(
4H, dq, J 8.3, 7.1, 2 × CH O), 4.67 (2H, dd, J 46.9, 4.8,
General procedure for the preparation of the morpholidate
4,17
2
PCH F); δ (100 MHz, CDCl ) 16.40 (d, J 5.7, CH ), 63.01 (d,
(24)
2
C
3
3
J 6.3, CH O), 76.59 (dd, J 180.7, 169.3, PCH F); δ (162 MHz,
2
2
P
This compound was made up immediately prior to coupling
with the bisphosphonate residues. A typical procedure for the
preparation of the morpholidate 24 is as follows: To a solution
12
CDCl ) 17.01 (d, J 63.2) (lit., 16.3); δ (376 MHz, CDCl )
3
F
3
1
2
Ϫ85.77 (dt, J 63.2, 46.7) (lit., Ϫ250.4 (CFCl external stand-
ard)) [Found: (EI) M , 170.05076. C H FO P requires
70.05081]; m/z 170 (1.9%, M ), 149 (38.3), 86 (50.7), 57 (100).
3
ϩ
5
12
3
4
of the phosphonate 23 (17.1 mg, 0.052 mmol) in 50% aqueous
ϩ
1
3
2
-methylpropan-2-ol (1.5 cm ) was added morpholine (0.036
3
cm , 0.41 mmol) which was stirred at room temperature for 1 h.
Assessment of the thermal stability of lithiated diethyl
This was brought to reflux and a solution of DCC (84.7 mg,
11
fluoromethylphosphonate (18)
3
0
.41 mmol) in 2-methylpropan-2-ol (3 cm ) was added dropwise
3
over 4 h via a syringe pump and was left under reflux until the
reaction had gone to completion (by TLC). After allowing the
To a solution of LDA (1.20 mmol) in anhydrous THF (1.5 cm ),
pre-cooled to Ϫ78 ЊC, was added a solution of 18 (186 mg, 1.09
3
mixture to cool, solvents were evaporated and water was added
mmol) in THF (1 cm ) dropwise. This was stirred at Ϫ78 ЊC for
3
(
(
(
5 cm ). The suspension was filtered and the residue washed
6
0 min before allowing to warm to 0 ЊC and quenching with a
3
H O, 8 cm ). Aqueous extracts were combined and washed
ether, 3 × 5 cm ) and solvents were then removed from the
saturated solution of aqueous KH PO . The mixture was
2
2
4
3
extracted (EtOAc × 3) and the combined organics were dried
MgSO ) before solvents were removed to give a crude sample
combined aqueous layers to give a pale brown gum. Residual
water was removed as an azeotropic mixture (pyridine 3 × 1
(
4
of 18 as a yellow–brown oil (103 mg). The crude mixture was
3
3
31
cm followed by benzene 1 cm ) to give the morpholidate 24 as
approximately 88 mol% pure (by P NMR) showing that only
9% of the starting material 18 was recovered.
an off white solid.
4
Ethyl 2-fluoro 2-(diethoxyphosphoryl)acetate (21)
Enzyme assays
To a slurry of sodium hydride (pre-washed with Et O) in
anhydrous THF (20 cm ) at 0 ЊC was added a solution of ethyl
Testing of compounds as inhibitors of HIV-rt catalysed DNA
synthesis was carried out using the DuPont RT-Detect assay
2
3
diethoxyphosphorylacetate 20 (1.02 g, 4.5 mmol) in THF
(cat. No. NEK-070A). Recombinant HIV-1-rt was also pur-
chased from DuPont (cat. no. NEI-490) . All the reference
standards, inhibitor-free controls and test compound concen-
trations were assayed in at least two parallel runs and the results
were averaged. Activity values are expressed as the concen-
tration of test substrate (µM) which is required to inhibit HIV-
rt catalysed cDNA synthesis by 50% (IC50). The IC50 values are
relative to the values of the external standards, AZTTP 2 and
3
(
20 cm ) dropwise which was stirred at 0 ЊC for 30 min and at
room temperature for 60 min. Following the addition of
3
anhydrous DMF (13 cm ), (F-TEDA-BF ) (145 mg, 4.83 mmol)
4
was added as one portion and the solution was stirred at room
temperature for 140 min. The reaction mixture was then poured
3
into ether (30 cm ) and the organics were washed with 5%
3
aqueous sulfuric acid (25 cm ) followed by saturated aqueous
3
20
sodium hydrogen carbonate (25 cm ). The organic layer was
5, which were also tested in the same assay. AZTTP 2 and
4
then dried (MgSO ) and solvents were removed. The crude
5 were prepared according to literature procedures.
