Highly water-soluble prodrugs of anthelmintic benzimidazole carbamates: Synthesis, pharmacodynamics, and pharmacokinetics

Article abstract of DOI:10.1021/jm701456r

Highly water-soluble prodrugs 1a-g of anthelmintic benzimidazole carbamates 2a-g were synthesized. These prodrugs combine high aqueous solubility and stability with high lability in the presence of alkaline phosphatases. The veterinary utility of 1a was shown by a pharmacodynamic and pharmacokinetic study performed in swine. Comparable anthelmintic efficacy was observed with prodrug 1a or the parent fenbendazole 2a. The pharmacokinetic results showed that 2a is better absorbed when derived from 1a than when applied as such.

Full text of DOI:10.1021/jm701456r

J. Med. Chem. 2008, 51, 1111–1114
1111
Scheme 1. Retrosynthetic approaches to 1
Highly Water-Soluble Prodrugs of
Anthelmintic Benzimidazole Carbamates:
Synthesis, Pharmacodynamics, and
Pharmacokinetics
C. Chassaing,* M. Berger, A. Heckeroth, T. Ilg, M. Jaeger,
C. Kern, K. Schmid, and M. Uphoff
InterVet InnoVation GmbH, Zur Propstei 55270 Schwabenheim, Germany
ReceiVed NoVember 19, 2007
Abstract: Highly water-soluble prodrugs 1a-g of anthelmintic ben-
zimidazole carbamates 2a-g were synthesized. These prodrugs combine
high aqueous solubility and stability with high lability in the presence
of alkaline phosphatases. The veterinary utility of 1a was shown by a
pharmacodynamic and pharmacokinetic study performed in swine.
Comparable anthelmintic efficacy was observed with prodrug 1a or
the parent fenbendazole 2a. The pharmacokinetic results showed that
2a is better absorbed when derived from 1a than when applied as such.
In cases where the discovery phase of a drug is completed,
prodrug design constitutes a method of choice to influence the
physicochemical properties of a drug.8 With this approach, some
efforts have been dedicated to the development of water-soluble
anthelmintic benzimidazole prodrugs. In the majority of cases,
the prodrug strategy relies on the attachment of promoieties to
the benzimidazole core which included acyl groups,9,10 alkoxy-
carbonyl moieties,11,12 or Mannich bases.13,14 However, since
these approaches imply a chemically driven conversion of the
prodrug into the drug, the optimal conversion conditions of the
prodrugs are often too close to those where the prodrug should
remain stable. This probably explains why none of these
prodrugs combined all prerequisites for veterinary use: a high
solubility in water for the preparation of a concentrate prior to
dilution and distribution to the animals, a sufficient aqueous
stability during the application (hours) in a pH range from 5 to
9,15 and a rapid conversion into the corresponding drug at the
site of action (typically the gastrointestinal tract). In the 1990s,
Stella and co-workers developed a prodrug concept for deriva-
tizing tertiary amines using a phosphonooxymethyl group as
promoiety,16,17 which was later extended to the derivatization
of hydroxy functions.18 In this concept, the release of the parent
drug involves a two-step process. Following a phosphatase-
catalyzed hydrolytic dephosphorylation step, the hydroxymethyl
intermediate obtained spontaneously decomposes into the parent
drug and formaldehyde.
In the present report we describe the synthesis of phos-
phonooxymethyl prodrugs derived from water-insoluble benz-
imidazole carbamates. Furthermore, we describe the investiga-
tion of the enzymatic in vitro conversion of one of the prodrugs
and the comparison of the pharmacodynamic and pharmaco-
kinetic profiles of this prodrug and of the parent drug in swine
after oral application. Since established protocols allowing the
large scale production of compounds 2a-g are available,
synthetic accesses based on the substitution of the parent drugs
were privileged for the preparation of the phosphonooxymethyl
prodrugs 1a-g. Therefore, the synthetic strategies envisaged
were based on the preparation of intermediates 3 followed by
the addition of a phosphorus nucleophile or on a nucleophilic
attack of alkali benzimidazole salts on the chloromethylphos-
phate triester 4 (Scheme 1).
Parasitic infection in animals is a major cause of animal
suffering, loss in production, and in extreme cases even death.
