Synthesis and in-vitro/in-vivo evaluation of orally administered entacapone prodrugs

Article abstract of DOI:10.1211/0022357011778025

Entacapone is a new inhibitor of catechol-O-methyltransferase (COMT) that is used as an adjunct to L-dopa therapy in the treatment of Parkinson's disease. The bioavailability of orally administered entacapone is, however, relatively low (29-46%). In this study we have prepared more lipophilic acyl and acyloxyacyl esters, an acyloxy alkyl ether and an alkyloxycarbonyl ester of entacapone, and we have evaluated them as potential prodrugs to enhance the oral bioavailability of entacapone. All the derivatives fulfilled prodrug criteria and released entacapone in human serum in-vitro. The oral bioavailability of monopivaloyl (1a) and dipivaloyl (1b) esters of entacapone were investigated further in rats. The lipophilicity of 1b was high (log P_app 4.0 at pH 7.4) but its oral bioavailability was low (F = 0.6%), most probably due to its low aqueous solubility. The monopivaloyl ester of entacapone (1a) had a higher lipophilicity (log P_app 0.80) than entacapone (log P_app 0.18) at pH 7.4 while maintaining an aqueous solubility equal to entacapone. However, oral bioavailability was not increased when compared with the parent drug entacapone (F = 7.0% and 10.4%, respectively).

Full text of DOI:10.1211/0022357011778025

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 1489–1498
# 2001 The Authors
Received February 21, 2001
Accepted July 10, 2001
ISSN 0022-3573
Synthesis and in-vitro/in-vivo evaluation of orally
administered entacapone prodrugs
Jukka Leppanen, Jouko Savolainen, Tapio Nevalainen, Markus Forsberg,
Juhani Huuskonen, Hannu Taipale, Jukka Gynther, Pekka T. Mannisto
and Tomi Jarvinen
Abstract
Entacapone is a new inhibitor of catechol-O-methyltransferase (COMT) that is used as an
adjunct to L-dopa therapy in the treatment of Parkinson’s disease. The bioavailability of orally
administered entacapone is, however, relatively low (29–46%). In this study we have prepared
more lipophilic acyl and acyloxyacyl esters, an acyloxy alkyl ether and an alkyloxycarbonyl ester
of entacapone, and we have evaluated them as potential prodrugs to enhance the oral
bioavailability of entacapone. All the derivatives fulfilled prodrug criteria and released
entacapone in human serum in-vitro. The oral bioavailability of monopivaloyl (1a) and
dipivaloyl (1b) esters of entacapone were investigated further in rats. The lipophilicity of 1b was
high (log Papp 4.0 at pH 7.4) but its oral bioavailability was low (F l 0.6%), most probably due
to its low aqueous solubility. The monopivaloyl ester of entacapone (1a) had a higher
lipophilicity (log Papp 0.80) than entacapone (log Papp 0.18) at pH 7.4 while maintaining an
aqueous solubility equal to entacapone. However, oral bioavailability was not increased when
compared with the parent drug entacapone (F l 7.0% and 10.4%, respectively).
Department of Pharmaceutical
Chemistry, University of Kuopio,
PO Box 1627, 70211, Kuopio,
Finland
Jukka Leppanen, Jouko
Savolainen, Tapio Nevalainen,
Juhani Huuskonen, Hannu
Taipale, Tomi Jarvinen, Jukka
Gynther
Department of Pharmacology
and Toxicology, University of
Kuopio, PO Box 1627, 70211,
Kuopio, Finland
Markus Forsberg, Pekka T.
Mannisto
Introduction
Finncovery Ltd, Kuopio, Finland
Entacapone (Backstrom et al 1995) [(E)-2-cyano-N,N-diethyl-3-(3,4-dihydroxy-5-
nitrophenyl)propenamide] (1) is a new 3,4-dihydroxy-5-nitrobenzylidine derivative
and a potent inhibitor of catechol-O-methyltransferase (COMT) (Backstrom et al
1989; Mannisto et al 1992). -Dopa (3,4-dihydroxyphenyl--alanine) is mainly
metabolized by COMT and aromatic amino acid decarboxylase (AADC)
(Mannisto & Kaakkola 1990). -Dopa is a precursor to dopamine, of which there
is a shortage in the brains of patients suffering from Parkinson’s disease.
Jukka Gynther, Pekka T.
Mannisto, Tomi Jarvinen
Correspondence: J. Leppanen,
Department of Pharmaceutical
Chemistry, University of Kuopio,
PO Box 1627, 70211, Kuopio,
Finland. E-mail:
Traditional Parkinson’s disease treatment consists of -dopa administration
combined with an AADC inhibitor. During treatment, COMT remains the main
enzyme to metabolize -dopa although some new pathways may be activated. Even
after this combination therapy, no more than 5–10% of the orally administered
-dopa enters the brain (Mannisto & Kaakkola 1990). The administration of
entacapone, together with -dopa and an AADC inhibitor, leads to increased -
dopa bioavailability and prolonged elimination of -dopa (Kaakkola et al 1994).
Entacapone is used clinically as an adjunct to -dopa therapy in Parkinson’s
disease (Mannisto & Kaakkola 1999); however, the bioavailability of entacapone
is low (29–46%) after oral administration and characterized by large inter-
jukka.leppanen!uku.fi
Acknowledgement and
Funding: The authors are
grateful to Ms Tarja Ihalainen,
Mrs Helly Rissanen, Ms Pirjo
Hakkarainen and Mrs Jaana
Leskinen for their skilful
technical assistance. This work
was supported by the Academy
of Finland and the National
Technology Agency (Finland).
