Design, synthesis and in vitro/in vivo evaluation of orally bioavailable prodrugs of a catechol-O-methyltransferase inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.02.057

Compound 1 is an investigational, nanomolar inhibitor of catechol-O-methyltransferase (COMT) that suffers from poor oral bioavailability, most probably due to its low lipophilicity throughout most of the gastrointestinal tract and, to a lesser extent, its rapid systemic clearance. Several lipophilic esters were designed as prodrugs and synthesized in an attempt to optimize presystemic drug absorption. A modest twofold increase in 6-h exposure of 1 was observed with two prodrugs, compared to that of 1, after oral treatment in rats.

Full text of DOI:10.1016/j.bmcl.2010.02.057

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2614–2616
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and in vitro/in vivo evaluation of orally bioavailable
prodrugs of a catechol-O-methyltransferase inhibitor
a
a
a
b
b
Jarkko Rautio a, , Jukka Leppänen , Marko Lehtonen , Krista Laine , Mikko Koskinen , Jarmo Pystynen ,
*
Jouko Savolainen c, Mikko Sairanen b
a University of Eastern Finland, School of Pharmacy, PO Box 1627, FI-70211 Kuopio, Finland
b Orion Corporation, Orion Pharma, Tengströminkatu 8, PO Box 425, FI-20101 Turku, Finland
c Fennopharma Ltd, Mikrokatu 1, FI-70210 Kuopio, Finland
a r t i c l e i n f o
a b s t r a c t
Article history:
Compound 1 is an investigational, nanomolar inhibitor of catechol-O-methyltransferase (COMT) that suf-
fers from poor oral bioavailability, most probably due to its low lipophilicity throughout most of the gas-
trointestinal tract and, to a lesser extent, its rapid systemic clearance. Several lipophilic esters were
designed as prodrugs and synthesized in an attempt to optimize presystemic drug absorption. A modest
twofold increase in 6-h exposure of 1 was observed with two prodrugs, compared to that of 1, after oral
treatment in rats.
Received 24 November 2009
Revised 16 February 2010
Accepted 16 February 2010
Available online 19 February 2010
Keywords:
Prodrug
Ó 2010 Elsevier Ltd. All rights reserved.
COMT inhibitor
Oral bioavailability
Parkinson’s disease (PD) is a slowly progressive, neurodegener-
ative disorder which is characterized by the destruction of dopami-
nergic cells in the pars compacta region of the substantia nigra in
the mid brain region, leading to a deficiency of dopamine.1 Current
PD treatment involves oral administration of levodopa, a precursor
of dopamine, combined with an inhibitor of DOPA decarboxylase
(DDC), such as carbidopa, to inhibit decarboxylation of levodopa
in the periphery.2 During such treatment, catechol-O-methyltrans-
ferase (COMT) becomes the main enzyme to metabolize levodopa.3
Compound 1 is an investigational, very potent, nanomolar inhibitor
of COMT (Fig. 1). However, 1 displays less than ideal pharmacoki-
netic properties, such as oral absorption, similar to another struc-
turally related COMT inhibitor, entacapone.4 The main barrier to
achieving high bioavailability for entacapone has been suggested
to be due to its high systemic clearance.4 Extensive ionization asso-
ciated with the poor lipophilicity (log D values) of entacapone at
intestinal pH and, to a lesser extent, its high systemic clearance,
are the main factors contributing to the poor bioavailability of 1
based in preliminary studies.
and also saturation of systemic clearance by optimizing presystem-
ic drug absorption. The prodrugs designed, synthesized and evalu-
ated in this letter are alkyl esters (2–6), acyloxyalkyl esters (7–10),
aminoacyloxy alkyl esters (11) and cyclic carbonate esters (12).
Synthetic pathways to prodrugs are shown in the Scheme 1. The
alkyl esters 2–5 were prepared by heating 1 in the corresponding
alcohol in the presence of thionyl chloride. All other prodrugs were
synthesized by starting from the acetate-protected compound 1b
to avoid alkylation to the catechol. The acetate proved to be the most
convenient protecting group for the catechol and was easily hydro-
lyzed in most cases in a ACN–water solution by adding a few drops
of 25% aqueous ammonia, with only low to moderate losses of the
desired promoiety. Prodrugs 6 and 12 were synthesized by reacting
1b with benzyl bromide and 4-(bromomethyl)-5-methyl-1,3-diox-
ol-2-one,7 respectively, using potassium carbonate as a base. Acy-
loxyalkyl chlorides used in the preparation of 7–11 were
synthesized as previously described8,9, and reacted with compound
1b in presence of sodium iodide and cesium carbonate. The overall
reaction yields were low to moderate, partly due to tailing of the tar-
get compounds during the flash chromatography, which allowed the
collection of only a narrow window of pure fractions.
Prodrugs have been utilized to overcome several drug delivery
problems by introducing lipophilicity and masking hydrogen
bonding groups.5,6 In the present study, several prodrug esters
were studied in an attempt to improve the oral absorption of 1.
The rational for embarking on a prodrug approach to address the
poor bioavailability of 1 is based on increasing its lipophilicity,
Chemical and enzymatic stabilities of selected prodrugs were as-
sessed in aqueous buffers, fasted state simulated intestinal fluid
(FaSSIF), human serum and rat liver homogenate. As seen in Table
1, the prodrugs showed varying levels of stability to hydrolysis rela-
tive to the structure of promoiety. Esterification of an acidic func-
tional group with a simple alkyl or aryl alcohol is the most
commonly used approach in successful prodrugs when increasing
* Corresponding author.
E-mail address: Jarkko.Rautio@uku.fi (J. Rautio).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.057

