Synthesis and stability studies of phosphonoformate-amino acid conjugates: A new class of slowly releasing foscarnet prodrugs

Article abstract of DOI:10.1016/j.bmcl.2004.01.016

Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be susceptible to P-C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA P-N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or basic conditions, which led, respectively, to premature cleavage of the P-N linkage (with incomplete deprotection of the PFA ester moiety), or to P-C cleavage. Here we report that novel, fully deprotected PFA-amino acid P-N conjugates 4 can be prepared via coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic intermediate 3 that resists P-C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37°C, the resulting new PFA-amino acid (Val, Leu, Phe) conjugates (4a-c) undergo P-N cleavage near neutral pH, cleanly releasing PFA. A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [4a] with respective t_1/2 values of 1.4, 3.8, and 10.6 h.

Full text of DOI:10.1016/j.bmcl.2004.01.016

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1787–1790
Synthesis and stability studies of phosphonoformate–amino acid
conjugates: a new class of slowly releasing foscarnet prodrugs
Mong S. Marma, Boris A. Kashemirov and Charles E. McKenna*
Department of Chemistry, University of Southern California, Los Angeles, CA 90089-0744, USA
Received 20 November 2003; accepted 10 January 2004
Abstract—Prodrugs of phosphonoformic acid (PFA), an anti-viral agent used clinically as the trisodium salt (foscarnet), are of
interest due to the low bioavailability of the parent drug, which severely limits its utility. Neutral PFA triesters are known to be
susceptible to P–C bond cleavage under hydrolytic de-esterification conditions, and it was previously found that P,C-dimethyl PFA
P–N conjugates with amino acid ethyl esters did not release PFA at pH 7, and could not be fully deprotected under either acid or
basic conditions, which led, respectively, to premature cleavage of the P–N linkage (with incomplete deprotection of the PFA ester
moiety), or to P–C cleavage. Here we report that novel, fully deprotected PFA-amino acid P–N conjugates 4 can be prepared via
coupling of C-methyl PFA dianion 2 with C-ethyl-protected amino acids using aqueous EDC, which gives a stable monoanionic
ꢀ
intermediate 3 that resists P–C cleavage during subsequent alkaline deprotection of the two carboxylate ester groups. At 37 C, the
resulting new PFA-amino acid (Val, Leu, Phe) conjugates (4a–c) undergo P–N cleavage near neutral pH, cleanly releasing PFA.
A kinetic investigation of 4a hydrolysis at pH values 6.7, 7.2, and 8.5 showed that PFA release was first-order in [4a] with respective
t1/2 values of 1.4, 3.8, and 10.6 h.
#
2004 Elsevier Ltd. All rights reserved.
Foscarnet, the trisodium salt of phosphonoformic acid
(
vage of the PFA–amino acid linkage occurs in vivo. It
was recently reported that a fully alkyl-protected, neu-
tral P–N linked PFA–amino acid phosphoramide
PFA), has been used as an antiviral agent1 to treat
À4
AIDS-related cytomegalovirus infection, and also exerts
5
an inhibitory action against HIV. The antiviral effect
of PFA is attributed to inhibition of viral nucleic acid
produced by coupling (MeO)(Cl)P(O)CO Me with an
2
l-amino acid Et ester, decomposed with P–C cleavage
under basic deprotection conditions.
6
,7
17À19
The same
polymerases. Because of its multiple negative charges
at physiological pH, PFA has low oral bioavailability;
this poor ability to penetrate cell membranes has long
been a major impediment to its overall effectiveness as
an anti-viral drug.8 Past efforts to address this short-
coming by prodrug strategies have included partial
workers found that acidic conditions applied to the
same protected conjugate hydrolyzed its P–N linkage
1
7
instead of dealkylating the PFA diester. This perplex-
ing result is not unanticipated, given that the P–C bond
in PFA esters is known to be highly activated due to
juxtaposition of the carbonyl and phosphoryl groups,
and is highly susceptible to cleavage under hydrolytic
conditions to produce a phosphate or phosphite,
depending on whether the water nucleophile attacks at
,9
8
À13
esterification of PFA,
1
including its incorporation
into cyclic esters, and the replacement of an oxygen
1
atom in PFA by a sulfur atom.1
4À16
It has been shown
that all three acidic groups of PFA must be available for
maximal nucleic acid polymerase inhibition activity, and
thus the ultimate antiviral activity of PFA prodrugs
wherein one or more of these groups has been deriva-
tized by esterification (or amidation) is likely to depend
on a hydrolytic activation process in vivo to release the
2
0À24
carbon or phosphorus.
We reasoned that an inter-
mediate product from condensation between C-methyl
PFA and a C-alkyl-protected amino acid should be sta-
bilized to unwanted P–C bond cleavage at high pH by
virtue of its retention of one negative charge on the
phosphonate group, thereby permitting alkaline depro-
tection of both carboxylate esters.
8
active drug. Thus, PFA–amino acid conjugates might
be useful as prodrugs of foscarnet, provided that such
compounds are synthetically accessible and facile clea-
We report here successful application of this simple
strategy to synthesis of the first examples of PFA–
amino acid conjugates (4), and describe the surprisingly
*
Corresponding author. Tel.: +1-213-740-7007; fax: +1-213-740-
930; e-mail: mckenna@usc.edu
0
0
960-894X/$ - see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.01.016

