Synthesis and Biological Evaluation of Halogenated Naphthyridone

Bioconjugate Chem. 2003, 14, 629−641

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Synthesis and Biological Evaluation of Halogenated Naphthyridone Carboxamides as Potential Ligands for in Vivo Imaging Studies of Substance P Receptors Caroline Bagot-Gue´ret,† Marie-Delphine Le Bas,‡ Sylvie Tymciu,§ Mircea Darabantu,‡ Patrick Emond,| Denis Guilloteau,| Marie Claire Lasne,‡ Anne Wijkhuisen,§ Louisa Barre´,*,† and Cećile Perrio*,‡ Groupe de De´veloppements Me´thodologiques en Tomographie par E Ä mission de Positons, UMR CEA, Universite´ de Caen-Basse Normandie, Centre Cyceron, 15 Boulevard Henri Becquerel, 14070 Caen Cedex, France, Laboratoire de Chimie Molećulaire et Thioorganique, CNRS UMR 6507, ENSICAEN, Universite´ de Caen-Basse Normandie, 6 Boulevard Marećhal Juin, 14050 Caen Cedex, France, CEA-Universite´ Paris 7, DRM/SPI, CEA-Saclay, 91 191 Gif sur Yvette, France, and Laboratoire de Biophysique Me´dicale et Pharmaceutique, INSERM U 316, Universite´ Franç ois Rabelais, 31 Avenue Monge, 37 200 Tours, France. Received December 19, 2002; Revised Manuscript Received February 19, 2003

With the aim of developing new radioligands for in vivo studies of substance P receptors using positron emission tomography or single photon emission computed tomography, 2- and 3-halo naphthyridone6-carboxamide derivatives were synthesized. Their affinities toward the target receptors were evaluated on CHO cells and compared to the unsubstituted analogue EP 00652218 (IC50 ) 100 nM ( 20). The IC50 value was not altered in the case of 2-chloro compound 1 (IC50 ) 100 nM ( 15) and only slightly reduced for the 2-fluoro and -iodo analogues 6 and 8 (IC50 ) 500 nM ( 80). A drastic reduction in binding (IC50 > 1000 nM) was observed for the halogenated compounds 2-5, 7, and 9.

INTRODUCTION

Substance P (SP) is an important neuropeptide of the mammalian tachykinin family besides neurokinin A (NKA) and neurokinin B (NKB) (1). These neurotransmitters share the common C-terminal amino acid sequence “-Phe-X-Gly-Leu-Met-NH2” and are involved in a wide variety of physiological activities both in the central (CNS) and peripheral (PNS) nervous system, such as contraction of muscles, vasodilatation, stimulation of salivary secretion, and activation of pain transmission and stress signals (1, 2). The biological responses induced by the tachykinins are mediated by three distinct receptors termed as NK1 (SP-preferring), NK2 (NKA-preferring), and NK3 (NKB-preferring). Characterization of these receptors, carried out by pharmacological studies and binding assays, has been finally supported by isolation and cloning of the three receptor proteins. The distribution of these latter has mainly been investigated by autoradiography or by in situ hybridization histochemistry and immunocytochemistry. NK1 receptors are widely distributed in various brain areas (2-4) and in PNS, while expression of NK2 and NK3 receptors is primarily confined in PNS and in CNS, respectively. Increasing interest in central NK receptors is based on reports describing their possible role in various pathological conditions. NK antagonists (5, 6) have been proposed for the treatment of migraine, inflammation, asth* To whom correspondence should be addressed. E-mail: [email protected]. † Groupe de De ´ veloppements Me´thodologiques en Tomographie par E Ä mission de Positons, Universite´ de Caen-Basse Normandie. ‡ Laboratoire de Chimie Mole ´ culaire et Thioorganique, Universite´ de Caen-Basse Normandie. § CEA-Universite ´ Paris 7. | Laboratoire de biophysique me ´ dicale et pharmaceutique INSERM U 316, Tours.

ma, emesis, cancer, and psychiatric disorders (depression, anxiety, schizophrenia). Their involvment in neurodegenerative diseases has also been suggested (7-11). Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are unique and complementary imaging techniques for in vivo assessment of drug distribution and interaction with biochemical target systems (12-16). They are well adapted for the exploration of brain function in healthy and disease conditions and crucial tools for clinical research and diagnosis. PET is a quantitative and very sensitive method, but it requires high cost facilities due to the use of tracers labeled with cyclotron-produced positron emitters (carbon-11, t1/2 ) 20.4 min; fluorine-18, t1/2 ) 109.7 min; bromine-76, t1/2 ) 16 h). SPECT has coarser spatial and temporal resolution, reduced sensitivity and less quantification capability; however, it uses commercially available gamma emitter radionuclides of relatively long half-lives (iodine-123, t1/2) 13.2 h; technetium-99m, t1/2) 6 h) and thus is routinely employed in nuclear medicine centers. A few radioligands have been synthesized for PET or SPECT studies of NK1 receptors (Figure 1). The first ones had quinuclidine {[11C]CP-96,345 (17, 18) and [125I]L 703606 (19)} or piperidine structures {[11C]CP-99,994 (20), [18F]FTP (21), [11C]GR 203040 (22), [11C]GR 205171 (22), [18F]L 829,165 (23), and [11C]CP-643,051 (24)}. More recently, a tryptophan derivative {[11C]LY 303870 (25)} and a substituted piperazine {[11C]R 116301 (26)} were also described. All radioligands, except [18F]L 829,165, for which biological studies in human are promising, suffer from low brain uptake, nonspecific binding, and extensive metabolism. The search for highly selective radioligands as well as the development of a unique tracer for PET and SPECT is still needed. This challenge requires the presence, in the same structure, of atoms (C, F, I, Br) that can be substituted for their positron (11C, 18F, 77Br) or gamma (123/125I) isotope.

10.1021/bc025656r CCC: $25.00 © 2003 American Chemical Society Published on Web 04/19/2003

630 Bioconjugate Chem., Vol. 14, No. 3, 2003

Bagot-Gue´ret et al.

Figure 1. Radioligands for in vivo NK1 receptor imaging.

Figure 2. EP00652218 and target molecules.

In 1995, Takeda company described a series of 6-pyrido[3,4-b]pyridinecarboxamide derivatives as a novel class of highly potent, selective, and orally active NK1 receptor antagonists (Figure 2) (27-30). The lead compound, EP 00652218, was shown to exhibit subnanomolar affinity (IC50: 0.21 nM, in vitro inhibition of [125I]-(BH)SP binding in human IM-9 cells) and selectivity toward the NK1 receptor (NK1/NK2 and NK1/NK3 > 1000). SAR studies demonstrated that the bis(trifluoromethylbenzyl) moiety was essential for the affinity and that structural modifications on the pyridine ring have a relatively small influence on this value. For instance, introduction of a chlorine atom on the pyridine ring in the 3-position maintained a nanomolar affinity and a good selectivity. This result, with the other reported data (28), suggested that halogenated derivatives of EP 00652218 could display a similar affinity toward the NK1 receptor. Herein we describe the synthesis and the in vitro evaluation of halogenated 6-pyrido[3,4-b]pyridinecarboxamide derivatives 1-9. EXPERIMENTAL SECTION

All reagents were purchased from ACROS, Aldrich, or Fluka and used without further purification. Solvents

were freshly distilled under nitrogen prior to use: 1,4dioxane from sodium, N,N-dimethylformamide (DMF), and acetonitrile from calcium hydride, tetrahydrofuran (THF), from sodium benzophenone ketyl. Haloquinolinic acids 11-13 were prepared according to (31). [125I]Bolton-Hunter (BH)-SP was purchased from Amersham France. CHO cells expressing the human NK1 receptor were a generous gift from Dr. L. Prodier, Rhoˆne-Poulenc Rorer, Yvry, France. 1H (200 MHz), 13C (62.8 MHz), and 19 F (235.4 MHz) NMR spectra were recorded on a Bruker AC instrument. Chemical shifts (δ) are reported in parts per million downfield from internal tetramethylsilane (for 1 H and 13C NMR) or from external freon (for 19F NMR) and referenced from solvent resonances. NMR coupling constants are reported as absolute values in hertz. All the new structures were assigned by 1H and 13C NMR using heteronuclear correlation experiments. IR spectra were recorded on a Perkin-Elmer 684. Low resolution (EI or CI) mass spectra were recorded on a Nermag R 10 or a JEOL Gcmate instrument at 70 eV. Relative intensities are given in brackets. High resolution (HRMS, EI or FAB) mass spectra were obtained on a JEOL Gcmate instrument. Elemental analyses were performed using a ThermoQuest analyzer CHNS-O and were within (0.4% of the calculated values. Melting points were determined using Koffler bank and are uncorrected. Flash chromatography was performed using Merck silica gel Si 60 (0.040-0.063 mm) packing. Reverse phase chromatography was performed using C-18 silica gel Macherey-Nagel PolygoPrep 60-50. HPLC analysis was carried out by means of a Waters 501 pump, an UV detector set to 254 nm, and a Valco valve injector. General Procedure for the Synthesis of Quinolinic Anhydrides 14-16. A solution of quinolinic acid (31) 11-13 (8-12 mmol) in thionyl chloride (10-20 mL)

