Design, synthesis and biological evaluation of peptide derivatives of l-dopa as anti-parkinsonian agents

Article abstract of DOI:10.1016/j.bmcl.2013.08.007

A series of dipeptide derivatives of l-dopa were synthesized and investigated for their pharmacological activity using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an experimental model of Parkinson's disease. Among them, (S)-isopropyl 2-(2-amino-2-methylpropanamido)-3-(3,4- dihydroxyphenyl)propanoate (4g) was found to be the most active compound, with 106% AUC activity and 149% peak activity of l-dopa after oral administration.

Full text of DOI:10.1016/j.bmcl.2013.08.007

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5279–5282
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of peptide derivatives
of L-dopa as anti-parkinsonian agents
b
Tao Zhou a, , Robert C. Hider , Peter Jenner c,d, Bruce Campbell d, Christopher J. Hobbs d, Sarah Rose c,d
,
⇑
Mark Jairaj d, Kayhan A. Tayarani-Binazir d, Alexander Syme d
a School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310035, PR China
b Division of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
c NDRC, School of Biomedical and Health Sciences, King’s College, London SE1 1UL, UK
d Proximagen Neuroscience plc, Hodgkin Building, King’s College, London SE1 1UL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of dipeptide derivatives of
L
-dopa were synthesized and investigated for their pharmacological
Received 14 June 2013
Revised 15 July 2013
Accepted 2 August 2013
Available online 11 August 2013
activity using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an experimental model of
Parkinson’s disease. Among them, (S)-isopropyl 2-(2-amino-2-methylpropanamido)-3-(3,4-dihydroxy-
phenyl)propanoate (4g) was found to be the most active compound, with 106% AUC activity and 149%
peak activity of L-dopa after oral administration.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
L
-Dopa
Prodrug
Parkinson’s disease
6-Hydroxydopamine (6-OHDA) lesioned rat
Parkinson’s disease is a neurodegenerative disorder that is char-
acterized by symptoms affecting smooth and well controlled mus-
cle movement.1 It is an age-related disorder of the central nervous
system primarily attacking people over the age of 60. The disease
results from degeneration of dopaminergic neurons in the substan-
tia nigra, and the resultant decrease in interstitial dopamine levels
leads to alterations in the activity of neural circuits within the ba-
sal ganglia that regulate movement.2 However, peripheral admin-
istration of dopamine cannot represent an useful therapeutic
treatment since dopamine hardly penetrates into the brain being
unable to cross the blood–brain barrier (BBB) because of its hydro-
philicity and the absence of specific transport system within the
endothelial cell membranes. Many treatments have been tried
since James Parkinson first described the condition in 1817. Cur-
rent therapy for Parkinson’s disease is generally by administration
emerge such as extensive metabolism and plasma level
fluctuations.5,6
Numerous attempts have been made to overcome the problem
of low BBB permeability, rapid metabolism and short half-life7–9
,
among which the prodrug approach, commonly used to improve
physicochemical, biopharmaceutical, and drug delivery properties
of therapeutic agents,10–12 has been the most promising.13–16 Con-
jugation of potential CNS-active drugs with nutrients such as ami-
noacids has been demonstrated to lead to active transport across
the BBB, thus improving brain delivery.17,18 Encouraged by these
results and in continuation of our research interest in the develop-
ment of novel prodrugs of
thesized a series of dipeptide derivatives of
L
-dopa,19 in the present study we syn-
-dopa (4a–4i, 5a-5d,
L
6, 7, and 9), and evaluated their pharmacological activity using
the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an
experimental model of Parkinson’s disease.20,21
of dopamine precursor levodopa (L-dopa) or dopaminergic com-
pounds3, which enters the central nervous system through the
large neutral amino acid transporter and is enzymatically cleaved
in the brain to release dopamine.4 However, this treatment is far
In order to avoid the generation of L-dopa from derivatives
while they are present in the lumen of the gut and systematic cir-
culation, non-natural amino acids were employed in dipeptide
construction. Amide bonds formed with non-natural amino acids
are frequently less easily cleaved by peptidases. Lipophilicity is
also an important factor controlling the interaction of drugs with
biological membranes, as the amount of dopamine generated in
from ideal, L-dopa is highly effective in reversing the motor symp-
toms of the illness but on chronic treatment and with disease pro-
gression, its effectiveness declines and a variety of problems may
the brain will depend on the amount of L-dopa peptide crossing
the blood brain barrier. Thus, a range of ester groups were also
introduced into the dipeptide structure.