1489

1490
Jukka Leppanen et al
individual variation (Keranen et al 1994). The low oral DC-Alufolien, Merck). The synthesis products were
bioavailability of entacapone may be due to several purified without attempts to maximize yields.
reasons. Firstly, entacapone (weak acid; pKa 4.5) is un-
ionizedat lowpH (i.e. inthe stomach)with pooraqueous
solubility and slow dissolution, which may limit its
Synthesis methods
absorption (Savolainen et al 2000a). Secondly, at higher
pH (i.e. in the small intestine) entacapone is mostly Method A
ionized and its solubility and dissolution properties Entacapone (1.0 g, 3.3 mmol) was dissolved in dry pyri-
are improved. Unfortunately, ionized drug molecules dine (7 mL) and cooled to 0mC when valeroyl chloride
having low lipophilicity are generally poorly absorbed (1.2 g, 8.3 mmol) was added to the mixture. The mixture
from the small intestine (Lee et al 1997). Thirdly, high was stirred at room temperature for 15 h, the pyridine
first-pass metabolism of phenols and catechols may hydrochloride was filtered off and the filtrate was evapo-
decrease the bioavailability of entacapone (Wikberg et rated. The residue was purified by column chromato-
al 1993; Friis & Bundgaard 1996).
graphy using ethyl acetate\petroleum ether (10:1) as
The aim of the study was to investigate whether the an eluent to give (E)-2-cyano-N,N-diethyl-3-[3,4-diva-
oral bioavailability of entacapone could be improved by leroyloxy-5-nitrophenyl]propenamide (1f) (0.17 g, 11%
lipophilic prodrugs. Therefore, we synthesized acyl and of theoretical), as a clear yellowish oil. 1H NMR δ: 0.91
acyloxyacyl esters, an acyloxy alkyl ether and an alky- (3H, J l 7.3 Hz, t, CH2CH2CH3), 0.97 (3H, J l 7.3 Hz,
oxycarbonyl ester of entacapone and evaluated the t, CH2CH2CH3), 1.24 (6H, br, NCH2CH3), 1.35 (2H,
derivatives as potential prodrugs of entacapone.
J l 7.4 Hz, m, CH2CH2CH3), 1.46 (2H, J l 7.4 Hz, m,
CH2CH2CH3), 1.61 (2H, J l 7.5 Hz, m, CH2CH2CH2),
1.77 (2H, J l 7.5 Hz, m, CH2CH2CH2), 2.34 (2H, J l
7.4 Hz, t, COCH2), 2.65 (2H, J l 7.4 Hz, t, COCH2),
3.48 (4H, br, NCH2), 7.58 (1H, s, CHlC), 8.06 (1H, J
l 2.2 Hz, d, ArH[2]), 8.43 (1H, J l 2.2 Hz, d, ArH[6]).
13C NMR δ: 12.9, 13.0, 13.6, 17.8, 18.2, 35.5, 40.8, 43.4,
110.9, 114.7, 124.1, 128.5, 130.3, 139.0, 143.0, 144.8,
146.2, 161.8, 169.4, 170.1. ESI-MS: 474.0 (Mj1).
Materials and Methods
Materials
Entacapone ((E)-2-cyano-N,N-diethyl-3-(3,4-di-
hydroxy-5-nitrophenyl)propenamide, OR-611), Z-enta-
capone and tolcapone (4h-methyl-3,4-dihydroxy-5- Method B
nitrobenzophenone, Ro 40-7592) were obtained from Entacapone (1.5 g, 4.9 mmol), dimethylaminopyridine
Orion Pharma (Espoo, Finland). All other materials (DMAP) (15 mg) and valeroyloxy acetic acid (3.2 g,
and solvents were commercially available and used as 19.7 mmol) were dissolved in dry tetrahydrofuran
received. All melting points are uncorrected. Structures (30 mL). Dicyclohexylcarbodiimide (DCC) (2.1 g,
1
of the synthesized compounds were confirmed by H 10.1 mmol) was added and the solution was stirred at
NMR, 13C NMR and electrospray ionization mass room temperature overnight. The mixture was filtered
spectrometry (ESI-MS). 1H and 13C NMR spectra were and the filtrate was evaporated. The residue was purified
obtained by a Bruker AM 400 WB spectrometer (opera- by column chromatography using ethyl acetate\hexane
ting at 400.14 and 100.6 MHz, respectively) using CDCl3 1:1 as an eluent to give (E)-2-cyano-N,N-diethyl-3-[3,4-
as a solvent and tetramethylsilane as internal reference. divaleryloxyacetyloxy-5-nitrophenyl]propenamide (2c)
1
The following abbreviations were used: s l singlet, d l (1.5 g, 52% of theoretical) as a clear yellow oil. H
doublet, t l triplet, q l quartet, qv l quintet, sx l NMR δ: 0.86 (6H, J l 7.6 Hz, t, CH2CH3), 1.25 (6H, br,
sextet, br l broad. Electrospray ionization mass spectra NCH2CH3), 1.36 (4H, J l 7.7 Hz, sx, CH2CH3), 1.63
were acquired using a LCQ ion trap mass spectrometer (4H, J l 7.7 Hz, qv, CH2CH2CH2), 2.39 (4H, J l
equipped with an electrospray ionization source 7.6 Hz, t, CH2CO), 3.44 (4H, br, NCH2), 4.90 (2H, s, O-
(Finnigan MAT, San Jose, CA). The samples were CH2-CO), 4.93(2H, s, O-CH2-CO), 7.55(1H, s, CHlC),
1
−
diluted with methanol to 20 µg mL and injected di- 8.01 (1H, J l 2.2 Hz, d, ArH[2]), 8.47 (1H, J l 2.2 Hz,
rectly to the eluent flow via a 5-µL loop injector (the s, ArH[6]). 13C NMR δ: 12.6, 13.8, 14.3, 22.1, 26.9, 33.5,
total amount of the sample was approximately 100 ng). 41.1, 43.7, 60.3, 60.8, 108.8, 115.5, 124.6, 128.9, 130.2,
Full scan mass spectra were recorded. The synthesis 140.6, 142.8, 147.3, 149.9, 162.6, 164.9, 165.6, 173.3,
reactions were monitored by TLC (Kieselgel 60 F 254, 173.4. ESI-MS: 590.0 (Mj1).

