Enzymatically ActiVated cycloSal-d4TMPs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1665
brought to boil with a heat gun for 30 s. This procedure was
repeated until the deprotection was complete (monitoring by TLC,
CH2Cl2). The reaction mixture was diluted with CH2Cl2 and water
and the aqueous layer was extracted with CH2Cl2. The combined
organic layers were washed with water twice, dried with sodium
sulfate and concentrated under reduced pressure. The product was
purified by preparative TLC [Chromatotron; petroleum ether 50-
70/CH2Cl2 gradient (50-100%) and CH2Cl2/CH3OH gradient (0-
5%)]. Yield: 903 mg (4.34 mmol, 68%) of an orange oil. TLC Rf
for further purification the saligenyl chlorophosphite was not storred
in dry THF overnight at -20 °C but in dry Et2O for a complete
precipitation of pyridinium chloride. Quantities (for saligenyl
chlorophosphite synthesis): 4-Formyl-6-methylsalicyl alcohol (7b,
750 mg, 4.51 mmol) dissolved in 35 mL of dry THF, freshly
destilled phosphorus(III)chlorid (0.46 mL, 5.3 mmol), dry pyridine
(0.81 mL, 10 mmol) in dry THF. Yield: 623 mg (2.72 mmol).
Quantities (for cycloSal-d4T-monophosphate synthesis): Saligenyl
chlorophosphite (620 mg, 2.71 mmol) dissolved in dry CH3CN,
d4T (1, 285 mg, 1.27 mmol) dissolved in 24 mL of dry CH3CN,
DIPEA (0.35 mL, 2.0 mmol), tert-butyl hydroperoxide (5.5 M in
n-nonane; 0.70 mL, 3.9 mmol). Yield: 282 mg (0.649 mmol, 51%)
of a diastereomeric mixture (ratio 0.9:1.0) as a colorless foam. TLC
Rf value 0.44 (CH2Cl2/CH3OH, 9:1). 1H NMR (500 MHz, DMSO-
d6): δ ) 11.37 (s, 1H, 1 X NH), 11.34 (s, 1H, 1 X NH), 9.91 (s,
1H, 1 X formyl-H), 9.90 (s, 1H, 1 X formyl-H), 7.84-7.81 (m,
2H, 2 X aryl-H-4), 7.72-7.68 (m, 2H, 2 X aryl-H-6), 7.19 (d, J )
1.0 Hz, 1H, 1 X thymine-H-6), 7.16 (d, J ) 1.0 Hz, 1H, 1 X
thymine-H-6), 6.81-6.76 (m, 2H, 2 X 1′-H), 6.44-6.41 (m, 1H, 1
X 3′-H), 6.40-6.36 (m, 1H, 1 X 3′-H), 6.05-6.00 (m, 2H, 2′-H),
5.60 (dd, J ) 14.8, 9.8 Hz, 1H, 1 X benzyl-H), 5.57 (dd, J ) 15.1,
8.8 Hz, 1H, 1 X benzyl-H), 5.48 (dd, J ) 13.9, 6.9 Hz, 1H, 1 X
benzyl-H), 5.45 (dd, J ) 14.2, 8.2 Hz, 1H, 1 X benzyl-H), 4.98-
4.93 (m, 2H, 2 X 4′-H), 4.38-4.25 (m, 4H, 2 X 5′-H), 2.29 (s, 3H,
1 X CH3), 2.26 (s, 3H, 1 X CH3), 1.67 (s, 6H, 2 X thymine-CH3)
ppm. 13C NMR (101 MHz, DMSO-d6): δ ) 191.49 (1 X formyl-
C), 191.46 (1 X formyl-C), 163.63 (1 X thymine-C-4), 163.60 (1
X thymine-C-4), 152.24 (2 X aryl-C-2), 150.62 (2 X thymine-C-
2), 135.62 (2 X thymine-C-6), 132.71 (1 X C-3′), 132.61 (1 X C-3′),
132.36 (2 X aryl-C-4), 131.94 (2 X aryl-C-5), 127.45 (2 X aryl-
C-3), 127.30 (2 X C-2′), 125.17 (1 X aryl-C-6), 125.14 (1 X aryl-
C-6), 121.92 (2 X aryl-C-1), 109.62 (1 X thymine-C-5), 109.53 (1
X thymine-C-5), 89.26 (1 X C-1′), 89.21 (1 X C-1′), 84.05 (1 X
C-4′), 83.98 (1 X C-4′), 68.84 (1 X C-5′), 68.77 (1 X C-5′), 67.95
(1 X benzyl-C), 67.87 (1 X benzyl-C), 14.80 (1 X CH3), 14.71 (1
X CH3), 11.79 (1 X thymine-CH3), 11.75 (1 X thymine-CH3) ppm.
