Degradation kinetics of oxycarbonyloxymethyl prodrugs of phosphonates in solution

Full text of DOI:10.1023/A:1011044804823

Pharmaceutical Research, Vol. 18, No. 2, 2001
Short Communication
MATERIALS AND METHODS
Materials
Degradation Kinetics of
Oxycarbonyloxymethyl Prodrugs of
Phosphonates in Solution
The crystalline fumarate salts of bis-POC PMEA and
bis-POC PMPA and the corresponding mono-POC esters
were synthesized by the Process Chemistry Departments, Gi-
lead Sciences, Inc. Sodium chloride was available from
Mallinckrodt. All salts and solvents were either reagent or
1
,2
1
Lung-Chi Yuan, Terrence C. Dahl, and
1
,3
Reza Oliyai
18
HPLC grade and used as received. O heavy water (95%
enriched) was obtained from Aldrich Chemical Corporation.
Deionized water was used for buffer and HPLC mobile phase
preparation.
Received July 27, 2000; accepted November 3, 2000
KEY WORDS: prodrugs; phosphonates; degradation; kinetics; teno-
fovir; adefovir.
Solution Stability
Buffers were prepared and the pHs measured at 50°C.
The pH 1 buffer was prepared from 0.1 N HCl; pH 2–3
buffers were prepared from H PO4 and KH PO ; pH 4–5
INTRODUCTION
3
2
4
Phosphonate analogs of nucleotides have recently re-
ceived considerable attention as potential antiviral agents.
The ionic character of these agents limits their permeability
across the human intestinal mucosa, resulting in low bioavail-
ability after oral administration (1,2). We have previously
demonstrated the utility of the bis-isopropyloxycarbonyl-
methyl (bis-POC) moiety in improving the oral bioavailability
of phosphonate nucleotides (3–5). The bis-POC promoiety
utilizes the oxycarbonyloxymethyl spacer group. The lipo-
philicity of the prodrug can be adjusted by varying the chain
length of the alcohol. Scheme I depicts the putative enzymatic
steps involved in the bioconversion of bis-POC prodrugs of a
phosphonate moiety to the corresponding phosphonate
monoester. The initial enzymatic catalysis is believed to occur
at the site remote from phosphorus, thus avoiding enzymatic
phosphorylation.
buffers were prepared from CH COOH and CH COOK; pH
3
3
6
- 8 buffers were prepared from KH PO and K HPO . Total
2 4 2 4
buffer concentrations were 10, 50, and 100 mM, and the total
ionic strength was adjusted to 0.30 M with KCl. Bis-POC
PMEA fumarate and bis-POC PMPA fumarate stock solu-
tions (20 mM) were prepared using a diluent comprised of
8
0% aqueous/20% acetonitrile and were stored at −20°C.
Working solutions were prepared by transferring 0.5 mL of
the stock solution to a 25 mL volumetric flask and adding the
appropriate buffer solution. The stability samples had a final
concentration of 0.40 mM. The solution stability samples were
filled in two mL, 13 mm, flint type I tubing vials (West Com-
pany). These vials were capped with 4416/50 gray butyl, 13
mm, Teflon-faced stoppers (West Company) and sealed with
13 mm, flip-off aluminum seals (West Company). The stabil-
ity was monitored at 50°C. Samples removed from the stabil-
ity chambers were stored at −20°C until HPLC analysis. The
hydrolysis of pH 8 stability samples (10, 50, and 100 mM)
were quenched to pH 3.0 using 1.0 N HCl solutions and then
stored at −20°C until HPLC analysis. The pseudo-first order
rate constant, kobs, was obtained by following the disappear-
ance of the peak area of the prodrugs as a function of time for
at least two half-lives. The ionization constant determined
using potentiometric titration of the free base of bis-POC
PMEA was determined to be 1.58 × 10 (pKa ס 3.8) and was
used for the curve fitting of the pH-rate profile.
From linear regression analysis of the plots of kobs versus
the total buffer concentration at a fixed pH and ionic strength,
the intercepts yielded the first-order buffer independent rate
The oxycarbonyloxymethyl promoiety has been previ-
ously applied to amines and hindered alcohols (6,7). Safadi
and co-workers utilized this spacer group promoiety to en-
hance the water solubility of various compounds by chemi-
cally linking inorganic phosphates to hindered alcohols and
amines (6). These prodrugs were chemically unstable and
were not suitable as commercially viable pharmaceuticals. Al-
exander and co-workers also applied this spacer group to alter
the lipophilicity of amine containing agents (7). To our
knowledge, the degradation kinetics and hydrolytic pathway
of oxycarbonyloxymethyl spacer group applied to phospho-
nates have not been reported.
−
4
In the present study, we have applied the bis-POC pro-
moiety to 9-((R)-2-(phosphonomethoxy)ethyl)adenine (Ad-
efovir, PMEA) and to 9-((R)-2-(phosphonomethoxy)propyl)
adenine (Tenofovir, PMPA). The chemical stability of bis-
POC PMEA and bis-POC PMPA (Scheme II) in solution
constant, k . The respective slopes yielded the second-order
o
buffer dependent rate constants, kcat. The buffer independent
pH-rate profile was curve fit to a semiempirical equation.
