An Efficient Synthesis of γ-Aminobutyric Acid-Derived Phospholipase A₂ Inhibitors from Acyl Cyanophosphoranes and Amine Derivatives

아실 시아노포스포레인과 아민 유도체로 부터 γ-아미노부틸산에서 유도된 포스포리파제 A₂ 저해제의 효과적인 합성

Abstract

A series of ${\gamma}$-aminobutyric acid-derived, potent human cytosolic phospholipase A$_2$ inhibitors have been prepared from acyl cyanophosphoranes and amine derivatives in a convergent manner. The ${\alpha}$-keto amide functionalities in the inhibitors have been introduced as electrophilic fragments via direct coupling reactions between the labile ${\alpha},{\beta}$-diketo nitriles and ${\gamma}$-aminobutyric acid t-butyl ester derivatives at -78 $^{\circ}C$ in moderate to good yields.

일련의 유효한 ${\gamma}$-아미노부틸산에서 유도된 인간 시토솔릭 포스포리파제 A$_2$ 저해제를 아실 시아노포스포레인과 아민 유도체로 부터 수렴적으로 합성하였다. 저해제 내의 친전자적인 단편인 알파-케토 아미드 작용기는 불안정한 ${\alpha},{\beta}$-디케토 니트릴과 ${\gamma}$-아미노부틸산 삼차-부틸에스테르 유도체와의 직접 융합반응에 의하여 -78 $^{\circ}C$에서 양호한 수율로 합성하였다.

INTRODUCTION

There has been a considerable research interest in phospholipase A2 (PLA2) inhibitors over the years since these compounds are known to have antiinflammatory properties,1 and therefore are considered to play key roles in controlling inflammatory diseases.2 Several types of inhibitors e.g., arachidonyl trifluoromethyl ketone,3a methyl arachidonyl fluorophosphonate,3b fatty acid tricarbonyls,3c amides of amino acids with long-chain amines,2b pyrrolidinebased inhibitors,3d,e and very recently lipophilic 2-oxoamide butyric acids3f have been reported. Among the electron-deficient carbonyl residues incorporated in the inhibitors, α-keto amide unit is of special interest since it is the most frequently encountered electrophilic ketone pharmacophore found in many potent inhibitors of proteolytic enzymes such as proteases, lipases and serine esterases.4 A number of synthetic routes to a-keto amide unit including oxidation of α-hydroxy amide have been reported in the literature,5 however, these approaches may have some limitations such as lengthy procedures, harsh reaction conditions, or limited scope. Wasserman et. al. recently reported an elegant synthetic approach to this unit utilizing cyanophosphorane chemistry under mild conditions in a convergent manner (Eq. 1).6

This approach has been well exemplified in the syntheses of various biologically important natural products.7 The same approach has been successfully applied to the synthesis of tricarbonyl unit.8

Very recently we have reported a convergent synthesis of 2-oxo amide triacylglycerol analogs of human gastric lipase inhibitors based on acyl cyanophosphorane chemistry.9 As our continuing effort in this chemistry, we herein wish to report a successful synthesis of lipophilic 2-oxoamide g-aminobutyric acids, recently reported human cytosolic phospholipase A2 inhibitors and a new analog, in a convergent manner.

 

EXPERIMENTAL SECTION

All reactions were carried out in oven-dried glassware under an argon atmosphere. THF was purified by distillation from Na/benzophenone, and CH2Cl2 was dried over CaH2. Melting points were determined on an Electrothermal melting-point apparatus and were uncorrected. Optical rotations were measured on Jasco P-1020 Auto Polarimeter. FT IR spectra were obtained on a Jasco FT-IR/410 using KBr or as CHCl3 solution. 1H (400 MHz), 13C NMR (100 MHz) and 19F NMR (376 MHz) spectra were recorded on Jeol JNM-EX400 FT NMR spectrometer using CDCl3 as solvent, and chemical shifts (δ) are given in ppm downfield with respect to the solvent or tetramethylsilane as an internal standard (for 1H and 13C NMR) or CFCl3 as an external standard (for 19F NMR). Mass spectra were measured with a VG Autospec Ultima instrument in EI (70 eV) mode. Flash column chromatography was carried out on silica gel (Merck, 230-400 mesh) and solvents were eported as V/V ratio mixtures. (Cyanomethylene)-triphenylphosphorane was synthesized from (cyanomethylene) tri-phenylphosphonium chloride according to the known procedure.7a IBX (o-Iodoxybenzoic acid) was prepared from 2-iodobenzoic acid following the literature procedure.10 (Cyanomethylene)triphenylphosphonium chloride and N-Cbz-L-norleucine were purchased from Lancaster Synth. Inc.. EDCI, DMAP, NMM, HOBT, TFA (trifluoroacetic acid) and DME (1,2-dimethoxyethane) were purchased from Aldrich Chem. Co., and used without further purification. Other commercial reagents were purchased from commercial sources and used as received unless otherwise stated.

