Structure–activity relationship studies on 1-heteroaryl-3-phenoxypropan-2-ones acting as inhibitors of cytosolic phospholipase A₂α and fatty acid amide hydrolase: replacement of the activated ketone group by other serine traps

Abstract

Cytosolic phospholipase A₂α (cPLA₂α) and fatty acid amide hydrolase (FAAH) are serine hydrolases. cPLA₂α is involved in the generation of pro-inflammatory lipid mediators, FAAH terminates the anti-inflammatory effects of endocannabinoids. Therefore, inhibitors of these enzymes may represent new drug candidates for the treatment of inflammation. We have reported that certain 1-heteroarylpropan-2-ones are potent inhibitors of cPLA₂α and FAAH. The serine reactive ketone group of these compounds, which is crucial for enzyme inhibition, is readily metabolized resulting in inactive alcohol derivatives. In order to obtain metabolically more stable inhibitors, we replaced this moiety by α-ketoheterocyle, cyanamide and nitrile serine traps. Investigations on activity and metabolic stability of these substances revealed that in all cases an increased metabolic stability was accompanied by a loss of inhibitory potency against cPLA₂α and FAAH, respectively.

• Cytosolic phospholipase A₂α
• fatty acid amide hydrolase
• inhibitor
• metabolic stability
• serine trap

Introduction

Serine hydrolases are one of the largest families of enzymes in eukaryotes. Around 240 of such enzymes exist in humans. They can be divided into the serine proteases with about 125 members and the metabolic serine hydrolases with about 115 members1^,2. The latter enzyme group hydrolyzes ester, thioester, and amide bonds predominantly in smaller molecules, like lipids and peptides. A common feature of the serine hydrolases is the presence of a nucleophilic serine in the active site. Assisted by one or more other amino acid groups, this structural element cleaves amide or ester bonds in substrates via a covalent acyl-enzyme intermediate. Serine hydrolases play prominent roles in physiological and pathophysiological processes. Several marketed drugs are inhibitors of such enzymes, including thrombin, factor Xa, dipeptidyl peptidase IV, and acetylcholine esterase.

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, and platelet-activating factor) by cleaving membrane phospholipids at the sn-2 position into arachidonic acid and lysophospholipids3. FAAH is an important enzyme of the endocannabinoid metabolism4^,5. It hydrolyzes the endocannabinoid anandamide into arachidonic acid and ethanolamine, thus terminating its anti-inflammatory and analgesic activity. For these reasons, inhibitors of cPLA₂α6^,7 and FAAH8^,9 are expected to be new treatment options for inflammatory diseases.

We have found that certain 1-heteroarylpropan-2-ones like compounds 1–6 (Figure 1) inhibit cPLA₂α and FAAH, respectively, with high efficacy10–16. Structure–activity relationship studies have revealed that a carboxylic acid moiety at a certain position of the heterocyclic part of the molecules is essential for a pronounced inhibition of cPLA₂α. FAAH inhibitors with high activity can be obtained with nitrogen-rich, acid-free heterocyclic backbones like benzotriazole or tetrazole.

Figure 1. Heteroaryl-substituted propan-2-ones with preferential inhibition of cPLA₂α (1 and 2) and FAAH (3–6).

An important part of the pharmacophore of these substances is the activated electrophilic ketone moiety of their propan-2-one substructure. This structural element is supposed to form reversible covalent binding interactions with a serine residue of the active site of cPLA₂α and FAAH, respectively, and is, therefore, a so-called “serine trap”.

Unfortunately, these inhibitors are extensively metabolized in vitro as well as in vivo11^,12^,15^,17. Particularly, the activated ketone is reduced to an alcohol leading to inactivity against cPLA₂α and FAAH. Thus, this kind of compounds is not suitable for a systemic application. In order to get inhibitors, which are not inactivated in organism so quickly, we substituted the scissile ketone function with phenoxy-carbamate moieties, which in principle, can also act as serine traps. These variations resulted in substances with higher metabolic in vitro stability but significantly lower inhibitory potency18^,19. In the present study, we describe the effect of the replacement of the activated electrophilic ketone by further serine traps such as α-ketoheterocycle, cyanamide, and nitrile20^,21 on enzyme inhibition and metabolic stability.

Methods

Chemistry

General

Column chromatography was performed on silica gel 60, a particle size of 0.040–0.063 mm, from Macherey & Nagel (Düren, Germany). Melting points were determined on a Büchi B-540 apparatus (Büchi Inc., Flawil, Switzerland) and are uncorrected. ¹H NMR spectra were recorded on an Agilent DD2 spectrometer (Agilent Technologies, Richardson, TX) (400 MHz) or an Agilent DD2 spectrometer (600 MHz). ¹³C NMR spectra were measured on an Agilent DD2 spectrometer (Agilent Technologies, Richardson, TX) (101 MHz) or an Agilent DD2 spectrometer (151 MHz). Electron ionization (EI) mass spectra were obtained on a Finnigan GCQ apparatus (Finnigan Corporation, San Jose, CA). The high-resolution mass spectra (HRMS) were recorded on a Bruker micrOTOF-Q II spectrometer using electrospray chemical ionization (ESI) or atmospheric pressure chemical ionization (APCI). ATR-FTIR spectra were measured on a Prestige 21 system from Shimadzu (Shimadzu Corporation, Tokyo, Japan). Preparative reversed phase HPLC was performed using a Knauer Azura pump P2.1L equipped with a Knauer RP18 Eurospher II 5 μm column (Knauer, Berlin, Germany) (20 mm (I.D.) × 250 mm) protected with a RP18 Eurospher II 5 μm guard column (Knauer, Berlin, Germany) (20 mm (I.D.) × 30 mm) and eluting at a flow rate of 25 mL/min. Detection was conducted with a Shimazu SPD-6A UV detector at 254 nm (Shimadzu Corporation, Tokyo, Japan). Chromatograms were recorded with MacDAcq32 Control Software from Bischoff (Leonberg, Germany). The compounds were dissolved in DMSO and the injected sample volume was 0.5–1 mL. The substances were obtained after distilling off the organic solvent and freeze-drying the remaining aqueous phase using a Christ alpha 1–2 LD plus apparatus (Christ, Osterode am Harz, Germany). The purity of the target compounds was determined by reversed phase HPLC on a Nucleosil 100 RP18 3 µm column (Macherey & Nagel, Düren, Germany) (3 mm (I.D.) × 125 mm) at a flow rate of 0.4 mL/min with a gradient consisting of acetonitrile/water/trifluoroacetic acid (42:58:0.1–86:14:0.1, v/v/v). The samples were prepared by mixing 20 μL of a 5 mM solution of the compound in DMSO with 180 μL of acetonitrile. About 5 μL of this solution was injected into the HPLC-system. UV-absorbance was measured at 254 nm. Purities of the target compounds were greater than 96%.

Allyl 1-(ethoxycarbonylmethyl)indole-5-carboxylate (8)

Ethyl bromoacetate (2.75 g, 1.82 mL, 16.5 mmol), caesium carbonate (2.68 g, 8.23 mmol), and potassium iodide (0.23 g, 1.39 mmol) were added to a solution of allyl indole-5-carboxylate (7) (1.11 g, 5.52 mmol) in acetonitrile (40 mL). Then the mixture was stirred at room temperature for 16 h. After addition of ethyl acetate, the reaction mixture was washed with water, dried (Na₂SO₄), and concentrated under reduced pressure. The residue was chromatographed on silica gel (hexane/ethyl acetate, 8:2) to yield 8 as an oil (1.57 g, 99%). ¹H NMR (400 MHz, CDCl₃): δ 1.22–1.32 (m, 3H), 4.18–4.27 (m, 2H), 4.82–4.89 (m, 4H), 5.25–5.33 (m, 1H), 5.39–5.47 (m, 1H), 6.08 (ddt, J = 16.7 Hz, 10.4 Hz, and 5.5 Hz, 1H), 6.66 (dt, J = 3.3 Hz and 0.8 Hz, 1H), 7.16 (d, J = 3.3 Hz, 1H), 7.23–7.30 (m, 1H), 7.96 (dd, J = 8.7 Hz and 1.6 Hz, 1H,), 8.43 (dt, J = 1.5 Hz and 0.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 14.3, 48.1, 62.0, 65.4, 104.1, 108.8, 117.9, 122.2, 123.7, 124.3, 128.3, 130.0, 132.8, 139.2, 167.3, and 168.2; HRMS (APCI, direct probe) [M + H]⁺ calculated: 288.1230, found: 288.1234.

