Journal of Medicinal Chemistry
Article
(R)-4-Benzyl-3-butyryloxazolidin-2-one ((R)-9), (S)-4-Benzyl-3-
4-((S)-2-((R)-4-Benzyl-2-oxooxazolidine-3-carbonyl)butyl)-N-(4-
methoxy-2-(trifluoromethyl)benzyl)-benzamide ((4S,2R)-12), 4-
((R)-2-((S)-4-Benzyl-2-oxooxazolidine-3-carbonyl)butyl)-N-(4-me-
1
butyryloxazolidin-2-one ((S)-9). H NMR (300 MHz, DMSO-d6) δ
= 7.36−7.17 (m, 5H, Har), 4.71−4.60 (m, 1H, CH), 4.32 (t, 3JHH= 8.5
1
3
Hz, 1H, CH), 4.17 (dd, JHH= 8.8 Hz, 4JHH = 2.9 Hz, 1H, CH), 3.01
thoxy-2-(trifluoromethyl)benzyl)benzamide ((4R,2S)-12). H NMR
(500 MHz, DMSO-d6): δ = 8.98 (t, 3JHH = 5.8 Hz, 1H, NH), 7.89 (d,
3JHH = 8.2 Hz, 2H, Har), 7.39−7.34 (m, 3H, Har), 7.20 (d, 4JHH = 2.6
2
3
2
(dd, JHH = 13.5 Hz, JHH = 3.5 Hz, 1H, CH), 2.92 (dd, JHH= 13.7
Hz, 3JHH = 7.5 Hz, 1H, CH), 2.76 (dd, 2JHH = 12.3 Hz, 3JHH = 4.8 Hz,
Hz, 1H, Har), 7.18−7.14 (m, 3H, Har), 7.12 (d, 3JHH = 8.6 Hz, 4JHH
=
2H, CH2), 1.70−1.53 (m, 2H, CH2), 0.93 (dd, 2JHH = 10.2 Hz, 3JHH
=
3
4
4.6 Hz, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO-d6) δ = 172.4,
153.4, 135.7, 129.5 (2 × C) 128.6 (2 × C) 126.9, 66.1, 54.2, 36.7,
17.3, 13.6 ppm.
2.5 Hz, 1H, Har), 6.81 (dd, JHH = 7.2 Hz, JHH = 2.0 Hz, 2H, Har),
3
4.70−4.65 (m, 1H, N-CH), 4.56 (d, JHH = 5.5 Hz, 2H, NH-CH2),
2
4.31 (t, 3JHH = 8.6 Hz, 1H, CH), 4.12 (dd, JHH = 8.9 Hz, 3JHH = 2.6
Hz, 1H, CH), 4.02 (dt, 2JHH = 13.0 Hz, 3JHH = 6.6 Hz, 1H, CH), 3.80
General Method for Asymmetric Alkylation. A solution of the
(R)-9 or (S)-9 (1.0 equiv) in dry THF (15 mL/mmol) was cooled to
−78 °C, and 1 M NaHMDS in THF (1.1 equiv) was added slowly.
After additional 30 min of stirring at −78 °C a solution of 7 (1.5
equiv) in THF was added. The solution was allowed to warm up to
room temperature overnight. The reaction mixture was quenched by
addition of saturated, aqueous NH4Cl solution. The product was
extracted with ethyl acetate. The organic phase was washed with brine
and dried over MgSO4. After removal of the solvent, the crude
product was purified by flash chromatography (hexane/ethyl acetate
100:0−80:20) and the product could be isolated as colorless oil
((4R,2S)-10: 566 mg, 1.2 mmol, 67%; (4S,2R)-10: 1.78 g, 3,8 mmol,
66%).
2
3
(s, 3H, O-CH3), 3.04 (dd, JHH = 13.2 Hz, JHH = 8.7 Hz, 1H, CH),
2.85−2.77 (m, 2H, 2 × CH), 2.71 (dd, 2JHH = 13.6 Hz, 3JHH = 6.9 Hz,
1H, CH), 1.73−1.63 (m, 1H, CH), 1.53−1.43 (m, 1H, CH), 0.89 (t,
3JHH = 7.4 Hz, 3H, CH3) ppm. 13C NMR (126 MHz, DMSO-d6): δ =
174.6, 166.2, 158.0, 153.1, 143.3, 135.3, 132.0, 130.1, 129.5 (2 × C),
129.2 (2 × C), 129.1, 128.4 (2 × C), 127.4 (2 × C), 126.8, 125.3,
123.1, 117.6, 111.5, 65.7, 55.6, 54.3, 45.5, 38.8, 36.7, 36.3, 24.7, 11.3
ppm. 19F-NMR (471 MHz, DMSO-d6): δ = −59.11 (Ar-CF3) ppm.
