3930 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 18
Demange et al.
ethyl acetate:chexane ratio) gave 4a (690 mg, 88%) and 4b
nitrile:acetic acid (0.25% in water) 25:75 to 75:25 in 30 min)
(580 mg, 92%).
to give 7a (121 mg, 77%) and 7b (128 mg, 82%).
7a : tR 12.59 (>95%); H NMR (CDCl3) δ 8.02 (d, J ) 9.2,
1
4a : 1H NMR (CDCl3) δ 8.10 (d, J ) 7.5, 2H) + 7.88 (d, J )
9.0, 2H) + 7.75 (d, J ) 7.3, 2H) + 7.54 (d, J ) 7.2, 2H) + 7.42-
6.80 (bm, 9H), 5.48-5.45 (m, 1H), 4.97-4.93 (m, 1H), 4.41-
4.30 + 4.20-3.92 (2bm, 8H), 3.47-3.44 + 3.22-3.07 (2bm, 4H),
1.97-1.80 + 1.75-1.53 (2b, 4H), 1.38-1.20 (bm, 6H); 13C NMR
(CDCl3) δ 173.8 + 173.5 + 173.4 + 171.3, 156.9 + 156.2,
2H), 7.51 (bd, J ) 7.95, 1H), 7.33-7.13 (m, 5H), 6.50 (d, J )
9.2, 2H), 6.30 (bd, J ) 7.1, 1H), 6.19 (bt, J ) 5.6, 1H), 4.44 (t,
J ) 7.0, 1H), 4.40-4.31 (m, 1H), 4.02-3.88 (m, 5H), 3.41-
3.25 + 3.10-3.01 + 2.97-2.93 (3m, 6H), 1.99 (s, 3H), 2.04-
1.94 + 1.81-1.62 (2m, 4H), 1.36 (d, J ) 7.0, 3H), 1.26 (t, J )
7.0, 3H); 13C NMR (CDCl3) δ 173.8 + 172.7 + 172.6 + 170.5,
137.4 + 129.1 + 128.6 + 128.5 + 127.7 + 126.8 + 126.4 +
144.3-118.8 (complex), 67.3, 62.1, 61.4 (d, J 2
) 6.6), 54.9,
C-P
) 7.3), 18.0, 16.5 (d, J 3
C-P C-P
C-P
53.7 + 47.0, 50.4 + 49.1 + 46.7, 37.2, 36.3 (d, J 1
) 147.3),
126.3 + 110.9, 61.6 (d, J 2
) 4.15), 61.2 (d, J 2
) 6.8), 50.3
) 3.6), 46.2, 38.1, 34.8 (d, J 1
) 143.3),
C-P
31.4 (d, J 3
) 7.2), 25.8 (d, J 3
C-P
C-P
C-P
+ 49.1, 47.5 (d, J 2
) 5.9); 31P NMR (CDCl3, decoupled) δ 26.5 (minor) + 27.1
(major); DCI-MS (NH3) m/z 797 (MH+, 30%), 415 ([Pro-Phe-
pNA]H+, 35%), 383 ([Fmoc-Ala-CH2PO2Et]H+, 100%).
C-P
31.1 (d, J 3
C-P
) 6.5), 25.1 (d, J 3
) 6.9), 23.0, 17.9, 16.3 (d,
C-P
C-P
J 3
) 6.3); 31P NMR (CDCl3, decoupled) δ 27.1; ES-MS 602.4.
1
4b: 1H NMR (CDCl3) δ 8.08 (d, J ) 9.3), 7.91 (d, J ) 9.3),
7.78-7.64 + 7.57-7.50 + 7.46-7.11 (bm, 13H), 5.40-5.31 (m,
1H), 4.97-4.87 (m, 1H), 4.49-3.96 (2bm, 8H), 3.61-3.32 +
3.11-2.98 (2bm, 4H); 13C NMR (CDCl3) δ 173.4 + 172.9 +
170.3, 157.4 + 156.1 + 155.7, 144.2-110.8 (complex), 67.2, 63.8
7b: tR 11.90 (>95%); H NMR (CDCl3) δ 8.00 (d, J ) 10.0,
2H), 7.55-7.46 (b, 1H), 7.34-7.15 (m, 5H), 6.51 (d, J ) 10.0,
2H), 6.44 (d, J ) 7.5, 1H), 6.16-6.08 (b, 1H), 4.50-4.29 (m,
2H), 4.23-3.84 (m, 5H, Ha), 3.68-3.55 + 3.43-3.22 + 3.18-
2.97 (3m, 6H), 2.04 (minor) + 2.02 (major) (s, 3H), 2.11-1.99
+ 1.81-1.62 (2m, 4H), 1.34 (d, J ) 7.5, major) + 1.30 (d, J )
5.0, minor), 1.26 (t, J ) 5.0, major) + 1.25 (t, J ) 5.0, minor);
13C NMR (CDCl3) δ 174.5 + 173.0 + 172.8 + 170.4, 137.6 +
(d, J 2
) 6.2), 62.0 (d, J 2
) 4.5) + 61.0 (d, J 2
) 6.9),
C-P
C-P
C-P
53.9, 50.9 + 47.7 + 47.0 + 47.0 (d, J 2
) 9.2), 36.9, 34.1 (d,
C-P
J 1
) 141.8), 31.6 (d, J 3
) 6.9), 25.5 (d, J 3
) 8.2), 18.1,
C-P
C-P
C-P
137.4 + 129.1 + 128.5 + 126.7 + 110.9, 61.8 (d, J 2
) 6.8),
16.2 (d, J 3
) 6.8); 31P NMR (CDCl3, decoupled) δ 27.6; DCI-
C-P
C-P
60.9 (d, J 2
) 3.6), 50.2 + 49.0, 47.3 (d, J 2
) 3.4), 46.4,
C-P
MS (NH3) m/z 797 (MH+, 13%), 415 ([Pro-Phe-pNA]H+, 29%),
C-P
38.1, 36.2 (d, J 1
) 146.0), 31.5 (d, J 3
) 7.1), 25.7 (d, J 3
C-P C-P
383 ([Fmoc-Ala-CH2PO2Et]H+, 100%).
