Design, synthesis and biological evaluation of l-dopa amide derivatives as potential prodrugs for the treatment of Parkinson's disease

Article abstract of DOI:10.1016/j.ejmech.2010.05.062

A range of amide derivatives of l-dopa were synthesized and investigated for their pharmacological activity and their ability to be converted to l-dopa using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat, as an experimental model of Parkinson's

Full text of DOI:10.1016/j.ejmech.2010.05.062

European Journal of Medicinal Chemistry 45 (2010) 4035e4042
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of
L
-dopa amide derivatives
as potential prodrugs for the treatment of Parkinson’s disease
,
,
c
,
,
**
Tao Zhou a b, Robert C. Hider b , Peter Jenner 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, P. R. 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 range of amide derivatives of
activity and their ability to be converted to
L
-dopa were synthesized and investigated for their pharmacological
-dopa using the unilaterally 6-hydroxydopamine (6-OHDA)-
lesioned rat, as an experimental model of Parkinson’s disease. The diacetyl derivative of -dopa amide
(11b) was found to be more active than -dopa after its oral administration and generated plasma levels
of -dopa in the therapeutic range for an antiparkinsonian effect in man.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Received 26 March 2010
Received in revised form
25 May 2010
Accepted 26 May 2010
Available online 2 June 2010
L
L
L
L
Keywards:
L-dopa
Dopamide
Parkinson’s disease
Prodrug
6-hydroxydopamine (6-OHDA) lesioned rat
1. Introduction
peaks and troughs in its plasma levels that leads to pulsatile
stimulation of striatal post-synaptic dopamine receptors [7]. This
Parkinson’s disease (PD) is a common neurodegenerative
disorder resulting in motor disability, primarily associated with
progressive degeneration of pigmented neurones in the sub-
stantia nigra pars compacta, resulting in a reduction of striatal
dopamine levels and the formation of intracellular proteinous
inclusions, termed Lewy bodies mainly composed of fibrillar
coupled to the rapid metabolism of L-dopa and its short plasma
half-life causes a non-physiological response in the basal ganglia
that causes the motor complications that make the later treat-
ment of PD complex.
Numerous attempts have been made to overcome the problem
of erratic absorbtion, rapid metabolism and short half-life, among
which prodrug design has been the most promising [8]. Recent
examples have included amide derivatives linked to glutathione or
other antioxidants but where the objective was to achieve
a symptomatic effect in PD associated with modification of the
disease progression through inhibition of oxidative stress [9,10].
a
-synuclein [1,2]. Dopamine replacement therapy using L-dopa or
dopamine agonist drugs, such as ropinirole and pramipexole, is
the mainstay of treatment resulting in an initial normalisation of
motor function [3,4].
L-dopa is the most effective of these
medications and all patients require its use at some point in the
course of the illness. However, on chronic use and with disease
These derivatives did not, however, lead to the release of
a manner that was more prolonged than that achieved by
itself. Indeed, simple amide derivatives made hold more promise
and recently activity was reported with a class of -dopamides [11].
But to date, there has been no detailed investigation of the struc-
ture-activity relationship of -dopa amide derivatives. We report
herein the pharmacological and pharmacokinetic behaviour of
series of -dopamide prodrugs in the 6-hydroxydopamine
L-dopa in
progression, the effects of
L
-dopa become shorter (wearing-off),
L-dopa
unpredictable (on-off) and involuntary movements termed
dyskinesia can appear [5,6]. Wearing off and dyskinesia are in
part due to the erratic absorption of L-dopa from the gut and
L
L
* Corresponding author. Tel.: þ44 207 8484882; fax: þ44 207 8484800.
** Corresponding author. Tel.: þ44 207 8488177; fax: þ44 207 8486034.
E-mail addresses: robert.hider@kcl.ac.uk (R.C. Hider), chris.hobbs@proximagen.
com (C.J. Hobbs).
a
L
(OHDA)-lesioned rat that acts as a functional model of drug effect in
PD [12].
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.062