1788
M. S. Marma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1787–1790
facile release of PFA from these isolable prodrugs at
physiological pH and temperature.
To circumvent the decomposition observed previously
in attempted hydrolytic deprotection of a neutral l-
1
7
amino acid C-ester-PFA P-diester P–N conjugate, we
investigated coupling of a dianionic PFA C-monoester
such as 2 with a C-blocked amino acid. The single
negative charge remaining on the phosphoryl group in
the product (3) was expected to impede P–C bond clea-
vage during subsequent alkaline hydrolysis of the pro-
tecting alkyl groups. To form the P-N linkage, we chose
EDC-promoted coupling of 2 with C-ethyl protected
l-amino acids (Val, Leu, or Phe) in aqueous solution
Figure 1.
plished by removal of remaining EDC/by-products with
acetone, followed by washing with MeOH or EtOH to
remove excess NaOH. The structural integrity of the
conjugate during work upand pu rification was mon-
2
5
under neutral to slightly alkaline conditions. The
intermediate 2 was obtained by quantitative and regio-
selective silyldealkylation of the P,P-dimethyl groups of
3
1
itored by the CH–N–P P NMR doublet (dP 3–4,
2
6,27
3
trimethyl PFA with bromotrimethylsilane (BTMS).
JPH=ꢁ8.5 Hz) (Scheme 1).
3
1
The amidation reaction was followed by P NMR,
which revealed replacement over several hours of the
singlet resonance of 2 by a doublet due to CH–N–P
coupling. The condensation reaction went to comple-
tion without detectable formation of double condensa-
tion by-product, even with excess EDC and amino acid.
The intermediate reaction mixture was processed to
remove unreacted amino acid and NaCl, and the inter-
mediate product (3a–c) was then hydrolyzed with
NaOH to yield the target PFA conjugates (4a–c). Grat-
ifyingly, 3 was deprotected under these conditions
without any trace of P–C or P–N bond cleavage. Care-
ful control of pH, and appropriate choice of washing/
extraction solvents were important for success. Specifi-
cally, the coupling reaction was done within a pH range
of 7.0–7.5; excess amino acid and NaCl were removed
from the reaction mixture by extraction with EtOAc
and acetone, successively, after adjustment of the pH to
The structure and purity of the products were confirmed
1
13
31
by NMR ( H, C, and P), combustion microanalysis
8
2
and HRMS. The compounds were stable at room
temperature in the solid state.
The potential of the conjugates 4 as prodrugs depends
on their ability to release the active drug PFA under
mild conditions. Interestingly, they all cleanly released
PFA and amino acid at physiological pH and temper-
ature, with no P–C bond cleavage observed. The kinetics
of pH-dependent release of PFA from 4a was studied at
several pH values near neutrality (6.7, 7.4, 8.5; all at
ꢀ
31
37 C) by P NMR. P–N bond cleavage liberating PFA
was exclusively observed in a process characterized by
pseudo-first order kinetics, with t1/2 values of 1.4, 3.8
and 10.6 h, respectively (Fig. 1 and Scheme 2). The
other two analogues (4b–c) also cleanly released the
parent drug within this pH range (data not shown). All
conjugates were found to be unstable at low pH; at pH
9.5; dealkylation of 3a–c was optimized at pH ꢁ12.5;
and purification of the final products 4a–c was accom-
Scheme 1.
Scheme 2. Hydrolytic pathways of the conjugates at different pH values.