Allyl 1-(2-oxoethyl)indole-5-carboxylate (9)

A solution of diisobutylaluminium hydride in dry cyclohexane (1 M, 3.48 mL, 3.48 mmol) was added dropwise to a solution of 8 (0.505 g, 1.76 mmol) in dry THF (20 mL) at −70 °C. The mixture was stirred at this temperature for 4 h. After addition of 1 M hydrochloric acid (5 mL), the reaction mixture was allowed to warm up to room temperature and extracted exhaustively with ethyl acetate. The combined organic phases were dried (Na₂SO₄), evaporated, and chromatographed on silica gel (hexane/ethyl acetate, 7:3–1:1) to yield 9 as an oil (0.176 mg, 41%).¹H NMR (400 MHz, CDCl₃): δ 4.85–4.88 (m, 2H), 4.90 (d, J = 1.1 Hz, 2H), 5.29 (dq, J = 10.5 Hz and 1.4 Hz, 1H), 5.44 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.08 (ddt, J = 17.1 Hz, 10.9 Hz and 5.6 Hz, 1H), 6.72 (dd, J = 3.3 Hz and 0.8 Hz, 1H), 7.16 (d, J = 3.2 Hz, 1H), 7.22 (dt, J = 8.7 Hz and 0.8 Hz, 1H), 7.98 (dd, J = 8.6 Hz and 1.6 Hz, 1H), 8.46 (dd, J = 1.6 Hz and 0.7 Hz, 1H), 9.73 (s, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 55.9, 65.5, 104.8, 108.7, 118.0, 122.2, 124.0, 124.5, 128.5, 129.6, 132.8, 139.1, 167.2, and 196.4. HRMS (ESI+) [M + H]⁺ calculated: 244.0968, found: 244.0961.

Allyl 1-(2-cyano-2-hydroxyethyl)indole-5-carboxylate (10)

A solution of 9 (0.500 g, 2.06 mmol) in dry CH₂Cl₂ (50 mL) was treated with acetone cyanhydrin (563 µL, 6.17 mmol) followed by triethylamine (171 µL, 1.23 mmol) and stirred at room temperature for 4 h. Then the solution was evaporated to dryness and dissolved in diethyl ether (30 mL). After repeated washing with brine, the organic phase was dried with Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 8:2–6:4) to yield 10 as a solid (0.374 g, 67%). Mp 113–114 °C; ¹H NMR (400 MHz, CDCl₃): δ 3.20 (s_broad, 1H), 4.50 (dd, J = 14.9 Hz and 6.6 Hz, 1H), 4.57 (dd, J = 14.9 Hz and 4.6 Hz, 1H), 4.83 (dt, J = 5.6 Hz and 1.5 Hz, 3H), 5.30 (dq, J = 10.5 Hz and 1.4 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.07 (ddt, J = 17.1 Hz, 10.3 Hz, and 5.6 Hz, 1H), 6.67 (dd, J = 3.3 Hz and 0.8 Hz, 1H), 7.26 (d, J = 3.4 Hz, 1H), 7.38 (dt, J = 8.8 Hz and 0.8 Hz, 1H), 7.92 (dd, J = 8.7 Hz and 1.6 Hz, 1H), 8.39 (dd, J = 1.6 Hz and 0.7 Hz, 1H). ¹³C NMR (101 MHz, CDCl₃): δ 50.1, 61.1, 65.6, 104.6, 109.0, 117.8, 118.2, 122.4, 123.9, 124.4, 128.5, 130.0, 132.6, 139.0, and 167.4. HRMS (APCI, direct probe) [M + H]⁺ calculated: 271.1077, found: 271.1086.

Allyl 1-[2-hydroxy-2-(5-phenoxybenzoxazol-2-yl)ethyl]indole-5-carboxylate (11)

Under a nitrogen atmosphere, a mixture of dry chloroform (4 mL) and dry ethanol (1.07 mL, 18.3 mmol) was treated dropwise with acetyl chloride (1.19 mL, 16.7 mmol) at 0 °C over a 15 min period. Next, a solution of 10 (0.150 g, 0.56 mmol) in dry chloroform (4 mL) was added and stirring was continued at 0 °C for 1 h. The solution was evaporated to dryness at room temperature, dissolved in dry ethanol (6 mL), and treated with 2-amino-4-phenoxyphenol (0.112 g, 0.56 mmol). After heating to reflux for 6 h, the reaction mixture was evaporated to dryness and dissolved in ethyl acetate. After washing with 1 M hydrochloric acid, saturated sodium bicarbonate solution and brine, the organic phase was dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield 11 as an oil (0.144 g, 57%). ¹H NMR (400 MHz, CDCl₃): δ 4.65 (dd, J = 14.8 Hz and 6.8 Hz, 1H), 4.80 (dd, J = 14.8 Hz and 3.8 Hz, 1H), 4.84 (dt, J = 5.6 Hz and 1.5 Hz, 2H), 5.25–5.33 (m, 2H), 5.43 (dq, J = 17.1 Hz and 1.6 Hz, 1H), 6.07 (ddt, J = 17.2 Hz, 10.6 Hz and 5.4 Hz, 1H), 6.60 (dd, J = 3.2 Hz and 0.8 Hz, 1H), 6.99 (dd, J = 8.7 Hz and 1.1 Hz, 2H), 7.07–7.16 (m, 2H), 7.21 (d, J = 3.2 Hz, 1H), 7.30 (d, J = 2.4 Hz, 1H), 7.34 (dd, J = 8.5 Hz and 7.6 Hz, 2H), 7.39 (d, J = 8.9 Hz, 1H), 7.48 (dt, J = 8.8 Hz and 0.5 Hz, 1H), 7.89 (dd, J = 8.7 Hz, J = 1.6 Hz, 1H), 8.39 (d, J = 1.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 50.7, 65.4, 68.1, 103.9, 109.1, 110.5, 111.5, 118.0, 118.0, 118.7, 122.0, 123.5, 123.6, 124.3, 128.4, 130.0, 130.2, 132.8, 139.1, 141.4, 147.3, 154.8, 157.7, 165.4, and 167.3. HRMS (APCI, direct probe) [M + H]⁺ calculated: 455.1601, found: 455.1592.

Allyl 1-[2-oxo-2-(5-phenoxybenzoxazol-2-yl)ethyl]indole-5-carboxylate (12)

Under a nitrogen atmosphere, a solution of 11 (0.104 g, 0.23 mmol) in dry CH₂Cl₂ (10 mL) was treated with Dess–Martin periodinane (0.146 g, 0.34 mmol) and stirred for 4 h. Then aqueous sodium thiosulfate solution (5%) and saturated aqueous sodium bicarbonate solution were added. After stirring for 10 min, the reaction mixture was exhaustively extracted with CH₂Cl₂. The combined organic layers were dried (Na₂SO₄), concentrated and purified by chromatography on silica gel (hexane/ethyl acetate, 9:1) to yield 12 as a solid (0.072 g, 69%). Mp 83–84 °C (decomp.); ¹H NMR (400 MHz, CDCl₃): δ 4.85 (dt, J = 5.5 Hz and 1.5 Hz, 2H), 5.29 (dq, J = 10.4 Hz and 1.4 Hz, 1H), 5.43 (dq, J = 17.3 Hz and 1.6 Hz, 1H), 5.74 (s, 2H), 6.00–6.15 (m, 1H), 6.72 (dd, J = 3.2 Hz and 0.9 Hz, 1H), 7.04–7.09 (m, 2H), 7.16–7.22 (m, 1H), 7.25 (d, J = 3.3 Hz, 1H), 7.28–7.36 (m, 2H), 7.37–7.43 (m, 2H), 7.49 (dt, J = 2.5 Hz and 0.5 Hz, 1H), 7.65 (dt, J = 9.0 Hz and 0.5 Hz, 1H), 7.95 (dd, J = 8.7 Hz and 1.7 Hz, 1H), 8.45 (d, J = 1.5 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 52.6, 65.4, 104.6, 108.9, 111.0, 112.9, 118.0, 119.2, 121.9, 122.4, 123.9, 124.2, 124.4, 128.4, 130.2, 130.2, 132.8, 139.3, 141.4, 147.2, 156.3, 156.6, 157.0, 167.3, and 183.0. HRMS (APCI, direct probe) [M + H]⁺ calculated: 453.1445, found: 453.1500.