J. Rautio et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2614–2616
2615
human serum. Although bioconversion of 2–6 took place in rat li-
ver homogenate, double esters 7–10 were also prepared to increase
the recognition by esterases through the second ester. Previous
examples of such double prodrugs include orally active acy-
loxyalkyl esters of b-lactam antibiotics and nucleoside monophos-
phate anti-viral agents.11–14 The isopropyloxycarbonylmethyl (7)
and propyloxycarbonylmethyl (9) analogues were synthesized
along with their homologated derivatives with a 1-ethyl group (8
and 10). Upon hydrolysis by esterases, prodrugs 8 and 10 released
acetaldehyde instead of formaldehyde, which was released by the
prodrugs 7 and 9. Although the amount of formaldehyde generated
from prodrugs would be small, formaldehyde is a probable human
carcinogen.15
Figure 1. Structure and selected properties of 1.
lipophilicity is the desired objective; for example, angiotensin-con-
verting enzyme (ACE) inhibitors.10
In the present set of prodrugs, however, the simple alkyl (2–5)
and aryl (6) esters were not susceptible to esterase activity in
As expected, the acyloxyalkyl esters 7–10 were susceptible to
enzymatic hydrolysis in human serum and hydrolyzed quantita-
tively to
1 with half-lives ranging from 5.1 to 32.3 h. The
Scheme 1. Synthesis routes of compounds 1b–12. Reagents: (a) thionyl chloride, R–OH, reflux; (b) acetic anhydride, pyridine; (c) Cs2CO3, acyloxyalkyl chloride, NaI, ACN or
DMF; (d) K2CO3, alkyl bromide, acetone; (e) ACN–water, 25% ammonia.
Table 1
Physicochemical, pharmacokinetic and pharmacologic properties for prodrugs of 1
Compound
COMT inhibition
(Ki) (nM)
Uncoupling
(lM)
T1/2
AUC, fold increasee
pH 4.0 (days)
pH 7.4 (h)
FaSSIF (h)
Human serum (h)
Rat liver homogenate (min)
1
2
3
4
5
6
7
8
9
<1
2.2
9.2
1.3
>50
>10
>5
>2.5
>1
d
b
Stablea
31
—
Stablea
Stablea
Stablea
Stablea
21.6
7.6
48
2.5
4.5
2.0
—
—
—
—
—
—
—
—
—
—
—
—
19.8
20.4
41.9
130.9
51.9
61
d
b
d
—
—
—
—
c
d
b
d
d
b
d
1.1
23
c
d
b
d
10.2
17.0
17.0
10.6
16.6
6.0
>10
>50
>50
>50
>50
25
—
—
b
8.4
5.1
32.3
5.3
25.0
0.06
0.03
2.0
0.5
c
b
—
61
c
d
d
—
61
—
—
c
d
d
10
11
12
—
—
61
1.7
3.9
31.7
167
61
0.4
2.1
d
1.6
—
11.7
61
a
b
c
No degradation was detected during one week.
No degradation was detected within 8 h.
Not determined due to poor aqueous solubility.
Not determined.
d
e
Adult male Wistar rats were orally administered the test compounds at 10 lmol/kg, and area under the curve (AUC) was determined over a 6-h interval relative to the
parent compound, 1.

