S. Arns et al. / Bioorg. Med. Chem. 20 (2012) 2131–2140
2137
50% MeOH/ 50% ammonium formate (0.01 M, pH 6.5), 35 °C,
3.5 mL/min, Phenomenex reverse phase column (P/N 00G-4337-
N0). Product retention time: variable from 8.0 to 9.8 min. Com-
pound 11a characterization data: 1H NMR (600 MHz, D2O):
d = 7.40–7.55 (m, 5H), 6.42 (s, 2H), 5.55–5.75 (m, 2H), 4.62–4.73
(m, 1H), 4.15–4.23 (m, 1H), 3.13–3.21 (m, 1H), 3.09–3.13 (m,
2H), 2.90–2.95 (t, 2H), 2.48–2.56 (m, 1H), 2.34–2.40 (t, 2H), 2.15–
2.25 (m, 1H), 1.86–1.98 (m, 2H), 1.63–1.85 (m, 5H), 1.11–1.35
(m, 6H). 13C NMR (150 MHz, D2O): d = 178.0,167.6, 155.6, 136.1,
133.5 (t, JC,F = 25 Hz), 130.6, 128.6, 127.6, 125.6, 121.3 (t,
JC,F = 245 Hz), 74.0 (t, JC,F = 29 Hz), 73.8 (t, JC,P = 130 Hz), 72.4,
60.9, 41.2, 40.6, 30.8, 30.0, 27.4, 26.6, 25.9, 25.3, 24.4, 24.2, 23.7.
31P NMR (243 MHz, D2O) d = 20.7 (s).
125.9 (CH, t, JF = 25.2 Hz), 124.2 (CH), 123.8 (CH, t, JC,F = 11.5 Hz),
122.4 (C4), 119.7 (C4, t, JC,F = 245.6 Hz), 74.7 (CH, t, JC,F = 31.6 Hz),
60.3 (CH2), 59.8 (CH), 40.6 (CH2), 34.4 (CH2), 30.0 (CH2), 28.9
(CH2), 27.3 (CH2), 26.6 (CH2), 25.3 (CH2), 25.0 (CH2), 21.1 (CH3),
14.4 (CH3). HRMS (ESI): Found (M+H)+ at 586.2625 for
C32H38F2NO7. Calculated at 586.2616.
3.9. (R,E)-4-((R)-1-(7-ethoxy-7-oxoheptyl)-5-oxopyrrolidin-2-
yl)-1,1-difluoro-1-phenylbut-3-en-2-yl 2-hydroxybenzoate (17a)
A solution of 17 (264 mg, 0.451 mmol, 1.0 equiv) in absolute
ethanol (4.5 mL, 0.1 M) was cooled to 0 °C and bubbled with dry
HCl gas for 10 min. The mixture was slowly allowed to warm to
room temperature for 3.5 h after which the reaction was bubbled
with argon for 20 min. The solvent was removed by evaporation
at temperatures that do not exceed 30 °C. Purification by flash
chromatography (EtOAc/hexanes) afforded phenol 17a (177 mg,
0.325 mmol, 72%, Rf = 0.35 in 80% EtOAc/hexanes) as a colorless
oil. 1H NMR (400 MHz, CDCl3) d = 10.32 (s, 1H), 7.82 (dd, J = 8.0,
1.7 Hz, 1H), 7.52–7.49 (m, 3H), 7.47–7.41 (m, 3H), 6.97 (d,
J = 8.4 Hz, 1H), 6.91 (dd, J = 7.1, 7.1 Hz, 1H), 5.94 (td, J = 10.3,
6.0 Hz, 1H), 5.79–5.65 (m, 2H), 4.11 (q, J = 7.1 Hz, 2H), 4.07–4.02
(m, 1H), 3.42 (ddd, J = 13.8, 7.0, 7.0 Hz, 1H), 2.65 (ddd, J = 13.7,
8.4, 5.3 Hz, 1H), 2.42–2.13 (m, 3H), 2.27 (t, J = 7.5 Hz, 2H), 1.71–
1.64 (m, 1H), 1.59 (dt, J = 15.2, 7.5 Hz, 2H), 1.45–1.19 (m, 6H),
1.24 (t, J = 7.2 Hz, 3H).
HRMS (ESI): Found (M+H)+ at 725.2271 for C27H41F2N6O11P2.
Calculated at 725.2277.