(Triphenylphosphoranylidene)pentadecanoylacetonitrile (2a). This ylide was prepared from pentadecanoic acid and (cyanomethylene)triphenylphosphorane (1.10 equiv) using EDCI (1.10 equiv)/DMAP (0.10 equiv) in 88% yield according to the literature procedure.9 Rf = 0.32 (hexane/EtOAc, 2/1); mp 123.0-124.0 ℃; IR (KBr) 3066, 2949, 2922, 2173, 1581 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 3H, J=6.8 Hz, CH3-), 1.25 (m, 22H, CH3(CH2)11-), 1.66 (m, 2H, -CH2CH2C(=O)-), 2.68 (t, 2H, J=7.3 Hz, -CH2C(=O)-), 7.45-7.73 (m, 15H, aromatic); 13C NMR (CDCl3) δ 14.13, 22.69, 25.62, 29.36, 29.44, 29.53, 29.62, 29.67, 29.71, 31.93, 39.66 (d, J= 6.7 Hz), 48.34 (d, J= 126.4 Hz), 122.85 (d, J=16.6 Hz), 123.48 (d, J=93.2 Hz), 129.08 (d, J=12.5 Hz), 133.00 (d, J=2.5 Hz), 133.58 (d, J=9.9 Hz), 197.68 (d, J=3.3 Hz).

(Triphenylphosphoranylidene)undecanoylacetonitrile (2b). Compound 2b was prepared from undecanoic acid according to the same procedures described for 2a in 85% yield. A white solid; Rf = 0.47 (Hexane/EtOAc, 3/2); mp 126.0-127.0 ℃; IR (KBr) 3072, 3026, 2924, 2172, 1583 cm−1; 1H NMR (CDCl3) d 0.88 (t, 3H, J=6.8 Hz, CH3-), 1.26 (m, 14H, CH3(CH2)7-), 1.66 (m, 2H, -CH2CH2C(=O)-), 2.68 (t, 2H, J=7.6 Hz, -CH2C(=O)-), 7.42-7.70 (m, 15H, aromatic); 13C NMR (CDCl3) d 14.13, 22.69, 25.62, 29.35, 29.44, 29.51, 29.61, 31.92, 39.66 (d, J=6.7 Hz), 48.34 (d, J=125.6 Hz), 122.85 (d, J=17.4 Hz), 123.48 (d, J=93.2 Hz), 129.08 (d, J=13.3 Hz), 133.00 (d, J=2.5 Hz), 133.58 (d, J=9.9 Hz), 197.68 (d, J=3.3 Hz); MS (EI) m/z 183, 252, 262, 301, 318, 328, 343, 469; HRMS calcd for C31H36NOP 469.2535, found 469.2551.

t-Butyl N-(benzyloxycarbonyl)-γ-aminobutyrate (5). A stirred solution of N-(benzyloxycarbonyl)-γ-aminobutyric acid 411 (834 mg, 3.52 mmol) in THF (10 mL) was treated successively with Et3N (491 mL, 1.0 equiv) and 2,4,6-trichlorobenzoyl chloride (550 mL, 1.0 equiv), and the resulting mixture was stirred for 40 min at rt under Ar. The reaction mixture was filtered, and the filter-cake was washed with dry THF (10 mL). The solvent was evaporated under Ar to afford the mixed anhydride as a white gummy residue, which was dissolved again in dry benzene (10 mL). To this solution was transferred a solution of t-BuOH (673 μL, 2.0 equiv) and DMAP (860 mg, 2.0 equiv) in dry benzene (5 mL) via cannula, and the resulting mixture was stirred for 1.5 h at rt under Ar. The reaction mixture was diluted with Et2O (30 mL), washed successively with 0.1 N HCl, H2O, and saturated NaHCO3, dried over Na2SO4, filtered, and concentrated in vacuo to provide an oily residue. Flash column chromatography of the residue on SiO2 using (Hexane/EtOAc, 3/1) gave pure compound 511 (675 mg, 65%) as a colorless oil. Rf =0.43 (Hexane/ EtOAc, 2/1); IR (CHCl3) 3452, 3019, 2981, 1720, 1517 cm−1; 1H NMR (CDCl3) δ 1.44 (s, 9H, -C(CH3)3), 1.79 (m, 2H, -CH2CH2CH2-), 2.27 (t, 2H, J=7.3 Hz, -CH2CO2-), 3.23 (q, 2H, J=6.5 Hz, -NHCH2-), 4.93 (br s, 1H, -NH-), 5.09 (s, 2H, PhCH2-), 7.35 (m, 5H, aromatic); 13C NMR (CDCl3) δ 25.19, 28.07, 32.80, 40.51, 66.63, 80.53, 128.09, 128.35, 128.51, 136.57, 156.41, 172.61; MS (EI) m/z 57 , 91, 107, 108, 237, 293; HRMS calcd for C16H23NO4 293.1627, found 293.1611.

