Peptidyl Boronic Acids as Chymotrypsin Inhibitors
J . Org. Chem., Vol. 62, No. 3, 1997 521
[(S)-(R*,R*)]-2-[(Ch lor oa cetyl)oxy]-3-(2-m eth ylp r op yl)-
1.46 (m, 11), 1.28 (m, 1), 1.16 (d, 3, J ) 6.6), 0.98 (d, 3, J )
6.4), 0.94 (d, 3, J ) 6.4), 0.88 (d, 3, J ) 6.9), 0.87 (d, 3, J )
6.9); 13C NMR (CD3OD) δ 175.66, 175.33, 174.73, 174.61,
173.53, 173.25, 160.75, 74.06, 53.09, 52.81, 51.24, 48.66, 45.09,
43.63, 42.00, 41.64, 39.03, 36.87, 29.53, 27.08, 25.78, 24.07,
23.35, 22.50, 22.14, 22.04, 20.45; HRMS (FAB) calcd for
C28H51N8O11: m/z 675.3677 (MH+), found 675.3695.
bu ta n ed ioic Acid , 1-Meth yl 4-ter t-Bu tyl Ester , 28.
A
slurry of 10% Pd/C (0.67 g, 1.8 mmol) and ester 27 (1.0 g, 3.1
mmol) in anhydrous MeOH (31 mL, 0.1 M) in a Parr bottle
was placed under hydrogen at 45 psi. After 18 h at 25 °C, the
slurry was filtered through Celite and concentrated to give 1.02
g (96%) of crude 28, which was used without further purifica-
tion: TLC Rf 0.49 (2:1 hexanes/Et2O); [R] ) -8.9° (c 2.2,
CHCl3); 1H NMR (CDCl3) δ 5.47 (d, 1, J ) 5.0), 4.16 (d, 1, J )
15.5), 4.15 (d, 1, J ) 15.5), 3.77 (s, 3), 2.93 (ddd, 1, J ) 4.0,
5.0, 10.2), 1.79 (ddd, 1, J ) 4.9, 10.2, 13.7), 1.63 (m, 1), 1.45
(s, 9), 1.22 (ddd, 1, J ) 4.0, 9.2, 13.7), 0.94 (d, 3, J ) 6.6), 0.88
(d, 3, J ) 6.5); 13C NMR (CDCl3) δ 170.5, 168.3, 166.5, 81.6,
73.9, 52.6, 45.7, 40.4, 36.2, 27.9, 26.0, 23.2, 21.4; IR 3056, 2958,
2872, 1754, 1736 (br), 1469, 1437, 1368, 1251, 1158, 1050, 914
cm-1; HRMS (FAB) calcd for C15H26ClO6: m/z ) 336.1418
(MH+), found 336.1419.
N2-[N-[(2S,3S)-3-[[[(1S,3S)-3-(Acetyla m in o)-4-h yd r oxy-
1-m eth yl-4-oxobu tyl]a m in o]ca r bon yl]-2-h yd r oxy-5-m eth -
yl-1-oxoh exyl]-L-leu cyl]-L-a r gin in e, Meth yl Ester , 34. A
slurry of crude 33 (20 mg, 0.03 mmol) and 20% Pd(OH)2 (2
mg, 0.003 mmol) in anhydrous MeOH (1.0 mL) was stirred
under a hydrogen atmosphere for 17 h, filtered through a 0.2-
µm nylon filter, and concentrated in vacuo. The residue was
washed with hexanes (0.5 mL) to give 18 mg (100%) of 34.