4036
T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
2. Results
acetyl group as it is sensitive to strong acid conditions. When
Boc-protected diacetyl -dopa amide is treated with hydrochloric
L
2.1. Chemistry
acid in dioxane, the acetyl groups are partially hydrolysed, leading
to a product of low purity.
In order to investigate the inference of different substitutents on
the amide and catechol functions, on the pharmacological proper-
Conversion of the carboxylic acid function of L-dopa to the
simple amide (10b) led to a marked increase in the clogP value. The
clogP value is a useful measure of the ability of a compound to
penetrate biological membranes by simple diffusion. LogP 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 membrane permeability [14]. Thus the marked
ties on
-dopamide derivatives (9a-9e, 10b-10g, 11b-11f). A range of pro-
tected amide derivatives of -dopa (6a-6g) were synthesized as
shown in Scheme 1. The methyl ester 2 was obtained by reaction of
-dopa with thionyl chloride in methanol. The protection of the
L-dopamide, we have designed and synthesized a series of
L
L
L
amino group of 2 was achieved by treating 2 with either di-tert-
butyl-dicarbonate or benzyl chloroformate to obtain 3a or 3b.
Further protection of the hydroxyl groups on 3a and 3b was ach-
ieved by treatment with benzyl bromide in the presence of potas-
sium carbonate resulting in 4a and 4b, which generated acids 5a
and 5b, respectively, on hydrolysis in lithium hydroxide. Coupling
of 5a or 5b with various amines was carried out in the presence of
N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N0-
ethylcarbodiimide hydrochloride (EDC) at room temperature,
increase of the clogP value between L-dopa and the simple amide
10b would suggest improved membrane permeability of the latter
molecule. As the size of the alkyl substituent on the amide function
increase there is a parallel increase in the clogP value [15] (Table 1).
A surprising feature of this study is that in every case the conver-
sion of both the catechol hydroxyl functions to acetyl groups leads
to an increase in hydrophilicity (that is a decrease in clogP value
(Table 1). Thus the introduction of the two acetyl functions is
unlikely to enhance the membrane penetration of these molecules
by non facilitated transport.
providing a range of protected amide derivatives of -dopa (6ae6g).
L
The advantages of using the coupling conditions in this reaction
are high yield, simplicity of manipulation and ease of product
purification.
2.2. Pharmacological evaluation
As
L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine
produces
a sustained
L
-dopa plasma level and large -dopa bioavailability
L
The potential anti-parkinsonian activity of
drugs was assessed in the unilaterally 6-OHDA-lesioned rat where
the administration of -dopa leads to a marked contraversive
rotational response. -Dopa and the -dopamides 10b, 10c, 10d, 10g
were administered both orally and intraperitoneally (ip). In
6-OHDA-lesioned rats, 10b was found to be the most active, with
oral administration being less effective than ip administration.
L-dopa and the pro-
after oral dosing in rats and dogs [13], the pivaloyl group and
several other groups possessing similar lipophilicity were
employed to protect the hydroxyl groups on catechol. The synthesis
L
L
L
of 3,4-diacyl L-dopa amide is presented in Scheme 2. Debenzylation
of 6a was achieved by hydrogenation in the presence of Pd/C to
generate 7. Acylation of catechol hydroxyl functions on 7 was
carried out by treatment with a series of acyl chlorides to form
8ae8e, which when subjected to deprotection of the Boc group in
hydrogen chloride solution provided the final products 9ae9e.
Hydrogenation of 6be6g in the presence of Pd/C and hydro-
Activity was more pronounced than seen with L-dopa but was of
similar duration. 10c had very low activity when administered by
both routes and was of short duration, and 10d possessed moderate
activity but this was less intense and of shorter duration than seen
with L-dopa. In contrast good activity was associated with the ip
administration of 10g, in fact much higher activity than experi-
enced following oral administration of 10g but the response was
chloric acid provided the hydrochloride salts of the L-dopa amide
derivatives 10be10g. Diacetylation of 10be10f was carried out by
treatment with acetyl chloride in the presence of hydrogen chloride
to produce 11be11f, respectively (Scheme 3). This step was used for
not of longer duration than occurred with L-dopa.
O
P
O
O
HO
HO
HO
HO
a
HO
HO
b
O
O
OH
HN
NH3
Cl
NH2
3a: P =Boc
3b: P =Cbz
2
1
O
P
O
P
BnO
BnO
BnO
BnO
e
OH
O
c
d
HN
HN
5a: P =Boc
5b: P =Cbz
4a: P =Boc
4b: P =Cbz
6a: P = Boc, R = R' = H
6b: P = Cbz, R = R' = H
O
6c: P = Cbz, R = H, R' = Me
6d: P = Cbz, R = H, R' = Et
6e: P = Cbz, R = H, R' = i-Pr
6f: P = Cbz, R = R' = Me
BnO
BnO
NRR'
HN
P
6g: P = Cbz, R = H, R' = CH2CH2Ph
Scheme 1. Reagents and conditions: (a) MeOH, SOCl2, ꢀ5 ꢁC then reflux; (b) (Boc)2O, or CbzCl, DMF, 0 ꢁC; (c) BnBr, K2CO3, acetone; (d) LiOH, THF/MeOH; (e) i) NHS, EDC, ii) RR0NH.