Halogenated Naphthyridone Carboxamides

was refluxed for 3 h. After concentration under vacuum, the residue was heated in chloroform for 0.5 h. The resulting suspension was filtered off, and the filtrate was concentrated to give the expected anhydride 14-16. 6-Chloroquinolinic Anhydride (14). Anhydride 14 was obtained from 11 (2.54 g, 11.57 mmol): white solid, mp 127 °C; 93% (1.97 g). 1H NMR (CDCl3) δ 7.97 (d, 3JHH ) 8.2 Hz, 1H), 8.34 (d, 3JHH ) 8.2 Hz, 1H). 13C NMR (CDCl3) δ 125.5, 130.7, 135.5, 150.8, 159.2, 159.4, 161.2. IR (KBr) 1868, 1786. m/z (EI) 185 (M+•, C7H237ClNO3, 1), 183 (M+•, C7H235ClNO3, 4), 139 (100). 5-Bromoquinolinic Anhydride (15). Anhydride 15 was obtained from 12 (2.14 g, 8.7 mmol): white solid, mp 138-140 °C (lit. (32) 137-140 °C); 90% (1.8 g). 1H NMR (CDCl3) δ 8.49 (d, 4JHH ) 2.0 Hz, 1H), 9.22 (d, 4JHH ) 2.0 Hz, 1H). 13C NMR (CDCl3) δ 127.8, 128.1, 136.1, 148.2, 151.7, 159.1, 159.7. IR (KBr) 1870, 1774. 5-Iodoquinolinic Anhydride (16). Anhydride 16 was obtained from 13 (0.612 g, 2.1 mmol): white solid, mp 140 °C; 73% (0.422 g). 1H NMR (CDCl3) δ 8.72 (d, 4JHH ) 2.0 Hz, 1H), 9.44 (d, 4JHH ) 2.0 Hz, 1H). 13C NMR (CDCl3) δ 101.1, 128.4, 142.2, 149.1, 156.8, 160.5, 164.7. IR (KBr) 1750, 1718, 1684. m/z (EI) 275 (M+•, 19), 249 (100). HRMS (EI) (M+•, C7H2NO3I) calcd 274.9083, found 274.9090. Anal. (C7H2NO3I) C, H, N. General Procedure for Friedel-Crafts Reactions. Aluminum chloride (2.5 equiv) was added to anhydride 14-16 (1 equiv) in halobenzene or to a mixture of anhydride 14-16 (1 equiv) and halobenzene (1.5 equiv) in 1,2-dichloroethane. The solution was refluxed for 3 to 6 h or stirred at room temperature for 24 h. After cooling to room temperature, cold water (10 mL) and HCl 36% (10 mL) were added. The solution was stirred until precipitation (1 h to 3 d). The precipitate was filtered off, washed with water (2 × 4 mL), and dried under vacuum to give the expected acid 17-21. 3-(4-Bromobenzoyl)-6-chloropyridine-2-carboxylic Acid Hydrochloride (17). Acid 17 was obtained from 14 (2.3 g, 12.53 mmol) and bromobenzene (2.0 mL, 18.7 mmol) after reaction in 1,2-dichloroethane (12 mL) for 4 h under reflux and recrystallization from methanol: beige solid, mp 178 °C; 65% (2.77 g). 1H NMR (CDCl3) δ 7.54-7.61 (m, 4H), 7.72 (d, 3JHH ) 8.0 Hz, 1H), 7.80 (d, 3 JHH ) 8.0 Hz, 1H). 13C NMR (CDCl3) δ 127.4, 128.1, 130.9, 132.6, 134.9, 136.2, 138.2, 142.7, 150.0, 160.9, 190.8. IR (KBr) 3372, 1710, 1658. m/z (EI) 343 (M+•, C13H781Br37ClNO3, 1), 341 (M+•, C13H779Br37ClNO3 and C13H781Br35ClNO3, 5), 339 (M+•, C13H779Br35ClNO3, 4), 76 (100). 6-Chloro-3-(4-fluorobenzoyl)pyridine-2-carboxylic Acid Hydrochloride (18). Acid 18 was obtained from 14 (3.51 g, 19.1 mmol) and fluorobenzene (2.68 mL, 28.7 mmol) after reaction in 1,2-dichloroethane (50 mL) under reflux for 6 h: beige solid, mp 181 °C; 85% (5.12 g). 1H NMR (DMSO-d6) δ 7.25 (t, 3JHH ) 3JHF ) 8.7 Hz, 2H), 7.69 (dd, 3JHH ) 8.7 Hz, 4JHF ) 5.6 Hz, 2H), 7.78 (d, 3JHH ) 8.2 Hz, 1H), 7.97 (d, 3JHH ) 8.2 Hz, 1H). 13C NMR (CDCl3) δ 116.5 (d, 2JCF ) 22.2 Hz), 129.1, 132.4 (d, 3JCF ) 9.6 Hz), 132.9 (d, 4JCF ) 2.9 Hz), 136.9, 140.0 144.1, 151.6, 162.7, 166.6 (d, 1JCF ) 257.0 Hz), 192.0. 19F NMR (CDCl3) δ -103.4. 6-Chloro-3-(4-iodobenzoyl)pyridine-2-carboxylic Acid Hydrochloride (19). Acid 19 was obtained in a mixture (0.34 g, 34%) with the deiodinated compound (19/deiodinated compound ratio of 75/25) from 14 (0.50 g, 2.94 mmol) and iodobenzene (0.46 mL, 4.08 mmol) after reaction in 1,2-dichloroethane (10 mL) for 24 h at room temperature and was used in the next step without further purification. 1H NMR (CDCl3) δ 7.43 (d, 3JHH )

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6.4 Hz, 2H), 7.61 (d, 3JHH ) 7.3 Hz, 1H), 7.73-7.85 (m, 3H). 13C NMR (CDCl3) δ 102.8, 129.2, 129.8, 130.9, 135.8, 138.6, 139.7, 144.2, 151.7, 162.4, 192.1. 5-Bromo-3-(4-fluorobenzoyl)pyridine-2-carboxylic Acid Hydrochloride (20). Acid 20 was obtained from 15 (1.80 g, 7.9 mmol) after reaction in fluorobenzene (20 mL) under reflux for 3 h: white solid, mp 190 °C; 100% (2.9 g). 1H NMR (CDCl3) δ 7.06 (t, 3JHH ) 3JHF ) 8.5 Hz, 2H), 7.69 (dd, 3JHH ) 8.5 Hz, 4JHF ) 5.4 Hz, 2H), 7.89 (d, 4J 4 13 HH ) 1.6 Hz, 1H), 8.78 (d, JHH ) 1.6 Hz, 1H). C NMR (CDCl3) δ 115.1 (d, 2JCF ) 22.2 Hz), 124.4, 131.1 (d, 3JCF ) 9.6 Hz), 131.6, 137.7, 138.1, 141.5, 149.9, 162.1, 165.2 (d, 1JCF ) 257.0 Hz), 189.8.19F NMR (CDCl3) δ -103.6. IR (KBr) 1712, 1674. m/z (EI) 325 (M+•, C13H7NO3F81Br, 12), 323 (M+•, C13H7NO3F79Br, 13), 123 (100). HRMS (FAB+) (MH+, C13H8NO3F81Br) calcd 325.9670, found 325.9675, (MH+, C13H8NO3F79Br) calcd 323.9671, found 325.9670. Anal. (C13H7NO3FBr‚HCl) C, H, N. 3-(4-Fluorobenzoyl)-5-iodopyridine-2-carboxylic Acid Hydrochloride (21). Acid 21 was obtained from 16 (1.0 g, 3.6 mmol) after reaction in fluorobenzene (15 mL) under reflux for 3 h: white solid, mp 176-177 °C; 55% (0.731 g). 1H NMR (CDCl3) δ 7.14 (t, 3JHH ) 3JHF ) 8.5 Hz, 2H), 7.76 (dd, 3JHH ) 8.5, 4JHF ) 5.3 Hz, 2H), 8.18 (d, 4JHH ) 1.6 Hz, 1H), 8.99 (d, 4JHH ) 1.6 Hz, 1H). 13C NMR (CDCl ) δ 98.7, 116.2 (d, 2J 3 CF ) 22.6 Hz), 132.1 (d, 3JCF ) 10.1 Hz), 132.5 (d, 4JCF ) 3.1 Hz), 138.7, 142.1, 144.9, 155.3, 162.9, 166.2 (d, 1JCF ) 268.6 Hz), 190.7.19F NMR (CDCl3) δ -103.3. IR (KBr) 1720, 1690, 1600, 1290. m/z (EI) 370 (M+•, 22), 50 (100). Anal. (C13H8NO3FI‚HCl) C, H, N. General Procedure for Amidation of Acids 17 and 20 with a N-Methylglycinate. A mixture of acid 17 or 20 (1 equiv), oxalyl chloride (3-3.5 equiv), and DMF (0.1 equiv) in CH2Cl2 (20 or 40 mL) was stirred at room temperature for 3 h and then concentrated under vacuum. The residue was diluted in CH2Cl2 (25 or 50 mL), and sarcosine ester (1.8 equiv) and Et3N (3 equiv) were added. The mixture was stirred at room temperature for 16 h and then concentrated under vacuum. After addition of ethyl acetate (30 mL) to the residue and filtration, the organic layer was washed with brine (2 × 20 mL), dried over MgSO4, and concentrated under vacuum to give a crude oil which was purified by column chromatography. N-Methyl-N-[(methyloxycarbonyl)methyl]-3-(4bromobenzoyl)-6-chloropyridine-2-carboxamide (22). Amide 22 was obtained from 17 (1 g, 2.92 mmol) and sarcosine methyl ester hydrochloride (0.73 g, 5.25 mmol): white solid, mp 129 °C; 52% (0.65 g). Rf 0.38 (AcOEt/pentane 30/70). 1H NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 3.04 and 3.13 (s, 3H), 3.66 and 3.77 (s, 3H), 4.10 and 4.17 (s, 2H), 7.38 and 7.40 (d, 3JHH ) 8.2 Hz, 1H), 7.52-7.58 (m, 4H), 7.64 and 7.70 (d, 3JHH ) 8.2 Hz, 1H). 13C NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 35.0 and 36.9, 48.2 and 51.3, 51.4 and 52.1, 123.3 and 123.7, 128.0 and 128.1, 130.2 and 130.3, 130.9 and 131.0, 132.5 and 133.9, 134.0 and 134.1, 137.8 and 138.1, 149.7 and 150.8, 152.0 and 153.2, 164.5 and 165.7, 167.6 and 168.4, 191.7 and 191.9. IR (KBr) 1746, 1670, 1654. m/z (EI) 428 (M+•, C17H1481Br37ClN2O4, 4), 426 (M+•, C17H1481Br35ClN2O4 and C17H1479Br37ClN2O4, 13), 424 (M+•, C17H1479Br35ClN2O4, 10), 102 (100). N-Methyl-N-[(ethyloxycarbonyl)methyl]-3-(4-bromobenzoyl)-6-chloropyridine-2-carboxamide (23). Amide 23 was obtained from 17 (1 g, 2.92 mmol) and sarcosine ethyl ester hydrochloride (0.80 g, 5.25 mmol): yellow oil; 51% (0.65 g). Rf 0.61 (AcOEt/pentane 30/70). 1 H NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 1.17 and 1.23 (t, 3JHH ) 7.2 Hz, 3H), 3.00 and 3.09 (s, 3H), 4.05