Among the parasitic diseases, a survey has shown that infection
by endo parasitic helminths is perceived as the most important
when compared to infections by ectoparasites such as ticks,
mange mites, flies, or lice.1 The following figures help to get
an understanding on the economic impact of helminthiasis on
livestock production. Losses induced by gastrointestinal parasites
to sheep production in Australia were estimated at $175
millions2 in 1995 and about $350 millions for ruminants in the
U.S.3 The most common intestinal nematode parasite of pig
worldwide, Ascaris suum, was solely responsible for condemna-
tion of livers valued at an estimated $17.5 million in Eastern
Europe in 1999, while an estimated loss of $60.1 million for
extra feed needed to attain the desired bodyweight was incurred
in the U.S. for the same period.4 The development of potent
chemotherapies against veterinary relevant helminths started
about 40 years ago5 with the discovery of Levamisole. Since
then, even better drugs have been introduced on the market,
and two classes of compounds have gained prevalence in the
field: the macrocyclic lactones and the benzimidazoles.6 The
benzimidazoles are characterized by a broad spectrum of activity
associated with a wide safety margin combined with ovicidal
larvicidal and adulticidal effects. The most effective representa-
tives of the group are fenbendazole 2a (Scheme 1, R1 ) PhS)
and albendazole 2c (Scheme 1, R1 ) n-PrS) because of their
particularly long in vivo half-lives. The anthelmintic benzimi-
dazoles are generally poorly soluble in water and are given orally
as suspension, paste, powder, or intraruminal bolus.7 Among
them, 2-aminobenzimidazole carbamates have particularly low
aqueous solubility probably because of the presence of an
intramolecular hydrogen bond interaction between the benz-
imidazole NH and the carbonyl of the carbamate moiety. This
characteristic has been until now a major obstacle for the
development of more convenient application forms, such as via
the drinking water.
Our initial efforts focused on preparation of intermediates 3
via hydroxymethylbenzimidazoles usually obtained by conden-
sation with formaldehyde19 and were unsuccessful when applied
to 2-aminobenzimidazole carbamates. Finally, chloromethyl-
benzimidazole 3a was obtained in one step by reacting 2a in
* To whom correspondence should be addressed. Phone: +49 (0) 6130
948 372. Fax: +49 (0) 6130 948 517. E-mail: christophe.chassaing@
intervet.com.
10.1021/jm701456r CCC: $40.75
2008 American Chemical Society
Published on Web 02/14/2008

1112 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Scheme 2. Synthesis of 1a-ga
Letters
1
Scheme 3. H NMR Signal Assignments for 5a
Table 1. Aqueous Solubility and Stability of 1a-g
compd
R1
aqueous solubilitya (mM) aqueous stabilityb (h)
1a
1b
1c
1d
1e
1f
PhS
PhSO
n-PrS
PhCO
4F-PhCO
4F-PhSO3
n-Bu
130
>50
>50
>50
>50
>50
>50
>8
>8
>8
>8
>8
>8
>8
1g
a Determined at pH 7.4. b >95% UV-HPLC, determined at pH 5 and 9.
to both isomers. Since H-C1′ exhibited long-range proton-
carbon correlations to C2 and C7a but not to the carbon of the
carbamate carbonyl, the formation of the N-2′ substituted
derivative could be excluded. Second, nuclear Overhauser
correlations were observed between H-C7 and H-C1′ allowing
the distinction between N-1 and N-3 substituted regioisomers.
Interestingly, the remaining proton attached to one of the
nitrogen atoms in regioisomer N-1 displayed a long-range
carbon-proton coupling to C7a. This observation can only
be rationalized by assuming that this proton is located at the
benzimidazole N-3, thus implying an exocyclic double bond
and supporting our hypothesis on the presence of an intramo-
lecular hydrogen bond in unsubstituted benzimidazole carbam-
ates 2. No similar correlation was observed in the case of the
other regioisomer (Scheme 3).