4
material was purified by chromatography using EtOAc–petrol
(
1:1) as eluent to give 21 as a colourless oil (188 mg, 17%).
Ϫ1
Acknowledgements
νmax(neat)/cm 1769 (C᎐O), 1278 (P᎐O); δ (300 MHz, CDCl )
H
3
1
4
5
.34 (3H, t, J 7.2, CH ), 1.37 (6H, ddt, J 7.0, 3.0, 0.5, 2 × CH ),
3
3
This paper is dedicated to the memory of Professor Ralph
Raphael, a truly inspirational synthetic organic chemist. We
thank the Medical Research Council (U.K.) for a research
studentship to C. J. H.
.19–4.30 (4H, m, 2 × CH OP), 4.35, (2H, q, J 7.5, CO CH ),
2
2
2
13
.19, (1H, dd, J 47.7, 12.0, PCFH) (lit., 5.2, J 48, α-proton);
δ (100 MHz, CDCl ) 14.03, (CH ), 16.31 (d, J 5.7, CH ), 62.42
C
3
3
3
(
CH ), 64.19 (d, J 6.7, CH ), 64.22 (d, J 6.7, CH ), 85.04 (dd,
2
2
2
J 196.6, 158.6, CFH), 164.81 (d, J 20.9, C᎐O); δ (162 MHz,
᎐
P
CDCl ) 10.77 (d, J 72.0); δ (376 MHz, CDCl ) Ϫ46.50 (dd,
3
F
3
References
13
J 72.0, 47.0) (lit., Ϫ210, J 70.5 (CFCl external standard))
3
ϩ
1 (a) E. De Clercq, J. Med. Chem., 1995, 38, 2491; (b) H. Mitsuya,
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[
Found: (EI) M , 242.07218. C H FO P requires 242.07194];
8 16 5
ϩ
m/z 242 (3.1%, M ), 214 (31.6, MH Ϫ Et), 197 (39.2, M Ϫ OEt),
1
37 (26.2, M Ϫ CFHCO Et), 109 (100).
2
2
(a) E. J. De Clercq, J. Med. Chem., 1986, 29, 1561; (b) C. Kim,
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1
987, 30, 867; (c) R. Vince and M. Hua, J. Med. Chem., 1990, 33, 17;
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3
3
(a) S. M. Daluge, S. S. Good, M. B. Faletto, W. H. Miller, M. H.
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stirring until the mixture was sufficiently acidified (pH 1). The
Dowex resin was removed and the filtrate was lyophilised to
give 22 as a sticky yellow–white lyophilate (393 mg, 98%). Trace
(
b) R. H. Foster and D. Faulds, Drugs, 1998, 55, 729.
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6
7
C. J. Hamilton, S. M. Roberts and A. Shipitsin, Chem. Commun.
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1
impurities were evident by H NMR but the highly polar nature
T. Rozovskaya, A. Mischenko, N. Tarusova, M. Kukhanova,
A. Krayevsky and R. Bibilashvilli, Mol. Biol.1993, 27, 649.
(a) B. Eriksson, A. Larsson, E. Helgstrand, N.-G. Johanson and
B. Öberg, Biochim. Biophys. Acta, 1980, 607, 53; (b) B. Eriksson,
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J. M. Reno, L. F. Lee, A. F. Isbell and J. A. Boezi, Biochemistry,
of the monoester made further purification very difficult so the
isolated material was carried through, to be purified at a later
step. δ (300 MHz, D 0) 1.37 (3H, t, J 7.5, CH ), 4.14 (2H, dt,
H
2
3
J 6.6, 6.6, CH ), 5.40 (1H, dd, J 47.1, 12.3, PCFH); δ (100
2
C
MHz, D O) 15.96 (d, J 5.5, CH ), 62.97 (t, J 6.3, CH ), 86.68
2
3
2
1
2
976, 15, 426; (d) C. Crumpacker, Am. J. Med., 1992, 92 (suppl. 2A),
(
dd, J 187.6, 143.3, CFH), 170.57 (C᎐O); δ (162 MHz, D O)
᎐
P
2
A-3S and references therein.
8
.33 (d, J 64.0); δ (376 MHz, D O) Ϫ42.53 (dd, J 64.0, 47.0)
F 2
ϩ
8
For mechanistic studies on the role of phosphonoformic and
phosphonoacetic acid as irreversible non-competitive inhibitors of
DNA polymerases see: (a) B. A. Larder, D. J. M. Purifoy, K. L.
[
Found: (FAB) M Ϫ H , 185.00135. C H FO P requires
4
7
5
ϩ
1
85.00152]; m/z 185 (97%, M Ϫ H ), 153 (100).
J. Chem. Soc., Perkin Trans. 1, 1999, 1051–1056
1055