and 4.13 (s, 2H), 4.08 and 4.19 (q, 3JHH ) 7.2 Hz, 2H), 7.36 and 7.37 (d, 3JHH ) 8.1 Hz, 1H) 7.52-7.53 (m, 4H), 7.63 and 7.69 (d, 3JHH ) 8.1 Hz, 1H). 13C NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 16.1 and 16.2, 38.0 and 39.9, 51.3 and 55.2, 63.3 and 63.5, 126.2 and 126.6, 131.0 and 131.1, 133.3 and 133.4, 134.0 and 134.1, 135.5 and 136.9, 137.0 and 137.1, 139.2 and 139.4, 151.2 and 152.2, 153.5 and 154.8, 165.9 and 167.1, 168.5 and 169.3, 193.1 and 193.3. IR (KBr) 1742, 1690, 1652. m/z (EI) 442 (M+•, C18H1681Br37ClN2O4, 5), 440 (M+•, C18H1681Br35ClN2O4 and C18H1679Br37ClN2O4, 19), 438 (M+•, C18H1679Br35ClN2O4, 12), 324 (100). N-Methyl-N-[(tert-butyloxycarbonyl)methyl]-3-(4bromobenzoyl)-6-chloropyridine-2-carboxamide (24). Amide 24 was obtained from 17 (1 g, 2.92 mmol) and sarcosine tert-butyl ester hydrochloride (0.95 g, 5.25 mmol): beige solid, mp 104 °C; 54% (0.73 g). Rf 0.80 (AcOEt/pentane 30/70). 1H NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 1.46 and 1.53 (s, 9H), 3.07 and 3.18 (s, 3H), 4.04 and 4.13 (s, 2H), 7.46 and 7.47 (d, 3JHH ) 8.1 Hz, 1H), 7.58-7.66 (m, 4H), 7.73 and 7.79 (d, 3JHH ) 8.1 Hz, 1H). 13C NMR (CDCl3) (2 rotamers in a 1/1 ratio) δ 26.0 and 26.1, 33.8 and 35.8, 48.0 and 51.7, 80.0 and 80.3, 122.2 and 122.6, 126.9 and 127.0, 129.3 and 129.9, 130.0 and 130.2, 131.5 and 132.8, 132.9 and 133.0, 136.8 and 137.0, 149.0 and 149.7, 151.5 and 152.3, 163.4 and 163.5, 165.1 and 165.7, 190.7 and 190.9. IR (KBr) 1718, 1684, 1646. m/z (EI) 470 (M+•, C20H2081Br37ClN2O4, 1), 468 (M+•, C20H2081Br35ClN2O4 and C20H2079Br37ClN2O4, 3), 466 (M+•, C20H2079Br35ClN2O4, 2), 44 (100). Anal. (C20H20BrClN2O4) C, H, N. N-Methyl-N-[(ethyloxycarbonyl)methyl]-5-bromo3-(4-fluorobenzoyl)pyridine-2-carboxamide (25). Amide 25 was obtained from 20 (4 g, 11.1 mmol) and sarcosine ethyl ester hydrochloride (3.0 g, 19.8 mmol): brown oil; 95% (4.46 g). Rf 0.4 (CH2Cl2/EtOH/H2O/EtNH2 95/4.7/0.1/0.2). 1H NMR (CDCl3) (2 rotamers) δ 1.27 (t, 3 JHH ) 7.1 Hz, 1.8H), 1.32 (t, 3JHH ) 7.1 Hz, 1.2H), 3.09 (s, 1.2H), 3.19 (s, 1.8H), 4.16 (s, 1.2H), 4.26 (s, 0.8H), 4.19 (q, 3JHH ) 7.1 Hz, 1.2H), 4.27 (q, 3JHH ) 7.1 Hz, 0.8H), 7.15 (t, 3JHH ) 3JHF ) 8.5 Hz, 0.8H), 7.16 (t, 3JHH ) 3JHF ) 8.5 Hz, 1.2H), 7.82 (dd, 3JHH ) 8.8 Hz, 4JHF ) 5.3 Hz, 0.8H), 7.83 (dd, 3JHH ) 8.8 Hz, 4JHF ) 5.3 Hz, 1.2H), 7.87 (d, 4JHH ) 2.2 Hz, 0.8H), 7.92 (d, 4JHH ) 2.2 Hz, 1.2H), 8.66 (d, 4JHH ) 2.2 Hz, 0.8H), 8.78 (d, 4JHH ) 2.2 Hz, 1.2H). 13C NMR (CDCl3) (2 rotamers) δ 13.9 and 14.0, 35.6 and 37.7, 49.1 and 52.9, 61.0 and 61.1, 115.6 and 115.7 (d, 2JCF ) 22.6 Hz), 120.4 and 120.9, 132.4 and 132.6 (d, 3JCF ) 7.5 Hz), 136.4 and 137.8, 138.3 and 138.4, 149.6 and 150.6, 150.9 and 151.9, 165.8 and 165.9 (d, 1JCF ) 254.7 Hz), 166.0 and 167.2, 167.9 and 168.7, 191.4 and 191.5. 19F NMR (CDCl3) δ -(103.7-104.0) (m). m/z (EI) 424 (M+•, C18H1681BrFN2O4, 8), 422 (M+•, C18H1679BrFN2O4, 9), 116 (100). HRMS (EI) (M+•, C18H1681BrFN2O4) calcd 424.0253, found 424.0269 (M+•, C18H1679BrFN2O4) calcd 422.0274, found 422.0267. General Procedure for the Cyclization-Dehydration of Pyridine Carboxamides 22-25. A mixture of ester 22-25 (1 equiv) and DBU (2.3 equiv for esters 2224 or 3.15 equiv for ester 25) in toluene (90-100 mL) was refluxed for 4 h (for esters 22-24) or for 20 h (for ester 25). After cooling to room temperature, HCl 1 N (80 mL) was added until pH 1. The pH was then adjusted to 4-5 by addition of aqueous Na2CO3 (10%). After extraction with ethyl acetate/THF 1/1 (2 × 50 mL), the combined organic layers were washed with brine (2 × 20 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was purified by column chromatography.


5-(4-Bromophenyl)-2-chloro-7-methyl-8-oxo-7,8dihydro-[1,7]naphthyridine-6-carboxylic Methyl Ester (26). Ester 26 was obtained from 22 (1.42 g, 3.43 mmol): white solid, mp 40-50 °C; 30% (0.40 g). Rf 0.40 (AcOEt/pentane 50/50). 1H NMR (CDCl3) δ 3.56 (s, 6H), 7.12 (d, 3JHH ) 8.4 Hz, 2H), 7.42 (d, 3JHH ) 8.7 Hz, 1H), 7.49 (d, 3JHH ) 8.7 Hz, 1H), 7.56 (d, 3JHH ) 8.4 Hz, 2H). 13 C NMR (CDCl3) δ 34.4, 53.4, 114.6, 123.7, 128.7, 131.7, 132.2, 132.5, 132.6, 135.9, 137.1, 141.5, 152.5, 159.5, 163.5. IR (KBr) 1736, 1672. m/z (EI) 410 (M+•, C17H1281Br37ClN2O3, 22), 408 (M+•, C17H1281Br35ClN2O3 and C17H1279Br37ClN2O3, 100), 406 (M+•, C17H1279Br35ClN2O3, 76). Anal. (C17H12BrClN2O3) C, H, N. 5-(4-Bromophenyl)-2-chloro-7-methyl-8-oxo-7,8dihydro-[1,7]naphthyridine-6-carboxylic Ethyl Ester (27). Ester 27 was obtained from 23 (1.47 g, 3.34 mmol): white solid, mp 30-40 °C; 49% (0.69 g). Rf 0.46 (AcOEt/pentane 50/50). 1H NMR (CDCl3) δ 1.03 (t, 3JHH ) 7.1 Hz, 3H), 3.66 (s, 3H), 4.11 (q, 3JHH ) 7.1 Hz, 2H), 7.19 (d, 3JHH ) 8.6 Hz, 2H), 7.48 (d, 3JHH ) 8.4 Hz, 1H), 7.54 (d, 3JHH ) 8.4 Hz, 1H), 7.62 (d, 3JHH ) 8.6 Hz, 2H). 13 C NMR (CDCl3) δ 12.5, 32.9, 61.7, 113.1, 122.2, 127.2, 128.4, 129.7, 130.4, 130.7, 134.7, 135.7, 140.0, 151.0, 158.2, 161.5. IR (KBr) 1730, 1670. m/z (EI) 424 (M+•, C18H1481Br37ClN2O3, 26), 422 (M+•, C18H1481Br35ClN2O4 and C18H1479Br37ClN2O4, 100), 420 (M+•, C18H1479Br35ClN2O4, 77). 5-(4-Bromophenyl)-2-chloro-7-methyl-8-oxo-7,8dihydro-[1,7]naphthyridine-6-carboxylic tert-Butyl Ester (28). Ester 28 was obtained from 24 (1.56 g, 3.34 mmol): white solid, mp 160 °C; 45% (0.67 g). Rf 0.53 (AcOEt/pentane 50/50). 1H NMR (CDCl3) δ 1.27 (s, 9H), 3.67 (s, 3H), 7.22 (d, 3JHH ) 8.2 Hz, 2H), 7.48 (d, 3JHH ) 8.6 Hz, 1H), 7.53 (d, 3JHH ) 8.6 Hz, 1H), 7.64 (d, 3JHH ) 8.2 Hz, 2H). 13C NMR (CDCl3) δ 28.5, 34.4, 85.8, 113.7, 123.9, 129.0, 132.4, 132.8, 132.9, 133.6, 137.3, 137.5, 141.6, 152.5, 160.0, 162.3; IR (KBr) 1728, 1672. m/z (EI) 452 (M+•, C20H1881Br37ClN2O3, 0.1), 450 (M+•, C20H1881Br35ClN2O3 and C20H1879Br37ClN2O3, 0.4), 448 (M+•, C20H1879Br35ClN2O3, 0.2), 56 (100). 3-Bromo-5-(4-fluorophenyl)-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridine-6-carboxylic Ethyl Ester (29). Ester 29 was obtained from 25 (1.8 g, 4.25 mmol): white solid, mp 188 °C; 85% (1.45 g). Rf 0.50 (AcOEt). 1H NMR (CDCl3) δ 1.03 (t, 3JHH ) 7.1 Hz, 3H), 3.67 (s, 3H), 4.11 (q, 3JHH ) 7.1 Hz, 2H), 7.11 (dd, 3JHH ) 8.6, 4JHF ) 5.4 Hz, 2H), 7.21 (t, 3JHH ) 3JHF) 8.6 Hz, 2H), 7.69 (d, 4 JHH ) 2.0 Hz, 1H), 8.93 (d, 4JHH ) 2.0 Hz, 1H). 13C NMR (CDCl3) δ 13.9, 34.2, 63.0, 114.0, 116.5 (d, 2JCF ) 21.6 Hz), 125.1, 128.8 (d, 4JCF ) 3.6 Hz), 133.0 (d, 3JCF ) 8.2 Hz), 134.4, 135.9, 137.2, 140.0, 151.9, 160.4, 163.0, 163.4 (d, 1JCF ) 249.3 Hz). 19F NMR (CDCl3) δ -(112.3-112.4) (m). IR (KBr) 1742, 1676, 1512, 1226. m/z (CI, isobutane) 407 (MH+, C18H1581BrFN2O3, 92), 405 (MH+, C18H1579BrFN2O3, 100). HRMS (CI, isobutane) (MH+, C18H1581BrFN2O3) calcd 407.0231, found 407.0229, (MH+, C18H1579BrFN2O3) calcd 405.0250, found 405.0212. Hydrolysis of Esters 26-29. 5-(4-Bromophenyl)6-carboxy-2-chloro-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridinium Trifluoroacetate (34). A mixture of ester 28 (1 g, 2.2 mmol) and trifluoroacetic acid (7 mL) in CH2Cl2 (14 mL) was stirred at room temperature for 7 h. After concentration, acid 34 was obtained as a yellow solid, mp > 260°C; 98% (1.1 g). 1H NMR (DMSO-d6) δ 3.61 (s, 3H), 7.37 (d, 3JHH ) 8.3 Hz, 2H), 7.65 (d, 3JHH ) 8.6 Hz, 1H), 7.78 (d, 3JHH ) 8.3 Hz, 2H), 7.80 (d, 3JHH ) 8.6 Hz, 1H). 13C NMR (DMSO-d6) δ 34.0, 111.9, 119.1 (q, 1 JCF ) 295.5 Hz), 122.5, 128.8, 132.0, 132.1, 132.7, 133.2, 137.9, 138.3, 140.3, 150.4, 158.6, 162.6 (q, 2JCF ) 32.3


