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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 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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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Design, palladium-catalyzed synthesis, and biological investigation of 2-substituted 3aroylquinolin-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.

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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.

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. 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 2substituted 3-aroylquinolin-4(1H)-ones 2.

On the basis of the carbonylative cyclization of related 2-substituted N-(2-haloaryl)-2propenoates 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.

Table 1. Palladium-catalyzed carbonylative cyclization of 1a. Optimization studies.a Entry Pd/ligand

CO Solvent Time (atm) (h)

1

Pd(PPh3)4

1

MeCN

24

‒d

2

Pd(PPh3)4

5

MeCN

72

tracese

3

Pd(PPh3)4

10

MeCN

72

tracesf

4

Pd(PPh3)4

20

MeCN

39

53

5

Pd2(dba)3/SPhos

20

MeCN

48

56

6

Pd2(dba)3/SPhos

20

MeCN

72

64

7

Pd2(dba)3/Xantphos 20

MeCN

48

59

8

Pd2(dba)3/dppf

20

MeCN

72

36g

9

Pd2(dba)3/XPhos

20

MeCN

72

68

10

Pd2(dba)3/XPhos

20

THF

68

41h

11

Pd2(dba)3/XPhos

20

DMF

24

54i

12

Pd2(dba)3/XPhos

20

MeCN

72

42j

13

Pd2(dba)3/XPhos

30

MeCN

48

44k

Yield % of 2ab,c


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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 of carbon monoxide (see Table). b Yields are given for isolated products. c Unless otherwise stated, no evidence of 1a was obtained after the allotted reaction time. d The corresponding indole was isolated in 78% yield. e 1a was recovered in 93% yield. f 1a was recovered in 96% yield. g 1a was recovered in 35% yield. h 1a was recovered in 50% yield. i 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. 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

Ar1

R

Ar2

1

H

Ph

Ph

2a (68)

2

H

Ph

3-CF3-C6H4

2b (65)d

3

H

Ph

3-F-C6H4

2c (73)e

4

H

Ph

3-MeO-C6H4

2d (82)

5

H

Ph

3-Me-C6H4

2e (62)

6

H

Ph

4-Me-C6H4

2f (82)

7

H

Ph

4-Cl-C6H4

2g (62)

8

H

Ph

4-MeO-C6H4

2h (57)

9

H

Ph

4-CN-C6H4

2i (74)

10

H

4-MeCO-C6H4

Ph

2j (91)

11

H

4-MeCO2-C6H4

Ph

2k (83)

12

H

4-Me-C6H4

Ph

2l (60)f

13

H

4-MeO-C6H4

Ph

2m (58)g

14

H

4-HO-C6H4

Ph

2n (78)

15

H

4-MeCO-C6H4

4-Cl-C6H4

2o (47)

16

4-MeO

Ph

Ph

2p (60)


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17

4-Me

Ph

Ph

2q (88)

18

4-F

Ph

Ph

2r (70)h

19

4-Cl

Ph

Ph

2s (71)

20

4-Br

Ph

Ph

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. b Yields are given for isolated products. c Unless otherwise stated, no evidence of the starting material was obtained after the allotted reaction time. d The starting material was recovered in 15% yield. e The starting material was recovered in 10% yield. f 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). 3Aroylquinolin-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 µM, 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 curve of 2d and 2t in NIH 3T3 Shh-Light II cells. Treatment time was 48 h, and normalization was against Renilla luciferase. Data show the mean ±SD of three independent experiments. *P

Design, Palladium-Catalyzed Synthesis, and ... - ACS Publications

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