Compound 11b 1,5-regioisomer characterization data: 1H NMR
(600 MHz, D2O): d = 7.40–7.55 (m, 5H), 6.26 (s, 2H), 5.55–5.75
(m, 2H), 4.62–4.73 (m, 1H), 4.15–4.23 (m,1H), 3.15–3.25 (m, 1H),
3.08–3.14 (t, 2H), 3.03–3.08 (t, 2H), 2.53–2.63 (m, 1H), 2.34–2.42
(t, 2H), 2.15–2.27 (m, 1H), 1.85–1.97 (m, 2H), 1.75–1.85 (m, 3H),
1.65–1.74 (m, 1H), 1.25–1.39 (m, 4H), 1.15–1.25 (m, 2H). 13C
NMR (150 MHz, D2O): d = 177.8, 170.8, 157.2, 155.4, 135.8, 133.3
(t, JC,F = 25 Hz) 130.3, 128.3, 127.4, 125.5, 121.1 (t, JC,F = 245 Hz),
74.1 (t, JC,F = 29 Hz), 73.83 (t, JC,P = 130 Hz), 67.6, 60.8, 41.2, 40.4,
31.0, 29.7, 27.4, 25.7, 25.2, 24.2, 23.7, 22.0. 31P NMR (243 MHz,
D2O) d = 19.6 (s).
HRMS (ESI): Found (M+Na)+ at 747.2128 for C27H40F2N6O11P2-
Na. Calculated at 747.2096.
13C NMR (100 MHz, CDCl3) d = 174.8 (C4), 173.8 (C4), 168.3 (C4),
162.0 (C4), 138.5 (CH), 136.6 (CH), 133.6 (C4, t, JC,F = 25.3 Hz), 130.8
(CH), 130.0 (CH), 128.6 (CH), 125.9 (CH, t, JC,F = 6.3 Hz), 123.8 (CH, t,
JC,F = 2.8 Hz), 119.6 (CH), 119.5 (C4, t, JC,F = 247.1 Hz), 117.9 (CH),
111.7 (C4), 74.9 (CH, t, JF = 32.4 Hz), 60.3 (CH2), 59.9 (CH), 40.7
(CH), 34.3 (CH), 30.0 (CH), 28.9 (CH), 27.3 (CH), 26.6 (CH), 25.3
(CH), 24.9 (CH), 14.4 (CH3). HRMS (ESI): Found (M+H)+ at
544.2479 for C30H36F2NO6. Calculated at 544.2511.
3.7. Preparation of [3H]-11a and [3H]-11b
[3H]-Tetrazole 6 (a kind gift from Merck; tritium labelled in
the para position of the phenyl moiety) (30 mg; 1.03 mCi;
15 mCi/mmol) was converted as above to a mixture of [3H]-10a
and 10b (0.45 mCi) after purification by column chromatography
on silica gel. The mixture was converted as above to yield a
mixture of [3H]-11a and 11b (6 mg; 0.1 mCi; nominally 15 mCi/
mmol).
3.10. (R,E)-4-((R)-1-(7-ethoxy-7-oxoheptyl)-5-oxopyrrolidin-2-
yl)-1,1-difluoro-1-phenylbut-3-en-2-yl-((4-nitrophenoxy)-
carbonyloxy)benzoate (18)
A solution of phenol 17a (117 mg, 0.326 mmol, 1.0 equiv) in
dichloromethane (5 mL) was treated with triethylamine (136 lL,
3.8. Phenol intermediate en route to 17a: (R,E)-4-((R)-1-(7-
ethoxy-7-oxoheptyl)-5-oxopyrrolidin-2-yl)-1,1-difluoro-1-
phenylbut-3-en-2-yl 2-acetoxybenzoate (17)
0.978 mmol, 3.0 equiv) and 4-nitrophenylchloroformate (99 mg,
0.489 mmol, 1.5 equiv). This mixture was stirred at room temper-
ature for 5 h after which the reaction is quenched with aqueous
NH4Cl. The layers were separated and the aqueous phase was ex-
tracted with dichloromethane (3Â). The organic layers were com-
bined, dried over MgSO4, filtered and concentrated and the product