Hz), 170.6. 19F NMR (DMSO-d6) δ -75.6 (s). IR (KBr) 1732, 1668, 1578. m/z (EI) 396 (M+•, C16H1081Br37ClN2O3, 0.1), 394 (M+•, C16H1081Br35ClN2O3 and C16H1079Br37ClN2O3, 0.3), 392 (M+•, C16H1079Br35ClN2O3, 0.2), 42 (100). 3-Bromo-5-(4-fluorophenyl)-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridine-6-carboxylic Acid (35). Ester 29 (2.2 g, 5.4 mmol) in a mixture of EtOH/THF/ aqueous NaOH 1 N 1/1/1 (84 mL) was refluxed for 5 h. The solvents were removed under vacuum, and the residue was dissolved in water. The aqueous layer was washed with ethyl acetate and acidified with HCl 36% to pH 1. After extraction with ethyl acetate (2 × 20 mL), the organic layer was dried over MgSO4 and then concentrated under vacuum to give acid 35 as a yellow solid, mp 190-192 °C; 72% (1.6 g). 1H NMR (DMSO-d6) δ 2.54 (s, 3H), 7.35 (t, 3JHH ) 3JHF ) 8.6 Hz, 2H), 7.40 (dd, 3JHH ) 8.6 Hz, 4JHF ) 5.6 Hz, 2H), 7.60 (d, 4JHH ) 2.1 Hz, 1H), 8.93 (d, 4JHH ) 2.1 Hz, 1H). 13C NMR (DMSO-d6) δ 34.4, 111.9, 116.6 (d, 2JCF ) 20.1 Hz), 124.8, 128.7 (d, 4JCF ) 4.0 Hz), 133.9 (d, 3JCF ) 8.0 Hz), 134.9, 135.7, 139.1, 139.6, 151.3, 159.8, 163.0 (d, 1JCF ) 245.5 Hz), 164.5. 19F NMR (DMSO-d6) δ -113.5 (s). IR (KBr) 1734, 1662, 1636, 1574. m/z (EI) 334 (C16H1081BrFN2O3 -CO2, 49), 332 (C16H1079BrFN2O3 - CO2, 61), 42 (100). HRMS (EI) (M+•, C16H1081BrFN2O3) calcd 377.9830, found 377.9841, (M+•, C16H1079BrFN2O3) calcd 375.9859, found 375.9820. 5-(4-Bromophenyl)-6-carboxy-2-ethoxy-7-methyl8-oxo-7,8-dihydro-[1,7]naphthyridinium Chloride (36). To ester 27 (0.5 g, 1.18 mmol) in a mixture of EtOH/ THF 1/1 (100 mL) was added aqueous NaOH 5 N (5 mL). The mixture was heated under reflux for 2 h. After evaporation to dryness, HCl 5 N was added to the residue, and the final suspension was filtered off to give 36 as a yellow solid, mp 218 °C; 64% (0.33 g). 1H NMR (DMSO-d6) δ 1.37 (t, 3JHH ) 7.0 Hz, 3H), 3.54 (s, 3H), 4.47 (q, 3JHH ) 7.0 Hz, 2H), 7.13 (d, 3JHH ) 8.9 Hz, 1H), 7.29 (d, 3JHH ) 8.4 Hz, 2H), 7.44 (d, 3JHH ) 8.9 Hz, 1H), 7.70 (d, 3JHH ) 8.4 Hz, 2H). 13C NMR (DMSO-d6) δ 14.7, 33.9, 62.3, 112.6, 117.5, 122.1, 127.8, 131.9, 133.2, 133.5, 137.1, 138.5, 159.1, 162.7, 164.4, 177.0. IR (KBr) 1724, 1636. m/z (EI) 404 (M+•, C18H1581BrN2O4, 89), 402 (M+•, C18H1579BrN2O4, 88), 126 (100). 5-(4-Bromophenyl)-6-carboxy-2-hydroxy-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridinium Chloride (37). Acid 36 (0.13 g, 0.30 mmol) in HCl 6 N (6.5 mL) was refluxed for 2 h. After cooling to room temperature and filtration, acid 37 was isolated as a beige solid, mp > 265 °C; 60% (0.073 g). 1H NMR (DMSO-d6) δ 3.56 (s, 3H), 6.69 (d, 3JHH ) 9.7 Hz, 1H), 7.16 (d, 3JHH ) 9.7 Hz, 1H), 7.27 (d, 3JHH ) 8.4 Hz, 2H), 7.70 (d, 3JHH ) 8.4 Hz, 2H). 13C NMR (DMSO-d6) δ 34.0, 113.7, 119.7, 122.3, 128.0, 130.0, 131.9, 133.0, 133.1, 136.9, 154.3, 161.2, 164.1, 177.2. IR (KBr) 3330, 1664, 1654. m/z (EI) 376 (M+•, C16H1181BrN2O4, 45), 374 (M+•, C16H1179BrN2O4, 45), 330 (100). 5-(4-Bromophenyl)-2-hydroxy-7-methyl-8-oxo-7,8dihydro-[1,7]naphthyridine-6-carboxylic Ethyl Ester (38). Ester 27 (0.05 g, 0.11 mmol) and HCl (6 N, 1 mL) in 1,4-dioxane (1 mL) was refluxed for 24 h. After evaporation of solvent, water (3 mL) was added to the residue. The suspension was filtered off to give a mixture of compounds 37 and 38 in a 1/1 ratio. 1H NMR (DMSOd6) δ 0.89 (t, 3JHH ) 7.0 Hz, 3H), 3.53 (s, 3H), 4.04 (q, 3 JHH ) 7.0 Hz, 2H), 6.70 (d, 3JHH ) 9.7 Hz, 1H), 7.18 (d, 3 JHH ) 9.7 Hz, 1H), 7.24 (d, 3JHH ) 8.0 Hz, 2H), 7.68 (d, 3 JHH ) 8.0 Hz, 2H). Synthesis of N-Methylbenzylammonium Trifluoroacetate 41. N-[3,5-Bis(trifluoromethyl)benzyl]car-


bamic Acid tert-Butyl Ester (39). To 3,5-bis(trifluoromethyl)benzylamine (10 g, 41.1 mmol) in CH2Cl2 (125 mL) were added triethylamine (0.577 mL, 4.11 mmol) and di-tert-butyl dicarbonate (9.4 g, 43.2 mmol) at room temperature. The mixture was stirred for 4 h, and then aqueous saturated NaHCO3 (70 mL) was added. After extraction with CH2Cl2 (2 × 30 mL), the organic layers were combined, washed with HCl 1 N (50 mL) and then with water (2 × 50 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was recrystallized from pentane: white solid, mp 72-73 °C; 95% (14 g). 1H NMR (CDCl3) δ 1.46 (s, 9H), 4.42-4.44 (m, 2H), 5.30 (br s, 1H), 7.73 (s, 2H), 7.77 (s, 1H). 13C NMR (CDCl3) δ 28.6, 44.1, 80.6, 121.2 (sept, 3JCF ) 3.8 Hz), 123.2 (q, 1JCF ) 273.0 Hz), 127.6-127.7 (m), 132.2 (q, 2JCF ) 33.1 Hz), 142.3, 156.3. 19F NMR (CDCl3) δ -63.6 (s). IR (KBr) 1684, 1442, 1278. m/z (CI, isobutane) 344 (MH+, C14H16F6NO2, 100). HRMS (CI, isobutane) (MH+, C14H16F6NO2) calcd 344.1091, found 344.1085. Anal. (C14H16F6NO2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methylcarbamic Acid tert-Butyl Ester (40). To a suspension of NaH (60% dispersion in mineral oil, 0.7 g, 17.5 mmol) in DMF (80 mL) was added carbamate 39 (3 g, 8.75 mmol). The mixture was stirred at room temperature for 1 h, and methyl iodide (1.1 mL, 17.5 mmol) was added. After stirring for 15 h, water (120 mL) and then ethyl acetate (180 mL) were added. The aqueous layer was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed successively with HCl 1 N (60 mL), aqueous saturated NaHCO3, and water (60 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was purified by column chromatography: yellow oil; 80% (2.45 g). Rf 0.4 (AcOEt/pentane 1/9). 1H NMR (CDCl3) δ 1.50 (s, 9H), 2.93 (s, 3H), 4.58 (s, 2H), 7.43 (s, 2H), 7.81 (s, 1H). 13C NMR (CDCl3) δ 28.4, 34.6, 52.3, 80.6, 121.4 (sept, 3JCF ) 3.8 Hz), 123.6 (q, 1JCF ) 267.5 Hz), 127.6, 133.2 (q, 2JCF ) 33.1 Hz), 141.5, 156.4. 19 F NMR (CDCl3) δ -63.7 (s). IR (NaCl) 1698, 1394, 1136. m/z (CI, isobutane) 358 (MH+, C15H18F6NO2, 40), 302 (100). HRMS (CI, isobutane) (MH+, C15H18F6NO2) calcd 358.1242, found 358.1246. Anal. (C15H18F6NO2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methylammonium Trifluoroacetate (41). A mixture of N-methylcarbamate 40 (2.35 g, 6.58 mmol) and trifluoroacetic acid (9 mL, 116.7 mmol) in CH2Cl2 was stirred at room temperature for 15 h. After concentratrion under vacuum, the crude product was recrystallized from Et2O: white solid, mp 149-150 °C; 87% (2.12 g). 1H NMR (DMSOd6) δ 2.70 (s, 3H), 4.40 (s, 2H), 8.20 (s, 1H), 8.30 (s, 2H), 9.50 (br s, 2H). 13C NMR (DMSO-d6) δ 32.6, 50.4, 117.4 (q, 1JCF ) 298.1, Hz), 122.9 (sept, 3JCF ) 3.8 Hz), 123.5 (q, 1JCF ) 272.9 Hz), 125.7, 131.0 (q, 2JCF ) 32.8 Hz), 135.7, 159.2 (q, 2JCF ) 31.7 Hz). 19F NMR (DMSO-d6) δ -62.1 (s, 6F), -74.5 (s, 3F). m/z (EI) 371 (M+•, C12H10F9NO2, 0.5), 257 (100). Anal. (C12H10F9NO2) C, H, N. Synthesis of N-Methylglycine Amide 43. N-{N[3,5-Bis(trifluoromethyl)benzyl]-N-methylcarbamoylmethyl}-N-methylcarbamic Acid tert-Butyl Ester (42). To a mixture of DCC (0.66 g, 3.2 mmol), Bocsarcosine (0.51 g, 2.7 mmol), and DMAP (0.03 g, 0.27 mmol) in chloroform (30 mL) were added benzylammonium trifluoroacetate 41 (1 g, 2.7 mmol) and diisopropylethylamine (0.56 mL, 3.2 mmol) in chloroform (10 mL). The mixture was stirred at room temperature for 15 h, and the precipitate was filtered off. The filtrate was washed successively with aqueous saturated Na2CO3 (20 mL), HCl 1 M (20 mL), and water (20 mL), dried over MgSO4, and then concentrated under vacuum. After