1
8
were investigated. In addition, the O incorporation studies
were conducted to elucidate the degradation pathway(s) for
the hydrolysis of the prodrug in aqueous solution.
HPLC Analysis
All prodrugs and their degradation products were ana-
lyzed by a reverse phase HPLC method using the modular
system described in the instrumentation section. The HPLC
method employed an Inertsil ODS-2, 5 ␮, 4.6 × 150 mm col-
umn (Keystone Scientific). Elutions were performed at am-
bient temperature using mixtures of mobile phases consisting
1
Gilead Sciences, 333 Lakeside Drive, Foster City, California 94404.
Present address: Praecis Pharmaceutical, One Hampshire Street,
2
Cambridge, Massachusetts 02139.
3
To whom correspondence should be addressed. (e-mail: of (A) 5% acetonitrile/95% 20 mM sodium phosphate pH 6.8
reza_oliyai@gilead.com)
v/v and (B) 65% acetonitrile/35% 20 mM sodium phosphate
0
724-8741/01/0200-0234$19.50/0 © 2001 Plenum Publishing Corporation
234

Oxycarbonyloxymethyl Prodrugs of Phosphonates
235
pH 6.8 v/v. The flow rate was set at 1.0 mL/min with UV
detection at 260 nm. The mobile phase was set at 100% mo-
bile phase (A) from 0 to 5 min, followed by an 18 minute
linear gradient to 100% mobile phase (B) and a 7 minute
re-equilibration with 100% mobile phase (A). The elution
times of bis-POC PMEA, bis-POC PMPA, mono-POC
PMEA, and mono-POC PMPA were 18.6 min, 19.2 min, 11.8
min, and 12.4 min respectively.
Determination of the Site of Bond Cleavage (C-O
and/or P-O)
The site of nucleophilic attack was determined by hydro-
1
8
16
lyzing bis-POC PMEA in H2 O and H2 O and then per-
forming mass spectral analysis of the hydrolytic product,
mono-POC PMEA. Complete hydrolysis of bis-POC PMEA,
confirmed by HPLC, was performed at 37°C in 460 ␮L of
1
8
9
␮
5% enriched H2 O, which was adjusted to pH 7.0 using 20
1
8
L of 0.5 M ammonium acetate. The final content of O in
water was approximately 91%. The control samples were pre-
1
6
pared similarly, except H2 O was used as the solvent to es-
timate the uncertainties of the measurements. The mass spec-
tra samples were analyzed using the liquid secondary ion mass
spectrometry technique (LSIMS, Negative FAB-MS). The
mass spectrometry samples were prepared by mixing 1 ␮L of
matrix with 1 ␮L of sample solution on the LSIMS probe tip
and introducing the probe into the source of the mass spec-
trometer. Glycerol was used as the matrix to perform all the
1
8
sample analysis. The incorporation of O to the phosphorous
for bis-POC PMEA was determined based on the ratio of
(
M+1)/(M−1) of the corresponding mono-POC PMEA by
1
8
negative FAB-MS, corrected for the final content of O in
water.
To determine if oxygen exchange between the solvent
and the hydrolytic products was occurring, additional control
experiments, under identical conditions, were performed us-
ing mono-POC PMEA as starting materials. The incorpora-
1
8
tion of O label into monoesters was examined using nega-
tive FAB-MS.
RESULTS AND DISCUSSION
Scheme I. Bioconversion of bis-POC ester to the corresponding
mono-POC ester.
Solution Hydrolysis of bis-POC PMEA
The kinetics of hydrolysis bis-POC PMEA were studied
at 50°C and ␮ ס 0.30 M as functions of pH and buffer con-
centration. Hydrolysis of bis-POC PMEA leads to the forma-
tion of one mole of each formaldehyde, isopropanol, carbon
dioxide, and mono-POC PMEA (Scheme I). Mono-POC
PMEA was the only degradation product observable by
HPLC. Mass balance was obtained for all pH values studied.
The plausible intermediates shown in Scheme I were not
chromatographically detected and are considered to be rela-
tively unstable.
fraction of free base, kH is the microscopic second-order rate
constant for the hydronium ion catalyzed hydrolysis, kH2O is
the first-order rate constant for water catalyzed or spontane-
ous hydrolysis of the conjugate acid, kЈH2O is the first-order
rate constant for water catalyzed or spontaneous hydrolysis of
the free base, and kOH is the microscopic second-order rate
constant for hydroxide ion catalyzed hydrolysis. The ioniza-
tion of the adenine ring appears to modestly perturb the pH-
−1
rate profile. The value of kЈ
(0.015 hr ) is only 1.5 times
H2O
−1
The pH-dependency of the buffer-independent rate con-
higher than the kH2O (0.01 hr ), indicating that the free base
is slightly more reactive than the conjugate acid.
stants, k , for bis-POC PMEA is shown in Figure 1. Over the
o
pH range studied, the rates of hydrolysis of bis-POC PMEA
were described by the following equation:
Hydrolysis of bis-POC PMEA was subjected to buffer
catalysis in the pH range of 2–8. The rate of hydrolysis in-
creased with increasing buffer concentration while maintain-
ing constant pH and ionic strength. The extent of buffer ca-
talysis increased with increasing the solution pH, indicating
+
−
ko ס kH fAH (H ) + k
fAH+ kЈH2O fA + kOH fA (OH )
H2O
Eq (1)
where, fAH is the fraction of conjugate acid and fA is the general base catalysis.