C-P
) 6.8), 23.1, 17.9, 16.3 (d, J 3
decoupled) δ 26.7; ES-MS m/z 602.3.
) 6.2); 31P NMR (CDCl3,
C-P
Su c-Ala -GlyΨ(P O2Et-N)P r o-P h e-pNA (1a a n d 1b). Com-
pounds 4a (500 mg, 0.79 mmol) and 4b (450 mg, 0.58 mmol)
were treated with 20% diisopropylamine in DMF for 3 h at
room temperature. After evaporation of the solvent under
reduced pressure, methanol was chased over the product (3
times). The crude product was treated with succinic anhydride
(2.0 equiv) and trietylamine (3.0 equiv) overnight at room
temperature. After evaporation of the solvent under reduced
pressure, the crude product was purified by RP-HPLC (semi-
preparative column Vydac C18; rate, 4 mL min-1; linear
gradient, acetonitrile:acetic acid (0.25% in water) 25:75 to 75:
25 in 30 min).
Ac-Ala -GlyΨ(P O2Bn -N)P r o-P h e-pCMA (11). Compound
10 (85 mg, 87 µmol) was treated with 20% diisopropylamine
in DMF for 3 h at room temperature. After evaporation of the
solvent under reduced pressure, methanol was chased over the
product (3 times). The crude product was treated with acetyl
chloride (13 mL, 2.0 equiv) and DIPEA (45 mL, 3.0 equiv)
overnight at room temperature. After evaporation of the
solvent under reduced pressure, the crude product was purified
by preparative thin-layer chromatography (eluent, 94:6 chlo-
roform:methanol ratio) to give 11 (31 mg, 52%): 1H NMR
(CDCl3) δ 7.98 (d, J ) 10.0, 2H) + 7.84 (d, J ) 10.0, 2H), 7.68
(d, J ) 7.5, 1H) + 7.37-7.18 (m, 9H), 5.96 (d, J ) 7.3, 1H),
1
1a (50 mg, 12%): tR 11.09 min (95%); H NMR (CD3OD) δ
8.19 (d, J ) 9.0) + 7.84 (d, J ) 9.2), 7.36-7.16 (bm, 5H), 4.90-
4.82 (m, 1H), 4.39 (q, J ) 7.2, 1H), 4.28-4.17 (m, 1H), 4.06-
3.90 (m, 2H), 3.83-3.69 (m, 2H), 3.30-3.10 (bm, 4H), 2.68-
2.55 + 2.55-2.41 (bm, 4H), 2.19-2.03 + 1.84-1.52 (2bm, 4H),
1.35 (d, J ) 7.2, 3H), 1.26 (t, J ) 7.0, 3H); 13C NMR (CD3OD)
δ 176.4 + 175.3 + 175.2 + 174.6 + 172.4, 145.7 + 144.7 +
5.00 (AB, δA ) 5.07, δB ) 4.94, J AB ) 11.8) + 5.00 (AB, δA
)
5.03, δB ) 4.97, J AB ) 11.7) (m, 2H), 4.85-4.96 + 4.42-4.34
+ 4.08-4.05 + 3.94-3.84 (4m, 5H), 3.87 (s, 3H), 3.51-2.95
(m, 4H), 1.79 (s, 3H), 1.80-1.73 + 1.54-1.48 (2m, 4H), 1.26
(d, J ) 7.1, 3H); 13C NMR (CDCl3) δ 173.7 + 173.2 + 171.0 +
170.7 + 166.6, 142.6 + 137.7 + 136.0 + 135.9 + 130.6 + 129.3
+ 128.8 + 128.4 + 128.0 + 126.7 + 125.4 + 119.3, 66.7 (d,
138.5 + 130.5 + 129.5 + 127.9 + 125.7 + 120.7, 62.7 (d, J 2
C-P
) 3.6), 62.3 (d, J 2
142.7), 32.7 (d, J 3
) 6.8), 56.7, 50.5, 38.5, 37.3 (d, J 1
)
C-P
J 2
) 7.0), 61.3 (d, J 2
) 2.3), 54.9 + 52.0 + 48.6 + 47.0,
C-P
C-P
C-P
) 6.9), 31.4 + 30.2, 26.6 (d, J 3
) 6.5),
C-P
36.6, 36.6 (d, J 1
) 146.0), 31.1 (d, J 3
) 7.0), 25.2 (d, J 3
C-P C-P
C-P
C-P
17.7, 16.6 (d, J 3
) 6.4); 31P NMR (CD3OD, decoupled) δ 25.7;
) 7.0), 22.8, 16.9; 31P NMR (CDCl3, decoupled) δ 28.1; ES-MS
m/z 691.5.