1H
NMR (CD3OD) δ 4.48-4.19 (m, 3), 4.20 (d, 1, J ) 4.2), 4.02
(m, 1), 3.70 (s, 3), 3.18 (m, 2), 2.66 (d, 1, J ) 13.9), 2.62 (m, 1),
2.51 (d, 1, J ) 13.9), 1.98 (s, 3), 1.99-1.44 (m, 9), 1.26 (m, 1),
1.15 (d, 3, J ) 6.6), 0.98 (d, 3, J ) 6.4), 0.94 (d, 3, J ) 6.4),
0.88 (d, 3, J ) 6.9), 0.87 (d, 3, J ) 6.9); 13C NMR (CD3OD) δ
177.88, 175.31, 174.81, 174.64, 173.43, 172.75, 158.66, 74.12,
52.83, 52.74, 52.63, 49.85, 48.71, 45.35, 43.44, 41.86, 41.65,
39.36, 36.49, 29.59, 27.15, 26.22, 25.79, 24.09, 23.39, 22.80,
22.17, 22.06, 21.08; HRMS (FAB) calcd for C28H52N7O9: m/z
630.3827 (MH+), found 630.3829.
[(S)-(R*,R*)]-2-Hyd r oxy-3-(2-m eth ylp r op yl)bu ta n ed io-
ic Acid , 1-Meth yl 4-ter t-Bu tyl Ester , 29. A mixture of crude
28 (1.02 g, e3.1 mmol) and NaHCO3 (53 mg, 0.6 mmol) in
anhydrous MeOH (10 mL) was stirred at room temperature
for 4 h, diluted with EtOAc (35 mL), and washed with H2O
(15 mL) and brine (15 mL). The organic layer was dried
(MgSO4), filtered, and concentrated in vacuo, and the residue
was chromatographed (3:1 hexanes/Et2O) to give 0.69 g (85%)
[3a S-[2[S*[1R*(2R*,3R*),3R*]],3a r,4â,6â,7a r]]-N2-[N-
[(2S,3S)-3-[[[(1S,3S)-3-(Acet yla m in o)-4-[[1-h exa h yd r o-
3a ,5,5-t r im et h yl-4,6-m et h a n o-1,3,2-b en zod ioxa b or ol-2-
yl)-2-p h en ylet h yl]a m in o]-1-m et h yl-4-oxob u t yl]a m in o]-
ca r b on yl]-2-h yd r oxy-5-m et h yl-1-oxoh exyl]-L-leu cyl]-L-
a r gin in e, Meth yl Ester , 35. To a solution containing acid
34 (18 mg, 0.03 mmol) and the hydrochloride of amino boronate
1220-23 (11 mg, 0.03 mmol) in CH2Cl2 (0.9 mL) and DMF (0.1
mL) were added EDC (7 mg, 0.04 mmol), HOBT (5 mg, 0.04
mmol), and DIEA (6 µL, 0.03 mmol), and the mixture was
stirred under N2 at 25 °C for 16 h. The mixture was diluted
with CH2Cl2, filtered through glass wool, and concentrated in
vacuo. The crude product was washed with EtOAc (2 × 1 mL)
and purified by reverse phase HPLC (gradient of 9:1 H2O/CH3-
CN f 9:1 CH3CN/H2O) to give 7 mg (29%) of the boronate ester
35: 1H NMR (CD3OD) δ 9.69 (s, NH), 8.62 (d, J ) 7.3, NH),
8.57 (d, J ) 6.8, NH), 7.28-7.10 (m, 5), 4.35 (m, 1), 4.27 (m,
1), 4.15 (d, 1, J ) 8.6), 4.08 (d, 1, J ) 9.0), 4.07 (m, 1), 3.69 (s,
3), 3.07 (m, 2), 2.91-2.75 (m, 4), 2.39 (m, 1), 2.27 (m, 1), 1.93
(s, 3), 2.04-1.44 (m, 16), 1.34 (s, 3), 1.24 (s, 3), 1.14 (d, 3, J )
6.4), 1.09 (d, 1, J ) 10.2), 0.95 (d, 3, J ) 6.4), 0.92 (d, 3, J )
6.4), 0.89 (m, 6), 0.85 (s, 3); 13C NMR (CD3OD) δ 177.66, 175.26,
174.96 174.93, 173.43, 172.71, 158.6, 142.04, 130.20, 129.29,
127.10, 84.45, 77.44, 73.81, 53.51, 53.38, 53.28, 53.01, 52.72,
50.85, 49.50, 48.91, 42.79, 41.90, 41.80, 41.33, 39.16, 39.00,
38.27, 37.20, 29.67, 29.51, 27.73, 27.14, 26.87, 26.15, 25.64,
24.54, 24.44, 22.88, 22.85, 22.41, 21.90, 21.85; HRMS (FAB)
calcd for C46H76BN8O10: m/z ) 911.5777 (MH+), found 911.5793.
