Biphenyl Pyridazinone Derivatives as Inhaled PDE4 Inhibitors

Article pubs.acs.org/jmc

Biphenyl Pyridazinone Derivatives as Inhaled PDE4 Inhibitors: Structural Biology and Structure−Activity Relationships Jordi Gràcia,*,† Maria Antonia Buil,† Jordi Castro,† Peter Eichhorn,‡ Manel Ferrer,† Amadeu Gavaldà,§ Begoña Hernández,† Victor Segarra,† Martin D. Lehner,†,⊥ Imma Moreno,† Lluís Pagès,† Richard S. Roberts,†,# Jordi Serrat,† Sara Sevilla,† Joan Taltavull,† Miriam Andrés,† Judit Cabedo,† Dolors Vilella,§ Elena Calama,§ Carla Carcasona,§ and Montserrat Miralpeix∥ †

Medicinal Chemistry and Screening, ‡Pharmacokinetics and Metabolism, §Experimental Dermatology, and ∥Licensing and Corporate Development, Centro de Investigación y Desarrollo, Almirall S.A., Crta. Laureà Miró 408-410, Sant Feliu de Llobregat, 08980 Barcelona, Spain S Supporting Information *

ABSTRACT: Cyclic nucleotide cAMP is a ubiquitous secondary messenger involved in a plethora of cellular responses to biological agents involving activation of adenylyl cyclase. Its intracellular levels are tightly controlled by a family of cyclic nucleotide degrading enzymes, the PDEs. In recent years, cyclic nucleotide phosphodiesterase type 4 (PDE4) has aroused scientific attention as a suitable target for anti-inflammatory therapy in respiratory diseases, particularly in the management of asthma and COPD. Here we describe our efforts to discover novel, highly potent inhaled inhibitors of PDE4. Through structure based design, with the inclusion of a variety of functional groups and physicochemical profiles in order to occupy the solventfilled pocket of the PDE4 enzyme, we modified the structure of our oral PDE4 inhibitors to reach compounds down to picomolar enzymatic potencies while at the same time tackling successfully an uncovered selectivity issue with the adenosine receptors. In vitro potencies were demonstrated in a rat lung neutrophilia model by administration of a suspension with a Penn-Century MicroSprayer Aerosolizer.

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INTRODUCTION Intracellular levels of the secondary messengers cAMP and cGMP are tightly controlled by the phosphodiesterase (PDE) family of enzymes. PDE4 catalyzes the hydrolysis of cAMP by its four isotypes PDE4A−D.1 The cAMP pathway is central in several inflammatory cell types and has a key role in pathologies with an inflammatory component such as asthma and chronic obstructive pulmonary disease (COPD), as well as inflammatory bowel disease (IBD), atopic dermatitis (AD), psoriasis, and rheumatoid arthritis (RA).2,3 The presence of PDE4A and PDE4B in eosinophils, neutrophils, and monocytes suggests that they are potential targets for the anti-inflammatory actions of PDE4-selective inhibitors.4,5 PDE4 inhibitors also have a relevant role in the CNS, including antidepressant and memoryenhancing effects.6,7 Despite this potential, the clinical development of PDE4 inhibitors has been greatly hampered by cardiac8 and emetic9 © 2016 American Chemical Society

side effects, both attributed to the PDE4D isoform. Few isoformselective inhibitors with sufficient selectivity to individually determine the roles of PDE4B or PDE4D have been reported.10 Isoform selectivity remains a formidable challenge given the apparent similarities of the active sites of the various isoforms.11 Nevertheless, roflumilast (1) was the first PDE4 inhibitor to be launched for the oral treatment of COPD (Figure 1). Recently, apremilast (2) has been approved in the U.S. and Canada for the oral treatment of plaque psoriasis.12 The clinical development of PDE4 inhibitors has been reviewed by us and by others.13−16 In order to circumvent the potential side effects of PDE4 inhibitors in respiratory disorders, many companies have turned to inhaled delivery to the lung. For inhaled delivery, as oral absorption is neither necessary nor in fact relevant, other kinds of Received: June 14, 2016 Published: November 9, 2016 10479

DOI: 10.1021/acs.jmedchem.6b00829 J. Med. Chem. 2016, 59, 10479−10497

Journal of Medicinal Chemistry

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internal hydrogen bond between the acetyl carbonyl and the 6amino NH group. X-ray analysis of the bound conformation of 6 revealed that this was not the case and that the acetyl group was not involved in binding, neither internally in the structure of 6 nor externally to the PDE4 enzyme. Therefore, we removed this group in all subsequent analogues. Second, we could incorporate the dichloropyridine metal-binding motif of roflumilast with suitable spacing. Simple overlay of the X-ray structures of 6 and 1 suggested that a urea link would be optimum as a spacer between the 3,5-dichloropyridine group and the pyridazinone. Third, the meta position of the pyridazinone 6-phenyl ring was orientated toward the solvent filled pocket. Here we planned to freely attach groups to specifically modify the physicochemical properties of the series, without significantly changing the binding potency (Figure 4).

Figure 1. Structures of approved oral drugs roflumilast 1 and apremilast 2.

“druglike” properties are needed: molecules can be especially large with molecular weights of >500, often accompanied by a low solubility. Molecules can incorporate metabolically labile groups, susceptible to either oxidative phase I or conjugative phase II metabolism to reduce systemic exposure. Molecules can also incorporate long, lipophilic-linked basic groups with the aim of increasing lung tissue retention. However, since PDE4 is an intracellular target, molecular properties should still be controlled, as cellular permeability is necessary for efficacy. Whole-cell assays are routinely employed to detect any such deficiencies in permeation and target access. Compounds representative of these strategies which have reached clinical trials are the quinolone GSK256066 317 from GSK, the dual PDE3/PDE4 inhibitor RPL554 418 from Verona Pharma, and Pfizer’s UK-500,001 519 (Figure 2). The development of compounds 3 and 5 has since been discontinued. Here we describe our efforts to discover novel, highly potent (double-digit picomolar) inhaled inhibitors of PDE4 for the treatment of respiratory diseases. The use of structural X-ray crystallography has contributed greatly to the understanding of the catalytic domains of PDE4A− D and is central to the design of newer PDE4 inhibitors.20−22 Indeed, the binding modes for several classes of PDE4 inhibitors have been described.23−28 Structure-based design (SBD) has allowed the discovery of very potent compounds, often with IC50 values below 0.1 nM.26 From our own extensive oral PDE4 program we had obtained crystal structure data for a series of pyridazinone compounds such as 629 (PDB code 5K1I, Figure 3). From the X-ray structure of 6 (Figure 3) the pyridazinone nucleus was held as expected in the π-clamp region [1] and formed a hydrogen bond to the glutamine switch [2]. On the basis of earlier SAR around this series, we fixed the pyridazinone 2-substituent as ethyl. This was also backed up by the X-ray structure showing a close fit of the ethyl group in the small lipophilic pocket [3]. The phenyl ring in the 6-position of the pyridazinone filled the large lipophilic pocket [4]. Finally, the benzoate was directed toward the metal-binding zone, interacting with the outer solvation sphere. We could see three areas open to optimization of the binding mode: first, we had originally thought that 6 would maintain an

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CHEMISTRY Pyridazinones 7 were synthesized as shown in Scheme 1. The route started with 3-bromoacetophenone 8 to provide a suitable handle for functionalization in the last step. Claisen condensation of 8 with ethyl acetate gave diketone 9. This was condensed with ethyl chlorooximidoacetate under basic conditions.32 This reaction represents a formal [3 + 2] cycloaddition between the nitrile oxide of the chlorooxime and the enolate of 9, followed by elimination of water to give 10. Although diketone 9 is asymmetrical, the selectivity of cyclization was always very good with effectively none of the regioisomer of 10 detected. Ester 10 was condensed with hydrazine hydrate to form the pyridazinone nucleus 11. N-Alkylation with ethyl iodide proceeded smoothly (11 → 12) followed by reductive ringopening of the isoxazolo[3,4-d]pyridazin-7(6H)-one core of 12 to give aminopyridazinone 13. The acetyl group was removed by a retro-Claisen-type reaction to give 14. The amine of 14 was first reacted with triphosgene and then condensed with 4-amino-3,5dichloropyridine. Despite the apparent steric hindrance and low nucleophilicity of this aminopyridine, the addition proceeded in high yield to give key intermediate 15. Suzuki coupling with a wide range of commercial and bespoke boronic acids gave the desired pyridazinones 7 (see Experimental Section).

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RESULTS AND DISCUSSION Our desired compound profile was as follows: a potent PDE4 inhibitor (IC50 in a cellular assay of 1000 33 28 5 22 9 33 105 41 59 86

172 111 222 n.d. 263 >1000 120 77 n.d. 169 n.d. 145 90 206 459 94

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EXPERIMENTAL SECTION

General. All test compounds have purity of >95%. HPLC analysis was conducted according to the described method, with the retention time expressed in minutes. UV chromatograms were processed at 210 nm with blank subtraction. HPLC was performed on a Symmetry C18 10485



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Table 6. SAR of Doubly Substituted Biphenyls

a

Ratio of most potent adenosine IC50/PDE4 IC50 in PBMCs.

column (100 mm × 2.16 mm, 3.5 μm). The mobile phase, at a flow of 0.4 mL/min, was a 20 min binary gradient of water (containing 0.01 M ammonium formate at pH 3.0) and a mixture of acetonitrile−methanol 50:50 (containing 0.01 M ammonium formate) (0−95%). The total run time was 26 min. Reaction products were purified, when necessary, by flash chromatography on silica gel (40−63 μm) with the solvent system

indicated. Purifications in reverse phase were made in a Biotage SP1 automated purification system equipped with a C-18 column and using a standard gradient of water−acetonitrile/MeOH (1:1) (0.1% v/v ammonium formate for both phases) from 0% to 100% acetonitrile/ MeOH (1:1) in 80 column volumes. Other conditions are stated explicitly in the text. Preparative HPLC−MS was performed on a Waters 10486



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instrument equipped with a 2767 injector/collector, a 2525 binary gradient pump, a 2996 PDA detector, a 515 pump as a makeup pump, and a ZQ4000 mass spectrometer detector. 1H nuclear magnetic resonance spectra were recorded on a Gemini 200 MHz spectrometer, Gemini-2000 300 MHz spectrometer, or a Varian Mercury plus operating at a frequency of 400 MHz. Tetramethylsilane was used as reference. Chemical shifts δ are in ppm, and the following abbreviations are used: singlet (s), doublet (d), triplet (t), quartet (q), double doublet (dd), quintet (quin), multiplet (m), broad signal (br s), apparent signal (app). “Partial” generally refers to spectra where compound peaks are hidden below solvent peaks and are therefore unassignable. Mass spectra (m/z) were recorded on Micromass ZMD or Waters ZQ mass spectrometer using ESI ionization. Isotopic distributions are quoted for the mass ion of the lowest molecular weight isotope (35Cl and 79Br) and the number of isotopes detected. PdCl2dppf·CH2Cl2 refers to [1,1bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex. The following general synthetic procedures are exemplified: Suzuki reaction (16), pinacol boronate ester formation (22a), ester hydrolysis (26a), amide coupling (38). 1-(3-Bromophenyl)butane-1,3-dione (9). Sodium hydride (50 g, 60% dispersion in oil, previously washed with pentane, 1.25 mol) was suspended in 920 mL of dry tetrahydrofuran. Ethyl acetate (123 mL, 1.25 mol) was added followed by 15 drops of absolute ethanol. The mixture was cooled in an ice−water bath. A solution of 1-(3bromophenyl)ethanone 8 (125 g, 0.63 mol) dissolved in 330 mL of tetrahydrofuran was added with stirring, maintaining the reaction temperature below 20 °C. Upon completion of addition 18-crown-6 (3.5 g, 0.01 mol) was added. The mixture was stirred at room temperature for 15 min, at 40 °C for 15 min, then at reflux for 5.5 h, and then overnight, cooling to room temperature. The mixture was evaporated and the residue taken up in 1.5 L of water. The aqueous portion was extracted with 5 × 250 mL of ether and was then cooled in an ice-bath and acidified to pH 1−2 with concentrated hydrochloric acid. The aqueous phase was extracted with 3 × 250 mL of ether. The combined organics were washed twice with water and once with brine. The organics were dried over sodium sulfate, filtered, and evaporated to give 9 (124.9 g, 0.52 mol, 82%) as an oil, contaminated with several small impurities. It was used as such without further purification. 1H NMR (300 MHz, CDCl3) enol tautomer: δ ppm 2.22 (s, 3H), 6.15 (s, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 8.01 (s, 1H). Ethyl 4-(3-Bromobenzoyl)-5-methylisoxazole-3-carboxylate (10). Sodium metal (13 g, previously washed with pentane, 0.57 mol) was mechanically stirred in 50 mL of absolute ethanol until completely dissolved. The solution was cooled to between 0 and −5 °C with an ice− salt bath, and a solution of 9 (124 g, 0.51 mol) dissolved in 325 mL of ethanol was added dropwise with stirring, maintaining the reaction temperature below 0 °C. The mixture was stirred for 15 min, and then a solution of ethyl 2-chloro-2-(hydroxyimino)acetate (85 g, 0.56 mol) dissolved in 180 mL of ethanol was added dropwise, maintaining the reaction temperature below 0 °C. The mixture was stirred for 30 min at 0 °C and then overnight, warming to room temperature. The solution (at pH 7) was acidified to pH 5 with 25 mL of acetic acid and was evaporated to dryness. The residue was taken up in 750 mL of ice−water and was extracted with 3 × 300 mL of ether. The combined organics were washed successively with 4% sodium bicarbonate solution, water, and brine. The organics were dried over sodium sulfate, decolorized at reflux with activated carbon, and filtered. Evaporation under high vacuum gave 10 (155.4 g, 0.46 mol, 89%) as an oil which solidified upon standing. It was used as such without further purification. 1H NMR (300 MHz, CDCl3) δ ppm 1.14 (t, J = 7.0 Hz, 3H), 2.57 (s, 3H), 4.18 (q, J = 7.0 Hz, 2H), 7.36 (t, J = 7.8 Hz, 1H), 7.66 (d, J = 7.4 Hz, 1H), 7.74 (d, J = 7.7 Hz, 1H), 7.90 (s, 1H). 4-(3-Bromophenyl)-3-methylisoxazolo[3,4-d]pyridazin7(6H)-one (11). Pyridazinone 10 (155.4 g, 0.46 mol) was suspended in 1.15 L of ethanol. Hydrazine hydrate (34.1 mL, 0.70 mol) was slowly added with stirring, dissolving the remaining solid. The mixture was stirred at room temperature overnight, precipitating a solid. The mixture was cooled in an ice−water bath for 15 min and then filtered. The solid

