1-(5-Carboxyindazol-1-yl)propan-2-ones as dual inhibitors of cytosolic phospholipase A₂α and fatty acid amide hydrolase: bioisosteric replacement of the carboxylic acid moiety

Abstract

Indazole-5-carboxylic acids with 3-aryloxy-2-oxopropyl residues in position 1 were previously reported to be potent dual inhibitors of cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH). In continuation of our structure-activity studies on cPLA₂α and FAAH inhibitors, a number of derivatives of these substances characterized by bioisosteric replacement of the carboxylic acid functionality by inverse amides, sulfonylamides, carbamates and ureas were prepared. The biological evaluation of the obtained compounds showed that the carboxylic acid functionality of the lead compounds is of special importance for a pronounced inhibition of cPLA₂α and FAAH.

Keywords:

• Bioisosteric replacement
• cytosolic phospholipase A₂α
• fatty acid amide hydrolase
• indazol-1-ylpropan-2-ones
• inhibitor

Introduction

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are two metabolic serine hydrolases, which play important roles in inflammatory processes. cPLA₂α has been recognized as the key enzyme in the synthesis of several pro-inflammatory lipid mediators (prostaglandins, leukotrienes, platelet activating factor) by cleaving membrane phospholipids at the sn-2 position into arachidonic acid and lysophospholipids1,2. FAAH is an important enzyme of the endocannabinoid metabolism3,4. It hydrolyzes the endocannabinoid anandamide into arachidonic acid and ethanolamine thus terminating its anti-inflammatory and analgesic activity. For these reasons, inhibitors of cPLA₂α5–7 and FAAH8,9 are expected to be new treatment options for inflammatory diseases.

We have found that certain carboxy-substituted 1-heteroarylpropan-2-ones like compounds 1–3 (Figure 1) inhibit cPLA₂α and/or FAAH with high efficacy10–14.

Figure 1. Indole- and indazole-5-carboxylic acid inhibitors of cPLA₂α and/or FAAH.

Pharmacokinetic experiments with the indole-5-carboxylic acid derivative 2 (Figure 1) revealed an excessive biliary excretion of the compound in form of its carboxylic acid glucuronide12. A similar in vivo fate was observed for the indazole-5-carboxylic acid 415. The rapid glucuronidation and biliary excretion appeared to be one important factor for the very low plasma levels found for 2 and 4 after peroral and intravenous administration, respectively. Therefore, this type of compound is not suitable for a systemic application.

One possibility to prevent glucuronidation of a drug is the replacement of the scissile moiety by bioisosteric non-glucuronidable functionalities16. Recently, we have examined the substitution of the carboxylic acid group of 1 by carboxamide, sulfonamide, oxadiazole, thiazolidinedione and tetrazole residues11. In the present study, we extended this bioisosteric approach by exchange of the carboxylic acid moiety of the potent cPLA₂α and FAAH inhibitor 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazole-5-carboxylic acid (8) (Scheme 1) with inverse amides, carbamates, ureas and sulfonylamides.

Methods

Chemistry

General

Column chromatography was performed on silica gel 60, particle size 0.040–0.063 mm (Macherey & Nagel Düren, Germany). Melting points were determined on a Büchi B-540 apparatus and are uncorrected (Büchi Inc., Flawil, Switzerland). ¹H NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer (400 MHz), a Bruker AV300 spectrometer (300 MHz), a Bruker AV400 Spectrometer (400 MHz) (Bruker Daltronic, Bremen, Germany) or an Agilent VNMRS-600 spectrometer (600 MHz) (Agilent Technologies, Richardson, TX). ¹³C-NMR spectra were measured on a Varian Mercury Plus 400 spectrometer (101 MHz) or an Agilent VNMRS-600 spectrometer (151 MHz) (Agilent Technologies, Richardson, TX). Electron ionization (EI) mass spectra (MS) were obtained on a Finnigan GCQ apparatus (Finnigan Corporation, San Jose, CA). Electrospray ionization (ESI) MS were measured with a Shimadzu LCMS-2020 single quadrupole mass spectrometer (Shimadzu Corporation, Tokyo, Japan). High resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II spectrometer (Bruker Daltronic, Bremen, Germany) using ESI or atmospheric pressure chemical ionization (APCI). For preparative reversed phase HPLC a RP18 Eurospher II 5 μm column (20 mm inside diameter × 250 mm) (Knauer, Berlin, Germany) protected with a RP18 Eurospher II 5 μm guard column (20 mm inside diameter × 30 mm) was used. Elution was performed in isocratic mode using acetonitrile/water as mobile phase (composition in dependence of the lipophilicity of the substance). The compounds were dissolved in DMSO and the injected sample volume was 0.5–1 mL. The flow rate was 25 mL/min and detection was carried out at 254 nm. The substances were obtained after distilling off the organic solvent and freeze-drying the remaining aqueous phase. The purity of the target compounds was assessed by reversed phase HPLC on a Nucleosil 100RP18 3 μm column (3 mm inside diameter × 125 mm) (Macherey & Nagel, Düren, Germany) with a gradient consisting of acetonitrile/water/trifluoroacetic acid (42:58:0.1 to 86:14:0.1, v/v/v) or, in case of the amines 61 and 63, of acetonitrile/10 mM ammonium acetate, adjusted to pH 5 with formic acid (10:90 to 90:10, v/v) at a flow rate of 0.40 mL/min. UV-absorbance was measured at 254 nm. Purities of the target compounds were greater or equal 95%.

Methyl 1-[2-hydroxy-3-(4-phenoxyphenoxy)propyl]-1H-indazole-5-carboxylate (6)

A mixture of methyl 1-(oxiran-2-ylmethyl)-1H-indazol-5-carboxylate14 (5) (980 mg, 4.22 mmol), 4-phenoxyphenol (868 mg, 4.66 mmol) and 4-(dimethylamino)pyridine (525 mg, 4.30 mmol) was heated at 120 °C for 45 min. The reaction mixture was dissolved in a small amount of toluene and chromatographed on silica gel (hexane/ethyl acetate, 8:2 to 7:3 to 1:1) to give 6 as a solid (334 mg, 19%). C₂₄H₂₂N₂O₅; mp 114–116 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.88 (dd, J = 9.5 Hz and 5.9 Hz, 1H), 3.94 (s, 3H), 3.99 (dd, J = 9.5 Hz and 5.3 Hz, 1H), 4.48–4.55 (m, 1H), 4.61 (dd, J = 14.2 Hz and 6.0 Hz, 1H), 4.69 (dd, J = 14.2 Hz and 4.2 Hz, 1H), 6.83–6.88 (m, 2H), 6.90–6.98 (m, 4H), 7.01–7.09 (m, 1H), 7.27–7.33 (m, 2H), 7.49 (d, J = 8.9 Hz, 1H), 8.05 (d, J = 8.9 Hz, 1H), 8.15 (d, J = 0.9 Hz, 1H), 8.53 (s, 1H); MS (EI, 70 eV) m/z (%): 418 (10) M⁺, 233 (100).

Methyl 1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazole-5-carboxylate (7)

A solution of 6 (338 mg, 0.81 mmol) in dry CH₂Cl₂ (20 mL) was treated with Dess-Martin periodinane (517 mg, 1.22 mmol) and stirred under a nitrogen atmosphere at room temperature for 2.5 h. After addition of a mixture (1:1, v/v) of saturated aqueous sodium bicarbonate solution and 5% aqueous sodium thiosulfate solution, the reaction mixture was extracted with ethyl acetate. The organic layer was separated, and dried (Na₂SO₄). The solvent was distilled off, and the residue was purified by silica gel chromatography (hexane/ethyl acetate, 8:2 to 1:1) to give 7 as a solid (293 mg, 87%). C₂₄H₂₀N₂O₅; mp 129–132 °C; ¹H NMR (300 MHz, CDCl₃): δ 3.95 (s, 3H), 4.68 (s, 2H), 5.52 (s, 2H), 6.85–6.92 (m, 2H), 6.93–7.04 (m, 4H), 7.03–7.12 (m, 1H), 7.27–7.37 (m, 3H), 8.09 (dd, J = 8.8 Hz and 1.5 Hz, 1H), 8.19 (d, J = 1.0 Hz, 1H), 8.55 (dd, J = 1.5 Hz and 0.8 Hz, 1H); MS (EI, 70 eV) m/z (%): 416 (27) M⁺, 231 (100).