En zym e Assa ys. Inhibitor and buffer solutions were
prepared using doubly distilled water and filtered through
0.45-µm nylon filters. Bovine R-chymotrypsin stock solutions
were prepared in 0.1 M sodium acetate buffer, pH 5.0,
containing 0.5 M NaCl, and stored at -20 °C. Dilutions were
made with the pH 5.0 acetate buffer containing 0.2 mg/mL
bovine serum albumin (BSA). Substrate (Suc-Ala-Ala-Pro-
Phe-p-nitroanilide) solutions were made in 0.1 M HEPES
buffer, pH 7.0, containing 0.5 M NaCl. The substrate concen-
tration was determined from the absorbance at 315 nm (ꢀ )
14000 cm-1 mol-1 L). Boronic acid inhibitor solutions were
made by incubating the corresponding pinanediol ester in
potassium phosphate buffer, pH 7.5, containing 0.5 M NaCl,
for 1-2 h, and dilutions were made with the pH 7.0 HEPES
buffer. Assays were conducted at 25 °C in the pH 7.0 HEPES
buffer.
1
of the alcohol 29: TLC Rf 0.36 (1:1 hexanes/Et2O); H NMR
(CDCl3) δ 4.38 (d, 1, J ) 4.6), 3.79 (s, 3), 3.10 (br s, 1), 2.73
(ddd, 1, J ) 4.3, 4.8, 10.2), 1.74 (ddd, 1, J ) 5.0, 10.2, 13.7),
1.54 (m, 1), 1.45 (s, 9), 1.24 (ddd, 1, J ) 4.3, 9.1, 13.7), 0.90 (d,
3, J ) 6.6), 0.87 (d, 3, J ) 6.6); 13C NMR (CDCl3) δ 173.8, 172.5,
81.2, 71.7, 52.5, 48.2, 36.3, 28.0, 26.0, 23.2, 21.6; IR 3491 (br),
2957, 2871, 1737 (br), 1455, 1368, 1250, 1155, 1102, 847 cm-1
;
HRMS (FAB) calcd for C13H25O5: m/z ) 260.1702 (MH+), found
261.1698.
N2-[N-[(2S,3S)-3-(ter t-Bu t oxyca r b on yl)-2-h yd r oxy-5-
m eth yl-1-oxoh exyl]-L-leu cyl]-N8-n itr o-L-a r gin in e, Meth yl
Ester , 30. Acid 13 (0.15 g, 0.47 mmol) and the TFA salt of
amine 14 (0.20 g, 0.56 mmol) were coupled in CH2Cl2 according
to procedure B. The crude product was chromatographed (97:3
CH2Cl2/MeOH) to give 0.18 g (65%) of peptide 30: TLC Rf 0.29
1
(95:5 CH2Cl2/MeOH); H NMR (CD3OD) δ 4.49-4.43 (m, 2),
4.31 (d, 1, J ) 4.4), 3.71 (s, 3), 3.23 (br m, 2), 2.74 (m, 1), 1.91
(m, 1), 1.77-1.52 (m, 9), 1.46 (s, 9), 1.13 (ddd, 1, J ) 3.2, 9.4,
13.6), 0.97 (d, 3, J ) 6.5), 0.95 (d, 3, J ) 6.5), 0.88 (d, 3, J )
6.5), 0.84 (d, 3, J ) 6.5); HRMS (FAB) calcd for C25H47N6O9:
m/z 575.3405 (MH+), found 575.3407.
