Journal of Medicinal Chemistry
ARTICLE
Next, the sample was subjected to 31P NMR experiments at 37 °C and
the spectra were recorded every 12 min over 14 h.
added dropwise NMI (0.40 mL, 5.07 mmol). The reaction mixture was
allowed to stir for 30 min, and then a solution of appropriate phosphor-
ochloridate (5) (3.04 mmol) dissolved in anhydrous THF (3 mL) was
added dropwise. The reaction mixture was stirred at room temperature
for 16ꢀ18 h and then evaporated in vacuo to give a residue that was
redissolved in CH2Cl2 and washed twice with 0.5 M HCl (2 ꢁ 5 mL).
The organic phase was purified by column chromatography on silica gel,
eluting with CH2Cl2ꢀMeOH as a gradient (0ꢀ5% MeOH) to afford
the products as white solid.
Carboxypeptidase Y (EC 3.4.16.1) Assay. The experiment was
carried out by dissolving FUDR ProTide 7d (3.0 mg) in acetone-d6
(0.15 mL) and by adding 0.30 mL of Trizma buffer (pH 7.6). After the
31P NMR data were recorded at 25 °C as a control, a previously
defrosted carboxypeptidase Y (0.1 mg dissolved in 0.15 mL of Trizma)
was added to the sample. Next, the sample was submitted to 31P NMR
experiments (at 25 °C) and the spectra were recorded every 7 min over
14 h. 31P NMR recorded data were processed and analyzed with the
Bruker Topspin 2.1 program.
Stability Assay in Human Serum. The experiment was carried
out by dissolving FUDR ProTide 7a (5.0 mg) in DMSO (0.050 mL) and
D2O (0.15 mL). After the 31P NMR data were recorded at 37 °C as a
control, a previously defrosted human serum (0.30 mL) was added to
the sample. Next, the sample was submitted to 31P NMR experiments at
37 °C and the spectra were recorded every 15 min over 14 h. 31P NMR
recorded data were processed and analyzed with the Bruker Topspin 2.1
program.
Chemistry. General. Anhydrous solvents were obtained from
Aldrich and used without further purification. Amino acid esters were
purchased from Carbosynth. Carboxypeptidase Y, human serum, and
buffers were from Sigma-Aldrich. All reactions were carried out under an
argon atmosphere. Reactions were monitored with analytical TLC on
silica gel 60-F254 precoated aluminum plates and visualized under UV
(254 nm) and/or with 31P NMR spectra. Column chromatography was
performed on silica gel (35ꢀ70 μM). Proton (1H), carbon (13C),
phosphorus (31P), and fluorine (19F) NMR spectra were recorded on a
Bruker Avance 500 spectrometer at 25 °C. Spectra were autocalibrated
to the deuterated solvent peak, and all 13C NMR and 31P NMR were
proton-decoupled. The purity of final compounds was verified to be
>95% by HPLC analysis using Varian Polaris C18-A (10 μM) as an
analytic column with a gradient elution of H2O/MeOH from 100/0 to
0/100 in 45 min (method 1) and with a gradient elution of H2O/
CH3CN from 100/0 to 0/100 in 35 min (method 2). The HPLC
analysis was conducted by Varian Prostar (LC Workstation-Varian
prostar 335 LC detector). Low and high resolution mass spectra were
performed as a service by Birmingham University, Birmingham, U.K.,
using electrospray mass spectrometry (ESMS). CHN microanalysis was
performed as a service by MEDAC Ltd., Surrey, U.K.