t-Butyl g-aminobutyrate (3a). A stirred suspension of compound 5 (337 mg, 1.15 mmol) and 10% Pd/C (65mg, ca. 20%) in EtOH (10mL) was hydrogenated using H2 balloon (1 atm) for 1.5 h at rt. The mixture was filtered over Celite, and the filter-cake was washed with EtOH, then Et2O. The solvent was carefully evaporated under reduced pressure while maintaining the bath temperature below 20 ℃ to afford pure amine 3a11,12 (173 mg, 95%) as a colorless oil. IR (CHCl3) 3446, 2980, 1719 cm−1; 1H NMR (CDCl3) δ 1.45 (s, 9H, -C(CH3)3), 1.74 (m, 2H, -CH2CH2CH2-), 1.85 (br s, 2H, NH2-), 2.27 (t, 2H, J=7.3 Hz, -CH2CO2-), 2.74 (br s, 2H, NH2CH2-); 13C NMR (CDCl3) δ 28.02, 28.79, 32.92, 41.35, 80.16, 172.84; MS (EI) m/z 55, 57, 71, 84, 86, 91, 102, 159; HRMS calcd for C8H17NO2 159.1259, found 159,1258.

(2S)-2-[(Benzyloxycarbonyl)amino]pentan-1-ol (7). To a stirred, precooled (-15 ℃) solution of N-Cbz- L-norleucine (408 mg, 1.54 mmol) in DME (10 mL) was added N-methylmorpholine (190 mL, 1.0 equiv), followed by i-butyl chloroformate (226 mL, 1.0 equiv) and the resulting mixture was stirred for 1 min under Ar. The white solid of N-methylmorpholine. HCl formed immediately. The reaction mixture was filtered, and the filter-cake was washed with DME (10 mL). The combined filtrate was cooled again in an ice bath, then treated successively with NaBH4 (98.1 mg, 1.5 equiv) in H2O (2 mL) then H2O (30 mL) immediately. The reaction mixture was stirred for additional 5 min at 0 ℃, quenched with 0.1N HCl (15 mL), and extracted with EtOAc (15 mL × 3). The combined organic layers were washed with saturated NaHCO3, brine, dried over MgSO4, filtered, and concentrated in vacuo. Purification of the crude product by flash column chromatography on SiO2 (Hexane/EtOAc, 3/2) gave 277 mg (72%) of 7 as a white solid. Rf = 0.53 (Hexane/EtOAc, 1/1); mp 91.0-92.0 ℃; -22.0o (c 0.78, CH2Cl2); IR (KBr) 3319, 3065, 2952, 2858, 1687, 1542 cm−1; 1H NMR (CDCl3) δ 0.89 (br s, 3H, -CH3), 1.23-1.48 (m, 5H, -CHCHH- overlapped with -(CH2)2CH3), 1.52 (m, 1H, -CHCHH-), 2.44 (br s, 1H, -OH), 3.55 (br s, 1H, -NHCH-), 3.68 (d, 2H, J=8.4 Hz, -CH2OH), 4.92 (d, 1H, J=6.4 Hz, -NH-), 5.10 (s, 2H, PhCH2O-), 7.35 (m, 5H, aromatic); 13C NMR (CDCl3) δ 13.96, 22.56, 28.13, 31.13, 53.31, 65.62, 66.87, 128.12, 128.18, 128.55, 136.40, 156.86; MS (EI) m/z 91, 176, 220, 251; HRMS calcd for C14H21NO3 251.1521, found 251.1521.

(2S)-2-[(Benzyloxycarbonyl)amino]pentanal (8). IBX (393 mg, 1.5 equiv) was added to a solution of amino alcohol 7 (235mg, 0.935mmol) in dry DMSO (5 mL) and the resulting mixture was stirred for 5h at rt under Ar. The solution was diluted with H2O (20 mL), filtered over Celite, and extracted with Et2O (15mL × 3). The combined organic layers were washed with brine, dried over MgSO4, filtered, and evaporated in vacuo. Filtration of the crude product through short column (SiO2, Hexane/EtOAc, 3/1) provided aldehyde 8 (182 mg, 78%) as a colorless oil, which was pure enough for the next reaction. This almost pure aldehyde 8 was subjected immediately to the next reaction without further purification. Rf = 0.38 (Hexane/EtOAc, 2/1); [α]23D+26.6o (c 0.80, CH2Cl2); IR (CHCl3) 3435, 2960, 2863, 1717, 1508 cm−1; 1H NMR (CDCl3) δ 0.90 (t, 3H, J=6.1 Hz, -CH3), 1.33 (m, 4H, -(CH2)2CH3), 1.89 (m, 1H, -CHCHH-), 2.17 (m, 1H, -CHCHH-), 4.31 (dd, 1H, J1=13.0 Hz, J2=7.0 Hz, -NHCH-), 5.12 (s, 2H, PhCH2O-), 5.38 (d, 1H, J=6.0 Hz, -NH-), 7.36 (m, 5H, aromatic), 9.58 (s, 1H, -CH(=O)); 13C NMR (CDCl3) δ 13.81, 22.47, 27.13, 28.88, 60.24, 67.11, 128.14, 128.27, 128.58, 136.17, 156.10, 199.39; MS (EI) m/z 91, 92, 176, 220, 249; HRMS calcd for C14H19NO3 249.1365, found 249.1351.