The deprotection of the phosphate di-tert-butyl diesters 5a-g
was then accomplished under acidic conditions to afford the
desired phosphates 7a-g, which were finally converted into the
corresponding disodium salts 1a-g by addition of sodium
methoxide. These transformations had no effect on the ratio of
the two regioisomers. Aqueous solubility of the prodrugs 1a-g
was then ensured at 50 mM corresponding to the targeted
conditions, and the stability was determined at pH 5 and 9. For
all compounds, the desired stability (>95% after 8 h at room
temperature) was achieved (Table 1). In addition, aqueous
solubility of 130 mM was determined for 1a, thus representing
a 195.000-fold increase when compared to the parent drug
fenbendazole 2a.
Under these conditions prodrugs 1a-g also showed high
stability for at least 8 h because no conversion into 2a-g was
observed during this period. In the following part of our work,
the in vitro conversion of prodrugs 1 was studied. To this end,
1a was used as a representative example. The lability of this
prodrug was studied in the presence of chicken or porcine
alkaline phosphatases by varying the enzyme concentrations,
incubation times, and conditions in pH (Figures 1 and 2). As
expected, an increase in pH promoted higher conversion rates.
However, prolonging the incubation time from 15 to 30 min
resulted only in a moderate increase of the conversions (Figure
2). From these results we concluded that under physiologically
relevant pH conditions, both enzymes achieve complete conver-
sion of 1a into 2a within either 15 or 30 min when concentrated
at 25 or 10 mU/mL respectively.
a Reagents: (i) NaHCO3, n-Bu4NHSO4, CH2Cl2-H2O, 0 °C to room
temp; (ii) NaH, DMF, room temp; (iii) HCl, dioxane, room temp; (iv) MeNa,
MeOH, room temp.
the presence of a large excess of formaldehyde and phosphor
trichloride. Because of the low stability of this intermediate, it
was directly engaged in the next step without isolation.
Unfortunately, nucleophilic substitution attempts performed with
a wide range of phosphorus nucleophiles, optionally in the
presence of Lewis acids or of phase transfer catalysts, resulted
in the absence of reaction or in the degradation of the starting
material. The introduction of the phosphate promoiety on the
benzimidazole via an electrophilic phosphate reagent was then
studied. To this end chloromethyl phosphate triester 4 was
prepared according to a literature protocol.20 During this work,
we found that the incomplete conversion of di-tert-butyl
phosphate due to the hydrolysis of chloromethyl chlorosulfate
can be improved by adding a second crop of electrophile after
20 h reaction time or by using iodochloromethane as an
alternative electrophile.21 Condensations of the obtained elec-
trophile 4 with 2-aminobenzimidazole carbamates 2a-g were
then performed by adding the electrophile to solutions of the
alkali benzimidazole salts obtained by the addition of 3 equiv
of base. Under these conditions the desired phosphate triesters
5a-g were successfully obtained, generally in the presence of
varying amounts of methylene-bis-benzimidazoles 6a-g. Al-
though the need of a third equivalent of the base is still not
fully understood, it proved to be critical to the reaction. The
use of less base resulted in lower conversion rates, while an
increase in the stoichiometry favored the formation of larger
amounts of compounds 6a-g. Similarly, we observed that the
temperature has a strong influence on the reaction course. In
general, higher temperatures resulted in the isolation of a larger
amount of dimers 6 and lower temperatures led to lower
conversion. The intermediates 5a-g were obtained as a nearly
1 to 1 mixture of regioisomers resulting from the undifferentiated
substitution of both benzimidazole nitrogen atoms. The presence
of the N-2′ substituted isomer was not detected (Scheme 2).
In the case of derivative 5a, the two regioisomers formed
were separated by high-performance liquid chromatography
(HPLC), and their structures could be successfully assigned by
1
spectroscopic analysis on the basis of H, 13C, H-H COSY,
H-H NOESY, H-C HSQC, and H-C HMBC NMR experi-
ments. The signals of the protons H-C1′ were easily attributed
The alkaline phosphatase being a membrane-bound protein
of the microvillar enterocytes,22 incubations were conducted in

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5 1113
Figure 4. Conversion of prodrug 1a to 2a after incubation over porcine
intestine mucosa disks.
Figure 1. Conversion of prodrug 1a to 2a in the presence of chicken
or porcine alkaline phosphatase after 15 min of incubation time.