1-[2-Oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazole-5-carboxylic acid (8)

A solution of 7 (293 mg, 0.70 mmol) in ethanol (54 mL) was treated with aqueous KOH solution (10%, m/m) (18 mL) and stirred at room temperature for 14 h. The reaction mixture was acidified with dilute HCl and exhaustively extracted with CH₂Cl₂. The combined organic layers were concentrated, and the residue was chromatographed on silica gel (hexane/ethyl acetate/formic acid, 8:2:0.1 to 5:5:0.1) to give 8 as a solid (201 mg, 71%). C₂₃H₁₈N₂O₅; mp 162–164 °C; ¹H NMR (400 MHz, CDCl₃): δ 4.70 (s, 2H), 5.55 (s, 2H), 6.85–6.92 (m, 2H), 6.94–7.04 (m, 4H), 7.05–7.13 (m, 1H), 7.28–7.36 (m, 3H), 8.13 (dd, J = 8.9 Hz and 1.5 Hz, 1H), 8.23 (s, 1H), 8.64 (d, J = 1.3 Hz, 1H); MS (EI, 70 eV) m/z (%): 402 (10) M⁺, 217 (100).

1-(1H-Indazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-ol (11)

A mixture of indazole (9) (262 mg, 2.22 mmol), 2-[(4-phenoxyphenoxy)methyl]oxirane16 (10) (1.07 g, 4.42 mmol), cesium carbonate (2.17 g, 6.66 mmol) and dry DMF (10 mL) was stirred at room temperature for 23 h. Then DMF was distilled off under reduced pressure. The residue was diluted with water and exhaustively extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by silica gel chromatography (hexane/ethyl acetate, 9:1) to give 11 as a solid (470 mg, 59%). C₂₂H₂₀N₂O_3; mp 104–107 °C; ¹H-NMR (600 MHz, CDCl₃): δ 3.86 (dd, J = 9.5 Hz and 6.1 Hz, 1H), 4.00 (dd, J = 9.5 Hz, J = 5.3 Hz, 1H), 4.49–4.54 (m, 1H), 4.61 (dd, J = 14.3 Hz and 6.1 Hz, 1H), 4.68 (dd, J = 14.2 Hz and 4.1 Hz, 1H), 6.84–6.88 (m, 2H), 6.91–6.94 (m, 2H), 6.94–6.97 (m, 2H), 7.03–7.07 (m, 1H), 7.15–7.19 (m, 1H), 7.28–7.32 (m, 2H), 7.36–7.43 (m, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 8.06 (s, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 361.1547, found: 361.1610. The isomeric 1-(2H-indazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-ol was obtained as additional product during this reaction (330 mg, 41%); ¹H NMR (600 MHz, CDCl₃): δ 3.79 (dd, J = 9.5 Hz and 6.8 Hz, 1H), 4.02 (dd, J = 9.5 Hz and 5.1 Hz, 1H), 4.48–4.54 (m, 1H), 4.66 (dd, J = 13.9 Hz and 6.5 Hz, 1H), 4.78 (dd, J = 13.8 Hz and 3.2 Hz, 1H), 6.85–6.88 (m, 2H), 6.93–6.95 (m, 2H), 6.95–6.98 (m, 2H), 7.03–7.07 (m, 1H), 7.11–7.14 (m, 1H), 7.28–7.31 (m, 2H), 7.33–7.36 (m, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 8.7 Hz, 1H), 8.01 (s, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 361.1547, found: 361.1581.

1-(1H-Indazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (12)

Compound 11 (200 mg, 0.55 mmol) was oxidized by the procedure described for 7 and purified by silica gel chromatography (hexane/ethyl acetate, 9:1) followed by reversed phase HPLC (acetonitrile/water, 7:3) to afford 12 as a solid (35 mg, 18%). C₂₂H₁₈N₂O_3; mp 105–108 °C; ¹H NMR (400 MHz, CDCl₃): δ 4.63 (s, 2H), 5.45 (s, 2H), 6.82–6.87 (m, 2H), 6.94–7.01 (m, 4H), 7.05–7.10 (m, 1H), 7.21 (dd, J = 8.3 Hz and 6.8 Hz, 1H), 7.24–7.29 (m, 1H), 7.29–7.35 (m, 2H), 7.42 (dd, J = 8.5 Hz and 6.9 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 8.10 (s, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 55.7, 72.4, 108.6, 115.7, 118.0, 120.7, 121.2, 121.4, 122.9, 124.3, 127.1, 129.7, 134.7, 140.3, 151.5, 153.5, 158.0, 201.2; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 359.1390, found: 359.1401.

N-(1H-Indazol-5-yl)acetamide (14)

To a solution of indazol-5-amine (13) (400 mg, 3.0 mmol) in dry THF (15 mL) was added triethylamine (0.75 mL) followed by acetic anhydride (0.28 mL, 3.0 mmol). The mixture was stirred at room temperature for 1.5 h. Then the solvent was distilled off and the crude product recrystallized from acetone to give 14 as a solid (360 mg, 68%). C₉H₉N₃O; ¹H NMR (400 MHz, DMSO-d₆): δ 2.05 (s, 3H), 7.38 (dd, J = 8.9 Hz and 1.9 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 8.00 (s, 1H), 8.10 (d, J = 1.8 Hz, 1H), 9.91 (s_broad, 1H), 12.94 (s_broad, 1H); MS (EI, 70 eV) m/z (%): 175 (41) M⁺, 133 (100).

N-[1-(Oxiran-2-ylmethyl)-1H-indazol-5-yl)acetamide (15)

To 14 (220 mg, 1.26 mmol) and cesium carbonate (592 mg, 1.82 mmol) was added dry acetonitrile (15 mL) followed by epichlorohydrin (258 μL, 3.3 mmol). The mixture was heated under reflux for 3 h. Then the solvent was distilled off and the residue purified by chromatography on silica gel (ethyl acetate/CH₂Cl₂, 2:1) to yield 15 as a solid (113 mg, 39%). C₁₂H₁₃N₃O₂; mp 99–101 °C; ¹H NMR (400 MHz, CDCl₃): δ 2.17 (s, 3H), 2.54 (dd, J = 4.7 Hz and 2.6 Hz, 1H), 2.83 (dd, J = 4.7 Hz and 4.0 Hz, 1H), 3.30–3.39 (m, 1H), 4.39 (dd, J = 15.2 Hz and 5.5 Hz, 1H), 4.67 (dd, J = 15.1 Hz and 3.3 Hz, 1H), 7.32 (dd, J = 8.9 Hz and 1.9 Hz, 1H), 7.36–7.44 (m, 1H), 7.70 (s, 1H), 7.93 (d, J = 1.0 Hz, 1H), 7.96–8.02 (m, 1H); MS (EI, 70 eV) m/z (%): 231 (100) M⁺, 189 (63).

N-{1-[2-Hydroxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}acetamide (16)

To 15 (110 mg, 0.48 mmol), 4-phenoxyphenol (98 mg, 0.53 mmol), cesium carbonate (196 mg, 0.60 mmol) and tetrabutylammonium bromide (155 mg, 0.48 mmol) was added dry acetonitrile (15 mL). The mixture was heated under reflux for 7 h. Then the solvent was distilled off and the residue purified by chromatography on silica gel (ethyl acetate/CH₂Cl₂, 1:1) to yield 16 as a solid (147 mg, 73%). C₂₄H₂₃N₃O₄; mp 147–148 °C; ¹H NMR (400 MHz, CDCl₃): δ 2.05 (s, 3H), 3.84 (dd, J = 9.4 Hz and 6.0 Hz, 1H), 3.97 (dd, J = 9.5 Hz and 5.3 Hz, 1H), 4.45–4.53 (m, 1H), 4.57 (dd, J = 14.2 Hz and 5.9 Hz, 1H), 4.64 (dd, J = 14.2 Hz and 4.1 Hz, 1H), 6.83–6.87 (m, 2H), 6.91–6.97 (m, 4H), 7.02–7.07 (m, 1H), 7.27–7.33 (m, 3H), 7.41 (d, J = 8.9 Hz, 1H), 7.99 (d, J = 0.9 Hz, 1H), 8.02–8.04 (m, 1H); MS (EI, 70 eV) m/z (%): 417 (5) M⁺, 232 (100).

