Carbon-14 labelled Tribendimidine, a broad-spectrum anthelmintic drug

Article abstract of DOI:10.1002/jlcr.3070

The preparation of [¹⁴C] tribendimidine, a broad-spectrum anthelmintic agent related to amidantel, and its use during excretion and metabolism studies in the rat are described in this paper. Copyright

Full text of DOI:10.1002/jlcr.3070

Special Issue Article
Received 02 April 2013,
Revised 15 May 2013,
Accepted 15 May 2013
Published online 9 July 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3070
Carbon-14 labelled Tribendimidine, a
broad-spectrum anthelmintic drug†
a
a
Erminia Fontana,a,d Alberto Pignatti, Alberto Ghiglieri,
*
Rosangela Battaglia,a Flavio Cinato,a Chong Wang,b and Eugenio de Hostosc
The preparation of [14C]tribendimidine, a broad-spectrum anthelmintic agent related to amidantel, and its use during
excretion and metabolism studies in the rat are described in this paper.
Keywords: tribendimidine; carbon-14; ADME; rat
Netherlands) ratio to HPLC effluent: 2.5/1). Method B: Gemini C18 column
Introduction
(4.6ꢀ 100 mm, 5 mm) eluting with water : acetonitrile : triethylamine
More than a billion people in tropical and subtropical regions are 60:40:0.35 (by volume). Flow rate: 1 ml/min. Column temperature: 30ꢁC.
Analytical wavelength: 265 nm. Radiometric detection with 0.5 ml
homogeneous cell (liquid scintillation cocktail: Ultima Flo-M (PerkinElmer
Life Sciences, Groningen, The Netherlands) ratio to HPLC effluent: 2.5/1.
infected by parasitic intestinal roundworms. Soil-transmitted
helminths are considered by public-health officials to have a
debilitating impact on human populations that is equal to or
greater than malaria or tuberculosis.1 Currently only a few drugs
are available to combat soil-transmitted helminths infections.
Tribendimidine, namely N,N0-bis[40-(1-dimethylamino ethylidene
amino)phenyl]-1,4-phenylene-dimethylidyne amine, is a broad-
spectrum anthelmintic agent related to amidantel, developed
in China since the mid-1980s. The compound has been approved
for human use by the Chinese authorities for its good safety and
therapeutic profile against soil-transmitted helminthiasis.2,3
Preliminary metabolism studies indicated that the compound is
quickly cleaved to p-(1-dimethylamino ethylimino)aniline (dADT)
and terephthalaldehyde (TPAL). Furthermore, the acetylated
dADT (adADT) and terephtalic acid (TPAC) were identified as
main metabolites of tribendimidine4 (Figure 1). A radiolabeled
version of tribendimidine was needed to support in vitro and
Method of synthesis
The preparation of [14C]tribendimidine labelled in the benzylideneamino
moiety was accomplished following a three-step synthetic procedure as
shown in Scheme 1. [14C]Copper cyanide 2 (0.476 mmol; 1 GBq) was
refluxed with 4-iodobenzaldheyde
1
(218.6 mg; 0.94 mmol) in
dimethylformamide (DMF; 2 ml) for 1.5 h obtaining intermediate 3. Water
(5 ml) was added to the reaction mixture that, after transferring into a
separating funnel, was extracted with EtOAc (3ꢀ 3 ml). The organic
extracts were combined, washed with water (6ꢀ 10 ml), dried (IST phase
separator column) and evaporated to dryness. After purification by flash
chromatography on a silica gel column (Eluting system: n-hexane: EtOAc
6:1 by volume), the obtained cyano-derivative 3 [0.333 mmol; 0.71 GBq;
radiochemical purity >98% (method A; retention time (Rt) = 8.1 min );
yield: 71%] was converted into the corresponding aldehyde 4 by
treatment with PtO2 (24.2 mg) in 80% aqueous formic acid (0.9 ml) at
60ꢁC for 10 h. The solid material was separated by filtration then washed
with water (3 ml) and Et2O (5 ml). The filtrate with washings were
transferred into a separating funnel, and the aqueous phase was
extracted with Et2O (2ꢀ 5 ml). The organic phases were combined, dried
(IST phase separator column), evaporated to dryness and purified by
in vivo ADME studies. In particular,
a more extensive
investigation regarding the possible aldehyde-derivatives
formation was of main interest for their potential impact on
the compound safety profile. Therefore the introduction of
carbon-14 in the benzilideneamino moiety was required. The
preparation of carbon-14 labelled tribendimidine in the
benzilideneamino moiety and its use during in vivo ADME
studies in the rat are described in this paper.
aAccelera S.r.l., viale Pasteur 10 20014, Nerviano, MI, Italy
bShandong Xinhua Pharmaceutical Company Ltd., Shandong, Zibo, China
cOneWorld Health, South San Francisco, CA, USA
Experimental
Carbon-14 labelled tribendimidine
dIsotope Chemistry, Accelera S.r.l., viale Pasteur 10 20014, Nerviano, MI, Italy
Analytical HPLC Methods
*Correspondence to: Erminia Fontana, Isotope Chemistry, Accelera S.r.l., viale
Pasteur 10, 20014 Nerviano (MI), Italy.
E-mail: erminia.fontana@accelera.org
Method A: XBridge C18 column (4.6ꢀ 100 mm, 5 mm) eluting with H2O :
CH3CN : TFA (trifluoroacetic acid) 90:10:0.1 by volume (A) and H2O :
CH3CN : TFA 10:90:0.1 by volume (B): from 100% A to 0% A in 15 min;
1 min at 0% A; from 0% A to 100% A in 1 min; 4 min at 100% A. Flow rate:
1 ml/min. Column temperature: 25ꢁC. Analytical wavelength: 254 nm.
Radiometric detection with 0.5 ml homogeneous cell (liquid scintillation
cocktail: Ultima Flo-M (PerkinElmer Life Sciences, Groningen, The
† This article is published in Journal of Labelled Compounds and
Radiopharmaceuticals as a special issue on IIS 2012 Heidelberg Conference,
edited by Jens Atzrodt and Volker Derdau, Isotope Chemistry and Metabolite
Synthesis, DSAR-DD, Sanofi-Aventis Deutschland GmbH, Industriepark Höchst
G876, 65926 Frankfurt am Main, Germany.
J. Label Compd. Radiopharm 2013, 56 471–474
Copyright © 2013 John Wiley & Sons, Ltd.