1491
Synthesis and evaluation of entacapone prodrugs
(E)-2-Cyano-N,N-diethyl-3-[3,4-dibenzoyloxy-5-
Method C
nitrophenyl]propenamide (1d)
Entacapone (1.0 g, 3.3 mmol) was added to pivaloyl
anhydride (5 mL containing a drop of conc. H2SO4) and
the mixture was stirred at 50mC for 2 h. The mixture was
poured into water and extracted twice with diethyl ether.
The organic phase was washed with 5% NaHCO3, dried
with Na2SO4 and evaporated. The residue was purified
by recrystallization from ethyl acetate\petroleum ether
to give (E)-2-cyano-N,N-diethyl-3-[3-pivaloyloxy-4-
hydroxy-5-nitrophenyl]propenamide (1a) (0.62 g, 48%
of theoretical) as a yellow crystalline powder; mp
Method A
Yield 0.14 g (10% of theoretical); mp 143.0mC. Excep-
tions in the method: 2.5 equiv. benzoyl chloride was
1
used (dimethylaminopyridine was not used). H NMR
δ: 1.24 (6H, br, NCH2CH3), 3.52 (4H, br, NCH2), 7.40
(4H, m, ArH), 7.56 (2H, m, ArH), 7.66 (1H, s, CHlC),
8.04 (4H, m, ArH), 8.28 (1H, J l 2.2 Hz, d, ArH[2]),
8.45 (1H, J l 2.2 Hz, d, ArH[6]). 13C NMR δ: 12.77,
14.01, 41.1, 43.7, 111.4, 115.1, 124.5, 127.5, 127.7, 128.9,
128.9, 130.6, 130.6, 130.9, 134.7, 134.7, 139.9, 143.6,
145.6, 146.6, 162.2, 163.1, 163.8. ESI-MS: 514.1 (Mj1).
1
125.3mC. H NMR (CDCl3) δ: 1.26 (6H, br, CH2CH3),
1.41 (9H, s, CCH3), 3.50 (4H, br, NCH2), 7.59 (1H, s,
CHlC), 8.03 (1H, J l 2.2 Hz, d, ArH[2]), 8.46 (1H,
J l 2.2 Hz, d, ArH[6]), 10.9 (1H, br, ArOH). 13C NMR
δ: 12.7, 13.65, 27.1, 39.35, 41.0, 43.5, 108.1, 115.4, 123.8,
124.6, 129.9, 134.3, 141.9, 147.3, 150.3, 162.4, 175.9.
ESI-MS: 390.0 (Mj1).
(E)-2-Cyano-N,N-diethyl-3-[3,4-diacetoxyacetyloxy-
5-nitrophenyl]propenamide (2a)
Method A
Yield 0.23 g (28% of theoretical); oil. Exceptions in the
method: 1.6 mmol entacapone, 4 equiv. acetoxyacetyl
chloride and 4 equiv. triethylamine in tetrahydrofuran
In the following syntheses, 3.3 mmol (1.0 g) entacapone
was used as the starting material.
1
were used (dimethylaminopyridine was not used). H
(E)-2-Cyano-N,N-diethyl-3-[3,4-dipivaloyloxy-5-
nitrophenyl]propenamide (1b)
NMR δ: 1.24 (6H, br, NCH2CH3), 2.17 (6H, s, CH3CO),
3.54 (4H, br, NCH2), 4.96 (4H, s, OCH2CO), 7.60 (1H,
s, CHlC), 8.06 (1H, J l 2.2 Hz, d, ArH[2]), 8.43 (1H,
J l 2.2 Hz, d, ArH[6]). 13C NMR δ: 12.4, 13.8, 20.2,
42.1, 43.0, 60.2, 60.3, 111.7, 114.7, 124.4, 128.6, 131.3,
138.8, 142.8, 144.3, 145.8, 161.7, 164.5, 165.2, 170.3.
ESI-MS: 506.1 (Mj1).
Method A
Yield 0.69 g (45% of theoretical); mp 106.5mC. Excep-
tions in the method: 4 equiv. pivaloyl chloride and
1
10 mg dimethylaminopyridine were used. H NMR δ:
1.26 (6H, br, NCH2CH3), 1.41 (18H, s, CCH3), 3.49 (4H,
br, NCH2), 7.59 (1H, s, CHlC), 7.99 (1H, J l 2.2 Hz,
d, ArH[2]), 8.33 (1H, J l 2.2 Hz, d, ArH[6]). 13C NMR
δ: 12.3, 13.7, 26.4, 27.0, 39.2, 39.3, 40.0, 43.3, 112.8,
115.8, 123.1, 128.7, 130.0, 138.9, 141.7, 143.3, 145.1,
160.3, 174.3, 175.2. ESI-MS: 474.0 (Mj1).