N-{1-[2-Oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}-acetamide (17)

Dry DMSO (2 mL) was treated with acetic anhydride (0.45 mL, 4.8 mmol) and stirred under a nitrogen atmosphere at room temperature for 10 min. Then a solution of 16 (50 mg, 0.12 mmol) in dry DMSO (2 mL) was added and stirring at room temperature was continued for 16 h. The reaction mixture was poured into a mixture of 5% aqueous NaHCO₃ and brine (1:1). After 10 min, the mixture was exhaustively extracted with diethyl ether. The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by chromatography on silica gel (hexane/ethyl acetate, 1:9) to yield 17 as a solid (24 mg, 48%). C₂₄H₂₁N₃O₄; mp 179–180 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 2.06 (s, 3H), 5.02 (s, 2H), 5.60 (s, 2H), 6.90–6.95 (m, 2H), 7.00 (s, 4H), 7.04–7.11 (m, 1H), 7.32–7.37 (m, 2H), 7.42 (dd, J = 1.9 Hz and 9.0 Hz, 1H), 7.51 (dt, J = 0.9 Hz and 9.0 Hz, 1H), 8.06 (d, J = 0.9 Hz, 1H), 8.12 (dd, J = 0.7 Hz and 1.9 Hz, 1H), 9.96 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 24.6, 55.7, 71.7, 110.4, 116.6, 2C, 118.1, 121.0, 121.3, 123.4, 124.3, 130.6, 133.5, 134.1, 134.2, 137.9, 150.6, 154.6, 158.5, 168.8, 201.5; MS (EI, 70 eV) m/z (%): 415 (3) M⁺, 186 (100). HRMS (ESI+) m/z [M + H]⁺calculated: 416.1605, found: 416.1605.

1-(5-Nitro-1H-indazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-ol (41)

A mixture of 5-nitroindazole (40) (1.00 g, 6.13 mmol), 2-[(4-phenoxyphenoxy)methyl]oxirane (10) (3.00 g, 12.4 mmol), cesium carbonate (6.05 g, 18.6 mmol) and dry DMF (200 mL) was stirred at room temperature for 4.5 d. Then DMF was distilled off under reduced pressure. The residue was diluted with water and exhaustively extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄) and concentrated. The residue was purified by silica gel chromatography (cyclohexane/ethyl acetate, 8:2) to give 41 as an oil (1.10 g, 44%). C₂₂H₁₉N₃O₅; ¹H NMR (400 MHz, CDCl₃): δ 3.93 (dd, J = 9.6 Hz and 5.5 Hz, 1H), 3.98 (dd, J = 9.6 Hz and 5.5 Hz, 1H), 4.50–4.58 (m, 1H), 4.65 (dd, J = 14.3 Hz and 6.1 Hz, 1H), 4.73 (dd, J = 14.3 Hz and 4.1 Hz, 1H), 6.83–6.89 (m, 2H), 6.91–6.98 (m, 4H), 7.02–7.09 (m, 1H), 7.27–7.34 (m, 2H), 7.57 (d, J = 9.3 Hz, 1H), 8.26 (d, J = 0.8 Hz, 1H), 8.26 (dd, J = 9.2 Hz and 2.1 Hz, 1H), 8.71–8.76 (m, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 406.1397, found: 406.1407. The isomeric 1-(5-nitro-2H-indazol-2-yl)-3-(4-phenoxyphenoxy)propan-2-ol was obtained as additional product during this synthesis (672 mg, 27%); ¹H NMR (400 MHz, CDCl₃): δ  3.89 (dd, J = 9.5 Hz and 6.1 Hz, 1H), 4.03 (dd, J = 9.5 Hz and 5.3 Hz, 1H), 4.50 – 4.59 (m, 1H), 4.68 (dd, J = 13.9 Hz and 6.7 Hz, 1H), 4.80 (dd, J = 13.8 Hz and 3.2 Hz, 1H), 6.85–6.90 (m, 2H), 6.92–6.96 (m, 2H), 6.96–7.00 (m, 2H), 7.03–7.09 (m, 1H), 7.27 – 7.33 (m, 2H), 7.77 (d, J = 9.5 Hz, 1H), 8.14 (dd, J = 9.5 Hz and 2.2 Hz, 1H), 8.32 (d, J = 0.8 Hz, 1H), 8.75 (dd, J = 2.2 Hz and 0.7 Hz, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 406.1397, found: 406.1440.

1-{2-[(tert-Butyldimethylsilyl)oxy]-3-(4-phenoxyphenoxy)-propyl}-5-nitro-1H-indazole (42)

A solution of 41 (906 mg, 2.23 mmol) and tert-butyldimethylsilyl chloride (514 mg, 3.41 mmol) in dry THF (7 mL) was treated with imidazole (322 mg, 4.73 mmol) and stirred at room temperature for 2 d. The reaction mixture was diluted with water and extracted exhaustively with ethyl acetate. The combined organic layers were dried (Na₂SO₄) and concentrated. The crude product was purified by chromatography on silica gel (cyclohexane/ethyl acetate, 9:1 to 8:2) to give 42 as an oil (738 mg, 64%). C₂₈H₃₃N₃O₅Si. ¹H NMR (400 MHz, CDCl₃): δ − 0.47 (s, 3H), −0.05 (s, 3H), 0.71 (s, 9H), 3.91–4.02 (m, 2H), 4.48–4.62 (m, 2H), 4.65–4.78 (m, 1H), 6.86–6.91 (m, 2H), 6.93–6.97 (m, 2H), 6.97–7.01 (m, 2H), 7.03–7.08 (m, 1H), 7.28–7.34 (m, 2H), 7.56 (d, J = 9.3 Hz, 1H), 8.25 (s, 1H), 8.25 (dd, J = 9.3 Hz and 2.1 Hz, 1H), 8.73 (d, J = 2.1 Hz, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 520.2262, found: 520.2263.

1-{2-[(tert-Butyldimethylsilyl)oxy]-3-(4-phenoxyphenoxy)-propyl}-1H-indazol-5-amine (43)

A solution of 42 (738 mg 1.42 mmol) in dry THF (12 mL) was treated with palladium (10%) on charcoal (284 mg) and stirred under a balloon filled with H₂ at room temperature for 5 h. The reaction mixture was filtered through Celite and the filtrate was evaporated to dryness to yield 43 as an oil (687 mg, 99%). C₂₈H₃₅N₃O₃Si; ¹H NMR (400 MHz, CDCl₃): δ − 0.32 (s, 3H), −0.04 (s, 3H), 0.79 (s, 9H), 3.85–3.97 (m, 2H), 4.36–4.45 (m, 1H), 4.49–4.61 (m, 2H), 6.83–6.90 (m, 3H), 6.92–6.98 (m, 5H), 7.01–7.07 (m, 1H), 7.27–7.33 (m, 3H), 7.82 (s, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 490.2520, found: 490.2516.

Phenyl N-(1-{2-[(tert-butyldimethylsilyl)oxy]-3-(4-phenoxyphenoxy)propyl}-1H-indazol-5-yl)carbamate (44)

Phenyl chloroformate (47 mg, 0.30 mmol) was added to a solution of 43 (139 mg, 0.28 mmol) in dry THF (10 mL). After stirring at room temperature for 1 h, the mixture was concentrated and the residue chromatographed on silica gel (cyclohexane/ethyl acetate, 7:3) to give 44 as a solid (163 mg, 95%). C₃₅H₃₉N₃O₅Si; mp 112–115 °C; ¹H NMR (600 MHz, CDCl₃): δ − 0.38 (s, 3H), −0.05 (s, 3H), 0.76 (s, 9H), 3.91 (dd, J = 9.6 Hz and 5.2 Hz, 1H), 3.94 (dd, J = 9.5 Hz and 5.2 Hz, 1H), 4.46 (dd, J = 14.1 Hz and 7.5 Hz, 1H), 4.51–4.58 (m, 1H), 4.64 (dd, J = 14.1 Hz and 4.4 Hz, 1H), 6.85–6.88 (m, 2H), 6.93–6.95 (m, 2H), 6.96–6.98 (m, 2H), 7.03–7.06 (m, 1H), 7.20–7.23 (m, 2H), 7.23–7.26 (m, 1H), 7.28–7.32 (m, 2H), 7.35 (dd, J = 9.0 Hz and 2.0 Hz, 1H), 7.38–7.42 (m, 2H), 7.45 (d, J = 8.9 Hz, 1H), 7.91 (s_broad, 1H), 7.98 (s, 1H); MS (ESI) m/z: 610.25 [M + H]⁺.