was washed with ethanol, then with ether and was dried in a stream of air and then at 50 °C under vacuum to give 11 (101.5 g, 0.33 mol, 72%) as a solid. 1H NMR (300 MHz, DMSO-d6) δ ppm 3.35 (s, 3H), 7.53 (t, J = 7.9 Hz, 1H), 7.66 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.83 (s, 1H). 4-(3-Bromophenyl)-6-ethyl-3-methylisoxazolo[3,4-d]pyridazin-7(6H)-one (12). Compound 11 (18.9 g, 61.8 mmol) was dissolved in 200 mL of anhydrous dimethylformamide. Potassium carbonate (25.7 g, 186 mmol) was added and the mixture stirred for 20 min. Bromoethane (13.9 mL, 186 mmol) was added dropwise with stirring, maintaining the reaction temperature below 30 °C, and the mixture was stirred at room temperature overnight. The mixture was evaporated to dryness and the residue taken up in 200 g of ice−water and extracted several times with ethyl acetate. The combined organics were washed successively with water, 4% sodium bicarbonate solution, water, brine−water, and brine. The organics were dried over sodium sulfate, decolorized at reflux with activated carbon, and filtered. Evaporation gave a solid which was broken up in ether and collected by filtration. The solid was washed with ether and was dried in a stream of air and then at 50 °C under vacuum to give 12 (17.6 g, 51 mmol, 85%) as a solid. 1H NMR (300 MHz, CDCl3) δ ppm 1.42 (t, J = 7.1 Hz, 3H), 2.58 (s, 3H), 4.28 (q, J = 7.1 Hz, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.72 (s, 1H). HPLC/MS (15 min) retention time 8.83 min. LRMS: m/z 334 (M + H+, 1 × Br). 5-Acetyl-4-amino-6-(3-bromophenyl)-2-ethylpyridazin3(2H)-one (13). Isoxazole 12 (34.4 g 103 mmol) was dissolved in 500 mL of tetrahydrofuran and 100 mL of ethanol. A heaping spoonful of Raney nickel was added and the mixture agitated under hydrogen atmosphere (14 psi) overnight. The mixture was filtered and the catalyst washed with tetrahydrofuran. The combined filtrate was evaporated, and the solid residue was broken up in 100 mL of hot ethanol. Upon cooling to room temperature, the solid was collected by filtration, was washed with ether, and was dried in a stream of air and then at 50 °C under vacuum to give 13 (31.2 g, 92.8 mmol, 90%) as a solid. 1H NMR (300 MHz, CDCl3) δ ppm 1.42 (t, J = 7.1 Hz, 3H), 1.83 (s, 3H), 4.25 (q, J = 7.2 Hz, 2H), 7.29−7.40 (m, 2H), 7.60 (d, J = 7.4 Hz, 1H), 7.66 (s, 1H). HPLC/MS (15 min) retention time 7.77 min. LRMS: m/z 336 (M + H+, 1 × Br). 4-Amino-6-(3-bromophenyl)-2-ethylpyridazin-3(2H)-one (14). Acetylpyridazinone 13 (29.4 g, 87.5 mmol) was suspended in 215 mL of 48% hydrobromic acid and was heated at 130 °C for 1.25 h. The mixture was allowed to cool and was carefully diluted with ice−water and basified with solid sodium carbonate. Once basic, the aqueous portion was extracted with 2 × 300 mL of ethyl acetate. The combined organics were washed successively with water and brine. The organics were dried over sodium sulfate, decolorized at reflux with activated carbon, and filtered. Evaporation gave a residue which was broken up in 100 mL of hot diisopropyl ether. Upon cooling, the solid was collected by filtration, washed with diisopropyl ether, and dried in a stream of air and then at 50 °C under vacuum to give 14 (21.1 g, 71.7 mmol, 82%) as a solid. 1H NMR (300 MHz, CDCl3) δ ppm 1.45 (t, J = 7.3 Hz, 3H), 4.30 (q, J = 7.1 Hz, 2H), 5.02 (br s, 2H), 6.67 (s, 1H), 7.30 (t, J = 7.7 Hz, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.67 (d, J = 8.0 Hz, 1H), 7.93 (s, 1H). HPLC/ MS (15 min) retention time 8.58 min. LRMS: m/z 294 (M + H+, 1 × Br). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(3-bromophenyl)3-oxo-2,3-dihydropyridazin-4-yl]urea (15). Solution 1. Triphosgene (6.0 g, 20.2 mmol) was dissolved in 94 mL of dichloromethane in a 500 mL flask. A filtered solution of 14 (16.9 g, 57.5 mmol) and triethylamine (8.5 mL, 60.9 mmol) in 165 mL of dichloromethane was added and the mixture placed under argon atmosphere and stirred for 3 h. The mixture was evaporated to dryness and was re-evaporated twice more from tetrahydrofuran. The residue was taken up in 420 mL of tetrahydrofuran and was filtered to remove triethylamine hydrochloride. Solution 2. Sodium hydride (5.7 g, 60% dispersion in oil, previously washed with pentane, 142 mmol) was suspended in 120 mL of dry tetrahydrofuran in a 1 L flask. A solution of 4-amino-3,5-dichloropyridine (18.8 g, 115 mmol) dissolved in 140 mL of tetrahydrofuran was added with stirring. The mixture was stirred for 2.5 h, forming a white suspension. The filtrate from solution 1 was added and the mixture 10487



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d6) δ ppm 1.37 (t, J = 7.1 Hz, 3H), 4.26 (d, J = 7.0 Hz, 2H), 6.67−6.83 (m, 2H), 7.33 (dd, J = 8.8, 7.2 Hz, 1H), 7.45−7.63 (m, 2H), 7.73 (d, J = 6.7 Hz, 1H), 7.90 (s, 1H), 8.38 (s, 1H), 8.69 (s, 2H), 9.68 (s, 1H), 9.93 (br s, 1H), 10.19 (br s, 1H). HPLC/MS (30 min) retention time 18.45 min. LRMS: m/z 514 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(5′-fluoro-2′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (20). Suzuki reaction of 15 (2.69 g, 5.40 mmol) with 2-(5,5-dimethyl1,3,2-dioxaborinan-2-yl)-4-fluorophenol (3.3 g, 8.10 mmol) according to the general method gave 20 (1.65 g, 3.21 mmol, 59%) after crystallization from dichloromethane−methanol (6:4). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.1 Hz, 3H), 4.26 (q, J = 7.2 Hz, 2H), 6.94 (dd, J = 8.8, 5.0 Hz, 1H), 7.03 (td, J = 8.5, 3.1 Hz, 1H), 7.15 (dd, J = 9.5, 3.0 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.61 (d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.7 Hz, 1H), 7.97 (s, 1H), 8.38 (s, 1H), 8.68 (s, 2H), 9.61−9.73 (m, 3H). HPLC/MS (30 min) retention time 18.31 min. LRMS: m/z 514 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(2′-fluoro-3′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (21). Step 1. 2-Fluoro-3-methoxyphenylboronic acid (1.1 g, 6.5 mmol) was dissolved in 30 mL of dichloromethane and cooled to 5 °C under nitrogen. Boron tribromide solution (32 mL, 1 M in dichloromethane, 32 mmol) was added dropwise with stirring. Upon addition, the mixture was stirred for 1 h at room temperature. The mixture was added dropwise to 50 mL of cold ethanol. The mixture was then neutralized with portionwise addition of excess solid sodium bicarbonate with cooling. The mixture was stirred for 15 min, filtered and the filtrate evaporated. The residue was taken up in tetrahydrofuran, refiltered, and re-evaporated to give 2-(fluoro-3-hydroxyphenyl)boronic acid 21a (0.96 g, 6.2 mmol, 96%). 1H NMR (200 MHz, DMSO-d6) δ ppm 6.91 (m, 3H), 8.13 (br s, 2H), 9.53 (s, 1H). Step 2. Suzuki reaction of 15 (250 mg, 0.52 mmol) with 21a (125 mg, 0.80 mmol) according to the general method and purification by the SP1 automated purification system gave 21 (25 mg, 0.049 mmol, 9%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.2 Hz, 3H), 4.26 (q, J = 7.1 Hz, 2H), 6.87−7.04 (m, 2H), 7.09 (t, J = 8.0, 7.4 Hz, 1H), 7.59 (app d, J = 4.5 Hz, 2H), 7.81 (td, J = 4.6, 1.8 Hz, 1H), 7.87 (s, 1H), 8.39 (s, 1H), 8.69 (s, 2H), 9.67 (br s, 1H), 9.90 (br s, 1H), 9.99 (s, 1H). HPLC/ MS (30 min) retention time 17.77 min. LRMS: m/z 514 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(3′-fluoro-4′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (22). Step 1. General Procedure for Synthesis of Pinacol Boronates. In a Schlenk tube, a mixture of 4-bromo-2-fluorophenol (0.50 g, 4.57 mmol), bis(pinacolato)diboron (1.33 g, 5.23 mmol), and potassium acetate (1.37 g, 14 mmol) was dissolved in dimethylsufoxide (25 mL). The mixture was purged (vacuum−argon three times), and PdCl2dppf· CH2Cl2 (0.10 g, 0.13 mmol) was added. The mixture was purged again (vacuum−argon three times) and stirred at 80 °C for 4 h. The suspension was filtered and the filtrate diluted with water and extracted three times with ethyl acetate. The combined organic layers were washed with water and brine and dried over anhydrous sodium sulfate. Solvent was removed in vacuo and the residue was partially purified by flash chromatography (ethyl acetate−hexane, 20:80) and then reversephase chromatography using the SP1 automated purification system to give 2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol 22a (0.25 g, 85% pure, 0.89 mmol, 19% yield). 1H NMR (200 MHz, CD3OD) δ ppm 1.31 (s, 12H), 6.87 (t, J = 8.3 Hz, 1H), 7.26−7.41 (m, 2H). Step 2. Suzuki reaction of 15 (250 mg, 0.52 mmol) with 22a (220 mg, 0.78 mmol) according to the general method and purification by chromatography (methanol−dichloromethane, 5:95) gave 22 (40 mg, 0.078 mmol, 16%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.1 Hz, 3H), 4.28 (d, J = 7.3 Hz, 2H), 7.06 (t, J = 8.7 Hz, 1H), 7.36 (ddd, J = 8.2, 2.1, 0.9, 1H), 7.51 (dd, J = 12.6, 2.3 Hz, 1H), 7.56 (d, J = 7.3 Hz, 1H), 7.63−7.74 (m, 2H), 7.93 (t, J = 1.3 Hz, 1H), 8.38 (s, 1H), 8.70 (s, 2H), 9.69 (br s, 1H), 9.91 (br s, 1H), 10.06 (br s, 1H). HPLC/MS (30 min) retention time 17.73 min. LRMS: m/z 514 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(2′-fluoro-4′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (23). Step 1. Pinacol boronate synthesis with 4-bromo-3-fluorophenol