t-Butyl (E, 4S)-4-[(benzyloxycarbonyl)amino]oct-2-enoate (9). A solution of amino aldehyde 8 (169 mg, 0.679 mmol) and (t-butoxycarbonylmethylene) triphenyl-phosphorane (282 mg, 1.10 equiv) in dry THF (10 mL) was heated at reflux for 1.5 h under Ar. After evaporation of the solvent in vacuo, the oily residue was purified by flash column chromatography on SiO2 (Hexane/Et2O, 2/1) to afford 9 (171 mg, 73%) as a colorless oil. Rf = 0.32 (Hexane/ Et2O, 2/1); [α]23D-10.8o (c 1.08, CH2Cl2); IR (CHCl3) 3439, 3019, 2980, 2934, 2862, 1712, 1508 cm−1; 1H NMR (CDCl3) δ 0.89 (br s, 3H, -(CH2)3CH3), 1.20-1.75 (m, 15H, -(CH2)3CH3 overlapped with -C(CH3)3), 4.33 (m, 1H, -NHCH-), 4.76 (d, 1H, J=8.3 Hz, -NH-), 5.11 (dd, 2H, J1=18.0 Hz, J2=12.0 Hz, PhCH2O-), 5.84 (d, 1H, J=15.5 Hz, -CH=CHCO2-), 6.73 (dd, 1H, J1=15.5 Hz, J2=5.6 Hz -CH=CHCO2-), 7.35 (m, 5H, aromatic); 13C NMR (CDCl3) δ 13.88, 22.38, 27.72, 28.11, 34.38, 51.95, 66.94, 80.57, 122.66, 128.16, 128.20, 128.56, 136.32, 146.74, 155.68, 165.61; MS (EI) m/z 91, 156, 190, 200, 234, 290, 291, 347; HRMS calcd for C20H29NO4 347.2097, found 347.2066.

t-Butyl (4S)-4-aminooctanoate (3b). Compound 3b was prepared from 9 according to the same procedures described for 3a12 in 100% yield. A colorless oil; [α]23D-2.8o (c 0.85, CH2Cl2); IR (CHCl3) 2964, 1719 cm−1; 1H NMR (CDCl3) δ 0.90 (t, 3H, J=6.3 Hz, -CH3), 1.15-1.62 (m, 16H, -CHCHH- overlapped with -CH2CH2CO2-,-(CH2)2CH3, -C(CH3)3), 1.74 (br s, 3H, -CHCHH- overlapped with -NH2), 2.29 (m, 2H, -CH2CO2H), 2.71 (br s, 1H, -NH2CH-); 13C NMR (CDCl3) δ 13.96, 22.66, 27.99, 28.17, 32.29, 32.86, 37.52, 50.63, 80.02, 173.10; MS (EI) m/z 55, 57, 71, 84, 86, 91, 102, 215; HRMS calcd for C12H25NO2 215.1885, found 215.1880.

S-MTPA amide (10a). To an ice-cooled solution of amine 3b (15.0 mg, 0.0698 mmol) and HOBT (11.3 mg, 1.20 equiv) in CH2Cl2 (5 mL) was added S-(-)-MTPA (19.6 mg, 1.20 equiv), followed by EDCI (16.1 mg, 1.20 equiv), and the resulting mixture was stirred for 1 h at 0 ℃ then overnight at rt under Ar. The reaction mixture was diluted with CH2Cl2 (10 mL), washed with H2O and saturated NaHCO3, dried over MgSO4, filtered, and concentrated. Purification of the crude product was done by preparative-TLC (SiO2, Hexane/Et2O, 3/1). Rf = 0.41 (Hexane/EtOAc, 5/1); 1H NMR (CDCl3) δ 0.84 (t, 3H, J=6.8 Hz, -CH3), 1.16-1.74 (m, 16H, -CHCHH(CH2)2- overlapped with -CH2CH2CO2-, -(CH2)2CH3, -C(CH3)3), 1.87 (m, 1H, -CHCHH(CH2)2-), 2.29 (m, 2H, -CH2CO2-), 3.41 (s, 3H, -OCH3), 3.96 (m, 1H, -NHCH-), 6.64 (d, 1H, J=9.3 Hz, -NH-), 7.40 (br t, 3H, J=3.2 Hz, Ar-Hm,p), 7.52 (d, 1H, J=3.9 Hz, Ar-Ho); 19F NMR (CDCl3) d 11.10 (s, -OCF3)