Phenyl N-{1-[2-hydroxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}carbamate (45)

To 44 (150 mg, 0.25 mmol) and a catalytic amount of copper(II)chloride dihydrate was added acetone (4.75 mL) and water (0.25 mL). The mixture was refluxed for 40 h. After cooling, the reaction mixture was diluted with brine and saturated aqueous NaHCO₃ solution and extracted with ethyl acetate. The organic layer was dried (Na₂SO₄) and concentrated. The crude product was purified by chromatography on silica gel (cyclohexane/ethyl acetate, 6:4) to give 45 as a solid (97 mg, 80%). C₂₉H₂₅N₃O₅; mp 162–165 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.85 (dd, J = 9.5 Hz and 6.0 Hz, 1H), 3.98 (dd, J = 9.5 Hz and 5.2 Hz, 1H), 4.46–4.54 (m, 1H), 4.58 (dd, J = 14.2 Hz and 5.9 Hz, 1H), 4.65 (dd, J = 14.1 Hz and 4.1 Hz, 1H), 6.83–6.88 (m, 2H), 6.91–6.98 (m, 4H), 7.02–7.07 (m, 1H), 7.19–7.27 (m, 3H), 7.27–7.33 (m, 2H), 7.36 (dd, J = 8.9 Hz and 2.0 Hz, 1H), 7.38–7.43 (m, 2H), 7.44 (d, J = 9.0 Hz, 1H), 7.93 (s_broad, 1H), 8.00 (s, 1H); MS (ESI) m/z: 496.20 [M + H]⁺.

Phenyl N-{1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}carbamate (46)

Compound 45 (97 mg, 0.20 mmol) was oxidized by the procedure described for 7 and purified by silica gel chromatography (cyclohexane/ethyl acetate, 7:3) to give 46 as a solid (53 mg, 54%). C₂₉H₂₃N₃O₅; mp 165–166 °C; ¹H NMR (600 MHz, CDCl₃): δ 4.63 (s, 2H), 5.45 (s, 2H), 6.82–6.89 (m, 2H), 6.94–6.97 (m, 2H), 6.97–7.01 (m, 2H), 7.04–7.10 (m, 1H), 7.19–7.25 (m, 4H), 7.29–7.34 (m, 2H), 7.37–7.44 (m, 3H), 7.95 (s_broad, 1H), 8.05 (s, 1H); ¹³C NMR (151 MHz, CDCl₃): δ 56.0, 72.5, 109.3, 111.1, 115.8, 118.0, 120.9, 121.0, 121.8, 123.0, 124.7, 125.9, 129.6, 129.9, 131.5, 134.7, 137.9, 150.7, 151.7, 152.2, 153.6, 158.1 and 201.2; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 494.1710, found: 494.1719.

1-(1-{2-[(tert-Butyldimethylsilyl)oxy]-3-(4-phenoxyphenoxy)-propyl}-1H-indazol-5-yl)-3-phenylurea (47)

To a solution of 43 (200 mg, 0.41 mmol) in dry THF (4 mL) was slowly added with stirring at room temperature phenyl isocyanate (48 mg, 0.40 mmol). After 30 min, the solvent was distilled off and the residue chromatographed on silica gel (cyclohexane/ethyl acetate, 8:2) to give 47 as a solid (222 mg, 91%). C₃₅H₄₀N₄O₄Si; mp 183–185 °C; ¹H NMR (400 MHz, CDCl₃): δ − 0.38 (s, 3H), −0.05 (s, 3H), 0.75 (s, 9H), 3.86–3.97 (m, 2H), 4.39–4.68 (m, 3H), 6.84–6.88 (m, 2H), 6.92–6.98 (m, 4H), 7.02–7.11 (m, 2H), 7.27–7.36 (m, 7H), 7.43 (d, J = 8.5 Hz, 1H), 7.74 (s, 1H), 7.95 (s, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 609.2892, found: 609.2929.

1-{1-[2-Hydroxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}-3-phenylurea (48)

The procedure described for the preparation of 45 was applied to 47 (222 mg, 0.36 mmol). The reaction time was 23 h. Purification by silica gel chromatography (cyclohexane/ethyl acetate, 1:1, followed by ethyl acetate and ethyl acetate/methanol, 8:2) gave 48 as a solid (163 mg, 92%). C₂₉H₂₆N₄O₄; mp 196–199 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 3.86 (dd, J = 10.0 Hz and 5.5 Hz, 1H), 3.94 (dd, J = 10.0 Hz and 4.7 Hz, 1H), 4.22–4.32 (m, J = 5.3 Hz, 1H), 4.44 (dd, J = 14.2 Hz and 6.6 Hz, 1H), 4.55 (dd, J = 14.2 Hz and 5.3 Hz, 1H), 5.39 (d, J = 5.4 Hz, 1H), 6.89–6.93 (m, 2H), 6.93–7.01 (m, 5H), 7.02–7.09 (m, 1H), 7.24–7.30 (m, 2H), 7.30–7.37 (m, 3H), 7.44–7.49 (m, 2H), 7.56–7.60 (m, 1H), 7.89 (dd, J = 2.0 Hz and 0.7 Hz, 1H), 7.99 (d, J = 0.9 Hz, 1H), 8.71 (s_broad, 2H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 495.2027, found: 495.2040.

1-{1-[2-Oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}-3-phenylurea (49)

A solution of 48 (160 mg, 0.32 mmol) in dry DMSO (15 mL) was treated with Dess-Martin periodinane (207 mg, 0.49 mmol) and stirred under nitrogen at room temperature for 4 h. After addition of a mixture of saturated aqueous sodium bicarbonate solution and 5% aqueous sodium thiosulfate solution (1:1, v/v), the reaction mixture was exhaustively extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄), and the solvent was distilled off. The residue was purified by silica gel chromatography (cyclohexane/ethyl acetate/methanol, 7.5:0.5) followed by preparative reversed phase chromatography (acetonitrile/water, 6:4) to give 49 as a solid (49 mg, 31%). C₂₉H₂₄N₄O₄; mp 201– 204 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 5.02 (s, 2H), 5.60 (s, 2H), 6.91–6.95 (m, 2H), 6.94–6.99 (m, 1H), 7.00 (s, 4H), 7.05–7.10 (m, 1H), 7.25–7.30 (m, 2H), 7.32–7.39 (m, 3H), 7.45–7.49 (m, 2H), 7.49–7.53 (m, 1H), 7.92 (dd, J = 1.9 Hz and 0.7 Hz, 1H), 8.04 (d, J = 1.0 Hz, 1H), 8.67 (s_broad, 2H); ¹³C NMR (101 MHz, DMSO-d₆): δ 55.0, 71.0, 108.8, 109.9, 115.9, 117.4, 118.1, 120.5, 120.6, 121.7, 122.7, 123.9, 128.8, 129.9, 133.1, 133.2, 136.9, 139.9, 150.0, 152.9, 154.0, 157.8, 200.8; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 493.1870, found: 493.1872.

