Synthesis of Deamido Bleomycin A
A R T I C L E S
2
trated under diminished pressure. The residue was purified by flash
chromatography on a silica gel column (8 × 1 cm). Elution with 4:1
ethyl acetate-hexane gave 9 as an amorphous solid: yield 80 mg
(s, 9H), 1.95 (s, 3H), 2.81 (dd, 1H, J ) 11.0, 2.0 Hz), 3.20 (dd, 1H, J
) 12.0, 4.5 Hz), 3.42 (dd, 1H, J ) 15.0, 5.5 Hz), 3.69 (br d, 1H, J )
5.5 Hz), 3.88 (dd, 1H, J ) 15.0, 9.5 Hz), 4.14 (q, 2H, J ) 7.0 Hz),
4.35 (dq, 1H, J ) 8.0, 6.5 Hz), 4.41-4.53 (m, 1H), 4.87 (d, 1H, J )
7.5 Hz), 5.56 (br s, 1H), 6.07 (d, 1H, J ) 8.0 Hz), and 6.96-7.05 (m,
5H). 13C NMR (C6D6): δ 11.6, 14.1, 14.6, 28.0, 28.4, 41.5, 49.1, 54.9,
55.4, 61.5, 62.0, 78.6, 78.9, 81.1, 110.3, 126.0, 134.2, 153.2, 155.0,
156.0, 164.1, 166.4, 168.0, 171.0, and 171.3. Mass spectrum (FAB),
m/z 671.4 (M + H)+. Mass spectrum (FAB), m/z 671.3399 (M + H)+
(C33H47N6O9 requires 671.3404).
Nr-(tert-Butoxycarbonyl)-Nâ-[1-amino-3(S)-(4-amino-6-(carbo-
ethoxy)-5-methypyrimidin-2-yl)propion-3-yl]-(S)-â-aminoalanine tert-
Butyl Ester (15). To a solution containing 25.1 mg of 14 (37 µmol) in
2 mL of absolute ethanol at 0 °C was added 5 mL of saturated ethanolic
NH3 solution. The reaction mixture was stirred at 0 °C for 1.5 h and
concentrated under diminished pressure. The residue was purified by
flash chromatography on a silica gel column (15 × 1 cm). Gradient
elution with 3 f 10% methanol in dichloromethane gave 15 as a
1
(40%). Silica gel TLC Rf 0.40 (93:7 dichloromethane-methanol). H
NMR (CDCl3): δ 1.44 (t, 3H, J ) 7.0 Hz), 2.33 (s, 3H), 4.48 (q, 2H,
J ) 7.0 Hz), 6.49 (br s, 2H), and 9.89 (s, 1H). Mass spectrum (chemical
ionization), m/z 210 (M + H)+.
Nr-(tert-Butoxycarbonyl)-Nâ-[[(4-amino-6-(carboethoxy)-5-meth-
ylpyrimidin-2-yl)methylene]amino]-(S)-â-aminoalanine tert-Butyl
Ester (11). To a solution containing 135 mg (0.645 mmol) of 9 and
168 mg (0.645 mmol) of 10 in 15 mL of dry acetonitrile was added
3.7 g of 3 Å molecular sieves. The reaction mixture was stirred under
N2 for 24 h and then filtered through Celite. The filtrate was
concentrated under diminished pressure to give imine 11 as a colorless
1
foam: yield 282 mg (97%). [R]25 -20.6 (c 0.85, CH2Cl2). H NMR
D
(C6D6): δ 1.32 (s, 9H), 1.40 (s, 9H), 1.43 (t, 3H, J ) 7.0 Hz), 2.34 (s,
3H), 3.84 (dd, 1H, J ) 14.0, 3.0 Hz), 4.00 (q, 2H, J ) 7.0 Hz), 4.02-
4.06 (m, 1H), 4.56-4.59 (m, 1H), 5.88 (d, 1H, J ) 9.0 Hz), and 8.27
(s, 1H). 13C NMR (C6D6): δ 12.48, 14.17, 54.06, 79.78, 82.66, 114.63,
152.93, 155.30, 158.00, 163.77, 164.64, 166.04, and 171.61. Mass
spectrum (FAB), m/z 452.3 (M + H)+. Mass spectrum (FAB), m/z
452.2493 (M + H)+ (C21H34N5O6 requires 452.2509).
