Novel triclabendazole prodrug: A highly water soluble alternative for the treatment of fasciolosis

Article abstract of DOI:10.1016/j.bmcl.2016.12.004

In this work we present the synthesis, aqueous solubility and stability, hydrolysis by alkaline phosphatase, and in vivo fasciolicidal activity in sheep of a highly water soluble phosphate salt prodrug of triclabendazole (MFR-5). The aqueous solubility of MFR-5 at pH 7 was 88,000-fold that of triclabendazole. MFR-5 showed excellent aqueous stability (>95% after 26?h) at pH 7, making it ideal for developing pharmaceutical compositions in the form of solutions that can easily be hydrolyzed by the enzyme alkaline phosphatase (t?=?13.6?s) to liberate the precursor compound. An aqueous solution of MFR-5 administered intramuscularly to sheep at concentrations of 4, 6 and 8?mg/kg presented a fasciolicidal efficiency of 96.5%, 98.4% and 99.2%, respectively. In the in vivo experiments, MFR-5 reduced 100% the excretion of eggs in all of the above concentrations.

Full text of DOI:10.1016/j.bmcl.2016.12.004

Accepted Manuscript
Novel triclabendazole prodrug: A highly water soluble alternative for the treat-
ment of fasciolosis
Miguel Flores-Ramos, Froylán Ibarra-Velarde, Helgi Jung-Cook, Alicia
Hernández-Campos, Yolanda Vera-Montenegro, Rafael Castillo
PII:
S0960-894X(16)31270-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.004
BMCL 24493
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
15 November 2016
30 November 2016
1 December 2016
Please cite this article as: Flores-Ramos, M., Ibarra-Velarde, F., Jung-Cook, H., Hernández-Campos, A., Vera-
Montenegro, Y., Castillo, R., Novel triclabendazole prodrug: A highly water soluble alternative for the treatment
of fasciolosis, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.004
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Novel triclabendazole prodrug: A highly water soluble alternative for the treatment of
fasciolosis
Miguel Flores-Ramosa, Froylán Ibarra-Velardeb, Helgi Jung-Cooka, Alicia Hernández-
Camposa, Yolanda Vera-Montenegrob, Rafael Castilloa*
aFacultad de Química, Departamento de Farmacia, Universidad Nacional Autónoma de
México, Av. Universidad 3000, CDMX, México 04510, Mexico.
bFacultad de Medicina Veterinaria y Zootecnia, Departamento de Parasitología,
Universidad Nacional Autónoma de México, Av. Universidad 3000, CDMX, México 04510,
Mexico
*Corresponding author. Tel.: +52 55 56 22 52 87; fax: +52 55 56 22 53 29.
E-mail address: rafaelc@unam.mx (R. Castillo).
Keywords
Triclabendazole; Fasciola hepatica; Prodrug; Benzimidazole; Phosphonooxymethyl;
Fasciolicidal activity.
1

Abstract
In this work we present the synthesis, aqueous solubility and stability, hydrolysis by alkaline
phosphatase, and in vivo fasciolicidal activity in sheep of a highly water soluble phosphate
salt prodrug of triclabendazole (MFR-5). The aqueous solubility of MFR-5 at pH 7 was
88,000 fold that of triclabendazole. MFR-5 showed excellent aqueous stability (> 95% after
26 hours) at pH 7, making it ideal for developing pharmaceutical compositions in the form
of solutions that can easily be hydrolyzed by the enzyme alkaline phosphatase (t=13.6 s) to
liberate the precursor compound. An aqueous solution of MFR-5 administered
intramuscularly to sheep at concentrations of 4, 6 and 8 mg/kg presented a fasciolicidal
efficiency of 96.5%, 98.4% and 99.2%, respectively. In the in vivo experiments, MFR-5
reduced 100% the excretion of eggs in all of the above concentrations.
2

