Synthesis of a potent inhibitor of HIV reverse transcriptase

Article abstract of DOI:10.1039/a801127c

The newly synthesised P(β),-P(γ)-difluoromethylenebisphosphonate analogue 2 of nor-carbovir triphosphate is a potent inhibitor of HIV reverse transcriptase; it also exhibits a greatly enhanced stability to dephosphorylation, in foetal blood serum, relative to AZTTP and other nucleoside triphosphates.

Full text of DOI:10.1039/a801127c

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Synthesis of a potent inhibitor of HIV reverse transcriptase
Chris J. Hamilton,a Stanley M. Robertsb† and Alexander Shipitsinc
a Department of Chemistry, Exeter University, Stocker Road, Exeter, Devon, UK EX4 4QD
b Robert Robinson Laboratories, Department of Chemistry, Liverpool University, Liverpool, UK L69 3BX
c Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilov Street, Moscow 117984, Russia
O
O
O
The newly synthesised Pb,–Pg-difluoromethylenebisphos-
phonate analogue 2 of nor-carbovir triphosphate is a potent
inhibitor of HIV reverse transcriptase; it also exhibits a
greatly enhanced stability to dephosphorylation, in foetal
blood serum, relative to AZTTP and other nucleoside
triphosphates.
i
ii, iii
v, iii
[(HO)2P]2CF2•NBu3
(EtO)2PCF2H
[(EtO)2P]2CF2
5
6
O
O
O
iv
[(EtO)2P]2CH2
[(EtO)2P]2CFH
[(HO)2P]2CFH•NBu3
8
7
O
O
P
O
O
vii
The in vivo lability of the P–O–P phosphate ester bonds in
nucleoside triphosphates (NTPs) possessing interesting biol-
ogical activity in vitro has prompted the search for more stable
analogues. A great deal of effort has centred on the substitution
of the phosphate ester oxygen atoms with carbon to give the
corresponding phosphonates. These phosphonates are generally
more stable to hydrolytic cleavage whilst being isosteric with
their parent phosphates. The lower electronegativity of the
methylene group, compared to oxygen, leads to a significant
decrease in the acid dissociation constants of these phosphonic
acids, which is often reflected in a reduction of biological
activity. Through the seminal work of Blackburn and co-
workers, halogenoalkylphosphonates have been shown to have
improved potential as phosphate mimetics, as they more closely
resemble the steric and electronic features of their parent
units.1
HO
P
O
P
P
O
G
G
6 (→ 2)
HO
X
O
X
8 (→ 3)
HO
OH
OH
9 (→ 4)
2 X = CF2
3 X = CFH
4 X = CH2
10 X = OH
11 X =
vi
N
O
O
O
N
NH
G =
[(HO)2P]2CH2•NBu3
N
N
NH2
9
Scheme 1 Reagents and conditions: i, LDA, THF, 270 °C, 1 h, then
ClP(O)(OEt)2, 270 °C, 1 h (34%); ii, Me3SiBr, room temp., 18 h (83%); iii,
NBu3, EtOH, H2O, room temp., 90 min; iv, KHMDS, THF, 278 °C, 1 h,
then N-fluorodibenzenesulfonimide, THF–toluene, 278 °C, 90 min (26%);
v, Me3SiBr, room temp., 72 h (98%); vi, morpholine, DCC, ButOH, H2O,
reflux, 5 h; vii, DMSO, room temp., 0.5–7 days (24–32%)
O
N
NH
exchange, followed by reverse phase chromatography, and
isolated as their ammonium salts.8
The efficacies of 2–4 as inhibitors of recombinant HIV-1-rt
(Du Pont cat. No. NEI-490) were examined using the Du Pont
RT-DetectTM Reverse Transcriptase Assay (cat. No. NEK-070)
and the results are shown in Table 1.
O
P
O
O
1 X = O
N
N
NH2
2 X = CF2
3 X = CFH
4 X = CH2
P
P
O
HO
X
O
HO
OH
OH
Compounds 2–4 showed an expected correlation of increased
activity with an increase in fluoro-substitution, culminating in
the CF2 analogue 2 being just an order of magnitude less active
than the parent compound 1.
The stability of the CF2 compound 2 in human foetal blood
serum was assessed and compared with AZTTP, some natural
nucleoside triphosphates, and the pyrophosphoryl phospho-
nates’ natural enantiomer, 1-ent (Table 2). Blood serum is an
appropriate medium in which to perform this assay as it contains
numerous dephosphorylating enzymes and so provides a good
model system of the extracellular environment in vivo. The half-
We have previously shown that the pyrophosphoryl phos-
phonate 1 shows potent inhibition of HIV reverse transcriptase
(HIV-rt), comparable to that of both AZT and carbovir
triphosphates.2 It is noteworthy that the active compound 1 has
an absolute configuration that is the mirror image of that
expected for a natural nucleotide.3 Previous assessments of
other such ‘unnatural’ nucleosides have shown them to exhibit
reduced cytoxicity relative to their natural enantiomers.4
Further progress in this area would result from structural
modifications of the diphosphate unit in 1 to further enhance its
stability in vivo whilst still retaining good biological activity. To
these ends we have prepared a series of bisphosphonates 2–4
with progressive fluoro-substitution within the Pb,Pg-methylene
linker group, as described in Scheme 1.
Noteworthy features of the chemical syntheses include the
preparation of the tetraethyl difluoromethylenebisphosphonate
5 by coupling diethyl difluoromethylphosphonate with diethyl
chlorophosphate,5 and the preparation of tetraethyl fluor-
omethylenebisphosphonate 7 using the easy to handle and
readily available fluorinating reagent N-fluorodibenzene-
sulfonimide.6 The tetraesters were hydrolysed using bromo-
trimethylsilane and subsequently converted into their respective
tributylammonium salts 6, 8 and 9. The bisphosphonates were
coupled to the nucleoside monophosphonate 10 via the
activated morpholidate 11.2,7 Products were purified by anion
Table 1 Relative efficacy of substrates 1–4 as inhibitors of HIV-rta
Compound
IC50b/mm
1
2
0.5
5.8
3
4
34.8
> 100
1.0
AZTTP
a The IC50 values shown are the results from enzyme assays carried out in
reaction mixtures containing 0.1 units ml21 of HIV-rt, which were
incubated at 37 °C for 110 min. b IC50 = substrate concentration required
to inhibit to HIV-rt by 50%.
Chem. Commun., 1998
1087

