp: 287-289 °C; IR (KBr): 3254, 1687, 1509 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
12.15 (bs, 1H), 7.78 (d, J = 7.6 Hz, 2 H), 7.73 (d, J = 9.2 Hz, 1 H), 7.54 (t, J = 7.6 Hz, 1 H), 7.47
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(s, 1 H), 7.46-7.39 (m, 8 H), 3.86 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 196.4, 174.8,
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156.6, 149.0, 138.5, 135.0, 134.2, 133.4, 130.3, 129.4, 129.1, 129.0, 128.9, 126.4, 123.3, 121.2,
119.7, 104.5, 55.9; MS m/z (%) N-methyl derivative: 369 (M+, 67), 354 (43), 207 (18), 105 (46),
77 (100); HRMS [M+H]+: calcd for C23H18NO3 356.12812, found 356.12784. HPLC purity:
99.47%; tR = 3.40 min.
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3-Benzoyl-6-methyl-2-phenylquinolin-4(1H)-one (2q). Pale yellow solid; 89.6 mg, yield:
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88%; mp: 250-252 °C; IR (KBr): 3260, 1671, 1498, (cm-1); H NMR (400.13 MHz, DMSO d6):
δ 12.11 (bs, 1 H), 7.90 (bs, 1 H), 7.78 (d, J = 7.2 Hz, 2 H), 7.69-7.67 (m, 1 H), 7.59-7.54 (m, 2
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H), 7.45-7.43 (m, 7 H), 2.44 (s, 3 H); C NMR (100.6 MHz, DMSO d6): δ 196.3, 175.4, 149.6,
138.5, 138.4, 134.2, 134.2, 133.9, 133.4, 130.4, 129.4, 129.1, 129.0, 128.9, 125.2, 124.5, 120.4,
119.3, 21.3; MS m/z (%) N-methyl derivative: 353 (M+, 30), 338 (22), 324 (40), 276 (21), 207
(34), 105 (56), 77 (100); HRMS [M+H]+: calcd for C23H18NO2 340.13321, found 340.13312.
HPLC purity: 97.05%; tR = 3.55 min.
3-Benzoyl-6-fluoro-2-phenylquinolin-4(1H)-one (2r). Pale yellow solid; 72.1 mg, yield:
70%; mp: 261-263 °C; IR (KBr): 3251, 1676, 1572, 1506 (cm-1); 1H NMR (400.13 MHz, DMSO
d6): δ 12.32 (bs, 1 H), 7.85-7.75 (m, 4 H), 7.69 (t, J = 8.4 Hz, 1 H), 7.56 (t, J = 6.8 Hz, 1 H),
7.47-7.42 (m, 7 H); 13C NMR (100.6 MHz, DMSO d6): δ 195.9, 174.7, (d, J = 2.4 Hz), 159.2 (d,
J = 243 Hz), 150.1, 138.3, 137.1, 133.9, 133.5, 130.5, 129.4, 129.1, 129.0, 128.9, 126.5 (d, J =
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6.7 Hz), 122.2 (d, J =8.0 Hz), 121.8 (d, J = 25.4 Hz), 120.0, 109.4 (d, J =22.3 Hz); F (376.5
MHz, DMSO d6): δ - 116.7; MS m/z (%) N-methyl derivative: 357 (M+, 34), 342 (30), 280 (20),
207 (17), 105 (72), 77 (100); HRMS [M+H]+: calcd for C22H15FNO2 344.10813, found
344.10785. HPLC purity: 98.95%; tR = 3.48 min.
3-Benzoyl-6-chloro-2-phenylquinolin-4(1H)-one (2s). Pale yellow solid; 76.6 mg, yield:
71%; mp: 231-233°C; IR (KBr): 3251, 1671, 1499 (cm-1); 1H NMR (400.13 MHz, DMSO d6): δ
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12.35 (bs, 1 H), 8.00 (s, 1 H), 7.81-7.78 (m, 4 H), 7.57 (t, J = 7.2 Hz, 1 H), 7.48-7.41 (m, 7 H);
13C NMR (100.6 MHz, DMSO d6): δ 195.9, 174.6, 150.3 139.0, 138.2, 133.8, 133.5, 133.0,
130.6, 129.4, 129.1 (overlapping), 129.0, 128.9, 126.3 124.3 ,121.8 120.9; MS m/z (%) N-methyl
derivative: 373 (M+, 24), 359 (19), 296 (20), 281 (15), 207 (40), 105 (72), 77 (100); HRMS
[M+H]+: calcd for C22H15ClNO2 360.07858, found 360.07850. HPLC purity: 95.65%; tR = 3.75
min.