C-P
ES-MS (negative ionization) m/z 674.19.
1
1b (50 mg, 12%): tR 12.45 min (92%); H NMR (CD3OD) δ
8.19 (d, J ) 9.15, 2H) + 7.87 (d, J ) 9.15, 2H), 7.33-7.17 (bm,
5H), 4.81-4.73 (m, 2H), 4.34 (q, J ) 7.2, 1H), 4.15-3.96 (m,
4H), 3.70-3.56 + 3.26-3.13 (2m, 4H), 2.64-2.47 (bm, 4H),
2.21-2.02 + 1.91-1.69 (2bm, 4H), 1.35 (d, J ) 7.5, 3H), 1.24
(t, J ) 7.05, 3H); 13C NMR (CD3OD) δ 176.3 + 175.2 + 175.1
+ 174.7 + 172.5, 145.7 + 144.8 + 138.1 + 130.4 + 129.6 +
128.0 + 125.7 + 125.6 + 120.8 (complex), 62.6-62.3 (m), 56.5,
Ac-Ala -GlyΨ(P O2H-N)P r o-P h e-pCMA (12). Compound
11 (23 mg, 33 µmol) dissolved in a 50:50 methanol/water
mixture (7 mL) was treated with sodium bicarbonate (8 mg,
2.8 equiv) and 10% Pd-C (11 mg) under 4 bar of hydrogen for
2 h. The catalyst was removed by filtration, and the solvent
was evaporated under reduced pressure. The crude product
was dissolved in a 50:50 water/acetonitrile mixture and freeze-
dried. Compound 12 was used without further purification:
1H NMR (CD3OD) δ 8.01-7.92 (m, 4H), 7.32-7.19 (m, 5H),
4.90-4.78 (m), 4.32 (q, J ) 7.0, 1H), 4.00-3.93 (m, 1H), 3.88
(s, 3H), 3.64 (dd, J ) 15.2, J ′ ) 11.8, 1H) + 3.52 (dd, J ) 13.9,
J ′ ) 4.0, 1H) + 3.30-3.19 (m, 1H) + 3.01-2.91 (m, 1H), 1.96
(s, 3H), 1.90-1.75 + 1.65-1.50 (2m, 4H), 1.29 (d, J ) 5.0, 3H);
13C NMR (CD3OD) δ 178.5 + 174.4 + 174.3 + 173.1 + 173.0 +
168.3, 144.3 + 139.1 + 131.1 + 130.2 + 129.5 + 127.7 + 126.6
50.2 (d, J 2
) 7.3), 38.4, 35.6 (d, J 1
) 144.9), 32.8 (d, J 3
C-P
C-P
C-P
) 7.0), 31.4 + 30.3, 26.3 (d, J 3
) 7.5), 17.7, 16.6 (d, J 3
)
C-P
C-P
5.8); 31P NMR (CD3OD, decoupled) δ 25.8; ES-MS (negative
ionization) m/z 674.30.
Ac-Ala -GlyΨ(P O2Et-N)P r o-P h eY(CH2-NH)pNA (7a a n d
7b). Compounds 6a (210 mg, 0.26 mmol) and 6b (208 mg, 0.26
mmol) were treated with 20% diisopropylamine in DMF for 3
h at room temperature. After evaporation of the solvent under
reduced pressure, methanol was chased over the product (3
times). The crude product was treated with acetyl chloride (2.0
equiv) and DIPEA (3.0 equiv) overnight at room temperature.
After evaporation of the solvent under reduced pressure, the
crude product was purified by preparative thin-layer chroma-
tography (eluent, chloroform:methanol with 98:2, then 97:3,
and finally 95:5 ratios) or by RP-HPLC (semipreparative
column Vydac C18; rate, 4 mL min-1; linear gradient, aceto-
+ 121.7, 63.6, 56.3 + 52.5 + 50.5, 38.3, 37.3 (d, J 1
) 128.9),
C-P
32.6, 26.3 (d, J 3
) 5.5), 22.5, 17.9; 31P NMR (CD3OD,
C-P
decoupled) δ 16.4; ES-MS (negative ionization) m/z 601.4.
Bioch em ica l Eva lu a tion . (1) Deter m in a tion of Kd .
Fluorimetric titration was carried out as previously re-
ported.12,33
(2) En zym a tic Assa ys. The PPIase uncoupled assay was
carried out in a 35 mM Hepes buffer (pH 7.8) at 10 ( 0.5 °C
using Suc-Ala-Ala-Pro-Phe-DFA as a substrate.34 The PPIase