⇑
Corresponding author. Tel.: +86 571 88071024 7587; fax: +86 571 88905733.
E-mail address: taozhou@zjgsu.edu.cn (T. Zhou).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.08.007

5280
T. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5279–5282
The synthetic route of 4a–4i and 5a–5d is presented in
Scheme 1. Methyl ester 2a was prepared by the treatment of meth-
anol solution of -dopa with thionyl chloride, and benzyl ester 2b
was prepared by the treatment of a solution of -dopa and benzyl
alcohol with 6 M HCl.22 Other
-dopa esters (2c–2e) were prepared
by esterification of -dopa in various alcohol solutions with hydro-
gen chloride in good yield (82–89%). Coupling of various -dopa es-
ter with N-Boc protected amino acids was carried out in the
presence of 1-hydroxybenzotriazole (HOBt) and 1-ethyl-3-(30-
dimethylaminopropyl)carbodiimide (EDC) at room temperature,
providing the protected dipeptide derivatives 3 in moderate to
solution in dioxane, providing final product 9 in 87% yield.27 All the
final compounds were fully characterized by 1H NMR, ESI-MS and
HRMS.
L
L
The 6-OHDA-lesioned rat model has been extensively used to
study dopamine neurons in the brain, to investigate the role of
these dopamine neurons with respect to behaviour, to examine
the brain0s capacity to recover from or compensate for specific neu-
rochemical depletions and to investigate the promotive effects of
experimental and clinical approaches which are relevant for the
treatment of Parkinson0s disease.20,28 In this study, the 6-OHDA-le-
sioned rat model was employed to assess the anti-parkinsonian
L
L
L
good yield (73–87%), which in turn generated
L-dopa dipeptide
activity of the peptide prodrugs and compared with L
-dopa itself.29
derivatives (4a–4i)23 in 86–93% yield by the deprotection of Boc
in 4 M HCl in dioxane. Diacetylation products (5a–5d)24 were ob-
tained in 71–78% yield by treating compounds 3a–3d with acetyl
chloride in HCl-acetic acid solution. In this reaction both acetyla-
tion and deprotection of Boc were carried out in a one-pot
approach.
The synthesis of 6 and 7 is presented in Scheme 2. Compound
4e was subjected to hydrogenation in the presence of palladium/
active charcoal, providing the debenzylated product 6 in 96%
yield.25 Diacetylation of 6 was achieved by treating with acetyl
chloride in HCl–acetic acid solution to provide 7 in 82% yield.26
The synthesis of 9 is presented in Scheme 3. Debenzylation of 3f
was achieved by hydrogenation in the presence of Pd/C to generate
8 in 93% yield, which was subsequently treated with hydrochloride
In this model, Male Wistar rats were treated with desipramine
hydrochloride (25 mg/kg ip, 30 min prior to 6-OHDA) to prevent
the destruction of noradrenergic fibres. The neurotoxin 6-OHDA
was injected into the left median forebrain bundle to produce a
targeted degeneration of midbrain DA neurons in the pars com-
pacta of the substantia nigra. 6-OHDA-induced neurotoxicity
causes a dramatic deficit in DA neurons that is accompanied by
denervation-induced supersensitivity of postsynaptic DA receptors
in the striatum of the lesioned side. An imbalance in DA activity be-
tween the two sides of the brain causes asymmetry in motor
behavior, which can be enhanced by drug treatment. Drugs that
stimulate postsynaptic DA receptors produce an imbalance in DA
signaling that favors the lesion side and induces rotation behavior
(turning) toward the side opposite (contralateral) the lesion side.
