EUROPEAN JOURNAL OF DRUG METABOLISM AND PHARMACOKINETICS, 1983, Vol. 8, No 4, p. 329-334

4-Amino-3-(3'-Methoxycarbonyl-2'-Thioureido)Benzophenone,a prodrug of Mebendazole.

MICHAEL DAWSON AND THOMAS R. WATSONDepartment of Pharmacy, University of Sydney, Australia

Received for publication : September 14, 1981

Key-words Mebendazole, prodrug

SUMMARY

The compound 4-amino-J-(J'-methoxycarbonyl-2'-thioureido) benzophenone has shown promise as a prodrug of the anthel­mintic mebendazole. The compound is stable in acid and neutral media and is rapidly hydrolysed in base. An HPLC assayprocedure for mebendazole, the prodrug and their known or expected metabolites and degradation products in aqueous mediaand rat blood has been developed. The prodrug administered orally to rats is rapidly converted to mebendazole. The area underthe blood level versus time curve of mebendazole, in rats dosed with the prodrug, is more than twice that obtained after dosingrats with an equimolar amount of mebendazole. Only the prodrug, mebendazole and known metabolites of mebendazole aredetected in rats dosed with the prodrug.

INTRODUCTION

The broad spectrum anthelmintic mebendazole iscurrently undergoing clinical trials as a chemothera­peutic agent active against the cestodes Echinococcusgranulosus and Echinococcus multi/ocularis whichcause hydatid disease in humans (1,2,3).

Mebendazole is extremely insoluble in water (ca.0.5 mg/L) and all other pharmaceutical vehicles. Itis poorly absorbed after oral administration (4) andno injectable form is available. We have examineddrug latentiation as a possible way of overcomingthe oral bioavailability problems associated with thistherapeutic application of mebendazole.

Douche (5) showed that alkoxycarbonylthioureidoderivatives of ortho-phenylenediamine undergo cy­c1ization in sheep to their corresponding benzimida­zole derivatives.

Send reprint request to : Professor T.R. Watson TheUniversity of Sydney, Pharmacy Department NSW 2006,Sydney. Australia.

4-Amino-3-(3'-methoxycarbonyl- 2' - thioureido)benzophenone has been synthesised as a possibleprodrug of mebendazole. In this paper the pHstability profile of the compound and a comparisonof blood level versus time, and urinary excretionprofiles of mebendazole in rats dosed with equimo­lar amounts of mebendazole and its prodrug, arereported.

MATERIALS AND .METHODSChemicals:

4- Amino -3- (3' - methoxycarbonyl-2' - thioureido)benzophenone (I, Figure I) was synthesized byrefluxing 3,4-diaminobenzophenone with methoxy­carbonylisothiocyanate (I, 1.5 mole ratio) in acetoni­trile for 2 hours. The product was recrystallizedfrom ethanol (yield 60%; M.P. 195-198 QC).

4-Amino-3-thioureidobenzophenone (II, Figure I)was prepared by aqueous alkaline (I M NaOH)hydrolysis of 4-amino- 3-(3' -methoxycarbonyl- 2'­thioureido) benzophenone. The product was recry­stallized from ethanol/water (yield 88 %; M.P.178-180 QC).

330 European Journal of Drug Metabolism and Pharmacokinetics, 1983. No 4

Mebendazole (methyl-5-(6)-benzoylbenzimidazo­le-2-ylcarbamate (III, Figure I) was supplied byJanssen Pharmaceutica, Sydney, Australia (M.P.> 300°C with decomposition).

2-Amino- 5(6)-benzoylbenzimidazole (IV, FigureI) and methyl-5(6)-(a-hydroxybenzyl)-2-benzimida­zole-2-yl-carbamate (Y, Figure I) were supplied byR.J. Allan of this department.

Ethyl- 5(6)-benzoylbenzimidazole-2-yl-carbamate(VI, Figure I) was synthesized by refluxing 3,4­diaminobenzophenone with I,3-bis(ethoxycarbonyl)­S-methylisothiourea (I : 1.5 mole ratio) in a mixtureof ethanol/water/acetic acid (10 : 10 : 0.4) for onehour. The product was recrystallized from aceticacid/water (yield 37 %; M.P. ± 300°C with de­composition).

All other chemicals used were of analyticalreagent grade.

