Patent ID: 12221434

EXAMPLES—COMPOUND SYNTHESIS

All solvents, reagents and compounds were purchased and used without further purification unless stated otherwise.

Abbreviations

2-MeTHF 2-methyltetrahydrofuranAc2O acetic anhydrideAcOH acetic acidaq aqueousBoc tert-butyloxycarbonylbr broadCbz carboxybenzylCDI 1,1-carbonyl-diimidazoleconc concentratedd doubletDABCO 1,4-diazabicyclo[2.2.2]octaneDCE 1,2-dichloroethane, also called ethylene dichlorideDCM dichloromethaneDIPEA N,N-diisopropylethylamine, also called Hünig's baseDMA dimethylacetamideDMAP 4-dimethylaminopyridine, also called N,N-dimethylpyridin-4-amineDME dimethoxyethaneDMF N,N-dimethylformamideDMSO dimethyl sulfoxideeq or equiv equivalent(ES+) electrospray ionization, positive modeEt ethylEtOAc ethyl acetateEtOH ethanolh hour(s)HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphateHPLC high performance liquid chromatographyLC liquid chromatographym multipletm-CPBA 3-chloroperoxybenzoic acidMe methylMeCN acetonitrileMeOH methanol(M+H)+ protonated molecular ionMHz megahertzmin minute(s)MS mass spectrometryMs mesyl, also called methanesulfonylMsCl mesyl chloride, also called methanesulfonyl chlorideMTBE methyl tert-butyl ether, also called tert-butyl methyl etherm/z mass-to-charge ratioNaOtBu sodium tert-butoxideNBS 1-bromopyrrolidine-2,5-dione, also called N-bromosuccinimideNCS 1-chloropyrrolidine-2,5-dione, also called N-chlorosuccinimideNMP N-methylpyrrolidineNMR nuclear magnetic resonance (spectroscopy)Pd(dba)3tris(dibenzylideneacetone) dipalladium(o)Pd(dppf)C2[1,1′-bis(diphenylphosphino)ferrocene] dichloropalladium(II)PE petroleum etherPh phenylPMB p-methoxybenzyl, also called 4-methoxybenzylprep-HPLC preparative high performance liquid chromatographyprep-TLC preparative thin layer chromatographyPTSA p-toluenesulfonic acidq quartetRP reversed phaseRT room temperatures singletSept septupletsat saturatedSCX solid supported cation exchange (resin)t tripletT3P propylphosphonic anhydrideTBME tert-butyl methyl ether, also called methyl tert-butyl etherTEA triethylamineTFA 2,2,2-trifluoroacetic acidTHF tetrahydrofuranTLC thin layer chromatographywt % weight percent or percent by weight

EXPERIMENTAL METHODS

Nuclear Magnetic Resonance

NMR spectra were recorded at 300, 400 or 500 MHz. Spectra were measured at 298 K, unless indicated otherwise, and were referenced relative to the solvent resonance. The chemical shifts are reported in parts per million. Spectra were recorded using one of the following machines:a Bruker Avance III spectrometer at 400 MHz fitted with a BBO 5 mm liquid probe,a Bruker 400 MHz spectrometer using ICON-NMR, under TopSpin program control,a Bruker Avance III HD spectrometer at 500 MHz, equipped with a Bruker 5 mm SmartProbe™,an Agilent VNMRS 300 instrument fitted with a 7.05 Tesla magnet from Oxford instruments, indirect detection probe and direct drive console including PFG module, oran Agilent MercuryPlus 300 instrument fitted with a 7.05 Tesla magnet from Oxford instruments, 4 nuclei auto-switchable probe and Mercury plus console.
LC-MS

LC-MS Methods: Using SHIMADZU LCMS-2020, Agilent 1200 LC/G1956A MSD and Agilent 1200\G6110A, Agilent 1200 LC & Agilent 6110 MSD. Mobile Phase: A: 0.025% NH3.H2O in water (v/v); B: acetonitrile. Column: Kinetex EVO C18 2.1×30 mm, 5 μm.

Reversed Phase HPLC Conditions for the LCMS Analytical Methods

Methods 1a and 1b: Waters Xselect CSH C18 XP column (4.6×30 mm, 2.5 μm) at 40° C.; flow rate 2.5-4.5 mL min−1eluted with a H2O-MeCN gradient containing either 0.1% v/v formic acid (Method 1a) or 10 mM NH4HCO3in water (Method 1b) over 4 min employing UV detection at 254 nm. Gradient information: 0-3.00 min, ramped from 95% water-5% acetonitrile to 5% water-95% acetonitrile; 3.00-3.01 min, held at 5% water-95% acetonitrile, flow rate increased to 4.5 mL min−1; 3.01-3.50 min, held at 5% water-95% acetonitrile; 3.50-3.60 min, returned to 95% water-5% acetonitrile, flow rate reduced to 3.50 mL min−1; 3.60-3.90 min, held at 95% water-5% acetonitrile; 3.90-4.00 min, held at 95% water-5% acetonitrile, flow rate reduced to 2.5 mL min−1.