E. Fontana et al.
Figure 1. Preliminary metabolism studies of tribendimidine.
flash chromatography on a silica gel column (Eluting system: n-hexane: and ID dosing. Quantification of TPAL and other radioactive metabolites
EtOAc 6:1 by volume). The obtained aldehyde 4 [0.156 mmol; 329 MBq; was performed by HPLC with on-line radiochemical detection [Gemini
radiochemical purity >96% (method A; Rt = 7.3 min); yield: 46%] was C18 column (4.6ꢀ 150 mm, 3 mm) eluting with 0.2% formic acid in
reacted with intermediate 55 (55 mg; 0.31 mmol) in tetrahydrofuran 10 mM ammonium formate (A) and 0.1% formic acid in CH3CN (B):
(THF; 1 ml) at 40ꢁC for 24 h. A precipitate was formed and after filtration, 1 min at 98% A; from 98% A to 80% A in 15 min; from 80% A to 5% A
[
14C]tribendimidine (0.086 mmol; 180 MBq; yield: 55%) was obtained as a
in 5 min; 6 min at 5% A; from 5% A to 98% A in 0.5 min; 7.5 min at 98%
yellow solid with a radiochemical purity >98 by radio-HPLC (Method B; A. Flow rate: 1 ml/min. Column temperature: 40ꢁC. Radiometric detection
Rt = 6.2 min) and a specific activity of 2.1 GBq/mmol. MS (ESI-MS): with 0.5 ml homogeneous cell (liquid scintillation cocktail: Ultima Flo-M
m/z 455 ([MH]+). The overall radiochemical yield from Cu[14C]CN 2 (PerkinElmer Life Sciences, Groningen, The Netherlands) ratio to HPLC
was 18%.
effluent: 2.5/1]. Structure elucidation of radiolabelled metabolites was
carried out by LC-MS/MS in negative ion mode [Mass spectrometer:
Waters QTof 2 equipped with an ESI source. Data were acquired from
70 to 600 Da, by using a source temperature of 120ꢁC, a desolvation
In vivo excretion balance in the rat
Male Sprague Dawley rats (n = 3/administration route), cannulated for gas temperature of 250ꢁC, a cone voltage of 25 V and a collision energy
serial blood sampling, were given a single intravenous (IV) dose and a of 10 V in full scan mode and 15 V in MS/MS mode. Data were centroided
single intraduodenal (ID) dose of
14C]tribendimidine (10 mg/kg; during acquisition using an external reference].
approximately 3.7 MBq/kg) in 50% PEG400 dextrose aqueous solution. Urine and selected plasma samples were also analysed by mass
[
The elimination of radioactive drug-related material was determined in spectrometry in positive ion mode, thus allowing the identification of
urine, faeces and cage washes collected from each animal at pre-dose dADT and other unlabelled metabolites [Mass spectrometer: Waters
and at the intervals of 0–8, 8–24, 24–48, 48–72 and 72–96 h after QTof 2 equipped with an ESI source. Data were acquired from 70 to
administration. Radioactivity levels were measured in whole blood 600 Da, by using a source temperature of 120ꢁC, a desolvation gas
and plasma collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 24, 48, 72 and 96 h after
administration. The carcasses and the skin were analysed for 10 V in full scan mode, and 15, 20, 25 V in MS/MS mode. Data were
temperature of 250ꢁC, a cone voltage of 30 V and a collision energy of
residual radioactivity. Total radioactivity was also evaluated in liver and
heart. The radioactivity content in the excreta, cage washes, blood,
plasma, collected organs and carcasses were determined by liquid
scintillation counting.
centroided during acquisition using an external reference].
Results
In vivo excretion balance in the rat
In vivo metabolism in the rat
[
14C]tribendimidine were
The excretion profiles of drug-related material after IV and ID
administration were very similar. The radioactivity excreted via
The radiochemical metabolite profiles of
determined in pooled urine samples collected in the 0–24-h interval
and in pooled plasma samples collected at 5–15 and 30–60 min after IV urine within 96 h after administration ranged on average from
Scheme 1. Synthesis of [14C]Tribendimidine.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 471–474

