Design, Palladium-Catalyzed Synthesis, and Biological Investigation

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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 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 ‡

S Supporting Information *

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 FDAapproved 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 toward the crystallographic structure of the Smo receptor.8 Among tested molecules, 2-substituted 3aroylquinolin-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, Figure S1). 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. © 2017 American Chemical Society

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 3Aroylquinolin-4(1H)-ones 2

On the basis of the carbonylative cyclization of related 2substituted N-(2-haloaryl)-2-propenoates described by Torii et al.,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 Received: November 16, 2016 Published: January 25, 2017 1469

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synthetic point of view, were obtained prolonging the reaction time to 72 h (2a, 30%; recovered 1a, 53%; indole derivative, 13%). Apparently, the presence of a ketonic group (our work) instead of an ester group (Torii work) in the β-amino-α,βunsaturated carbonyl fragment of the substrate makes it more difficult for 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

Table 2. Synthesis of 3-Aroylquinolin-4(1H)-ones 2 via Palladium-Catalyzed Carbonylative Cyclization of N-(2Iodoaryl)enaminones 1a 1 entry

R

Ar

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

H H H H H H H H H H H H H H H 4-MeO 4-Me 4-F 4-Cl 4-Br

Ph Ph Ph Ph Ph Ph Ph Ph Ph 4-MeCO-C6H4 4-MeCO2-C6H4 4-Me-C6H4 4-MeO-C6H4 4-HO-C6H4 4-MeCO-C6H4 Ph Ph Ph Ph Ph

Table 1. Palladium-Catalyzed Carbonylative Cyclization of 1a: Optimization Studiesa entry

Pd/ligand

CO (atm)

solvent

time (h)

yield, % of 2ab,c

1 2 3 4 5 6 7 8 9 10 11 12 13

Pd(PPh3)4 Pd(PPh3)4 Pd(PPh3)4 Pd(PPh3)4 Pd2(dba)3/SPhos Pd2(dba)3/SPhos Pd2(dba)3/Xantphos Pd2(dba)3/dppf Pd2(dba)3/XPhos Pd2(dba)3/XPhos Pd2(dba)3/XPhos Pd2(dba)3/XPhos Pd2(dba)3/XPhos

1 5 10 20 20 20 20 20 20 20 20 20 30

MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN MeCN THF DMF MeCN MeCN

24 72 72 39 48 72 48 72 72 68 24 72 48

d tracese tracesf 53 56 64 59 36g 68 41h 54i 42j 44k

1

Ar2

2a (yield %)b,c

Ph 3-CF3-C6H4 3-F-C6H4 3-MeO-C6H4 3-Me-C6H4 4-Me-C6H4 4-Cl-C6H4 4-MeO-C6H4 4-CN-C6H4 Ph Ph Ph Ph Ph 4-Cl-C6H4 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) 2j (91) 2k (83) 2l (60)f 2m (58)g 2n (78) 2o (47) 2p (60) 2q (88) 2r (70)h 2s (71) 2t (37)

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 20 atm of carbon monoxide. bYields are given for isolated products. cUnless otherwise stated, no evidence of the starting material was obtained after the allotted reaction time. dThe starting material was recovered in 15% yield. eThe starting material was recovered in 10% yield. fThe starting material was recovered in 14% yield. gThe starting material was recovered in 12% yield. hThe starting material was recovered in 8% yield.

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 of carbon monoxide (see table). bYields are given for isolated products. cUnless otherwise stated, no evidence of 1a was obtained after the allotted reaction time. dThe corresponding indole was isolated in 78% yield. e 1a was recovered in 93% yield. f1a was recovered in 96% yield. g1a was recovered in 35% yield. h1a was recovered in 50% yield. iThe corresponding indole was isolated in 14% yield. jThe reaction was carried out using 2 equiv of K2CO3. 1a was recovered in 33% yield. k1a was recovered in 4% yield.

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).

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. 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 metalcatalyzed 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-(2iodoaryl) 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

Scheme 2. Sequential Synthesis of 2a from 2-Iodoaniline and 1,3-Diphenylprop-2-yn-1-one

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 Gliresponsive 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 μM, molecules 2b, 2j, 2h, 2n, and 2s showed mild activity as Hh inhibitor (Supporting Information, Figure S2A), while 2d and 1470

