Synthesis of L-(+)-3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine as potential prodrug of L-dopa

Article abstract of DOI:10.1016/S0960-894X(00)00249-3

The synthesis and in vitro chemical and enzymatic stability of L-(+)-3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine (9) as L-Dopa prodrug are described. Prodrug 9 possesses a good lipophilicity (log P=2.153 ± 0.017), is stable in aqueous buffer solutions (pH 1.3 and 7.4), and in 80% rat and human plasma it is turned into L-Dopa. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00249-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1385±1388
Synthesis of L-(+)-3-(3-Hydroxy-4-pivaloyloxybenzyl)-
2,5-diketomorpholine as Potential Prodrug of L-Dopa
Gian Mario Cingolani,a,* Antonio Di Stefano,b Barbara Mosciatti,a Fabrizio Napolitani,a
Gianfabio Giorgioni,a Massimo Ricciutellia and Francesco Claudia
a
Á
Dipartimento di Scienze Chimiche, Universita di Camerino, 62032 Camerino, Italy
Dipartimento di Scienze del Farmaco, Universita G. D'Annunzio, 66100 Chieti, Italy
b
Á
Received 22 February 2000; accepted 25 April 2000
AbstractÐThe synthesis and in vitro chemical and enzymatic stability of l-(+)-3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketomor-
pholine (9) as l-Dopa prodrug are described. Prodrug 9 possesses a good lipophilicity (log P=2.153 Æ 0.017), is stable in aqueous
bu€er solutions (pH 1.3 and 7.4), and in 80% rat and human plasma it is turned into l-Dopa. # 2000 Elsevier Science Ltd. All
rights reserved.
l-( )-3-(3,4-Dihydroxyphenyl)alanine (l-Dopa, 1), the
immediate biological precursor of dopamine, is still con-
sidered the drug of choice in the treatment of Parkinson's
disease. Substitution therapy with l-Dopa is, however,
associated with a number of acute problems. The drug
undergoes extensive decarboxylation to dopamine by
amino acid decarboxylase (AADC) in the gastrointestinal
tract before entering the systemic circulation and is con-
verted by catechol-O-methyltransferase (COMT) into the
inactive metabolite 3-O-methyldopa before crossing the
blood-brain barrier.
partition, and the high susceptibility to chemical and
enzymatic degradation. In order to improve the bio-
availability the prodrug approach appeared to be the most
promising and some l-Dopa prodrugs have been prepared
in an e€ort to solve these problems.3 6 An ideal prodrug of
l-Dopa should be soluble in water and in lipids, completely
absorbed by the gastrointestinal tract without any chemical
degradation or metabolism, and thus deliver intact l-
Dopa in the blood stream at a reproducible therapeutic
level.
In this study we designed and synthesized the l-3-(3-
hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine (9)
as l-Dopa prodrug. In this compound the carboxylic
and amino groups were masked in the 2,5-diketomor-
pholine skeleton as amide and ester groups, respectively.
The 2,5-diketomorpholine system has been chosen in
order to obtain a lipophilic prodrug with enhanced
absorption and to protect the l-Dopa aminoacidic moi-
ety toward amino acid decarboxylase (AADC). More-
over, enzymatic hydrolysis by esterase and peptidases
could restore l-Dopa. The catechol was protected by the
pivaloyl group considering that the l-3-(3-hydroxy-4-
pivaloyloxyphenyl)alanine produces a sustained l-Dopa
plasma level and large l-Dopa bioavailability after oral
dosing in rats and dogs.7
The peripheral conversion of l-Dopa to dopamine is
responsible for the typical gastrointestinal (nausea,
emesis) and cardiovascular (arrhythmia, hypotension)
side e€ects. To minimize the conversion to dopamine
outside the central nervous system l-Dopa is usually given
in combination with peripheral inhibitors of AADC (car-
bidopa and benserazide). In spite of that other central
nervous side e€ects such as dyskinesia, on-o€ phenom-
enon and end-of-dose deterioration still remain. These
e€ects might be reduced by attenuating peaks and rapid
¯uctuations of l-Dopa plasma levels.1,2
The main factors responsible for the poor bioavailability
and the wide range of inter- and intra-patient variations of
plasma levels are the drug's physical-chemical properties:
low water and lipid solubility, resulting in unfavorable
The synthesis of compound 9 was carried out as shown in
Scheme 1. The N-Boc derivative 2 was transformed into the
benzyl derivative 3. The carboxylic and the amino groups
were deprotected with NaOH and tri¯uoroacetic acid,
respectively. The tri¯uoroacetate salt 5 was acylated
*Corresponding author. Tel.: +39-0737-402267; fax: +39-0737-
637345; e-mail: giemmeci@camserv.unicam.it
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00249-3