Table 2. Pharmacokinetic Parameters after Oral Application of 6.8
mg/kg 1a and of 5.0 mg/kg 2a to Swine
FBZ
FBZ-SO
1a
FBZ-SO2
1a
2a
2a
1a
2a
Cmax
(µg/mL)
Tmax(h)
AUC
0.14
0.05
1.41
1.09
0.78
0.38
6.5
1.77
5.4
0.31
15
58.77
17
35.00
36
31.15
34
12.73
(µg·h/mL)
zole 2a (FBZa) and of its metabolites fenbendazole sulfoxide
a
(FBZ-SOa) and sulfone (FBZ-SO2 ) were determined (plasma
concentration of 1a was not measured). In terms of anthelmintic
efficacy, identical results were obtained with the prodrug 1a
and for fenbendazole 2a. In both cases, 6–7 days after treatment,
complete fecal egg shedding reduction and complete reduction
of worm burden were observed.
Figure 2. Conversion of prodrug 1a to 2a in the presence of chicken
or porcine alkaline phosphatase after 30 min of incubation time.
The pharmacokinetic results showed that 2a is significantly
better absorbed into the bloodstream when derived from the
prodrug 1a than when applied as such (Table 2, Figure 5).24
Cmax and AUC values for FBZ and its active metabolite FBZ-
SO were significantly higher after application of the prodrug
1a than after application of the parent drug 2a. In the case of
1a, maximal plasma concentrations of 0.14 and 1.41 ng/mL were
reached for FBZ and FBZ-SO, respectively, whereas only 0.05
and 1.09 µg/mL were measured for 2a. Similarly, areas under
curves of 0.31 and 35 µg·h/mL were calculated for FBZ and
FBZ-SO after treatment with 2a, while 1.77 and 58.8 µg·h/mL
were reached when the prodrug 1a was applied. Finally,
concerning the Tmax values, no significant differences were
noticed among all treatment groups. We thus concluded that
2a derived from the metabolism of the prodrug 1a is absorbed
to a markedly higher extent but follows the same curve profile
in comparison to 2a administered as such in powder form. Since
it is a common understanding that the intestinal absorption of
a drug is a function of the dissolution rate, which is in turn
dependent on the particle size,25 we believe that the metabolism
of prodrug 1a generating molecular particles of 2a at the mucosa
surface constitutes the principal driver of the observed higher
bioavailability. Achievement of local drug supersaturation in
the neighborhood of the intestinal mucosa following enzymatic
conversion or increased solubility of the parent drug in the
presence of the prodrug has been also cited in the literature as
other potential factors contributing to a better oral absorption
of drugs derived from phosphate prodrugs.26
Figure 3. Conversion of prodrug 1a to 2a after incubation over chicken
intestine mucosa disks.
the presence of chicken and swine duodenum and jejunum
mucosa disks to mimic in vivo conditions in an in vitro setting
(Figures 3 and 4).
Conversion of 1a into 2a was observed under every experi-
mental condition studied. In the case of porcine duodenum and
jejunum mucosa disks, particularly high conversions were
observed already after 30 min of incubation time (for example,
80% and 70%, respectively, at pH 6.5, Figure 4).
These results prompted us to perform a combined pharma-
codynamic and pharmacokinetic study in swine with 1a and
the parent drug fenbendazole 2a.23 Both compounds were
applied orally as aqueous solution or as a suspension at single
doses of 6.8 and 5.0 mg/kg body weight for 1a and 2a,
respectively. The anthelmintic efficacy against the parasite
Oesophagostomum dentatum was assessed by coproscopic
examination of egg shedding and post mortem by determination
of the worm burden. The pharmacokinetic profiles of fenbenda-
In conclusion, we have shown that N-phosphonooxymethyl
prodrugs 1a-g can be synthesized in a straightforward way from
a Abbreviations: FBZ, fenbenbazole; FBZ-SO, fenbendazole sulfoxide;
FBZ-SO2, fenbendazole sulfone.

1114 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 5
Letters
Department, Food and Agriculture Organization of the United Nations:
Rome, 2004.
(4) Stewart, T. B. Economics of endoparasitism of pigs. PigNews Inf. 2001,
22, 29N–30N.