E. Fontana et al.
Figure 2. Proposed biotrasformation of [14C]tribendimidine in urine and plasma of male rats following single intravenous and intraduodenal administrations of [14C]
tribendimidine at 10 mg/kg.
87% to 90% (including cage washes) of the administered assigned. In plasma, two major radioactive peaks were
dose after both administration routes. The mean amount of detected, TPAC and MX2, and their sum was in the 70–90%
radioactivity recovered in faeces was approximately 2.3% and range of radioactivity. The percentage of MX2 decreased from
4.2% of the dose respectively after IV and ID dosing. The 5–15 to 30–60 min, whereas the percentage of TPAC
excretion of the compound and/or its metabolites was rapid with increased with time. The m/z of MX2 could not be
most of the radioactivity (>80% of the dose) excreted in the determined because of low MS response and ion suppression
urine within the first 24 h. Negligible to null amounts of from the biological matrices. Analyses in MS positive ion
radioactivity were found in the collected organs (liver and heart) mode of urine showed that dADT accounted for
96 h after dosing. After IV dosing, blood and plasma data approximately 40% of the total unlabelled drug-related
indicated that the total radioactivity distributed into material after both IV and ID dosing, as well as the sum of
tissues and disappeared from circulation with a low-moderate the acetyl derivative adADT and the acetylated-hydroxylated
clearance of 1.61 and 1.13 L/h/Kg, respectively. Following ID metabolite M5. In plasma, metabolite dADT accounted for
administration, the total radioactivity was rapidly absorbed more than 50% in the 5–15 min samples after IV and ID
reaching its maximal concentration within 15 min post dosing. administration and was not detected at 30–60 min. The
The bioavailability of drug-related material was 72% and 60% metabolites, adADT and M5 were in the range of 9–16% of
in blood and plasma, respectively. After both routes of total drug-related material.
administration, the red blood cells to plasma partition coefficient
(Kp) at the first sampling times was lower than 1, indicating
that the drug related material had limited association with red
Conclusions
blood cells.
A method to introduce carbon-14 in the benzilideneamino
moiety of tribendimidine considered of main interest from a
metabolic point of view was successfully developed and
implemented. An excretion balance study performed on a
In vivo metabolism in the rat
The proposed metabolic pathway is shown in Figure 2. TPAC rat by using radiolabelled tribendimidine showed that the
accounted for about 20% of radioactivity in urine after both excretion of the compound and/or its metabolites was rapid
routes of administration. In urine, the major radiolabelled with most of the radioactivity (>80% of the dose) excreted
metabolite MX1 accounting for 44% (IV) and 64% (ID) of the in the urine within the first 24 h after both IV and ID
radioactivity showed the same m/z, elemental composition and dosing. The biotransformation products of tribendimidine
fragmentation pattern as TPAC, but different retention time in observed in urine and plasma were similar after IV and
gradient elution. Therefore a definitive structure could not be ID administration.
[
14C]Tribendimidine was rapidly and
J. Label Compd. Radiopharm 2013, 56 471–474
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

E. Fontana et al.
extensively transformed into TPAL and dADT. Both metabolites
were further transformed into several additional metabolites:
TPAL was metabolized toTPAC and other unknown metabolites;
dADT was metabolized mainly by acetylation to adADT,
followed by oxidation or desmethylation. In addition, conjugates
of dADT with glucose (M4) were detected.
References
[1] P. J. Hotez, D. H. Molyneux, A. Fenwick, J. Kumaresan, S. E. Sachs, J. D.
Sachs, L. Savioli, N. Engl. J. Med. 2007, 357, 1018–1027.
[2] X. Shu-Hua, W. Hui-Ming, M. Tanner, J. Huntzinger, W. Chong, Acta
Trop. 2005, 94, 1–14.
[3] J. Kaiser, X. Shu-Hua, J. Chollet, M. Tanner, J. Hutzinger, Antimicrob.
Agents Chemother. 2007, 51, 1096–1098.
[4] Y. Guiyan, W. Benjie, W. Chummin, Z. Rui, G. Ruichen,
Chromatographia 2008, 46, 1016–1019.
[5] Y. Cui, X. Yu, Y. Xu, Intermediate 5 was prepared at Accelera S.r.l.
according to the procedure described in China Patent 1510027A; July,
2004.
Conflict of Interest
The authors did not report any conflict of interest.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2013, 56 471–474