addition of Et2O (50 mL) to the residue, stirring for 10 min, and filtration of the resulting suspension, the organic layer was concentrated under vacuum to give a crude product which was recrystallized from pentane: white solid, mp 59-61 °C; 65% (0.75 g). 1H NMR (CDCl3) (2 rotamers) δ 1.40 (s, 5.4H), 1.48 (s, 3.6H), 2.95 (s, 1.2H), 2.98 (s, 3H), 2.99 (s, 1.8H), 4.06 (s, 0.8H), 4.13 (s, 1.2H), 4.64 (s, 1.2H), 4.70 (s, 0.8H), 7.72 (s, 2H), 7.79 (s, 1H). 13 C NMR (CDCl3) δ 28.6, 34.6, 36.2, 52.9, 80.5, 122.0122.1 (m), 123.5 (q, 1JCF ) 297.8 Hz), 128.4 (m), 132.4 (q, 2JCF ) 33.2 Hz), 140.2, 156.7, 169.8. 19F NMR (CDCl3) δ -63.4 (s). IR (KBr) 1694, 1652, 1282, 1174, 1130. m/z (EI) 428 (M+•, C18H22F6N2O3, 0.1), 372 (3), 44 (100). Anal. (C18H22F6N2O3) C, H, N. N-{N-[3,5-Bis(trifluoromethyl)benzyl]-N-methylcarbamoylmethyl}-N-methylammonium Trifluoroacetate (43). To carbamate 42 (2.03 g, 4.74 mmol) in chloroform (6 mL) was added trifluoroacetic acid (5.5 mL, 71.1 mmol) at 0 °C. After stirring at room temperature for 15 h, the mixture was concentrated under vacuum to give a crude product which was recrystallized from Et2O: white solid, mp 100-101 °C; 66% (1.38 g). 1H NMR (DMSO-d6) δ 2.55 (s, 3H), 2.94 (s, 3H), 4.13 (s, 2H), 4.70 (s, 2H), 6.83 (br s, 2H), 7.90 (s, 2H), 7.93 (s, 1H). 13C NMR (DMSO-d6) δ 33.0, 34.5, 48.9, 50.1, 116.6 (q, 1JCF ) 292.5 Hz), 121.4-121.5 (m), 123.6 (q, 1JCF ) 270.6 Hz), 128.7128.8 (m), 130.8 (q, 2JCF ) 32.5 Hz), 141.1, 158.9 (q, 1JCF ) 34.4 Hz), 166.7. 19F NMR (DMSO-d6) δ -61.9 (s, 6F), -75.1 (s, 3F). IR (KBr) 1690, 1670, 1282, 1150. m/z (EI) 328 (M+•, C13H14F6N2O, 0.5), 44 (100). HRMS (FAB+) (MH+, C13H15F6N2O) calcd 329.1089, found 329.1077. Anal. (C15H15F9N2O3) C, H, N. N-{N-[3,5-Bis(trifluoromethyl)benzyl]carbamoylmethyl}-N-methylcarbamic Acid tert-Butyl Ester (44). To a mixture of 3,5-bis(trifluoromethyl)benzylamine (1.5 g, 6.17 mmol), Boc-sarcosine (1.17 g, 6.17 mmol), and DMAP (0.075 g, 0.62 mmol) in chloroform (30 mL) was added DCC (1.59 g, 7.71 mmol). The mixture was stirred at room temperature for 23 h, and the formed precipitate was filtered off. The filtrate was washed successively with aqueous saturated Na2CO3 (40 mL), HCl 1 M (40 mL), and water (40 mL), dried over MgSO4, and then concentrated under vacuum. After addition of Et2O (50 mL) to the residue, stirring for 10 min, and filtration of the resulting suspension, the organic layer was concentrated under vacuum to give a crude product which was recrystallized from pentane: white solid, mp 103-104 °C; 78% (1.98 g). 1H NMR (CDCl3) δ 1.41 (s, 9H), 2.97 (s, 3H), 3.92 (s, 2H), 4.58 (m, 2H), 7.00 (br s, 1H), 7.72 (s, 2H), 7.78 (s, 1H). 13C NMR (CDCl3) δ 27.8, 35.6, 41.9, 52.9, 80.6, 121.3 (sept, 3JCF ) 3.7 Hz), 123.5 (q, 1JCF ) 272.4 Hz), 127.6, 132.0 (q, 2JCF ) 33.1 Hz), 141.5, 162.7. 19F NMR (CDCl3) δ -63.3 (s, 6F). IR (KBr) 1696, 1654, 1278, 1134. m/z (EI) 414 (M+•, C17H20F6N2O3, 0.2), 44 (100). Anal. (C17H20F6N2O3) C, H, N. 3-[3,5-Bis(trifluoromethyl)benzyl]-1-methylimidazolidine-2,4-dione (45). To a suspension of NaH (60% dispersion in mineral oil, 0.11 g, 2.75 mmol) in DMF (50 mL) was added carbamate 44 (1 g, 2.41 mmol) in DMF (3 mL). The mixture was stirred at room temperature for 1 h, and methyl iodide (0.165 mL, 2.65 mmol) was added. After stirring for 18 h, water (50 mL) and then ethyl acetate (50 mL) were added. The aqueous layer was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with water (2 × 25 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was purified by chromatography followed by recrystallization in pentane: white solid, mp


69-70 °C; 60% (0.48 g); Rf 0.7 (pentane/AcOEt 2/1). 1H NMR (CDCl3) δ 2.94 (s, 3H), 3.85 (s, 2H), 4.67 (s, 2H), 7.73 (s, 1H), 7.79 (s, 2H). 13C NMR (CDCl3) δ 30.1, 42.1, 52.1, 122.5, 123.5 (q, 1JCF ) 271.3 Hz), 129.5, 132.4 (q, 2J 19F NMR (CDCl ) δ CF ) 33.1 Hz), 138.8, 156.4, 169.7. 3 -63.5 (s). IR (KBr) 1780, 1712, 1294, 1174, 1136. m/z (EI) 340 (M+•, C13H10F6N2O2, 100). Anal. (C13H10F6N2O2) C, H, N. General Procedure for Amidation of Acids 1721 with Sarcosine Amide 43. A mixture of acid 1721 (1 equiv), oxalyl chloride (3.3 equiv), and DMF (0.1 equiv) in CH2Cl2 (10-40 mL) was stirred at room temperature for 2-4 h and then concentrated under vacuum. After dilution of the residue in CH2Cl2 (20-60 mL), sarcosine amide 43 (1.0-1.8 equiv) and then Et3N (4 equiv) were added. The mixture was stirred at room temperature for 16 h and then concentrated under vacuum. After addition of ethyl acetate (30 mL) to the residue and filtration, the organic layer was washed with brine (2 × 20 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was purified by column chromatography. N-{N-[(3,5-Bis(trifluoromethyl)benzyl)-N-methylcarbamoylmethyl}-N-methyl-3-(4-bromobenzoyl)-6chloropyridine-2-carboxamide (46). Diamide 46 was obtained from 17 (1.7 g, 5.0 mmol) and 43 (2.3 g, 5.10 mmol): yellow oil; 65% (2.1 g). Rf 0.55 (CH2Cl2/Et2O 5.5/ 0.5). 1H NMR (CDCl3) δ 2.96 (s, 3H), 3.38 (s, 3H), 4.28 (s, 2H), 4.69 (s, 2H), 7.08-7.78 (m, 6H), 7.65 (s, 2H), 7.86 (s, 1H). 13C NMR (CDCl3) δ 35.8, 37.9, 45.2, 46.0, 115.4, 120.6, 122.3 (q, 1JCF ) 273.2 Hz), 122.4, 126.5, 126.9, 129.5 (q, 2JCF ) 32.7 Hz), 130.4, 132.4, 134.0, 135.3, 139.7, 141.5, 149.2, 157.0, 157.2, 171.6, 190.5. 19F NMR (CDCl3) δ -61.4 (s). N-{N-[(3,5-Bis(trifluoromethyl)benzyl)-N-methylcarbamoylmethyl}-N-methyl-6-chloro-3-(4-fluorobenzoyl)pyridine-2-carboxamide (47). Diamide 47 was obtained from 18 (0.5 g, 1.58 mmol) and 43 (0.7 g, 1.60 mmol): yellow oil; 61% (0.57 g). Rf 0.49 (CH2Cl2/Et2O 5/1). 1 H NMR (CDCl3) δ 3.05 (s, 3H), 3.31 (s, 3H), 4.30 (s, 2H), 4.73 (s, 2H), 7.12-7.80 (m, 6H), 7.69 (s, 2H), 7.82 (s, 1H). 13 C NMR (CDCl3) δ 35.7, 38.1, 45.0, 45.9, 115.3 (d, 2JCF ) 28.3 Hz), 120.5, 122.0 (q, 1JCF ) 273.3 Hz), 122.2, 126.8, 129.4 (q, 2JCF ) 33.1 Hz), 131.6, 133.8, 138.2, 139.7, 140.1, 149.3, 157.0, 157.2, 164.4 (d, 1JCF ) 261.2 Hz), 171.8, 190.7. 19F NMR (CDCl3) δ -63.4 (s, 6F), -111.3 (s, 1F). N-{N-[(3,5-Bis(trifluoromethyl)benzyl)-N-methylcarbamoylmethyl}-N-methyl-6-chloro-3-(4-iodobenzoyl)pyridine-2-carboxamide (48). Diamide 48 was obtained from 19 (0.235 g, 0.54 mmol) and 43 (0.25 g, 0.55 mmol): yellow oil; 29% (0.11 g). Rf 0.49 (CH2Cl2/ Et2O 5/0.5). 1H NMR (CDCl3) δ 3.00 (s, 3H), 3.22 (s, 3H), 4.28-4.75 (m, 4H), 7.41-7.85 (m, 9H). 13C NMR (CDCl3) δ 35.7, 38.0, 45.2, 45.9, 100.8, 120.5, 122.2 (q, 1JCF ) 273.2 Hz), 122.4, 126.8, 129.4 (q, 2JCF ) 32.8 Hz), 131.6, 133.9, 135.3, 138.1, 139.7, 141.4, 149.2, 156.9, 157.0, 171.6, 190.6. N-{N-[(3,5-Bis(trifluoromethyl)benzyl)-N-methylcarbamoylmethyl}-N-methyl-5-bromo-3-(4-fluorobenzoyl)pyridine-2-carboxamide (49). Diamide 49 was obtained from 20 (1.26 g, 3.50 mmol) and 43 (1.65 g, 3.87 mmol): yellow solid, mp 59-64 °C; 81% (1.8 g). Rf 0.50 (CH2Cl2/Et2O 5/1). 1H NMR (CDCl3) (2 rotamers) δ 2.93 (s, 0.9H), 2.98 (s, 2.1H), 3.07 (s, 0.9H), 3.14 (s, 2.1H), 4.36 (s, 1.4H), 4.52 (s, 0.6H), 4.69 (s, 1.4H), 4.76 (s, 0.6H), 7.14 (t, 3JHF ) 3JHH ) 8.5 Hz, 2H), 7.62 (s, 1.4H), 7.71 (s, 0.6H), 7.78 (s, 0.3H), 7.79 (s, 0.7H), 7.82 (dd, 3JHH ) 8.5 Hz, 4JHF ) 5.5 Hz, 2H), 7.92 (d, 4JHH ) 2.2 Hz, 1H), 8.47 (d, 4JHH ) 2.2 Hz, 0.3H), 8.79 (d, 4JHH ) 2.2 Hz, 0.6H). 13C NMR