1-[2-Oxo-2-(5-phenoxybenzoxazol-2-yl)ethyl]indole-5-carboxylic acid (13)

Under a nitrogen atmosphere, a solution of 12 (0.060 g, 0.13 mmol) in dry THF (5 mL) was treated with tetrakis(triphenylphosphine) palladium (0) (15 mg, 0.013 mmol). After bubbling nitrogen through the solution for 10 min, acetic acid (0.2 mL) was added and the mixture was stirred at room temperature. Upon completion of the reaction, a small amount of silica gel was added and the solvent was evaporated. The residue was directly purified by chromatography on silica gel (hexane/ethyl acetate/acetic acid, 9:1:0.02) followed by semi-preparative RP HPLC (acetonitrile/water/formic acid, 70:30:0.1) to yield 13 as a solid (0.008 g, 15%). Mp 128–129 °C (decomp.); ¹H NMR (600 MHz, DMSO-D₆): δ 5.98 (s, 2H), 6.66 (dd, J = 3.2 Hz and 0.8 Hz, 1H), 7.06 (dd, J = 8.7 Hz and 1.1 Hz, 2H), 7.17 (tt, J = 7.4 Hz and 1.1 Hz, 1H), 7.38 (dd, J = 8.9 Hz and 2.5 Hz, 1H,), 7.40–7.44 (m, 2H), 7.46 (d, J = 3.2 Hz, 1H), 7.56 (dt, J = 8.7 Hz and 0.8 Hz, 1H), 7.69 (d, J = 2.5 Hz, 1H), 7.72 (dd, J = 8.6 Hz and 1.6 Hz, 1H), 7.98 (d, J = 8.9 Hz, 1H), 8.26 (dd, J = 1.7 Hz, J = 0.6 Hz, 1H), 12.46 (s_broad, 1H); ¹³C NMR (151 MHz, DMSO-D₆): δ 52.9, 102.8, 110.1, 111.4, 113.2, 118.2, 121.1, 121.9, 122.4, 122.9, 123.6, 127.7, 130.2, 131.3, 139.1, 141.0, 146.3, 154.5, 157.1, 157.2, 168.3, and 183.8; HRMS (APCI, direct probe) [M + H]⁺ calculated: 413.1132, found: 413.1105.

tert-Butyl 2,2-dimethyl-4-[(4-phenoxyphenoxy)methyl]oxazolidine-3-carboxylate (21)

Sodium hydride (60% dispersion in mineral oil) (0.533 g, 13.3 mmol) was added to a solution of 4-phenoxyphenol (2.48 g, 13.3 mmol) in dry DMF (50 mL) and the mixture was stirred for 30 min. Then a solution of tert-butyl 2,2-dimethyl-4-[(tosyloxy)methyl]oxazolidine-3-carboxylate (20)22 (5.65 g, 14.7 mmol) in dry DMF (50 mL) was added dropwise and the mixture was heated at 70 °C for 3 h. After addition of water (50 mL), the solution was exhaustively extracted with ethyl acetate. The organic layers were dried with Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 19:1) to yield 21 as an oil (4.50 g, 85%). ¹H NMR (600 MHz, CDCl₃): δ 1.47–1.63 (m, 15H), 3.78–4.31 (m, 5H), 6.88–6.98 (m, 6H), 7.00–7.06 (m, 1H), 7.26–7.31 (m, 2H); HRMS (APCI, direct probe) [M + H]⁺ calculated: 400.2118, found: 400.2143.

tert-Butyl [1-hydroxy-3-(4-phenoxyphenoxy)propan-2-yl] carbamate (22)

p-Toluenesulfonic acid monohydrate (1.05 g, 5.52 mmol) was added to a solution of 21 (4.40 g, 11.0 mmol) in methanol (50 mL) and the resulting mixture was stirred at room temperature for 4 h. After neutralization with saturated sodium bicarbonate solution (50 mL), the organic solvent was distilled off and the aqueous residue was exhaustively extracted with ethyl acetate. The combined organic layers were dried with Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 8:2) to yield 22 as an oil (1.09 g, 28%). ¹H NMR (600 MHz, CDCl₃): δ 1.45 (s, 9H), 3.80 (dd, J = 11.1 Hz and 5.0 Hz, 1H), 3.90 (dd, J = 11.2 Hz and 4.5 Hz, 1H), 3.96–4.03 (m, 1H), 4.05 (dd, J = 9.1 Hz and 4.9 Hz, 1H), 4.08–4.15 (m, 1H), 5.16 (s, 1H), 6.88 (d, J = 9.0 Hz, 2H), 6.93 (dd, J = 8.6 Hz and 0.9 Hz, 2H), 6.96 (d, J = 9.0 Hz, 2H), 7.04 (t, J = 7.4 Hz, 1H), 7.29 (dd, J = 8.5 Hz and 7.5 Hz, 2H); MS (EI, 70 eV) m/z (%): 359 (1) M⁺, 186 (100), 118 (79).

2-[(tert-Butoxycarbonyl)amino]-3-(4-phenoxyphenoxy)propyl 4-methylbenzenesulfonate (23)

A solution of 22 (2.20 g, 6.12 mmol) in dry CH₂Cl₂ (25 mL) was added dropwise to a solution of tosyl chloride (2.34 g, 12.3 mmol) and 4-dimethylaminopyridine (0.375 g, 3.07 mmol) in dry CH₂Cl₂ (25 mL). The mixture was treated with triethylamine (1.70 mL, 12.2 mmol) and stirred at room temperature for 12 h. After washing three times with 1 M KHSO₄ and brine, the organic layer was dried over Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1–8:2) to yield 23 as an oil (2.58 g, 82%). ¹H NMR (600 MHz, CDCl₃): δ 1.42 (s, 9H), 2.38 (s, 3H), 3.86 (dd, J = 9.0 Hz and 5.7 Hz, 1H), 3.98 (dd, J = 9.3 Hz and 3.2 Hz, 1H), 4.14–4.20 (m, 2H), 4.21–4.25 (m, 1H), 4.95 (d_broad, J = 4.9 Hz, 1H), 6.73 (d, J = 9.0 Hz, 2H), 6.91–6.95 (m, 4H), 7.05 (t, J = 7.4 Hz, 1H), 7.23–7.27 (m, 2H), 7.30 (dd, J = 8.5 Hz and 7.5 Hz, 2H), 7.74 (d, J = 8.3 Hz, 2H); HRMS (APCI, direct probe) [M + H]⁺ calculated: 514.1894, found: 514.1929.

Allyl 1-{2-[(tert-butoxycarbonyl)amino]-3-(4-phenoxyphenoxy) propyl}indole-5-carboxylate (24)

Sodium hydride (60% dispersion in mineral oil) (0.117 g, 2.92 mmol) was added to a solution of allyl indole-5-carboxylate (7) (0.588 g, 2.92 mmol) in dry DMF (15 mL) and the resulting mixture was stirred at room temperature for 30 min. A solution of 23 (1.50 g, 2.92 mmol) in dry DMF (15 mL) was added dropwise and the mixture was heated at 80 °C for 3 h. After addition of water (50 mL), the solution was extracted exhaustively with ethyl acetate. The combined organic layers were dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield 24 as an oil (1.16 g, 73%). ¹H NMR (600 MHz, CDCl₃): δ 1.47 (s, 9H), 3.73 (d, J = 8.8 Hz, 1H), 3.88 (dd, J = 9.4 Hz and 3.5 Hz, 1H), 4.30–4.39 (m, 1H), 4.42 (dd, J = 13.8 Hz and 8.6 Hz, 1H), 4.47 (dd, J = 14.2 Hz and 4.7 Hz, 1H), 4.84 (d, J = 5.4 Hz, 2H), 5.13 (d, J = 8.3 Hz, 1H), 5.29 (dq, J = 10.4 Hz and 0.9 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.4 Hz, 1H), 6.07 (ddd, J = 22.6 Hz, 10.8 Hz, and 5.5 Hz, 1H), 6.59 (d, J = 3.0 Hz, 1H), 6.85 (d, J = 8.9 Hz, 2H), 6.94–7.01 (m, 4H), 7.07 (t, J = 7.4 Hz, 1H), 7.13 (d, J = 2.6 Hz, 1H), 7.32 (dd, J = 8.3 Hz and 7.6 Hz, 2H), 7.51 (d, J = 8.6 Hz, 1H), 7.91 (dd, J = 8.7 Hz and 1.0 Hz, 1H), 8.41 (s, 1H); HRMS (APCI, direct probe) [M + H]⁺ calculated: 543.2490, found: 543.2476.