stirred overnight at room temperature, precipitating a solid. The mixture was filtered, keeping both the solid and the filtrate. The solid was dissolved in water and the solution acidified to pH 6−7 with 2 M hydrochloric acid, precipitating a solid. The solid was collected by filtration, washed with water, then with ether and was dried in a stream of air and then at 50 °C under vacuum to give 15 (12.2 g). The filtrate was evaporated to dryness. The residue was taken up in 700 mL of water and acidified to pH 6−7 with 2 M hydrochloric acid, precipitating a solid. The solid was collected by filtration, washed with water, then with ether and was dried in a stream of air and then at 50 °C under vacuum. The solid was dissolved in 500 mL of hot methanol−dichloromethane (30:70) and was decolorized with activated carbon. Filtration and concentration to approximately 120 mL precipitated a solid. The solid was collected by filtration, washed with methanol and was dried in a stream of air and then at 50 °C under vacuum to give a further 7.2 g of 15. Total yield, 19.4 g (40.2 mmol, 70%). Mp 176−178 °C. 1H NMR (300 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.2 Hz, 1H), 4.26 (q, J = 7.2 Hz, 1H), 7.47 (t, J = 7.9 Hz, 1H), 7.67 (ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.77 (dd, J = 6.6, 1.3 Hz, 1H), 7.95 (t, J = 1.8 Hz, 1H), 8.32 (s, 1H), 8.70 (s, 1H), 9.71 (s, 1H), 9.90 (s, 1H). HPLC/MS (15 min) retention time 10.30 min. LRMS: m/z 480 (M − H+, 2 × Cl, 1 × Br). General Procedure for Suzuki Coupling. Exemplified for N(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(3′-hydroxybiphenyl3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (16). In a Schlenk tube, aryl bromide 15 (3.00 g, 6.21 mmol) and 3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenol (2.05 g, 6.21 mmol) were dissolved in dioxane (100 mL). Cesium carbonate solution (2 M, 9.31 mL, 18.6 mmol) was added, and the mixture was purged (vacuum−argon three times). PdCl2dppf·CH2Cl2 (0.30 g, 0.37 mmol) was added and the mixture purged again (vacuum−argon three times). The mixture was stirred at 90 °C for 3 h. The reaction mixture was cooled to room temperature, diluted with water, and extracted three times with ethyl acetate. The combined organics were washed with water and brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuum and the residue was purified by the SP1 automated purification system to give 16 (1.26 g, 2.54 mmol, 41%) as a solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 4.28 (q, J = 7.3 Hz, 2H), 6.80 (ddd, J = 8.1, 2.4, 0.9 Hz, 1H), 7.05 (t, J = 2.1 Hz, 1H), 7.09 (ddd, J = 7.7, 1.6, 0.9 Hz, 1H), 7.29 (t, J = 7.9 Hz, 1H), 7.57 (t, J = 7.8 Hz, 1H), 7.68 (dt, J = 7.7, 1.1 Hz, 1H), 7.76 (dt, J = 7.7, 1.4 Hz, 1H), 7.93 (t, J = 1.7 Hz, 1H), 8.40 (s, 1H), 8.69 (s, 2H), 9.61 (s, 1H), 9.64 (s, 1H). HPLC/MS (30 min) retention time 15.94 min. LRMS: m/z 496 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(4′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (17). Suzuki reaction 15 (200 mg, 0.41 mmol) with 4-hydroxyphenylboronic acid (85 mg, 0.62 mmol) according to the general method gave 17 (30 mg, 0.060 mmol, 15%) after crystallization from ethanol. 1H NMR (200 MHz, DMSO-d6) δ ppm 1.47 (t, J = 7.1 Hz, 3H), 4.35 (d, J = 6.7 Hz, 2H), 6.93 (d, J = 8.4 Hz, 2H), 7.34 (s, 1H), 7.38−7.61 (m, 4H), 7.71 (d, J = 7.3 Hz, 1H), 7.97 (s, 1H), 8.52 (s, 2H), 8.74 (s, 1H), 9.53 (s, 1H), 9.63 (s, 1H). HPLC/MS (30 min) retention time 17.33 min. LRMS: m/z 496 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(2′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (18). Suzuki reaction of 15 (200 mg, 0.41 mmol) with 2-(4,4,5,5-tetramethyl-1,3,2dioxaborolan-2-yl)phenol (61 mg, 0.28 mmol) according to the general method gave 18 (50 mg, 0.10 mmol, 35%) after crystallization from ethanol. 1H NMR (200 MHz, DMSO-d6) δ ppm 1.38 (t, J = 7.1 Hz, 3H), 4.26 (q, J = 7.2 Hz, 2H), 6.90 (t, J = 7.3 Hz, 1H), 6.95 (d, J = 7.7 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 6.9 Hz, 1H), 7.51 (t, J = 7.2 Hz, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.94 (s, 1H), 8.39 (s, 1H), 8.69 (s, 2H), 9.61 (s, 1H), 9.69 (s, 1H), 9.90 (s, 1H). HPLC/MS (30 min) retention time 18.12 min. LRMS: m/z 496 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-[2-ethyl-6-(4′-fluoro-2′-hydroxybiphenyl-3-yl)-3-oxo-2,3-dihydropyridazin-4-yl]urea (19). Suzuki reaction of 15 (0.20 g, 0.42 mmol) with 4-fluoro-2hydroxyphenylboronic acid (0.10 g, 0.64 mmol) according to the general method and purification by the SP1 automated purification system gave 19 (0.06 g, 0.12 mmol, 28%). 1H NMR (200 MHz, DMSO10488



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J = 7.7, 1H), 7.77 (d, J = 2.0 Hz, 1H), 7.83−7.90 (m, 2H), 8.37 (s, 1H), 8.70 (s, 2H), 9.69 (s, 1H), 9.90 (s, 1H), 11.86 (s, 1H). HPLC/MS (30 min) retention time 14.66 min. LRMS: m/z 497 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-2-carboxylic Acid (26). Step 1. Suzuki reaction of 15 (700 mg, 1.41 mmol) with ethyl 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (600 mg, 2.11 mmol) according to the general scheme gave ethyl 3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-2-carboxylate 26a (440 mg, 0.80 mmol, 57%) after triturating with diethyl ether. HPLC/MS (9 min) retention time 7.57 min. LRMS: m/z 552 (M + H+, 2 × Cl). Step 2. General Method for Ester Hydrolysis. A solution of lithium hydroxide monohydrate (150 mg, 3.57 mmol) in 5 mL of water was added dropwise to a stirred solution of 26a (440 mg, 0.71 mmol) in 12 mL of methanol−tetrahydrofuran (2:1). The mixture was stirred at room temperature for 24 h, then diluted with water and acidified with 2 N hydrochloric acid precipitating a solid, which was collected by filtration and dried in a stream of air to give 26 (130 mg, 0.25 mmol, 34%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.2 Hz, 3H), 4.25 (q, J = 7.2 Hz, 2H), 7.25−7.99 (m, 8H), 8.38 (br s, 1H), 8.69 (br s, 2H), 9.71 (br s, 1H), 9.90 (br s, 1H), 12.81 (br s, 1H). HPLC/MS (30 min) retention time 17.28 min. LRMS: m/z 524 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylic Acid (27). Step 1. Suzuki reaction of 15 (250 mg, 0.50 mmol) with [3(ethoxycarbonyl)phenyl]boronic acid (0.150 g, 0.77 mmol) according to the general method ethyl 3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3carboxylate 27a (0.159 g, 0.29 mmol, 58%) after trituration with methanol. HPLC/MS (9 min) retention time 7.69 min. LRMS: m/z 552 (M + H+, 2 × Cl). Step 2. Ester hydrolysis of 27a (0.159 g, 0.29 mmol) according to the general method gave 27 (0.043 g, 0.082 mmol, 28%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (t, J = 7.20 Hz, 3H), 4.28 (q, J = 7.20 Hz, 2H), 7.56−7.65 (m, 2H), 7.79 (t, J = 8.41 Hz, 2H), 7.90 (d, J = 7.63 Hz, 1H), 7.96 (d, J = 7.63 Hz, 1H), 8.02 (s, 1H), 8.20 (s, 1H), 8.41 (s, 1H), 8.69 (s, 2H), 9.70 (s, 1H). HPLC/MS (30 min) retention time 18.01 min. LRMS: m/z 524 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-4-carboxylic Acid (28). Step 1. Suzuki reaction of 15 (350 mg, 0.72 mmol) with 4(ethoxycarbonyl)phenylboronic acid (211 mg, 1.09 mmol) according to the general method gave ethyl 3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-4-carboxylate 28a (190 mg, 0.34 mmol, 46%) after triturating with diethyl ether. HPLC/MS (9 min) retention time 7.71 min. LRMS: m/z 552 (M + H+, 2 × Cl). Step 2. Ester hydrolysis of 28a (880 mg, 1.12 mmol) according to the general method gave 28 (440 mg, 0.84 mmol, 75%) after crystallization with dichloromethane−methanol (7:3). 1H NMR (200 MHz, DMSOd6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 4.28 (q, J = 7.2 Hz, 2H), 7.54−8.27 (m, 8H), 8.41 (br s, 1H), 8.70 (br s, 2H), 9.71 (br s, 1H), 9.91 (br s, 1H), 12.99 (br s, 1H). HPLC/MS (30 min) retention time 17.61 min. LRMS: m/z 524 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-6-fluorobiphenyl-3carboxylic Acid (29). Step 1. Pinacol boronate synthesis from methyl 3-bromo-4-fluorobenzoate (1.05 g, 4.51 mmol) and bis(pinacolato)diboron (1.14 g, 4.49 mmol) according to the general method gave crude methyl 4-fluoro-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 29a (1.52 g). It was used as such without further purification. HPLC/MS (9 min) retention time 4.47 min. LRMS: 281 (M + H+). Step 2. Suzuki reaction of compound 15 (1.00 g, 2.07 mmol) with crude 29a (1.40 g) according to the general method gave methyl 3′-(5(3-(3,5-dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl)-6-fluorobiphenyl-3-carboxylate 29b (0.55 g, 0.99 mmol, 48%). HPLC/MS (9 min) retention time 7.53 min. LRMS: m/z 556 (M + H+, 2 × Cl). Step 3. Ester hydrolysis of 29b (550 mg, 0.99 mmol) according to the general method (1:1) gave 29 (274 mg, 0.51 mmol, 51%) after

(1.00 g, 5.24 mmol) and bis(pinacolato)diboron (2.66 g, 10.47 mmol) according to the general method gave 3-fluoro-4-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)phenol 23a (0.37 g, 1.55 mmol, 30%) after purification by reverse-phase chromatography. 1H NMR (200 MHz, CDCl3) δ ppm 1.26 (s, 12H), 6.55 (m, 1H), 6.62 (m, 1H), 7.61 (m, 1H). HPLC/MS (9 min) retention time 8.85 min. LRMS: m/z 239 (M + H+). Step 2. Suzuki reaction of 15 (0.30 g, 0.62 mmol) with 23a (0.37 g, 1.55 mmol) according to the method gave 23 (0.075 g, 0.15 mmol, 37%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.37 (t, J = 7.2 Hz, 3H), 4.26 (q, J = 7.2 Hz, 2H), 6.65−6.78 (m, 2H), 7.38 (t, J = 9.1 Hz, 1H), 7.55 (app d, J = 5.1 Hz, 2H), 7.71−7.78 (m, 1H), 7.84 (t, J = 1.3 Hz, 1H), 8.37 (s, 1H), 8.70 (s, 2H), 9.70 (br s, 1H), 9.88 (br s, 1H), 10.10 (br s, 1H). HPLC/MS (30 min) retention time 17.81 min. LRMS: m/z 514 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-{2-ethyl-6-[3-(5-hydroxypyridin-3-yl)phenyl]-3-oxo-2,3-dihydropyridazin-4-yl}urea (24). Step 1. Pinacol boronate synthesis with 3-bromo-5-methoxypyridine (2.0 g, 10.3 mmol) and bis(pinacolato)diboron (2.88 g, 11.4 mmol) according to the general method gave 3-methoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine 24a (1.38 g) as an oil. It was used as such without further purification. 1H NMR (200 MHz, CDCl3) δ ppm 1.36 (br s, 12H), 3.87 (s, 3H), 7.27 (s, 1H), 8.37 (d, J = 2.34 Hz, 1H), 8.55 (s, 1H). Step 2. Suzuki reaction of 15 (250 mg, 0.50 mmol) with 24a (177 mg, 0.75 mmol) according to the general method gave N-(3,5-dichloropyridin-4-yl)-N′-{2-ethyl-6-[3-(5-methoxypyridin-3-yl)phenyl]-3-oxo-2,3dihydropyridazin-4-yl}urea 24b (50 mg, 0.098 mmol, 19%) after purification by flash chromatography (hexane−ethyl acetate, 2:1). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 3.91 (s, 3H), 4.28 (q, J = 7.0 Hz, 2H), 7.59−7.71 (m, 2H), 7.82 (d, J = 7.8 Hz, 2H), 8.06 (s, 1H), 8.33 (d, J = 2.7 Hz, 1H), 8.40 (s, 1H), 8.50 (s, 1H), 8.69 (s, 2H), 9.69 (s, 1H), 9.90 (s, 1H). HPLC/MS (30 min) retention time 16.92 min. LRMS: m/z 511 (M + H+, 2 × Cl). Step 3. 24b (150 mg, 0.28 mmol) was added to a solution of boron tribromide−methyl sulfide complex (0.71 g, 2.27 mmol) in 11 mL of 1,2-dichloroethane. The mixture was stirred at reflux for 10 h. Additional boron tribromide−methyl sulfide complex (2.49 g, 7.97 mmol) was added and the reaction followed by HPLC until the starting material had disappeared. The reaction mixture was diluted with water and neutralized with ammonium hydroxide solution to pH 7−8. Dichloromethane was added precipitating a solid, which was collected by filtration and dried in a stream of air. Purification by preparative HPLC gave 24 (12 mg, 0.024 mmol, 9%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.1 Hz, 3H), 4.28 (q, J = 7.0 Hz, 2H), 7.42 (t, J = 2.0 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.73 (d, J = 8.2 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.98 (t, J = 1.4 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.37 (d, J = 1.6 Hz, 1H), 8.40 (s, 1H), 8.69 (s, 2H), 9.70 (s, 1H), 10.10 (br s, 2H). HPLC/ MS (30 min) retention time 14.23 min. LRMS: m/z 497 (M + H+, 2 × Cl). N-(3,5-Dichloropyridin-4-yl)-N′-{2-ethyl-6-[3-(6-hydroxypyridin-3-yl)phenyl]-3-oxo-2,3-dihydropyridazin-4-yl}urea (25). Step 1. Suzuki reaction of 15 (0.30 g, 0.62 mmol) with 6methoxypyridin-3-ylboronic acid (0.19 g, 1.24 mmol) according to the general scheme gave N-(3,5-dichloropyridin-4-yl)-N′-{2-ethyl-6-[3(6-methoxypyridin-3-yl)phenyl]-3-oxo-2,3-dihydropyridazin-4-yl}urea 25a (0.23 g, 0.45 mmol, 74%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 3.90 (s, 3H), 4.30 (q, J = 7.3 Hz, 2H), 6.94 (d, J = 8.6 Hz, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.72−7.82 (m, 2H), 7.98 (t, J = 1.6 Hz, 1H), 8.06 (dd, J = 8.7, 2.5 Hz, 1H), 8.40 (s, 1H), 8.53 (d, J = 2.3 Hz, 1H), 8.70 (s, 2H), 9.70 (br s, 1H), 9.90 (s, 1H). HPLC/MS (30 min) retention time 18.98 min. LRMS: m/z 511 (M + H+, 2 × Cl). Step 2. 25a (153 mg, 0.30 mmol) was suspended in acetonitrile (10 mL) in a microwave vial. Sodium iodide (225 mg, 1.50 mmol) was added. The vial was sealed, purged with argon, and then trimethylsilyl chloride (190 μL, 1.50 mmol) was added. The mixture was microwaved at 100 °C for 5 h. The solvent was evaporated under reduced pressure and the residue purified directly by the SP1 automated purification system to give 25 (60 mg, 0.121 mmol, 40%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.38 (t, J = 7.1 Hz, 3H), 4.27 (q, J = 7.1 Hz, 2H), 6.45 (d, J = 9.5 Hz, 1H), 7.52 (t, J = 7.8 Hz, 1H), 7.64 (d, J = 7.7, 1H), 7.69 (d, 10489