O
O
O
HO
HO
R1
R1
O
O
b
a
HO
HO
HO
HO
OH
O
HN
R2
NH2
NH3
Cl
Boc
N
H
R3
3a: R1=R2=R3=Me
1
2a: R1=Me
1
2
3
3b:
3c:
3d:
R =Me, R R =c-Hexyl
R =Me, R R =c-Pentyl
1
2b:
2c:
2d:
R =Bn
1
2 3
1
R =i-Pr
1
2
3
R =Me, R =H, R =c-Hexylmethyl
1
R =c-Hexyl
3e: R1=Bn, R2=R3=Me
2e: R1=i-Pentyl
3f: R1=Bn, R2=Me, R3=Bn
1
2
3
3g:
3h:
3i:
R =i-Pr, R =R =Me
1
2
3
R =c-Hexyl, R =R =Me
1
2
3
R =i-Pentyl, R =R =Me
d
O
HO
HO
R1
c
O
O
O
HN
R2
O
O
Cl
O
O
NH3
R3
HN
R2
O
Cl
4a: R1=R2=R3=Me
O
NH3
R3
4b: R1=Me, R2R3=c-Hexyl
4c: R1=Me, R2R3=c-Pentyl
2
3
5a:
R =R =Me
1
2
3
5b: R2R3=c-Hexyl
5c: R2R3=c-Pentyl
4d:
4e:
R =Me, R =H, R =c-Hexylmethyl
1
2
3
R =Bn, R =R =Me
4g: R1=i-Pr, R2=R3=Me
5d: R2=H, R3=c-Hexylmethyl
4h: R1=c-Hexyl, R2=R3=Me
4i: R1=i-Pentyl, R2=R3=Me
Scheme 1. Reagents and conditions: (a) (i) for 2a: MeOH, SOCl2; (ii) for 2b: BnOH, 6 M HCl; (iii) for 2c–2e: ROH, HCl, reflux, 6 h; (b) N-Boc protected amino acid, HOBt, EDC,
DMF, rt; (c) 4 M HCl in dioxane; (d) acetyl chloride, acetic acid, HCl, 50 °C 1 h, then rt, overnight.
O
O
O
O
O
HO
HO
HO
HO
Bn
O
O
a
OH
O
b
O
O
OH
O
HN
HN
HN
Me
NH3
Cl
NH3
Cl
NH3
Cl
Me
4e
7
6
Scheme 2. Reagents and conditions: (a) H2, 5% Pd/C (10% W/W), MeOH; (b) acetyl chloride, acetic acid, HCl, 50 °C 1 h, then rt, overnight.

T. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5279–5282
5281
O
O
O
HO
HO
Bn
HO
HO
HO
HO
a
O
O
OH
O
b
OH
O
HN
HN
HN
Boc
Boc
N
H
N
H
NH3
Cl
Ph
Ph
Ph
3f
8
9
Scheme 3. Reagents and conditions: (a) H2, 5% Pd/C (10% W/W), MeOH; (b) 4 M HCl in dioxane.
Table 1
Pharmacological analysis results
Prodrugs
Route of administration
AUC activity
Peak activity
Duration of activity
clogP
Rot/test period
As % of
L
-dopa
Rot/10 min
As % of
L
-dopa
Duration (min)
As % of L-dopa
p.o.
ip
522
71
119
160
ꢀ2.20
L
L
-Dopa
-Dopa
1021
104
4a
4b
4c
4d
4e
4g
4h
4i
5a
5b
5c
5d
6
ip
704
459
407
209
240
606
548
412
287
219
204
327
339
219
136
69
88
78
40
80
76
75
38
50
106
58
42
49
39
43
53
57
47
26
77
107
105
53
71
149
82
59
69
55
61
101
100
115
54
45
86
83
58
87
42
52
43
68
54
48
63
84
97
45
38
72
70
49
73
42
44
27
57
45
40
0.324
1.49
p.o.
p.o.
p.o
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
ip
0.984
1.709
1.919
1.063
2.229
1.977
ꢀ0.071
1.096
0.59
1.314
ꢀ1.749
ꢀ2.143
ꢀ0.321
46
116
105
79
55
42
39
32
65
42
26
51
80
66
37
p.o.
p.o.
p.o.