OONH2

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III

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II Ho

IV

VI

kley, California, USA), equipped with a fixed wave­length UV-detector (254 nm), a Hewlett-Packardmodel 3380A integrator/recorder (Hewlett-Packard,Avondale, Pennsylvania, USA), and a Waters Intel­ligent Sample Processor model 710B (Waters Asso­ciates, Sydney, Australia). Gradient analyses werecarried out on a 250 mm X 4.6 mm 1.0. chromato­graphic column packed in this laboratory withLichrosorb RP8 10Jl Packing meterial (Merck, Dar­mstadt, W. Germany). Isocratic analyses were car­ried out on a 150 mm x 4.6 mm 1.0. chromato­graphic colum packed in this laboratory with 5JlLichrosorb RP8 (Merck, Darmstadt, W. Germany).A precolumn, 35 mm X 3.2 mm 1.0., dry packedwith Corasil C 18 (Waters Associates, Sydney, Au­stralia) was fitted to both systems. The mobile phaseconsisted of methanol: distilled water (53 : 47, pumpA) and methanol : aqueous ammonium phosphatebuffer 0.01 M, pH 3.5 (53:47, pump B). For gradientelution the flow rate was 1.7 ml/min. which pro­duced a column pressure of 82 to 84 bar. Gradientelution was commenced with pump A providing100 % of the mobile phase. After seven minutes acurved gradient began which brought pump B to 100% at 13 minutes, and held these conditions for up to20 minutes, when the column was pumped free ofions by passing 25 column volumes of the e1ectrolyte­free mobile phase through the system. Isocraticelution was performed with pump B providing 100%of the mobile phase, at a flow rate of 1.0 ml/minute,which produced a column pressure of 300-310 bar.

Determination of the pH stability profile ofcompound I

Fig. 1: 4-Amino-3-(3'-methoxycarbonyl-2'-thioureido)ben­zophenone, (I); 4-Amino-3-thioureidobenzophe­none, (II); mebendazole, (III); metabolites of me­bendazole, (IV) & (V); ethyl-5(6}-benzoylbenzi­midazole-Z-yl-carbamate, (VI).

HPLC Analyses :

Two HPLC analyses were used, an ionic gradientelution system for the analysis of urine, modifiedfrom the procedure of Allan et al., (6), for thedetermination of mebendazole in human plasma,and a simple isocratic elution system for the analysisof buffer solutions and rat blood.

Chromatographic analyses were performed on anAltex model 322MP gradient elution high perfor­mance liquid chromatograph (Altex Scientific, Ber-

The following buffer solutions of ionic strength0.1 were prepared : hydrochloric acid-potassiumchloride, pH 1.4; potassium dihydrogen phosphate ­disodium hydrogen phosphate, pH 6.4; potassiumdihydrogen phosphate - disodium hydrogen phospha­te, pH 7.5; tris - tris hydrochloride, pH 9.9; glycine­sodium glycinate, pH 9.3; glycine - sodium glycinate,pH 10.0; sodium bicarbonate - sodium carbonate,pH 10.6; sodium bicarbonate - sodium carbonate,pH 11.0. The rate constant of degradation of 4­amino -3-(3'- methoxycarbonyl-2'-thioureido) benzo­phenone at 37°C in each of the buffers was determi­ned as follows; 10 ml of each buffer was equilibratedin a water bath at 37°C. 6.25 ug of 4-amino-3-(3'­methoxycarbonyl-2'-thioureido) benzophenone in 100JlI of DMSO was added to each buffer solution. Atappropriate time intervals 20 JlI of the reactionmixture was analysed by isocratic HPLC and the log

M. Dawson and T. R. Watson. Prodrug of Mebendazo/e 331

of the amount remaining was plotted versus time.Lines of best fit were determined using a linearregression programme and the half-life and rateconstant at each pH was determined.

Animals

Male Wistar rats weighing 350 to 450 g wereused in all experiments. Each animal was used onlyonce, and the animals were allowed food and waterad lib.

Urine Collection

Rats were individually housed in all-glassMetabowls (Jencons Laboratory Furnishings,Hemel Hempstead, UK) during the course of theexperiments. Urine samples were collected from eachrat before, and 24 hours after dosing.

Urine Assay Procedure.

A 10 ml aliquot of urine was adjuxted to pH 6,and 12.5 ug of ethyl-5(6)benzoylbenzimidazole-2­ylcarbamate was added as the internal standard. Themixture was shaken with 10 ml of diethyl ether for60 seconds on a vortex mixer, after which the diethylether layer was transferred to an evaporation tubeand evaporated to dryness. The residue was dis­solved in 100 Jll of OMSO, and a 20 ul aliquot wasassayed by gradient elution HPLC. The amount ofeach compound in each sample was obtained fromthe integrator/recorder after it had been suitablycalibrated.