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crystallization from ethanol−chloroform. 1H NMR (200 MHz, DMSOd6) δ ppm 1.38 (t, J = 7.0 Hz, 3H), 4.27 (q, J = 6.9 Hz, 2H), 7.38−7.58 (m, 1H), 7.66 (s, 2H), 7.75−8.22 (m, 4H), 8.39 (s, 1H), 8.69 (s, 2H), 9.70 (s, 1H), 9.90 (s, 1H), 13.20 (s, 1H). HPLC/MS (30 min) retention time 17.87 min. LRMS: m/z 542 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-4-fluorobiphenyl-3carboxylic Acid (30). Step 1. Pinacol boronate synthesis from methyl 5-bromo-2-fluorobenzoate (0.50 g, 2.15 mmol) and bis(pinacolato)diboron (1.09 g, 4.29 mmol) according to the general gave methyl 2fluoro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 30a (1.04 g) as an oil which was used as such without further purification. 1 H NMR (200 MHz, CDCl3) δ ppm 1.26 (s, 12H), 3.92 (s, 3H), 7.04− 7.18 (m, 1H), 7.85−8.01 (m, 1H), 8.36 (d, J = 7.8 Hz, 1H). Step 2. Suzuki reaction of 15 (535 mg, 1.07 mmol) with crude 30a (1.0 g) according to the general method gave methyl 3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-4-fluorobiphenyl-3-carboxylate 30b (420 mg, 0.75 mmol, 47%) after crystallization from dichloromethane−methanol (8:2). HPLC/MS (9 min) retention time 7.54 min. LRMS: m/z 556 (M + H+, 2 × Cl). Step 3. Ester hydrolysis of 30b (420 mg, 0.51 mmol) according to the general method gave 30 (38 mg, 0.072 mmol, 14%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.38 (t, J = 7.0 Hz, 3H), 4.28 (q, J = 6.9 Hz, 2H), 7.19−7.47 (m, 1H), 7.51−7.68 (m, 1H), 7.67−7.90 (m, 3H), 8.00 (d, J = 8.1 Hz, 2H), 8.39 (s, 1H) 8.69 (s, 2H), 9.71 (br s, 1H). HPLC/ MS (30 min) retention time 15.39 min. LRMS: m/z 525 (M + H+, 2 × Cl). 5-{3-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]phenyl}nicotinic Acid (31). Step 1. Pinacol boronate synthesis from methyl 5-bromonicotinate (1.0 g, 4.63 mmol) and bis(pinacolato)diboron (1.29 g, 5.08 mmol) according to the general method gave methyl 5-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)nicotinate 31a (1.12 g) as an oil which was used as such without further purification. 1H NMR (200 MHz, CDCl3) δ ppm 1.21−1.31 (m, 12H), 3.95 (s, 3H), 8.67 (br s, 1H), 9.09 (br s, 1H), 9.27 (br s, 1H). Step 2. Suzuki reaction of 15 (500 mg, 1 mmol) with crude 31a (396 mg, 1.51 mmol) according to the general method gave methyl 5-{3-[5({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo1,6-dihydropyridazin-3-yl]phenyl}nicotinate 31b (160 mg, 0.30 mmol, 27%) after crystallization from dichloromethane−methanol (8:2). HPLC/MS (9 min) retention time 6.83 min. LRMS: m/z 537 (M − H+, 2 × Cl). Step 3. Ester hydrolysis of 31b (160 mg, 0.27 mmol) according to the general method gave 31 (52 mg, 0.10 mmol, 36%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 6.6 Hz, 3H), 4.29 (q, J = 6.6, 2H), 7.56− 7.78 (m, 1H), 7.87 (m, 2H), 8.10 (br s, 1H), 8.41 (br s, 1H), 8.49 (br s, 1H), 8.69 (s, 2H), 9.09 (br s, 1H), 9.14 (br s, 1H), 9.71 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 15.39 min. LRMS: m/z 525 (M + H+, 2 × Cl). {3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-yl}acetic Acid (32). Step 1. Pinacol boronate synthesis from methyl (3bromophenyl)acetate (2.05 g, 7.43 mmol) and bis(pinacolato)diboron (4.55 g, 18.0 mmol) according to the general method gave methyl [3(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]acetate 32a (2.0 g). It was used as such without further purification. HPLC/MS (9 min) retention time 6.59 min. LRMS: m/z 277 (M + H+). Step 2. Suzuki reaction of 15 (1.00 g, 2.07 mmol) with crude 32a (0.85 g) according to the general method gave methyl {3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-yl}acetate 32b (0.76 g, 1.38 mmol, 67%). 1 H NMR (400 MHz, DMSO-d6) δ ppm 1.36 (t, J = 7.2 Hz, 3H), 3.59 (s, 3H), 3.75 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 7.27 (d, J = 7.8 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.51−7.61 (m, 3H), 7.69 (d, J = 8.2 Hz, 1H), 7.74 (d, J = 7.8 Hz, 1H), 7.95 (s, 1H), 8.37 (s, 1H), 8.67 (s, 2H), 9.67 (s, 1H), 9.87 (s, 1H). HPLC/MS (9 min) retention time 7.33 min. LRMS: m/z 550 (M − H+, 2 × Cl).

Step 3. Ester hydrolysis of 32b (0.76 g, 1.38 mmol) according to the general method gave 32 (0.33 g, 0.61 mmol, 44%), after crystallization with dichloromethane−methanol (7:3). 1H NMR (400 MHz, DMSOd6) δ ppm 1.36 (t, J = 7.1 Hz, 3H), 3.64 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 7.26 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.50−7.60 (m, 3H), 7.69 (d, J = 8.2 Hz, 1H), 7.73 (d, J = 7.8.2 Hz, 1H), 7.95 (s, 1H), 8.36 (s, 1H), 8.66 (s, 2H), 9.67 (s, 1H), 9.88 (s, 1H), 12.33 (s, 1H). HPLC/MS (30 min) retention time 17.47 min. LRMS: m/z 538 (M + H+, 2 × Cl). {3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-4-yl}acetic Acid (33). Step 1. Pinacol boronate synthesis from ethyl (4bromophenyl)acetate (1.0 g, 4.1 mmol) with bis(pinacolato)diboron (2.1 g, 8.3 mmol) according to the general method gave ethyl [4(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]acetate 33a (0.86 g, 2.97 mmol, 72%). 1H NMR (200 MHz, CDCl3) δ ppm 1.23 (t, J = 6.8 Hz, 3H), 1.34 (s, 12H), 3.62 (s, 2H), 4.13 (q, J = 7.0 Hz, 2H), 7.22−7.34 (m, 2H), 7.77 (d, J = 7.4 Hz, 2H). HPLC/MS (9 min) retention time 6.86 min. LRMS: m/z 308 (M + NH4+). Step 2. Suzuki reaction of 15 (1.0 g, 2.07 mmol) with 33a (0.86 g, 2.97 mmol) according to the general method ethyl {3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-4-yl}acetate gave 33b (0.65 g, 1.15 mmol, 55%). HPLC/MS (9 min) retention time 7.56 min. LRMS: m/z 566 (M + H+, 2 × Cl). Step 3. Ester hydrolysis of 33b (0.65 g, 1.15 mmol) according to the general method gave 33 (0.303 g, 0.56 mmol, 49%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.3 Hz, 3H), 3.62 (s, 2H), 4.28 (q, J = 7.3 Hz, 2H), 7.38 (d, J = 8.3 Hz, 2H), 7.59 (t, J = 7.7 Hz, 2H), 7.64 (d, J = 8.3 Hz, 2H), 7.70−7.79 (m, 2H), 7.99 (s, 1H), 8.40 (s, 1H), 8.70 (s, 2H), 9.70 (s, 1H), 9.90 (s, 1H), 12.37 (s, 1H). HPLC/MS (15 min) retention time 9.10 min. LRMS: m/z 538 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxamide (34). Suzuki reaction of 15 (0.40 g, 0.83 mmol) with [3(aminocarbonyl)phenyl]boronic acid (0.204 g, 1.05 mmol) according to the general method gave 34 (0.055 g, 13%) after crystallization with dichloromethane−methanol. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 4.29 (q, J = 7.2 Hz, 2H), 7.45 (s, 1H), 7.60 (dt, J = 17.8, 7.7 Hz, 2H), 7.75−7.93 (m, 4H), 8.05 (s, 1H), 8.13 (s, 1H), 8.19 (s, 1H), 8.40 (s, 1H), 8.70 (s, 2H), 9.71 (s, 1H), 9.90 (s, 1H). HPLC/ MS (30 min) retention time 15.98 min. LRMS: m/z 523 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-4-carboxamide (35). Suzuki reaction of 15 (300 mg, 0.60 mmol) with 4carbamoylphenylboronic acid (149 mg, 0.90 mmol) according to the general method gave 35 (130 mg, 0.25 mmol, 14%) after purification by chromatography (neutral alumina, methanol−ethyl acetate gradient, 1:99 rising to 4:96). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 6.8 Hz, 3H) 4.28 (q, J = 7.0, 2H), 7.42 (bs, 1H), 7.54−7.70 (m, 1H), 7.72−7.89 (m, 3H), 7.72−7.89 (m, 3H), 7.97−8.04 (m, 5H), 8.41 (s, 1H), 8.69 (s, 2H), 9.69 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 16.11 min. LRMS: m/z 523 (M + H+, 2 × Cl). N-Cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxamide (36). Suzuki reaction of 15 (0.20 g, 0.41 mmol) with N-cyclopropyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)benzamide (0.142 g, 0.50 mmol) according to the general method gave 36 (0.072 g, 0.13 mmol, 31%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.55−0.62 (m, 2H), 0.66−0.75 (m, 2H), 1.39 (t, J = 7.0 Hz, 3H), 2.79−2.99 (m, 1H), 4.28 (q, J = 7.0 Hz, 2H), 7.53−7.66 (m, 2H), 7.74− 7.88 (m, 4H), 8.03 (s, 1H), 8.10 (s, 1H), 8.40 (s, 1H), 8.57 (d, J = 4.3 Hz, 1H), 8.70 (s, 2H), 9.71 (s, 1H), 9.90 (s, 1H). HPLC/MS (30 min) retention time 17.51 min. LRMS: m/z 563 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N-[2-(dimethylamino)ethyl]biphenyl-3-carboxamide (37). Step 1. N,N-Dimethylethane1,2-diamine (0.71 g, 8.06 mmol) was added to a solution of 3-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic acid (1.00 g, 4.03 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 0.82 g, 6.07 mmol), and Nethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 10490



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(s, 1H), 8.67 (t, J = 5.5 Hz, 1H), 8.68 (s, 1H). HPLC/MS (30 min) retention time 12.39 min. LRMS: m/z 634 (M + H+, 2 × Cl). 2-(3′-(5-(3-(3,5-Dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo1,6-dihydropyridazin-3-yl)biphenyl-4-yl)-N-(2-(piperidin-1-yl)ethyl)acetamide (42). Amide coupling of 33 (300 mg, 0.56 mmol) with 2-(piperidin-1-yl)ethanamine (160 μL, 1.12 mmol) according to the general method gave 42 (185 mg, 0.29 mmol, 51%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.27 (t, J = 7.3 Hz, 3H), 1.48−2.06 (m, 8H), 3.09−3.35 (m, 4H), 3.66 (s, 2H), 3.73 (bs, 2H), 4.05 (q, J = 7.3 Hz, 2H), 7.03 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 7.6 Hz, 3H), 7.42−7.53 (m, 1H), 7.53−7.67 (m, 3H), 7.67−7.82 (m, 4H), 7.96 (s, 1H), 8.20 (d, J = 8.2 Hz, 1H), 8.33 (s, 1H), 8.40 (s, 1H), 8.70 (s, 1H), 9.72 (s, 1H), 9.93 (s, 1H). HPLC/MS (30 min) retention time 12.64 min. LRMS: m/z 648 (M + H+, 2 × Cl). 3′-(5-(3-(3,5-Dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo-1,6dihydropyridazin-3-yl)-N-(7-((2-hydroxyethyl)(methyl)amino)heptyl)biphenyl-3-carboxamide (43). Step 1. 2-(Methylamino)ethanol (1.90 mL, 23.7 mmol) was dissolved in 90 mL of methyl isobutyl ketone, and potassium carbonate (3.27 g, 23.7 mmol) was added. To this mixture was added a solution of 7-bromoheptanenitrile (3.00 g, 15.8 mmol) in 10 mL of methyl isobutyl ketone. The reaction was stirred overnight at 70 °C. The solvent was evaporated under vacuum and the residue taken up in ethyl acetate. The organics were washed with water and brine, dried over magnesium sulfate, filtered, and evaporated to give 7-[(2-hydroxyethyl)(methyl)amino]heptanenitrile 43a (2.81 g, 15.3 mmol, 97%) as a yellow oil. HPLC/MS (9 min) retention time 0.67 min. LRMS: m/z 185 (M + H+). Step 2. Lithium aluminum hydride (1.74 g, 45.7 mmol) was suspended in 90 mL of dry tetrahydrofuran under nitrogen atmosphere. A solution of 43a (2.81 g, 15.3 mmol) in 10 mL of dry tetrahydrofuran was added. The reaction was stirred for 3 h at room temperature. The reaction was quenched with successive additions of 1.75 mL of water, 1.75 mL of 4 N sodium hydroxide, and 5.25 mL of water. The mixture was filtered through Celite, and the filtrates were evaporated under vacuum. The residue was dissolved in dichloromethane and the organics were dried over magnesium sulfate, filtered, and evaporated to give 2[(7-aminoheptyl)(methyl)amino]ethanol 43b (2.32 g, 12.3 mmol, 81%). HPLC/MS (9 min) retention time 0.64 min. LRMS: m/z 189 (M + H+). Step 3. Amide coupling of 27 (300 mg, 0.57 mmol) with 43b (215 mg, 1.14 mmol) according to the general method gave 43 (169 mg, 0.24 mmol, 43%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.12−1.48 (m, 8H), 1.49−1.88 (m, 3H), 2.76 (s, 3H), 2.86−3.20 (m, 3H), 3.44 (q, J = 6.4 Hz, 2H), 3.77−4.07 (m, 2H), 4.25 (q, J = 7.3 Hz, 2H), 4.91 (s, 4H), 6.67 (t, J = 5.7 Hz, 1H), 7.48 (t, J = 7.6 Hz, 3H), 7.55−7.89 (m, 3H), 8.02 (d, J = 2.0 Hz, 2H), 8.49 (br s, 1H), 8.57 (s, 2H), 8.66 (s, 1H), 9.59 (br s, 1H). HPLC/MS (30 min) retention time 13.44 min. LRMS: m/z 694 (M + H+, 2 × Cl). 2-(3′-(5-(3-(3,5-Dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo1,6-dihydropyridazin-3-yl)biphenyl-3-yl)-N-(7-((2hydroxyethyl)(methyl)amino)heptyl)acetamide (44). Amide coupling of 32 (300 mg, 0.56 mmol) with 43b (209 mg, 1.11 mmol) according to the general method gave 44 (89 mg, 0.125 mmol, 23%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.13−1.53 (m, 10H), 1.56−1.85 (m, 2H), 2.76 (s, 3H), 2.85−3.00 (m, 2H), 3.02−3.12 (m, 2H), 3.20 (q, J = 6.6 Hz, 2H), 3.64 (s, 2H), 3.86−4.04 (m, 2H), 4.26 (q, J = 7.3 Hz, 2H), 5.65 (s, 2H), 5.81 (t, J = 5.9 Hz, 1H), 7.19−7.31 (m, 2H), 7.32− 7.66 (m, 4H), 7.81 (d, J = 7.8 Hz, 1H), 7.93 (br s, 1H), 8.52 (br s, 1H), 8.55 (s, 2H), 8.63 (s, 1H), 9.60 (br s, 1H). HPLC/MS (30 min) retention time 13.21 min. LRMS: m/z 708 (M + H+, 2 × Cl). 2-(3′-(5-(3-(3,5-Dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo1,6-dihydropyridazin-3-yl)biphenyl-4-yl)-N-(7-((2hydroxyethyl)(methyl)amino)heptyl)acetamide (45). Amide coupling of 33 (300 mg, 0.56 mmol) with 43b (209 mg, 1.11 mmol) according to the general method gave 45 (97 mg, 0.137 mmol, 25%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.08−1.79 (m, 11H), 2.38−2.54 (m, 3H), 2.56−2.70 (m, 2H), 2.71−2.85 (m, 2H), 3.02−3.41 (m, 4H), 3.62 (s, 2H), 3.67−3.83 (m, 2H), 4.25 (q, J = 7.3 Hz, 2H), 5.60 (t, J = 5.7 Hz, 1H), 7.33 (d, J = 8.6 Hz, 2H), 7.41−7.68 (m, 4H), 7.83 (d, J = 7.8 Hz, 2H), 7.93−8.04 (m, 2H), 8.57 (s, 2H), 8.70 (s, 1H), 9.61 (br s, 1H).