(E)-2-Cyano-N,N-diethyl-3-[(3,4-diacetyloxy-5-
nitrophenyl)]propenamide (1c)
Method C
Yield 0.95 g (74% of theoretical); mp 135.0mC. Excep-
tions in the method: 4 equiv. acetic acid anhydride
(E)-2-Cyano-N,N-diethyl-3-[3,4-dibutyryloxy-5-
nitrophenyl]propenamide (1e)
1
dissolved in ethyl acetate was used. H NMR δ: 1.26
(6H, d, NCH2CH3), 2.36 (3H, s, CH3CO), 2.39 (3H, s,
CH3CO), 3.49 (4H, br, NCH2), 7.61 (1H, s, CHlC),
8.1(1H, J l 2.2 Hz, d, ArH[2]), 8.35 (1H, J l 2.2 Hz, d,
ArH[6]). 13C NMR δ: 12.4, 13.6, 20.2, 20.3, 40.7, 43.3,
111.0, 114.7, 124.1, 128.4, 130.4, 138.8, 142.8, 144.7,
146.0, 161.8, 166.8, 167.5. ESI-MS: 389.9 (M).
Method A
Yield 1.36 g (93% of theoretical); oil. Exceptions in the
method: 4 equiv. butyroyl chloride and 4 equiv. tri-
ethylamine in dry tetrahydrofuran were used. 1H NMR
δ: 1.04 (6H, J l 7.2 Hz, t, CH2CH2CH3), 1.24 (6H, br,
NCH2CH3), 1.76 (2H, J l 7.2 Hz, m, CH2CH2CH3),
1.80 (2H, J l 7.2 Hz, m, CH2CH2CH3), 2.55 (2H, J l
7.3 Hz, t, COCH2), 2.60 (2H, J l 7.3 Hz, t, COCH2),
3.48 (4H, br, NCH2), 7.60 (1H, s, CHlC), 8.07 (1H, J
l 2.1 Hz, d, ArH[2]), 8.32 (1H, J l 2.1 Hz, d, ArH[6]).
(E)-2-Cyano-N,N-diethyl-3-[3,4-
dipivaloyloxyacetyloxy-5-nitrophenyl]-
propenamide (2b)
13C NMR δ: 12.9, 13.0, 17.8, 18.2, 35.5, 40.8, 43.4, 110.9, Method B
114.7, 124.1, 128.5, 130.3, 139.0, 143.0, 144.8, 146.2, Yield 0.70 g (85% of theoretical); oil. Exceptions in the
161.8, 169.4, 170.1. ESI-MS: 446.0 (Mj1).
method: 1.4 mmol entacapone and 3.1 mmol piva-

1492
Jukka Leppanen et al
Determination of aqueous solubilities
loyloxy acetic acid and ethyl acetate as a solvent were
used. 1H NMR δ: 1.25 (6H, br, NCH2CH3), 1.38 (9H, s,
CCH3), 1.39 (9H, s, CCH3), 2.17 (6H, s, CH3CO), 3.50
(4H, br, NCH2), 4.72 (2H, s, O-CH2-CO), 4.82 (2H, s,
OCH2CO), 7.62 (1H, s, CHlC), 8.04 (1H, J l 2.2 Hz,
d, ArH[2]), 8.35 (1H, J l 2.2 Hz, d, ArH[6]). 13C NMR
δ: 12.5, 13.4, 27.3, 39.6, 41.1, 43.0, 60.4, 61.0, 111.1,
115.1, 124.1, 128.7, 130.4, 139.8, 145.5, 146.7, 153.2,
132.2, 167.4, 167.6, 175.6. ESI-MS: 590.0 (Mj1).
The aqueous solubilities of entacapone and its prodrugs
were determined in phosphate buffer solution (0.16 ,
pH 5.0 and 7.4) at room temperature. An excess amount
of each compound was added to the buffered solution.
The suspensions were shaken 30–60 min and filtered
(Millipore 0.45 µm). Clear solutions were diluted, if
necessary, and analysed by HPLC. All determinations
were made in triplicate. The pH of the suspension was
held constant throughout the study.
(E)-2-Cyano-N,N-diethyl-3-[3,4-
dipivaloyloxy(methoxy)-5-
nitrophenyl]propenamide (3a)
Determination of apparent partition
coefficients
The apparent partition coefficient (Papp) of entacapone
and 1a were determined from the distribution of the
compound between a mixture of 1-octanol and phos-
phate buffer (0.16 ), where the buffer solution and 1-
octanol were saturated before the experiment by shaking
overnight. A known concentration of derivative in phos-
phate buffer solution was shaken for 30 (1a) or 60 min
(entacapone) with a suitable volume of the 1-octanol
buffer solution. The phases were separated by centri-
Chloromethyl pivalate (0.75 g, 5 mmol) was dissolved
in dry acetone (20 mL) and sodium iodide (0.75 g,
5 mmol) was added. The mixture was placed in the dark
and stirred at room temperature for 3 h. Entacapone
(0.75 g, 2.5 mmol) and potassium carbonate (1.4 g,
10 mmol) were added and stirring was continued over-
night. The mixture was filtered and the filtrate was
evaporated. The residue was purified by column chro-
matography using ethyl acetate as an eluent to give 3a
(1.5 g, 56%) as a clear yellow oil. 1H NMR δ: 1.15 (9H,
s, CCH3), 1.19 (9H, s, CCH3), 1.27 (6H, br, NCH2CH3),
3.48 (4H, br, NCH2), 5.77 (2H, s, O-CH2-O), 5.83 (2H,
s, O-CH2-O), 7.55 (1H, s, CHlC), 7.82 (1H, J l 2.2 Hz,
d, ArH[2]), 8.1 (1H, J l 2.2 Hz, d, ArH[6]). 13C NMR δ:
12.6, 13.8, 26.8, 39.0, 39.2, 40.9, 43.3, 85.7, 88.8, 109.4,
115.3, 119.2, 120.2, 128.6, 143.6, 144.4, 147.3, 150.6,
1
−
fugation (3 min at 1500 rev min ) and the concen-
trations of the derivative were determined from the
buffer phase by HPLC. Log Papp values of poorly water-
soluble derivatives (1b–e, 2b and 3a) were determined
from capacity factors (kh) by reversed-phase HPLC
(Lambert 1993). A Purospher C18e column was used as
the stationary phase and the mobile phase consisted of
25 m phosphate\10 m citrate buffer (pH 2.2) and
methanol (28:72, v\v). The flow rate (isocratic) was
+
162.2, 177.0, 177.7. ESI-MS: 556.1 (Mj1jNa ).