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G. M. Cingolani et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1385±1388
with chloroacetylchloride. Cyclization of the N-chloro-
acetyl derivative 6 was accomplished using triethylamine
to give the l-3-(3,4-dibenzyloxy-benzyl)-2,5-diketomor-
pholine 7. By treatment with H2-Pd/C 10% the benzyl
groups were removed. The ®nal conversion of the catechol
8 into 9 was obtained by selective acylation with pivaloyl
chloride.5 In no synthesis step was racemization observed
and optical activity was retained in the ®nal compounds
8 and 9.8
The prodrug 9 was also assayed in vitro to evaluate its
chemical and enzymatic stability. The kinetic of chemical
hydrolysis was studied at 37 ꢁC in aqueous bu€er solu-
tions of pH 1.3 and pH 7.4 (Fig. 1) as well as in 80% rat
and human plasma.10
The results obtained showed pseudo-®rst-order kinetics,
and the decomposition product was the catechol 8,
unequivocally identi®ed by HPLC and NMR analysis.
Lipophilicity is an important factor controlling the
interaction of drugs with biological membranes. It is
generally accepted that good absorption of an orally
administered drug could be obtained when the value of
the octanol/water partition coecient is ꢀ100 (log Pꢀ2)
and the aqueous solubility is more than 10 mg/mL.9 To
assess this potential, the apparent partition coecient
(log P) of the studied prodrug 9 was determined in n-
octanol/phosphate bu€er of pH 7.4. The concentration
of compound 9 in octanol and bu€er layer was deter-
mined by correlating the peak areas in HPLC to a
known concentration of the compound. The mean value
obtained from experiments conducted in triplicate was:
log P=2.153Æ0.017 (ÆSEM; n=3).
Figure 2 shows the time course for disappearance of 9
and for appearance of 8 in phosphate of pH 7.4. The
rate constant (Kobs) for chemical and enzymatic hydro-
lysis and the corresponding half-life times are listed in
Table 1. The hydrolysis of pivaloyl ester moiety in
compound 9 at pH 7.4 is faster than that in bu€er of pH
1.3. The half-life time of 9 at pH 7.4 (t1=2=92.8 min) is
higher than that reported for l-3-(3-hydroxy-4-pivaloy-
loxyphenyl)alanine (t1=2=22.3 min) which seem to be
stable at pH 2.7
Data show that the prodrug 9 is stable in aqueous bu€er
solution of pH 1.3 (t1=2=52.2 h). This stability implies
that the compound passes unhydrolyzed through the
The aqueous solubility was 50 mg/mL. Thus, the data
indicate that 9 possesses the requirements for good oral
absorption.
Figure 1. First-order kinetic plots for hydrolysis of prodrug 9 in
phosphate bu€er of pH 7.4 (&) and in hydrochloric acid bu€er of pH
1.3 (^) at 37 ꢁC.
Scheme 1. Reagents: (a) (t-BuCO2)2O, H2O-dioxane, Et3N, 0 ꢁC, then
rt, 10 h, 95%; (b) C6H5CH2Br, K2CO3, acetone, re¯ux 5 h; (c) NaOH
1N, H2O-dioxane, rt, 24 h; (d) CH2Cl2, CF3COOH, rt, 15 min, 41%;
(e) NaOH, ClCH2COCl, 0 ꢁC, 77%; (f) DMF, Et3N, 100 ꢁC, 12 h,
38%; (g) EtOH, HCl 36%, H2-Pd/C 10%, 20 psi, 40 min, 95%; (h)
CF3COOH, (CH3)3CCOCl, 1.2 equiv, rt, 6 h, 29%.
Figure 2. Time courses for disappearance of 9 (&) and appearance of
8 (^) during hydrolysis in phosphate bu€er of pH 7.4 at 37 ꢁC.