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2t showed high activity in this assay (Figure 1A). The remaining 15 compounds proved to be inactive (2c, 2g, 2m, Supporting Information, 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). Moreover, compounds 2d and 2t suppressed Hh genes signature in genetically defined Ptch1−/− mouse embryonic fibroblasts (Ptch1−/− MEFs) (Supporting Information, 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 to molecular modeling, these molecules bind the antagonist’s 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 with the Ar2 ring (Scheme 2 and Table 2) stacked with Trp480 or Tyr394. The Ar1 ring is docked in a hydrophobic subpocket bounded by Phe222 and Val386. The peculiar m-methoxy group of 2d is projected toward the solvent area at the upper part of Smo, whereas the bromine atom of 2t is projected toward the lower intracellular portion of Smo. These chemical moieties interact in hydrophobic subpockets 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 (Supporting Information, Figure S1). 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 wildtype and drug-resistant Smo (2d on mSmo-WT IC50 = 13.76 μM, on mSmo-D477G IC50 = 12.41 μM; 2t on mSmo-WT IC50 = 23.96 μM, on mSmo-D477G IC50 = 29.35 μM) (Supporting Information, 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

Figure 1. Inhibition of Hh signaling and predicted binding mode of 2d and 2t. (A) Dose−response curve of 2d and 2t in NIH3T3 Shh-Light II cells. Treatment time was 48 h, and normalization was against Renilla luciferase. Data show the mean ± SD of three independent 1471

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Figure 1. continued 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 code 4JKV).17 Smo is shown as green transparent surface. Residues interacting with small molecules are shown as green sticks and are labeled. 2d and 2t are shown as cyan sticks. Polar interactions are highlighted by black dashed lines.

cell-derived Shh and is required for intense GCPs proliferation and proper development of the granule neuron lineage. After the 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 compounds 2d and 2t to interfere with Hh-dependent growth events, we tested their ability to suppress Hh signaling in SAGactivated GCPs isolated from 4-day-old mice. As shown in Supporting Information, Figure S5, these compounds significantly inhibited both mRNA (Supporting Information, Figure S5A) and protein level (Supporting Information, Figure S5B) of several Hh target genes and marker related to cell proliferation. 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 Compounds 2d and 2t significantly arrested the proliferation of Ptch± MB cells (Figure 2A) and inhibited Hh target gene mRNA levels (Figure 2B). 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 μM concentration, both molecules impaired ASZ001 BCC cell proliferation in a dosedependent manner (Figure 3A), also in agreement with the significant decrease of Hh signature genes observed after drug treatment, as shown in Figure 3B.

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 24 h 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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discovery of anticancer lead compounds targeting the Hh pathway and overcoming drug resistance.

CONCLUSIONS 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 2iodoanilines and α,β-ynones omitting the isolation of N-(2iodoaryl)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

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EXPERIMENTAL SECTION

General Procedures. All of the reagents, catalysts, bases, and solvents are commercially available and were used as purchased, without further purification. 1H NMR (400.13 MHz), 13C NMR (100.6 MHz), and 19F spectra (376.5 MHz) were recorded with a Bruker Avance 400 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. 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. 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 μL, 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 (5 mL) and extracted by diethyl ether (3 × 15 mL). The combined organic phases were dried 1472

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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 μL, 6.0 mmol), PdCl2(PPh3)2 (70 mg, 0.1 mmol), and Et3N (607.1 mg, 834 μL, 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 μL, 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 μm), 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)-1,3diphenylprop-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 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 μm), eluting with a 95/5 (v/v) n-hexane/AcOEt mixture to afford 1.44 g (85% yield) of 1a. 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 (d, J = 8.0 Hz, 1 H), 6.21 (s, 1 H). 13C 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-1one (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 μm), 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 (m, 4 H), 7.56 (t, J = 7.6 Hz, 1 H), 7.48−7.41 (m, 8 H). 13C NMR (100.6 MHz, DMSO-d6): δ 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. Sequential Synthesis of 2-Substituted 3-Aroylquinolin4(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 5 mL 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

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 a 48 h treatment with compound 2d or 2t or DMSO only, as control. Results were normalized to endogenous control (β2microglobulin and HPRT). All data show the mean ± SD of three independent experiments: (∗) P < 0.05, (∗∗) P < 0.01 versus CTR (DMSO). 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 into 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 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 Molecular Modeling. The crystallographic structure of the Smoothened receptor in complex with an antitumor agent (PDB code 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 1473

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Journal of Medicinal Chemistry

Article

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 Hz, 2 H), 3.80 (s, 1H). 13C 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. 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 (s, 2 H), 7.46−7.44 (m, 6H). 13C 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), 2.58 (s, 3 H). 13C 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. Methyl 4-(3-Benzoyl-4-oxo-1,4-dihydroquinolin-2-yl)benzoate (2k). Pale yellow solid; 95.5 mg, yield 83%; mp 253−254 °C. IR (KBr): 3254, 1727, 1629, 1508 (cm−1). 1H NMR (400.13 MHz, DMSO-d6): δ 12.26 (bs, 1 H), 8.11 (d, J = 8.0 Hz, 1 H), 7.98 (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 H). 13C 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), 7.45−7.35 (m, 5 H), 7.22 (d, J = 8.0 Hz, 2 H), 2.29 (s, 3 H). 13 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 (m, 5 H), 6.98 (d, J = 8.4 Hz, 2 H), 3.77 (s, 3 H). 13 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