1.16 g, 6.05 mmol) in 8 mL of dimethylformamide. The mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue dissolved in 4% sodium bicarbonate solution. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuum to give N-[2-(dimethylamino)ethyl]-3-(4,4,5,5-tetramethyl1,3,2-dioxaborolan-2-yl)benzamide 37a (0.81 g, 2.55 mmol, 63%). HPLC/MS (9 min) retention time 4.23 min. LRMS: m/z 319 (M + H+) . Step 2. Suzuki reaction of 15 (0.50 g, 1.04 mmol) with 37a (0.493 g, 1.55 mmol) according to the general method gave 37 (0.320 g, 0.54 mmol, 51%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 2.25 (s, 6H), 2.49−2.54 (m, 2H), 3.32−3.48 (m, 2H), 4.28 (q, J = 7.2 Hz, 2H), 7.61 (dt, J = 15.7, 7.7 Hz, 2H), 7.80 (dt, J = 7.8, 1.7 Hz, 2H), 7.86 (t, J = 7.1 Hz, 2H), 8.04 (s, 1H), 8.14 (s, 1H), 8.19 (s, 1H), 8.40 (s, 1H), 8.59 (t, J = 5.6 Hz, 1H), 8.69 (s, 2H), 9.71 (s, 1H). HPLC/MS (30 min) retention time 12.32 min. LRMS: m/z 594 (M + H+, 2 × Cl). General Method for Amide Coupling Reaction, Exemplified for 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N-(2-piperidin-1ylethyl)biphenyl-3-carboxamide (38). Compound 27 (170 mg, 0.29 mmol) and 2-(piperidin-1-yl)ethanamine (75 mg, 0.59 mmol) were added to a solution of 1-hydroxybenzotriazole hydrate (60 mg, 0.44 mmol) and N-ethyl-N′-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (85 mg, 0.44 mmol) in dimethylformamide (3.5 mL). The mixture was stirred at room temperature for 3 h. The solvent was removed under reduced pressure and the residue dissolved in 4% sodium bicarbonate solution. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were washed with water and brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuum and the residue purified by crystallization from dichloromethane−methanol (8:2) to give 38 (70 mg, 0.11 mmol, 37%). 1 H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 6.6 Hz, 3H), 1.44− 1.60 (m, 6H), 2.32−2.52 (m, 6H), 3.39−3.48 (m, 2H), 4.28 (q, J = 6.7 Hz, 2H), 7.83 (m, 2H), 8.04 (br s, 1H), 8.13 (br s, 1H), 8.41 (s, 1H), 8.53 (br s, 1H), 8.68 (s, 2H), 9.68 (br s, 1H), 9.94 (br s, 1H). HPLC/MS (30 min) retention time 11.85 min. LRMS: m/z 634 (M + H+, 2 × Cl). 3′-(5-(3-(3,5-Dichloropyridin-4-yl)ureido)-1-ethyl-6-oxo-1,6dihydropyridazin-3-yl)-N-(2-(piperazin-1-yl)ethyl)biphenyl-3carboxamide (39). Amide coupling of 27 (200 mg, 0.35 mmol) with 2(piperidin-1-yl)ethanamine (69 μL, 0.53 mmol) according to the general method gave 39 (60 mg, 0.095 mmol, 27%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 6.6 Hz, 3H), 1.44−1.60 (m, 6H), 2.32−2.52 (m, 6H), 3.39−3.48 (m, 2H), 4.28 (q, J = 6.7 Hz, 2H), 7.83 (t, J = 8.4 Hz, 6H), 8.04 (br s, 1H), 8.13 (br s, 1H), 8.41 (s, 1H), 8.53 (br s, 1H), 8.68 (s, 2H), 9.68 (br s, 1H), 9.94 (br s, 1H). HPLC/MS (30 min) retention time 11.85 min. LRMS: m/z 635 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N-(2-morpholin-4ylethyl)biphenyl-3-carboxamide (40). Amide coupling of 27 (170 mg, 0.29 mmol) and 2-morpholinoethanamine (77 mg, 0.59 mmol) according to the general method gave 40 (70 mg, 0.11 mmol, 37%) after crystallization from dichloromethane−methanol (8:2). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.39 (t, J = 6.6 Hz, 3H), 2.35−2.45 (m, 8H), 3.56 (br s, 4H), 4.28 (q, J = 6.7 Hz, 2H), 7.51−7.72 (m, 2H), 7.72−7.94 (m, 5H), 8.08 (d,, J = 19.1 Hz, 3H), 8.40 (s, 1H), 8.58 (br s, 1H), 8.70 (s, 2H), 9.72 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 12.54 min. LRMS: m/z 636 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N-(2-(1-methylpyrrolidin-2-yl)ethyl)biphenyl-3-carboxamide (41). Amide coupling of 27 (300 mg, 0.57 mmol) and 2-(1-methylpyrrolidin-2-yl)ethylamine (169 mg, 1.15 mmol) according to the general method gave 41 (190 mg, 0.30 mmol, 52%) after reverse phase chromatography. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 1.42−1.52 (m, 2H), 1.58−1.72 (m, 2H), 1.84−2.04 (m, 2H), 2.07−2.22 (m, 2H), 2.25 (s, 3H), 2.94−3.04 (m, 1H), 3.32 (q, J = 6.9 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 7.59 (t, J = 8.1 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.80 (d, J = 7.7 Hz, 2H), 7.83−7.88 (m, 2H), 8.03 (s, 1H), 8.13 (s, 1H), 8.31 (s, 2H), 8.41 10491



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1H), 8.59 (t, J = 1.9 Hz, 1H). HPLC/MS (9 min) retention time 5.69 min. LRMS: m/z 244 (M − H+, 1 × Br). Step 2. Concentrated hydrochloric acid (30 mL) was gradually added to a solution of 47a (7.2 g, 28.5 mmol) dissolved in 150 mL of ethanol, and the mixture was stirred at room temperature for 5 min. Tin(II) chloride dehydrate (22.5 g, 100 mmol) was added, and the solution was stirred at 50 °C for 2 h. The solution was allowed to cool and was basified to pH 9 with 8 N sodium hydroxide solution, precipitating a solid. The mixture was filtered and the filtrate was acidified with hydrochloric acid to pH 5, forming a precipitate. The solid was collected by filtration and was dried under vacuum to give 3-amino-5-bromobenzoic acid 47b (5.8 g, 26.7 mmol, 94%). HPLC/MS (9 min) retention time 4.67 min. LRMS: m/z 214 (M − H+, 1 × Br). Step 3. 47b (5.8 g, 26.7 mmol) was dissolved in 50 mL of water and 5 mL of sulfuric acid. Sodium nitrite (1.85 g, 26.8 mmol) was added followed by 15 mL of water and 15 mL of sulfuric acid. The mixture was stirred at reflux for 100 min. The solution was allowed to cool, forming a precipitate. The solid was collected by filtration and dried under vacuum to give 3-bromo-5-hydroxybenzoic acid 47c (3.83 g, 17.7 mmol, 66%). HPLC/MS (9 min) retention time 5.06 min. LRMS: m/z 215 (M − H+, 1 × Br). Step 4. 47c (1.12 g, 5.16 mmol) and potassium carbonate (3.57 g, 25.8 mmol) were suspended in 100 mL of acetonitrile. Iodoethane (0.91 mL, 11.4 mmol) was added, and the mixture was stirred at reflux overnight. The solvent was evaporated, and the residue was partitioned between chloroform and water. The aqueous phase was extracted twice with chloroform. The combined organics were washed with brine, dried over magnesium sulfate, and filtered. The solvent was evaporated to give ethyl 3-bromo-5-ethoxybenzoate 47d (1.15 g, 4.21 mmol, 82%). 1H NMR (200 MHz, CDCl3) δ ppm 1.39 (t, J = 7.1 Hz, 3H), 1.42 (t, J = 7.1 Hz, 3H), 4.06 (q, J = 7.1 Hz, 2H), 4.37 (q, J = 7.1 Hz, 2H), 7.22 (t, J = 2.0 Hz, 1H), 7.49 (dd, J = 2.1, 1.3 Hz, 1H), 7.74 (t, J = 1.3 Hz, 1H). HPLC/ MS (9 min) retention time 7.27 min. LRMS: no ionization. Step 5. Pinacol boronate synthesis from 47d (0.95 g, 3.27 mmol) and bis(pinacolato)diboron (1.25 g, 4.92 mmol) according to the general method gave ethyl 3-ethoxy-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)benzoate 47e (0.97 g, 3.03 mmol, 52%) after purification by chromatography (ethyl acetate−hexane gradient, 0:100 to 30:70). HPLC/MS (9 min) retention time 7.46 min. LRMS: m/z 321 (M + H+). Step 6. Suzuki reaction of 15 (0.98 g, 2.03 mmol) with 47e (0.97 g, 3.03 mmol) according to the general method gave ethyl 3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5-ethoxybiphenyl-3-carboxylate 47f (0.94 g, 1.57 mmol, 39%) after purification by chromatography ethyl acetate−hexane gradient, 0:100 to 50:50). 1H NMR (200 MHz, CDCl3) δ ppm 1.28 (t, J = 7.2 Hz, 3H), 1.38 (t, J = 7.2 Hz, 3H), 1.46 (t, J = 7.2 Hz, 3H), 4.15 (q, J = 7.0 Hz, 2H), 4.27 (q, J = 7.2 Hz, 2H), 4.40 (q, J = 7.2 Hz, 2H), 7.34 (br s, 1H), 7.45−7.60 (m, 2H), 7.65 (d, J = 7.5 Hz, 1H), 7.77−7.94 (m, 2H), 8.06 (s, 1H), 8.62 (s, 2H), 8.74 (s, 1H), 9.19 (br s, 1H), 9.64 (br s, 1H). HPLC/MS (9 min) retention time 7.86 min. LRMS: m/z 596 (M + H+, 2 × Cl). Step 7. Ester hydrolysis of 47f (0.95 g, 1.45 mmol) according to the general method gave 3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5-ethoxybiphenyl-3-carboxylic acid 47g (0.304 g, 0.53 mmol, 69%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (q, J = 7.0 Hz, 6H), 4.17 (q, J = 6.9 Hz, 2H), 4.28 (q, J = 7.1 Hz, 2H), 7.45 (s, 1H), 7.46 (s, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.75−7.83 (m, 3H), 8.00 (s, 1H), 8.39 (s, 1H), 8.69 (s, 2H), 9.70 (s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 17.38 min. LRMS: m/z 568 (M + H+, 2 × Cl). Step 8. Amide coupling of 47g (160 mg, 0.24 mmol) and cyclopropanamine (33 μL, 0.48 mmol) according to the general method gave 47 (102 mg, 0.168 mmol, 70%) after purification by SP1 automated purification system. 1H NMR (200 MHz, DMSO-d6) δ ppm 0.54−0.63 (m, 2H), 0.66−0.77 (m, 2H), 1.37 (t, J = 7.1 Hz, 3H), 1.39 (t, J = 7.1 Hz, 3H), 2.80−2.92 (m, 1H), 4.15 (q, J = 7.1 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 7.34 (s, 1H), 7.38 (s, 1H), 7.63 (t, J = 7.3 Hz, 1H), 7.68 (s, 1H), 7.79 (app d, J = 7.4 Hz, 2H), 8.02 (s, 1H), 8.40 (s, 1H), 8.52 (d, J = 3.9 Hz, 1H), 8.68 (s, 2H), 9.67 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 17.17 min. LRMS: m/z 607 (M + H+, 2 × Cl).