236
Yuan, Dahl, and Oliyai
Table I. Pseudo First Order Rate Constants of POC-PMPA (kPMPA
)
and POC-PMEA (kPMEA) as a Function of pH at 50°C
a
−1
2
−1
2
pH
kPMEA (hr ) × 10
kPMPA (hr ) × 10
kPMEA/kPMPA
1
2
3
4
5
6
7
8
.1
4.42
1.72
1.55
1.61
2.7
9.15
39.17
243.5
2.67
1.30
1.21
1.65
2.11
3.95
17.91
109.0
1.7
1.3
1.3
1.0
1.3
2.3
2.2
2.2
.0
.0
.0
.0
.0
.0
.0
a
Total buffer concentration was 100 mM, and total ionic strength was
adjusted to 0.30 M.
nucleophilic attack of water on the carbonyl moiety leads to
1
8
O incorporation into carbon dioxide. Hydrolysis of bis-POC
1
8
16
PMEA was carried out in H2 O and H2 O at pH 7.0, 37°C.
Mono-POC PMEA was analyzed for isotopic composition us-
ing negative FAB mass spectrometry. In addition, the mono-
POC PMEA was also incubated with H2 O under the iden-
tical condition as bis-POC PMEA. The extent of P-O bond
cleavage was estimated to be 85% ± 5% for bis-POC PMEA
at pH 7.0, 37°C.
1
8
These observations suggest that hydrolysis of bis-POC
PMEA primarily proceeds via P-O bond cleavage with an
additional minor pathway involving C-O bond cleavage.
1
8
There was minimal incorporation of O into mono-POC
1
8
Fig. 1. pH-rate profile for the degradation of bis-POC PMEA at PMEA during incubation with the H2 O enriched buffer sys-
5
0°C. The pH-rate profile was fitted to the following equation: k ס
o
tem.
+
−
kH fAH(H ) + k
M
1
fAH+ k’
fA + kOH fA (OH ), where k ס 0.40
H2O
H2O
−1
H
−
1
−1
−1
hr , k
ס 0.01 hr , kЈH2O ס 0.015 hr , and kOH ס 1.65 × Solution Hydrolysis of bis-POC PMPA
H2O
−1
−
5
−1
0
M
hr .
The kinetics of bis-POC PMPA hydrolysis were also
studied at 50°C and ␮ ס 0.30 M as functions of pH. Mono-
POC PMPA was the only degradation product observable by
HPLC and mass balance was obtained for all the pH values.
Table I compares the rate of hydrolysis of bis-POC PMPA to
bis-POC PMEA. Interestingly, bis-POC PMPA demonstrated
better chemical stability than bis-POC PMEA. At pH values
above 6.0, the rate of hydrolysis of bis-POC PMPA is about 2
times slower than bis-POC PMEA. The side chain methyl
group may stericly hinder the nucleophilic attack of water at
the phosphorus center. This is consistent with the O incor-
poration studies indicating that the attack at the phosphorus
is the primary degradation pathway for bis-POC PMEA. In
addition, the methyl moiety may reduce the extent of intra-
molecular hydrogen bonding of nucleophilic water to the side
chain ␤-oxygen.
Site of Nucleophilic Attack for bis-POC PMEA
Hydrolysis of bis-POC PMEA may proceed via two dis-
tinct pathways as shown in Scheme II. The first involves the
nucleophilic attack of water at the carbonyl center to form the
tetrahedral intermediate (C-O bond cleavage) and the second
involves the nucleophilic attack of water on the phosphorus
atom (P-O bond cleavage). Both pathways lead to the forma-
tion of the mono-POC PMEA. As shown in Scheme II, the
1
8
1
8
nucleophilic attack of water at phosphorus results in O in-
corporation into the phosphonate monoester, whereas, the
In summary, the degradation kinetics and the hydrolytic
pathway of bis-POC PMEA and bis-POC PMPA were stud-
1
8
ied in solution. The H2 O incorporation studies indicated
that chemical hydrolysis of bis-POC PMEA predominately
occurs via P-O bond cleavage with an additional minor path-
way involving the C-O bond cleavage. The data suggest that
the oxycarbonyloxymethyl spacer group can be successfully
applied to phosphonates resulting in prodrugs with adequate
chemical stability to allow for formulation development and
delivery. Bis-POC PMPA is currently in Phase III clinical
testing for the treatment of AIDS.
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