(CDCl3) (2 rotamers) δ 34.7 and 35.1, 36.8 and 38.2, 49.6, 51.2, 116.3 and 116.4 (d, 2JCF ) 22.0 Hz), 120.9, 122.0 (sept, 3JCF ) 3.8 Hz), 123.6 (q, 1JCF ) 273.0 Hz), 128.5 (br s), 132.4 (q, 2JCF ) 33.1 Hz), 132.6, 133.1 and 133.2 (d, 3JCF ) 9.4 Hz), 136.5, 138.8 and 139.1, 140.2 and 140.3, 149.7 and 151.3, 152.9, 166.5 (d, 1JCF ) 257.3 Hz), 167.7, 168.2, 192.1. 19F NMR (CDCl3) δ -63.3 (s, 6F), -103.4 (s, 1F). IR (KBr) 1670, 1654, 1648, 1598, 1280. m/z (EI) 635 (M+•, C26H1981BrF7N3O3, 1), 633 (M+•, C26H1979BrF7N3O3 4), 44 (100). Anal. (C26H19BrF7N3O3) C, H, N. N-{N-[(3,5-Bis(trifluoromethyl)benzyl)-N-methylcarbamoylmethyl}-N-methyl-3-(4-fluorobenzoyl)-5iodopyridine-2-carboxamide (50). Diamide 50 was obtained from 21 (0.33 g, 0.80 mmol) and 43 (0.64 g, 1.44 mmol): white solid, mp 67-68 °C; 35% (0.19 g). Rf 0.60 (CH2Cl2/Et2O 5/1). 1H NMR (CDCl3) (2 rotamers) δ 2.95 (s, 0.9H), 3.02 (s, 2.1H), 3.16 (s, 0.9H), 3.24 (s, 2.1H), 4.38 (s, 1.4H), 4.54 (s, 0.6H), 4.70 (s, 1.4H), 4.78 (s, 0.6H), 7.18 (t, 3JHF ) 3JHH ) 8.4 Hz, 2H), 7.73 (s, 2H), 7.83 (dd, 3JHH ) 8.8 Hz, 4JHF ) 5.6 Hz, 2H), 7.84 (s, 1H), 8.03 (d, 4JHH ) 2.0 Hz, 0.3H), 8.12 (d, 4JHH ) 2.0 Hz, 0.7H), 8.65 (d, 4 JHH ) 2.0 Hz, 0.3H), 8.97 (d, 4JHH ) 2.0 Hz, 0.7H). 13C NMR (CDCl3) (2 rotamers) δ 34.7 and 35.1, 36.6 and 38.1, 49.6, 51.3 and 53.3, 93.2, 116.3 and 116.4 (d, 2JCF ) 22.1 Hz), 122.2, 123.6 (q, 1JCF ) 276.7 Hz), 128.5, 132.4 (q, 2 JCF ) 33.2 Hz), 132.6, 133.1 and 133.2 (d, 3JCF ) 9.1 Hz), 136.7, 140.2, 144.3 and 144.6, 151.7 and 153.3, 154.7 and 166.3, 167.6 (d, 1JCF ) 250.6 Hz), 167.8, 168.2, 192.4. 19 F NMR (CDCl3) δ -63.3 (s, 6F), -103.7 (s, 1F). IR (KBr) 1670, 1650, 1280. m/z (EI) 681 (M+•, C26H19FIN3O2, 37), 354 (100). Anal. (C26H19FIN3O2) C, H, N. Synthesis of Compounds 1-5. General Procedure for Amidation of Acids 34 and 35 with Benzylammonium Trifluoroacetate 41. A mixture of acid 34 or 35 (1 equiv), oxalyl chloride (13.5 equiv), and DMF (0.1 equiv) in CH2Cl2 (10-40 mL) was stirred at room temperature for 2.5 h and then concentrated under vacuum. To the residue were added CH2Cl2 (20-60 mL), benzylammonium trifluoroacetate 41 (1.35 equiv), and Et3N (5.35 equiv). The mixture was stirred at room temperature for 16 h and then concentrated under vacuum. After addition of ethyl acetate (30 mL) to the residue and filtration of the suspension, the organic layer was washed successively with brine (50 mL), HCl 1 N (50 mL), brine (50 mL), saturated NaHCO3 (50 mL), and brine (50 mL), dried over MgSO4, and concentrated under vacuum to give a crude oil which was purified by column chromatography. General Procedure for Cyclization-Dehydration of Polyamides 46-50 with DBU. A mixture of polyamides 46-50 (1 equiv) and DBU (3.7 equiv) in toluene (90-100 mL) was refluxed for 8 days. After cooling to room temperature, the solution was acidified with HCl 1 N to pH 1 and then pH was adjusted to 4-5 by addition of aqueous Na2CO3 (10%). After extraction with ethyl acetate (2 × 100 mL), the combined organic layers were washed with brine (2 × 20 mL), dried over MgSO4, and concentrated under vacuum to give a crude oil which was purified by chromatography. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-(4bromophenyl)-2-chloro-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (1). Naphthyridone 1 was obtained from acid 34 (1 g, 1.97 mmol) and benzylamine 41 (0.99 g, 266 mmol): white solid, mp 187 °C; 66% (0.82 g). Rf (2 rotamers) 0.72 (major) and 0.42 (minor) (AcOEt). 1H NMR (CDCl3) (2 rotamers) δ 2.79 (s, 0.38H), 2.91 (s, 2.62H), 3.63 (s, 2.62H), 3.67 (s, 0.38H), 4.23 and 4.87 (AB, 2JAB ) 14.4 Hz, 0.24H), 4.41 and 4.69 (AB, 2JAB ) 14.4 Hz, 1.76H), 7.08-7.52 (m, 6H), 7.65 (s,


2H), 7.90 (s, 1H). 13C NMR (CDCl3) δ 33.2, 35.9, 50.0, 112.4, 122.3, 122.5, 123.4 (q, 1JCF ) 273.0 Hz), 128.3, 130.9, 131.5, 131.9, 132.4 (q, 2JCF ) 32.8 Hz), 132.6, 132.9, 136.3, 136.4, 137.6, 140.8, 151.8, 159.4, 163.4.19F NMR (CDCl3) δ -63.3 (s). IR (KBr) 1654, 1648. m/z (EI) 635 (M+•, C26H1781Br37ClF6N3O2, 28), 633 (M+•, C26H1781BrF635ClN3O2 and C26H1779Br37ClF6N3O2, 100), 631 (M+•, C26H1779Br35ClF6N3O2, 74). Anal. (C26H17BrClF6N3O2) C, H, N. Naphthyridone 1 was also obtained from diamide 46 (2.1 g, 3.24 mmol) and DBU (1.8 mL, 12.0 mmol): 53% (1.1 g). N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-3-bromo-5-(4-fluorophenyl)-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (2). Naphthyridone 2 was obtained from acid 35 (1 g, 2.42 mmol) and benzylamine 41 (0.75 g, 2.90 mmol): white solid, mp 206-208 °C; 86% (1.3 g). Rf 0.1 (AcOEt). 1H NMR (CDCl3) δ 2.82 (s, 3H), 3.64 (s, 3H), 4.23 and 4.83 (AB, 2JAB ) 15.0 Hz, 2H), 7.01 (t, 3JHH ) 3JHF ) 8.0 Hz, 1H), 7.03 (t, 3 JHH ) 3JHF ) 8.0 Hz, 1H), 7.13 (dd, 3JHH) 7.6 Hz, 4JHF ) 4.8 Hz, 1H), 7.35 (dd, 3JHH ) 8.0 H, 4JHF ) 5.2 Hz, 1H), 7.54 (s, 2H), 7.63 (d, 4JHH ) 2.1 Hz, 1H), 7.84 (s, 1H), 8.91 (d, 4JHH ) 2.1 Hz, 1H). 13C NMR (CDCl3) δ 33.5, 36.2, 50.2, 112.5, 116.0 (d, 2JCF ) 21.2 Hz), 116.9 (d, 2JCF ) 21.2 Hz), 122.6-122.7 (m), 123.3 (q, 1JCF ) 272.8 Hz), 125.2, 127.7 (d, 4JCF ) 3.7 Hz), 129.3 (br s), 131.5 (d, 3JCF ) 8.2 Hz), 132.6 (q, 2JCF ) 33.6 Hz), 133.8 (d, 3JCF ) 8.2 Hz), 134.3, 135.5, 137.7, 137.8, 139.7, 151.8, 160.6, 163.1 (d, 1JCF ) 250.3 Hz), 163.7. 19F NMR (CDCl3) δ -63.3 (s, 6F), -111.1 (s, 1F). IR (KBr) 1672, 1650, 1616, 1282, 1162. m/z (EI) 618 (MH+, C26H1781BrF7N3O2, 35), 617 (M+•, C26H1781BrF7N3O2, 100), 616 (MH+, C26H1779BrF7N3O2, 33), 615 (M+•, C26H1779BrF7N3O2, 100). Anal. (C26H17BrF7N3O2) C, H, N. Naphthyridone 2 was also obtained from diamide 49 (1.67 g, 2.6 mmol) and DBU (2.3 g, 15.6 mmol): 62% (1 g) or from diamide 49 and NaH according to the following procedure. To a suspension of NaH (60% dispersion in mineral oil, 1.9 mg, 0.08 mmol) in DMF (10 mL) was added dropwise, at 0 °C, a solution of diamide 49 (0.05 g, 0.08 mmol) in THF. The mixture was heated at 100 °C for 4 h, cooled to 0 °C, and then poured onto ice (5 g). After extraction with ethyl acetate (3 × 5 mL), the combined organic layers were washed with water (3 × 10 mL), dried over MgSO4, and concentrated under vacuum to give a crude product which was purified by column chromatography: 100% (0.051 g). N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-(4fluorophenyl)-3-iodo-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (3). Naphthyridone 3 was obtained from diamide 50 (0.9 g, 0.27 mmol) and DBU (0.25 mL, 1.70 mmol): white solid, mp 114116 °C; 17% (0.03 g). Rf 0.3 (AcOEt). 1H NMR (CDCl3) δ 2.83 (s, 3H), 3.65 (s, 3H), 4.24 and 4.86 (AB, 2JAB ) 14.4 Hz, 2H), 7.02 (t, 3JHH ) 3JHF ) 8.5 Hz, 1H), 7.03 (t, 3JHH ) 3JHF ) 8.3 Hz, 1H), 7.13 (dd, 3JHH ) 9.0 Hz, 4JHF ) 5.7 Hz, 1H), 7.35 (dd, 3JHH ) 9.0 Hz, 4JHF ) 5.7 Hz, 1H), 7.55 (s, 2H), 7.86 (s, 1H), 7.86 (d, 4JHH ) 1.7 Hz, 1H), 9.08 (d, 4 JHH ) 1.7 Hz, 1H). 13C NMR (CDCl3) δ 33.6, 36.2, 50.2, 98.9, 112.5, 116.0 (d, 2JCF ) 22.0 Hz), 116.9 (d, 2JCF ) 21.2 Hz), 122.7-122.8 (m), 123.3 (q, 1JCF ) 272.0 Hz), 127.7 (d, 4JCF ) 3.4 Hz), 129.3-129.4 (m), 131.5 (d, 3JCF ) 8.2 Hz), 132.6 (q, 2JCF ) 33.5 Hz), 133.8 (d, 3JCF ) 8.1 Hz), 134.3, 137.5, 137.9, 140.0, 141.6, 156.5, 160.9, 163.1 (d, 1JCF ) 250.3 Hz), 163.7. 19F NMR (CDCl3) δ -63.3 (s, 6F), -111.1 (s, 1F). IR (KBr) 1680, 1650, 1500, 1280. m/z (EI) 663 (M+•, C26H17F7IN3O2, 41), 91 (100). HRMS (EI) (M+•, C26H17F7IN3O2) calcd 663.3255, found 663.3253. Anal. (C26H17F7IN3O2) C, H, N.