HPLC/MS (30 min) retention time 13.22 min. LRMS: m/z 708 (M + H+, 2 × Cl). 5-Cyano-N-cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3yl]biphenyl-3-carboxamide (46). Step 1. 3-Bromo-5-iodobenzoic acid (2.00 g, 6.12 mmol) was dissolved in 25 mL of ethanol. Sulfuric acid (2.1 mL) was added, and the mixture was heated at 90 °C for 5 h. The mixture was allowed to cool and was evaporated. The residue was taken up in water and basified to pH 9 with 32% sodium hydroxide solution. The aqueous was extracted five times with dichloromethane. The organic phase was dried over magnesium sulfate and evaporated to give crude ethyl 3-bromo-5-iodobenzoate 46a (2.18 g, purity 87%). It was used as such without further purification. 1H NMR (200 MHz, CDCl3) δ ppm 1.40 (t, J = 7.0 Hz, 3H), 4.38 (q, J = 7.0 Hz, 2H), 8.03 (t, J = 1.8 Hz, 1H), 8.13 (t, J = 1.6 Hz, 1H), 8.29 (t, J = 1.4 Hz, 1H). HPLC/MS (9 min) retention time 3.54 min. LRMS: m/z 355 (M + H+, 1 × Br). Step 2. Iodide 46a (1.59 g, 4.48 mmol) was dissolved in 10 mL of dimethylformamide under argon atmosphere. Zinc cyanide (0.58 g, 4.9 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.52 g, 0.45 mmol) were added, and the mixture was stirred at 80 °C for 1 h. The mixture was allowed to cool and was diluted with ethyl acetate. The organics were washed three times with water, brine and dried over magnesium sulfate. Filtration and evaporation gave ethyl 3-bromo-5cyanobenzoate 46b (0.66 g, 2.60 mmol, 58%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.34 (t, J = 7.0 Hz, 3H), 4.36 (q, J = 7.2 Hz, 2H), 8.34 (br s, 2H), 8.48 (t, J = 1.9 Hz, 1H). HPLC/MS (9 min) retention time 6.43 min. LRMS: m/z 254 (M + H+, 1 × Br). Step 3. Pinacol boronate synthesis with 46b (660 mg, 2.60 mmol) and bis(pinacolato)diboron (1.32 g, 5.20 mmol) according to the general method gave ethyl 3-cyano-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan2-yl)benzoate 46c (650 mg, 2.17 mmol, 84%). 1H NMR (200 MHz, DMSO-d6) δ ppm 1.15−1.57 (m, 15H), 4.37 (q, J = 7.2 Hz, 2H), 8.21 (br s, 1H), 8.44 (m, 2H). HPLC/MS (9 min) retention time 5.32 min. LRMS: m/z 302 (M + H+). Step 4. Suzuki reaction of 15 (850 mg, 1.76 mmol) with 46c (635 mg, 2.12 mmol) according to the general method gave ethyl 5-cyano-3′-[5({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylate 46d (1.00 g, 1.73 mmol, 98%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.35 (t, J = 7.0 Hz, 3H), 1.40 (t, J = 7.1 Hz, 3H), 4.29 (q, J = 7.2 Hz, 2H), 4.38 (q, J = 6.9 Hz, 2H), 7.66 (t, J = 7.1 Hz, 1H), 7.88 (app d, J = 7.4 Hz, 2H), 8.10 (s, 1H), 8.34 (s, 1H), 8.42 (s, 1H), 8.46 (s, 1H), 8.54 (s, 1H), 8.69 (s, 2H), 9.69 (br s, 1H), 9.89 (br s, 1H). HPLC/MS (15 min) retention time 10.17 min. LRMS: m/z 575 (M − H+, 2 × Cl). Step 5. Ester hydrolysis of 46d (1.00 g, 1.77 mmol) according to the general method gave 5-cyano-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylic acid 46e (0.87 g, purity 91%) after triturating with acetonitrile. It was used as such without further purification. HPLC/MS (30 min) retention time 6.83 min. LRMS: m/z 549 (M + H+, 2 × Cl). Step 6. Amide coupling of 46e (200 mg, 0.36 mmol) with cyclopropylamine (50 μg, 0.73 mmol) according to the general method gave 46 (74 mg, 0.125 mmol, 34%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.52−0.66 (m, 2H), 0.67−0.83 (m, 2H), 1.39 (t, J = 7.2 Hz, 3H), 2.89 (tt, J = 7.9, 4.0 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.86 (t, J = 7.4 Hz, 2H), 8.11 (t, J = 1.4 Hz, 1H), 8.23 (t, J = 1.3 Hz, 1H), 8.37−8.40 (m, 2H), 8.41 (s, 1H), 8.68 (s, 2H), 8.75 (d, J = 4.3 Hz, 1H), 9.66 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 9.15 min. LRMS: m/z 588 (M + H+, 2 × Cl). N-Cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5ethoxybiphenyl-3-carboxamide (47). Step 1. A mixture of 3nitrobenzoic acid (10 g, 0.06 mol), concentrated sulfuric acid (120 mL), silver sulfate (9.33 g, 0.03 mol), and bromine (3.68 mL, 0.07 mol) was stirred at 100 °C for 6 h. The mixture was poured on ice, forming a precipitate, and the solid was collected by filtration. The solid was dissolved in sodium carbonate solution. Acidification gave a precipitate which was collected by filtration and dried in a stream of air to give 3bromo-5-nitrobenzoic acid 47a (7.11 g, 0.029 mol, 47%). 1H NMR (200 MHz, DMSO-d6) δ ppm 8.38 (t, J = 1.8, 1.3 Hz, 1H), 8.52 (t, J = 1.5 Hz, 10492



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N,N′-Dicyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3,5-dicarboxamide (48). Step 1. Cyclopropanamine (1.0 mL, 14.5 mmol) was added to a solution of 5-bromoisophthalic acid (710 mg, 2.90 mmol), 1-hydroxybenzotriazole hydrate (1.17 g, 8.69 mmol), and N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.66 g, 8.69 mmol) in 20 mL of dimethylformamide. The mixture was stirred at room temperature for 2 h. The solvent was removed under reduced pressure and the residue suspended in 4% sodium bicarbonate solution precipitating a solid. The solid was collected by filtration and dried under vacuum to give 5-bromo-N,N′dicyclopropylisophthalamide 48a (0.65 g, 2.00 mmol, 70%). HPLC/MS (9 min) retention time 5.03 min. LRMS: m/z 321 (M − H+, 1 × Br). Step 2. Pinacol boronate synthesis with 48a (0.78 g, 2.41 mmol) and bis(pinacolato)diboron (1.22 g, 4.83 mmol) according to the general method gave N,N′-dicyclopropyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)isophthalamide 48b (0.64 g, 1.73 mmol, 72%). HPLC/MS (9 min) retention time 5.77 min. LRMS: m/z 371 (M + H+). Step 3. Suzuki reaction of 15 (0.20 g, 0.41 mmol) with 48b (0.199 g, 0.54 mmol) according to the general method gave 48 (0.123 g, 0.19 mmol, 46%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.43−0.67 (m, 4H), 0.71 (td, J = 7.1, 5.0 Hz, 4H), 1.39 (t, J = 7.2 Hz, 3H), 2.88 (tq, J = 8.0, 4.1 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 8.07 (t, J = 1.9 Hz, 1H), 8.21 (d, J = 1.6 Hz, 2H), 8.27 (t, J = 1.5 Hz, 1H), 8.41 (s, 1H), 8.68 (d, J = 3.2 Hz, 2H), 8.69 (s, 2H), 9.71 (br s, 1H), 9.90 (br s, 1H). HPLC/MS (15 min) retention time 8.66 min. LRMS: m/z 646 (M + H+, 2 × Cl). Methyl 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5-({[3(dimethylamino)propyl]amino}carbonyl)biphenyl-3-carboxylate (49). Step 1. A solution of potassium hydroxide (0.29 g, 5.13 mmol) in 3.7 mL of water was added to a solution of dimethyl 5bromoisophthalate (2.80 g, 10.3 mmol) in 19 mL of methanol. The mixture was stirred overnight under reflux. The mixture was partitioned between water and ethyl ether. The aqueous phase was washed twice with ether. The aqueous portion was then acidified to acid pH with 5 N hydrochloric acid and was extracted three times with ethyl ether. The combined organics were washed with brine and dried over sodium sulfate. Purification by chromatography (ethyl acetate−hexane gradient, 0:100 to 100:0) gave 3-bromo-5-(methoxycarbonyl)benzoic acid 49a (0.70 g, 2.70 mmol, 26%). HPLC/MS (9 min) retention time 5.79 min. LRMS: m/z 257 (M − H+, 1 × Br). Step 2. Amide coupling of 49a (3.14 g, 12.1 mmol) with N,Ndimethylpropane-1,3-diamine (2.48 g, 24.2 mmol) according to the general method gave methyl 3-bromo-5-({[3-(dimethylamino)propyl]amino}carbonyl)benzoate 49b (3.98 g, 11.4 mmol, 94%). HPLC/MS (9 min) retention time 3.82 min. LRMS: m/z 343 (M + H+, 1 × Br). Step 3. Pinacol boronate synthesis with 49b (3.98 g, 11.4 mmol) with bis(pinacolato)diboron (5.76 g, 22.7 mmol) according to the general method gave methyl 3-({[3-(dimethylamino)propyl]amino}carbonyl)5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 49c (3.00 g, 7.69 mmol, 68%). HPLC/MS (9 min) retention time 3.82 min. LRMS: m/z 391 (M + H+). Step 4. Suzuki reaction of 15 (3.13 g, 6.45 mmol) with 49c (3.00 g, 7.69 mmol) according to the general method gave 49 (0.93 g, 1.40 mmol, 22%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.2 Hz, 3H), 1.70 (quin, J = 7.1 Hz, 2H), 2.15 (s, 6H), 2.35 (t, J = 7.1 Hz, 2H), 3.33 (q, J = 6.7 Hz, 2H), 3.92 (s, 3H), 4.29 (q, J = 7.1 Hz, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.83 (s, 1H), 7.85 (s, 1H), 8.06 (s, 1H), 8.24 (s, 1H), 8.32 (s, 1H), 8.40 (s, 2H), 8.45 (s, 1H), 8.68 (s, 1H), 8.89 (t, J = 5.5 Hz, 1H), 9.69 (br s, 2H). HPLC/MS (30 min) retention time 13.10 min. LRMS: m/z 666 (M + H+, 2 × Cl). N-Ethyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N′-(2dimethylaminopropyl)biphenyl-3,5-dicarboxamide (50). Step 1. Ester hydrolysis of 49 (930 mg, 1.40 mmol) according to the general method gave 50a (900 mg, 1.38 mmol, 99%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.39 (t, J = 7.1 Hz, 3H), 1.92 (quin, J = 7.1 Hz, 2H), 2.70 (s, 6H), 3.02 (t, J = 7.7 Hz, 2H), 3.38 (q, J = 6.4 Hz, 2H), 4.29 (q, J = 7.2 Hz, 2H), 7.66 (t, J = 7.8 Hz, 1H), 7.84 (d, J = 7.8 Hz, 2H), 7.85 (s,