colorless foam: yield 11 mg (58%). Silica gel TLC Rf 0.34 (9:1
1
dichloromethane-methanol). [R]25 -25.9 (c 0.37, EtOH). H NMR
D
(CD3OD): δ 1.31 (t, 3H, J ) 7.5 Hz), 1.36 (s, 9H), 1.37 (s, 9H), 2.06
(s, 3H), 2.47 (dd, 1H, J ) 15.0, 8.0 Hz), 2.54 (dd, 1H, J ) 14.5, 5.5
Hz), 2.74 (d, 2H, J ) 5.5 Hz), 3.89 (dd, 1H, J ) 8.0, 5.5 Hz), 4.03 (t,
1H, J ) 5.5 Hz), and 4.33 (q, 2H, J ) 7.5 Hz). 13C NMR (CD3OD):
δ 11.8, 14.2, 28.0, 28.5, 42.0, 49.0, 55.5, 61.2, 61.6, 79.3, 81.3, 110.9,
154.0, 156.2, 164.6, 166.4, 167.3, 171.3, and 174.6. Mass spectrum
(FAB), m/z 511.3 (M + H)+. Mass spectrum (FAB), m/z 511.2885 (M
+ H)+ (C23H39N6O7 requires 511.2880).
Nr-(tert-Butoxycarbomyl)-Nâ-[3(S)-(4-amino-6-carboethoxy-5-
methylpyrimidin-2-yl)-1-[(4S, 5R)-4-methyl-5-phenyl-2-oxazolidi-
nyl]-2-methylthiopropion-3-yl]-(S)-â-aminoalanine tert-Butyl Ester
(13). To a solution containing 407 mg (976 µmol) of stannous
trifluoromethanesulfonate in 1.5 mL of THF at -78 °C was added
slowly a solution containing 129 mg (488 µmol) of oxazolidinone 12
in 1.5 mL of THF followed by 187 µL (138.8 mg, 1.07 mmol) of
diisopropylethylamine. The reaction mixture was allowed to warm to
-20 °C and was then stirred for 1 h. The reaction mixture was again
cooled to -78 °C. To this mixture was added slowly a solution
containing 110 mg (244 µmol) of imine 11 in 1.5 mL of THF. The
reaction mixture was warmed to -5 °C and stirred for 2 h. The reaction
mixture was poured into 20 mL of 2:1 CH2Cl2-saturated sodium
bicarbonate solution and then filtered through Celite. The filtrate was
washed with 20 mL of brine, dried (MgSO4), and concentrated under
diminished pressure. The residue was purified by flash chromatography
on a silica gel column (25 × 4 cm). Elution with 1:2 ethyl acetate-
hexane gave 13 as a colorless solid: yield 79 mg (45%). Silica gel
Nr-(tert-Butoxycarbonyl)-Nâ-[1-amino-3(S)-(4-amino-6-carboxy-
5-methylpyrimidin-2-yl)propion-3-yl]-(S)-â-aminoalanine tert-Butyl
Ester (16). To a solution containing 33.7 mg (66 µmol) of 15 in 1 mL
of 3:1:1 THF-CH3OH-H2O was added 132 µL of 1 N LiOH. The
mixture was stirred at 0 °C for 2 h. After evaporation of most of the
THF and CH3OH, the aqueous phase was acidified to pH 4-5 with
10% citric acid. The residue was applied to an Amberlite XAD-2
column (8 × 1 cm). The column was washed successively with water
and methanol. The combined methanol fraction was concentrated to
dryness. The residue was dissolved in 1 mL of water, frozen, and
lyophilized to give 16 as a colorless powder: yield 23.8 mg (75%).
Silica gel TLC Rf 0.45 (4:1:1 BuOH-AcOH-H2O). [R]25 -33.1 (c
D
TLC Rf 0.47 (1:1 ethyl acetate-hexane). [R]25 -19.3 (c 0.41, CH2-
0.22, H2O). 1H NMR (CD3OD): δ 1.44 (s, 9H), 1.46 (s, 9H), 2.21 (s,
3H), 2.82 (dd, 1H, J ) 16.0, 8.0 Hz), 2.90 (dd, 1H, J ) 16.0, 4.0 Hz),
3.09 (dd, 1H, J ) 12.5, 8.0 Hz), 3.31 (m, 1H), and 4.27-4.34 (m,
2H). 13C NMR (D2O): δ 10.8, 26.6, 27.1, 36.2, 46.1, 51.7, 58.7, 81.7,
84.4, 107.3, 157.0, 158.6, 159.4, 163.7, 169.4, 172.3, and 173.6. Mass
spectrum (FAB), m/z 483.3 (M + H)+. Mass spectrum (FAB), m/z
483.2561 (M + H)+ (C21H35N6O7 requires 483.2567).