Method 1c: Agilent 1290 series with UV detector and HP 6130 MSD mass detector using Waters XBridge BEH C18 XP column (2.1×50 mm, 2.5 μm) at 35° C.; flow rate 0.6 mL/min; mobile phase A: ammonium acetate (10 mM); water/MeOH/acetonitrile (900:60:40); mobile phase B: ammonium acetate (10 mM); water/MeOH/acetonitrile (100:540:360); over 4 min employing UV detection at 215 and 238 nm. Gradient information: 0-0.5 min, held at 80% A-20% B; 0.5-2.0 min, ramped from 80% A-20% B to 100% B.

Reversed Phase HPLC Conditions for the UPLC Analytical Methods

Methods 2a and 2b: Waters BEH C18 (2.1×30 mm, 1.7 μm) at 40° C.; flow rate 0.77 mL min−1eluted with a H2O-MeCN gradient containing either 0.1% v/v formic acid (Method 2a) or 10 mM NH4HCO3in water (Method 2b) over 3 min employing UV detection at 254 nm. Gradient information: 0-0.11 min, held at 95% water-5% acetonitrile, flow rate 0.77 mL min−1; 0.11-2.15 min, ramped from 95% water-5% acetonitrile to 5% water-95% acetonitrile; 2.15-2.49 min, held at 5% water-95% acetonitrile, flow rate 0.77 mL min−1; 2.49-2.56 min, returned to 95% water-5% acetonitrile; 2.56-3.00 min, held at 95% water-5% acetonitrile, flow rate reduced to 0.77 mL min−1.

Preparative Reversed Phase HPLC General Methods

Method 1 (Acidic Preparation): Waters X-Select CSH column C18, 5 μm (19×50 mm), flow rate 28 mL min−1eluting with a H2O-MeCN gradient containing 0.1% v/v formic acid over 6.5 min using UV detection at 254 nm. Gradient information: 0.0-0.2 min, 20% MeCN; 0.2-5.5 min, ramped from 20% MeCN to 40% MeCN; 5.5-5.6 min, ramped from 40% MeCN to 95% MeCN; 5.6-6.5 min, held at 95% MeCN.

Method 2 (Basic Preparation): Waters X-Bridge Prep column C18.5 μm (19×50 mm), flow rate 28 mL min−1eluting with a 10 mM NH4HCO3-MeCN gradient over 6.5 min using UV detection at 254 nm. Gradient information: 0.0-0.2 min, 10% MeCN; 0.2-5.5 min, ramped from 10% MeCN to 40% MeCN; 5.5-5.6 min, ramped from 40% MeCN to 95% MeCN; 5.6-6.5 min, held at 95% MeCN.

Method 3: Phenomenex Gemini column, 10 μm (150×25 mm), flow rate=25 mL/min eluting with a water-acetonitrile gradient containing 0.04% NH3at pH 10 over 9 minutes using UV detection at 220 and 254 nm. Gradient information: 0-9 minutes, ramped from 8% to 35% acetonitrile; 9-9.2 minutes, ramped from 35% to 100% acetonitrile; 9.2-15.2 minutes, held at 100% acetonitrile.

Method 4: Revelis C18 reversed-phase 12 g cartridge [carbon loading 18%; surface area 568 m2/g; pore diameter 65 Angstrom; pH (5% slurry) 5.1; average particle size 40 μm], flow rate=30 mL/min eluting with a water-methanol gradient over 35 minutes using UV detection at 215, 235, 254 and 280 nm. Gradient information: 0-5 minutes, held at 0% methanol; 5-30 minutes, ramped from 0% to 70% methanol; 30-30.1 minutes, ramped from 70% to 100% methanol; 30.1-35 minutes, held at 100% methanol.

SYNTHESIS OF INTERMEDIATES

Intermediate P1: 1-(Pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Step A: Lithium 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-sulfinate

A solution of BuLi (100 mL, 250 mmol, 2.5M in hexanes) was added slowly to a solution of 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole (36.2 g, 238 mmol) in THF (500 mL) keeping the temperature below −65° C. The mixture was stirred for 1.5 hours, then sulfur dioxide was bubbled through for 10 minutes. The mixture was allowed to warm to room temperature, the solvent evaporated and the residue triturated with TBME (300 mL) and filtered. The solid was washed with TBME and isohexane and dried to afford the crude title compound (54.89 g, 99%).

1H NMR (DMSO-d6) δ 7.26 (d, J=1.6 Hz, 1H), 6.10 (d, J=1.7 Hz, 1H), 5.99 (dd, J=10.0, 2.5 Hz, 1H), 3.92-3.87 (m, 1H), 3.56-3.49 (m, 1H), 2.25-2.15 (m, 1H), 2.00-1.91 (m, 1H), 1.75-1.69 (m, 1H), 1.66-1.46 (m, 3H).

LCMS; m/z 215 (M−H)−(ES−).