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3-Benzoyl-6-bromo-2-phenylquinolin-4(1H)-one (2t). Pale yellow solid; 44.8 mg, yield:
37%; mp: 265-267 °C; IR (KBr): 3256, 1670, 1567, 1494 (cm-1); 1H NMR (400.13 MHz, DMSO
d6): δ 12.30 (bs, 1 H), 8.19 (s, 1 H), 7.92 (d, J = 8.8 Hz, 1 H), 7.79 (d, J = 7.6 Hz, 2 H) 7.73 (d, J
= 8.8 Hz, 1 H), 7.57 (t, J = 7.6 Hz, 1 H), 7.48-7.42 (m, 7 H); 13C NMR (100.6 MHz, DMSO d6):
δ 195.7, 174.2,150.3, 139.3, 138.2, 135.6, 133.8, 133.6, 130.6, 129.4, 129.10, 129.06, 129.00,
127.5, 126.7, 121.9, 121.1, 117.1; MS m/z (%) N-methyl derivative: 417 (81Br, 29), 415 (79Br,
30), 80 (32), 207 (55), 105 (74), 77 (100) HRMS [M+H]+: calcd for C22H15BrNO2 404.02807,
found 404.02800. HPLC purity: 99.22%; tR = 3.87 min.
Biological Assays
Plasmids. Mouse Smoothened Flag-tagged mutant (Smo D477G) was generated using the
QuickChange II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) and
verified by sequencing.
mRNA expression analysis. Total RNA was isolated with Trizol (InvitrogenTMLife
Technologies, Carlsbad, CA, USA) and reverse transcribed with SensiFASTTM cDNA
Synthesis Kit (Bioline Reagents Limited, London, UK). Quantitative real-time PCR (Q-PCR)
analysis of Gli1, Gli2, Ptch1, CycD1, CycD2, HIP1, BMP2, Igf2, N-Myc, β-2 microglobulin and
HPRT mRNA expression was performed on each cDNA sample using the VIIATM7 Real Time
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PCR System employing Assay-on-Demand Reagents (Life Technologies, Carlsbad, CA, USA).
A reaction mixture containing cDNA template, SensiFASTTM Probe Lo-ROX Kit (Bioline
Reagents Limited, London, UK) and primer probe mixture was amplified using FAST Q-PCR
thermal cycler parameters. Each amplification reaction was performed in triplicate and the
average of the three threshold cycles was used to calculate the amount of transcript in the sample
(using SDS version 2.3 software). mRNA quantification was expressed, in arbitrary units, as the
ratio of the sample quantity to the quantity of the calibrator. All values were normalized with two
endogenous controls, β-2 microglobulin and HPRT, which yielded similar results.
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Western Blot Analysis. Cells were lysed in standard radioimmunoprecipitation assay buffer
plus protease inhibitors (Roche, Welwyn Garden City, UK). Lysates were separated on SDS-
PAGE and immunoblotted using standard procedures. Mouse anti-Gli1 antibody was from Cell
Signaling (Beverly, MA, USA); mouse anti-NMyc, rabbit anti-Cyclin D1, goat anti-Actin and
HRP-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). All antibodies were used at different dilutions followed by enhanced
chemiluminescence (ECL, Amersham Pharmacia).
Cell culture, Transfection and treatments. HEK293T, MEFs Ptch-/- and Shh-Light II, were
cultured in DMEM plus 10% FBS. All media contained L-glutamine and antibiotics. ASZ001
BCC cells were cultured in 154CF medium (Gibco-BRL, Grand Island, NY, USA) plus 2% FBS
chelated with Chelex 100® sodium (Sigma Aldrich, St. Louis, MO, USA), calcium chloride
0.05 mM (Gibco-BRL) and antibiotics. Cerebellar GCPs (from 4-days old mice) were isolated
and cultured as previously described.36 Murine MBs were isolated from Ptch1+/- mice
(EMMA, Monterotondo, Italy). Tissues were collected as previously described37 and
immediately prepared cell suspensions were used for short-term cultures to keep Hh-
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sensitivity in vitro.24-26 Transient transfections were performed using DreamFectTM Gold
transfection reagent (Oz Biosciences SAS, Marseille, FR). Cells were treated with SAG (200
nM, Alexis Biochemicals Farmingdale, NY, USA), Bodipy-Cyclopamine (5nM, BioVision Inc.,
San Francisco, CA, USA).