Allyl 1-[2-amino-3-(4-phenoxyphenoxy)propyl]indole-5- carboxylate (25)

Trifluoroacetic acid (5.64 mL, 73.7 mmol) was added dropwise to a solution of 24 (1.00 g, 1.84 mmol) in dry CH₂Cl₂ (35 mL). Then the mixture was stirred at room temperature for 2 h and evaporated to dryness. The residue was dissolved in ethyl acetate and washed with 2 M aqueous sodium hydroxide. The aqueous phase was extracted three times with ethyl acetate. The combined organic layers were dried (Na₂SO₄) and concentrated to yield 25 as a solid (0.72 g, 88%). Mp 92–93 °C; ¹H NMR (600 MHz, DMSO-D₆): δ 3.93 (dd, J = 10.5 Hz and 4.7 Hz, 1H), 4.00–4.05 (m, 1H), 4.10 (d_broad, J = 4.4 Hz, 2H), 4.16 (dd, J = 10.6 Hz and 3. Hz, 1H), 4.58 (dd, J = 14.9 Hz and 6.3 Hz, 1H), 4.63 (dd, J = 14.9 Hz and 7.3 Hz, 1H), 4.82 (d, J = 5.3 Hz, 2H), 5.29 (dq, J = 10.5 Hz and 1.4 Hz, 1H), 5.42 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.08 (ddt, J = 16.9 Hz, 10.6 Hz, and 5.3 Hz, 1H), 6.73 (d, J = 3.1 Hz, 1H), 6.93 (dd, J = 8.6 Hz and 0.9 Hz, 2H), 7.01–7.04 (m, 4H), 7.10 (t, J = 7.4 Hz, 1H), 7.37 (dd, J = 8.6 Hz and 7.4 Hz, 2H), 7.53 (d, J = 3.2 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 7.81 (dd, J = 8.7 Hz and 1.5 Hz, 1H), 8.33 (d, J = 0.9 Hz, 1H); HRMS (APCI, direct probe) [M + H]⁺ calculated: 443.1965, found: 443.1981.

Allyl 1-[2-cyanamido-3-(4-phenoxyphenoxy)propyl] indole-5-carboxylate (26)

A solution of 25 (0.200 g, 0.45 mmol) and sodium bicarbonate (0.114 g, 1.36 mmol) in dry THF (5 mL) was treated dropwise with a solution of cyanogen bromide in CH₂Cl₂ (3 M, 180 µL, 0.54 mmol) at 0 °C and stirred at the same temperature for 2 h. After addition of water, the mixture was extracted exhaustively with diethyl ether. The combined organic layers were dried over Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield 26 as a solid (0.102 g, 48%). Mp 58–59 °C; ¹H NMR (600 MHz, CDCl₃): δ 3.87 (dd, J = 9.5 Hz and 4.2 Hz, 1H), 3.89–3.95 (m, 1H), 4.01 (dd, J = 9.5 Hz and 4.2 Hz, 1H), 4.10 (d, J = 6.9 Hz, 1H), 4.49–4.58 (m, 2H), 4.83 (dt, J = 5.7 Hz and 1.4 Hz, 2H), 5.28 (dq, J = 10.5 Hz and 1.3 Hz, 1H), 5.42 (dq, J = 17.2 Hz and 1.5 Hz, 1H), 6.06 (ddt, J = 17.2 Hz, 10.8 Hz, and 5.6 Hz, 1H), 6.65 (dd, J = 3.3 Hz and 0.9 Hz, 1H), 6.84 (d, J = 9.0 Hz, 2H), 6.94 (dd, J = 8.8 Hz and 1.1 Hz, 2H), 6.97 (d, J = 9.0 Hz, 2H), 7.06 (tt, J = 7.3 Hz and 1.1 Hz, 1H), 7.17 (d, J = 3.2 Hz, 1H), 7.31 (dd, J = 8.7 Hz and 7.3 Hz, 2H), 7.37 (d, J = 8.7 Hz, 1H), 7.93 (dd, J = 8.7 Hz and 1.6 Hz, 1H), 8.41 (d, J = 1.1 Hz, 1H); IR: ν (cm⁻¹) 2222 (CN); HRMS (APCI, direct probe) [M + H]⁺ calculated: 468.1918, found: 468.1955.

1-[2-Cyanamido-3-(4-phenoxyphenoxy)propyl] indole-5-carboxylic acid (27).

The allyl ester group of 26 (0.081 g, 0.17 mmol) was cleaved following the procedure described for the preparation of 13. Chromatography on silica gel (hexane/ethyl acetate, 8:2–6:4 followed by ethyl acetate) and subsequent purification with preparative RP HPLC (acetonitrile/water/formic acid, 70:30:0.1) yielded 27 as a solid (0.031 g, 42%). Mp 68–69 °C; ¹H NMR (600 MHz, DMSO-D₆): δ 4.14 (dd, J = 10.9 Hz and 7.2 Hz, 1H), 4.30 (dd, J = 11.2 Hz and 3.5 Hz, 1H), 4.55 (dd, J = 15.0 Hz and 8.4 Hz, 1H), 4.65 (dd, J = 15.1 Hz and 5.4 Hz, 1H), 4.90–5.00 (m, 1H), 6.62 (d, J = 3.2 Hz, 1H), 6.90 (dd, J = 8.7 Hz and 1.2 Hz, 2H), 6.98 (s, 4H), 7.05 (tt, J = 7.4 Hz and 1.2 Hz, 1H), 7.32 (dd, J = 8.6 Hz and 7.4 Hz, 2H), 7.46 (d, J = 3.3 Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.75 (dd, J = 8.6 Hz and 1.6 Hz, 1H), 8.21 (d, J = 1.5 Hz, 1H), 12.39 (s_broad, 1H); ¹³C NMR (151 MHz, DMSO-D₆): δ 44.2, 53.4, 66.1, 103.3, 109.4, 116.0, 116.1, 117.5, 120.6, 122.2, 122.6, 122.7, 123.3, 127.8, 129.9, 130.4, 138.3, 150.2, 153.9, 157.8, and 168.2; IR: ν (cm⁻¹) 2210 (CN); HRMS (ESI+) [M + H]⁺ calculated: 428.1605, found: 428.1615.

2,2-Dimethyl-5-[(4-phenoxyphenoxy)methyl]-1,3-dioxane (31)

Sodium hydride (60% dispersion in mineral oil) (0.80 g, 20.0 mmol) was added to a solution of 4-phenoxyphenol (4.09 g, 22.0 mmol) in dry DMF (50 mL). The resulting mixture was stirred at room temperature for 30 min. Then a solution of 2,2-dimethyl-1,3-dioxan-5-ylmethyl 4-methylbenzenesulfonate (30)23 (6.00 g, 20.0 mmol) in dry DMF (50 mL) was added dropwise and the mixture was heated at 80 °C for 3 h. After addition of water (50 mL), the solution was exhaustively extracted with ethyl acetate. The combined organic layers were dried over Na₂SO₄, concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield 31 as a solid (5.31 g, 84%). Mp 113–114 °C; ¹H NMR (400 MHz, DMSO-D₆): δ 1.33 (s, 3H), 1.35 (s, 3H), 2.01–2.09 (m, 1H), 3.75 (dd, J = 11.8 Hz and 5.9 Hz, 2H), 3.96–4.03 (m, 4H), 6.92 (dd, J = 8.7 Hz and 1.1 Hz, 2H), 6.98 (s, 4H), 7.07 (tt, J = 7.4 Hz and 1.1 Hz, 1H), 7.34 (dd, J = 8.7 Hz and 7.3 Hz, 2H); ¹³C NMR (101 MHz, DMSO-D₆): δ 23.4, 24.4, 33.6, 66.6, 97.4, 115.7, 117.3, 120.6, 122.6, 129.8, 149.5, 154.8, and 157.9; HRMS (APCI, direct probe) [M + H]⁺ calculated: 315.1591, found: 315.1591.

