A R T I C L E S
Champeil et al.
through a Sep-Pak cartridge with water using a gradient of 0% to
40% methanol in water. The eluting purple solution was collected
and lyophilized to give 15 (7 mg, 0.027 mmol, 77%) as a purple
solid. 1H NMR (methanol-d4) δ 1.83 (s, CH3, 3H), 2.63 (dd, H1, J
) 4.5, 16.3 Hz, 1H), 3.13 (dd, H1, J ) 7.5, 16.3 Hz, 1H), 3.95 (dd,
H3, J ) 3.5, 12.5 Hz, 1H), 3.97 (s, H10, 2H), 4.20 (m, H2, 1H),
4.39 (dd, H3, J ) 6.5, 12.9 Hz, 1H). HRMS m/z calcd for
C13H17N4O2 [M + H]+: 261.1346, found: 261.1348.
2,7-Diamino-10-decarbamoyl-10-azido-mitosene (22). A solu-
tion of 21 (30 mg, 0.074 mmol) in MeOH (50 mL) and 0.05 M
potassium phosphate buffer pH 8.5 (100 mL) was treated with
penicillin amidase (0.540 mL, 500 units). The resulting solution
was gently stirred for 18 h at room temperature, when TLC analysis
(5% MeOH in CH2Cl2) showed the disappearance of 21 and the
formation of a single new compound. The mixture was partially
concentrated to remove MeOH. The resulting aqueous solution was
acidified with 1 M HCl and extracted with EtOAc-hexane (1:1, 2
× 40 mL) to remove traces of 21. The aqueous phase was treated
with saturated aqueous Na2CO3 to pH 10-11, then extracted with
CH2Cl2 (3 × 40 mL). The combined extracts were dried and
concentrated, to give 22 as a purple solid (20 mg, 0.0699 mmol,
94%). TLC and NMR analysis showed that the product was of high
purity, and was used in the next step without further purification.1H
NMR (methanol-d4): δ 1.82 (s, CH3, 3H), 2.62 (dd, H1, J ) 4.5,
16.1 Hz, 1H), 3.21 (dd, H1, J ) 6.9, 16.1 Hz, 1H), 3.96 (dd, H3, J
) 4.4, 12.9 Hz, 1H), 4.26 (m, H2, 1H); 4.43 (dd, H3, J ) 6.5, 12.9
Hz, 1H), 4.45 and 4.50 (AB quartet, H10, J ) 14.0 Hz, 2H), 4.87
(br s, NH2, 2H). 13C NMR (methanol-d4) δ 8.1 (1C, CH3), 29.7
(1C), 33.7 (2C), 45.6 (1C), 55.8 (1C), 107.3 (1C, Ph), 110.7 (1C,
Ph), 122.1 (1C, Ph), 128.7 (1C, Ph), 139.8 (1C, Ph), 145.5 (1C,
Ph), 177.7 (1C, CO), 178.6 (1C, CO). HRMS m/z calcd for
C13H15N6O2 [M + H]+: 287.1251, found: 287.1250.
Synthesis of Protected Adduct 17. 2-Fluoro-O6-(2-p-nitrophen-
ylethyl)deoxyinosine19 (16) (18.8 mg, 0.0448 mmol) was dissolved
in dry DMF (40 µL). Compound 14 (17 mg, 0.0449 mmol) and
diisopropylethylamine (10 µL, 7.42 mg, 0.958 mmol) were added
to the reaction mixture which was incubated at 45° for 18 h. The
resulting crude material was diluted with water (100 µL) and
lyophilized. The desired product was isolated by preparative thin
layer chromatography (SiO2:40% acetone:60% EtOAc) to give 15
1
mg (43% yield) of 17. H NMR (methanol-d4): δ 1.80 (s, CH3,
3H), 2.34 (ddd, H2′, J ) 3, 6.2, 13.4 Hz, 1H), 2.64 (dd, H1, J )
4.6, 16 Hz, 1H), 2.82 (ddd, H2′′, J ) 2, 6.2, 13.4 Hz, 1H), 3.13
(dd, H1, J ) 7.1, 15.5 Hz, 1H), 3.19 (t, CH2, J ) 4.6 Hz, 2H), 3.42
and 3.43 (AB quartet, CH2, J ) 14 Hz, 2H), 3.67 (bd, H5′, J )
12.2 Hz, 1H) and 3.77 (bd, H5′′, J ) 8.5 Hz, 1H), 3.99 (d, H3, J )
4.6 Hz, 1H), 4.02 (d, H4′, J ) 4.4 Hz, 1H), 4.39 (dd, H3, J ) 7.1,
12.9 Hz, 1H), 4.52 (m, H3′, 1H), 4.67 (s, CH2, 2H), 4.75 (t, CH2,
J ) 6.6 Hz, 2H), 4.87 (m, H2, 1H), 6.35 (t, H1′, J ) 3.3 Hz, 1H),
7.20-7.27 (m, Har, 5 H), 7.52 (d, Har, J ) 8.5 Hz, 2H), 8.13 (d,
Har, J ) 8.5 Hz, 2H), 8.25 (bs, 1H). HRMS m/z calcd for
C39H40N9O9 [M + H]+: 778.2949, found: 778.2922.