ESI-MS (m/z): 567.20 [M − H]−.
General Method for Oxidative Cleavage of Evans Auxiliar.
(4S,2R)-12 or (4R,2S)-12 (1.0 equiv) was dissolved in a THF:H2O
mixture (10 mL/mmol, 3:1 v/v) and cooled to 0 °C. Thereafter H2O2
(w = 30% in H2O, 8.0 equiv) and LiOH (2.0 equiv) were added. The
reaction mixture was allowed to warm up overnight. Solvent was
removed under reduced pressure, and pH was adjusted to 2. The
residue was extracted three times with ethyl acetate, and the
combined organic phase was dried over MgSO4. The crude product
was purified by preparative HPLC, and the two enantiomers could be
isolated as colorless solids ((S)-3: 1.04, 2.54 mmol, 87%; (R)-3:
0.91g, 2.22 mmol, 92%).
Benzyl 4-((S)-2-((R)-4-Benzyl-2-oxooxazolidine-3-carbonyl)-
butyl)benzoate ((4S,2R)-10), Benzyl 4-((R)-2-((S)-4-Benzyl-2-oxoox-
azolidine-3-carbonyl)butyl)benzoate ((4R,2S)-10). 1H NMR (250
MHz, DMSO-d6): δ = 7.95 (d, 2H, Har), 7.47−7.32 (m, 5H, Har),
3
4
7.17−7.10 (m, 3H, Har), 6.80 (dd, JHH = 6.4 Hz, JHH= 3.1 Hz, 2H,
3
Har), 5.33 (s, 2H, O-CH2), 4.72−4.60 (m, 1H, CH), 4.30 (t, JHH
=
3
4
8.5 Hz, 1H, CH), 4.11 (dd, JHH = 8.9 Hz, JHH = 2.7 Hz, 1H, CH),
4.06−3.95 (m, 1H, CH), 3.05 (dd, 2JHH = 13.2 Hz, 3JHH = 8.7 Hz, 1H,
2
3
CH), 2.90−2.82 (m, 1H, CH), 2.79 (dd, JHH = 10.2 Hz, JHH = 3.3
(S)-2-(4-((4-Methoxy-2-(trifluoromethyl)benzyl)carbamoyl)-
benzyl)butanoic Acid ((R)-3), (R)-2-(4-((4-Methoxy-2-
(trifluoromethyl)benzyl)carbamoyl)benzyl)butanoic Acid ((S)-3).
2
3
Hz, 1H, CH), 2.69 (dd, JHH = 13.6 Hz, JHH = 6.7 Hz, 1H, CH),
1.79−1.58 (m, 1 H, CH), 1.57−1.38 (m, 1H, CH), 0.88 (t, 3JHH = 7.4
Hz, 3H, CH3) ppm. 13C NMR (75 MHz, DMSO-d6): δ = 174.4,
165.5, 153.0, 145.6, 136.2, 135.2, 127.7, 129.7 (2 × C), 129.4 (2 ×
C), 129.3 (2 × C), 128.5 (2 × C), 128.3 (2 × C), 128.1, 127.9 (2 ×
C), 126.8, 65.7, 65.0, 54.2, 45.4, 36.8, 36.2, 24.7, 11.3 ppm.
General Method for Hydrogenolysis of Benzyl Esters.
(4S,2R)-10 or (4R,2S)-10 (1.0 equiv) was dissolved in MeOH (5
mL/mmol) and treated with palladium (w = 10%) on charcoal (1 mol
% Pd). The reaction mixture was stirred at room temperature for 5 h
in an autoclave under a hydrogen atmosphere of 5 bar. The
purification was realized by column chromatography (hexane/ethyl
acetate 3:1 + 1% acetic acid), and both enantiomers could be isolated
as colorless oils ((4S,2R)-11: 1.23 g, 3.24 mmol, 86%; (4R,2S)-11:
1.32 g, 3.47 mmol, 76%).
3
1H NMR (500 MHz, MeOD-d4) δ = 7.79 (d, JHH = 8.3 Hz, 2H,
3
3
Har), 7.46 (d, JHH = 8.6 Hz, 1H, Har), 7.32 (d, JHH = 8.2 Hz, 2H,
Har), 7.21 (d, 4JHH = 2.6 Hz, 1H, Har), 7.13 (dd, 3JHH = 8.6 Hz, 4JHH
2.6 Hz, 1H, Har), 4.69 (s, 2H, N-CH2), 3.83 (s, 3H, O-CH3), 2.96
=
2
3
2
(dd, JHH = 13.6 Hz, JHH = 8.9 Hz, 1H, CH), 2.83 (dd, JHH = 13.7
3
3
3
Hz, JHH = 6.1 Hz, 1H, CH), 2.60 (tt, JHH = 8.7 Hz, JHH = 5.8 Hz,
1H, CH), 1.70−1.55 (m, 2H, CH2), 0.96 (t, 3JHH = 7.4 Hz, 3H, CH3)
ppm. 13C NMR (126 MHz, MeOD-d4) δ = 177.6, 168.8, 158.8, 144.0,
132.0, 130.3, 128.8 (2 × C), 128.4 (2JCF = 30.7 Hz), 128.2, 127.1 (2 ×
C), 124.3 (1JCF = 273.3 Hz), 116.6, 111.8 (3JCF = 5.9 Hz), 54.7, 49.0,
38.5, 38.4, 25.0, 10.6 ppm. 19F-NMR (471 MHz, MeOD-d4): δ =
−61.6 (Ar-CF3) ppm. ESI-MS (m/z): 408.13 [M − H]−.