2-[(4-Phenoxyphenoxy)methyl]propane-1,3-diol (32)

Hydrochloric acid (5 M, 10 mL) was added to a solution of 31 (4.70 g, 15.0 mmol) in methanol (100 mL). After stirring the mixture at room temperature overnight, the solvent was distilled off and the residue chromatographed on silica gel (hexane/ethyl acetate, 1:1) to yield 32 as a solid (4.09 g, 99%). Mp 119–120 °C; ¹H NMR (400 MHz, DMSO-D₆): δ 1.96 (hept, J = 5.9 Hz, 1H), 3.45–3.59 (m, 4H), 3.95 (d, J = 5.9 Hz, 2H), 4.51 (t, J = 5.2 Hz, 2H), 6.87–6.95 (m, 2H), 6.96 (d, J = 2.3 Hz, 4H), 7.06 (tt, J = 7.3 and 1.2 Hz, 1H), 7.34 (dd, J = 8.4 Hz and 7.5 Hz, 2H); ¹³C NMR (101 MHz, DMSO-D₆): δ 43.9, 59.0, 66.2, 115.6, 117.2, 120.7, 122.5, 129.8, 149.2, 155.2, and 158.0; HRMS (APCI, direct probe) [M + H]⁺ calculated: 275.1278, found: 275.1293.

3-Hydroxy-2-[(4-phenoxyphenoxy)methyl]propyl 4-methylbenzenesulfonate (33)

A solution of 32 (2.50 g, 9.11 mmol) in dry THF (50 mL) was added dropwise to a solution of tosyl chloride (1.74 g, 9.11 mmol) and 4-dimethylaminopyridine (0.557 g, 4.56 mmol) in dry THF (75 mL). Then the mixture was treated with triethylamine (1.27 mL, 9.11 mmol) and stirred at room temperature for 12 h. The reaction mixture was washed three times with 1 M KHSO₄ and with brine, dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1–8:2) to yield 33 as an oil (2.61 g, 67%). ¹H NMR (400 MHz, CDCl₃): δ 2.37 (hept, J = 5.8 Hz, 1H), 2.42 (s, 3H), 3.81 (dd, J = 5.5 Hz and 1.8 Hz, 2H), 3.98 (dd, J = 5.7 Hz and 3.7 Hz, 2H), 4.27 (dd, J = 5.8 Hz and 3.7 Hz, 2H), 6.78 (d, J = 8.8 Hz, 2H), 6.91–6.98 (m, 4H), 7.05 (tt, J = 7.5 Hz and 1.0 Hz, 1H), 7.28–7.34 (m, 4H), 7.78 (d, J = 8.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 21.8, 41.2, 60.7, 66.1, 68.2, 115.6, 117.9, 120.8, 122.7, 128.1, 129.8, 130.1, 132.8, 145.1, 150.8, 154.7, and 158.4; HRMS (APCI, direct probe) [M + H]⁺ calculated: 429.1366, found: 429.1361.

3-[(tert-Butyldimethylsilyl)oxy]-2-[(4-phenoxyphenoxy)methyl]propyl 4-methylbenzenesulfonate (34)

tert-Butyldimethylsilyl chloride (0.677 g, 4.49 mmol) was added to a solution of 33 (1.61 g, 3.76 mmol) in dry CH₂Cl₂ (20 mL). The mixture was treated with triethylamine (622 µL, 4.46 mmol) and stirred at room temperature for 4 h. The reaction mixture was washed three times with brine, dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield 34 as an oil (1.81 g, 89%). ¹H NMR (400 MHz, CDCl₃): δ 0.00 (s, 3H), 0.01 (s, 3H), 0.84 (s, 9H), 2.33 (hept, J = 5.8 Hz, 1H), 2.42 (s, 3H), 3.69 (d, J = 5.8 Hz, 2H), 3.90 (qd, J = 9.4 Hz and 5.9 Hz, 2H), 4.20 (d, J = 5.9 Hz, 2H), 6.76 (d, J = 8.5 Hz, 2H), 6.91–6.97 (m, 4H), 7.05 (tt, J = 7.5 Hz and 0.8 Hz, 1H), 7.27–7.34 (m, 4H), 7.77 (d, J = 8.1 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ −5.4, 18.3, 21.8, 25.9, 41.5, 60.2, 65.3, 68.3, 115.6, 117.8, 120.8, 122.7, 128.1, 129.8, 130.0, 132.9, 144.9, 150.5, 155.0, and 158.5; HRMS (APCI, direct probe) [M + H]⁺ calculated: 543.2231, found: 543.2292.

Allyl 1-{3-[(tert-butyldimethylsilyl)oxy]-2-[(4-phenoxyphenoxy)methyl]propyl}indole-5-carboxylate (35)

Sodium hydride (60% dispersion in mineral oil) (0.122 g, 3.05 mmol) was added to a solution of allyl indole-5-carboxylate (7) (0.612 g, 3.04 mmol) in dry DMF (15 mL). The mixture was stirred at room temperature for 30 min. Then a solution of 34 (1.50 g, 2.76 mmol) in dry DMF (15 mL) was added dropwise and the mixture was heated at 70 °C for 3 h. After addition of water (50 mL), the reaction mixture was exhaustively extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 19:1) to yield 35 as an oil (1.47 g, 93%); ¹H NMR (400 MHz, CDCl₃): δ 0.06 (s, 3H), 0.06 (s, 3H), 0.93 (s, 9H), 2.52 (hept, J = 5.6 Hz, 1H), 3.64 (dd, J = 10.3 Hz and 4.8 Hz, 1H), 3.72 (dd, J = 10.3 Hz and 5.4 Hz, 1H), 3.88 (d, J = 5.6 Hz, 2H), 4.30–4.44 (m, 2H), 4.85 (dt, J = 5.6 Hz and 1.5 Hz, 2H), 5.29 (dq, J = 10.4 Hz and 1.4 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.08 (ddt, J = 17.2 Hz, 10.4 Hz and 5.6 Hz, 1H), 6.59 (dd, J = 3.2 Hz and 0.8 Hz, 1H), 6.85 (d, J = 9.0 Hz, 2H), 6.92–7.00 (m, 4H), 7.05 (tt, J = 7.5 Hz and 1.1 Hz, 1H), 7.16 (d, J = 3.2 Hz, 1H), 7.28–7.34 (m, 2H), 7.41 (dt, J = 8.7 Hz and 0.8 Hz, 1H), 7.91 (dd, J = 8.7 Hz and 1.6 Hz, 1H), 8.42 (dd, J = 1.6 Hz and 0.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ −5.4, −5.3, 18.4, 26.0, 42.7, 44.5, 60.9, 65.3, 66.4, 103.2, 109.3, 115.6, 117.8, 117.9, 120.9, 121.6, 122.7, 123.2, 124.2, 128.2, 129.8, 130.2, 132.9, 139.0, 150.6, 155.0, 158.5, and 167.5; HRMS (APCI, direct probe) [M + H]⁺ calculated: 572.2827, found: 572.2863.