2,7-Diamino-N2-(2-trimethylsilyl)ethoxycarbonyl-10-decar-
bamoyl-10-azido-mitosene (23). 4-Nitrophenyl-2-(trimethylsilyl)-
ethyl carbonate (29 mg, 0.10 mmol) and i-Pr2NEt (0.021 mL, 16
mg, 0.10 mmol) were added to a solution of 22 (16 mg, 0.056
mmol) in i-PrOH (0.70 mL). The reaction mixture was stirred for
3 h at room temperature. TLC analysis (5% MeOH in CH2Cl2)
showed the disappearance of 23, and the formation of a single new
compound. The reaction was quenched by addition of 0.050 mL
of concentrated aqueous ammonia. The resulting mixture was
concentrated in vacuum, and the residue was dissolved in EtOAc
(25 mL) and washed with saturated Na2CO3 (5 × 10 mL) to remove
4-nitrophenol. The organic phase was dried and concentrated in
vacuum. The residue was purified by flash chromatography using
a gradient of 10% to 0% hexane in CH2Cl2 to give 23 (22 mg,
Synthesis of 18. The N2- and O6-protected mitosene-nucleoside
adduct 17 (6 mg, 0.00771 mmol) was dissolved in acetonitrile/
DMF (100 µL/200 µL) and was treated with DBU (30 µL, 30.54
mg, 0.201 mmol). TLC showed the disapearance of all starting
material after 1.5 h. Water was added to the sample (1 mL), and
the solution was lyophilized. The desired product was isolated by
preparative thin layer chromatography (SiO2:3% triethylamine:15%
MeOH:82% CH2Cl2) to give 4 mg (82% yield) of 18 as a red solid.
1H NMR (methanol-d4): δ ) 1.81 (s, CH3, 3H), 2.40 (ddd, H2′, J
) 3.5, 6.2, 13.5 Hz, 1H), 2.69 (m, H2′′, 1H), 2.83 (dd, H1, J ) 4.5,
16 Hz, 1H), 3.19 (m, H1, 1H), 3.67 (dd, H5′, J ) 4.8, 12.8 Hz, 1H)
and 3.75 (dd, H5′′, J ) 3.9, 8.1 Hz, 1H), 3.99 (m, H4′, 1H), 4.02
(dd, H3, J ) 4.8, 13.2 Hz, 1H), 4.45 (dd, H3, J ) 7.5, 13.2 Hz,
1H), 4.51 (bm, H3′, 1H), 4.58 and 4.64 (AB quartet, CH2, J ) 15
Hz, 2H), 4.96 (m, H2, 1H), 6.35 (t, H1′, J ) 7.1 Hz, 1H), 7.20-7.29
(m, Har, 5 H), 8.00 (s, H8, 1H), 8.11 (s, H8, 1H). HRMS m/z calcd
for C31H33N8O7 [M + H]+: 629.2472, found: 629.2485.
1
0.051 mmol, 91%) as a purple solid. H NMR (methanol-d4): δ
0.07 (s, TMS, 9H), 1.02 (t, CH2Si, J ) 8.2 Hz, 2H), 1.79 (s, 3H,
CH3), 2.80 (dd, H1, J ) 5.2, 16.3 Hz, 1H), 3.24 (dd, H1, J ) 7.8,
16.3 Hz, 1H), 4.04 (dd, H3, J ) 5.2, 12.9 Hz, 1H), 4.18 (t, CH2O,
J ) 8.1 Hz, 2H), 4.45 (s, H10, 2H), 4.47 (dd, H3, J ) 7.2, 12.9 Hz,
1H), 4.80 (m, H2, 1H). 13C NMR (methanol-d4) δ-1.3 (3C, TMS),
8.1 (1C, CH3), 17.8 (1C), 31.1 (1C), 45.6 (1C), 55.3 (1C), 54.1
(1C), 63.6 (1C), 107.3 (1C, Ph), 110.9 (1C, Ph), 122.2 (1C, Ph),
128.8 (1C, Ph), 136.7 (1C, Ph), 145.5 (1C, Ph), 155.9 (1C, CO),
174.4 (1C, CO), 178.6 (1C, CO). HRMS m/z calcd for
C19H26N6O4NaSi [M + Na]+: 453.1677, found: 453.1681.