1-(5-Nitro-1H-indazol-1-yl)-3-(4-phenoxyphenoxy)propan-2-one (50)

Compound 41 (958 mg, 2.36 mmol) was oxidized with Dess-Martin periodinane by the procedure described for 7 (reaction time: 3 h) and purified by silica gel chromatography (cyclohexane/ethyl acetate, 8:2) to give 50 as a solid (679 mg, 71%). C₂₂H₁₇N₃O₅; mp 85–87 °C; ¹H NMR (600 MHz, CDCl₃): δ 4.72 (s, 2H), 5.59 (s, 2H), 6.89–6.93 (m, 2H), 6.96–6.99 (m, 2H), 7.01–7.04 (m, 2H), 7.08–7.11 (m, 1H), 7.29 (d, J = 9.2 Hz, 1H), 7.31–7.35 (m, 2H), 8.28–8.30 (m, 2H), 8.76 (d, J = 2.1 Hz, 1H); ¹³C NMR (151 MHz, CDCl₃): δ 56.3, 72.8, 109.3, 115.8, 118.3, 119.3, 120.9, 122.2, 123.2, 123.7, 129.9, 137.2, 142.4, 142.9, 152.1, 153.3, 157.9, 200.3; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 404.1241, found: 404.1242.

1-[2,2-Diethoxy-3-(4-phenoxyphenoxy)propyl]-5-nitro-1H-indazole (51)

A solution of 50 (920 mg 2.28 mmol) in dry ethanol (52 mL) was treated with triethyl orthoformate (4 mL, 24 mmol) followed by conc. H₂SO₄ (8 drops) and heated under reflux for 12 h. The reaction mixture was slowly poured into a saturated aqueous NaHCO₃ solution and extracted with ethyl acetate. The organic phase was dried (Na₂SO₄) and concentrated. The residue was chromatographed on silica gel (cyclohexane/ethyl acetate, 9:1) to give 51 as an oil (588 mg, 54%). C₂₆H₂₇N₃O₆; ¹H NMR (400 MHz, CDCl₃): δ 1.24 (t, J = 7.0 Hz, 6H), 3.61–3.72 (m, 2H), 3.70 (s, 2H), 3.86–3.95 (m, 2H), 4.78 (s, 2H), 6.76–6.84 (m, 2H), 6.92–6.98 (m, 4H), 7.03–7.09 (m, 1H), 7.30–7.35 (m, 2H), 7.52 (d, J = 9.3 Hz, 1H), 8.04 (dd, J = 9.3 Hz and 2.1 Hz, 1H), 8.18–8.22 (m, 1H), 8.67 (dd, J = 2.1 Hz and 0.6 Hz, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 478.1973, found: 478.1942.

1-[2,2-Diethoxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-amine (52)

To compound 51 (588 mg, 1.23 mmol) and palladium (10%) on charcoal (246 mg) was added THF (15 mL). The mixture was stirred under a balloon filled with H₂ at room temperature for 5.5 h. After addition of Celite, the mixture was filtered and the filtrate evaporated to dryness to yield 52 as an oil (522 mg, 95%). C₂₆H₂₉N₃O₄; ¹H NMR (400 MHz, CDCl₃): δ 1.24 (t, J = 7.0 Hz, 6H), 3.65 (dq, J = 8.9 Hz and 7.0 Hz, 2H), 3.72 (s, 2H), 3.93 (dq, J = 8.9 Hz and 7.0 Hz, 2H), 4.66 (s, 2H), 6.70 (dd, J = 8.9 Hz and 2.1 Hz, 1H), 6.82–6.86 (m, 2H), 6.93–6.97 (m, 4H), 7.01 (dd, J = 2.1 Hz and 0.7 Hz, 1H), 7.02–7.07 (m, 1H), 7.27–7.33 (m, 3H), 7.81–7.83 (m, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 448.2231, found: 448.2207.

1-{1-[2,2-Diethoxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}-3-ethylurea (53)

To a solution of 52 (317 mg 0.71 mmol) in dry THF (20 mL) was added with stirring at room temperature ethyl isocyanate (52 mg, 0.73 mmol). After 24 h, water was added and the mixture extracted with ethyl acetate. The organic layer was separated, dried (Na₂SO₄), and concentrated. The residue was purified by silica gel chromatography (cyclohexane/ethyl acetate, 7:3) to give 52 as a solid (284 mg, 77%). C₂₉H₃₄N₄O₅; mp 117–119 °C; ¹H NMR (400 MHz, CDCl₃): δ 1.04 (t, J = 7.2 Hz, 3H), 1.13 (t, J = 7.0 Hz, 6H), 3.10 (qd, J = 7.2 Hz and 5.6 Hz, 2H), 3.58 (dq, J = 9.1 Hz and 7.0 Hz, 2H), 3.69 (s, 2H), 3.84 (dq, J = 9.1 Hz and 7.0 Hz, 2H), 4.63 (s, 2H), 6.02 (t_broad, J = 5.6 Hz, 1H), 6.90–6.94 (m, 2H), 6.94–6.97 (m, 2H), 6.98–7.03 (m, 2H), 7.05–7.10 (m, 2H), 7.33–7.40 (m, 3H), 7.76 (dd, J = 2.0 Hz and 0.7 Hz, 1H), 7.95 (d, J = 0.9 Hz, 1H), 8.33 (s_broad, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 519.2602, found: 519.2659.

1-Ethyl-3-{1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}urea (54)

A solution of 53 (100 mg, 0.19 mmol) in a mixture of acetone (5 mL) and water (0.5 mL) was treated with conc. H₂SO₄ (0.5 mL) and stirred at room temperature for 2 d. The reaction mixture was carefully poured into a saturated aqueous NaHCO₃ solution and extracted with ethyl acetate. The organic phase was dried (Na₂SO₄) and concentrated. The residue was chromatographed on silica gel (cyclohexane/ethyl acetate, 3:7) to give 54 as a solid (46 mg, 54%). C₂₅H₂₄N₄O₄; mp 183–185 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 1.05 (t, J = 7.2 Hz, 3H), 3.10 (qd, J = 7.2 Hz and 5.5 Hz, 2H), 5.00 (s, 2H), 5.56 (s, 2H), 6.50 (t_broad, J = 5.6 Hz, 1H), 6.91–6.95 (m, 2H), 6.96–7.02 (m, 4H), 7.05–7.10 (m, 1H), 7.29–7.38 (m, 3H), 7.41–7.45 (m, 1H), 7.86 (dd, J = 1.9 Hz and 0.7 Hz, 1H), 7.97 (d, J = 0.9 Hz, 1H), 8.79 (s_broad, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 16.0, 34.4, 55.4, 71.4, 108.0, 110.0, 116.3, 117.9, 120.6, 121.0, 123.1, 124.4, 130.3, 133.4, 134.8, 136.9, 150.4, 154.4, 156.2, 158.2 and 201.3; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 445.1870, found: 445.1881.

1-Cyclohexyl-3-{1-[2,2-diethoxy-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}urea (55)

To a solution of 52 (411 mg, 0.92 mmol) in dry THF (10 mL) was added with stirring at room temperature cyclohexyl isocyanate (121 mg, 0.97 mmol). After 5 d, water was added and the mixture extracted with ethyl acetate. The organic layer was separated, dried (Na₂SO₄), and concentrated. The residue was purified by silica gel chromatography (cyclohexane/ethyl acetate, 7:3) to give 55 as a solid (428 mg, 81%). C₃₃H₄₀N₄O₅; mp 122–125 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 1.13 (t, J = 7.0 Hz, 6H), 1.18–1.34 (m, 5H), 1.50–1.56 (m, 1H), 1.63–1.69 (m, 2H), 1.76–1.83 (m, 2H), 3.42–3.50 (m, 1H), 3.58 (dq, J = 9.2 Hz and 7.0 Hz, 2H), 3.68 (s, 2H), 3.84 (dq, J = 9.2 Hz and 7.1 Hz, 2H), 4.63 (s, 2H), 5.97 (d, J = 7.9 Hz, 1H), 6.90–6.93 (m, 2H), 6.94–6.97 (m, 2H), 6.98–7.02 (m, 2H), 7.05–7.07 (m, 1H), 7.07–7.08 (m, 1H), 7.34–7.37 (m, 2H), 7.37–7.40 (m, 1H), 7.74 (dd, J = 1.9 Hz and 0.7 Hz, 1H), 7.94 (d, J = 0.9 Hz, 1H), 8.22 (s, 1H); HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 573.307, found: 573.3131.