Step B: N,N-Bis(4-methoxybenzyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-sulfonamide

NCS (12.0 g, 90 mmol) was added to a suspension of lithium 1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-sulfinate (20 g, 90 mmol) in DCM (250 mL) cooled in an ice bath. The mixture was stirred for 4 hours, quenched with water (100 mL), and then partitioned between DCM (300 mL) and water (200 mL). The organic phase was washed with water (200 mL), dried (MgSO4), filtered and evaporated to ˜50 mL. The solution was added to a mixture of bis(4-methoxybenzyl)amine (24 g, 93 mmol) and triethylamine (40 mL, 287 mmol) in DCM (300 mL) cooled in an ice bath. After stirring for 1 hour, the mixture was warmed to room temperature, and then partitioned between DCM (300 mL) and water (250 mL). The organic layer was washed with water (250 mL), aq 1M HCl (2×250 mL), water (250 mL), dried (MgSO4), filtered, and evaporated to afford the crude title compound (41.02 g, 97%) as a brown oil.

LCMS; m/z 494.2 (M+Na)+(ES+).

Step C: N,N-Bis(4-methoxybenzyl)-1H-pyrazole-3-sulfonamide

A mixture of N,N-bis(4-methoxybenzyl)-1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazole-5-sulfonamide (41 g, 87 mmol) and aq 1M HCl (30 mL) in THF (300 mL) and MeOH (50 mL) was stirred at room temperature for 18 hours. The solvent was evaporated and the residue partitioned between EtOAc (400 mL) and aq 1M HCl (200 mL). The organic layer was washed with 10% brine (200 mL), dried (MgSO4), filtered and evaporated. The residue was triturated with TBME, filtered and dried to afford the title compound (24.87 g, 69%) as an off white solid.

1H NMR (CDCl3) δ 7.88 (d, J=2.4 Hz, 1H), 7.06-7.02 (m, 4H), 6.79-6.75 (m, 4H), 6.63 (d, J=2.4 Hz, 1H), 4.31 (s, 4H), 3.78 (s, 6H). Exchangeable proton not visible.

LCMS; m/z 388 (M+H)+(ES+); 386 (M−H)−(ES−).

Step D: N,N-Bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide

K2CO3(0.535 g, 3.87 mmol) was added to a solution of N,N-bis(4-methoxybenzyl)-1H-pyrazole-3-sulfonamide (0.5 g, 1.290 mmol) and 2-(chloromethyl)pyrimidine hydrochloride (0.213 g, 1.290 mmol) in DMF (8 mL). The reaction mixture was heated to 70° C. and stirred for 16 hours. Then the reaction mixture was washed with saturated brine (3×20 mL), the washings were combined and extracted with DCM (3×20 mL). The organic extracts were combined, dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by chromatography on silica gel (24 g column, 0-100% EtOAc/isohexane), then purified by chromatography on silica gel (40 g column, 0-100% EtOAc/isohexane and 0-10% MeOH/DCM) to afford the title compound (62 mg, 6%) as a yellow oil.

1H NMR (DMSO-d6) δ 8.83 (d, J=4.9 Hz, 2H), 8.11 (d, J=2.3 Hz, 1H), 7.50 (t, J=4.9 Hz, 1H), 7.02-6.95 (m, 4H), 6.80-6.74 (m, 5H), 5.72 (s, 2H), 4.16 (s, 4H), 3.72 (s, 6H).

LCMS; m/z 502.4 (M+Na)+(ES+).

Step E: 1-(Pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide

N,N-Bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (60 mg, 0.079 mmol) was dissolved in DCM (1 mL) and TFA (1 mL) was added. The solution was stirred for 16 hours. The reaction mixture was concentrated in vacuo, suspended in toluene (5 mL) and concentrated again. The crude product was purified by chromatography on silica gel (12 g column, 0-5% MeOH/DCM) to afford the title compound (16 mg, 84%) as a brown solid.

1H NMR (DMSO-d6) δ 8.81 (d, J=4.9 Hz, 2H), 7.98 (d, J=2.3 Hz, 1H), 7.48 (t, J=4.9 Hz, 1H), 7.15 (s, 2H), 6.61 (d, J=2.3 Hz, 1H), 5.63 (s, 2H).

LCMS; m/z 240.2 (M+H)+(ES+).

Intermediate P2: 1-(Oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Step A: N,N-Bis(4-methoxybenzyl)-1-(oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N,N-bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step D) from N,N-bis(4-methoxybenzyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step C) and 2-(chloromethyl)oxazole to afford the title compound (523 mg, 83%) as a colourless crystalline solid.

1H NMR (CDCl3) δ 7.68 (d, J=0.9 Hz, 1H), 7.58 (d, J=2.4 Hz, 1H), 7.17 (d, J=1.0 Hz, 1H), 7.06-6.99 (m, 4H), 6.79-6.72 (m, 4H), 6.69 (d, J=2.4 Hz, 1H), 5.50 (s, 2H), 4.30 (s, 4H), 3.78 (s, 6H).

LCMS; m/z 491 (M+Na)+(ES+).

Step B: 1-(Oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of 1-(pyrimidin-2-ylmethyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step E) from N,N-bis(4-methoxybenzyl)-1-(oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide to afford the title compound (146 mg, 59%) as a colourless crystalline solid.

1H NMR (DMSO-d6) δ 8.14 (d, J=0.9 Hz, 1H), 8.03 (d, J=2.4 Hz, 1H), 7.45 (s, 2H), 7.25 (d, J=0.8 Hz, 1H), 6.65 (d, J=2.4 Hz, 1H), 5.64 (s, 2H).