1H), 8.05 (s, 1H), 8.33 (s, 1H), 8.39 (s, 2H), 8.47 (s, 1H), 8.69 (s, 2H), 8.97 (t, J = 5.6 Hz, 1H), 9.72 (s, 1H), 9.94 (br s, 1H). HPLC/MS (9 min) retention time 5.13 min. LRMS: m/z 652 (M + H+, 2 × Cl). Step 2. Amide coupling of 50a (175 mg, 0.27 mmol) with ethylamine (270 μL, 0.54 mmol) according to the general method gave 50 (73 mg, 0.11 mmol, 40%) after reverse-phase chromatography. 1H NMR (400 MHz, DMSO-d6) δ ppm 1.15 (t, J = 7.2 Hz, 3H), 1.40 (t, J = 7.1 Hz, 3H), 1.69 (quin, J = 7.0 Hz, 2H), 2.16 (s, 6H), 2.31 (t, J = 6.9 Hz, 2H), 3.13 (br s, 2H), 3.33 (quin, J = 6.0 Hz, 2H), 4.28 (q, J = 7.0 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.80 (d, J = 6.9 Hz, 1H), 7.82 (d, J = 7.5 Hz, 1H), 8.06 (s, 1H), 8.12−8.24 (m, 3H), 8.29 (s, 1H), 8.38 (s, 1H), 8.47 (br s, 1H), 8.54 (br s, 1H), 8.62 (s, 2H), 9.52 (s, 1H). HPLC/MS (30 min) retention time 12.00 min. LRMS: m/z 679 (M + H+, 2 × Cl). N-Cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N′{2-[(2R)-1-methylpyrrolidin-2-yl]ethyl}biphenyl-3,5-dicarboxamide (51). Step 1. Amide coupling of 49a (0.60 g, 2.32 mmol) with cyclopropanamine (322 μL, 4.63 mmol) according to the general method gave methyl 3-bromo-5-[(cyclopropylamino)carbonyl]benzoate 51a (0.66 g, 2.22 mmol, 96%). 1H NMR (200 MHz, CDCl3) δ ppm 0.59−0.73 (m, 2H), 0.83−1.00 (m, 2H), 2.91 (tq, J = 7.0, 3.5, 3.4 Hz, 1H), 3.94 (s, 3H), 6.46 (br s, 1H), 8.15 (t, J = 1.6 Hz, 1H), 8.22 (t, J = 1.4 Hz, 1H), 8.26 (t, J = 1.6 Hz, 1H). HPLC/MS (9 min) retention time 5.64 min. LRMS: m/z 296 (M − H+, 1 × Br). Step 2. Pinacol boronate synthesis from 51a (0.66 g, 2.22 mmol) and bis(pinacolato)diboron (1.12 g, 4.43 mmol) according to the general method) gave methyl 3-[(cyclopropylamino)carbonyl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 51b (0.572 g, 1.65 mmol, 75%). 1H NMR (400 MHz, CDCl3) δ ppm 0.61−0.69 (m, 2H), 0.89 (td, J = 7.0, 5.6 Hz, 2H), 1.36 (s, 12H), 2.92 (tq, J = 7.0, 3.8 Hz, 1H), 3.93 (s, 3H), 6.40 (br s, 1H), 8.29 (dd, J = 1.7, 1.1 Hz, 1H), 8.51 (t, J = 1.8 Hz, 1H), 8.56 (t, J = 1.7, 1.3 Hz, 1H). HPLC/MS (9 min) retention time 6.31 min. LRMS: m/z 346 (M + H+). Step 3. Suzuki reaction of 15 (0.62 g, 1.28 mmol) with 51b (0.572 g, 1.65 mmol) according to the general method gave methyl 5[(cyclopropylamino)carbonyl]-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylate 51c (0.54 g, 0.87 mmol, 68%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.58−0.65 (m, 2H), 0.69−0.77 (m, 2H), 1.39 (t, J = 7.0 Hz, 3H), 2.86−2.95 (m, 1H), 3.92 (s, 3H), 4.29 (q, J = 6.9 Hz, 2H), 7.66 (t, J = 7.7 Hz, 1H), 7.84 (d, J = 7.7 Hz, 2H), 8.06 (s, 1H), 8.32 (s, 1H), 8.37 (s, 1H), 8.40 (s, 1H), 8.43 (s, 1H), 8.69 (s, 2H), 8.78 (d, J = 3.5 Hz, 1H), 9.71 (br s, 1H), 9.90 (br s, 1H). HPLC/MS (9 min) retention time 7.08 min. LRMS: m/z 621 (M + H+, 2 × Cl). Step 4. Ester hydrolysis of 51c (540 mg, 0.87 mmol) according to the general method gave 5-[(cyclopropylamino)carbonyl]-3′-[5-({[(3,5dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylic acid 51d (520 mg, 0.86 mmol, 98%). It was used as such without further purification. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.50−0.66 (m, 2H), 0.67−0.83 (m, 2H), 1.39 (t, J = 7.0 Hz, 3H), 2.90 (tq, J = 8.1, 3.9 Hz, 1H), 4.29 (q, J = 7.0 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.79−7.90 (m, 2H), 8.06 (t, J = 1.6 Hz, 1H), 8.31 (t, J = 1.6 Hz, 1H), 8.35 (t, J = 1.7 Hz, 1H), 8.40 (s, 1H), 8.43 (t, J = 1.5 Hz, 1H), 8.70 (s, 2H), 8.78 (d, J = 4.3 Hz, 1H), 9.73 (s, 1H), 9.94 (s, 1H). HPLC/MS (9 min) retention time 6.60 min. LRMS: m/z 607 (M + H+, 2 × Cl). Step 5. Amide coupling of 51d (150 mg, 0.25 mmol) with {2-[(2R)-1methylpyrrolidin-2-yl]ethyl}amine (97 mg, 0.49 mmol) according to the general method gave 51 (21.6 mg, 0.030 mmol, 12%) after purification by reverse-phase chromatography. 1H NMR (400 MHz, DMSO-d6) δ ppm 0.57−0.65 (m, 2H), 0.67−0.75 (m, 2H), 1.38 (t, J = 7.0 Hz, 3H), 1.41−1.55 (m, 2H), 1.56−1.70 (m, 2H), 1.82−2.00 (m, 2H), 1.99−2.14 (m, 2H), 2.22 (s, 3H), 2.84−2.98 (m, 2H), 3.33 (q, J = 6.8 Hz, 2H), 4.27 (q, J = 7.2 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.78−7.83 (m, 2H), 8.07 (s, 1H), 8.20 (br s, 2H), 8.28 (s, 1H), 8.41 (br s, 1H), 8.45 (s, 1H), 8.56 (d, J = 3.9 Hz, 1H), 8.64 (t, J = 5.3 Hz, 1H). HPLC/MS (30 min) retention time 11.73 min. LRMS: m/z 717 (M + H+, 2 × Cl). 5-(Cyclopropylmethoxy)-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3yl]-N-(2-piperidin-1-ylethyl)biphenyl-3-carboxamide (52). Step 10493



Article

H NMR (400 MHz, DMSO-d6) δ ppm 0.55−0.64 (m, 2H), 0.67−0.78 (m, 2H), 1.34−1.38 (m, 2H), 1.39 (t, J = 7.0 Hz, 3H), 1.48 (quin, J = 5.5 Hz, 4H), 2.31−2.43 (m, 4H), 2.45 (t, J = 7.3 Hz, 2H), 2.89 (tq, J = 7.1, 4.0 Hz, 1H), 3.40 (q, J = 6.5 Hz, 2H), 4.29 (q, J = 7.0 Hz, 2H), 7.65 (t, J = 7.8 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 8.08 (s, 1H), 8.20−8.25 (m, 1H), 8.26−8.32 (m, 1H), 8.40 (s, 1H), 8.65 (t, J = 5.8 Hz, 1H), 8.67 (s, 1H), 8.69 (d, J = 4.2 Hz, 1H), 9.63 (br s, 1H). HPLC/MS (15 min) retention time 6.65 min. LRMS: m/z 717 (M + H+, 2 × Cl). 3′-[5-({[(3,5-Dichloropyridin-4-yl)amino]carbonyl}amino)-1ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N-(2-hydroxyethyl)-N′(2-piperidin-1-ylethyl)biphenyl-3,5-dicarboxamide (54). Step 1. Amide coupling of 49a (2.0 g, 7.72 mmol) with 2-(piperidin-1yl)ethylamine (2.2 mL, 15.5 mmol) according to the general method gave methyl 3-bromo-5-(2-(piperidin-1-yl)ethylcarbamoyl)benzoate 54a (2.80 g, 7.57 mmol, 96%). 1H NMR (200 MHz, CDCl3) δ ppm 1.40−1.54 (m, 2H), 1.54−1.70 (m, 4H), 2.39−2.51 (m, 4H), 2.58 (t, J = 6.0 Hz, 2H), 3.54 (q, J = 5.7 Hz, 2H), 3.95 (s, 3H), 7.20 (br s, 1H), 8.18 (t, J = 1.8 Hz, 1H), 8.27 (t, J = 1.6 Hz, 1H), 8.33 (t, J = 1.4 Hz, 1H). HPLC/MS (9 min) retention time 4.08 min. LRMS: m/z 369 (M + H+, 1 × Br). Step 2. Pinacol boronate synthesis with 54a (2.80 g, 7.57 mmol) and bis(pinacolato)diboron (2.90 g, 11.4 mmol) according to the general method gave methyl 3-(2-(piperidin-1-yl)ethylcarbamoyl)-5-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 54b (2.0 g, 4.80 mmol, 59%). HPLC/MS (9 min) retention time 4.78 min. LRMS: m/z 417 (M + H+). Step 3. Suzuki reaction of 15 (2.0 g, 4.14 mmol) with 54b (2.43 g, 4.96 mmol) according to the general method and purification by chromatography ((methanol−ethyl acetate, 20:80)−hexane gradient, 0:100 to 100:0) gave methyl 3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5-{[(2-piperidin-1-ylethyl)amino]carbonyl}biphenyl-3-carboxylate 54c (2.42 g, 3.49 mmol, 70%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.34−1.36 (m, 2H), 1.39 (t, J = 7.1 Hz, 3H), 1.48 (quin, J = 5.7 Hz, 4H), 2.34−2.42 (m, 4H), 2.45 (t, J = 7.4 Hz, 2H), 3.41 (q, J = 6.3 Hz, 2H), 3.92 (s, 3H), 4.29 (q, J = 6.8 Hz, 2H), 7.66 (t, J = 7.6 Hz, 1H), 7.84 (d, J = 8.2 Hz, 2H), 8.06 (s, 1H), 8.32 (s, 1H), 8.39 (s, 1H), 8.40 (s, 1H), 8.44 (s, 1H), 8.67 (s, 2H), 8.77 (t, J = 5.3 Hz, 1H), 9.66 (br s, 1H), 9.92 (br s, 1H). HPLC/ MS (30 min) retention time 11.77 min. LRMS: m/z 692 (M + H+, 2 × Cl). Step 4. Ester hydrolysis of 54c (2.30 g, 3.32 mmol) according to the general method gave 3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-5-{[(2-piperidin-1-ylethyl)amino]carbonyl}biphenyl-3-carboxylic acid 54d (1.40 g, 2.06 mmol, 61%). Partial 1H NMR (400 MHz, DMSO-d6) δ ppm 1.38 (t, J = 7.1 Hz, 3H), 1.38−1.45 (m, 2H), 1.57 (quin, J = 5.7 Hz, 4H), 2.63−2.70 (m, 2H), 2.71−2.77 (m, 2H), 3.52 (q, J = 6.3 Hz, 2H), 4.27 (q, J = 7.1 Hz, 2H), 7.60 (t, J = 7.7 Hz, 1H), 7.75 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H), 7.99 (s, 1H), 8.22 (s, 1H), 8.26 (s, 1H), 8.37 (s, 1H), 8.44 (s, 1H), 8.68 (s, 1H), 8.99 (s, 1H), 9.69 (s, 1H).7.60 (t, J = 7.9 Hz, 1H), 7.71−7.87 (m, 2H), 7.99 (s, 1H), 8.22 (s, 1H), 8.26 (s, 1H), 8.37 (s, 1H), 8.44 (s, 1H), 8.67 (s, 2H), 8.99 (br s, 1H), 9.69 (s, 1H), 9.92 (br s, 1H). HPLC/MS (30 min) retention time 10.73 min. LRMS: m/z 678 (M + H+, 2 × Cl). Step 5. Amide coupling of 54d (150 mg, 0.18 mmol) with 2aminoethanol (22.4 mg, 0.37 mmol) according to the general method and purification by reverse-phase chromatography gave 54 (67 mg, 0.093 mmol, 49%). 1H NMR (400 MHz, DMSO-d6) δ ppm 1.33−1.38 (m, 2H), 1.39 (t, J = 7.3 Hz, 3H), 1.45−1.53 (quin, J = 5.6 Hz, 4H), 2.39 (br s, 4H), 2.46 (t, J = 7.1 Hz, 2H), 3.38−3.45 (m, 4H), 3.55 (q, J = 5.8 Hz, 2H), 4.29 (q, J = 7.1 Hz, 2H), 4.76 (t, J = 5.2 Hz, 1H), 7.66 (t, J = 7.7 Hz, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 8.09 (s, 1H), 8.25 (s, 1H), 8.29 (s, 1H), 8.34 (s, 1H), 8.40 (s, 1H), 8.66 (t, J = 6.0 Hz, 1H), 8.68 (s, 2H), 8.71 (t, J = 5.6 Hz, 1H), 9.68 (br s, 1H), 9.91 (br s, 1H). HPLC/MS (30 min) retention time 10.12 min. LRMS: m/z 721 (M + H+, 2 × Cl). N-Cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N′(2-morpholin-4-ylethyl)biphenyl-3,5-dicarboxamide (55). . 1

1. Carboxylic acid 47c (2.32 g, 8.74 mmol) was dissolved in 25 mL of ethanol. Sulfuric acid (2.10 mL, 39.4 mmol) was added, and the mixture was heated at 90 °C for 5 h. The mixture was allowed to cool and was evaporated. The residue was taken up in water and basified to pH 9 with 32% sodium hydroxide solution. The aqueous was then extracted five times with dichloromethane. The organic phase was dried over magnesium sulfate. Purification by chromatography (ethyl acetate− hexane gradient, 0:100 to 100:0) gave ethyl 3-bromo-5-hydroxybenzoate 52a (2.02 g, 8.24 mmol, 94%). 1H NMR (200 MHz, CDCl3) δ ppm 1.40 (t, J = 7.1 Hz, 3H), 4.38 (q, J = 7.1 Hz, 2H), 8.03 (t, J = 1.5 Hz, 1H), 8.13 (t, J = 1.3 Hz, 1H), 8.29 (t, J = 1.2 Hz, 1H). HPLC/MS (9 min) retention time 6.08 min. LRMS: m/z 243 (M − H+, 1 × Br). Step 2. Phenol 52a (1.0 g, 4.08 mmol) was dissolved in 15 mL of dimethylformamide. Potassium carbonate (1.13 g, 8.18 mmol) and bromomethylcyclopropane (0.66 g, 4.89 mmol) were added, and the mixture was stirred at 100 °C overnight. The mixture was allowed to cool and partitioned between water and ethyl ether. The organic phase was washed twice with water, twice with brine, dried over magnesium sulfate, and evaporated to give ethyl 3-bromo-5-(cyclopropylmethoxy)benzoate 52b (0.98 g, 39.4 mmol, 75%). HPLC/MS (9 min) retention time 7.46 min. LRMS: m/z no ionization. Step 3. Pinacol boronate synthesis from 52b (0.98 g, 3.04 mmol) and bis(pinacolato)diboron (1.16 g, 4.56 mmol) according to the general method gave, after purification by chromatography (ethyl acetate− hexane gradient, 0:100 to 100:0), a 55:45 mixture of ethyl 3(cyclopropylmethoxy)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate 52c and (3-(cyclopropylmethoxy)-5-(ethoxycarbonyl)phenyl)boronic acid 52d (0.92 g). It was used as such without further purification. HPLC/MS (9 min) 52c retention time 7.57 min. LRMS: m/z 347 (M + H+). 52d retention time 6.00 min. LRMS: m/z 263 (M − H+). Step 4. Suzuki reaction of 15 (0.85 g, 1.74 mmol) with the mixture of 52c and 52d (0.92 g) according to the general method gave ethyl 5(cyclopropylmethoxy)-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3carboxylate 52e (0.89 g, 1.43 mmol, 76%). Partial 1H NMR (300 MHz, CDCl3) δ ppm 0.36−0.43 (m, 2H), 0.64−0.73 (m, 2H), 1.27 (t, J = 7.4 Hz, 3H), 1.41 (t, J = 7.1 Hz, 3H), 3.88−3.96 (m, 2H), 4.26 (q, J = 7.2 Hz, 2H), 4.40 (q, J = 7.1 Hz, 2H), 7.36 (dd, J = 2.4, 1.7 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H), 7.55 (dd, J = 2.5, 1.4 Hz, 1H), 7.65 (dt, J = 7.9, 1.3 Hz, 1H), 7.83 (dt, J = 8.1, 1.3 Hz, 1H), 7.88 (t, J = 1.5 Hz, 1H), 8.06 (t, J = 1.6 Hz, 1H), 8.62 (s, 2H), 8.76 (s, 1H), 9.26 (br s, 1H), 9.69 (br s, 1H). HPLC/ MS (9 min) retention time 7.90 min. LRMS: m/z 622 (M + H+, 2 × Cl). Step 5. Ester hydrolysis of 52e (0.89g, 1.43 mmol) according to the general method gave 5-(cyclopropylmethoxy)-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]biphenyl-3-carboxylic acid 52f (0.657 g, 1.11 mmol, 66%). 1H NMR (200 MHz, DMSO-d6) δ ppm 0.31−0.42 (m, 2H), 0.52−0.65 (m, 2H), 1.17−1.30 (m, 1H), 1.39 (t, J = 7.2 Hz, 3H), 3.96 (d, J = 7.0 Hz, 2H), 4.28 (q, J = 7.2 Hz, 2H), 7.41−7.51 (m, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.75−7.86 (m, 3H), 7.97−8.10 (m, 1H), 8.40 (s, 1H), 8.69 (s, 2H), 9.71 (br s, 1H), 9.91 (br s, 1H), 13.14 (br s, 1H). HPLC/MS (9 min) retention time 7.52 min. LRMS: m/z 592 (M − H+, 2 × Cl). Step 6. Amide coupling of 52f (160 mg, 0.23 mmol) with (2-piperidin1-ylethyl)amine (67 μL, 0.46 mmol) according to the general method and purification by reverse-phase chromatography gave 52 (105 mg, 0.15 mmol, 65%). Partial 1H NMR (200 MHz, DMSO-d6) δ ppm 0.31− 0.43 (m, 2H), 0.51−0.67 (m, 2H), 1.13−1.31 (m, 1H), 1.39 (t, J = 6.8 Hz, 3H), 1.34−1.59 (m, 6H), 2.39−2.46 (m, 6H), 3.95 (d, J = 7.0 Hz, 2H), 4.27 (q, J = 7.0 Hz, 2H), 7.37 (s, 1H), 7.40 (s, 1H), 7.61 (t, J = 7.6 Hz, 1H), 7.70 (s, 1H), 7.80 (d, J = 7.6 Hz, 2H), 8.02 (s, 1H), 8.20 (s, 1H), 8.39 (s, 1H), 8.54 (t, J = 4.8 Hz, 1H), 8.69 (s, 2H), 9.70 (br s, 1H). HPLC/MS (30 min) retention time 14.15 min. LRMS: m/z 704 (M + H+, 2 × Cl). N-Cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6-dihydropyridazin-3-yl]-N′(2-piperidin-1-ylethyl)biphenyl-3,5-dicarboxamide (53). Amide coupling of 51d (150 mg, 0.25 mmol) with (2-piperidin-1-ylethyl)amine (0.1 μL, 0.49 mmol) according to the general method and purification by reverse-phase chromatography gave 53 (88 mg, 0.123 mmol, 50%). 10494