1
−
1.0 mL min at 40mC. Column effluent was monitored
at 254 nm by UV detection. The calibration curve was
performed by using seven non-ionizable standard com-
pounds. The standard compounds and their log P values
were as follows: anisole 2.11 (Minick et al 1988), an-
thracene 4.54 (Hsieh & Dorsey 1995), budesonide 2.31
(Corwin et al 1990), hydrocortisone 1.53 (Singh &
Roberts 1996), naphthalene 3.30 (Minick et al 1988),
nitrobenzene 1.85 (Minick et al 1988) and progesterone
3.87 (Singh & Roberts 1996). The log Papp values of the
standard compounds were plotted against the measured
log kh values and a linear relationship (r2 l 0.938) was
found between these two parameters. The log Papp values
ofentacaponeprodrugsweredeterminedfromthislinear
relationship.
(E)-2-Cyano-N,N-diethyl-3-[3-t-
butyloxycarbonyloxy-4-hydroxy-5-nitrophenyl]-
propenamide (4a)
Entacapone (1.0 g, 3.3 mmol), sodium carbonate (0.7 g,
6.6 mmol), 15-crown-5 (catalytic amount) and di-tert-
butyldicarbonate (0.72 g, 3.3 mmol) were dissolved in
tetrahydrofuran (20 mL) and stirred for 12 h at 50mC.
The mixture was filtered and the filtrate was evaporated
to dryness in-vacuo. The product was recrystallized
from petroleum ether\ethyl acetate to give 4a (0.87g,
1
64%) as a yellow powder. H NMR δ: 1.25 (6H, br,
NCH2CH3), 1.52 (9H, s, CCH3), 3.50 (4H, br, NCH2),
7.50 (1H, s, CHlC), 7.84 (1H, J l 2.2 Hz, d, ArH[2]),
8.3 (1H, J l 2.2 Hz, d, ArH[6]). 13C NMR δ: 12.4, 13.3,
28.1, 42.0, 43.0, 84.2, 98.5, 113.3, 118.5, 124.5, 131.3,
Hydrolysis in aqueous solution
139.8, 148.1, 151.8, 153.0, 164.2, 167.0. ESI-MS: 427.9 Chemical hydrolysis rates of the entacapone prodrugs
+
(MjNa ).
were determined in phosphate buffer (0.16 ) solution

1493
Synthesis and evaluation of entacapone prodrugs
at pH 3.0 (1a–b, 3a) and pH 7.4 (all prodrugs) at 37mC. ing entacapone in a phosphate buffer solution (pH 7.4;
Solutions were prepared by dissolving an appropriate 50 m). The solution was filtered through a sterile
amount of compound in preheated (37mC) buffer sol- membrane (pore size 0.22 µm) before making it isotonic
ution. Due to the low aqueous solubility of some com- by the addition of an amount of NaCl. Entacapone
6
1
−
−
pounds, an addition of 5–10% ethanol was needed to content of all preparations was 3.73i10 mol mL .
achieve a detectable concentration. Samples were with-
In-vivo absorption studies
drawn at designated time intervals and analysed for
remaining prodrug by HPLC. Pseudo-first order half-
lives (t12) for the hydrolysis of the derivatives were
calculated from the linear slopes of logarithmic plots of
remaining entacapone prodrug over time.
Male Han\Wistar rats (7–9-weeks-old, 140–270 g;
National Laboratory Animal Centre, University of
Kuopio) were housed in stainless steel cages and kept on
a12-hlight–darkcycle(lightsonat0700 h)atanambient
temperature of 22p1mC. The relative air humidity was
50p10%. Pelleted food (Lactamin R36, Lactamin AB,
Sodertalje, Sweden) was removed 24 h before the ex-
periment but water was freely available.
Hydrolysis in human serum
The rate of enzymatic hydrolysis for the prodrugs was
determined in human serum diluted to 80% with 0.16 
phosphate buffer (pH 7.4) at 37mC. An appropriate
amount of prodrug was dissolved in preheated buffer
solution and preheated serum was added. Solutions
were kept in a water bath at 37mC. At designated time
intervals, samples of 0.5 mL were withdrawn and 1.0 mL
ethanol was added to each sample to terminate en-
zymatic activity. After immediate mixing and centri-
1
−
Entacapone and prodrugs (doses equal to 5.7 mg kg
entacapone) were administered orally by gavage in a
volume of 0.5 mL\100 g animal weight. Entacapone
was also administered intravenously to a tail vein
(0.5 mL\100 g). Vasodilatation of tail veins was induced
by warm water just before intravenous injection.
Ratsweredecapitated2.5, 5, 10, 20, 30, 60and120 min
(i.v.) or alternatively 10, 20, 30, 60 and 120 min (p.o.)
after drug administration. Blood samples were collected
in glass tubes containing EDTA to prevent coagulation.
Plasma was separated by centrifugation at j4mC at
1500 g for 10 min. Plasma samples were transferred into
plastic tubes and stored at k80mC until analysed. All
procedures with animals were reviewed and approved
by the Animal Ethics Committee at the University of
Kuopio.