R-MTPA amide (10b). Compound 10b was prepared from 3b and R-(+)-MTPA according to the same procedures described for 10a . Rf = 0.41 (Hexane/ EtOAc, 5/1); 1H NMR (CDCl3) δ 0.89 (t, 3H, J=6.8 Hz, -CH3), 1.15-1.74 (m, 16H, -CHCHH(CH2)2- overlapped with -CH2CH2CO2-, -(CH2)2CH3, -C(CH3)3), 1.86 (m, 1H, -CHCHH(CH2)2-), 2.16 (m, 2H, -CH2CO2-), 3.43 (s, 3H, -OCH3), 3.96 (m, 1H, -NHCH-), 6.69 (d, 1H, J=9.3 Hz, -NH-), 7.40 (br t, 3H, J=3.4 Hz, Ar-Hm,p), 7.54 (br d, 2H, J=3.9 Hz, Ar-Ho); 19F NMR (CDCl3) δ 11.10 (s, -OCF3)

t-Butyl 4-[(2-oxohexadecanoyl)amino]butanoate (11a). A precooled (-78 ℃) solution of phosphorane ylide 2a (228 mg, 1.3 equiv) in CH2Cl2 (25 mL) was bubbled with O3 for 5 min, then purged with Ar for 5 min to afford a pale yellow solution. To this solution was transferred a precooled (-78 ℃) solution of amine 3a (53.2 mg, 0.335 mmol) in CH2Cl2 (5 mL) via cannula, and the resulting solution was stirred for 30 min at -78 ℃, then allowed to warm to rt over 30 min under Ar. The solvent was evaporated under reduced pressure to afford a pale yellow solid, which was purified by flash column chromatography on SiO2 using (Hexane/EtOAc/ CH2Cl2, 16/2/1) to give the coupled product 11a (77.0 mg, 56%) as an off-white solid. Rf = 0.53 (Hexane/Et2O, 1/1); mp 59.0-60.5 ℃; IR (KBr) 3348, 2981, 2956, 2920, 2850, 1728, 1660, 1523 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 3H, J=6.8 Hz, -CH3), 1.25 (br s, 22H, -(CH2)11CH3), 1.45 (s, 9H, -C(CH3)3), 1.59 (m, 2H, -CH2CH2C(=O)-), 1.84 (m, 2H, -NHCH2CH2-), 2.28 (t, 2H, J=7.2 Hz, -CH2CO2-), 2.91 (t, 2H, J=7.4 Hz, -CH2C(=O)-), 3.33 (q, 2H, J=6.7 Hz, -NHCH2-), 7.12 (br s, 1H, -NH-); 13C NMR (CDCl3) δ 14.13, 22.70, 23.20, 24.49, 28.09, 29.08, 29.34, 29.37, 29.45, 29.60, 29.65, 29.68, 29.69, 31.93, 32.83, 36.75, 38.75, 80.73, 160.32, 172.28, 199.27; MS (EI) m/z 57, 86, 112, 130, 186, 225, 338, 411; HRMS calcd for C24H45NO4 411.3349, found 411.3316.

4-[(2-Oxohexadecanoyl)amino]butanoic acid (1a). To a solution of 11a (42.0 mg, 0.102 mmol) in CH2Cl2 (1.5 mL) was added TFA (1.5 mL) by syringe, and the resulting solution was stirred for 1.5 h at rt under Ar. The volatiles were evaporated in vacuo to provide a brown residue, which was flash column chromatographed on SiO2 using (CH2Cl2/EtOAc/ AcOH, 7/1/1%) as eluent. The fractions containing the desired product were combined, diluted with heptane, concentrated in vacuo while maintaining bath temperature below 20 ℃, and dried under high vacuum to afford 1a3f (35.8 mg, 99%) as an offwhite solid. mp 99-100 ℃; Rf = 0.53 (CH2Cl2/EtOAc/ AcOH, 2/1/1%); IR (KBr) 3334, 2916, 2850, 1717, 1658, 1525 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 3H, J=6.8 Hz, -CH3), 1.25 (br s, 22H, -(CH2)11CH3), 1.59 (m, 2H, -CH2CH2C(=O)-), 1.91 (m, 2H, -NHCH2CH2-), 2.43 (t, 2H, J=6.8 Hz, -CH2CO2H), 2.91 (t, 2H, J= 7.2 Hz, -CH2C(=O)-), 3.38 (q, 2H, J=6.4 Hz, -NHCH2-), 7.15 (br s, 1H, -NH-); 13C NMR (CDCl3) δ 14.13, 22.71, 23.20, 24.23, 28.08, 29.37, 29.46, 29.61, 29.67, 29.68, 29.70, 31.20, 31.94, 36.77, 38.59, 160.47, 178.06, 199.22; MS (EI) m/z 55, 57, 71, 85, 86, 112, 130, 225, 355; HRMS calcd for C20H37NO4 355.2723, found 355.2725.