1-Cyclohexyl-3-{1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazol-5-yl}urea (56)

Compound 55 (70 mg, 0.12 mmol) was treated with ethanol (5 mL), water (5 mL), THF (5 mL) and acetone (5 mL). Then conc. H₂SO₄ (0.5 mL) was added, and the solution was stirred at room temperature for 4 d. The reaction mixture was carefully poured into a saturated aqueous NaHCO₃ solution and extracted with ethyl acetate. The organic phase was dried (Na₂SO₄) and concentrated. The residue was chromatographed on silica gel (cyclohexane/ethyl acetate, 4:6) to give 56 as a solid (20 mg, 33%). C₂₉H₃₀N₄O₄; mp 196–198 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 1.11–1.37 (m, 5H), 1.49–1.58 (m, 1H), 1.62–1.71 (m, 2H), 1.77–1.86 (m, 2H), 3.42–3.53 (m, 1H), 5.00 (s, 2H), 5.56 (s, 2H), 6.02 (d, J = 7.9 Hz, 1H), 6.90–6.94 (m, 2H), 6.96–7.02 (m, 4H), 7.05–7.10 (m, 1H), 7.27 (dd, J = 9.0 Hz and 2.0 Hz, 1H), 7.32–7.38 (m, 2H), 7.44 (dd, J = 9.0 Hz and 0.9 Hz, 1H), 7.81–7.87 (m, 1H), 7.98 (d, J = 0.9 Hz, 1H), 8.27 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 24.8, 25.7, 33.5, 48.1, 55.4, 71.4, 108.0, 110.2, 116.3, 117.9, 120.4, 121.0, 123.1, 124.4, 130.3, 133.5, 134.5, 137.0, 150.4, 154.4, 155.3, 158.2, 201.3; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 499.2340, found: 499.2330.

N-(Methylsulfonyl)-1-[2-oxo-3-(4-phenoxyphenoxy)propyl]-1H-indazole-5-carboxamide (57)

A solution of 8 (70 mg, 0.17 mmol), 4-(dimethylamino)pyridine (46 mg, 0.38 mmol) and methane sulfonamide (37 mg, 0.39 mmol) in dry CH₂Cl₂ (1.5 mL) was treated at 0 °C with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (68 mg, 0.35 mmol) and stirred at room temperature for 42 h. Then the solvent was distilled off and the residue chromatographed on silica gel (hexane/ethyl acetate/formic acid, 7:3:0.2). The obtained product was further purified by preparative reversed phase HPLC (acetonitrile/water/trifluoroacetic acid, 55:45:0.1) to give 57 as a solid (9 mg, 11%). C₂₄H₂₁N₃O₆S; mp 199–201 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 5.04 (s, 2H), 5.68 (s, 2H), 6.88–6.92 (m, 2H), 6.98 (s, 4H), 7.03–7.07 (m, 1H), 7.29–7.35 (m, 2H), 7.65 (d, J = 8.9 Hz, 1H), 7.92 (dd, J = 8.9 Hz and 1.6 Hz, 1H), 8.25–8.31 (m, 1H), 8.44–8.48 (m, 1H), 12.07 (s_broad, 1H); ¹³C NMR (151 MHz, DMSO-d₆): δ 41.3, 55.2, 71.0, 109.8, 115.9, 117.4, 120.3, 122.7, 123.2, 123.2, 125.4, 126.1, 129.9, 135.5, 141.9, 150.0, 153.9, 157.8, 166.9, 200.4; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 480.1224, found: 480.1247.

1-[2-Oxo-3-(4-phenoxyphenoxy)propyl]-N-(phenylsulfonyl)-1H-indazole-5-carboxamide (58)

Compound 8 (50 mg, 0.12 mmol) was reacted with benzene sulfonamide (48 mg, 0.31 mmol) under the conditions described for the synthesis of 57. Purification by silica gel chromatography (hexane/ethyl acetate/formic acid, 9:1:0.2 to 7:3:0.2) followed by preparative reversed phase HPLC (acetonitrile/water/trifluoroacetic acid, 60:40:0.1) gave 58 as a solid (9 mg, 14%). C₂₉H₂₃N₃O₆S; mp 108–110 °C; ¹H NMR (400 MHz, DMSO-d₆): δ 5.05 (s, 2H), 5.70 (s, 2H), 6.90–6.94 (m, 2H), 7.00 (s, 4H), 7.04–7.11 (m, 1H), 7.31–7.38 (m, 2H), 7.59–7.73 (m, 4H), 7.84 (dd, J = 8.9 Hz and 1.6 Hz, 1H), 7.97–8.06 (m, 2H), 8.30 (d, J = 0.9 Hz, 1H), 8.45 (dd, J = 1.6 Hz and 0.8 Hz, 1H), 12.52 (s_broad, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 55.2, 71.0, 110.0, 115.9), 117.4, 120.6, 122.7, 123.1, 123.3, 124.8, 126.0, 127.6, 129.9, 133.3, 135.5, 140.2, 141.9, 150.0, 153.9, 157.8, 165.8, 200.4; HRMS (APCI, direct probe) m/z [M + H]⁺calculated: 542.1380, found: 542.1419.

1-[2-Oxo-3-(4-phenoxyphenoxy)propyl]-N-[(trifluoromethyl)sulfonyl]-1H-indazole-5-carboxamide (59)

Compound 8 (77 mg, 0.19 mmol) was reacted with trifluoromethanesulfonyl chloride (60 mg, 0.40 mmol) under the conditions described for the synthesis of 57. Purification by silica gel chromatography (hexane/ethyl acetate/formic acid, 9:1:0.2 to 7:3:0.2) gave 59 as a solid (70 mg, 69%). C₂₄H₁₈F₃N₃O₆S; mp 170–172 °C; ¹H NMR (600 MHz, DMSO-d₆): δ 5.04 (s, 2H), 5.65 (s, 2H), 6.91–6.95 (m, 2H), 7.00 (s, 4H), 7.05–7.10 (m, 1H), 7.32–7.38 (m, 2H), 7.52 (d, J = 8.8 Hz, 1H), 7.97 (dd, J = 8.8 Hz and 1.5 Hz, 1H), 8.23 (s, 1H), 8.40 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆): δ 55.1, 71.1, 108.8, 115.9, 117.4, 120.6, 122.4, 122.7, 123.4, 126.9, 129.9, 130.5, 135.1, 141.5, 150.0, 154.0, 157.8, 169.7, 200.6; HRMS (APCI, direct probe) m/z [M + H]⁺ calculated for C₁₉H₁₇N₅O₂: 534.0941, found: 534.0966.

Supplementary material includes the syntheses and analytical data of compounds 21, 29, 33, 37–39, and their intermediates.

Biological evaluation

Inhibition of cytosolic phospholipase A₂α (cPLA₂α)

Inhibition of cPLA₂α was measured according to a recently published procedure¹⁸. Briefly, cPLA₂α isolated from porcine platelets was incubated with co-vesicles consisting of the substrate 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (200 μM) and 1,2-dioleoyl-sn-glycerol (100 μM). Enzyme reactions were terminated after 60 min and cPLA₂α activity was determined by measuring the arachidonic acid released by the enzyme in absence and presence of a test compound with reversed phase HPLC and UV-detection at 200 nm after on-line solid phase extraction. The stock solution of compound 13 in DMSO was prepared freshly each time because of long-term instability of this compound in this solvent.

Inhibition of fatty acid amide hydrolase (FAAH)

Inhibition of FAAH was measured as described¹⁹. Briefly, the substrate N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide²⁰ (100 μM) solubilized with Triton X-100 (0.2%) was incubated with rat brain microsomes. Enzyme reactions were terminated after 60 min. FAAH activity was determined directly without further sample cleanup by measuring the amount of 4-pyren-1-ylbutanoic acid released by FAAH in absence and presence of a test compound with reversed phase HPLC and fluorescence-detection. The stock solution of compound 13 in DMSO was prepared freshly each time because of long term instability of this compound in this solvent.

Results and discussion

Chemistry

The lead compound 8 was prepared as outlined in Scheme 1. The epoxide ring of 5¹⁴ was opened by solvent free heating with 4-phenoxyphenol and 4-(dimethylamino)pyridine to give the hydroxy intermediate 6. Subsequent oxidation of the alcohol group of this compound with Dess-Martin reagent afforded the keto-ester 7. Upon deprotection of the ester group with aqueous KOH in ethanol at room temperature the target compound 8 was obtained.