N2-[N-[(2S,3S)-3-Ca r b oxy-2-h yd r oxy-5-m et h yl-1-oxo-
h exyl]-L-leu cyl]-N8-n it r o-L-a r gin in e, Met h yl E st er , 31.
tert-Butyl ester 30 (77 mg, 0.13 mmol) was deprotected
according to procedure C to give 68 mg (99%) of acid 31, which
was used without further purification. 1H NMR (CD3OD) δ
4.48-4.43 (m, 2), 4.41 (d, 1, J ) 3.9), 3.72 (s, 3), 3.28 (br m, 2),
2.87 (m, 1), 1.92 (m, 1), 1.81-1.54 (m, 9), 1.14 (ddd, 1, J ) 3.5,
9.3, 13.7), 0.98 (d, 3, J ) 6.5), 0.95 (d, 3, J ) 6.5), 0.89 (d, 3, J
) 6.6), 0.84 (d, 3, J ) 6.6); HRMS (FAB) calcd for C21H39N6O9:
m/z 519.2779 (MH+), found 519.2784.
N2-[N-[(2S,3S)-3-[[[(1S,3S)-3-(Acetylam in o)-4-ter t-bu toxy-
1-m eth yl-4-oxobu tyl]a m in o]ca r bon yl]-2-h yd r oxy-5-m eth -
yl-1-oxoh exyl]-L-leu cyl]-N8-n itr o-L-a r gin in e, Meth yl Es-
ter , 32. Hydroxy acid 31 (38 mg, 0.07 mmol) and amine 11
(17 mg, 0.07 mmol) were coupled according to procedure A to
give 35 mg (66%) of the intermediate 32: 1H NMR (CD3OD) δ
4.46 (m, 2), 4.30 (dd, 1, J ) 8.7, 6.2), 4.22 (d, 1, J ) 4.1), 3.98
(m, 1), 3.72 (s, 3), 3.25 (m, 2), 2.71 (m, 1), 1.98 (s, 3), 1.93-
1.48 (m, 11), 1.45 (s, 9), 1.28 (m, 1), 1.14 (d, 3, J ) 6.6), 0.98
(d, 3, J ) 6.4), 0.95 (d, 3, J ) 6.4), 0.88 (d, 3, J ) 6.8), 0.87 (d,
3, J ) 6.8); HRMS (FAB) calcd for C32H59N8O11: m/z 731.4303
(MH+), found 731.4304.
N2-[N-[(2S,3S)-3-[[[(1S,3S)-3-(Acetyla m in o)-4-h yd r oxy-
The initial rate of hydrolysis of Suc-Ala-Ala-Pro-Phe-p-
nitroanilide was monitored by observing the formation of
p-nitroaniline at 410 nm. Six substrate concentrations be-
tween 5 and 300 µM were used with several independent
determinations carried out at each concentration to determine
the Km value of 24 µM. Initial rates and Km values were
calculated using the Enzfitter program.41
1-m eth yl-4-oxobu tyl]a m in o]ca r bon yl]-2-h yd r oxy-5-m eth -
yl-1-oxoh exyl]-L-leu cyl]-N8-n it r o-L-a r gin in e,
Met h yl
Ester , 33. tert-Butyl ester 32 (23 mg, 0.03 mmol) was
deprotected according to procedure C to give 20 mg (99%) of
acid 33. 1H NMR (CD3OD) δ 4.38 (m, 3), 4.23 (d, 1, J ) 4.2),
4.05 (m, 1), 3.71 (s, 3), 3.24 (m, 2), 2.69 (m, 1), 1.99 (s, 3), 1.96-