t-Butyl (4S)-4-[(2-oxododecanoyl)amino] octanoate (11b). Compound 11b was prepared from 2b and 3b according to the same procedures described for 11a in 52% yield. A colorless oil; Rf = 0.38 (Hexane/ Et2O, 3/1); [α]26D -4.2o (c 1.05, CH2Cl2); IR (CHCl3) 3391, 2929, 2857, 1719, 1684, 1521 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 6H, J=6.6 Hz, 2 x (-CH3)), 1.20-1.75 (m, 32H, -CHCHH- overlapped with -CH2CH2CO2-, -(CH2)2CH3, -C(CH3)3, -(CH2)8CH3), 1.87 (m, 1H, -CHCHH-), 2.23 (m, 2H, -CH2CO2-), 2.91 (td, 2H, J1=7.4 Hz, J2=1.5 Hz, -CH2C(=O)-), 3.86 (m, 1H, -NHCH-), 6.75 (d, 1H, J=9.2 Hz, -NH-); 13C NMR (CDCl3) δ 13.94, 14.12, 22.49, 22.69, 23.23, 27.96, 28.08, 29.09, 29.31, 29.35, 29.45, 29.56, 29.92, 31.90, 32.15, 34.82, 36.81, 49.37, 80.55, 159.96, 172.53, 199.53; MS (EI) m/z 57, 125, 142, 143, 168, 186, 338, 411; HRMS calcd for C24H45NO4 411.3349, found 411.3357.

(4S)-4-[(2-Oxododecanoyl)amino]octanoic acid (1b). Compound 1b was prepared from 11b according to the same procedures described for 1a in 98% yield. An off-white solid; mp 57.0-59.0 ℃ (lit.3f mp 50-52 ℃); Rf=0.32 (CH2Cl2/EtOAc/AcOH, 7/1/1%); [α]23D-6.3o (c 0.74, CHCl3) (lit.3f [a]D -1.8o (c 0.5, CHCl3)); IR (KBr) 3312, 2953, 2850, 1733, 1717, 1698, 1661, 1541 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 6H, J=6.2Hz, 2 x (-CH3)), 1.20-1.62 (m, 22H, CH2CH2CO2H overlapped with -(CH2)2CH3, -(CH2)8CH3)), 1.74 (m, 1H, -CHCHH-), 1.95 (m, 1H, -CHCHH-), 2.37 (m, 2H, -CH2CO2H), 2.92 (td, 2H, J1=7.4 Hz, J2=1.3 Hz, -CH2C(=O)-), 3.92 (m, 1H, -NHCH-), 6.82 (d, 1H, J=9.6 Hz, -NH-); 13C NMR(CDCl3) δ 13.93, 14.12, 22.46, 22.69, 23.22, 27.98, 29.08, 29.32, 29.36, 29.46, 29.57, 29.97, 30.73, 31.90, 34.79, 36.85, 49.34, 160.19, 177.93, 199.46; MS (EI) m/z 55, 57, 71, 83, 97, 117, 125, 143, 168, 186, 355; HRMS calcd for C20H37NO4 355.2723, found 355.2723.

t-Butyl (4S)-4-[(2-oxohexadecanoyl)amino]octanoate (11c). Compound 11c was prepared from 2a and 3b according to the same procedures described for 11a in 50% yield. An off-white solid; Rf = 0.41 (Hexane/ Et2O, 4/1); mp 45.0-45.5 ℃; [α]26D-4.3o (c 0.65, CH2Cl2); IR (KBr) 3333, 2918, 2849, 1721, 1664, 1528 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 6H, J=6.6 Hz, 2 x (-CH3)), 1.18-1.75 (m, 40H, -CHCHH- overlapped with -CH2CH2CO2H, -(CH2)2CH3, -C(CH3)3, -(CH2)12CH3), 1.86 (m, 1H, -CHCHH-), 2.23 (m, 2H, -CH2CO2-), 2.91 (td, 2H, J1=7.4 Hz, J2=1.7 Hz, -CH2C(=O)-), 3.86 (m, 1H, -NHCH-), 6.74 (d, 1H, J=9.2 Hz, -NH-); 13C NMR (CDCl3) δ 13.94, 14.13, 22.49, 22.70, 23.23, 27.95, 28.08, 29.09, 29.37, 29.46, 29.61, 29.66, 29.68, 29.69, 29.91, 31.93, 32.15, 34.81, 36.82, 49.37, 80.56, 159.96, 172.53, 199.53; MS (EI) m/z 57, 73, 91, 117, 147, 207, 221, 265, 281, 295, 355, 369, 429, 467; HRMS calcd for C28H53NO4 467.3975, found 467.3952.