N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-2chloro-5-(4-fluorophenyl)-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridine-6-carboxamide (4). Naphthyridone 4 was obtained from diamide 47 (0.45 g, 0.76 mmol) and DBU (0.42 mL, 2.83 mmol): beige solid, mp 128-130 °C; 43% (0.19 g). Rf 0.52 (AcOEt). 1H NMR (CDCl3) δ 2.81 (s, 3H), 3.56 (s, 3H), 4.19 and 4.73 (AB, 2 JAB ) 14.4 Hz, 2H), 6.87-7.25 (m, 6H), 7.48 (s, 2H), 7.77 (s, 1H). 13C NMR (CDCl3) δ 33.6, 36.3, 50.2, 113.1, 116.7 (d, 2JCF ) 21.4 Hz), 122.3 (q, 1JCF ) 271.7 Hz), 128.0, 128.6, 129.3, 131.6, 132.4 (q, 2JCF ) 31.7 Hz), 132.7, 133.7, 136.9, 137.9, 139.2, 141.1, 159.8, 163.0 (d, 1JCF ) 250.1 Hz), 163.8, 169.2. 19F NMR (CDCl3) δ -63.3 (s, 6F), -111.3 (s, 1F). IR (KBr) 1654, 1648. HRMS (EI) (M+•, C26H17ClF7N3O2) calcd 571.8742, found 571.8748. Anal. (C26H17ClF7N3O2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-2chloro-5-(4-iodophenyl)-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (5). Naphthyridone 5 was obtained from diamide 48 (0.11 g, 0.16 mmol) and DBU (0.09 mL, 0.59 mmol): colorless oil; 64% (0.07 g). Rf 0.43 (AcOEt/pentane 85/15). 1H NMR (CDCl3) δ 2.77 (s, 3H), 3.53 (s, 3H), 4.38 and 4.52 (AB, 2JAB ) 14.6 Hz, 2H), 6.84-6.87 (m, 1H), 7.00-7.05 (m, 1H), 7.36-7.45 (m, 2H), 7.55-7.61 (m, 4H), 7.84 (s, 1H). 13C NMR (CDCl3) δ 33.4, 35.9, 50.2, 95.4, 112.4, 122.1, 123.4 (q, 1 JCF ) 273.1 Hz), 128.5, 130.8, 131.4, 132.5 (q, 2JCF ) 32.6 Hz), 132.7, 132.9, 136.3, 136.6, 137.7, 138.0, 140.8, 151.9, 159.6, 163.6. HRMS (EI) (M+•, C26H17ClF6IN3O2) calcd 679.7804, found 679.7807. Anal. (C26H17ClF6IN3O2) C, H, N. Synthesis of Compounds 6-9: Halogen-Halogen Exchanges. N-[3,5-Bis(trifluoromethyl)benzyl]-Nmethyl-5-(4-bromophenyl)-2-fluoro-7-methyl-8-oxo7,8-dihydro-[1,7]naphthyridine-6-carboxamide (6). A mixture of naphthyridone 1 (0.10 g, 0.16 mmol), potassium fluoride (0.027 g, 0.16 mmol), and Kryptofix 222 (0.060 g, 0.16 mmol) in DMSO (0.7 mL) was heated at 120 °C for 4 h. After cooling to room temperature, water (15 mL) was added and the mixture was extracted with ethyl acetate (40 mL). The organic layer was washed with water, dried over magnesium sulfate, filtered off, and concentrated under vacuum to give a crude product which was purified by chromatography: beige solid, mp 97-100 °C; 57% (0.055 g). Rf 0.65 (AcOEt). 1H NMR (CDCl3) δ 2.89 (s, 3H), 3.62 (s, 3H), 4.40 and 4.69 (AB, 2 JAB ) 14.4 Hz, 2H), 7.07-7.27 (m, 3H), 7.40-7.48 (m, 2H), 7.61-7.68 (m, 3H), 7.88 (s, 1H). 13C NMR (CDCl3) δ 33.2, 35.8, 50.0, 111.3 (d, 2JCF ) 28.4 Hz), 112.7, 122.3, 122.6, 123.3 (q, 1JCF ) 273.0 Hz), 128.4, 131.3, 131.7, 132.4 (q, 2JCF ) 32.8 Hz), 132.7, 132.8, 136.2, 136.4, 137.9, 141.2, 156.4 (d, 1JCF ) 255.2 Hz), 161.3, 163.4. 19F NMR (CDCl3) δ -62.1 (s, 1F), -63.3 (s, 6F). IR (KBr) 1654, 1648. m/z (EI) 617 (M+•, C26H1781BrF7N3O2, 19), 615 (M+•, C26H1779BrF7N3O2, 20), 334 (100). HRMS (EI) (M+•, C26H1779BrF7N3O2) calcd 615.0394, found 615.0392. Anal. (C26H17BrF7N3O2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-2-fluoro-5-(4-iodophenyl)-7-methyl-8-oxo-7,8-dihydro-[1,7]naphthyridine-6-carboxamide (7). A mixture of naphthyridone 5 (0.07 g, 0.10 mmol) and potassium fluoride (0.018 g, 0.31 mmol) in DMSO (1 mL) was heated at 120 °C for 6 h. After cooling to room temperature, water (15 mL) was added and the mixture was extracted with ethyl acetate (40 mL). The organic layer was washed with water, dried over magnesium sulfate, filtered off, and concentrated under vacuum to give a crude product which was purified by chromatography: white solid, 47% (0.032 g). Rf 0.46 (AcOEt). 1H NMR (CDCl3) δ 2.90 (s, 3H), 3.60


(s, 3H), 4.38 and 4.67 (AB, 2JAB ) 14.3 Hz, 2H), 7.077.22 (m, 3H), 7.42-7.49 (m, 2H), 7.54-7.68 (m, 3H), 7.86 (s, 1H). 13C NMR (CDCl3) δ 33.4, 35.7, 50.0, 95.3, 112.4, 113.9 (d, 2JCF ) 28.4 Hz), 122.1, 123.6 (q, 1JCF ) 273.0 Hz), 128.5, 130.8, 131.3, 132.5 (q, 2JCF ) 32.6 Hz), 132.6, 136.3, 136.5, 137.6, 138.2, 140.8, 156.2 (d, 1JCF ) 255.1 Hz), 159.4, 163.9. HRMS (EI) (M+•, C26H17F7IN3O2) calcd 663.3255, found 663.3257. Anal. (C26H17F7IN3O2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-(4fluorophenyl)-2-iodo-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (8). A mixture of naphthyridone 4 (0.1 g, 0.18 mmol), acetyl chloride (97 µL), and sodium iodide (0.15 g, 0.91 mmol) in CH3CN (2.5 mL) was stirred for 1 h at room temperature. After addition of aqueous K2CO3 10% (10 mL) and NaHSO3 5% (10 mL), the mixture was extracted with CH2Cl2 (3 × 15 mL). The combined organic layers were dried over MgSO4, filtered off, and concentrated under vacuum to give a crude product which was purified by HPLC (column Waters Symmetry C18, 300 × 7.8 mm i.d., size 7 µm; λ 254 nm; flow rate 2 mL/min; mobile phase: MeOH/H2O, 80/20, tR 13.8 min). After evaporation of the eluent, 8 was obtained as a yellow oil: 80% (0.097 g). 1H NMR (CDCl3) δ 2.83 (s, 3H), 3.62 (s, 3H), 4.25 and 4.82 (AB, 2JAB ) 14.3 Hz, 2H), 6.87-7.33 (m, 6H), 7.54 (s, 2H), 7.80 (s, 1H). 13C NMR (CDCl3) δ 33.6, 36.2, 50.2, 113.2, 116.7 (d, 2JCF ) 21.2 Hz), 122.5 (q, 1JCF ) 272.5 Hz), 128.0, 129.3, 131.6, 132.4 (q, 2JCF ) 31.6 Hz), 132.7, 134.9, 137.1, 133.7, 137.9, 138.7, 142.4, 159.7, 163.8, 163.1 (d, 1 JCF ) 250.5 Hz), 169.1. 19F NMR (CDCl3) δ -63.3 (s, 6F), -111.4 (s, 1F). IR (KBr) 1654, 1648. m/z (EI) 664 (MH+, C26H18F7IN3O2, 20), 663 (M+•, C26H17F7IN3O2, 36), 227 (100). HRMS (EI) (M+•, C26H17F7IN3O2) calcd 663.3255, found 663.3259. Anal. (C26H17F7IN3O2) C, H, N. N-[3,5-Bis(trifluoromethyl)benzyl]-N-methyl-5-(4bromophenyl)-2-iodo-7-methyl-8-oxo-7,8-dihydro[1,7]naphthyridine-6-carboxamide (9). A mixture of naphthyridone 1 (0.05 g, 0.08 mmol), acetyl chloride (42 µL, 0.59 mmol), and sodium iodide (0.059 g, 0.4 mmol) in CH3CN (2.5 mL) was stirred for 1 h at room temperature. After addition of aqueous K2CO3 10% (10 mL) and NaHSO3 5% (10 mL), the mixture was extracted with CH2Cl2 (3 × 15 mL). The combined organic layers were dried over magnesium sulfate, filtered off, and concentrated under vacuum to give a crude product which was purified by HPLC (column Waters µBondaPak C18, 300 × 7.8 mm i.d., size 10 µm; λ 254 nm; flow rate 3.5 mL/ min; mobile phase: MeOH/H2O, 70/30, tR 30.7 min). After evaporation of solvent, 9 was obtained as a yellow solid, mp 50-60 °C; 37% (0.021 g). 1H NMR (CDCl3) δ 2.86 (s, 3H), 3.60 (s, 3H), 4.39 and 4.66 (AB, 2JAB ) 14.4 Hz, 2H), 7.03-7.07 (m, 1H), 7.13 (d, 3JHH ) 8.5 Hz, 1H), 7.187.26 (m, 1H), 7.40-7.48 (m, 2H), 7.63 (s, 2H), 7.81 (d, 3 JHH ) 8.5 Hz, 1H), 7.89 (s, 1H). 13C NMR (CDCl3) δ 33.1, 36.0, 50.2, 112.4, 118.8, 122.2, 122.4, 123.4 (q, 1JCF ) 273.0 Hz), 128.2, 131.5, 131.8, 132.3 (q, 2JCF ) 32.8 Hz), 132.6, 132.8, 135.7, 136.2, 136.4, 137.7, 140.7, 159.2, 163.4. 19F NMR (CDCl3) δ -63.4 (s). IR (KBr) 1654, 1648. m/z (EI) 726 (MH+, C26H1881BrF6IN3O2, 46), 725 (M+•, C26H1781BrF6IN3O2, 100), 724 (MH+, C26H1879BrF6IN3O2, 48), 723 (M+•, C26H1779BrF6IN3O2, 99). HRMS (EI) (M+•, C26H1779BrF6IN3O2) calcd 722.9453, found 722.9441. Anal. (C26H17BrF6IN3O2) C, H, N. Cell Culture and Membrane Preparation. CHO cells expressing the human NK1 receptor were cultured in Ham’s F-12 nutrient mixture supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. Cells were seeded and grown to confluence in 100 nm diameter