Intermediate P3: 1-((1-Methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-3-sulfonamide

Step A: N,N-bis(4-Methoxybenzyl)-1-((1-methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N,N-bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step D) from N,N-bis(4-methoxybenzyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step C) and 2-(chloromethyl)-1-methyl-1H-imidazole, HCl salt to afford the title compound (270 mg, 37%) as a yellow oil.

1H NMR (DMSO-d6) δ 7.98 (d, J=2.4 Hz, 1H), 7.18 (d, J=1.2 Hz, 1H), 6.99-6.95 (m, 4H), 6.90 (d, J=1.2 Hz, 1H), 6.81-6.75 (m, 4H), 6.72 (d, J=2.4 Hz, 1H), 5.56 (s, 2H), 4.18 (s, 4H), 3.72 (s, 6H), 3.68 (s, 3H).

LCMS; m/z 482.8 (M+H)+(ES+).

Step B: 1-((1-Methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of 1-(pyrimidin-2-ylmethyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step E) from N,N-bis(4-methoxybenzyl)-1-((1-methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-3-sulfonamide to afford the title compound (105 mg, 85%) as a yellow oil.

1H NMR (DMSO-d6) δ 7.90 (d, J=2.4 Hz, 1H), 7.42 (s, 2H), 7.15 (d, J=1.2 Hz, 1H), 6.85 (d, J=1.2 Hz, 1H), 6.60 (d, J=2.3 Hz, 1H), 5.50 (s, 2H), 3.68 (s, 3H).

LCMS; m/z 242.3 (M+H)+(ES+).

Intermediate P4: 1-(Pyridin-2-yl)-1H-pyrazole-3-sulfonamide

Step A: N,N-Bis(4-methoxybenzyl)-1-(pyridin-2-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N,N-bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step D) from N,N-bis(4-methoxybenzyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step C) and 2-bromopyridine to afford the title compound (210 mg, 83%) as an oil.

LCMS; m/z 465 (M+H)+(ES+).

Step B: 1-(Pyridin-2-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of 1-(pyrimidin-2-ylmethyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step E) from N,N-bis(4-methoxybenzyl)-1-(pyridin-2-yl)-1H-pyrazole-3-sulfonamide to afford the title compound (82 mg, 67%) as a white solid.

LCMS; m/z 225 (M+H)+(ES+).

Intermediate P5: 1-(Thiazol-2-yl)-1H-pyrazole-3-sulfonamide

Step A: N,N-Bis(4-methoxybenzyl)-1-(thiazol-2-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N,N-bis(4-methoxybenzyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step D) from N,N-bis(4-methoxybenzyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step C) and 2-bromothiazole to afford the title compound (169 mg, 84%) as an oil.

1H NMR (CDCl3) δ 8.38 (d, J=2.6 Hz, 1H), 7.62 (d, J=3.5 Hz, 1H), 7.23 (d, J=3.4 Hz, 1H), 7.19-7.14 (m, 4H), 6.85-6.77 (m, 5H), 4.40 (s, 4H), 3.79 (s, 6H).

LCMS; m/z 471 (M+H)+(ES+).

Step B: 1-(Thiazol-2-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of 1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step E) from N,N-bis(4-methoxybenzyl)-1-(thiazol-2-yl)-1H-pyrazole-3-sulfonamide to afford the title compound (45 mg, 34%) as a white solid.

LCMS; m/z 231 (M+H)+(ES+).

Intermediate P6: 1-(Pyridin-3-yl)-1H-pyrazole-3-sulfonamide

Step A: N,N-Bis(4-Methoxybenzyl)-1-(pyridin-3-yl)-1H-pyrazole-3-sulfonamide

3-Bromopyridine (130 μL, 1.349 mmol) and N1,N2-dimethylethane-1,2-diamine (15 μL, 0.139 mmol) were added to a suspension of N,N-bis(4-methoxybenzyl)-H-pyrazole-3-sulfonamide (Intermediate P1, Step C) (495 mg, 1.278 mmol), K2CO3(350 mg, 2.53 mmol) and CuI (15 mg, 0.079 mmol) in dry DMF (8 mL). The resulting mixture was heated to 140° C. (bath temperature) for 3 days. The mixture was cooled to room temperature, diluted with EtOAc (50 mL) and filtered through Celite®. The solution was concentrated in vacuo to give a brown oil, which was purified by chromatography on silica gel (40 g column 0-50% EtOAc/isohexane) to afford the title compound as a colourless oil (127 mg, 20%).

1H NMR (CDCl3) δ 9.05 (d, J=2.6 Hz, 1H), 8.67 (dd, J=4.9, 1.4 Hz, 1H), 8.17 (ddd, J=8.4, 2.6, 1.4 Hz, 1H), 8.06 (d, J=2.6 Hz, 1H), 7.60 (dd, J=8.4, 4.9 Hz, 1H), 7.16-7.11 (m, 4H), 6.90 (d, J=2.6 Hz, 1H), 6.80-6.75 (m, 4H), 4.40 (s, 4H), 3.76 (s, 6H).

LCMS; m/z 465 (M+H)+(ES+).

Step B: 1-(Pyridin-3-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of 1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P1, Step E) from N,N-bis(4-methoxybenzyl)-1-(pyridin-3-yl)-1H-pyrazole-3-sulfonamide to afford the title compound (51 mg, 86%) as a white solid.