The derivative of 8 lacking the carboxy group (12) was synthesized by reaction of indazole (9) with 2-[(4-phenoxyphenoxy)methyl]oxirane (10)17 in DMF in presence of Cs₂CO₃ followed by Dess-Martin oxidation of the formed alcohol intermediate 11 (Scheme 2).

Scheme 3 shows the syntheses of the different inverse amide indazole derivatives 17, 21, 29, 33, 38 and 39, as well as of the indazole methyl carbamate 37. The preparations started from indazole-5-amine (13), which was first acylated and carbamylated, respectively, with the appropriate anhydride, acyl halogenide or chloroformate to yield the indazole derivatives 14, 18, 22, 26, 30 and 34. Then an oxiranyl methyl substituent was introduced in position 1 of the indazole heterocycle by reaction with epichlorohydrin either in DMF or in acetonitrile using Cs₂CO₃ as base. Reaction of obtained compounds 15, 19, 23, 27, 31 and 35 with 4-phenoxyphenol in DMF or in acetonitrile in presence of DABCO, Cs₂CO₃ or 4-(dimethylamino)pyridine resulted in the opening of the epoxide ring with formation of a hydroxy moiety. The target compounds 17, 21, 29, 33 and 37 were directly obtained from 16, 20, 28, 32 and 36 by Dess-Martin or Albright-Goldman oxidation of the alcohol group of these intermediates. For the synthesis of the benzamide derivative 38, the hydroxy substituent of the BOC-protected amine derivative 24 was oxidized to a ketone with Dess-Martin reagent. Then the BOC group was removed by treatment with trifluoroacetic acid and the resulting amine 25 acylated with benzoyl chloride to give the desired compound 38. The hydroxyacetamide 39 was prepared by hydrogenolytic cleavage of the benzylether group of the benzyloxyacetamide 33.

The phenyl carbamate 46 and the phenyl urea 49 were prepared by the route outlined in Scheme 4. First, 5-nitroindazole (40) was reacted with 2-[(4-phenoxyphenoxy)methyl]oxirane (10) to give the alcohol 41. The hydroxy group of this compound was protected by treatment with tert-butyldimethylsilyl chloride to yield 42. Reduction of the nitro substituent of 42 by catalytic hydrogenation with Pd/C gave the amine 43, which was reacted with either phenyl chloroformate or phenyl isocyanate to afford the phenyl carbamate derivative 44 and the phenyl urea derivative 47, respectively. The tert-butyldimethylsilyl protecting group of these compounds was removed by treatment with CuCl₂ in water/acetone to give the hydroxy derivatives 45 and 48. Finally, Dess-Martin oxidation of the hydroxy moieties of these intermediates led to the desired 2-oxopropyl-substitured phenyl carbamate and phenyl urea indazoles 46 and 49.

The corresponding ethyl and cyclohexyl urea derivatives could not be obtained via this route, because in the final oxidation step the substances degraded. Therefore, a new synthetic approach had to be developed, which started from the 5-nitroindazolyl-substituted propan-2-ol 41 (Scheme 5). This was oxidized to the ketone 50 with Dess-Martin reagent. Next, the ketone group was acetalized with orthoformic acid triethyl ester in the presence of catalytic amounts of H₂SO₄ to afford 51. Catalytic hydrogenation of the nitro group of this compound using Pd/C as catalyst led to the amine 52, which was converted to the ethyl and cyclohexyl urea derivatives 53 and 55 by reaction with ethyl and cyclohexyl isocyanate, respectively. Finally, the acetal protecting group was removed by H₂SO₄ in organic-aqueous solutions to yield the target compounds 54 and 56.

The N-methylsulfonyl-, N-phenylsulfonyl- and N-trifluoromethylsulfonyl-substituted indazole-carboxamides 57–59 were prepared by coupling indazole-5-carboxylic acid 8 with methanesulfonamide, benzenesulfonamide and trifluoromethanesulfonamide in CH₂Cl₂ in presence of 1-ethyl-3–(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 4-(dimethylamino)pyridine (Scheme 6).

Biological evaluation

Bioisosteric replacement is a well established medicinal chemistry technique. It consists of replacing one fragment in a bioactive molecule with another fragment possessing similar spatial and electronic character. Bioisosteric transformation has been extensively and successfully used in the optimization of lead candidates in drug discovery16. Because we had found that the carboxylic acid moiety in position 5 of the indole ring system of 1 has a great impact on the cPLA₂α inhibitory potency of this compound10, in an earlier study we have replaced this functionality by several bioisosteric groups like carboxamides, sulfonamides and tetrazole11. This investigation revealed that all these modifications led to a decrease of cPLA₂α inhibition. The most active bioisosteric derivative was the unsubstituted amide derivative of 1, which was fourfold less active than the lead compound. In contrast, inhibition of FAAH was influenced differently by these structural variations. While carboxamide and methylated carboxamide as well as tetrazole substituents led to a drop of activity, sulfonamide and methylated sulfonamide functionalities increased inhibitory potency against FAAH by 2–4 folds14.

As mentioned above, the carboxylic acid functionality of the indole-5-carboxylic acids and indazole-5-carboxylic acids synthesized in our group as cPLA₂α and FAAH inhibitors, such as that of compound 2 and 4, is very susceptible to metabolic glucuronidation in vivo. This is one reason for the low bioavailability of such type of compounds after peroral administration. These results prompted us to restart our investigations on bioisosteric replacement of the carboxylic acid moiety by functionalities, which are resistant to glucuronidation. Lead compound in this study was 1–(2-oxopropyl)indazole-5-carboxylic acid 8 (Scheme 1). Evaluation of an inhibition of cPLA₂α was measured by determining the amount of arachidonic acid released from a phospholipid substrate with HPLC and UV-detection at 200 nm after on-line solid-phase extraction in absence and presence of a test compound18. FAAH inhibition was determined by monitoring the cleavage of the fluorogenic substrate N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide by FAAH from rat brain with HPLC and fluorescence-detection19,20. Applying these methods, for the lead compound 8 an IC₅₀ of 0.005 μM against cPLA₂α and an IC₅₀ of 0.055 μM against FAAH was obtained (Table 1).

Scheme 1. Reagents and conditions: (a) 4-Phenoxyphenol, 4-(dimethylamino)pyridine, 120 °C, 45 min; (b) Dess-Martin periodinane, CH₂Cl₂, room temp., 2.5 h; (c) 10% aqueous KOH, ethanol, room temp., 14 h.

Scheme 2. Reagents and conditions: (a) Cs₂CO₃, DMF, room temp., 23 h; (b) Dess-Martin periodinane, CH₂Cl₂, room temp., 3 h.

Scheme 3. Reagents and conditions: (a) 15: Acetic anhydride, THF, triethylamine, room temp., 1.5 h; 18: isobutyryl chloride, pyridine, 0 °C, 2 h; 22: di-tert-butyl dicarbonate, methanol, Amberlyst® 15, room temp., 2 h; 26: 2-methoxyacetyl chloride, pyridine, 0 °C, 2 h; 30: 2-benzyloxyacetyl chloride, diisopropyl(ethyl)amine, DMF, 0 °C to room temp., 4 h; 34: methyl chloroformate, pyridine, 0 °C, 2 h; (b) 15: epichlorohydrin, Cs₂CO₃, acetonitrile, reflux, 3 h; 19, 23, 27, 31, 35: epichlorohydrin, Cs₂CO₃, DMF, room temp., 16–24 h; (c) 4-phenoxyphenol, 16, 32: Cs₂CO₃, tetrabutylammonium bromide, acetonitrile, reflux, 4–7 h; 20: 4-(dimethylamino)pyridine, DABCO, DMF, 120 °C, 1 h; 24: DABCO, DMF, 120 °C, 6 h; 28: DABCO, 4-(dimethylamino)pyridine, DMF, 120 °C, 4 h, 36: 4-(dimethylamino)-pyridine, 120 °C, 2 h; (d) 17, 21, 29, 37: acetic anhydride, DMSO, room temp., 12–16 h, 25: Dess-Martin periodinane, CH₂Cl₂, room temp., 4.5 h followed by trifluoroacetic acid, CH₂Cl₂, room temp., 18 h, 33: Dess-Martin periodinane, CH₂Cl₂, room temp., 3 h; (e) benzoyl chloride, ethyl acetate, pyridine, room temp., 2 h; (f) H₂, Pd/C, THF, room temp., 4 d.