5-Fluoro-20-deoxyuridine 50-O-[1-Naphthyl(benzyl-L-alaninyl)]
phosphate (7m). 7m was obtained from 5-fluoro-20-deoxyuridine and
5m as a white solid. Yield, 8% (47.0 mg). Rf = 0.19 (CH2Cl2ꢀMeOH,
95:5). (ES+) m/z, found: (M + Na+) 636.1520. C29H29N3O9FNaP
required: (M+), 613.15. Mixture of diastereoisomers (43%, 57%). 31P
NMR (202 MHz, MeOD): δP 4.61, 4.25. 19F NMR (470 MHz, MeOD):
1
δF ꢀ167.45, ꢀ167.25. H NMR (500 MHz, MeOD): δH 8.18ꢀ8.12
(m, 1H, ArH), 7.90ꢀ7.86 (m, 1H, ArH), 7.72ꢀ7.67 (m, 2H, ArH, H-6),
7.55ꢀ7.47 (m, 3H, ArH), 7.45ꢀ7.27 (m, 6H, ArH), 6.16ꢀ6.06 (m, 1H, H-10),
5.13, 5.08 (2 ꢁ AB system, 2H, J = 12.0 Hz, OCH2Ph), 4.36ꢀ4.24 (m,
3H, 2 ꢁ H-50, H-30), 4.15ꢀ4.03 (m, 2H, CHCH3, H-40), 2.17ꢀ2.08 (m,
1H, H-20), 1.79ꢀ1.67 (m, 1H, H-20), 1.38ꢀ1.34 (m, 3H, CHCH3).
13C NMR (125 MHz, MeOD):δC 174.9 (d, 3JCꢀP = 4.3 Hz, CdO, ester),
174.6 (d, 3JCꢀP = 5.0 Hz, CdO, ester), 159.3 (d, 2JCꢀF = 26.1 Hz, CdO,
base), 150.5 (d, 4JCꢀF = 4.0 Hz, CdO, base), 147.9 (d, 2JCꢀP = 7.4 Hz,
2
C-Ar, Naph), 147.8 (d, JCꢀP = 7.7 Hz, OC-Naph), 141.7, 141.6 (2d,
1JCꢀF = 234.0 Hz, CF-base), 137.2, 137.1, 136.2 (C-Ar), 129.7, 129.6,
129.5, 129.4, 129.0, 128.9, 128.1, 128.0 (CH-Ar), 127.9, 127.8 (C-Ar),
127.7, 127.6, 126.6, 126.5, 126.2 (CH-Ar), 125.6, 125.5 (2d, 2JCꢀF = 34.0
Hz, CH-base), 122.6 (CH-Ar), 116.5, 116.2 (2d, 3JCꢀP = 3.5 Hz, CH-
Ar), 87.0, 86.9 (C-10), 86.8, 86.7 (2d, 3JCꢀP = 8.1 Hz, C-40), 72.1, 72.0
(C-30), 68.1, 68.0 (CH2Ph), 67.8, 67.6 (2d, 2JCꢀP = 5.2 Hz, C-50), 51.9,
3
51.8 (CHCH3), 40.9, 40.8 (C-20), 20.5 (d, JCꢀP = 6.5 Hz, CHCH3),
20.3 (d, 3JCꢀP = 7.6 Hz, CHCH3). Reverse HPLC, eluting with H2O/
MeOH from 100/0 to 0/100 in 45 min, showed two peaks of the
diastereoisomers with tR = 34.23 min and tR = 34.59 min (47%, 51%).
Anal. Calcd for C29H29FN3O9P: C, 56.77; H, 4.76; N, 6.85. Found: C,
56.57; H, 5.06; N, 6.72. UV (0.05 M phosphate buffer, pH 7.4)
λmax = 271 nm (εmax = 7050). log P measured: 1.74.