31P NMR (162 MHz, DMSO-d6): δ ) -9.07, -9.53. HRMS
(ESI+) m/z ) calcd 457.0777 [M + Na+], found 457.0787 [M +
Na+]. UV/vis (water/CH3CN): λmax ) 260 nm.
5-Formyl-3-tert-butyl-cycloSal-d4T-monophosphate (6c). The
preparation of 6c was carried out as described above for 6a, but
dry Et2O was used as solvent. Quantities (for saligenyl chloro-
phosphite synthesis): 4-Formyl-6-tert-butylsalicyl alcohol (7c, 550
mg, 2.64 mmol) dissolved in 18 mL of dry Et2O, freshly destilled
phosphorus(III)chlorid (0.28 mL, 3.2 mmol), dry pyridine (0.50 mL,
6.2 mmol) in dry Et2O; yield: 308 mg (1.13 mmol). Quantities
(for cycloSal-d4T-monophosphate synthesis): Saligenyl chloro-
phosphite (130 mg, 0.477 mmol) dissolved in dry CH3CN, d4T (1,
50 mg, 0.22 mmol) dissolved in 4 mL of dry CH3CN, DIPEA (60
µL, 0.34 mmol), tert-butyl hydroperoxide (5.5 M in n-nonane; 0.12
mL, 0.67 mmol). Yield: 52 mg (0.11 mmol, 49%) of a diastere-
omeric mixture (ratio 0.7:1.0) as a colorless foam. TLC Rf value
0.56 (CH2Cl2/CH3OH, 9:1). 1H NMR (500 MHz, DMSO-d6): δ )
11.35 (s, 1H, 1 X NH), 11.32 (s, 1H, 1 X NH), 9.96 (s, 2H, 2 X
formyl-H), 7.94-7.91 (m, 2H, 2 X aryl-H-4), 7.78-7.75 (m, 2H,
2 X aryl-H-6), 7.22 (s, 1H, 1 X thymine-H-6), 7.19 (s, 1H, 1 X
thymine-H-6), 6.84-6.77 (m, 2H, 2 X 1′-H), 6.45-6.39 (m, 2H, 2
X 3′-H), 6.06-6.01 (m, 2H, 2′-H), 5.61-5.44 (m, 4H, 2 X benzyl-
H), 5.01-4.95 (m, 2H, 2 X 4′-H), 4.39-4.33 (m, 4H, 2 X 5′-H),
1.61 (s, 3H, 1 X thymine-CH3), 1.57 (s, 3H, 1 X thymine-CH3),
1.38 (s, 9H, 1 X t-Bu), 1.35 (s, 9H, 1 X t-Bu) ppm. 13C NMR (101
MHz, DMSO-d6): δ ) 192.23 (1 X formyl-C), 192.19 (1 X formyl-
C), 163.75 (1 X thymine-C-4), 163.70 (1 X thymine-C-4), 153.07
(2 X aryl-C-2), 150.72 (2 X thymine-C-2), 139.39 (1 X aryl-C-3),
139.32 (1 X aryl-C-3), 135.88 (2 X thymine-C-6), 132.94 (1 X
C-3′), 132.90 (1 X C-3′), 131.96 (2 X aryl-C-5), 129.33 (2 X aryl-
C-4), 127.47 (1 X C-2′), 127.32 (1 X C-2′), 125.77 (2 X aryl-C-6),
123.89 (1 X aryl-C-1), 123.80 (1 X aryl-C-1), 109.70 (1 X thymine-
C-5), 109.59 (1 X thymine-C-5), 89.35 (1 X C-1′), 89.22 (1 X C-1′),
84.11 (2 X C-4′), 69.28 (1 X C-5′), 69.23 (1 X C-5′), 67.89 (1 X
benzyl-C), 67.83 (1 X benzyl-C), 34.57 (1 X t-Bu-C), 34.50 (1 X
1
value 0.43 (CH2Cl2/CH3OH, 19:1). H NMR (400 MHz, DMSO-
d6): δ ) 9.81 (s, 1H, formyl-H), 7.68 (d, J ) 2.0 Hz, 1H, aryl-
H-5), 7.65 (d, J ) 2.0 Hz, 1H, aryl-H-3), 4.69 (s, 2H, benzyl-H),
1.39 (s, 9H, t-Bu) ppm. 13C NMR (101 MHz, DMSO-d6): δ )
191.61 (formyl-C), 159.92 (aryl-C-1), 136.97 (aryl-C-6), 128.37
(aryl-C-2), 127.97 (aryl-C-4), 127.58 (aryl-C-3), 127.32 (aryl-C-
5), 60.59 (benzyl-C), 34.45 (t-Bu-C), 29.25 (t-Bu-CH3) ppm. MS
(FAB) m/z ) calcd 209.1 [M + H+], found 209.1 [M + H+].