Fasciolosis, caused by the trematode Fasciola hepatica, is a worldwide parasitic disease
which hampers animal production, and also infects humans.1,2 Its importance lies in the great
economic losses ranging from the confiscation of the livers of infected animals, to weight
loss, growth retardation, reduced production of meat, milk or wool, decreased resistance to
other diseases, inhibited reproduction, miscarriages and even death.3 All the above
mentioned added to the high costs of deworming makes it one of the costliest parasites of
livestock worldwide. Human fasciolosis is considered by the WHO as an important human
parasitic disease, with estimates of 2.4 million4 up to 17 million infected people.5 Mammals,
including humans, get the parasite by the ingestion of infected aquatic plants or water. The
prevalence of fasciolosis is increasing in certain parts of the world due to different causes
such as climate change or man-made environmental modifications.6–8 The main strategy for
the effective control of fasciolosis is based on chemotherapy. Among all flukicides,9 one of
the most important chemical groups are the benzimidazoles. One of them, albendazole
(ABZ, Fig. 1), is recommended for the control of fasciolosis, and although its activity is
restricted to flukes older than 10 weeks,10 it removes up to 94-95% of adult flukes in
sheep.11,12 Currently, the most effective drug is another benzimidazole derivative introduced
in the early 1980s, triclabendazole (TCBZ, 6-chloro-2-(methylthio)-5-(2,3-
dichlorophenoxy)-1H-benzimidazole, Fig. 1); its efficacy is not only restricted to adult
flukes, as it kills immature worms as well.13 TCBZ eliminates between 99-100% flukes at 4-
14 weeks of age and between 90-99% against flukes at 2-3 weeks of age.9 TCBZ has also
been approved for its use in humans.14 This fasciolicide has a 2-methylthio group instead of
a 2-mehylcarbamate group, common to many anthelmintics with the benzimidazole nucleus,
fact that increases its lipophilicity, absorption and bioavailability.15,16
Figure 1. Structures of albendazole (ABZ) and triclabendazole (TCBZ).
3

TCBZ-resistant F. hepatica was first reported in sheep in Victoria, Australia in 1995,17 and
now resistance has been reported in several countries both in sheep18–22 and cattle.23–25 Due
to the importance of fasciolosis, various compounds have been sintesized9,14 and newer
treatment approaches to overcome or prevent resistance have been used.14,26–29 Clearly, any
potentially marketable product to replace TCBZ must have wide-spectrum of fasciolicidal
activity against TCBZ-sensitive and TCBZ-resistant isolates and all stages of F. hepatica;
but so far there is no candidate with these characteristics. Due to this, TCBZ is still the drug
of choice for the treatment of fasciolosis, and livestock health and production is highly
dependant on it, even though the resistant isolates problems persists and the presence of new
compounds.30,31 Additionally, as other benzimidazoles,10,15,32,33 TCBZ has very low
solubility in water and biological fluids, consequently has poor bioavailability.34–39 For this
reason, TCBZ is administered orally as suspensions, pastes, powders, or as intraruminal boli,
limiting other routes of administration such as the parenteral route.10,40 The poor water
solubility is an advantage when the parasite is located in the gastrointestinal tract, where a
high drug concentration is needed, but when the parasite is located in an organ, as in the case
of F. hepatica, absorption of the drug is needed. This feature is also present in several
benzimidazole derivatives41–46 and it is atributed to intermolecular interactions,41,47,48 this
results in high dosage treatments due to poor gastrointestinal absorption.49 Moreover, oral
administration is not always practical for large groups of animals. Consequently, new
administration routes are required. One of them is based on the observation that the use of
prodrugs is a good approach to increase water solubility by several orders of magnitude.50–53
Phosphate ester prodrugs are typically designed for the hydroxyl groups of poorly water
soluble drugs to increase the aqueous solubility and allow a better environment for the
parenteral administration.51 They also have excellent chemical stability54–56 and adequate
bioconversion to the parent drug by the alkaline phosphatases present in the intestine, liver
and blood stream.52,57,58 When a drug enters the body it must surpass several biological
barriers before reaching the target in a suitable concentration, depending on the route of
administration. The parenteral route of drug administration has several advantages when
compared with the oral route. The parenteral route has a higher effect speed, elevated
bioavailability and is not affected by the metabolism. Thus, the ideal prodrug will be
4