Cannulation Procedure.

Venous cannulas were inserted into the jugularvein of rats according to the method of Upton (7),and the rats were allowed to recover overnightbefore being dosed.

Blood Collection and Assay Procedure.

Approximately 125 ul of blood was withdrawnvia the cannula and transferred to a heparinizedtube. The animal's blood volume was maintained byreplacing the withdrawn blood with an equal volumeof normal saline containing heparin (10 units/ml). A

100 ul aliquot of blood was then transferred to anextraction tube which contained 500 ul of heparini­zed normal saline, and 400 ng of ethyl-5(6)benzoyl­benzimidazol-2-yl-earbamate was added as the in­ternal standard. The mixture was extracted with 6 mlof diethyl ether for 60 seconds and the organic layerwas transferred to an evaporation tube and evapora­ted to dryness. The residue was dissolved in 25 J.1l ofOMSO and a 20 JlI aliquot was analysed by isocraticHPLC. The amount of each compound in eachsample was obtained from the integrator/recorderafter it had been suitably calibrated.

Pharmacokinetic Analysis.

The elimination rate constant, kcl was calculatedfrem the slope of the linear terminal portion of thelog blood level versus time curve, obtained fromleast squares regression analysis. The areas under theblood level versus time curves were estimated by thetrapezoidal rule from zero time until the last sam­pling time (t), and extrapolated to infinite time bythe addition of C(t)/k cl '

RESULTS

In Vitro Stability Study

Upon incubation of 4-amino-3-{3'-methoxycar­bonyl-2'-thioureido) benzophenone in acidic, neutraland basic buffer solutions (Jl= O. I), it was foundthat the major product of degradation was thehydrolysis product 4-amino-3-thioureidobenzophe­none with less than 2 % of the cyclization product,mebendazole being formed. The half-life of 4-amino­3-{3'-methoxycarbonyl-2'-thioureido)benzophenone inthe buffer solutions at pH 1.4, 7.5 & 11.0 was3,399h, 290 hand 2.5 h (k, 2.1X 1O-4h- l, 2.4 X IO-Jh- I

and 0.28 h -I respectively). The graph of log half-lifeof 4-amino-3-{3'-methoxycarbonyl-2'-thioureido) ben­zophenone versus pH for each buffer solution usedwas plotted to give a pH stability profile, (Figure 2).

In Vivo

Five rats each fitted with a venous cannula weredosed orally with micronized 4-amino-3-{3'-metho­xycarbonyl-2'-thioureido) benzophenone in a methylcellulose-mucilage suspension (44.4 mg/kg; 135 umo­les/kg). Blood samples including a blank were takenat appropriate time intervals. All of the urine

332 European Journal of Drug Metabolism and Pharmacokinetics. 1983. No 4

410

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2 3 4 5 6 7 8 9 10 11 12

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Table I : Half-life, Ave and urinary excretion data forrats dosed with 4-amino-3-{3'-methoxycarbonyI2'thioureido)

benzophenone, (44 mg/kg; 135 I.l moles/kg).

rat number 2 3 4 5 average

tY2 prodrug (h) 3.1 3.9 3.0 3.2 3.2 3.3±0.4

t Y2 mebenda-zole (h) 4.9 6.3 6.6 6.2 5.4 5.9±0.7

AVe mebendazo-le (ng.h/mL) 14.2 17.3 15.3 15.0 16.1 15.6±1.2

% dose excretedin urine 1.6 1.0 1.1 2.1 0.9 1.3±0.5

Table II : Half-life, Ave and urinary excretion data forrats dosed with mebendazole, (40 mg/kg; 135umoles/kg),

rat number 2 3 4 5 average

t Y2 mebenda-zole (h) 3.0 5.2 3.8 3.7 5.4 4.2± 1.0

AVe mebendazo-Ie (ng.h/mL) 7.6 7.8 5.3 6.8 5.2 6.5± 1.2

% dose excretedin urine 0.7 0.4 0.6 0.3 0.6 0.5±0.2

Fig. 2: The pH stability profile of 4-amino-3-{3'-methoxy­carbonyl-2'-thioureido)benzophenone.

excreted over 24 h was collected. Another group offive rats, also fitted with venous cannulae was dosedwith micronized mebendazole in methyl cellulose­mucilage suspension (40 mg/kg; 135 umoles/kg)and blood and urine were collected as describedabove. The blood and urine samples from each ratwere analysed by HPLC. Typical blood level versustime curves obtained after rats were dosed with 4­amino-3-(3'-methoxycarbonyl-2'-thioureido) benzophe­none and mebendazole are shown in Figures 3 and 4respectively. Half-life data, area under the mebenda­zole blood level versus time data, and total 24 hoururinary excretion data for rats dosed with 4-amino­3-(3'-methoxycarbonyl-2'-thioureido) benzophenoneand mebendazole is shown in Tables I and 2.