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Table 2 Half-life time in fetal blood serum at 37 °C
Notes and References
† E-mail: sj11@liverpool.ac.uk
Compound
t1/2
1 G. M. Blackburn, D. Kent, F. Eckstein and T. Pere´e, Nucleosides
Nucleotides, 1985, 4, 165; G. M. Blackburn and D. Kent, J. Chem. Soc.,
Perkin Trans. 1, 1986, 913; G. M. Blackburn and D. Kent, J. Chem. Soc.,
Chem. Commun., 1981, 930.
2 D. Coe, S. M. Roberts and R. Storer, J. Chem. Soc., Perkin Trans. 1,
1992, 2695; V. Merlo, S. M. Roberts, R. Storer and R. Bethell, J. Chem.
Soc., Perkin Trans. 1, 1994, 1477.
3 Both enantiomers of carbovir triphosphate are equally effective as
inhibitors of HIV-rt; W. Miller, S. Daluge, E. Garvey, S. Hopkins,
J. Reardon, F. Boyd and F. Miller, J. Biol. Chem., 1992, 267, 21220.
4 T. Lin, M. Luo, M. Liu, Y. Zhu, E. Gullen, G. Dutschman and Y. Cheng,
J. Med. Chem., 1996, 39, 1757.
1-ent
2
dGTP
dATP
AZTTP
65 mina
45 ha
< 30 minb
< 30 minb
5 minb
a As determined by the appearance of the phosphonate 10. b As determined
by the disappearance of the nucleoside triphosphate.
life of compound 2 was found to be 90 times greater than those
of the natural purine NTPs, dGTP and dATP and also
significantly greater than that of the pyrophosphoryl phospho-
nate 1-ent. Evidently, the Pb,Pg-difluoromethylene group
greatly enhances the stability of compound 2 towards enzymatic
dephosphorylation of the terminal phosphate group. Cleavage
of the phosphate ester linkage at the Pa,Pb position could also be
reduced as a result of the CF2 analogue 2 being a poorer
substrate for those dephosphorylating enzymes which function
to hydrolyse NTPs at this position.
In conclusion, we have shown that it is possible to
incorporate two stabilising phosphonate linkages in the 5A-side
chain of the ‘unnatural’ enantiomer of carbovir triphosphate
with only a modest compromise in biological activity whilst
significantly enhancing biological stability. The use of more
lipophilic derivatives of compound 2, in order to facilitate drug
delivery into whole cells, is currently under investigation and
will be reported in due course.
5 M. Obayashi, E. Ito, K. Matsui and K. Kondo, Tetrahedron Lett., 1982,
23, 2323.
6 E. Differding, R. Duthaler, A. Krieger, R. Ru¨egg and C. Schmit, Synlett,
1991, 395; previous methodology employed perchloryl fluoride
(C. McKenna and P. Shen, J. Org. Chem., 1981, 46, 4573) or acetyl
hypofluorite (D. Hebel, K. Kirk, J. Kinjo, T. Kova´cs, L. Lesiak,
J. Balzarini, E. De Clercq and P. Torrence, Bioorg. Med. Chem. Lett.,
1991, 7, 357).
7 J. Moffatt and H. Khorana, J. Am. Chem. Soc., 1961, 83, 649; J. Moffatt,
Can. J. Chem., 1964, 42, 599.
8 Selected data for 2: nmax(KBr)/cm21 1269 (PNO); lmax(H2O)/nm 252.0;
dH(400 MHz, D2O) 1.89 (1 H, dt, J 15.6, 4.4, 5A-bH), 2.97 (1 H, dt, J 14.0,
7.2, 5A-aH), 3.84 (2 H, d, J 9.2, PCH2), 4.76–4.81 (1 H, br, 4A-H),
5.28–5.0 (1 H, br, 1A-H), 6.07–6.11 (1 H, m, 2A-H), 6.33–6.38 (1 H, m,
3A-H), 7.75–7.87 (1 H, br, 8-H); dF(376 MHz, D2O) 42.40–42.90 (br,
CF2); dP(162 MHz, D2O) 21.69 (br, Pb), 3.90 (br, Pg), 11.44 (d, J 31.0,
Pa); m/z (ES) 522 (6%, MH+), 328 (15, M 2 CH2F2O4P2). For 3:
nmax(KBr)/cm21 1236 (PNO); dP(162 MHz, D2O) 2.44 (br, Pb), 9.61 (br,
Pg), 10.34 (d, J 29.0, Pa); m/z (ES) 504 (57%, MH+), 328 (100, M 2
CFH3O4P2). For 4: nmax(KBr)/cm21 1216 (PNO); dP(162 MHz, D2O)
15.97 (1P, br, Pb), 10.08 (2P, br, Pa and Pg); m/z (ES) 486 (35%, MH+),
328 (100, M 2 CH4O4P2).
We thank the MRC for a research studentship (to C. J. H.) and
Wellcome Research Laboratories for the generous donation of
AZT, made available through the MRC AIDS Reagent
Project.
Received in Cambridge, UK, 9th February 1998; 8/01127C
1088
Chem. Commun., 1998