Scheme 4. Reagents and conditions: (a) 2-[(4-Phenoxyphenoxy)methyl]oxirane, Cs₂CO₃, DMF, room temp., 72 h; (b) tert-butyldimethylsilyl chloride, imidazole, THF, room temp., 2 d; (c) H₂, Pd/C, THF, 5 h; (d) 44: phenyl chloroformate, THF, room temp., 1 h; 47: phenyl isocyanate, THF, room temp., 30 min; (e) CuCl₂ dihydrate, acetone, H₂O, reflux, 40 h (45) or 8 h (48); (f) Dess-Martin periodinane, CH₂Cl₂, room temp., 1.5 h (48) or 4 h (49).

Scheme 5. Reagents and conditions: (a) Dess-Martin periodinane, CH₂Cl₂, room temp., 4 h; (b) triethyl orthoformate, H₂SO₄ conc., ethanol, reflux, 12 h; (c) H₂, Pd/C, THF, 5.5 h; (d) 53: ethyl isocyanate, THF, room temp., 1 d, 55: cyclohexyl isocyanate, THF, room temp., 6 d; (e) 54: H₂O, acetone, water, H₂SO₄ conc., room temp., 2 d, 56: ethanol, acetone, THF, water, H₂SO₄ conc., room temp., 4 d.

Scheme 6. Reagents and conditions: (a) Methanesulfonamide (57), benzenesulfonamide (58) or trifluoromethanesulfonamide (59), 4-(dimethylamino)pyridine, CH₂Cl₂, EDC, room temp., 20–42 h.

Table 1. Inhibition of cPLA₂α and FAAH.

Comp.	R	Inhibition of cPLA2α IC50 (μM)*	Inhibition of FAAH IC50 (μM)*
8	COOH	0.005	0.055
12	H	6.3	2.6
17	NHCOCH3	0.13	3.9
21	NHCOCH(CH3)2	n.a.	>10^¶
38	NHCOPhenyl	n.a.	>10^‡
29	NHCOCH2OCH3	0.59	6.3
39	NHCOCH2OH	0.66	5.4
33	NHCOCH2OCH2Phenyl	2.3	>10^§
37	NHCOOCH3	2.1	4.0
46	NHCOOPhenyl	>10†	2.1
50	NO2	0.55	0.55
54	NHCONHC2H5	n.a.	1.6
56	NHCONHCyclohexyl	n.a.	0.84
49	NHCONHPhenyl	n.a.	6.0
57	CONHSO2CH3	1.6	0.77
58	CONHSO2Phenyl	1.9	4.7
59	CONHSO2CF3	0.29	0.51

*Values are the means of at least two independent determinations, errors of the IC₅₀ values are within ± 20%; inhibition value of cPLA₂α reference inhibitor Axon 160921 (CAS 479422–22-5): IC₅₀=0.21 μM; inhibition value of FAAH reference inhibitor URB59722 (CAS 546141-08-6): IC₅₀=0.060 μM.

† 44% inhibition at 10 μM.

‡ 44% inhibition at 10 μM.

¶ 26% inhibition at 10 μM.

§ 37% inhibition at 10 μM.

For quantification of the impact of the carboxylic acid group of 8 on enzyme inhibition, first in 5-position unsubstituted indazole 12 was synthesized and tested. The results obtained again highlight the important role of the acidic functionality for activity. With IC₅₀ values of 6.3 μM and 2.6 μM against cPLA₂α and FAAH, respectively, 12 was a 1000-fold weaker inhibitor of cPLA₂α and a 50-fold weaker inhibitor of FAAH in comparison with the lead 8 (Table 1).

Next, we replaced the carboxylic acid group of 8 by different inverse amide functionalities. The most active cPLA₂α inhibitor of this series was the acetylamino substituted indazole 17. Although it was about 25-fold less active than the carboxylic acid 8, it still possessed a considerable activity against the enzyme (IC₅₀: 0.13 μM). Exchange of the terminal methyl group of the acetylamino moiety by a more bulky lipophilic isopropyl (21) or phenyl residue (38) rendered the compound inactive at the highest test concentration (10 μM). In contrast, more polar substituents at the acetylamino residue, such as hydroxy, methoxy and benzyloxy were still tolerated by cPLA₂α. The IC₅₀ values of the methoxy- and hydroxy-substituted derivatives 29 and 39 were about 0.60 μM, for the benzyloxy-substituted compound 33 an IC₅₀ against cPLA₂α of 2.3 μM was measured. The two carbamates 37 and 46 showed only weak inhibitory activity on cPLA₂α, with the methyl derivative again being more active than the corresponding phenyl derivative. The 5-nitroindazole 50, obtained as intermediate during the synthesis of the urea-substituted compounds 54 and 56, inhibited cPLA₂α with submicromolar activity. In contrast, the urea derivatives 49, 54 and 56 were inactive against the enzyme. Besides, the effect of the replacement of the carboxylic acid group of 8 by methyl-, phenyl- and trifluoromethylsulfonyl-substituted carboxamide functionalities (57–59) was examined. This bioisosteric replacement also led to a significant decrease of cPLA₂α inhibitory potency. With an IC₅₀ of 0.29 μM the compound with terminal trifluoromethyl group (59) was still the best inhibitor of this series.

Looking at the activity of the synthesized carboxylic acid bioisosters against FAAH, the following relationships between structure and activity can be pointed out. The inverse amides were also significantly less active against FAAH in comparison to the lead 8. The order of activity on FAAH was the same as observed for cPLA₂α inhibition. With an IC₅₀ of 2.6 μM the acetylamino derivative 17 was the best of the series, followed by the derivatives with the more polar hydroxyacetylamino (39) and methoxyacetylamino (29) substituents. The inverse amides with the lowest activity were the more lipophilic isobutyl-, benzyloxy- and phenyl-substituted compounds 21, 33 and 38. The two carbamate inhibitors 37 and 46 only showed weak activity too. Conversely, as observed for cPLA₂α inhibition, the phenyl carbamate was a slightly better inhibitor of FAAH than the methyl carbamate. The 5-nitroindazole 50 inhibited FAAH with the same IC₅₀ than cPLA₂α and, therefore, can be claimed as a “well balanced” dual inhibitor of these two enzymes. Interestingly, the three urea derivatives 49, 54 and 56, which were found to be inactive against cPLA₂α, are moderate inhibitors of FAAH with IC₅₀ values in the low micromolar range. In turn, the sulfonylcarboxamide derivatives 57–59 are dual cPLA₂α/FAAH inhibitors with comparable potencies. From the three sulfonylcarboxamides investigated, the trifluoromethyl compound 59 exerts the highest activity on both, cPLA₂α and FAAH.

In this context the questions arises, whether the nonacid bioisosters, such as compound 17 or 39 could be superior to the parent carboxylic acid 8 in regard to cell permeability, because they are – in contrast to 8 – permanently uncharged under physiological conditions. However, since 5-carboxyheteroaryl-substituted propan-2-ones structurally related to 8 were found to inhibit cPLA₂α in a cellular assay with comparable potency as in an isolated enzyme assay10, permeation of cell membranes does not seem to be a problem for this type of carboxylic acids.

Conclusion

In conclusion, in contrast to many other medicinal chemistry investigations, the bioisosteric replacement performed in this study failed to increase compound activity. Substitution of the carboxylic acid group of the lead 8 by inverse amide, carbamate, urea and sulfonylcarboxamide moieties resulted in a significant drop of inhibitory potency against cPLA₂α as well against FAAH. These results again emphasize the important role of the carboxylic acid group of the compound class investigated for cPLA₂α and FAAH inhibition. The results obtained can be helpful for the development of clinical active inhibitors of these two enzymes.

Supplementary material available online

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.