1H NMR (MeOH-d4) δ 9.12 (dd, J=2.7, 0.8 Hz, 1H), 8.59 (dd, J=4.9, 1.4 Hz, 1H), 8.49 (d, J=2.6 Hz, 1H), 8.33 (ddd, J=8.4, 2.7, 1.4 Hz, 1H), 7.62 (ddd, J=8.4, 4.8, 0.8 Hz, 1H), 6.95 (d, J=2.6 Hz, 1H). NH2not observed.

Intermediate P7: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt

Step A: Ethyl 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylate, sodium salt

2 M Sodium tert-butoxide in THF (1.005 mL, 2.009 mmol) was added to a solution of ethyl 1-methyl-3-sulfamoyl-1H-pyrazole-5-carboxylate (0.5 g, 1.914 mmol) in THF (15 mL) and stirred at room temperature for 1 hour to give a white suspension. Then 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) (0.419 g, 2.105 mmol) in THF (5 mL) was added and stirred at room temperature overnight. The resultant colourless precipitate was collected by filtration, washing with THF (4 mL), and dried in vacuo to afford the title compound (930 mg, 91%) as a colourless solid.

1H NMR (DMSO-d6) δ 7.51 (s, 1H), 6.96 (s, 1H), 6.77 (s, 1H), 4.28 (q, J=7.1 Hz, 2H), 4.05 (s, 3H), 2.74 (t, J=7.4 Hz, 4H), 2.66 (t, J=7.3 Hz, 4H), 1.90 (p, J=7.4 Hz, 4H), 1.30 (t, J=7.1 Hz, 3H).

LCMS; m/z 433.4 (M+H)+(ES+).

Step B: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt

Ethyl 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylate, sodium salt (3.15 g, 6.24 mmol) was dissolved in MeOH (20 mL), 2 M aqueous NaOH (3.12 mL, 6.24 mmol) was added and stirred for 6 hours. The reaction mixture was concentrated under reduced pressure to afford the title compound (2.80 g, 99%) as a colourless solid.

1H NMR (DMSO-d6) δ 7.57 (s, 1H), 6.76 (s, 1H), 6.44 (s, 1H), 4.02 (s, 3H), 2.74 (t, J=7.4 Hz, 4H), 2.65 (t, J=7.4 Hz, 4H), 1.89 (p, J=7.4 Hz, 4H).

LCMS; m/z 405.4 (M+H)+(ES+).

Intermediate A1: 4-Isocyanato-1,2,3,5,6,7-hexahydro-s-indacene

To a solution of phosgene (4.45 mL, 20% weight in toluene, 8.4 mmol) in EtOAc (90 mL) was added dropwise a solution of 1,2,3,5,6,7-hexahydro-s-indacen-4-amine (589 mg, 3.4 mmol) in EtOAc (45 mL) at ambient temperature. The resulting reaction mixture was then heated to reflux for 3 hours and upon cooling was filtered and concentrated in vacuo to afford the title compound as a brown oil (756 mg, 100%). The crude product was used directly in the next step without further purification.

1H NMR (CDCl3) δ 6.8 (s, 1H), 2.89 (m, 8H) and 2.09 (m, 4H).

PREPARATION OF EXAMPLES

Example 1: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Sodium tert-butoxide (2M in THF) (0.033 mL, 0.066 mmol) was added to a solution of 1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P1) (15 mg, 0.063 mmol) in THF (2 mL) and stirred at room temperature for 1 hour. Then 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) (14 mg, 0.070 mmol) was added and the reaction mixture was stirred at room temperature overnight. Volatiles were evaporated and the crude product was purified by reversed phase prep-HPLC (General Methods, basic prep) to afford the title compound (3.5 mg, 13%) as a white solid.

1H NMR (DMSO-d6) δ 10.81 (br s, 1H), 8.79 (d, J=4.9 Hz, 2H), 8.04 (d, J=2.4 Hz, 1H), 7.94 (s, 1H), 7.48 (t, J=4.9 Hz, 1H), 6.92 (s, 1H), 6.77 (d, J=2.4 Hz, 1H), 5.66 (s, 2H), 2.78 (t, J=7.4 Hz, 4H), 2.57 (t, J=7.4 Hz, 4H), 1.92 (p, J=7.4 Hz, 4H).

LCMS; m/z 439.4 (M+H)+(ES+).

Example 2: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-(oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Example 1) from 1-(oxazol-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Intermediate P2) and 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) to afford the title compound (27 mg, 25%) as a white solid.

1H NMR (DMSO-d6) δ 8.11 (d, J=0.9 Hz, 1H), 7.99 (s, 1H), 7.84 (s, 1H), 7.24 (d, J=0.9 Hz, 1H), 6.89 (s, 1H), 6.70 (s, 1H), 5.62 (s, 2H), 2.78 (t, J=7.4 Hz, 4H), 2.59 (t, J=7.4 Hz, 4H), 1.93 (p, J=7.4 Hz, 4H). NH not observed.

LCMS; m/z 428 (M+H)+(ES+); 426 (M−H)−(ES−).

