1474 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 8
Brief Articles
Sch em e 1
are more lipophilic than the parent clodronate, stable
against chemical hydrolysis, and hydrolyzed enzymati-
cally to clodronate in human serum. To our knowledge,
the clodronic acid dianhydrides are the first reported
bioreversible prodrugs of clodronate with the potential
to improve the oral bioavailability of clodronate.
Exp er im en ta l Section
Ch em istr y. 1H, 13C, and 31P NMR spectra were recorded
on a Bruker AM 400 spectrometer operating at 400.1, 100.6,
and 162.0 MHz, respectively; TSP was used as an internal
standard for 1H and 13C measurements and 85% H3PO4 used
as an external standard for 31P measurements. Normal J HH
3
> 2b . 2c > 2d (Table 1). The absorption of clodronate
is hindered by both its formation of poorly water-soluble
Ca2+ complexes15 and its low lipophilicity. A crystal
structure and ab initio studies have shown earlier
bidentate metal bonding in clodronate-calcium-water
complexes.16 The effect of Ca2+ ions on the aqueous
solubility of clodronic acid dianhydride 2d and clod-
ronate was determined at pH 6.0. The concentration of
clodronate decreased from 894 µg/mL (no calcium added)
to 123 µg/mL when 100 mM Ca2+ was added to the
solution (Figure 1). The concentration of 2d in buffer
solution was not affected by Ca2+ ions (Figure 1), which
verifies that masking two ionizable groups of clodronate
significantly decreases the complexation of clodronate
with cations. This may result in an improved oral
absorption of clodronate.
Ap p a r en t P a r tition Coefficien t. The log Papp val-
ues were determined using the 1-octanol-pH 7.4 buffer
system. The log Papp value for clodronate was not
successfully determined due to its very hydrophilic
character. However, it can be estimated to be less than
-5.4, which has been reported for clodronic acid mono-
ethyl ester at pH 7.4.17 Clodronic acid dianhydrides
2a -d are significantly more lipophilic than the parent
clodronate at pH 2.0 and 7.4 (Table 1). The change of
promoiety group in the structure did not have a signifi-
cant effect on log Papp values, and thus, more than two
phosphonic acid groups of clodronic acid may need to
be substituted in order to increase further the log Papp
values.
Hyd r olysis in Aqu eou s Solu tion . The hydrolysis
of 2a -d to clodronate followed first-order kinetics
(Figure 2). The dianhydrides 2a -d were more stable
toward chemical hydrolysis at pH 7.4 than at pH 2.0
(Table 1). The dianhydrides 2c,d were significantly more
stable in aqueous solution than 2a ,b (Table 1). This is
most probably due to the resonance stabilization of
benzoyl promoieties in 2d and the steric hindrance of
pivaloyl promoieties in 2c.
Hyd r olysis in Hu m a n Ser u m . Rates of enzymatic
hydrolysis of 2a -d were determined in 80% human
serum (pH 7.4) at 37 °C. These hydrolyses followed first-
order kinetics (Figure 2) and were substantially faster
than the chemical hydrolyses (Table 1). The dianhy-
drides 2a ,b,d hydrolyzed rapidly (<1 min) and com-
pletely to clodronate. Compound 2c was more resistant
toward enzymatic hydrolysis, having a half-life of 3.3
h, which is most probably due to its more hindered
structure.
couplings are indicated by the letter “J ”, and all J values are
given in Hz. All solvents and reagents were high-purity
reagent-grade materials, and the acetonitrile was distilled
before use. Synthesis and characterization of tetrasodium
clodronate has been reported earlier.18 HPLC measurements
were performed with a Merck LaChrom HPLC system consist-
ing of model L-7250 programmable autosampler, model L-7100
HPLC pump, model D-7000 interface module, and model
D-7000 HPLC system manager (Hitachi Ltd., Tokyo, J apan).