G. M. Cingolani et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1385±1388
1387
Table 1. Kinetic data for chemical and enzymatic hydrolysis of pro-
drug 9 at 37 ꢁC
was observed in rat plasma (t1=2=2.7 min). Hydrolysis
in 80% human plasma proceeds more slowly by a two
steps reaction; at ®rst the prodrug 9 was cleaved to cate-
chol diketomorpholine 8 which afterwards was turned
into l-Dopa.
1 a
Conditions
t1=2 (min)a
Kobs (min )
4
5
4
Bu€er pH 1.3
Bu€er pH 7.4
80% rat plasma
80% human plasma
80% human plasma
3135.7 Æ 22.5
92.8 Æ 1.49
2.7 Æ 0.09
147.6 Æ 2.71c
2.2Â10 Æ 1.1Â10
3
7.5Â10 Æ 1.2Â10
0.29 Æ 0.01
The faster hydrolysis in rat than in human plasma may be
ascribed to the di€erent enzyme systems. It is well repor-
ted that the esterases are highly ecient in rat plasma.11
2
4b
4c
36.7 Æ 1.1b
1.9Â10 Æ 3.6Â10
3
4.7Â10 Æ 1.4Â10
aValues represent the meanÆSEM; n=3.
bt1=2 and Kobs for the hydrolysis of the prodrug 9 to 8.
ct1=2 and Kobs for the hydrolysis of 8 to l-Dopa.
In conclusion, we designed and synthesized l-(+)-3-(3-
hydroxy-4-pivaloyloxybenzyl)-2,5-diketomorpholine (9)
as l-Dopa prodrug. The present ®ndings indicate that
prodrug 9 meets the requirements for gastrointestinal
absorption, shows good stability toward gastrointestinal
hydrolysis, and releases l-Dopa in human plasma after
enzymatic hydrolysis.
stomach after oral administration. At pH 7.4 the pro-
drug is stable enough (t1=2=1.5 h) to be absorbed intact
from the intestine.
Figures 3 and 4 show the plots of pseudo-®rst-order
kinetics for the hydrolysis in 80% rat and human
plasma at 37 ꢁC; the rate constants (Kobs) and the cor-
responding half-life times are shown in Table 1. In rat
and human plasma both the catechol ester and diketo-
morpholine ring of the prodrug 9 were cleaved and l-
Dopa was formed. A rapid conversion of 9 to l-Dopa
The 2,5-diketomorpholine ring seems to be a useful
system in the design of l-Dopa prodrugs. Further stu-
dies are in progress to investigate the bioavailability
and to evaluate the in vivo pharmacological activity of
this prodrug.
Acknowledgements
This work was supported by the Ministero della Uni-
versita e della Ricerca Scienti®ca e Tecnologica (Progetto
Nazionale Tecnologie Farmaceutiche). The authors
thank Prof. M. Cardellini for his collaboration.
References and Notes
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Figure 3. First-order kinetic plot for hydrolysis of prodrug 9 in 80%
rat plasma at 37 ꢁC.
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ter:methanol (60:40.5, v/v/v), (ee>97%); see ref 12. l-(+)-3-
(3,4-dihydroxybenzyl)-2,5-diketomorpholine 8: oil, puri®ed by
column chromatography with AcOEt as eluent, yield 95%,
Rf=0.33, [a]2d0=+14.28 (c=1, EtOH abs), H NMR (DMSO-
1
d6) d 8.80 (br s, 1H, OH), 8.70 (br s, 1H, OH), 7.72 (d, 1H, NH),
6.60 (m, 2H, ArH), 6.42 (m, 1H, ArH), 4.47 (m, 1H, CH-N), 3.80
(s, 2H, CH2CO), 2.84 (d, 2H, ArCH2). MS m/e 237 (M+). l-(+)-
Figure 4. Times courses for hydrolysis of prodrug 9 (&) to l-Dopa (&)
via the intermediate formation of 8 (~) in 80% human plasma at 37ꢁC.

1388
G. M. Cingolani et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1385±1388
3-(3-hydroxy-4-pivaloyloxybenzyl)-2,5-diketo-morpholine 9: oil,
puri®ed by column chromatography with AcOEt as eluent, yield
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32%, Rf=0.45, [a] 20=+9.46 (c=1, EtOH abs), 1H NMR
d
(DMSO-d6) d 9.57 (br s, 1H, OH), 7.88 (m, 1H, NH), 6.76 (m,
3H, ArH), 4.50 (m, 1H, CH-N), 3.82 (m, 2H, CH2CO), 2.98
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