M. S. Marma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1787–1790
1789
5
cleavage producing phosphate along with PFA.
.5 they underwent competitive P–C and P–N bond
2
22. Thatcher, G. R. J.; Krol, E. S.; Cameron, D. R. J. Chem.
Soc., Perkin Trans. 2 1994, 683.
9
2
2
2
2
3. Mitchell, A. G.; Nicholls, D.; Walker, I.; Irwin, W.;
Freeman, S. J. Chem. Soc., Perkin Trans. 2 1991, 1297.
4. Mitchel, M. G.; Nicholls, D.; Irwin, W. I.; Freeman, S.
J. Chem. Soc., Perkin Trans. 2 1992, 1145.
In conclusion, we have elaborated a practical method
for synthesis and isolation of deprotected P–N linked
PFA–amino acid conjugates. These phosphonopeptide
analogues are good candidates for evaluation as pro-
drugs of PFA because they release the drug quantita-
tively under physiological conditions, even in the
absence of amidases. These conjugates or related com-
pounds might also be susceptible to active transport by
5. Morr, M.; Wray, V. Angew. Chem., Int. Ed. Engl. 1994,
3
3, 1394.
6. McKenna, C. E.; Schmidhauser, J. J. Chem. Soc., Chem.
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27. McKenna, C. E.; Higa, M. T.; Cheung, N. H.; McKenna,
M. C. Tetrahedron Lett. 1977, 18, 155.
3
0À33
membrane peptide transporters.
28. Synthesis of N-[(hydroxycarbonyl)hydroxyphosphinyl]-l-
Valine, Trisodium Salt, 4a: to 2.00 mL trimethyl phos-
phonoformate (14.87 mmol) stirred magnetically under
nitrogen was added slowly 6.00 mL BTMS (45.46 mmol).
The reaction mixture was stirred overnight at room
temperature and excess BTMS was removed by rotary
evaporation. The residue was immediately treated with
Acknowledgements
This research was supported by the National Institutes
of Health.
1 N NaOH to obtain a pH of 9.4. Volatiles were removed
by rotary evaporation, and the residue was lyophilized to
obtain a white powder, 2 (2.33 g), which was used in
coupling reactions without further purification. NMR: d
References and notes
H
(
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with 590 mg l-valine ethyl ester hydrochloride (3.25
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was added to adjust the pH to 7.5, and the mixture stirred
overnight at room temperature; conversion to coupled
3
1
5
product 3a was quantitative by P NMR. The reaction
mixture pH was readjusted to 9.5 (1 N NaOH) then
extracted with EtOAc (3Â50 mL) to remove excess amino
acid. The aqueous phase was then evaporated to dryness
by rotary evaporation, and the conjugate separated from
NaCl by extraction with acetone. The acetone phase was
evaporated to dryness and the residue dissolved in 7 mL
6
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8
2
H O, then hydrolyzed by adding 5 N NaOH until the pH
was 12.5. Reaction was complete at room temperature
within 1/2 h. The reaction mixture was then evaporated to
dryness and washed repeatedly with acetone and MeOH
successively until a constant pH was attained (pH 9.5),
then washed once again with acetone to leave 4a as a
white powder (139 mg, 38% yield from 1). NMR: dH
9
. Neto, C. C.; Steim, J. M.; Sarin, P. S.; Sun, D. K.; Bhon-
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(D
2
O): 3.17 (1H, dd, J=9.3, 5.1 Hz), 1.63 (1H, m), 0.73
(D O):
180.3 (d, J =3.2 Hz), 179.4 (d, JCP=215 Hz), 60.3 (d,
(3H, d, J=7.3 Hz), 0.72 (3H, d, J=6.9 Hz); d
C
2
3
1
CP
2
3
J
(D
CP=8.4 Hz), 30.3 (d,
JCP=3.9 Hz) 16.7, 15.3; d
P
O): 3.7 (d, JPH=9.4 Hz). HRFAB-MS: m/z 267.9973
3
2
À
(M-Na) , calcd for C NPNa
.
for C H NO PNa H O: C 23.32, H 3.59, N 4.53; found:
C 23.29, H 3.36, N 4.37. Synthesis of N-[(hydroxy-
carbonyl)hydroxyphosphinyl]-l-Leucine, Trisodium Salt,
4b: Using the procedure described for 4a, 150 mg (0.82
mmol) of 2 produced 89 mg of 4b (31% yield from 1).
6
9
H O
6
2
: 267.9963. Anal calcd
6
9
6
3
2
1
1
1
1
4. McKenna, C. E.; Ye, T. G.; Levy, J. N.; Wen, T.;
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G.; Cheng, Y. C.; Kilkuskie, R. Phosphorus, Sulfur Silicon
Relat. El. 1993, 76, 399.
NMR: d
H 2
(D O): 3.37 (1H, m), 1.39 (1H, m), 1.21 (2H,
3
m), 0.63 (6H, m); d
C
(D
(d, JCP=220 Hz), 56.9 (d, JCP=8.4 Hz), 46.2 (d,
2
O): 184.7 (d, JCP=3.6 Hz), 182.2
1
2
3
J
J
CP=4.0 Hz), 25.6, 23.5, 23.4; d (D
PH=9.1 Hz). HRFAB-MS: m/z 282.0129 (M-Na) ,
P
2
O): 3.4 (d,
À
3
calcd for C
C H NO Na P.2H O: C 24.65, H 4.43, N 4.11 found: C
7 6 2
H11NO PNa 282.0120. Anal calcd for
7. Li, H.-Y.; Chen, R.-Y.; Ren, K.-T. Phosphorus, Sulfur
Silicon Relat. El. 1996, 119, 279.
7
11
6
3
2
24.70, H 4.11, N 4.04. Synthesis of N-[(hydroxycarbonyl)-
hydroxyphosphinyl]-l-phenylalanine, Trisodium Salt, 4c:
Using the procedure described for 4a, except coupling was
done at pH 7.0, and purification steps were modified as
specified below. Unlike 4a and 4b, this compound was
partially soluble in MeOH, therefore washing with this
solvent to remove excess base could not be applied. The
product was instead washed repeatedly with acetone fol-
1
1
8. Chen, R.; Li, H. Sci. China. B: Chem. 1996, 39, 371.
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Soc., Chem. Comm. 1991, 118.