perfectly stable until it reaches the target, and then it will be rapidly and quantitatively
converted to the active drug.
Trying to solve this issue a prodrug derivative from TCBZ (MFR-5) was designed by our
group. This novel compound, synthesized with an ionizable phosphoonoxymethtyl group at
position 1 of the benzimidazole nucleus, increased the aqueous solubility and allowed the
formation of an aqueous solution for parenteral administration. In the current study we
present the synthesis, aqueous solubility, aqueous stability, alkaline phosphatase hydrolysis
and in vivo evaluations of the novel prodrug MFR-5 against F. hepatica.
It is expected that the prodrug MFR-5 undergoes bioconversion to TCBZ in two steps. First,
a complete dephosphorilation with an alkaline phosphatase enzyme to generate the
hydroxymethyl intermediary (II) and in a second step, the spontaneous chemical
decomposition of II to give TCBZ and formaldehyde (Scheme 1).
Scheme 1. Prodrug strategy for MFR-5.
MFR-5 was synthesized from TCBZ (1) according to the synthetic pathway described in
Scheme 2. TCBZ was prepared using previously reported methodology.59 Freshly prepared
di-tert-butyl chloromethyl phosphate (2) was used to introduce the phosphate group.60,61
TCBZ was treated with NaH in DMF and consecutively with 2 in anhydrous conditions to
obtain 3 as a 1:1 mixture of regioisomers (3a-b). The resinous mixture recovered was
hydrolyzed with HCl in dioxane to obtain a mixture of dihydrogen phosphates (4a-b), which
were transformed into the desired disodium salts (MFR-5a-b) using NaOH in a mixture of
acetone/H2O. Since prodrug MFR, as a mixture of 5a and 5b, is expected to be hydrolyzed
enzymatically to afford 1, no attempts were made to isolate the regioisomers during the
synthetic sequence. All new structure was stablished using spectroscopic and spectrometric
methods.
5

Scheme 2. General procedure for the synthesis of prodrug MFR-5. Reagents and conditions:
(a) NaH, DMF, rt; (b) HCl 4M in dioxane, rt; (c) NaOH, acetone/H2O, rt.
The aqueous solubility of prodrug MFR-5 and TCBZ was determined using a procedure
reported by Yakolwsky.62 The aqueous solubility of TCBZ (1, 2.4 X10-4 mg/mL) was
improved greatly with prodrug MFR-5, with a mean solubility value of 21.12 mg/mL (Table
1). Thus, MFR-5 improved the aqueous solubility 88,000 fold, in comparison to its
precursor compound 1. In a previous study we reported a close related prodrug,60 but the
compound only showed an improvement of the aqueous solubility of its precursor drug by
50,000 times, compared to current 88,000 fold showed by MFR-5. This data demonstrate
that prodrugs with a phosphoonoxymethyl group, as in MFR-5, are useful to improve
aqueous solubility of non-polar drugs with a functionable nitrogen, which is consistent with
previous reports.52,63–67
Chemical stability is an important property, where precipitation is the limitant for the shelf
life of prodrug solutions.68–70 Chemical degradation of a prodrug can result in the formation
of the insoluble precursor drug, which can affect the solubility of the formulation by the
presence of insoluble particles.52,63 In a drug formulation, the compound must show suitable
chemical stability, specially for parenteral dosage forms. MFR-5 showed the desired
stability (> 95% after 26 horas) at neutral pH, which is ideal for the formulation from a
6