Allyl 1-{3-hydroxy-2-[(4-phenoxyphenoxy)methyl]propyl}indole-5-carboxylate (36)

A solution of tetrabutylammonium fluoride in dry THF (1 M, 1.96 mL, 1.96 mmol) was added to a solution of 35 (0.280 g, 0.49 mmol) in dry THF (5 mL) and the mixture was stirred at room temperature for 1 h. After evaporation, the residue was chromatographed on silica gel (hexane/ethyl acetate, 8:2–6:4) to yield 36 as an oil (0.220 g, 98%). ¹H NMR (400 MHz, CDCl₃): δ 2.48–2.62 (m, 1H), 3.74 (dd, J = 10.8 Hz and 5.3 Hz, 1H), 3.83 (dd, J = 10.9 Hz and 4.9 Hz, 1H), 3.92 (dd, J = 9.4 Hz and 4.8 Hz, 1H), 3.97 (dd, J = 9.4 Hz and 5.1 Hz, 1H), 4.43 (d, J = 7.1 Hz, 2H), 4.85 (dq, J = 5.6 Hz and 1.2 Hz, 2H), 5.29 (dq, J = 10.5 Hz and 1.3 Hz, 1H), 5.43 (dq, J = 17.1 Hz and 1.7 Hz, 1H), 6.08 (ddt, J = 17.3 Hz, 10.3 Hz and 6.0 Hz, 1H), 6.60 (dd, J = 3.2 Hz and 0.8 Hz, 1H), 6.82–6.89 (m, 2H), 6.92–7.01 (m, 4H), 7.06 (tt, J = 7.3 Hz and 1.1 Hz, 1H), 7.18 (d, J = 3.1 Hz, 1H), 7.27–7.36 (m, 2H), 7.41 (dd, J = 8.8 Hz and 0.9 Hz, 1H), 7.92 (dd, J = 8.7 Hz and 1.6 Hz, 1H), 8.42 (dd, J = 1.7 Hz and 0.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 42.2, 44.6, 61.6, 65.4, 67.1, 103.4, 109.2, 115.7, 117.9, 120.9, 121.7, 122.8, 123.3, 124.3, 128.2, 129.8, 130.1, 132.9, 139.0, 150.9, 154.7, 158.4, and 167.4; HRMS (APCI, direct probe) [M + H]⁺ calculated: 458.1962, found: 458.1971.

Allyl (E)-1-{3-(hydroxyimino)-2-[(4-phenoxyphenoxy)methyl]propyl}indole-5-carboxylate and allyl (Z)-1-{3-(hydroxyimino)-2-[(4-phenoxyphenoxy)methyl]propyl}indole-5-carboxylate (37)

To a solution of 36 (0.600 g, 1.31 mmol) in dry CH₂Cl₂ (20 mL) was added acetic acid (0.5 mL) followed by Dess–Martin periodinane (0.611 g, 1.44 mmol). The mixture was stirred at room temperature for 1.5 h. After addition of water (20 mL), the mixture was exhaustively extracted with CH₂Cl₂. The combined organic layers were dried (Na₂SO₄) and filtered. The filtrate was treated with hydroxylammonium chloride (1.50 g, 21.6 mmol) and pyridine (3 mL). After stirring at room temperature overnight, the reaction mixture was exhaustively washed with 1 M hydrochloric acid. The organic phase was dried (Na₂SO₄), concentrated, and chromatographed on silica gel (hexane/ethyl acetate, 9:1) to yield a 60:40 mixture of the E- and Z-isomers of 37 as an oil (0.315 g, 51%). ¹H NMR (400 MHz, CDCl₃): δ 3.22–3.33 (m, 0.6H), 3.73–3.98 (m, 2.4H), 4.43–4.62 (m, 2H), 4.85 (dt, J = 5.6 Hz and 1.5 Hz, 2H), 5.28 (dq, J = 10.4 Hz and 1.4 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.08 (ddt, J = 17.2 Hz and 10.5 Hz, J = 5.6 Hz, 1H), 6.58 (dd, J = 3.2 Hz and 0.8 Hz, 0.4H), 6.60 (dd, J = 3.2 Hz and 0.8 Hz, 0.6H), 6.82–6.88 (m, 2H), 6.92–7.01 (m, 4.4H), 7.06 (tq, J = 7.3 Hz and 1.1 Hz, 1H), 7.13 (t, J = 3.0 Hz, 1H), 7.31 (dd, J = 8.7 Hz and 7.5 Hz, 2H), 7.36 (dd, J = 8.7 Hz and 0.8 Hz, 0.6H), 7.48 (d, J = 8.7 Hz, 0.4H), 7.57 (d, J = 6.0 Hz, 0.6H), 7.92 (dt, J = 8.7 Hz and 1.5 Hz, 1H), 8.42 (d, J = 1.6 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 37.0, 41.1, 43.7, 45.3, 65.1, 65.2, 65.2, 66.3, 103.5, 103.5, 109.0, 109.2, 115.6, 115.6, 117.8, 117.8, 117.8, 117.8, 120.7, 121.7, 122.7, 122.7, 123.3, 124.1, 124.2, 128.1, 128.2, 129.6, 129.8, 129.8, 132.7, 138.7, 138.7, 149.4, 149.6, 150.9, 151.0, 154.3, 154.3, 158.2, 158.2, 167.2, and 167.3; HRMS (APCI, direct probe) [M + H]⁺ calculated: 471.1914, found: 471.1925.

Allyl 1-[2-cyano-3-(4-phenoxyphenoxy)propyl]indole-5-carboxylate (38)

Under a nitrogen atmosphere, 2-chloro-1-methylpyridinium iodide (0.155 g, 0.607 mmol) was added to a solution of 37 (0.220 g, 0.468 mmol) in dry THF (5 mL). The mixture was stirred at room temperature for 10 min. Then triethylamine (260 µL, 1.87 mmol) was added and stirring was continued for 20 h. After addition of 7 M hydrochloric acid (5 mL), the reaction mixture was exhaustively extracted with ethyl acetate. The combined organic layers were dried (Na₂SO₄), concentrated, and chromatographed on silica gel (dichloromethane) to yield 38 as an oil (0.053 g, 25%). ¹H NMR (400 MHz, CDCl₃): δ 3.41–3.51 (m, 1H), 3.96 (dd, J = 9.5 Hz and 4.2 Hz, 1H), 4.04 (dd, J = 9.6 Hz and 5.6 Hz, 1H), 4.64 (dd, J = 14.8 Hz and 6.1 Hz, 1H), 4.71 (dd, J = 14.8 Hz and 6.9 Hz, 1H), 4.85 (dt, J = 5.6 Hz and 1.5 Hz, 2H), 5.29 (dq, J = 10.4 Hz and 1.3 Hz, 1H), 5.43 (dq, J = 17.2 Hz and 1.6 Hz, 1H), 6.07 (ddt, J = 16.8 Hz, 10.4 Hz, and 5.6 Hz, 1H), 6.66 (dt, J = 3.3 Hz and 0.8 Hz, 1H), 6.84–6.91 (m, 2H), 6.91–7.02 (m, 4H), 7.08 (tt, J = 7.5 Hz and 1.1 Hz, 1H), 7.23 (d, J = 3.0 Hz, 1H), 7.29–7.35 (m, 2H), 7.37 (dd, J = 8.7 Hz and 0.8 Hz, 1H), 7.91–8.00 (m, 1H), 8.42 (dd, J = 1.5 Hz and 0.7 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 33.4, 44.4, 64.7, 65.5, 104.8, 108.7, 116.1, 117.8, 118.1, 118.2, 120.9, 122.6, 123.1, 124.0, 124.5, 128.5, 129.6, 129.9, 132.7, 138.6, 151.9, 153.5, 158.1, and 167.1; IR: ν (cm⁻¹) 2245 (CN). HRMS (ESI+) [M + H]⁺ calculated: 453.1809, found: 453.1837.

1-[2-Cyano-3-(4-phenoxyphenoxy)propyl] indole-5-carboxylic acid (39)

The allyl ester group of 38 (0.053 g, 0.12 mmol) was cleaved following the procedure described for the preparation of 13. After work up the residue was chromatographed on silica gel (hexane/ethyl acetate/acetic acid, 8:2:0.1) to yield 39 as a solid (0.036 g, 75%). Mp 148–149 °C; ¹H NMR (400 MHz, DMSO-D₆): δ 3.85–3.94 (m, 1H), 4.01 (dd, J = 9.7 Hz and 5.5 Hz, 1H), 4.19 (dd, J = 9.7 Hz and 4.5 Hz, 1H), 4.62–4.76 (m, 2H), 6.64 (d, J = 3.2 Hz, 1H), 6.90 (dd, J = 8.3 Hz and 1.2 Hz, 2H), 6.99 (s, 4H), 7.06 (t, J = 7.4 Hz, 1H), 7.33 (dd, J = 8.6 Hz and 7.3 Hz, 2H), 7.53 (d, J = 3.2 Hz, 1H), 7.65 (d, J = 8.7 Hz, 1H), 7.70 (dd, J = 8.6 Hz and 1.6 Hz, 1H), 8.21 (d, J = 1.5 Hz, 1H), 12.44 (s_broad, 1H); ¹³C NMR (151 MHz, DMSO-D₆): δ 32.9, 43.6, 65.2, 103.2, 109.8, 116.1, 117.5, 119.2, 120.7, 122.5, 122.7, 123.1, 127.7, 129.9, 130.4, 138.3, 150.2, 153.9, 157.8, and 168.2; IR: ν (cm⁻¹) 2245 (CN); HRMS (APCI, direct probe) [M + H]⁺ calculated: 413.1496, found: 413.1518. Supplementary material available: synthesis and analytical data of compounds 6, 19, 29, 43, and their intermediates; ¹H NMR spectra of all target compounds.