(5) Geary, T. G.; Conder, G. A.; Bishop, B. The changing landscape of
antiparasitic drug discovery for veterinary medicine. Trends Parasitol.
2004, 20, 449–455.
(6) Coles, G. C. The future of veterinary parasitology. Vet. Parasitol. 2001,
98, 31–39.
(7) Mc Kellar, Q. A.; Scott, E. W. The benzimidazole anthelmintic agents.
A review. J. Vet. Pharmacol. Ther. 1990, 13, 223–247.
(8) Ettmayer, P.; Amidon, G. L.; Clement, B.; Testa, B. Lessons learned
from marketed and investigational prodrugs. J. Med. Chem. 2004, 47,
1–12.
(9) Hernández-Luis, F.; Hernández-Campos, A.; Yépez-Mulia, L.; Ce-
dillob, R.; Castilloa, R. Synthesis and hydrolytic stability studies of
albendazole carrier prodrugs. Bioorg. Med. Chem. Lett. 2001, 11,
1359–1362.
(10) Dransch, G.; Mildenberger, H.; Düwel, D.; Kirsch, R. Substituted
2-Alkoxycarbonylamine-5-(6)-phenylmercapto-benzimidazoles. Patent
Application US3980796, 1976.
(11) Nielsen, L. S.; Sløk, F.; Bundgaard, H. N-Alkoxycarbonyl prodrugs
of mebendazole with increased water solubility. Int. J. Pharm. 1994,
102, 231–239.
(12) Nielsen, L. S.; Bundgaard, H.; Falch, E. Prodrugs of thiabendazole
with increased water-solubility. Acta Pharm. Nord. 1992, 4, 43–49.
(13) Dhaneshwar, S. R.; Khadikar, P. V.; Chaturvedi, S. C. Synthesis and
antimicrobial activity of some Mannich bases of fenbendazole. Indian
Drugs 1990, 28, 625–627.
(14) Röchling, H.; Härtel, K.; Kirsch, R.; Düwel, D. Bis-triazinobenz-
imidazoles and Their Preparation. Patent Application US3928345,
1975.
Figure 5. Pharmacokinetic profile after oral application of 6.8 mg/kg
1a and of 5.0 mg/kg 2a to swine.
(15) In order to cope with the requirements of the EMEA/CVMP/540/03
guidelines from the committee for medicinal products for veterinary
use concerning the quality aspects of pharmaceutical veterinary
medicines for administration via drinking water.
(16) Krise, J. P.; Zygmunt, J.; Georg, G. I.; Stella, V. J. Novel prodrug
approach for tertiary amines: synthesis and preliminary evaluation of
N-phosphonooxymethyl prodrugs. J. Med. Chem. 1999, 42, 3094–
3100.
(17) Krise, J. P.; Narisawa, S.; Stella, V. J. A novel prodrug approach for
tertiary amines. 2. Physicochemical and in vitro enzymatic evaluation
of selected N-phosphonooxymethyl prodrugs. J. Pharm. Sci. 1999, 88,
922–927.
(18) Mäntylä, A.; Garnier, T.; Rautio, F.; Nevalainen, T.; Vepsälainen, J.;
Koskinen, A.; Croft, S. L.; Järvinen, T. Synthesis, in vitro evaluation,
and antileishmanial activity of water-soluble prodrugs of buparvaquone.
J. Med. Chem. 2004, 47, 188–195.
(19) Sih, J. C.; Wha bin, I.; Robert, A.; Graber, D. R.; Blakeman, D. P.
Studies on (H+-K+)-ATPase inhibitors of gastric acid secretion.
Prodrugs of 2-[(2-pyridinylmethyl)sulfinyl]benzimidazole proton-pump
inhibitors. J. Med. Chem. 1991, 34, 1049–1062.
(20) Mäntylä, A.; Vepsäläinen, J.; Järvinen, T.; Nevalainen, T. A novel
synthetic route for the preparation of alkyl and benzyl chloromethyl
phosphates. Tetrahedron Lett. 2002, 43, 3793–3794.