Article

TNF-α Determination. The quantification of TNF-α in human serum was performed using commercial ELISA kit (DuoSet) obtained from R&D Systems, Inc., and following the manufacturer’s instructions. Plate Preparation. First, R&D DuoSet ELISA 96-well microplates were coated with 4.0 μg/mL mouse anti-human TNF-R diluted in PBS, overnight at room temperature. After washing, plates were then blocked with PBS containing 1% BSA for a minimum of 1 h at room temperature and then washed. Assay Procedure. 100 μL of samples or standard was added and incubated at 2 h at room temperature. After washing (ELX406 Select, BIO-TEK), biotinylated anti-hTNF-α antibody was added and incubated at room temperature for 2 h, followed by incubation with streptavidin−peroxidase for 20 min. Detection of bound hTNF-α was carried out with 100 μL of substrate solution (H 2 O 2 and tetramethylbenzidine) followed by measurement at 450 nm in a SPECTRA max Plus (Molecular Devices). These experiments were performed 2−3 times using the same experimental design. Duplicates from each series of experiments were averaged and expressed as hTNF-α levels in pg/mL. Inhibition of LPS-Induced Neutrophilia in Rats. Dry Powder Administration. A solution of LPS (Sigma, ref L-2630, 100 mg/vial) was prepared in 10 mL of PBS (CAMBREX BE17-512F). This was further diluted 1:100 with PBS. Solutions were prepared 24 h before use to ensure dissolution and stored at 4 °C. Test compounds were micronized before use and comixed with lactose to the correct dilution. Male Sprague-Dawley rats (230−280 g, fasted) were anesthetized with isoflurane. A 5 mg mixture of test compound and lactose was loaded into a Penn-Century Insufflator for Rat−Model DP-4 fitted with metal cannula. The cannula was carefully inserted into the trachea of the rat using a laryngoscope for guidance. Test compound was dispensed with 5 mL of air insufflation via a syringe. The cannula was carefully withdrawn and the animals allowed to regain consciousness. Inhibition of LPS-Induced Neutrophilia in Rats. Liquid Administration. A solution of LPS (Sigma, ref L-2630, 100 mg/vial) was prepared in 10 mL of PBS (CAMBREX BE17-512F). This was further diluted 1:100 with PBS. Solutions were prepared 24 h before use to ensure dissolution and stored at 4 °C. Test compounds were suspended in PBS containing 0.2% Tween 80 and homogenized in a mortar. Compounds were suspended at their maximum test concentration. Other concentrations were made by 1:10 dilutions with PBS containing 0.2% Tween 80. Male Sprague-Dawley rats (230−280 g, fasted) were anesthetized with isofluoroane. A suspension of 0.2 mL of the test compound was loaded into a Penn-Century MicroSprayer. The tip of the MicroSprayer was carefully inserted to about 1 cm from the tracheal carina using a laryngoscope for guidance. Test compound was dispensed rapidly to aerosolize the suspension. The cannula was carefully withdrawn, and the animals were allowed to regain consciousness. Nebulization of LPS. 1 or 18 h after test compound administration, animals were introduced into Perspex chambers and 30 mL of LPS solution was aerosolized using the DeVilbiss nebulizer during 40 min. After 4 h, the animals were euthanized with dolethal (2 g/mL, 10 mL/kg ip). 2 mL of PBS was introduced into the lungs via a cannula, and 0.8 mL of BAL solution was withdrawn. A further 2 mL of PBS was introduced and a further 1.8 mL of solution withdrawn. Neutrophils were counted directly by flow cytometry using the FACS. Control groups were formed by i.t. lactose only, i.t. lactose and LPS aerosol and using fluticasone (10% in lactose) and LPS aerosol as positive control. Animal Studies. All in vivo experiments were carried out in compliance with the European Committee Directive 2010/63/EU and the Spanish and Catalan laws. Experimental procedures were reviewed by The Animal-Welfare Body of Almirall and approved by the competent authority.

Amide coupling of 51d (150 mg, 0.25 mmol) with 2-morpholinoethanamine (61 mg, 0.47 mmol) according to the general method and purification by reverse-phase chromatography gave 55 (140 mg, 0.195 mmol, 83%). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.56−0.63 (m, 2H), 0.68−0.75 (m, 2H), 1.39 (t, J = 7.1 Hz, 3H), 2.42 (br s, 2H), 2.44− 2.50 (m, 4H), 2.89 (tq, J = 7.2, 3.9 Hz, 1H), 3.42 (q, J = 6.2 Hz, 2H), 3.56 (t, J = 4.2 Hz, 4H), 4.29 (q, J = 7.1 Hz, 2H), 7.65 (t, J = 7.7 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 8.08 (s, 1H), 8.22 (s, 1H), 8.24 (s, 1H), 8.30 (s, 2H), 8.41 (s, 1H), 8.67 (s, 1H), 8.68−8.73 (m, 2H). HPLC/MS (30 min) retention time 11.91 min. LRMS: m/z 719 (M + H+, 2 × Cl). N-(1-Benzylpiperidin-4-yl)-N′-cyclopropyl-3′-[5-({[(3,5-dichloropyridin-4-yl)amino]carbonyl}amino)-1-ethyl-6-oxo-1,6dihydropyridazin-3-yl]biphenyl-3,5-dicarboxamide (56). Amide coupling of 51d (150 mg, 0.25 mmol) with 1-benzylpiperidin-4-ylamine (95 mg, 0.50 mmol) according to the general method and purification by reverse-phase chromatography gave 56 (95 mg, 0.122 mmol, 49%). Partial 1H NMR (200 MHz, DMSO-d6) δ ppm 0.54−0.63 (m, 2H), 0.65−0.79 (m, 2H), 1.39 (t, J = 7.0 Hz, 3H), 1.62 (t, J = 11.0 Hz, 2H), 1.72−1.90 (m, 2H), 2.04 (t, J = 10.9 Hz, 2H), 2.83 (m, 3H), 4.29 (q, J = 6.8 Hz, 2H), 7.21−7.37 (m, 5H), 7.65 (t, J = 7.6 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 8.08 (s, 1H), 8.22 (br s, 3H), 8.28 (s, 1H), 8.41 (s, 1H), 8.53 (d, J = 7.4 Hz, 1H), 8.63−8.76 (m, 3H), 9.72 (br s, 1H). HPLC/MS (30 min) retention time 12.22 min. LRMS: m/z 779 (M + H+, 2 × Cl). PDE4 Activity Determination. PDE4 activity from human recombinant PDE4B1 subtype was monitored by measuring the hydrolysis of [3H]cAMP to [3H]AMP using a PDE-SPA kit from Amersham International as previously described.34 Enzyme extracts (∼4 μg of protein) were incubated in “low binding” plates (Costar 3604) for 60 min at room temperature. The assay mixture (80 μL) contained 15 nM [3H]cAMP (1 μCi/mL) in the assay buffer (50 mM Tris, pH 7.5, 8.3 mM MgCl2, 1.7 mM EGTA) and 10 μL of test compound. These compounds were resuspended in DMSO (the final DMSO concentration 5% (v/v)) at a stock concentration of 1 mM. The compounds were tested at different concentrations varying from 10 μM to 10 pM to calculate an IC50. These dilutions were done in 96-well plates. In some cases, plates containing diluted compounds were frozen before being assayed. In these cases, the plates were thawed at room temperature and agitated for 15 min. Hydrolysis of [3H]cAMP was initiated by adding 10 μL of a solution containing PDE4 enzyme, and the plate was then incubated under agitation at room temperature. The reaction was stopped after 60 min (with ∼10−20% substrate conversion) by addition of 50 μL of phosphodiesterase scintillation proximity assay (SPA) beads. All reactions were carried out in duplicate. [3H]AMP, captured by the SPA beads, was determined by counting the plates in a WallacMicrobeta Trilux scintillation counter 1 h after addition of the beads, although the signal was quite stable, and samples may be counted from 1 to 48 h after bead addition. LPS Induced TNF-α in Human Whole Blood (HWB-TNF-α). Human whole blood of healthy donors was collected in 50 mL Falcon tubes with heparin (5000 units/mL, heparin Mayne 5%, MAYNEPHARMA). LPS (lipopolysaccharide from Escherichia coli, Sigma, St. Louis, MO) dissolved in PBS (Dulbecco’s phosphate buffered saline, without calcium and magnesium chloride, Sigma, St. Louis, MO) was added to the tubes to give a final concentration in the assay of 1 μg/mL and preincubated at 37 °C for 10 min with rocking. Increasing concentrations of different inhibitors (2 μL), dissolved in 100% DMSO, were added to the 96-well plates, and an amount of 200 μL of blood containing LPS (except for controls) was then distributed into wells. Plates were shaken for 1−2 min, sealed with aluminum foil lid (Beckman Coulter), and then incubated for 24 h at 37 °C under agitation in Kelvitron T (Heraeus Instruments). After 24 h, plates were placed on ice, an amount of 50 μL of PBS was added, and the reaction was stopped by centrifugation of plates at 2000 rpm (800g) at 4 °C for 15 min. Serum obtained was then subjected to ELISA or kept at −80 °C until use. 10495



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00829. Molecular formula strings (CSV) Accession Codes

PDB code of compound 6: 5K1I (see Figure 3). Authors will release the atomic coordinates and experimental data upon article publication.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +34 93 291 3543. Fax: +34 93 291 3420. E-mail: jordi. [email protected]. ORCID

Jordi Gràcia: 0000-0002-7470-6933 Lluís Pagès: 0000-0002-3367-9529 Present Addresses

⊥ M.D.L.: Bionorica SE, Kerschensteinerstraße 11-15, 92318 Neumarkt, Germany. # R.S.R.: Mynorix Therapeutics S.L., TecnoCampus MataróMaresme, Av. Ernest Lluch 32, TCM3, 08302 Mataró, Barcelona, Spain.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the significant technical support provided by ́ Silvia Petit, Carmen Cabello, Agustina de la Cal, Dolores Marin, and Laura Estrella. We also acknowledge Montse Soler, Irena Bonin, and Joan Aymami for the crystal structure determination. We thank Sonia Espinosa and Josep M. Huerta for their support on physicochemical properties analysis and structural characterization of compounds. This work was funded by Almirall, Spain.

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ABBREVIATIONS USED cGMP, cyclic guanosine monophosphate; AD, atopic dermatitis; RA, rheumatoid arthritis; SBD, structure-based design; PYR, pyridazinone; UCR2, upstream conserved region 2; PBMC, polynuclear blood monocyte; hWB, human whole blood; PAPS, 3′-phosphoadenosine 5′-phosphosulfate; UDPGA, 5′-diphosphoglucuronic acid; HLM, human liver microsome; pM, picomolar; BAL, broncoalveolar lavage; A1, adenosine subtype 1; nM, nanomolar; SPA, scintillation proximity assay; DP, dry powder; FACS, fluorescence-activated cell sorting

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REFERENCES

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Biphenyl Pyridazinone Derivatives as Inhaled PDE4 Inhibitors

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