1
−
fugation for 10 min at 14000 rev min , the clear super-
natant was analysed by HPLC for remaining prodrug
and released entacapone. Pseudo-first order half-lives
(t12) for the hydrolysis of the derivative were calculated
from the linear slopes of logarithmic plots of remaining
entacapone prodrug over time.
Analytical procedure for the in-vitro samples
The HPLC system used for the determination of in-vitro
samples consisted of a Beckman System Gold Program-
mable Solvent Module 126, a Beckman System Gold
Detector Module 166 with variable wavelength UV
detector (set at 254–304 nm, depending on compound)
and a Beckman System Gold Autosampler 507e. Sepa-
rations were performed on a Purospher RP-18e reverse-
phase column (12.5 cmi4.0 mm i.d., 5 µm). The chro-
matographic conditions were as follows: loop injection
volume, 50 µL; column temperature, 40mC; flow rate,
Analytical procedure for the in-vivo samples
TheHPLC usedfor thedetermination of in-vivo samples
was similar to the one used for in-vitro samples, except
that the mobile phase contained a water\acetonitrile
mixture instead of water\methanol mixture. A linear
gradientelution was usedand the mobilephase consisted
of various percentages of an acetonitrile\water mixture
(0–7 min 40% acetonitrile; 7–10 min 48% acetonitrile;
10–20 min 87% acetonitrile) and citrate\phosphate
buffer (pH 2.2).
1
−
gradient\isocratic at 1.0 mL min . The mobile phase
consisted of various percentages of a methanol\water
mixture (90:10) and citrate\phosphate buffer (pH 2.2).
Before extraction, 100 µL 2  hydrochloric acid was
added to 1.0 mL plasma and 50 µL of a tolcapone
solution was added as an internal standard. Entacapone,
Preparation of dosage forms
Suspensions of entacapone and selected prodrugs (1a–b) Z-entacapone, prodrug and internal standard were ex-
were prepared by weighing an appropriate amount of tracted from plasma using Bond Elut C18 (3 CC\
compound, after which potassium biphthalate buffer 500 mg) solid phase extraction cartridges (Analytichem
(pH 3.0, USP 23) was added. Suspensions were soni- International, Harbor City, CA) and 2.5 mL acetonitrile
cated to obtain homogenous solutions. The entacapone as an eluent. The eluent was evaporated and the residues
intravenous solution (pH 7.4) was prepared by dissolv- were dissolved in 300 µL of a water\methanol mixture

1494
Jukka Leppanen et al
(50:50) and injected onto the HPLC. The results were acid chlorides and carboxylic acid anhydrides. The
calculated from peak-area ratios. structure of 1a was confirmed by X-ray crystallography,
A standard curve was prepared for entacapone by which showed that the pivaloyl moiety was at the C-3
mixing blank plasma with a known amount of enta- hydroxy position (Leppanen et al 2001). Acyl migration
1
capone (62.7–1960 ng mL ) and internal standard (tol- to the neighbouring hydroxyl group was not observed
−
capone). The standard curve showed excellent linearity (Ihara et al 1990).
(r2 l 0.999) which made one-point calibration feasible.
Acyloxyacyl esters 2a–b were synthesized from acid
Separate spiked plasma standard samples containing Z- chlorides in the presence of pyridine or via the route that
entacapone and two concentrations of entacapone and employed dicyclohexylcarbodiimide (DCC) in THF
spiked plasma samples containing two concentrations (2c). The synthesis of acyloxymethyl ether 3a was found
of prodrug were prepared daily. All the spiked plasma to be sensitive to moisture and the treatment of chloro-
samples contained tolcapone as an internal standard.
methyl pivaloate with NaI was essential, otherwise an
Intraday precision (coefficient of variation) of the unwanted acylated product (1a) was obtained (Bodor
method was assessed by extracting and analysing plasma et al 1983). Attempts to synthesize a monosubstituted
samples, containing entacapone and prodrugs (1a and acyloxymethyl ether primarily resulted in an acylated
1b), four to six times in one day. The intraday precision product. t-Butyloxycarbonyl ester 4a was synthesized
was 1.4, 3.9 and 6.3% for entacapone, 1a and 1b, from (Boc)2O using Na2CO3 and 15-crown-5 as a cata-
respectively.
lyst. Similarly to 1a entacapone was substituted merely
to the C-3 position.
In-vivo data analysis
Promoieties were primarily allowed to react only to
the other hydroxyl group of entacapone (1a, 4a) to
maintain adequate aqueous solubility and to increase
lipophilicity.
The maximum plasma concentrations (Cmax) and the
amount of time required to reach the maximum (tmax
)
were obtained directly from the plasma concentration
vs time data. The entacapone concentration in plasma
(C) after an intravenous administration was best de-
Unfortunately these derivatives have low chemical
stability at pH 7.4, which probably follows from the
intramolecular hydrolysis initiated by the unsubstituted
hydroxyl group. Therefore further syntheses of similar
derivatives were not carried out. Due to the low aqueous
solubilities of more stable disubstituted derivatives (e.g.
1b–f), amino acid derivatives were synthesized to main-
tain the stability and to increase aqueous solubility.
-Isoleucine and glycine have been used successfully to
improve the bioavailability of penciclovir (Kim et al
1999) and their Boc-protected derivatives of entacapone
were synthesized. However, after the cleavage of the
protective group these derivatives were very labile
(Jensen et al 1991) and were not evaluated for prodrug
purposes.
scribed by the biexponential equation, C l Ae λ1tj
−
Ae λ2t, where t is time. Results were obtained with the
−
Kaleida Graph (version 3.0.1) program (Macintosh).