5-Formyl-cycloSal-d4T-monophosphate (6a). Under nitrogen,
a solution of the salicyl alcohol derivate 7a (300 mg, 1.97 mmol)
in 15 mL of dry THF was cooled to -20 °C. After addition of
freshly destilled phosphorus(III)chlorid (0.21 mL, 2.4 mmol) and
stirring at -20 °C for 10 min, a solution of dry pyridine (0.37 mL,
4.6 mmol) in dry THF was added at the same temperature over a
period of 3 h. After completion of the addition the reaction mixture
was allowed to warm up to room temperature and stirred for 1.5 h.
It was kept at -20 °C overnight for best possible precipitation of
pyridinium chloride. Filtration under nitrogen and concentration
of the filtrate under reduced pressure afforded the phosphitylating
agent (saligenyl chlorophosphite) as a crude product to be directly
used for the synthesis of the cycloSal phosphate triester 6a without
further purification. The general synthesis of cycloSal-d4T-mono-
phosphates has been published before.4,6 To a solution of d4T (1,
180 mg, 0.804 mmol) in 15 mL of dry CH3CN was added DIPEA
(0.22 mL, 1.3 mmol). The resulting solution was cooled to -20 °C
and the saligenyl chlorophosphite (396 mg, 1.83 mmol) dissolved
in dry CH3CN was added. The reaction mixture was allowed to
warm up to room temperature and stirring was continued for 3 h.
Subsequently, tert-butyl hydroperoxide (5.5 M in n-nonane; 0.45
mL, 2.5 mmol) was added at -20 °C. After warming up to room
temperature and stirring for 1 h the reaction mixture was poured
over 1 M HOAc/ NaOAc buffer (pH ) 5). The aqueous layer was
extracted with EtOAc four times. The combined organic layers were
dried with sodium sulfate and concentrated under reduced pressure.
The resulting residue was purified by TLC [Chromatotron; CH2-
Cl2/CH3OH (0.1% HOAc) gradient (0-5%)]. The isolated product
was lyophilized from CH3CN/H2O 1:1 (v/v). Yield: 105 mg (0.250
mmol, 31%) of a diastereomeric mixture (ratio 0.8:1.0) as a colorless
1
foam. TLC Rf value 0.50 (CH2Cl2/CH3OH, 9:1). H NMR (500
MHz, DMSO-d6): δ ) 11.34-11.32 (m, 2H, 2 X NH), 9.95 (s,
2H, 2 X formyl-H), 7.97-7.90 (m, 2H, 2 X aryl-H-4), 7.89-7.85
(m, 2H, 2 X aryl-H-6), 7.35 (d, J ) 8.2 Hz, 1H, 1 X aryl-H-3),
7.32 (d, J ) 8.4 Hz, 1H, 1 X aryl-H-3), 7.18 (s, 1H, 1 X thymine-
H-6), 7.17 (s, 1H, 1 X thymine-H-6), 6.81-6.79 (m, 1H, 1 X 1′-
H), 6.78-6.76 (m, 1H, 1 X 1′-H), 6.44-6.41 (m, 1H, 1 X 3′-H),
6.39-6.36 (m, 1H, 1 X 3′-H), 6.05-6.00 (m, 2H, 2′-H), 5.63 (dd,
J ) 14.8, 6.9 Hz, 1H, 1 X benzyl-H), 5.60 (dd, J ) 14.8, 6.3 Hz,
1H, 1 X benzyl-H), 5.53-5.46 (m, 2H, 2 X benzyl-H), 4.99-4.94
(m, 2H, 2 X 4′-H), 4.40-4.27 (m, 4H, 2 X 5′-H), 1.67 (s, 3H, 1 X
thymine-CH3), 1.63 (s, 3H, 1 X thymine-CH3) ppm. 13C NMR (101
MHz, DMSO-d6): δ ) 191.47 (2 X formyl-C), 163.67 (2 X
thymine-C-4), 163.66 (2 X aryl-C-2), 150.73 (2 X thymine-C-2),
134.96 (2 X thymine-C-6), 131.90 (2 X C-3′), 131.03 (2 X aryl-
C-4), 127.29 (2 X aryl-C-5), 126.91 (2 X aryl-C-6), 126.69 (2 X
C-2′), 122.16 (2 X aryl-C-1), 118.45 (2 X aryl-C-3), 109.70 (2 X
thymine-C-5), 88.73 (2 X C-1′), 84.53 (2 X C-4′), 68.34 (2 X C-5′),
67.47 (2 X benzyl-C), 11.51 (2 X thymine-CH3) ppm. 31P NMR
(162 MHz, DMSO-d6): δ ) -9.84, -10.03. HRMS (ESI+) m/z )
calcd 443.0620 [M + Na+], found 443.0585 [M + Na+]. UV/vis
(water/CH3CN): λmax ) 258 nm.
5-Formyl-3-methyl-cycloSal-d4T-monophosphate (6b). The
preparation of 6b was carried out as described above for 6a, but