was isolated by flash chromatography (EtOAc/hexanes) to afford
compound 18 (223 mg, 0.315 mmol, 97%, Rf = 0.35 in 80% EtOAc/
hexanes) as a yellow oil. 1H NMR (400 MHz, CDCl3) d = 8.25–8.21
(m, 2H), 7.98 (dd, J = 7.0, 1.6 Hz, 1H), 7.60 (td, J = 7.9, 1.7 Hz, 1H),
7.28–7.16 (m, 8H), 7.24 (dd, J = 8.1, 8.1 Hz, 1H), 5.94–5.88 (m,
1H), 5.69–5.60 (m, 2H), 4.04 (q, J = 7.1 Hz, 2H), 3.99–3.93 (m,
1H), 3.35 (dt, J = 13.9, 7.8 Hz, 1H), 2.54 (ddd, J = 13.7, 8.2, 5.4 Hz,
1H), 2.29–2.15 (m, 2H), 2.19 (t, J = 7.4 Hz, 2H), 2.12–2.03 (m, 1H),
1.59–1.47 (m, 3H), 1.35–1.10 (m, 6H), 1.17 (t, J = 7.2 Hz, 3H). 13C
NMR (100 MHz, CDCl3) d = 174.7 (C4), 173.7 (C4), 162.3 (C4),
155.5 (C4), 150.64 (C4), 150.62 (C4), 145.7 (C4), 138.1 (CH), 135.0
(CH), 133.5 (C4, t, JC,F = 25.2 Hz), 132.1 (CH), 130.7 (CH), 128.6
(CH), 127.2 (CH), 126.1 (C4), 125.8 (CH, t, JC,F = 6.2 Hz), 125.3
(CH), 123.6 (CH, t, JC,F = 2.4 Hz), 123.5 (CH), 121.9 (CH), 119.5 (C4,
t, JC,F = 247.5 Hz), 74.8 (CH, t, JC,F = 31.5 Hz), 60.2 (CH2), 59.6 (CH),
40.5 (CH), 34.2 (CH), 29.8 (CH), 28.8 (CH), 27.1 (CH), 26.5 (CH),
25.1 (CH), 24.8 (CH), 14.3 (CH3). HRMS (ESI): Found (M+H)+ at
709.2525 for C36H37F2N2O10. Calculated at 709.2573.
A solution of alcohol 7 (214 mg, 0.505 mmol, 1.0 equiv) in
DCM (5 mL) was treated with triethylamine (352 lL, 2.53 mmol,
5.0 equiv) and 2-acetylsalicyloyl chloride (251 mg, 1.26 mmol,
2.5 equiv) and stirred at room temperature for 18 h. The reaction
was quenched with saturated aqueous NH4Cl, the layers were
separated and the aqueous phase was extracted with DCM
(3 Â 10 mL). The organic layers were combined, dried over
MgSO4, filtered and concentrated and the product was isolated
by flash chromatography (EtOAc/hexanes) to afford 17 (264 mg,
0.451 mmol, 89%, Rf = 0.30 in 80% EtOAc/hexanes) as a colorless
oil. 1H NMR (400 MHz, CDCl3) d = 7.94 (dd, J = 7.9, 1.7 Hz, 1H),
7.59 (ddd, J = 8.0, 8.0, 1.7 Hz, 1H), 7.53–7.49 (m, 2H), 7.45–7.40
(m, 3H), 7.32 (ddd, J = 7.8, 7.8, 1.2 Hz, 1H), 7.11 (dd, J = 8.1,
1.0 Hz, 1H), 5.94–5.87 (m, 1H), 5.69–5.67 (m, 2H), 4.11 (q,
J = 7.1 Hz, 2H), 4.06–4.00 (m, 1H), 3.44 (ddd, J = 14.9, 7.1,
7.1 Hz, 1H), 2.62 (ddd, J = 13.6, 8.2, 5.3 Hz, 1H), 2.41–2.23 (m,
4H), 2.29 (s, 3H), 2.21–2.13 (m, 1H), 1.75–1.55 (m, 3H), 1.44–
1.19 (m, 6H), 1.24 (t, J = 7.1 Hz, 3H).
13C NMR (100 MHz, CDCl3) d = 174.8 (C4), 173.9 (C4), 169.6 (C4),
162.7 (C4), 151.0 (C4), 138.0 (CH), 134.6 (CH), 133.8 (C4, t,
JC,F = 99.8 Hz), 131.9 (CH), 130.7 (CH), 128.6 (CH), 126.3 (CH),