2616
J. Rautio et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2614–2616
acyloxyethyl prodrugs 8 and 10 had 5–6-fold longer half-lives
compared to their acyloxymethyl analogues 7 and 9. These results
are in good agreement with a previously published study of oxym-
ethyl- and oxyethylphosphates, in which the latter prodrug
showed a twofold slower bioconversion rate.16 In rat liver homog-
enate 7–10 underwent rapid bioconversion to 1 with half-lives less
than 1 min.
experience with nucleoside-amino ester prodrugs17,18, the valinate
prodrug (11) did not appear to be a substrate for active transport,
and 11 did not enhance oral bioavailability. Therefore, our attempts
to improve drug absorption and address the poor bioavailability of 1
resulted in only modest success.
Acknowledgements
Amino acid prodrugs have previously been reported to be sub-
strates for the dipeptide intestinal transporter.17,18 The amin-
oacyloxyalkyl prodrug 11, which contains the amino acid valine,
was also prepared and evaluated. The cyclic carbonate prodrug
12 was designed to be labile only in plasma, while avoiding unde-
sired metabolism by esterases in the enterocytes during absorp-
tion. The clinically novel prodrugs olmesartan medoximil19 and
lenampicillin20 are examples of such cyclic carbonate prodrugs.
Both 11 and 12 hydrolyzed rapidly to 1 in both human serum
and rat liver homogenate, with the latter most probably metabo-
lized by the plasma enzyme called paraoxonase.21,22
The authors want to thank M.Sc. Mari Ryömä, M.Sc. Johanna
Kiviniemi, Mrs. Helly Rissanen, Mrs. Anne Riekkinen and Mrs. Miia
Reponen for their skillful technical assistance. The Academy of
Finland(Grant No. 132637, J.R.) has providedfinancial supportfor this
study.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.02.057.
Both 1 and prodrugs 2–12 were subjected to COMT inhibition
and uncoupling assays (Table 1).23,24 Although 2–12 were designed
to function as prodrugs; that is, to be inactive or less active deriv-
atives of 1, they all displayed nanomolar inhibition of COMT, indi-
cating that these prodrugs are also COMT inhibitors by themselves.
Moreover, based on its catechol structure, 1 is a potential mito-
chondrial uncoupler of oxidative phosphorylation, which may be
linked to its mechanism of hepatotoxicity, due to impairment of
energy production, which was a serious drawback seen with an-
other COMT inhibitor, tolcapone.25,26 While simple alkyl (2–5)
and aryl (6) esters and the aminoacyloxyalkyl ester (11) were
shown to be in vitro uncouplers at low micromolar concentrations,
high uncoupling efficacy was not seen either with 1 or acyloxyalkyl
ester prodrugs 7–10.
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coefficients (log D) were determined along with aqueous solubili-
ties in various buffer solutions. The aqueous solubility of 1 was sig-
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15,000
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cept for the ionized valinate ester 11 at pH 1.2 (Supplementary
data). As predicted, masking of the hydrophilic carboxylic acid
group by the addition of various lipophilic promoieties resulted
in prodrugs having log D values that were generally higher than
those of 1 within the pH range of 1.2–7.4. The log D values of these
derivatives typically varied between 1 and 3, which is in the opti-
mal range for good oral absorption by passive diffusion.
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orally administered to male Wistar rats at 10 lmol/kg. Because all
administered prodrugs showed reasonable good stability in FaSSIF
their conversion to 1 at the absorption should be negligible. Plasma
concentrations of prodrugs were undetectable during the 6-h expo-
sure, which is consistent with prodrug bioconversion by ubiquitous
esterases (7, 8 and 11) or the plasma enzyme paraoxonase (12) after
or during absorption. However, only prodrugs 7 and 12 presented a
very little benefit for the delivery of 1, and that being only a twofold
increase in plasma levels of 1. For example, in contrast to previous
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