(21) Ueda, Y.; Matiskella, J. D.; Golik, J.; Connolly, T. P.; Hudyma, T. W.;
Venkatesh, S.; Dali, M.; Kang, S.-H.; Barbour, N.; Tejwani, R.; Varia,
S.; Knipe, J.; Zheng, M.; Mathew, M.; Mosure, K.; Clark, J.; Lamb,
L.; Medin, I.; Gao, Q.; Huang, S.; Chena, C.-P.; Bronsona, J. J.
Phosphonooxymethyl prodrugs of the broad spectrum antifungal azole,
ravuconazole: synthesis and biological properties. Bioorg. Med. Chem.
Lett. 2003, 13, 3669–3672.
the parent anthelmintic benzimidazole carbamates 2a-g. These
prodrugs combine high solubility and stability in water and are
efficiently cleaved by intestinal alkaline phosphatases, making them
suitable for convenient veterinary application forms such as via
the drinking water. An in vivo study performed in swine with 1a
has shown that the application of the prodrug results in at least a
comparable level of anthelmintic efficacy when compared to the
parent drug 2a, thus attesting for the therapeutic potential of the
compounds developed. Furthermore, a comparative pharmacoki-
netic study has revealed that higher plasma concentrations of
fenbendazole and its active metabolite fenbendazole sulfoxide are
obtained when the prodrug 1a is applied. This observation opens
the opportunity to apply lower doses using prodrugs 1 than usually
recommended for the anthelmintic benzimidazoles 2. Since the link
between the plasma concentration of fenbendazole and its me-
tabolites and the efficacy is still not fully understood, further studies
are currently ongoing to verify this hypothesis. The application of
prodrugs 1a-g to other target animal species and via other modes
of application is also being investigated and will be reported in a
separate paper in due course.
Acknowledgment. We are grateful to many colleagues at
Intervet Innovation GmbH for their technical support: Daniel
Salanta, Meike Wohlleben, Kerstin Fleischauer, Karl-Heinz
Grimm, Diane Jungkunz, Thomas Heider, Margareth Werr, and
Petra Taggeselle.
(22) Moss, D. W. Perspectives in alkaline phosphatase research. Clin. Chem.
1992, 38, 2486–2492.
(23) For details on the study setup, refer to the following: Borgsteede,
F. H. M.; Gaasenbeck, C. P. H.; Nicoll, S.; Domangue, R. J.; Abbott,
E. M. A comparison of the efficacy of two ivermectin formulations
against larval and adult Ascaris suum and Oesophagostomum dentatum
in experimentally infected pigs. Vet. Parasitol. 2007, 146, 288–293.
(24) Petersen, M. B.; Friis, C. Pharmacokinetics of fenbendazole following
intravenous and oral administration to pigs. Am. J. Vet. Res. 2000,
61, 573–576.
(25) Jinno, J.-I.; Kamada, N.; Miyake, M.; Yamada, K.; Mukai, T.; Odomi,
M.; Toguchi, M.; Liversidge, G. G.; Higaki, K.; Kimura, T. Effect of
particle size reduction on dissolution and oral absorption of a poorly
water-soluble drug, cilostazol, in beagle dogs. J. Controlled Release
2006, 111, 56–64.
Supporting Information Available: Experimental details for
the preparation of 4 and 1a-g, their corresponding analytical
information, and details of the materials and methods used for the
characterization of the 5a regioisomers. This material is available
free of charge via the Internet at http://pubs.acs.org.
References
(1) Nari, A.; Hansen, J. W. Resistance of Ecto-Endo-Parasites: Current
and Future Solutions. Presented at the 67th General Session of the
International Committee of the OIE, Paris, May 17–21, 1999.
(2) Mc Leod, R. S. Costs of major parasites to the Australian livestock
industries. Int. J. Parasitol. 1995, 25, 1363–1367.
(3) Guidelines Resistance Management and Integrated Parasite Control
in Ruminants; Animal Production and Health Division, Agriculture
(26) Heimbach, T.; Oh, D.-M.; Li, L. Y.; Forsberg, M.; Savolainen, J.;
Leppänen, J.; Matsunaga, Y.; Flynn, G.; Fleisher, D. Absorption rate
limit considerations for oral phosphate prodrugs. Pharm. Res. 2003,
20, 848–856.
JM701456R