(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 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 μm), eluting with a nhexane/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, yield 65%; mp 238−240 °C. IR (KBr): 3255, 1683, 1670, 1508 (cm−1). 1H 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, 2 H), 7.69 (t, J = 7.6 Hz, 1 H), 7.47−7.45 (m, 6 H). 13C 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 Hz),129.6, 125.4, 125.3, 125.1 (q, J = 4.2 Hz), 124.3 (q, J = 272 Hz), 119.7, 119.5. 19F (376.5 MHz, DMSO-d6): δ −61.2. MS m/z (%) Nmethyl 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. 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), 125.36, 125.33, 124.5, 120.3 (d, J = 20.2 Hz), 120.2, 119.4, 115.4 (d, J = 22.1 Hz). 19F (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), 7.49−7.29 (m, 8 H), 2.31 (s, 3 H). 13C 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. 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 (m, 6 H), 7.23 (d, J = 8.0 Hz, 2 H), 2.34 (s, 3 H). 13 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 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). 13C NMR (100.6 MHz, DMSO-d6) δ 195.1, 175.5, 150.4, 140.3, 1474

DOI: 10.1021/acs.jmedchem.6b01135 J. Med. Chem. 2017, 60, 1469−1477

Journal of Medicinal Chemistry

Article

(M+, 37), 354 (40), 292 (26), 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. 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, 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). 13C 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 (%) Nmethyl 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 (s, 1 H), 7.46−7.39 (m, 8 H), 3.86 (s, 3 H). 13C NMR (100.6 MHz, DMSO-d6): δ 196.4, 174.8, 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. 3-Benzoyl-6-methyl-2-phenylquinolin-4(1H)-one (2q). Pale yellow solid; 89.6 mg, yield 88%; mp 250−252 °C. IR (KBr): 3260, 1671, 1498, (cm−1). 1H 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 H), 7.45−7.43 (m, 7 H), 2.44 (s, 3 H). 13C 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, DMSOd6): 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 = 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). 19F (376.5 MHz, DMSO-d6): δ −116.7. MS m/z (%) Nmethyl 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 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 (%) Nmethyl 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.

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 sitedirected 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, U.K.). 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 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, U.K.) 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. Western Blot Analysis. Cells were lysed in standard radioimmunoprecipitation assay buffer plus protease inhibitors (Roche, Welwyn Garden City, U.K.). 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 HRPconjugated 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-day-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 described,37 and immediately prepared cell suspensions were used for short-term cultures to keep Hh sensitivity in vitro.24−26 Transient transfections were performed using DreamFect Gold transfection reagent (Oz Biosciences SAS, Marseille, France). Cells were treated with SAG (200 nM, Alexis Biochemicals, Farmingdale, NY, USA), Bodipy-cyclopamine (5 nM, BioVision Inc., San Francisco, CA, USA). Hh-Dependent Luciferase Reporter Assay. The luciferase assay was performed in Shh-Light II cells, stably incorporating a Gliresponsive 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 dualluciferase assay system according to the manufacturer’s instructions (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% 1475

DOI: 10.1021/acs.jmedchem.6b01135 J. Med. Chem. 2017, 60, 1469−1477

Journal of Medicinal Chemistry

Article

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 Bodipycyclopamine (5 nM) and in 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× 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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broad singlet; DMF, N,N-dimethylformamide; MeCN, acetonitrile; THF, tetrahydrofuran; dba, dibenzylideneacetone; SPhos, 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl; Xantphos, 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; dppf, 1,1′ferrocenediyl-bis(diphenylphosphine); XPhos, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl

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(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. (3) Christodoulou, M. S.; Mori, M.; Pantano, R.; Alfonsi, R.; Infante, P.; Botta, M.; Damia, G.; Ricci, F.; Sotiropoulou, P. A.; Liekens, S.; Botta, B.; Passarella, D. Click Reaction as a Tool to Combine Pharmacophores: The Case of Vismodegib. ChemPlusChem 2015, 80, 938−943. (4) Infante, P.; Alfonsi, R.; Botta, B.; Mori, M.; Di Marcotullio, L. Targeting Gli Factors to Inhibit the Hedgehog Pathway. Trends Pharmacol. Sci. 2015, 36, 547−558. (5) Infante, P.; Mori, M.; Alfonsi, R.; Ghirga, F.; Aiello, F.; Toscano, S.; Ingallina, C.; Siler, M.; Cucchi, D.; Po, A.; Miele, E.; D’Amico, D.; Canettieri, G.; De Smaele, E.; Ferretti, E.; Screpanti, I.; Uccello Barretta, G.; Botta, M.; Botta, B.; Gulino, A.; Di Marcotullio, L. Gli1/ DNA Interaction Is a Druggable Target for Hedgehog-Dependent Tumors. EMBO J. 2015, 34, 200−217. (6) Infante, P.; Alfonsi, R.; Ingallina, C.; Quaglio, D.; Ghirga, F.; D’Acquarica, I.; Bernardi, F.; Di Magno, L.; Canettieri, G.; Screpanti, I.; Gulino, A.; Botta, B.; Mori, M.; Di Marcotullio, L. Inhibition of Hedgehog-Dependent Tumors and Cancer Stem Cells by a Newly Identified Naturally Occurring Chemotype. Cell Death Dis. 2016, 7, e2376. (7) Iovine, V.; Mori, M.; Calcaterra, A.; Berardozzi, S.; Botta, B. One Hundred Faces of Cyclopamine. Curr. Pharm. Des. 2016, 22, 1658− 1681. (8) Weierstall, U.; James, D.; Wang, C.; White, T. A.; Wang, D.; Liu, W.; Spence, J. C.; Bruce Doak, R.; Nelson, G.; Fromme, P.; Fromme, R.; Grotjohann, I.; Kupitz, C.; Zatsepin, N. A.; Liu, H.; Basu, S.; Wacker, D.; Han, G. W.; Katritch, V.; Boutet, S.; Messerschmidt, M.; Williams, G. J.; Koglin, J. E.; Marvin Seibert, M.; Klinker, M.; Gati, C.; Shoeman, R. L.; Barty, A.; Chapman, H. N.; Kirian, R. A.; Beyerlein, K. R.; Stevens, R. C.; Li, D.; Shah, S. T.; Howe, N.; Caffrey, M.; Cherezov, V. Lipidic Cubic Phase Injector Facilitates Membrane Protein Serial Femtosecond Crystallography. Nat. Commun. 2014, 5, 3309. (9) Bernini, R.; Cacchi, S.; Fabrizi, G.; Filisti, E.; Sferrazza, A. 3Aroylindoles Via Copper-Catalyzed Cyclization of N-(2-Iodoaryl)Enaminones. Synlett 2009, 2009, 1480−1484. (10) Torii, S.; Okumoto, H.; Long, H. X. A Direct Approach to 2Substituted 1,4-Dihydro-4-Oxo-Quinoline-3-Carboxylates by Palladium-Catalyzed Carbonylative Cyclization. Tetrahedron Lett. 1990, 31, 7175−7178. (11) Maligres, P. E.; Humphrey, G. R.; Marcoux, J. F.; Hillier, M. C.; Zhao, D.; Krska, S.; Grabowski, E. J. J. Practical, Highly Convergent, Asymmetric Synthesis of a Selective Ppar Gamma Modulator. Org. Process Res. Dev. 2009, 13, 525−534. (12) Kasahara, A.; Izumi, T.; Murakami, S.; Yanai, H.; Takatori, M. Synthesis of 3-Substituted Indoles by a Palladium-Assisted Reaction. Bull. Chem. Soc. Jpn. 1986, 59, 927−928. (13) Sakamoto, T.; Nagano, T.; Kondo, Y.; Yamanaka, H. Condensed Heteroaromatic Ring-Systems 0.17. Palladium-Catalyzed Cyclization of Beta-(2-Halophenyl)Amino Substituted Alpha,Beta-Unsaturated Ketones and Esters to 2,3-Disubstituted Indoles. Synthesis 1990, 1990, 215−218.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01135. 1 H, 13C, and 19F NMR spectra, HPLC chromatograms, and ESI-HRMS spectra of compounds 2b−t; plasmids; mRNA expression and Western blot analysis; molecular modeling; supplementary figures (PDF) Listing of PDB coordinates for computational models of Smo receptor and 2d and 2t (PDF) Molecular formula strings and some data (CSV) Structure coordinates (PDB) Structure coordinates (PDB)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone: +39 (06) 4969-3320. Fax: +39 (06) 4969-3316. Email: [email protected]. ORCID

Bruno Botta: 0000-0001-8707-4333 Antonella Goggiamani: 0000-0001-8339-4284 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.

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ACKNOWLEDGMENTS The authors 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 USED Hh, Hedgehog; Smo, Smoothened receptor; Gli, gliomaassociated oncogene; SAG, Smoothened agonist; Ptch1, Patched receptor; IC50, half maximal inhibitory concentration; RNA, ribonucleic acid; DNA, deoxyribonucleic acid; MEF, mouse embryonic fibroblast; BC, Bodipy-cyclopamine; GCP, granule cell progenitor; 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, 1476

DOI: 10.1021/acs.jmedchem.6b01135 J. Med. Chem. 2017, 60, 1469−1477

Journal of Medicinal Chemistry

Article

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