DISCUSSION

The chemical stability studies showed that themajor degradation product of the prodrug at eachpH studied was the thiourea with only very smallamounts of mebendazole being formed. The half lifeof the prodrug at pH 7.4 is 290 hours whereas itsapparent elimination half life in the rat is 3.3 hours.This observation, coupled with the fact that nothiourea but significant amounts of the prodrug andmebendazole are found in the blood of rats dosedwith the prodrug, suggests that the compound isstable in the gastrointestinal tract, is absorbed assuch, and that the conversion of the prodrug tomebendazole is enzymatically mediated.

After absorption from the gastrointestinal tractthe prodrug is rapidly converted to mebendazole.The apparent elimination half-life of the prodrug is3.3 hours and the apparent elimination half-life ofmebendazole is 4.2 hours (Tables I and 2).

M. Dawson and T. R. Watson, Prodrug of Mebendazole 333

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Fig. 3 : Blood concentration profile following a singleoraldose of 4-amino-3-(3'.methoxycarbonyl.2'-thiou­reido)benzophenone, (44mg/kg; 135 umoles/kg),4-Amino-3-(3'-methoxycarbonyl-2'-thioureido)ben­zophenone; .....Mebendazole; ____

Fig. 4: Blood concentration profile following a single oraldose of mebendazole, (40 mg/kg; 135 umoles/kg).Mebendazole; ......

There is a two and a half times increase inthe area under the mebendazole curve when ratsare dosed with prodrug compared with the corre­sponding area under the curve when rats are dosedwith mebendazole, which suggests that the prodrughas greater bioavailability than mebendazole.

No thiourea (II) and only prodrug (I), meben­dazole (III), the amine (IV), and the reduced meben­dazole (V) were detected in the blood of rats dosedwith prodrug, the latter two compounds beingknown metabolites of mebendazole. The prodrugtherefore fulfills the requirement that it is convertedonly to the active drug and then to metabolites ofthat drug, with no alternative metabolism occurring.

Total urinary excretion data accounts for lessthan 2 % of the dose of the two compoundsunder study and, further work is being carried out toelucidate the role of biliary excretion in the elimina­tion of these compounds and their metabolites.

ACKNOWLEDGEMENT

We acknowledge the financial support received for thiswork from the National Health and Medical ResearchCouncil of Australia.

REFERENCES

I. Goodman H.T. (1976): Mebendazole. Med. J. Aust. 2,662.

334 European Journal of Drug Metabolism and Pharmacokinetics. 1983. No 4

2. Bekhti, A., Schapps. J.-P., Capron, M., Dessaint, J.-P.,Santoro, F. & Capron, A. (1977): Treatment of hepatichydatid disease with mebendazole: preliminary resultsin four cases. Brit. Med. J. 2. 1047-1051.

3. Kern, P., Dietrich, M. & Volkmer, K.-J. (1979): Che­motherapy of echinocossosis with mebendazole: clinicalobservations of 7 patients. Tropenmed. Parasitol. 30.65-72.

4. Brugmans, J.P., Thienpont, D.C., van Wijngaarden, I.,Vanparijs, D.F., Schuermans, V.L. & Lauwers, H.L.( 1971): Mebendazole in enterobiasis: radiochemical &pilot clinical study in 1,278 subjects. J. Amer. Med.Assoc. 217, 313-316.

5. Douch, P.G.c. (1974): The metabolism of some thiou­reidobenzene fungicides in mice & sheep. Xenobiotica4. 457-475.

6. Allan, R.J., Goodman, H.T. & Watson, T.R. (1980):Two high performance liquid chromatographic deter­minations for mebendazole and its metabolites inhuman plasma using a rapid Sep Pak C 18 extraction.J. Chromatogr. 183,311-319.

7. Upton, R.A. (1975). Simple & reliable method for serialsampling of blood from rats. J. Pharm. Sci., 64 (I):112-114.