Areas under the concentration vs time curves from 0 to
infinity (AUC0-_) following intravenous injection were
estimated by using the equation AUC0-_ l A1\λ1j
A2\λ2 (Gibaldi & Perrier 1982). For oral dosage forms,
AUC2h-_ was the sum of AUC0–2h and AUC2h-_, where
AUC0–2h was calculated by using the linear trapezoidal
method and AUC2h-_ was estimated by dividing enta-
capone concentration at 2-h post-dosing by λ2 (Gibaldi
& Perrier 1982). The calculation of the bioavailability of
prodrugs was based on the sum of entacapone and
prodrug concentration in plasma.
In some reactions (1b, 1d, 2b and 3a) the parent
compound was partially converted to its Z-form. The
mechanism of the conversion and the chemical environ-
ment behind this process were not investigated in this
study.
A one-factor analysis of variance (for factorial meas-
urements) was used to test the statistical significance of
differences between formulations at each time point.
Significance in the differences of the means was tested
using Fisher’s protected least significant difference
(PLSD) method at the 95% confidence level.
Aqueous solubilities and apparent partition
coefficients
Results and Discussion
The aqueous solubilities and apparent partition co-
efficients of entacapone and its prodrugs 1a and 4a are
listed in Table 1. The aqueous solubilities of 1b–f, 2a–c
Chemistry
The synthetic routes are summarized in Figure 1. Acyl and 3a were not determined due to their low aqueous
esters 1a–f were synthesized from their corresponding solubility. Their apparent partition coefficients were

1495
Synthesis and evaluation of entacapone prodrugs
i
R1
R2
ii
1
i
1b
1d
1e
1f
1a
1c
iii
2a
3a
iv
v
ii 2b
2c
4a
Figure 1 Synthesis routes of entacapone prodrugs. i: acid chloride, pyridine, (DMAP); ii: carboxylic acid, DCC, THF, DMAP; iii: acid
anhydride, reflux; iv: 1. acyloxymethyl chloride, NaI, acetone; 2. K2CO3, 1; iv: (Boc)2O; Na2CO3, 15-Crown-5, THF.
Table 1 Apparent partition coefficients (log Papp) and aqueous
solubilities of entacapone (1) and its prodrugs 1a and 4a.
determined by using the capacity factor of reversed-
phase HPLC. Log Papp of 1f, 2a, 2c and 4a was not
determined due to inadequate chemical stability (Lam-
bert 1993).
Compound
log Papp
pH 5.0a
pH 7.4a
With columnb
The aqueous solubility of entacapone was very low at
acidic pH (e.g. 17 µg mL 1 at pH 1.2) but improved with
−
increasing pH (e.g. 1750 µg mL 1 at pH 7.4) (Savolainen
1
1.58p0.01
2.24p0.01
0.18p0.03
0.80p0.06
0.65
–
−
1a
1b
1c
1d
1e
2b
3a
et al 2000b). The large difference in the aqueous solu-
bility of entacapone was due to its low pKa value (4.5)
(Wikberg 1993).
–
–
–
–
–
–
–
–
–
–
–
–
4.00
1.23
3.44
3.15
3.99
3.86
The monosubstituted entacapone prodrug 1a had a
higher log Papp value than entacapone, while adequate
aqueous solubility was maintained at pH 7.4 (Table 1).
However, the aqueous solubility of 1a decreased more
rapidly with decreasing pH when compared with enta-
capone. Compound 4a improved aqueous solubility
threefold compared with entacapone.
1
a,c
−
Aqueous solubility (µg mL
)
pH 5.0
pH 7.4
1
77.3p2.3
5.2p0.6
–
1751.8p119.1
1623.0p76.0
6993.0p792.0
1a
4a
Chemical and enzymatical hydrolysis
aValues were meanps.d., n l 3. bApparent partition coefficients were
determined by using the capacity factor of reversed phase HPLC. cThe
aqueous solubilities of the other prodrugs were not determined due to
low chemical stability or low aqueous solubility (below detection limit
of the HPLC method).
The degradation kinetics of the prodrugs followed first-
order kinetics, and their half-lives (t12) are presented in
Table 2.
The chemical stability of the prodrugs was higher at
low pH than at neutral pH. In contrast, carbamate

1496
Jukka Leppanen et al
Table 2 Half-lives of entacapone prodrugs in phosphate buffer
(0.16 ) and 80% human serum (pH 7.4) at 37mC.
Compound
t12 (h)
t12 (h)
Phosphate buffer
Human serum
pH 3.0
11.1
pH 5.0
pH 7.4
pH 7.4
1a
1a
1b
1c
1d
1e
1f
2.0
71.6
7.2
2.1
7.6
0.4
4.8
0.72
c
87.5
a
c
c
c
c
c
c
c
a
a
a
a
a
a
4.1
a
1.6
a
b
b
2a
2b
2c
3a
4a
0.07
58.1
a
18.7
b
197.5
a
41.5
a
43.4
0.7
0.9d
1.0
aNot determined. bDegraded instantly to entacapone. cReleased enta-
capone, but the kinetics could not be determined due to low aqueous
solubility of the compound. dRelease of entacapone was incomplete.
Figure 2 Bioconversion of 1a to entacapone in 80% human serum
(pH 7.4) at 37mC.
prodrugs of entacapone had the opposite behaviour and
hydrolysed faster at low pH than at neutral pH (Savo-
lainen et al 2000b). The increased stability of the pro-
drugs in an acidic environment was a desired property
because they were protected against chemical hydrolysis
at the acidic pH (i.e. stomach).