Biological evaluation

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 cytosolic phospholipase A₂α (cPLA₂α)

Inhibition of cPLA₂α was measured according to a recently published procedure24. 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 the absence and presence of a test compound with reversed-phase HPLC and UV-detection at 200 nm after on-line solid phase extraction.

Inhibition of fatty acid amide hydrolase (FAAH)

Inhibition of FAAH was measured as described25. Briefly, the substrate N-(2-hydroxyethyl)-4-pyren-1-ylbutanamide26 (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 clean-up by measuring the amount of 4-pyren-1-yl-butanoic acid released by FAAH in the absence and presence of a test compound with reversed phase HPLC and fluorescence-detection.

Metabolic stability in rat liver S9 fractions

The metabolic stability was tested using S9 fractions of rat liver homogenate25. Briefly, the test compounds were incubated under aerobic conditions in the absence and presence of the co-factor NADPH. The metabolic reactions were terminated after 30 min. In deviation to the published procedure, the extent of metabolism was evaluated by reversed phase HPLC with UV-MS-detection. The HPLC/UV-MS system from Shimadzu (Tokyo, Japan) consisted of two LC-20ADXR HPLC-pumps, a SIL-30AC autosampler, a SPD-20A UV/Vis detector, and a LCMS-2020 single quad detector. Aliquots of 10 μL were injected onto a HICHROM ACE 3 C18 column (2.1 mm (I.D.) × 100 mm, particle size 3 µm) (HICHROM, Berkshire, UK) protected with a Phenomenex C18 guard column (Phenomenex, Torrance, CA) (3 mm (I.D.) × 4 mm). Autosampler temperature was 10 °C, column oven temperature was set to 30 °C. The mobile phase consisted of acetonitrile/10 mM ammonium acetate 20:80 (v/v), adjusted to pH 5 with formic acid (A) and acetonitrile/10 mM ammonium acetate 80:20 (v/v), adjusted to pH 5 with formic acid. The following gradient was applied (A%): 0 min: 90; 1 min: 90; 9 min: 0; 11 min: 0; 11.5 min: 90; and 16 min: 90. The flow rate was 0.3 mL/min and the UV detection was performed at 230 nm. The UV-chromatograms were used for quantification, and structural assignments were based on the MS-data.

Results and discussion

Chemistry

The indole-5-carboxylic acid 211 was chosen as lead compound for studying the effect of a replacement of the activated ketone group by other serine traps on cPLA₂α inhibition. The investigation of the impact of such structural modifications on inhibitory potency against FAAH started from the benzotriazole and tetrazole derivatives 4 and 6, respectively15.

The synthesis of the indole-5-carboxylic acid derivative with a α-ketobenzoxazole structural element (13) is outlined in Scheme 1. Reaction of allyl indole-5-carboxylate (7) with ethyl bromoacetate in acetonitrile in the presence of caesium carbonate and potassium iodide afforded the diester 8. The ethylester group of this compound was selectively reduced with diisobutylaluminium hydride to an aldehyde. The obtained intermediate 9 was treated with acetone cyanhydrin to obtain 10. Conversion of the cyanhydrin group of 10 to an imidate in a Pinner-reaction followed by condensation with 2–amino-4-phenoxyphenol27 led to the formation of the α-hydroxyethyl-substituted benzoxazole 11. Dess–Martin oxidation of the alcohol group and palladium catalyzed cleavage of the allyl ester functionality finally gave the target compound 13.

Scheme 1. Reagents and conditions: (a) Ethyl bromoacetate, Cs₂CO₃, KI, acetonitrile, room temperature, 16 h; (b) diisobutylaluminium hydride, cyclohexane, THF, −70 °C to room temperature, 4 h; (c) acetone cyanhydrin, triethylamine, CH₂Cl₂, room temperature, 4 h; (d) 1. acetyl chloride, ethanol, chloroform, 0 °C, 1 h; 2. 2–amino-4-phenoxyphenol, ethanol, reflux, 6 h; (e) Dess-Martin periodinane, CH₂Cl₂, room temperature, 4 h; (f) tetrakis(triphenylphosphine)palladium(0), acetic acid, THF, room temperature.

For the synthesis of the analogous benzotriazole 19 a similar route was applied (Scheme 2). Since the formation of the acetaldehyde 16 could not be achieved by reducing the corresponding acetic acid ethylester with diisobutylaluminium hydride, this intermediate was synthesized by reaction of benzotriazole (14) with bromoacetaldehyde diethyl acetal followed by acid catalyzed cleavage of the acetal group28. From the aldehyde 16, the α-ketobenzoxazole 19 was obtained using the same type of reactions as described above for the synthesis of 12 from 9.

Scheme 2. Reagents and conditions: (a) Bromoacetaldehyde diethyl acetal, DMF, NaHCO₃, reflux, 18 h; (b) conc. HCl, THF, room temperature, 5 h; (c) acetone cyanhydrin, triethylamine, CH₂Cl₂, room temperature, 5 h; (d) 1. acetyl chloride, ethanol, chloroform, 0 °C, 1 h; 2. 2–amino-4-phenoxyphenol, ethanol, reflux, 6 h; (e) Dess-Martin periodinane, CH₂Cl₂, room temperature, 4 h.

In Scheme 3, the synthesis of the indole-5-carboxylic acid derivative with a cyanamide group in the side chain is shown. tert-Butyl 2,2-dimethyl-4-[(tosyloxy)methyl]oxazolidine-3-carboxylate22 was reacted with 4-phenoxyphenolate to give the substitution product 21. Treatment with p-toluenesulfonic acid led to the BOC-protected aminoalcohol 22. The hydroxy group of this compound was esterified with p-toluenesulfonyl chloride. Reaction of obtained sulfonic acid ester 23 with allyl indole-5-carboxylate in DMF in the presence of NaH afforded the 1-(2-BOC-aminopropyl)indole 24. After removal of the BOC protecting group, the amine moiety of resulting compound 25 was reacted with cyanogen bromide in the presence of NaHCO₃ to yield the cyanamide 26. Subsequent cleavage of the allyl ester moiety using Pd(0) catalysis gave the desired target compound 27.

Scheme 3. Reagents and conditions: (a) 4-Phenoxyphenol, NaH, DMF, 70 °C, 3 h; (b) p-toluenesulfonic acid, methanol, room temperature, 4 h; (c) tosyl chloride, 4-dimethylaminopyridine, triethylamine, CH₂Cl₂, room temperature, 12 h; (d) allyl indole-5-carboxylate, NaH, DMF, 80 °C, 3 h; (e) trifluoroacetic acid, CH₂Cl₂, room temperature, 2 h; (f) cyanogen bromide, CH₂Cl₂, NaHCO₃, THF, 0 °C, 2 h; (g) tetrakis(triphenylphosphine)palladium(0), acetic acid, THF, room temperature.

The analogous cyanamide with tetrazole heterocycle (29) was prepared by reaction of the published amine 2829 with cyanogen bromide (Scheme 4).

Scheme 4. Reagents and conditions: (a) Cyanogen bromide, CH₂Cl₂, NaHCO₃, diethyl ether, 0 °C, 2 h.