A Sedex 55 evaporative light-scattering detector (Sedere, Vitry-
Sur-Seine, France) was used. The analytical column used was
a Kromasil 100 RP-C8 (250 × 4.6 i.d., 5 µm; Higgins Analytical
Inc., Mountain View, CA). The eluent consisted of 3% methanol
and 97% 0.10 M ammonium acetate buffer (pH 4.6) containing
0.23 M n-butylamine. After 1.5 min of elution, the organic
concentration was increased linearly from 3% to 60% for 2d
and from 3% to 70% for 2b,c in 5 min. pKa values were
measured with Mettler Toledo DL 50 (Schwerzenbach, Swit-
zerland).
P ,P ′-Dia cetyl (Dich lor om eth ylen e)bisp h osp h on a te Di-
sod iu m Sa lt (2a ). Anhydrous tetrasodium clodronate (1) (15.0
g, 45.1 mmol) and acetic anhydride (150.0 mL, 1589.8 mmol)
were heated in an oil bath at 135 °C for 24 h. The mixture
was chilled to 5 °C and allowed to stand overnight in the cold.
The mixture was filtered, and the solids were washed several
times with small portions of ether (130 mL) and dried to give
2a as a white solid (12.5 g, 74%). pKa1 ) 2.4, pKa2 ) 6.4. NMR
(D2O): δH 2.21 (6H, s); δP 2.91 s; δC 172.48 t (virtual triplet,19
due to poor signal/noise relation two small signals are covered),
77.37 t (1J CP ) 146.8), 24.85 q+t (virtual triplet). Anal. Calcd
(C5H6Cl2Na2O8P2): C, 16.10; H, 1.62. Found: C, 15.99; H, 1.90.
P ,P ′-Dibu tyr oyl (Dich lor om eth ylen e)bisp h osp h on a te
Disod iu m Sa lt (2b). Anhydrous 1 (1.0 g, 3.0 mmol) and
butyric anhydride (10.0 mL, 61.1 mmol) were heated in an oil
bath at 110 °C for 120 h and isolated as 2a . The preceding
reaction procedure was repeated a total of three times to give
2b as a white solid (0.68 g, 53%). NMR (D2O): δH 2.49 (4H, t,
J ) 7.3), 1.65 (4H, m), 0.95 (6H, t, J ) 7.49); δP 3.15 s; δC
175.08 t (virtual triplet), 77.64 t (1J CP ) 146.6), 39.95 t+t
(virtual triplet), 20.45 t, 15.62 q. Anal. Calcd (C9H14Cl2-
Na2O8P2): C, 25.20; H, 3.29. Found: C, 23.26; H, 3.02.
P ,P ′-Dip iva loyl (Dich lor om eth ylen e)bisp h osp h on a te
Disod iu m Sa lt (2c). Anhydrous 1 (1.33 g, 4.0 mmol), tri-
methylacetic anhydride (15.0 mL, 73.9 mmol), and acetonitrile
(30 mL) were heated in an oil bath at 100 °C for 120 h and
isolated as 2a . The preceding reaction procedure was repeated
a total of two times to give the crude product, which was
further washed with 35% 2-propanol (15 mL) and dried to give
2c as a white solid (0.70 g, 38%). pKa1 ) 2.3, pKa2 ) 5.6. NMR
(D2O): δH 1.25 (18H, s); δP 3.75 s; δC 179.95 t (virtual triplet),
77.84 t (1J CP ) 147.1), 42.78 t (virtual triplet), 28.88 q. Anal.
Calcd (C11H18Cl2Na2O8P2): C, 28.90; H, 3.97. Found: C, 26.21;
H, 3.92.
P ,P ′-Diben zoyl (Dich lor om eth ylen e)bisp h osp h on a te
Disod iu m Sa lt (2d ). Anhydrous 1 (1.0 g, 3.0 mmol), benzoic
anhydride (8.0 g, 35.4 mmol), and acetonitrile (20.0 mL) were
refluxed for 24 h and isolated as 2a . The product was mixed
with cold water (10.0 mL) for 5 min and filtered. The preceding
water wash (5.0 mL) was repeated, and the product was
further washed with acetonitrile (10 mL). The solids were dried
Con clu sion
The present clodronic acid dianhydrides, which are
shown to be novel bioreversible prodrugs of clodronate,