Example 3: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-((1-methyl-1H-imidazol-2-yl)methyl)-H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Example 1) from 1-((1-methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-3-sulfonamide (Intermediate P3) and 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) to afford the title compound (34 mg, 21%) as a white solid.

1H NMR (DMSO-d6) δ 10.85 (br s, 1H), 7.97 (s, 1H), 7.93 (d, J=2.4 Hz, 1H), 7.14 (d, J=1.1 Hz, 1H), 6.93 (s, 1H), 6.85 (d, J=1.2 Hz, 1H), 6.74 (d, J=2.4 Hz, 1H), 5.53 (s, 2H), 3.63 (s, 3H), 2.79 (t, J=7.4 Hz, 4H), 2.57 (t, J=7.4 Hz, 4H), 1.95 (p, J=7.5 Hz, 4H).

LCMS; m/z 441.4 (M+H)+(ES+).

Example 4: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyridin-2-yl)-1H-pyrazole-3-sulfonamide, sodium salt

Sodium tert-butoxide (2 M in THF) (0.103 mL, 0.206 mmol) was added to a solution of 1-(pyridin-2-yl)-1H-pyrazole-3-sulfonamide (Intermediate P4) (44 mg, 0.196 mmol) in THF (2 mL) and stirred at room temperature for 1 hour. Then 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) (41 mg, 0.206 mmol) was added and the reaction mixture was stirred at room temperature for 15 hours. EtOAc (6 mL) was added and the suspension stirred for 1 hour, filtered, and washed with EtOAc (1 mL). The collected solid was dried under reduced pressure to afford the title compound (15 mg, 16%) as a white solid.

1H NMR (DMSO-d6) δ 8.52 (d, J=2.6 Hz, 1H), 8.47 (ddd, J=4.9, 1.9, 0.9 Hz, 1H), 8.05-7.98 (m, 1H), 7.92-7.86 (m, 1H), 7.48 (s, 1H), 7.37 (ddd, J=7.3, 4.9, 1.0 Hz, 1H), 6.75 (s, 1H), 6.67 (d, J=2.6 Hz, 1H), 2.73 (t, J=7.4 Hz, 4H), 2.67 (t, J=7.3 Hz, 4H), 1.87 (p, J=7.5 Hz, 4H).

LCMS; m/z 424 (M+H)+(ES+).

Example 5: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-(thiazol-2-yl)-1H-pyrazole-3-sulfonamide, sodium salt

Prepared according to the general procedure of N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyridin-2-yl)-H-pyrazole-3-sulfonamide, sodium salt (Example 4) from 1-(thiazol-2-yl)-1H-pyrazole-3-sulfonamide (Intermediate P5) and 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) to afford the title compound (16 mg, 24%) as a white solid.

1H NMR (DMSO-d6) δ 8.40 (d, J=2.6 Hz, 1H), 7.64 (d, J=3.5 Hz, 1H), 7.57 (d, J=3.5 Hz, 1H), 7.48 (s, 1H), 6.77 (s, 1H), 6.70 (d, J=2.6 Hz, 1H), 2.81-2.70 (m, 8H), 1.93-1.87 (m, 4H).

LCMS; m/z 430 (M+H)+(ES+).

Example 6: N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyridin-3-yl)-1H-pyrazole-3-sulfonamide

Prepared according to the general procedure of N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)-1-(pyrimidin-2-ylmethyl)-1H-pyrazole-3-sulfonamide (Example 1) from 1-(pyridin-3-yl)-1H-pyrazole-3-sulfonamide (Intermediate P6) and 4-isocyanato-1,2,3,5,6,7-hexahydro-s-indacene (Intermediate A1) to afford the title compound (16 mg, 17%) as a white solid.

1H NMR (DMSO-d6) δ 11.12 (br s, 1H), 9.11 (d, J=2.0 Hz, 1H), 8.71 (d, J=2.6 Hz, 1H), 8.62 (dd, J=4.7, 1.4 Hz, 1H), 8.25 (ddd, J=8.4, 2.7, 1.5 Hz, 1H), 7.95 (s, 1H), 7.62 (ddd, J=8.3, 4.7, 0.7 Hz, 1H), 6.98 (d, J=2.6 Hz, 1H), 6.88 (s, 1H), 2.76 (t, J=7.4 Hz, 4H), 2.61 (t, J=7.4 Hz, 4H), 1.88 (p, J=7.4 Hz, 4H).

LCMS; m/z 424 (M+H)+(ES+); 422 (M−H)−(ES−).

Example 7: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-N,1-dimethyl-N-(thiazol-2-ylmethyl)-1H-pyrazole-5-carboxamide

HATU (58 mg, 0.151 mmol) was added to a solution of 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt (Intermediate P7) (65 mg, 0.145 mmol) and N-methyl-1-(thiazol-2-yl)methanamine (19 mg, 0.151 mmol) in DMF (1 mL) and stirred for 20 hours. Water (1 mL) was slowly added and the reaction mixture was stirred for 1 hour. The suspension was filtered and the collected solid triturated in water (3 mL) for 0.5 hour. The suspension was filtered and the collected solid was washed with water (0.5 mL) and TBME (1 mL). The solid was dried under reduced pressure for 6 hours to afford the title compound (18 mg, 26%) as a white solid.