Scheme 1a

a Reagents: (a) SOCl ; (b) YC H , AlCl (2.5 equiv); (c) (1) (COCl) , DMF, (2) sarcosine ester, Et N; (d) DBU; (e) for 34 from 28, 2 6 5 3 2 3 CF3CO2H; for 35 from 29, KOHaq 1 N; for 36, 37, or 38 from 26 or 27, NaOH or HCl; (f) (1) (COCl)2, DMF, (2) 3,5bis(trifluoromethyl)benzylamine, Et3N.

culture dishes. Cells were first washed with 8 mL of 50 mM Tris/HCl, pH 7.4, 0.15 M NaCl and incubated until detachment with 50 mM Tris/HCl, pH 7.4, 0.15 M NaCl containing EDTA (2 mM). Detaching cells were collected in Falcon tubes and centrifuged for 15 min (1000g). The pellet was then resuspended at 4 °C in 0.5 mL (per dish) of 10 mM Tris/HCl, pH 7.4, containing 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 40 µg/mL bacitracin, 5 µg/mL leupeptin, and 5 µg/mL soybean trypsin inhibitor. After 30 min at 4 °C, the pellets were homogenized with a Potter-Elveheim glass homogenizer (10-15 times) and centrifuged (1000g) to remove debris. The resulting supernatant was sedimented at 48000g for 30 min at 4 °C, the membrane pellet was stored at -80 °C in 50 mM Tris/HCl, pH 7.4, containing 1 mM EDTA, 10 mM MgCl2, 35 µg/mL phenylmethylsulfonyl fluoride (PMSF), 50 µg/mL bacitracin, 2 µg/ mL leupeptin, 300 µg/ mL of benzamidine, 2 µg/ mL of chymostatin, at a concentration of 2 mg protein/ mL which was determined using the BCA protein assay reagent (Pierce). Binding Assays. CHO cell membranes (10 µg of proteins) were preincubated for 90 min at room temperature in a binding buffer (50 mM TrisHCl, pH 7.4, containing 3 mM MnCl2, 0.1% bovine serum albumin, 35 µg/mL of phenylmethylsulfonyl fluoride, 50 µg/ mL of bacitracin, 2 µg/ mL of leupeptin, 300 µg/ mL of benzamidine, 2 µg/ mL of chymostatin). After centrifugation (4000g, 10 min) and removal of supernatants, the pellets were incubated for 3 h with [125I]-(BH)-SP (74 TBq/mmol, final concentration 6 pmol/tube) and with EP 00652218 or analogues 1-9 at different final concentrations ranged between 10-5 M and 10-11 M in a total assay volume of 200 µL. The reaction was stopped by filtration on GF/C filters (Whatman International, Maidstone) previously saturated for 5 h in polyethylenimine 2% (w/v). After four washes with 4 mL of cold 50 mM Tris-HCl, pH 7.4, radioactivity was directly counted. Nonspecific binding was estimated by adding an excess of SP (10-6 M).

RESULTS AND DISCUSSION

Syntheses of the target compounds 1-9 are shown in Schemes 1-4. The strategy was based on the described synthesis of EP 00652218 starting from quinolinic anhydride (27-30). Due to the presence of an halogen on the pyridine ring of the naphthyridones 1-9, this procedure required important modifications. Halogenated quinolinic anhydrides 14-16, not being commercially available, the corresponding quinolinic acids 11-13 were employed as starting material. These latter were obtained by oxidation of halogenoquinolines according to methods we previously developed (31). Acids 11-13 were transformed into anhydrides 14-16 by dehydration of the corresponding quinolinic acids. Thionyl chloride (33, 34) was shown to be the reagent of choice compared to acetic anhydride (32, 35) (overall yields: 7393% versus 30-60%). Reaction of anhydrides 14-16 with 4-halobenzenes under Friedel-Crafts conditions (27-30, 32) yielded the acids 17-21 with a total regioselectivity. The reaction conditions were set up with fluorobenzene. Initial attempts using stoichiometric amounts of reagents and substrate and no solvent failed (36). When fluorobenzene was the solvent of the reaction, the yields of 20 were strongly dependent on the amounts of aluminum chloride (42, 63, and 100%, respectively, with 1.67, 2, and 2.5 equiv). Under these conditions (haloarene as the solvent, 2.5 equiv of AlCl3), the bromo and fluoro compounds 17 and 21 were obtained in 46 and 55% yields, respectively. In 1,2-dichloroethane, the syntheses of 17 and 18 were achieved in 65 and 85% yields respectively whereas the iodo acid 19 was obtained in a low yield (25% from 1H NMR of the crude product) and was contaminated with its deiodinated analogue (19/byproduct: 75/25 ratio). Due to its instability, the iodo acid 19 was used without any purification.



Scheme 2a

a

Reagents: (a) t-BuOCO2t-Bu, Et3N; (b) (1) NaH, (2) MeI; (c) CF3CO2H; (d) Boc-sarcosine, DCC, DMAP, iPr2NEt; (e) CF3CO2H.

Scheme 3a

a

Reagents: (a) (1) (COCl)2, DMF, (2) sarcosine amide 43, Et3N; (b) DBU.

Amidation of acids 17 and 20 with N-methylglycine ester (R ) Et, Me, t-Bu) was carried out via the acid chlorides. The amides 22-25 were obtained in moderate to high yields (51-95%). Formation of the naphthyridoneesters 26-29 was achieved in 30-85% yield after purification by dehydration with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) in refluxing toluene. Hydrolysis of the 3-substituted ester 29 into the acid 35 was successfully carried out under basic conditions (27-30). These conditions as well as acidic ones (6 N HCl) (37) could not be used for the hydrolysis of the ethyl or methyl esters of 2-halo substituted pyridines 26 or 27 due to the competitive formation of the hydroxy- and alkoxynaphthyridones 36-38. After several attempts, the tert-butyl ester 28 was found the best substrate for an efficient hydrolysis (38) into the acid 34 (98%). Transformation of the acids 34 and 35 into the target compounds 1 and 2 required the preparation of the amine salt 41 (39, 40) for which no synthesis was reported. Attempts of direct methylation of (bistrifluoromethyl)benzylamine, reductive amination with formaldehyde (41) or reduction of an intermediate carbamate (42), failed. In all cases, a mixture of products was obtained due to either a polyalkylation or a partial reduction of the CF3 group. The amine salt 41 was obtained in 49% total yield in a three-step procedure from (bistrifluoromethyl)benzylamine (protection, methylation, and deprotection) (Scheme 2). Finally, its reaction with acids 34 and 35 afforded the compounds 1 and 2 in 10 and 15% overall yields, respectively, from the corresponding quinolinic acids 11 and 12.

To shorten the synthesis and to improve the overall yield, the synthesis of amides 46-50 was envisaged. With this aim, a synthesis of the salt 43 was studied. Attempt to methylate (NaH and then MeI) the amino-amide 44 led to the cyclic compound 45. To avoid this side-reaction, the salt 43 was prepared by amidation of 41 with Bocsarcosine followed by deprotection of the amine (overall yield: 66%). Reaction of 43 with the pyridine carboxylic acids 17-21 as their acid chlorides led to the amides 4650 in moderate to good yields (Scheme 3). Transformation of amides 46-50 into the target compounds was first carried out with DBU in toluene as for esters 22-25. Under these conditions, the cyclization required more than 8 days and naphthyridone carboxamides 1-5 were obtained in 17-64% yields from amides 46-50. To improve both the yield and the time, the reaction parameters (solvent, base, temperature, and time) were studied. The cyclization-dehydration sequence was found slightly faster by using DMF instead of toluene and by adding calcium hydride as desiccant. No reaction was observed when this reagent was used without DBU. Attempts to use t-BuOK (43) provided compounds that could not be identified. Finally, sodium hydride (44) was found to be an ideal base and the naphthyridone 2 was obtained in quantitative yield when amide 49 was heated for 4 h in DMF at 100 °C. This route afforded the target compounds 1 and 2 in three steps from quinolinic acids 11 and 12 in double yields compared to those previously obtained. Syntheses of compounds 6-9 were undertaken by direct halogen/halogen exchange reactions from the chlo-



Scheme 4a

a

Reagents: (a) KF, K222, DMSO; (b) NaI, AcCl, CH3CN. Table 1. Affinities (IC50, nM; mean ( SEM) of SP, CP-96,345, and Compounds 1-9 compd

Figure 3. [125I]-(BH)-SP Displacement studies with SP(O), EP 00652218 (2), and compound 1 (b) on membranes of human NK1 receptor-transfected CHO cells.

ronaphthyridones 1, 4, and 5 (Scheme 4). The fluoro compounds 6 and 7 were prepared in 57 and 47% yields, respectively, from 1 and 5 using anhydrous potassium fluoride in the presence of Kryptofix in DMSO (45), conditions easily adaptable to the introduction of a fluorine-18 atom on a pyridine ring (46). Sodium iodide in hydriodic acid (47, 48) was tested for the transformation of chloro-compound 1 to its iodo analogue 9 but poor conversion (35%) was observed. The couple sodium iodide/ acetyl chloride (49) was shown to be more efficient. The exchange was quantitative (HPLC analysis), and the compound 9 was isolated in only 37% yield. This procedure, applied to the naphthyridone 4, afforded its iodo analogue 8 in 80% yield. Binding efficiency of naphthyridones 1-9 was evaluated by analyzing the specific displacement of iodinelabeled substance P ([125I]-(BH)-SP) on CHO cells stably transfected with the human recombinant NK1 receptor cDNA. This cellular model was chosen due to the large amounts of NK1 receptors expressed on their surface (5 × 105 receptors/cell) (50). Results were compared with those obtained with CP-96,345 and EP 00652218 used as references. As shown in Figure 3 and Table 1, compound 1 and EP 00652218 displayed similar affinities; this suggested that the introduction of a chlorine atom in position-2 on the pyridine ring and the substitution of a fluorine by a bromine on the phenyl ring exerted a small influence on the IC50 value. The 2-fluoro and

SP CP-96,345 EP 00652218 1 2 3 4 5 6 7 8 9

IM-9a 0.6-0.7b

0.4 ( 0.2 (Ki)c 0.21 ( 0.03d -

CHOe 0.8 ( 0.1 3 ( 1.5f 100 ( 20 100 ( 15 >1000 >1000 >1000 >1000 500 ( 60 >1000 500 ( 80 >1000

a Data from literature. b Reported in ref 52. c Reported in ref 51. d Reported in refs 27, 28. e Our results; see Experimental Section for details. f This value was comparable to that previously reported (8.6 nM ( 0.19) (53).

2-iodo derivatives 6 and 8 exhibited a 5-fold lower affinity whereas the IC50 values for the 2-haloanalogues 4, 5, 7, and 9 were drastically reduced. The replacement of the hydrogen by a bromine or a iodine in the 3-substituted series (compounds 2 and 3) led to a drastic loss of the binding properties contrary to the reported results concerning the introduction of a chlorine atom (28). This could be attributed to the high disruption in the steric and electronic effects for the active site recognition (27). Surprisingly, the binding affinity of EP 00652218 we determined on CHO cells was much lower than that originally reported by Takeda company (27-30) on IM-9 cells (IC50(CHO)/IC50(IM9) ) 500), which naturally expressed the NK1 receptor (

Synthesis and Biological Evaluation of Halogenated Naphthyridone

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