physiological point of view (Table 1). This result demonstrates that MFR-5 has sufficient
aqueous stability to guarantee that the compound administered in vivo, as an inyectable
solution, is the prodrug and not the parent compound. Also, it guarantees the administration
to a herd of cattle or a flock of sheep without prodrug degradation.
Table 1. Aqueous solubility, chemical stability and enzymatic hydrolysis of prodrug MFR-5
Aqueous solubility
(mg/mL)a
pH 7
Aqueous stability
Enzymatic hydrolysis,
t1/2; alkaline phosphatase, 37 °C
seconds
(h)b
Compound
pH 7
>26
>26
21.12
13.6±4
MFR-5
TCBZ
2.4 X10-4
aDetermined at 25 °C, b>95% by UV-HPLC determined at room temperature.
It has been observed that phosphate ester prodrugs administered orally may have problems
due to enzymatic hydrolysis.60,71,72 On the other hand, those administered parenterally have
shown successful results.51,72 Additionally, not all compounds with a phosphate group and
an organic portion are substrates for the alkaline phosphatase enzyme.73 The enzymatic
hydrolysis of MFR-5 was determined in alkaline phosphatase solution at pH 7.4 and 37
°C.74–76 The ability of the prodrug MFR-5 to release to TCBZ in alkaline phosphatase
solution indicates that this prodrug, as a pair of regioisomers, is substrate for this enzyme
(Table 1), a fact that is promising for further drug development.
MFR-5 was evaluated in adult ramboullet sheep, against 10-week-old F. hepatica, using the
intramuscular route. Results showed that MFR-5 in aqueous solution had excellent
fasciolicidal activity at a dosage of 4, 6 y 8 mg/kg with a fluke reduction rate of 96.8%,
98.3% y 99.2%, respectively, similar to that obtained with a commercial suspension of
triclabendazole (Fasimec®, Novartis) administered orally at a dosage of 10 mg/kg with a
reduction of 99.9%. It is important to remark that MFR-5 at all dosages completely reduce
the excretion of F. hepatica eggs, same effect as TCBZ administered orally; these results are
shown in Table 2. It is important to mention that in all treated groups there were no post
treatment side effects; also, that animals did not show pain during the intramuscular
administration. Additionally, the environmental impact would be lower if a lower dose of
MFR-5 is used. These results are encouraging for future studies, such as the development of
a MFR-5 injectable formulation to improve fasciolicide activity.
7

Table 2. Efficacy of compound MFR-5 expressed as percentage of reduction of adult F.
hepatica and eggs in experimentally infected sheep
Mean ± SEc
Efficacy (%)
Groupa and route of
administration (n=6)
Dose
Egg
Fluke
Egg
Fluke
(mg/Kg/bwb)
retrieved
retrieved
reduction
reduction
1 (intramuscular)
2 (intramuscular)
3 (intramuscular)
4 TCBZ (1, oral)
5 (control group)
4
6
0±0
0±0
0±0
0±0
3.5±1.4
1.7±1.1
0.8±0.6
0.1±0.1
102.6±12.3
100
100
100
100
96.8
98.3
99.2
99.9
0
8
10
untreated
31.5±4.7
0
a200 metacercariae of F. hepatica/animal, bBody weight, cSE standard error.
In summary, a novel TCBZ prodrug (MFR-5) was synthesized as a phosphate disodium salt,
as a mixture of regioisomers. An 88,000-fold water solubility increase was obtained
compared with the precursor drug. This prodrug could be ideal for a parenteral formulation
due to his neutral pH making it easier for veterinary administration. In vivo results showed
that MFR-5 offers a good dosage reduction compared to TCBZ and conserves high
fasciolicidal activity. It is also important to remark that it removes 100% of F. hepatica eggs
from feces at 4, 6 and 8 mg/Kg.
Acknowledgments
To DGAPA PAPIIT for financial support of projects RT201015 and IT201113, to Jorge
Victoria Miguel for technical assistance in the analytic work. 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 acknowledge Lourdes Mayet her assistance in the solubility and enzymatic hydrolysis
tests. Study carried out in accordance with the current laws of Mexico.
Supplementary data
Supplementary data associated with this article can be found in the online version, at:
8

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Graphical abstract
A novel triclabendazole prodrug with fasciolicidal activity has been synthesized. The
prodrug showed an 88,000-fold increase in aqueous solubility in comparison to its precursor
compound. The prodrug was evaluated in vivo against Fasciola hepatica, demonstrating
high fasciolicidal activity.
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