1H NMR (DMSO-d6), rotamers; δ 10.95 (br s, 1H), 8.09-8.07 (2×s, 1H), 7.89-7.65 (m, 2H), 7.11-7.08 (2×s, 1H), 6.94 (s, 1H), 4.97-4.92 (2×s, 2H), 3.97-3.95 (2×s, 3H), 3.12-3.01 (2×s, 3H), 2.78 (t, J=7.6 Hz, 4H), 2.64-2.53 (m, 4H), 1.94-1.92 (m, 4H).

LCMS; m/z 515 (M+H)+(ES+).

Example 8: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-N,1-dimethyl-N-((1-methyl-1H-imidazol-2-yl)methyl)-1H-pyrazole-5-carboxamide

Prepared according to the general procedure of 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-N,1-dimethyl-N-(thiazol-2-ylmethyl)-1H-pyrazole-5-carboxamide (Example 7) from 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt (Intermediate P7) and N-methyl-1-(1-methyl-H-imidazol-2-yl)methanamine to afford the title compound (11 mg, 13%) as a white solid.

1H NMR (DMSO-d6), rotamers; δ 7.71 (s, 1H), 7.20 (s, 0.4H), 7.14 (s, 0.6H), 7.05 (s, 0.6H), 6.93 (s, 0.4H), 6.85-6.83 (m, 2H), 4.73-4.17 (m, 2H), 3.89 (s, 3H), 3.64-3.45 (2×s, 3H), 2.98-2.94 (2×s, 3H), 2.76 (t, J=7.4 Hz, 4H), 2.63-2.59 (m, 4H), 1.94-1.87 (m, 4H). NH not observed.

LCMS; m/z 512 (M+H)+(ES+).

Example 9: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-N,1-dimethyl-N-(thiazol-2-yl)-1H-pyrazole-5-carboxamide

Prepared according to the general procedure of 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-N,1-dimethyl-N-(thiazol-2-ylmethyl)-1H-pyrazole-5-carboxamide (Example 7) from 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt (Intermediate P7) and N-methylthiazol-2-amine to afford the title compound (4 mg, 7%) as a white solid.

1H NMR (DMSO-d6) δ 11.03 (br s, 1H), 8.11 (br s, 1H), 7.66 (d, J=3.6 Hz, 1H), 7.45 (d, J=3.6 Hz, 1H), 7.38 (s, 1H), 6.95 (s, 1H), 4.05 (s, 3H), 3.72 (s, 3H), 2.79 (t, J=7.4 Hz, 4H), 2.61 (t, J=7.3 Hz, 4H), 1.99-1.92 (m, 4H).

LCMS; m/z 501 (M+H)+(ES+).

Example 10: 3-(N-((1,2,3,5,6,7-Hexahydro-s-indacen-4-yl)carbamoyl) sulfamoyl)-N,1-dimethyl-N-((1-methyl-1H-pyrazol-5-yl)methyl)-1H-pyrazole-5-carboxamide, Partial Ammonium Salt

N-Methyl-1-(1-methyl-1H-pyrazol-5-yl)methanamine (23.45 mg, 0.187 mmol), NaHCO3(16 mg, 0.10 mmol) and HATU (72 mg, 0.189 mmol) were successively added to a suspension of 3-(N-((1,2,3,5,6,7-hexahydro-s-indacen-4-yl)carbamoyl)sulfamoyl)-1-methyl-1H-pyrazole-5-carboxylic acid, disodium salt (Intermediate P7) (70 mg, 0.156 mmol) in DMF (1 mL) and stirred for 2 days. The reaction was quenched with water (1 mL) and purified by reversed phase prep-HPLC (General Methods, basic prep) to afford the title compound (14 mg, 17%) as a white solid.

1H NMR (DMSO-d6), rotamers; δ 7.65 & 7.58 (2×s, 1H), 7.50-7.00 (br s, 1H), 7.36 (d, J=1.9 Hz, 1H), 6.81 (s, 1H), 6.81 & 6.60 (2×s, 1H), 6.27 & 6.14 (2×s, 1H), 4.76 & 4.70 (2×s, 2H), 3.87 & 3.80 (2×s, 3H), 3.80 & 3.58 (2×s, 3H), 2.99 (s, 3H), 2.75 (t, J=7.4 Hz, 4H), 2.63 (t, J=7.5 Hz, 4H), 1.89 (p, J=7.5 Hz, 4H).

LCMS; m/z 512.4 (M+H)+(ES+); 510.3 (M−H)−(ES−).

EXAMPLES—BIOLOGICAL STUDIES

NLRP3 and Pyroptosis

It is well established that the activation of NLRP3 leads to cell pyroptosis and this feature plays an important part in the manifestation of clinical disease (Yan-gang Liu et al., Cell Death & Disease, 2017, 8(2), e2579; Alexander Wree et al., Hepatology, 2014, 59(3), 898-910; Alex Baldwin et al., Journal of Medicinal Chemistry, 2016, 59(5), 1691-1710; Ema Ozaki et al., Journal of Inflammation Research, 2015, 8, 15-27; Zhen Xie & Gang Zhao, Neuroimmunology Neuroinflammation, 2014, 1(2), 60-65; Mattia Cocco et al., Journal of Medicinal Chemistry, 2014, 57(24), 10366-10382; T. Satoh et al., Cell Death & Disease, 2013, 4, e644). Therefore, it is anticipated that inhibitors of NLRP3 will block pyroptosis, as well as the release of pro-inflammatory cytokines (e.g. IL-1ρ) from the cell.