R-(3). MALDI-HRMS: m/z calculated for C21H23F3NO4 [M + H+]
410.15857 measured 410.15691 (Δm = 0.00166 g; 4.05 ppm).
S-(3). MALDI-HRMS: m/z calculated for C21H23F3NO4 [M + H+]
410.15857 measured 410.15698 (Δm = 0.00159 g; 3.88 ppm)
CD Spectrometry. CD spectra of (R)-3 and (S)-3 were recorded
on a spectropolarimeter Jasco J-810 (Jasco Labortechnik, Pfungstadt)
at a concentration of 0.5 mM in MeOH, using a 1 mm cuvette and a
total volume of 300 μL from 180 to 300 nm. Spectra were recorded by
accumulating three scans with a scan rate of 100 nm/min, and
background correction was accomplished by subtraction of methanol
spectrum.
4-((S)-2-((R)-4-Benzyl-2-oxooxazolidine-3-carbonyl)butyl)-
benzoic Acid ((4S,2R)-11), 4-((R)-2-((S)-4-Benzyl-2-oxooxazolidine-
1
3-carbonyl)butyl)benzoic Acid ((4R,2S)-11). H NMR (250 MHz,
DMSO-d6): δ = 12.34 (bs, 1H, COOH), 7.93−7.81 (m, 2H, Har),
7.43−7.31 (m, 2H, Har), 7.22−7.11 (m, 3H, Har), 6.86−6.78 (m, 2H,
Har), 4.71−4.62 (m, 1H, CH), 4.30 (t,3JHH = 8.5 Hz, 1H, CH), 4.11
(dd, 3JHH = 8.8 Hz, 4JHH = 2.6 Hz, 1H, CH), 4.06−3.95 (m, 1H, CH),
2
3
3.09−2.77 (m, 4H, CH2), 2.69 (dd, JHH = 13.6 Hz, JHH = 6.7 Hz,
1H, CH), 1.77−1.59 (m, 1H, CH), 1.55−1.38 (m, 1H, CH), 0.88 (t,
3JHH = 7.4 Hz, 3H, CH3) ppm.
General Method for Amide Coupling. (4S,2R)-11 or (4R,2S)-
11 (1.0 equiv) and HBTU (1.5 equiv) were dissolved in dry DMF (10
mL/mmol) under argon atmosphere and cooled to 0 °C. EDC·HCl
(1.5 equiv), 4-methoxy-2-(trifluoromethyl)benzylamine (1.1 equiv),
and N-methylmorpholine (6.0 equiv) were added, and the mixture
was allowed to warm up to room temperature overnight. After the
reaction was completed, the solvent was removed under reduced
pressure and ethyl acetate was added. The organic phase was washed
twice with H2O and brine. The organic phase was dried over MgSO4,
and solvent was removed under reduced pressure. The product could
be isolated after purification through flash chromatography (hexane/
ethyl acetate 100:0−50:50) as pale-yellow oil ((4S,2R)-12: 1.38 g,
2.43 mmol, 75%; (4R,2S)-12: 1.66 g, 2.9 mmol, 84%).
Smoothing of the spectra was accomplished under usage of Jasco
spectra analysis software with means movement method for
convulsion width of 25 data points.
sEH Activity Assay. Fluorescence-based sEH activity assay was
performed as described previously.40 The IC50 values of the
compounds were determined by a fluorescence-based assay system
in 96 well microtiter plates. Fluorogenic substrate 3-phenyl-cyano-(6-
methoxy-2-naphthalenyl)methyl ester-2-oxirane-acetic acid
(PHOME)41 was used, and the formation of the fluorescent 6-
methoxy-2-naphthaldehyde was measured (λem = 330 nm, λex = 465
nm) on a Tecan Infinite F200 Pro plate reader. Recombinant human
sEH was dissolved in pH 7 bis-Tris buffer with 0.1 mg/mL BSA
containing a 0.01% Triton-X 100 (final protein concentration 3 nM).
2823
J. Med. Chem. 2021, 64, 2815−2828