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Hh-Dependent Luciferase Reporter Assay. The luciferase assay was performed in Shh-Light
II cells, stably incorporating a Gli-responsive luciferase reporter and the pRL-TK Renilla
(normalization control), treated for 48 h with SAG (200 nM) and the studied compounds.
Luciferase and renilla activity were assayed with a dual-luciferase assay system according to the
manufacturer’s instruction (Biotium Inc., Hayward, CA). Results are expressed as
luciferase/renilla ratios and represent the mean SD of three experiments, each performed in
triplicate.
Bodipy-Cyclopamine binding assay. Mouse Flag-tagged Smo WT or SmoD477G mutant
transfected cells were washed in PBS supplemented with 0.5% fetal bovine serum, fixed in 4%
paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with Triton
X100 (Sigma) 0.2% and incubated for 3 h at 37 °C both in the same medium supplemented with
Bodipy-cyclopamine (5 nM) and the studied compounds. Dako Fluorescent mounting (Dako,
Carpinteria, CA, USA) was used as mounting medium and Hoechst reagent for staining of the
cell nuclei. Bodipy (green) and Hoechst (blue) signals were analyzed in three to four
representative fields per coverslip (20 X magnification; 1000 cells/field). Data were expressed as
percentage of BC incorporation observed with BC alone.
Cell proliferation assays. To determine the growth rate of viable cells, a trypan blue count
was performed after a treatment period of 24-48-72 h with studied compounds.
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ASSOCIATED CONTENT
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Supporting Information. The supporting information is available free of charge via the Internet
at http://pubs.acs.org.
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1H, C and F NMR spectra, HPLC chromatograms, and ESI-HRMS spectra of compounds
2b-t; Plasmids; mRNA expression and Western-Blot analysis; molecular modeling;
supplementary figures.
PDB coordinates for computational models of Smo receptor and 2d and 2t.
Molecular Formula strings.
AUTHOR INFORMATION
Corresponding Author
* Antonella Goggiamani, Dipartimento di Chimica e Tecnologie del Farmaco, La Sapienza,
Università di Roma, P.le A. Moro 5, 00185 Rome, Italy. Phone: +39 (06) 4969-3320, Fax: +39
(06) 4969-3316, E-mail: antonella.goggiamani@uniroma1.it
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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The authors wish also to thank the OpenEye Free Academic Licensing Program for providing a
free academic license for molecular modeling and chemoinformatics software. This research was
supported by grants AIRC IG14723, PRIN 2012-2013 (2012C5YJSK002), Progetti di Ricerca di
Università Sapienza di Roma, and Pasteur Institute/Cenci Bolognetti Foundation.
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ABBREVIATIONS
Hh, Hedgehog; Smo, Smoothened receptor; Gli, glioma-associated oncogene; SAG, Smoothened
Agonist; Ptch1, Patched receptor; IC50, half maximal inhibitory concentration; RNA,
Ribonucleic acid, DNA, Deoxyribonucleic acid; MEFs, mouse embryonic fibroblasts; BC,
Bodipy-Cyclopamine; GCPs, granule cell progenitors; EGL, external germinal layer; MB,
Medulloblastoma; BCC Basal cell carcinoma; CTR, control; DMSO, dimethyl sulfoxide; qRT-
PCR, Real-Time Quantitative polymerase chain reaction; NMR, Nuclear Magnetic Resonance; s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; bs, broad singlet; DMF, N,N-
dimethylformamide, MeCN, acetonitrile; THF, tetrahydrofuran; dba, dibenzylideneacetone;
SPhos, 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; Xanthphos, 4,5-
bis(diphenylphosphino)-9,9-dimethylxanthene; dppf, 1,1′-ferrocenediyl-bis(diphenylphosphine);
XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl.
REFERENCES
(1) Ruiz i Altaba, A.; Sanchez, P.; Dahmane, N. Gli and Hedgehog in Cancer: Tumours,
Embryos and Stem Cells. Nat. Rev. Cancer 2002, 2, 361-372.
(2) Ruat, M.; Hoch, L.; Faure, H.; Rognan, D. Targeting of Smoothened for Therapeutic
Gain. Trends Pharmacol. Sci. 2014, 35, 237-246.
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