D
1
Cl2). UV (MeOH) λmax 236 and 286 nm. H NMR (C6D6): δ 0.93 (t,
3H, J ) 7.5 Hz), 0.97 (d, 3H, J ) 6.0 Hz), 1.17 (s, 9H), 1.26 (s, 9H),
1.86 (s, 3H), 2.30 (s, 3H), 2.69 (dd, 1H, J ) 13.0, 4.5 Hz), 2.93 (dd,
1H, J ) 13.0, 4.0 Hz), 4.00 (q, 2H, J ) 7.0 Hz), 4.24 (m, 1H), 4.31
(dq, 1H, J ) 8.5, 7.5 Hz), 4.48 (d, 1H, J ) 11.0 Hz), 4.55 (d, 1H, J )
6.5 Hz), 5.43 (br s, 1H), 5.52 (d, 1H, J ) 11.0 Hz), 5.71 (d, 1H, J )
8.5 Hz), and 6.80-6.93 (m, 5H). 13C NMR (C6D6): δ 11.7, 11.9, 14.1,
14.8, 27.9, 28.4, 46.6, 49.3, 55.3, 55.5, 61.4, 65.8, 78.6, 78.8, 81.0,
111.2, 126.0, 134.0, 153.4, 154.5, 156.0, 164.3, 166.4, 166.6, 170.8,
and 171.0. Mass spectrum (FAB), m/z 717.4 (M + H)+. Mass spectrum
(FAB), m/z 717.3296 (M + H)+ (C34H49N6O9S requires 717.3281).
Nr-(tert-Butoxycarbonyl)-Nâ-[3(S)-4-amino-6-carboethoxy-5-me-
thylpyrimidin-2-yl)-1-[(4S, 5R)-4-methyl-5-phenyl-2-oxazolidinyl]-
propion-3-yl]-S-â-aminoalanine tert-Butyl Ester (14). To a solution
containing 36.5 mg (51 µmol) of 13 in 0.7 mL of dry benzene were
added 137 µL (148 mg, 510 µmol) of Bu3SnH and 4.5 mg (27.4 µmol)
of AIBN. The reaction mixture was heated to 80 °C and stirred for 2
h. The cooled reaction mixture was concentrated under diminished
pressure. The residue was purified by flash chromatography on a silica
gel column (15 × 1 cm). Gradient elution with 0 f 3% methanol in
dichloromethane gave 14 as a colorless foam: yield 17.3 mg (72%).
Silica gel TLC Rf 0.30 (95:5 dichloromethane-methanol). [R]25D -24.3
(c 0.30, CH2Cl2). UV (MeOH) λmax 236 and 286 nm. 1H NMR (C6D6):
δ 0.82 (d, 3H, J ) 6.5 Hz), 1.05 (t, 3H, J ) 7.0 Hz), 1.37 (s, 9H), 1.40
Deamido Bleomycin A2 (4). A solution of 8 mg (3.9 µmol) of
partially deprotected bleomycin A2 35 in 1 mL of methanol was treated
with 1 mL of 2 N NH4OH at 0 °C. The reaction mixture was stirred at
25 °C for 2 h and then concentrated under diminished pressure. The
solution was then acidified to pH 2-3 with 0.1 N HCl and purified by
flash chromatography on a C18 reversed-phase column (6 × 1 cm).
Gradient elution with 0 f 30% methanol-water gave 4 as a colorless
powder: yield 4.0 mg (68%). Silica gel TLC Rf 0.22 (10:9:1 methanol-
10% ammonium acetate solution-10% ammonium hydroxide). [R]25
+25.4 (c 0.13, methanol). UV λmax 238 (sh) and 291 nm. H NMR
D
1
(D2O): δ 1.10-1.12 (m, 9H), 2.02 (s, 3H), 2.22 (qn, 2H, J ) 7.0 Hz),
2.46-2.48 (m, 1H), 2.60-2.71 (m, 2H), 2.72-2.79 (m, 1H), 2.83-
2.88 (m, 1H), 2.93 (s, 6H), 3.27 (t, 2H, J ) 6.5 Hz), 3.40 (t, 2H, J )
6.5 Hz), 3.53-4.12 (m, 22H), 4.23 (d, 1H, J ) 4.5 Hz), 4.80 (m, 1H),
5.02 (br s, 1H), 5.04 (d, 1H, J ) 6.5 Hz), 5.26-5.29 (m, 2H), 7.28 (s,
1H), 7.80 (s, 1H), 8.04 (s, 1H), and 8.24 (s, 1H). Mass spectrum (FAB),
m/z 1415.5 (M+). Mass spectrum (FAB), m/z 1415.5030 (M+)
(C55H83N16O22S3 requires 1415.5046).
9
J. AM. CHEM. SOC. VOL. 124, NO. 32, 2002 9487