(4S)-4-[(2-Oxohexadecanoyl)amino]octanoic acid (1c). Compound 1c was prepared from 11c according to the same procedures described for 1a in 98% yield. An off-white solid; Rf = 0.40 (CH2Cl2/EtOAc/ AcOH, 5/1/1%); mp 77.0-78.0 ℃; [α]26D -5.8o (c 0.69, CHCl3); IR (KBr) 3327, 2954, 2916, 2849, 1733, 1717, 1699, 1655, 1541 cm−1; 1H NMR (CDCl3) δ 0.88 (t, 6H, J=6.4 Hz, 2 x (-CH3)), 1.18-1.65 (m, 30H, -CH2CH2CO2H overlapped with -(CH2)2CH3, -(CH2)12CH3), 1.75 (m, 1H, -CHCHH-), 1.94 (m, 1H, -CHCHH-), 2.36 (m, 2H, -CH2CO2H), 2.92 (td, 2H, J1=7.2 Hz, J2=2.1 Hz -CH2C(=O)-), 3.92 (m, 1H, -NHCH-), 6.83 (d, 1H, J=9.6 Hz, -NH-); 13C NMR (CDCl3) δ 13.93, 14.13, 22.46, 22.71, 23.22, 27.98, 29.09, 29.37, 29.47, 29.62, 29.67, 29.68, 29.70, 29.97, 30.72, 31.93, 34.79, 36.85, 49.34, 160.19, 177.93, 199.45; MS (EI) m/z 55, 57, 71, 83, 97, 125, 143, 168, 186, 225, 411; HRMS calcd for C24H45NO4 411.3349, found 411.3349.

 

RESULTS AND DISCUSSION

Two most active inhibitors 1a/b among the several inhibitors tested in the recent study3f have been chosen as target molecules in our synthesis, and a simple retrosynthetic analysis of 1a/b showed that they should be readily accessed from acyl cyanophosphoranes and the amine derivatives under mild conditions (Scheme 1).

Scheme 1.Retrosynthetic analysis of γ-aminobutyric acid-based phospholipase A2 inhibitors 1a, b, c.

The required acyl cyanophosphoranes 2a/b were readily prepared in high yields from the commercially available carboxylic acids and cyanomethylenetriphenyl- phosphorane13 according to the method described in the literature.6

To synthesize the appropriate amine derivatives for the key coupling reactions, t-butyl ester was adopted as an acid protecting group since it could be easily deblocked with TFA14 under mild conditions in the final step (Scheme 2).

Therefore, several t-butyl ester protecting methods e.g., t-BuOH/(EDCI, DMAP),15a t-BuOH/(CDI, DBU)15b and t-BuOH/(2,4,6-trichlorobenzoyl chloride, DMAP)15c were attempted for N-Cbz g-aminobutyric acid 4, and t-BuOH/(2,4,6-trichlorobenzoyl chloride, DMAP) gave the best result. This N-Cbz g-aminobutyric acid t-butyl ester 5 was then catalytically hydrogenated with Pd-C (10%)/H2 (1 atm)16 to provide g-aminobutyric acid t-butyl ester 3a in excellent yield.

The synthesis of another amine derivative 3b began with the commercially available N-Cbz-L-norleucine 6, which was treated with i-butyl chloroformate/ NaBH417 to afford amino alcohol 7 in 72% yield. In order to oxidize amino alcohol 7 to amino aldehyde 8, several oxidizing reagents such as Swern oxidation,18a TCT/(DMSO, Et3N),18b and IBX (2-iodoxybenzoic acid)18c were attempted. Among these, IBX was determined to be the best in terms of operational simplicity and overall yield (78%). This amino aldehyde 8 are known to be generally unstable and easily racemized, so it was subjected immediately under the Wittig reaction conditions with (t-butoxycarbonylmethylene)tri-phenylphosphorane to give 9 as a colorless oil in 73% yield. Catalytic hydrogenation of the double bond and simultaneous unmasking of N-Cbz group were accomplished by the use of Pd-C (10%)/H2 (1 atm) to provide the required amine derivative 3b as a colorless oil. At this stage, we have checked possible racemization that could have occurred during the last three steps: amine 3b was reacted with S-(-)- TPA, and R-(+)-MTPA in the presence of EDCI/ HOBT,19 and NMR (19F & 1H) spectra of each crude and purified MTPA amide were carefully compared. Unfortunately, CF3-peaksofboth MTPA amides (10a/b) appeared at the exactly same position (11.10 ppm) as a sharp singlet in 19F NMR. However, several proton peaks corresponding to NH- (d), CH3O- (s), CH3- (t) and especially -CH2COO- (m) of purified S-MTPA amide (10a) were well resolved from those of diastereomeric R-MTPA amide (10b) by 0.046 ppm (18.4 Hz), 0.018 ppm (7.2 Hz), 0.051 ppm (20.4 Hz) and 0.13 ppm (52.0 Hz), respectively. The 1H NMR spectra of crude S-MTPA and R-MTPA amide did not have any corresponding diastereomeric amide proton peaks at all, which demonstrated that no racemization took place during last three steps and optical integrity at the chiral center was retained.

Scheme 2.Synthesis of γ-aminobutyric acid t-butyl ester derivatives 3a, b.