5-Fluoro-20deoxyuridine 50-O-[(L-Alaninyl)]phosphate Am-
monium Salt (100). 5-Fluoro-20deoxyuridine 50-O-[1-naphthyl(benzyl-
L-alaninyl)]phosphate (7m) (0.08 g, 0.130 mmol) was dissolved in a
solution of triethylamine (5 mL) and water (5 mL). The reaction mixture
was stirred at 35 °C for 16 h, and then the solvents were removed under
reduced pressure. The residue was treated with water and extracted with
dichloromethane. The aqueous layer was concentrated and evaporated
under reduced pressure. Then the resulting crude material was purified by
column chromatography on silica, eluting with 2-propanolꢀH2OꢀNH3
(8:1:1) to afford the title compound 100 as a white solid. Yield, 30% (15.0
mg). Rf = 0.04 (2-propanolꢀH2OꢀNH3 (8:1:1)). 31P NMR (202 MHz,
D2O): δP 7.13. 19F NMR (470 MHz, D2O): δF ꢀ168.00. 1H NMR (500
MHz, D2O): δH 7.93 (d, 1H, 3JHꢀF = 6.1 Hz, H-6), 6.30ꢀ6.25 (m, 1H, H-
10), 4.49ꢀ4.44 (m, 1H, H-30), 4.11ꢀ4.06 (m, 1H, H-40), 3.94ꢀ3.83 (m,
2H, H-50), 3.53 (q, 1H, J = 7.5 Hz, CHCH3), 2.37ꢀ2.28 (m, 2H, H-20),
1.25ꢀ1.19 (m, 3H, CHCH3). 13C NMR (125 MHz, MeOD): δC 174.8 (d,
3JCꢀP = 4.6 Hz, CdO), 159.2 (d, 2JCꢀF = 26.2 Hz, CdO, base), 150.3 (d,
4JCꢀF = 4.0 Hz, CdO, base), 141.8 (d, 1JCꢀF = 233.8 Hz, CF-base), 125.6
(d, 2JCꢀF = 34.0 Hz, CH-base), 87.0 (C-10), 86.7 (d, 3JCꢀP = 7.5 Hz, C-40),
71.1 (C-30), 67.2 (d, 2JCꢀP = 5.5 Hz, C-50), 51.0 (CHCH3), 40.2 (C-20),
20.3 (d, 3JCꢀP = 7.2 Hz, CHCH3). m/z (ES) 396.1 (M ꢀ 2 NH4ꢀ + H]ꢀ,
100%). Reverse-phase HPLC, eluting with H2O/MeOH from 100/0 to
0/100 in 45 min, 1 mL/min, λ = 275 nm, showed a peak of the
diastereoisomer with tR = 3.65 min (95%).
General Method for the Preparation of Phosphorochlori-
dates (5). Anhydrous triethylamine (2.0 mol equiv) was added dropwise
at ꢀ78 °C to a stirred solution of the appropriate aryl dichlorophosphate
(1.0 mol equiv) and an appropriate amino acid ester (1.0 mol equiv) in
anhydrous DCM under argon atmosphere. Following the addition, the
reaction mixture was allowed to slowly warm to room temperature and was
stirred for 1ꢀ2 h. The formation of desired compound was monitored by
31P NMR. After the reaction was completed, the solvent was evaporated
under reduced pressure and the resulting residue was redissolved in
anhydrous Et2O and filtered. The filtrate was reduced to dryness to give
a crude product as an oil, which was in some cases used without further
purification in the next step. Most of aryl phosphorochloridates, in particular
those obtained from the amino acid tosylate salt, were purified by flash
column chromatography using EtOAc/hexane (7:3) as an eluent.
1-Naphthyl (Benzyl-L-alaninyl)phosphorochloridate (5m).
Yellowish oil; yield, 47% (1.82 g). Rf = 0.90 (hexaneꢀEtOAc, 7:3). 31
P
NMR (202 MHz, CDCl3, mixture of diastereoisomers): δP 7.92, 8.14
1
(int, 1.00:1.00). H NMR (500 MHz, CDCl3, mixture of diastereo-
isomers with a ratio of 1:1): δH 8.12ꢀ7.97 (m, 1H, ArH), 7.73ꢀ7.09 (m,
11H, ArH), 5.09 (s, 2H, OCH2Ph), 4.81ꢀ4.78 (m, 1H, NH), 4.23ꢀ4.20
(m, 1H, CHCH3), 1.45ꢀ1.43 (m, 3H, CHCH3).
General Method for the Preparation of FUDR ProTides
(6aꢀn and 7aꢀy). To a solution of 5-fluoro-20-deoxyuridine (0.25 g,
1.01 mmol) in dry THF (10 mL) at 0 °C under argon atmosphere was
7256
dx.doi.org/10.1021/jm200815w |J. Med. Chem. 2011, 54, 7247–7258
McGuigan, Christopher