1a
1b
The branched prodrugs 1a, 1b, 2b and 3a were more
stable against chemical hydrolysis than prodrugs 1e and
1f (Table 2), which was in good agreement with earlier
prodrug studies (Chien et al 1991; Geraldine et al 1992).
Diacyl ester prodrugs are chemically more stable than
monoacyl esters, due to the lacking intramolecular
hydrolysis. The carbonate structure of 4a was less stable
than ester structure 1a at pH 7.4, which may limit its
usefulness because entacapone might be released before
absorption in the small intestine.
Figure 3 The concentration of entacapone in plasma after oral
administration of entacapone, 1a and 1b as suspensions (pH 3.0) to
rats (mean value, n & 4). The administered dose was always equivalent
Chemical stability of the acyloxy acyl esters 2a–c was
very low. The most branched (2b) was the most stable.
The higher stability of 2b suggested that the chemical
hydrolysis rate of acyloxy acyl esters was mostly de-
pendent on the second ester group of the promoiety.
Prodrug 3a had the highest chemical stability due to
to 5.7 mg kg 1 entacapone.
−
the location of the ester group far from the electron However, due to their low aqueous solubility, accurate
withdrawing nitrocatechol structure via a methyl spacer. values of enzymatic hydrolysis rates were successfully
This stabilization was reflected in the half-lives of the determined for 1a, 3a and 4a only. Prodrugs 1a (Figure
acyl esters 1b and 3a, which were 7.6 and 43.4 h at pH 2) and 4a hydrolysed quantitatively to the parent enta-
7.4, respectively. capone in serum, but in the case of 3a only 20% of the
All entacapone prodrugs released entacapone in 80% parent drug was released after complete degradation of
human plasma and thus fulfilled the prodrug criteria. 3a. The reason for the incomplete bioconversion of 3a

1497
Synthesis and evaluation of entacapone prodrugs
Table 3 Mean pharmacokinetic parameters of entacapone in plasma after oral administration of entacapone and its prodrugs and after
intravenous administration of entacapone to rats (n & 4). Administered dose was always equivalent to 5.7 mg kg 1 entacapone.
−
1
−
Formulation
Cmax (ng mL
)
tmax (min)
AUC (0–2 h)
1
AUC (0–_)
F, (0–2 h) (%)
F, (0-_) (%)
1
−
−
(ng h mL
)
(ng h mL
)
1 suspensiona
1a suspensiona
1b suspensiona
1 solution (i.v.)b
& 1062.0
& 963.3
& 23.0
% 10
% 10
% 10
2.5
644.3
409.0
32.1
708.7
476.7
40.4
10.5
6.7
10.4
7.0
0.5
0.6
33496.2
6126.3
6806.6
100.0
100.0
apH 3.0. bpH 7.4.
during a 4-h enzymatic hydrolysis reaction might have decrease. The site of absorption was thus mainly in the
been due to a more stable intermediate prolonging the upper gastrointestinal-tract where pH was low and with
release of entacapone. The good chemical stability of 3a decreasing pH the aqueous solubility of 1a decreased
made it a possible prodrug of entacapone, but its low more rapidly than that of entacapone and thus its
aqueous solubility, together with the slow release of bioavailability was reduced. Therefore an ideal pro-
entacapone, decreased its usefulness.
moiety of entacapone should have, in addition to better
Prodrugs 1a, 3a and 4a were hydrolysed enzymatically chemical stability, an ionizable or polar group to in-
at least twice as rapidly as entacapone prodrugs crease aqueous solubility at low pH.
(Leppanen et al 2000; Savolainen et al 2000b). Rapid
enzymatic hydrolysis to release entacapone immediately
Conclusions
after the absorption enables effective COMT-inhibition
in the small intestine where COMT-activity is high
(Backstrom et al 1989).
The novel entacapone derivatives released the parent
compound, and thus fulfilled the prodrug criteria. All
disubstituted entacapone prodrugs were more lipophilic
than the parent entacapone, but their low aqueous
solubilities prevented their oral administration trials in
rats.
Bioavailability in-vivo
The absolute bioavailability of 1a, 1b and entacapone
was studied in rats, and the selection of the prodrugs
was based on their in-vitro properties. Their chemical
stability was adequate and they released entacapone in
80% human serum. The aqueous solubility of 1a was
comparable with the aqueous solubility of entacapone
at pH 7.4, but its log Papp was higher when compared
with entacapone. Prodrug 1b had a markedly higher log
Papp value when compared with entacapone.
The acyloxymethyl ether (3a) proved to be a useful
synthetic promoiety for the nitrocatechol structure, as
it was chemically more stable than the corresponding
acyl esters and its enzymatical hydrolysis was fast.
However, its poor aqueous solubility and incomplete
bioconversion to entacapone might limit its usefulness.
Prodrug 1a had a higher lipophilicity than entacapone
and while retaining the adequate aqueous solubility
equal to entacapone, its oral bioavailability in the rat
was lower than that of entacapone. This study showed
that lipophilic entacapone prodrugs were not able to
improve oral bioavailability in the rat.
The absolute bioavailability (AUC0-_) of entacapone,
1a and 1b (administered orally as suspensions) were
10.4%, 7.0% and 0.6%, respectively (Figure 3 and
Table 3). The poor bioavailability of 1b was considered
to be a result of its low aqueous solubility. In the case of
1b, no prodrug was detected in the plasma after oral
administration, but all of the detected drug was enta-
capone. This suggested that 1b was partly degraded in
the gastrointestinal-lumen and absorbed as entacapone.
The bioavailability of 1a was slightly lower than that
of entacapone. According to Figure 3 entacapone and
1a had rapid parallel absorption followed by a rapid
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