7
9
Vehicle: saline; test period: 240 min; dose: 63.4 lmol/kg.
Most of the prodrugs were administered orally (p.o.), with the
exception of 4a and 5d which were administered intraperitoneally
(ip). The results are presented in Table 1. In 6-OHDA-lesioned rat,
4g was found to be the most active, causing 606 rotations (AUC
activity) during the test period (4 h), which is 106% AUC activity
greater maximal effect, although with a shorter duration of action.
Our findings open up a novel possibility to develop a treatment for
L-dopa-induced dyskinesias. Further investigation on pharamac-
okinetics and in vivo toxicity of 4g will be carried out.
of
(106 rotations in 10 min) was more pronounced than seen with
-dopa (71 rotations in 10 min), namely 149% activity of -dopa.
But the effects of 4g were found to last for the shorter duration
of 86 min, which was only 72% of -dopa duration activity. 4b
and 4c had slightly higher peak activity than that of -dopa, how-
ever they had lower total activity than that of -dopa and were of
shorter or similar duration. Other tested compounds possessed
moderate activity, but the response was not of longer duration
than that observed with L-dopa.
LogP, a parameter of lipophilicity, is the log of the partition
coefficient of a non-charged compound between n-octanol and
aqueous phase and has been widely adopted as one of a number
of important parameters which can lead to the prediction of mem-
brane permeability.30 The clogP value is a useful measure of the
ability of a compound to penetrate biological membranes by sim-
ple diffusion. As the size of the alkyl substituent on the ester func-
tion increase there is a parallel increase in the clogP value
(Table 1).31 Although almost all the synthesized peptide deriva-
L
-dopa (
L
-dopa caused 522 rotations). The peak activity of 4g
References and notes
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tives are more lipophilic than L-dopa, most of them are less active.
Thus there are other factors affecting the pharmaceutical effect.
In conclusion, in the 6-OHDA-lesioned PD rat model, 4g elicited
the most efficient antiparkinsonian effect in the tested dipeptide
derivatives, when administered via the oral route. After oral
administration, this molecule resulted in a rotational response that
17. Polt, R.; Porreca, F.; Szabo‘, L. Z.; Bilsky, E. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91,
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Tayarani-Binazir, K. A.; Syme, A. Eur. J. Med. Chem. 2010, 45, 4035.
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was more than that seen after administration of L-dopa with a

5282
T. Zhou et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5279–5282
22. Bodor, N.; Sloan, K. B.; Higuchi, T.; Sasahara, K. J. Med. Chem. 1977, 20, 1435.
23. Data for 4g: 1H NMR (DMSO-d6)
1.14 (d, J = 6.2 Hz, 3H, CH3), 1.18 (d,
Carroll, K.; Lampron, L.; Westover, L.; Lim, H. K.; Russell, R.; Branum, S.; Wells,
K. M.; Damon, S.; Youells, S.; Li, X.; Beauchamp, D. A.; Rhodes, K.; Jackson, P. F. J.
Med. Chem. 2012, 55, 1402.
d
J = 6.2 Hz, 3H, CH3), 1.38 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.74–2.93 (m, 2H, CH2),
4.35 (m, 1H, CH), 4.88 (m, 1H, CH), 6.48 (d, J = 8.0, 1H), 6.61 (m, 2H), 8.21 (br,
3H, NHþ3 ), 8.59 (d, J = 8.0 Hz, 1H, NH), 8.80 and 8.82 (s, 2H, OH). ESI-MS: m/z
325 (MH+); HRMS: Calcd for C16H25N2O5: 325.1763 (MH+), found: 325.1782.
24. Data for 5d: 1H NMR (DMSO-d6) d 0.87 (m, 2H, CH2), 1.16 (m, 3H), 1.37 (m, 1H),
1.54–1.68 (m, 7H), 2.26 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.99–3.11 (m, 2H, CH2),
3.60 (s, 3H, CH3), 3.83 (t, J = 7.1 Hz, 1H, CH), 4.54 (m, 1H, CH), 7.20 (m, 3H), 8.26
(br, 3H, NHþ3 ), 9.13 (d, J = 7.2 Hz, 1H, NH). ESI-MS: m/z 449 (MH+); HRMS: Calcd
for C23H33N2O7: 449.2288 (MH+), found: 449.2305.