1790
M. S. Marma et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1787–1790
lowed by 1:1 EtOH/acetone until pH of the residue dis-
solved in H O was a constant (9.8); it was then washed
with acetone, leaving 4a as a white powder (281.0 mg,
the NMR signal versus PFA produced during hydrolysis.
The half-lives were calculated from the slopes of linear
plots of the logarithm of the remaining conjugate 4a
versus time. The stability of 4a was also studied at pH
5.5 using acetate buffer (0.6 M). Within 20 min, the
conjugate underwent complete decomposition to PFA
2
H 2
43% yield from 1). NMR: d (D O): 7.10 (5H, m), 3.67
(
1H, m), 2.83 (1H, m), 2.63 (1H, m); dC (D O): 182.5 (d,
2
1
3
JCP=215.5 Hz), 182.3 (d, JCP=3.8 Hz), 139.3, 131.2,
2
1
29.8, 127.9, 59.3 (d, JCP=5.3 Hz), 42.6; d
P
(D
d, JPH=8.3 Hz). HRFAB-MS: m/z 315.9963 (M–Na) ,
2
O): 3.2
À
2
and phosphate with the evolution of CO . PFA and
3
31
(
calcd for (C H NO PNa ), 315.9963. Anal calcd for
phosphate were identified by comparing P NMR after
spiking with the authentic compounds. The other con-
jugates 4b–c also released the parent drug PFA in the
neutral pH range.
1
0
9
6
2
6 2
3.
PNa 2H O: C 32.02, H 3.49, N 3.73; found: C
C
10
H
9
NO
2.44, H 3.16, N 3.75.
9. Hydrolysis kinetics of 4a: Buffer solutions with pH 6.7,
3
2
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7.4 or 8.5 (0.6 M borate–boric acid buffer) were prepared.
4a (10 mg) in an NMR tube was dissolved in 0.5 mL
buffer solution plus two drops of D
2
O, and immediately
placed in an thermostat at 37 C. The progress of
ꢀ
3
1
hydrolysis was monitored by P NMR at suitable inter-
vals. At the given pH values, PFA was the only phos-
phorus containing product. The product was determined
3
1
by comparison ( P NMR) with authentic PFA. The % of
a remaining was calculated from the relative height of
4