THP-1 Cells: Culture and Preparation

THP-1 cells (ATCC #TIB-202) were grown in RPMI containing L-glutamine (Gibco #11835) supplemented with 1 mM sodium pyruvate (Sigma #S8636) and penicillin (100 units/ml)/streptomycin (0.1 mg/ml) (Sigma #P4333) in 10% Fetal Bovine Serum (FBS) (Sigma #F0804). The cells were routinely passaged and grown to confluency (˜106cells/ml). On the day of the experiment, THP-1 cells were harvested and resuspended into RPMI medium (without FBS). The cells were then counted and viability (>90%) checked by Trypan blue (Sigma #T8154). Appropriate dilutions were made to give a concentration of 625,000 cells/ml. To this diluted cell solution was added LPS (Sigma #L4524) to give a 1 μg/ml Final Assay Concentration (FAC). 40 μl of the final preparation was aliquoted into each well of a 96-well plate. The plate thus prepared was used for compound screening.

THP-1 Cells Pyroptosis Assay

The following method step-by-step assay was followed for compound screening.1. Seed THP-1 cells (25,000 cells/well) containing 1.0 μg/ml LPS in 40 μl of RPMI medium (without FBS) in 96-well, black walled, clear bottom cell culture plates coated with poly-D-lysine (VWR #734-0317)2. Add 5 μl compound (8 points half-log dilution, with 10 μM top dose) or vehicle (DMSO 0.1% FAC) to the appropriate wells3. Incubate for 3 hrs at 37° C. and 5% CO24. Add 5 μl nigericin (Sigma #N7143) (FAC 5 μM) to all wells5. Incubate for 1 hr at 37° C. and 5% CO26. At the end of the incubation period, spin plates at 300×g for 3 mins and remove supernatant7. Then add 50 μl of resazurin (Sigma #R7017) (FAC 100 M resazurin in RPMI medium without FBS) and incubate plates for a further 1-1.5 h at 37° C. and 5% CO28. Plates were read in an Envision reader at Ex 560 nm and Em 590 nm9. IC50data is fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4-parameters)
96-Well Plate Map

123456789101112AHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowBHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowCHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowDHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowEHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowFHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowGHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowHHighComp 1Comp 2Comp 3Comp 4Comp 5Comp 6Comp 7Comp 8Comp 9Comp 10LowHighMCC950 (10 uM)Compound 8-point half-log dilutionLowDrug free control

The results of the pyroptosis assay performed are summarised in Table 1 below as THP IC50.

Human Whole Blood IL1β Release Assay

For systemic delivery, the ability to inhibit NLRP3 when the compounds are present within the bloodstream is of great importance. For this reason, the NLRP3 inhibitory activity of a number of compounds in human whole blood was investigated in accordance with the following protocol.

Human whole blood in Li-heparin tubes was obtained from healthy donors from a volunteer donor panel.1. Plate out 80 μl of whole blood containing 1 μg/ml of LPS in 96-well, clear bottom cell culture plate (Corning #3585)2. Add 10 μl compound (8 points half-log dilution with 10 μM top dose) or vehicle (DMSO 0.1% FAC) to the appropriate wells3. Incubate for 3 hrs at 37° C., 5% CO24. Add 10 μl nigericin (Sigma #N7143) (10 μM FAC) to all wells5. Incubate for 1 hr at 37° C., 5% CO26. At the end of the incubation period, spin plates at 300×g for 5 mins to pellet cells and remove 20 μl of supernatant and add to 96-well v-bottom plates for IL-1β analysis (note: these plates containing the supernatants can be stored at −80° C. to be analysed at a later date)7. IL-1β was measured according to the manufacturer protocol (Perkin Elmer-AlphaLisa IL-1 Kit AL220F-5000)8. IC50data is fitted to a non-linear regression equation (log inhibitor vs response-variable slope 4-parameters)

The results of the human whole blood assay are summarised in Table 1 below as HWB IC50.

TABLE 1NLRP3inhibitory activityExample NoTHP IC50HWB IC501++ND2++++*3+++++****4++ND5++ND6++ND7+++**8++++***9+++ND10++ND[THP IC50(≤0.04 μM = +++++, ≤0.16 μM = ++++, ≤0.64 μM = +++, ≤2.56 μM = ++, ≤10 μM = +, not determined = ND)] [HWB IC50(≤0.4 μM = *****, (≤0.8 μM = ****, ≤1.6 μM = ***, ≤3.2 μM = **, ≤10 μM = *, not determined = ND)]

As is evident from the results presented in Table 1, surprisingly in spite of the structural differences versus the prior art compounds, the compounds of the invention show high levels of NLRP3 inhibitory activity in the pyroptosis assay and in the human whole blood assay.

It will be understood that the present invention has been described above by way of example only. The examples are not intended to limit the scope of the invention. Various modifications and embodiments can be made without departing from the scope and spirit of the invention, which is defined by the following claims only.