29. Assessment of the induction of rotational activity: At least 1 week after
apomorphine administration, rats (n = 4–8 per treatment) were assessed for
rotational activity with either
L-dopa or a test compounds which were
administered either via the intraperitoneal (ip) route or orally by gavage
(po). Animals were treated with the peripheral decarboxylase inhibitor
benserazide (10 mg/kg) to prevent degradation of
peripheral tissues and placed in rotometers (Med Associates) for up to 30 min
to measure basal rotational activity. They were then treated with -dopa or a
test compound at a single fixed molar equivalent dose (63.4 mmole/kg ip or
po). Rotational behaviour was assessed for up to 4 h after test drug/ -dopa
L-dopa to dopamine in
L
25. Data for 6: 1H NMR (DMSO-d6) d 1.35 (s, 3H, CH3), 1.47 (s, 3H, CH3), 2.72–2.99
(m, 2H, CH2), 4.37 (m, 1H, CH), 6.49 (dd, J = 8.0, 1.8 Hz, 1H), 6.61 (d, J = 8.0 Hz,
1H), 6.64 (d, J = 1.8 Hz, 1H), 8.20 (br, 3H, NHþ3 ), 8.47 (d, J = 8.5 Hz, 1H, NH), 8.80
(br, 2H, OH). ESI-MS: m/z 283 (MH+), HRMS: Calcd for C13H19N2O5: 283.1294
(MH+); found: 283.1302.
L
administration. Animals were typically treated with a series of compounds for
comparative purposes with each treatment being administered at least 1 day
apart.
Data analysis. The number of rotations measured per 10 min over the 4-h
period was determined. Animals were considered active if they turned >10
turns per 10 min. From this data the following parameters were measured:
A: Total activity (AUC activity, where AUC = area under the locomotor activity/
time curve)
26. Data for 7: 1H NMR (DMSO-d6) d 1.32 (s, 3H, CH3), 1.48 (s, 3H, CH3), 2.25 (s, 3H,
CH3), 2.26 (s, 3H, CH3), 2.97–3.21 (m, 2H, CH2), 4.47 (m, 1H, CH), 7.19 (m, 3H),
8.24 (br, 3H, NHþ3 ), 8.68 (d, J = 8.4 Hz, 1H, NH). ESI-MS: m/z 367 (MH+); Calcd
for C17H23N2O7: 367.1505 (MH+); found: 367.1517.
27. Data for 9: 1H NMR (DMSO-d6) d 1.40 (s, 3H, CH3), 2.80 and 2.98 (m, 2H, CH2),
3.09 and 3.30 (d, 2H, J = 14.2 Hz, CH2), 4.45 (m, 1H, CH), 6.52 (dd, J = 8.0, 1.7 Hz,
1H), 6.62 (d, J = 8.0 Hz, 1H), 6.67 (d, J = 1.7 Hz, 1H), 7.27 (m, 3H, Ph), 7.32 (m,
2H, Ph), 8.02 (br, 3H, NHþ3 ), 8.83 (br, 2H, OH), 8.86 (s, 1H, NH). ESI-MS: m/z 359
(MH+); HRMS: Calcd for C19H23N2O5: 359.1607 (MH+); found: 359.1621.
28. Shook, B. C.; Rassnick, S.; Wallace, N.; Crooke, J.; Ault, M.; Chakravarty, D.;
Barbay, J. K.; Wang, A.; Powell, M. T.; Leonard, K.; Alford, V.; Scannevin, R. H.;
B: Peak activity
C: Duration of activity
30. Lipinski, C. A.; Lombardo, F.; Dominy, B. W.; Feeney, P. J. Adv. Drug Deliver. Rev.
1997, 23, 3.
31. http://www.molinspiration.com/cgi-bin/properties.