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M. Flores-Ramos et al. / Bioorg. Med. Chem. Lett. 24 (2014) 5814–5817
Table 3
Efficacy of compound 7 expressed as percentage of reduction of F. hepatica flukes and eggs in experimentally infected sheep
Group and route of administration (n = 6)
Dosea (mg/kg/bw)
Mean SD
Fluke reduction
Efficacy (%)
Fluke reduction
Egg reduction
Egg reduction
1 (Oral)
12
4
4
3.6 1.5
35.7 5.6
23.0 4.3
76.3 8.9
12.3 2.3
9.0 1.2
28.5 3.4
74.0 9.2
95.1
44.9
65.8
0
83.3
87.8
61.4
0
2 (Intramuscular)
3 (Subcutaneous)
4 (Control group)
Untreated
a
Body weight.
amines using a phosphonooxymethyl group as a promoiety.24,25
This was later extended to the derivatization of hydroxyl func-
tions.26 In this concept, the release of the parent drug involves a
two-step process. Following an initial alkaline phosphatase hydro-
lytic dephosphorylation, the hydroxymethyl intermediate (8)
obtained, spontaneously decomposes into the parent drug and
formaldehyde (Fig. 2).27,25
In the current study, we present the synthesis, water solubility,
chemical stability, in vitro and in vivo evaluations of the novel pro-
drug (7), a phosphate salt present as a mixture of regioisomers 7a
and 7b (this applies also for compounds 5 and 6 shown in Figure 3).
Prodrug 7 was designed using a phosphonooxymethyl group as a
moiety to increase solubility and bioavailability of the initial com-
pound alpha, favoring a parenteral administration at a reduced
dose.
Prodrug 7 was synthesized according to the synthetic pathway
described in Scheme 1 from compound alpha (4), which was
prepared by methods previously described by our research
group.11 The phosphate group was introduced using di-tert-butyl
chloromethyl phosphate (3) prepared from chloromethyl chloro-
sulfate28 (1) and tetramethylammonium di-tert-butyl hydrogen
phosphate (2), an approach adapted from methods reported in
the literature.29 Tetramethylammonium di-tert-butyl hydrogen
phosphate was synthesized using di-tert-butyl hydrogen phos-
phate, also an adapted method from those reported in the
literature.30
In the substitution reaction between 3 and 4, a 1 to 1 mixture of
regioisomers 5a and 5b was obtained. Compound (4) was treated
with sodium hydride in DMF and then with 3 to give a 1 to 1
mixture of two regioisomers of 5, resulting in the N-substituted
product as intended. 5a was successfully isolated and character-
ized by 1H NMR, 13C NMR, H-C HSQC, H-H COSY and NOE-1D. In
this step, the excess NaH was eliminated and the resinous product
recovered was subjected to hydrolysis to obtain 6. This was the
limiting step in the synthesis of prodrug 7, resulting in a 70% yield.
The de-protection of the di-tert-butyl chloromethyl phosphate (5)
was then accomplished under acidic conditions to afford the
desired phosphates (6), which were finally converted into the cor-
responding disodium salt (7) by addition of sodium hydroxide.31
These transformations had no effect on the ratio of the two regioi-
somers. The structures of the new compounds were established
from spectroscopic and spectrometric data.
neutral pH, which is ideal for the formulation from a physiological
standpoint of view.
Compound 7 was tested in vitro against recently excysted
metacercariae.34 It was evaluated at concentrations of 10 and
50 mg/L, see Table 2. At 50 mg/L and 24 h, compound 7 demon-
strated a fasciolicidal efficacy of 100%, at a concentration of
10 mg/L this efficacy was 95.83% at 24 and 48 h, obtaining 100%
efficacy until 72 hours post-treatment. These results compare with
the drug of choice Triclabendazole (FasinexÒ, Novartis) as the ref-
erence control. However, the fasciolicidal activity of compound 7
in the in vitro tests at 10 mg/L suggests that the parasite does
not express alkaline phosphatase that can release the active com-
pound from the prodrug and that this is hydrolyzed chemically
with time.
Compound 7 was evaluated in sheep against adult 10 week-old
F. hepatica using three different routes of administration (Table 3).
Results obtained show an efficacy of 87.8% at an intramuscular
dose of 4 mg/kg (Table 3). In previous studies the fasciolicidal
activity of compound alpha (4) was also determined, demonstrat-
ing an 86.9% efficacy against mature and juvenile fasciolas at an
oral dose of 15 mg/kg.11 It is evident then, that the fasciolicidal effi-
cacy of prodrug 7, at a lower dose via intramuscular injection, is
comparable with respect to orally administered compound alpha
(4), both demonstrating similar effects.
In summary, we have synthesized a new prodrug (7) as a
phosphate disodium salt, which shows a 50,000 fold increase in
aqueous solubility compared to its precursor compound alpha
(4). Compound 7 was stable at neutral pH, ideal for parenteral
formulation. Evaluations of compound 7 in vitro and in vivo
showed fasciolicidal activity. The in vivo results prove that 7 offers
a considerable reduction in dose administered compared to com-
pound alpha (4) while achieving similar effects via parenteral
administration.
Acknowledgments
This work was supported by the Dirección General de Asuntos
del Personal Académico (DGAPA) under the project IT201113. M.
Flores-Ramos is very grateful to CONACyT for the fellowship
granted (267703/223411). We are grateful to Rosa Isela del Villar,
Georgina Duarte and Margarita Guzmán for the analytical support
offered. We also thank Anuar Flores-Gahona from Facultad de Quí-
mica, UNAM, for the review and corrections of this paper. We
acknowledge Lourdes Mayet her assistance in the solubility tests.
The hydrosolubility of prodrug 7 was determined according to
the procedure of Yalkowski et al.32 The aqueous solubility of com-
pound alpha, determined as 2.6 Â 10À4 mg/mL, was substantially
increased in prodrug 7 with the introduction of the phosphono-
oxymethyl group, the latter with an observed solubility of
13.0 mg/mL at pH 7 (Table 1), demonstrating a 50,000 fold increase
in the solubility of the precursor compound 4. This result is consis-
tent with previous reports that the prodrug design of compounds,
such as 7, is useful for increasing the aqueous solubility of non-
polar drugs.21–26
Supplementary data
Supplementary data associated with this article can be found, in
References and notes
In formulating a prodrug, the compound should also demon-
strate adequate chemical stability, especially in parenteral dosage
forms. Compound 7 showed desired stability (>95% after 26 h) at