Synthesis of 8. The N2-protected mitosene-nucleoside adduct
18 (4 mg, 0.00636 mmol) was dissolved in a mixture of methanol
(0.6 mL) and a potassium phosphate buffer (1.8 mL, pH ) 8.5, M
) 0.05, KH2PO4). Penicillin amidase (37 units, 40 µL) was added,
and the reaction was incubated overnight at room temperature. The
crude mixture was diluted with methanol (15 mL), and a white
precipitate appeared which was discarded after centrifugation. The
solution was evaporated, and the resulting brown residue was
redissolved in methanol. A second white precipitate appeared and
was discarded as above. The methanol solution was then evapo-
rated to dryness. The black residue was taken up in water and
washed three times with chloroform. The aqueous layer was
collected and lyophilized to give 8 (3 mg, 92% yield) as a brown
solid. The compound was found to be pure by HPLC and NMR
analysis. 1H NMR (pyridine-d5-D2O): δ ) 1.53 (s, CH3, 3H), 2.19
(ddd, H2′, J ) 3.5, 6.2, 13.3 Hz, 1H), 2.38 (app quint, H2′′, J ) 6.9
Hz, 1H), 2.65 (dd, H1, J ) 4, 16 Hz, 1H), 2.77 (dd, H1, J ) 7.1,
16 Hz, 1H), 3.52 (dd, H5′, J ) 4.5, 12 Hz, 1H) and 3.59 (dd, H5′′,
J ) 3.9, 11.8 Hz, 1H), 3.68 (dd, H3, J ) 4.7, 12.5 Hz, 1H), 3.82
(m, H2, 1H), 3.96 (dd, H3, J ) 6.8, 12.8 Hz, 1H), 4.02 (app quartet,
H4′, J ) 5.7 Hz, 1H), 4.13 and 4.15 (AB quartet, CH2, J ) 15 Hz,
2H), 4.50 (app quint, H3′, J ) 3.4 Hz, 1H), 6.23 (t, H1′, J ) 6.8
Hz, 1H), 7.85 (s, H8, 1H). HRMS m/z calcd for C23H27N8O6 [M +
H]+: 511.2054, found: 511.2049.
2,7,10-Triamino-N2-(2-trimethylsilyl)ethoxycarbonyl-10-de-
carbamoyl-mitosene (24). Triphenylphosphine (35 mg, 0.13 mmol)
was added to a solution of 23 (8 mg, 0.018 mmol) in THF/H2O (1
mL; 1:1) and concentrated aqueous ammonia (0.5 mL). The reaction
mixture was stirred for 18 h under Ar at room temperature. TLC
analysis (1% concentrated NH4OH, 7% MeOH, 92% CH2Cl2)
showed the disappearance of 23, and the formation of a single new
compound. The reaction mixture was concentrated in vacuum, and
the resulting residue was purified by flash chromatography using a
gradient of 0 to 2% NH4OH in MeOH/CH2Cl2 (7:100) to give 24
1
(7 mg, 0.017 mmol, 93%) as a purple solid. H NMR (methanol-
d4): δ 0.07 (s, TMS, 9H), 1.07 (t, CH2Si, J ) 8.2 Hz, 2H), 1.80 (s,
CH3, 3H), 2.76 (dd, H1, J ) 5.1, 16.1 Hz, 1H), 3.21 (dd, H1, J )
7.6, 16.1 Hz, 1H), 3.79 (s, H10, 2H), 4.04 (dd, H3, J ) 5.0, 12.9
Hz, 1H), 4.18 (t, CH2O, J ) 8.1 Hz, 2H), 4.44 (dd, H3, J ) 7.0,
12.9 Hz, 1H), 4.78 (m, H2, 1H). 13C NMR (methanol-d4) δ-2.9
(3C, TMS), 6.8 (1C, CH3), 17.2 (1C), 29.1 (1C), 35.3 (1C), 55.4
(1C), 53.7 (1C), 62.7 (1C), 104.8 (1C, Ph), 114.6 (1C, Ph), 122.1
(1C, Ph), 129.2 (1C, Ph), 138.8 (1C, Ph), 147.4 (1C, Ph), 157.2
9
9564 J. AM. CHEM. SOC. VOL. 130, NO. 29, 2008