With both requisite starting materials 2,3 in hand we carried out the key coupling reaction according to the reported procedures (Scheme 3):6 A precooled (-78 ℃) solution of 2a (1.3 equiv) in CH2Cl2 was treated first with O3, then purged with dry Ar to afford a pale yellow solution which was reacted with amine derivative 3a under the mild reaction condition. TLC-analysis of the crude product clearly showed that one major compound considered to be the desired product formed together with several byproducts. Purification of the crude product by flash column chromatography (SiO2) afforded pure coupled product 11a in 56% yield. Attempts to increase the yield by using more amount of ylide 2a or extending O3-bubbling time/Ar-purging time turned out to be fruitless at all. The structure of the coupled product 11a was easily confirmed mainly by NMR: in 1H NMR, the amide proton (1H) appeared at 7.11 ppm as a broad singlet, and the methlene protons (2H) adjacent to dicarbonyl unit appeared at 2.90 ppm as a triplet, and t-butyl protons (9H) appeared at 1.45 ppm as a sharp singlet, etc.; in 13C NMR, three carbonyl carbons due to a-carbonyl carbon, amide carbon, and t-butyl ester carbon appeared at 199.27, 160.32, and 172.28 ppm, respectively. IR and LR/HR mass also strongly supported the proposed structure of 11a.

Scheme 3.Synthesis of γ-aminobutyric acid-based phospholipase A2 inhibitors 1a, b, c.

The next step for the final product 1a seemed to be straightforward. Deblocking of t-butyl ester group of 11a with TFA was smoothly progressed under mild conditions (rt, 1.5h), and after purification by flash column chromatography on SiO2 (CH2Cl2/EtOAc/ AcOH, 7/1/1%) the first target molecule 1a was obtained as a white solid in almost quantitative yield. All spectral data including 1H and 13C NMR unequivocally confirmed the proposed structure of pure acid 1a.

Having established the optimum reaction condition for coupling/deprotection of t-butyl ester group, we then attempted to synthesize the second target molecule 1b by following the similar reaction protocol as for 1a. Thus, treatment of 2b with O3/Ar to provide the diketo nitrile intermediate as a pale yellow solution, which was then reacted with amine 3b to afford the coupled product 11b as a colorless oil in 52% yield after flash column chromatography. 1H and 13C NMR were very informative for the proposed structure of 11b: in 1H NMR, the amide proton peak (1H) appeared at 6.75 ppm as a broad doublet, and the chiral proton peak (1H) appeared at 3.86 ppm as a multiplet, and the two methyl proton peaks (6H) appeared at 0.88 ppm as a triplet, etc.; in 13C NMR, three carbonyl carbon peaks corresponding to a-carbonyl carbon, amide carbon, and t-butyl ester carbon appeared at 199.53, 159.96, and 172.53 ppm, respectively, together with exactly 19 carbon peaks as expected. Having the coupled product 11b in hand, we then carried out the deprotection reaction of 11b with TFA, and the crude product was purified by flash column chromatography on SiO2 to provide pure acid 1b in 98% yield as an off-white solid. All spectral data of 1b including 1H and 13C NMR were matched well with those of the literature.3f Quite surprisingly, however, mp (57.0-59.0 ℃) and especially [α]24D (-6.3o, c 0.74, CHCl3) of our product 1b were considerably higher than reported values (mp 50.0-52.0 ℃; [α]D (-1.8o, c 0.5, CHCl3)),which implied that our product was much purer chemically and optically than the same compound reported in the literature. We are not sure of the plausible reasons for these big differences especially in [α]D value at this point, however, possible racemization during the peptide coupling step using EDCI/HOBT might be the source of the problem.20 Anyway, our synthetic approach turned out to be a racemization-free methodology for the synthesis of this kind of compounds.6

Some of the reported phospholipase A2 inhibitors were incorporating a longer aliphatic side chain than dodecanoic acid,2b,3c therefore it seemed worthwhile to attach a longer aliphatic side chain to the amine derivative 3b, and also in order to prove the generality of our approach for the synthesis of this kind of molecules, we next tried phosphorane ylide 2a for the coupling reaction with amine derivative 3b. By following the similar procedures as for 11a/b, the new coupled product 11c was obtained in 50% yield. This coupled product 11c was then deprotected with TFA as above to give a new, potentially active γ-aminobutyric acid-based phospholipase A2 inhibitor 1c in 98% yield.

 

CONCLUSION

We have shown that acyl cyanophosphoranes are efficient starting materials for the convergent synthesis of γ-aminobutyric acid-based, potent human cytosolic phospholipase A2 inhibitors. The conditions of the key coupling reaction between the diketo nitrile intermediates and the amine derivatives are mild, and the yields are moderate to good. The synthetic approach developed in this study should be applicable to the synthesis of a diverse group of γ-aminobutyric acid-based, potent human cytosolic phospholipase A2 inhibitor analogs simply by varying acyl cyanophosphoranes for a specific amine derivative or vice versa in a concise way. Furthermore the most active inhibitor 1b prepared by our synthetic route has higher mp and [α]D value than the same compound reported in the literature, which demonstrates that acyl cyanophosphorane approach is a racemization-free methodology.