The derivative of the lead 2, in which the ketone group was replaced by a cyanomethyl element, was prepared by the route shown in Scheme 5. The tosylate moiety of (2,2-dimethyl-1,3-dioxan-5-yl)methyl 4-methylbenzenesulfonate23 (30) was substituted with a 4-phenoxyphenoxy residue to give 31. Cleavage of the cyclic acetal structure of 31 was accomplished with aqueous HCl in methanol. One of the primary alcohol groups of resulting diol 32 was transformed into a sulfonic acid ester by the reaction with equimolar amounts of p-toluenesulfonyl chloride. Then the second alcohol functionality was protected as its tert-butyldimethylsilyl ether. Reaction of obtained intermediate 34 with allyl indole-5-carboxylate/NaH in DMF followed by deprotection of the silylated alcohol group by treatment with tetrabutylammonium fluoride gave the indole ester 36. The alcohol moiety of this compound was converted to an aldehyde by oxidation with Dess–Martin periodinane. Without further purification, the crude product was treated with hydroxylammonium chloride to yield the aldoxime 37 as a 60:40 mixture of the E- and Z-isomers. The oxime group of 37 was converted to a nitrile (38) by reaction with the dehydration agent 2-chloro-1-methylpyridinium iodide. Palladium-catalyzed cleavage of the allyl ester of 38 finally provided the target compound 39.

Scheme 5. Reagents and conditions: (a) 4-Phenoxyphenol, NaH, DMF, 80 °C, 3 h; (b) 5 M HCl, methanol, room temperature, overnight; (c) p-toluenesulfonyl chloride, 4-dimethylaminopyridine, triethylamine, THF, room temperature, 12 h; (d) tert–butyldimethylsilyl chloride, CH₂Cl₂, triethylamine, room temperature, 4 h; (e) allyl indole-5-carboxylate, NaH, DMF, 70 °C, 3 h; (f) tetrabutylammonium fluoride, THF, room temperature, 1 h; (g) 1. Dess-Martin periodinane, acetic acid, CH₂Cl₂, room temperature, 1.5 h; 2. hydroxylammonium chloride, pyridine, room temperature, overnight; (h) 2-chloro-1-methylpyridinium iodide, THF, triethylamine, room temperature, 20 h; (i) tetrakis(triphenylphosphine)palladium(0), acetic acid, THF, room temperature.

The corresponding tetrazole derivative 43 was obtained from the intermediate 34 in an analogous way (Scheme 6).

Scheme 6. Reagents and conditions: (a) Tetrazole, acetonitrile, DMF, NaOH, 100 °C, 2 h; (b) tetrabutylammonium fluoride, THF, room temperature, 2 h; (c) 1. Dess-Martin periodinane, acetic acid, CH₂Cl₂, room temperature, 1.5 h; 2. hydroxylammonium chloride, pyridine, room temperature, overnight; (d) 2-chlor-1-methylpyridinium iodide, THF, triethylamine, room temperature, 20 h.

Biological evaluation

Known inhibitors of serine hydrolases frequently contain highly electrophilic species, which can form covalent bonds with the catalytic serine of the active site of the enzymes. These so-called “serine traps” include fluorophosphonates, organoboric acids, carbamates, nitriles, cyanamides, and activated ketones, like α-diketones, trifluoromethyl ketones, α-ketoheterocyles and α-keto esters and amides20^,21. The high activity of our cPLA₂α inhibitors 1 and 2 and FAAH inhibitors 3–6 (Figure 1) is also based on the presence of an activated ketone serine trap. Among other things, this can be seen by the fact that the replacement of the reactive propan-2-one moiety by a propan-2-ol results in a loss of enzyme inhibitory potency. Since a metabolic reduction of the ketone group of such inhibitors to inactive alcohols rapidly occurs in vitro in rat liver homogenate as well as in vivo in mice, we tried to obtain metabolically more stable inhibitors by substitution of the central ketone groups of 2, 4, and 6, respectively, with other serine traps.

First, we incorporated an α-ketoheterocyclic system instead of the propan-2-one into the indole-5-carboxylic acid 2 and the benzotriazole 4. This structural variation led to a significant loss of activity in both cases. With an IC₅₀ value of 1.6 µM compound 13 inhibited cPLA₂α about 80-fold less effectively than the lead structure 2 (Table 1). The activity of the FAAH inhibitor 19 (IC₅₀ = 10 µM) was even 400-fold lower in comparison with that of the reference 4 (Table 2). Due to the neighboring oxazole ring system, in the α-ketoheterocyclic compounds 13 and 19 the keto group is more sterically hindered than the carbonyl moiety in the corresponding propan-2-one derivatives 2 and 4. Therefore, we expected that 13 and 19 are less good substrates of metabolic carbonyl reducing enzymes. However, incubation of these compounds in rat liver homogenate in the presence of NADPH also led to a complete metabolic transformation (Tables 1 and 2). Like in the case of 2 and 4, the analogous alcohol metabolites could be detected as main metabolites by LC/MS experiments.

Table 1. cPLA₂α inhibitory potency and metabolic stability in rat liver S9 fractions of (5-carboxyindol-1-yl)propan-2-ones.

Comp.	R	Inhibition of cPLA2α IC50 (µM)*	Metabolic stability (%)¶
2	=O	0.021	0
13		1.6	0
27	NHCN	>10†	14
39	CN	n.a. ‡	74

*Values are the means of at least two independent determinations, errors of the IC₅₀ values are within ± 20%; inhibition value of reference inhibitor Axon 160930 (CAS 479422-22-5): IC₅₀ = 0.21 µM.

†32% inhibition at 10 µM.

‡Not active at 10 µM.

¶Percentage of parent compound remaining after metabolism by rat liver microsomes in presence of NADPH; values are the means of at least two independent determinations. Metabolic stability of reference inhibitor 3-(5-carboxypentanoyl)-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid12 (CAS 1138028-71-3): 63 ± 6.7 % (n = 9).

Table 2. FAAH inhibitory potency and metabolic stability in rat liver S9 fractions of (1H-benzotriazol-1-yl)- and (2H-tetrazol-2-yl)propan-2-ones.

Comp.	R	Inhibition of FAAH IC50 (µM)*	Metabolic stability (%)‡
4		0.024	0
19		10	0
6	=O	0.008	0
29	NHCN	5.3	25
43	CN	n.a.†	57

*Values are the means of at least two independent determinations, errors of the IC₅₀ values are within ± 20%; inhibition value of reference inhibitor URB59731 (CAS 546141-08-6): IC₅₀ = 0.060 µM.

†Not active at 10 µM.

‡Percentage of parent compound remaining after metabolism by rat liver microsomes in presence of NADPH; values are the means of at least two independent determinations. Metabolic stability of reference inhibitor 3-(5-carboxypentanoyl)-1-[3-(4-octylphenoxy)-2-oxopropyl]indole-5-carboxylic acid12: 63 ± 6.7 % (n = 9).

Replacement of the carbonyl oxygen in compounds 2 and 6 by a cyanamide functionality led to a drop in activity, which was even more pronounced than in case of the α-ketoheterocyclic derivatives. The indole-5-carboxylic acid 27 inhibited cPLA₂α with an IC₅₀ value greater than 10 µM, the IC₅₀ value of the tetrazole 29 against FAAH was only 5.3 µM. In the metabolism experiments, both compounds showed modest metabolic stability. In contrast to the ketones described above, no main metabolites of 27 and 29 could be detected after the metabolic reactions by LC/MS.

For the nitrile derivatives 39 and 43, an inhibitory potency against cPLA₂α and FAAH could not be measured at the highest test concentration of 10 µM, while the metabolic stability of these compounds was the highest of all substances evaluated in this study. Just like in case of the cyanamide derivatives, a characteristic main metabolite could not be identified in the phase I metabolism studies with rat liver homogenate by LC/MS.

Conclusion

In summary, the replacement of the activated ketone functionality of the cPLA₂α inhibitor 2 and the FAAH inhibitors 4 and 6, respectively, by α-ketoheterocycle, cyanamide, and nitrile was not well tolerated. The resulting compounds were only weakly or even not active against the enzymes. A pronounced metabolic stability could only be detected for the nitrile compounds 39 and 43. The obtained results could be helpful for the development of clinical active cPLA₂α and FAAH inhibitors.

Supplementary material available online

Supplementary Information

Declaration of interest

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