T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
4037
O
treatment of Parkinson’s disease.[12] The injection of 6-OHDA
selectively damage mesencephalic dopamine neurons on one side
of the brain, causing the reduce of dopamine at the ipsilateral side
as the injection side of 6-OHDA. This leads to ipsiversive turning.
The dopamine receptor agonist apomorphine can induce asym-
metries in turning. This contraversive response is attributed to the
stimulation of supersensitive dopamine receptor mechanisms,
especially at the level of the dopamine-depleted neostriatum. In
animals with unilateral 6-OHDA lesions, the behavioural effects of
O
BnO
BnO
HO
HO
b
NH2
Boc
a
NH2
Boc
HN
HN
6a
7
R''
R''
O
R''
R''
O
O
O
O
O
O
O
O
NH2
c
NH2
Boc
L
-dopa are similar to those of the unselective dopamine receptor
agonist apomorphine, as -dopa generates dopamine and
compensates the loss of dopamine at the damaged side after it
NH3
Cl
HN
L
O
enters brain. Thus, the systemic administration of
induces intense contraversive turning[16].
L-dopa acutely
8a: R'' =C(CH3)3
8b: R'' =CH2CH(CH3)2
8c: R'' =C(CH3)2CH2CH3
8d: R'' =CH2C(CH3)3
8e: R'' =OCH2CH3
9a: R'' =C(CH3)3
9b: R'' =CH2CH(CH3)2
9c: R'' =C(CH3)2CH2CH3
9d: R'' =CH2C(CH3)3
9e: R'' =OCH2CH3
The results show that some of the dopamide derivatives
synthesized in this study are converted to -dopa in the 6-OHDA
L
lesioned rat in sufficient quantity to produce a behavioural
response. This is significant as this experimental model of PD is
highly predictive of drug effect in man. Compound 11b in particular
showed activity after oral administration that resulted in a greater
Scheme 2. Reagents and conditions: (a) 30 psi H2, Pd/C, EtOAc/MeOH; (b) R00COCl,
Et3N, CH2Cl2, 0 ꢁC; (c) HCl-dioxane, CH2Cl2.
pharmacological response and prolonged plasma levels of
compared to a molar equivalent dose of -dopa.
It is clear that the acetyl and unsubstituted amide functions
were rapidly cleaved, leading to the appearance of -dopa in
plasma. As there was a high yield of -dopa generated when 10b
L-dopa
L
In general there was lower activity observed with acylated
catechol derivatives both in terms of the overall rotational response
and the duration of effect, with the exception of 11b which was
active when administered by either the oral or intraperitoneal
routes and showed a response that was equivalent to or greater
L
L
was administered via the ip route there must be high peptidase
activity in the serum, which is capable of cleaving the unsubstituted
than that produced by -dopa. 11ce11f were all found to be largely
L
amide. In contrast, only low levels of -dopa resulted when 10c and
L
devoid of activity. The dicarbonate derivative of 10b (9e) was found
to be active, although with a lower activity than that of the corre-
sponding diacetyl derivative (Table 1).
10d were administered via ip route. This difference is probably
linked with the inability of the N-alkylated prodrugs to be cleaved
in the plasma [17,18]. As 11b produces a prolonged plasma t for
½
Although
accepted that the therapeutic effects of
correlated with the blood -dopa concentration resulting from
cleavage of the prodrug, as the amount of dopamine generated in
the brain depends on the amount of -dopa crossing the blood brain
barrier. Thus, we determined the -dopa concentration in the blood
L-dopa itself is a prodrug of dopamine, it is generally
L-dopa compared to 10b when both are administered via the oral
L-dopa prodrugs can be
route, there appears to be a beneficial effect in acetylating both the
phenolic functions. Presumably this is associated with the
increased ability of 11b to permeate the gut and to enter the general
circulation. Although double acetylation fails to increase the clogP
value (Table 1), the decreased hydrogen bonding potential of the
diesters, when compared with the H-donating catechol function is
probably responsible for the enhanced absorption of this molecule.
L
L
L
at various times after administration of equivalent doses of some of
the compounds. The results are summarised in Table 2. 10b and 11b
gave the largest AUC and Cmax values but only 11b had an extended
As the pKa value of the a-amine function is 8.9, there will always be
t1/2 compared to
phobic ester protecting groups (9a, 9b and 9e) gave rise to less
-dopa than the corresponding acetyl compound (11b) (Table 2).
L-dopa. The compounds with the more hydro-
an appreciable portion of the neutral form of 11b present in the
lumen of the intestine. The acetyl ester functions have been
demonstrated to be stable at both acid and neutral pH values (data
not presented) and are therefore likely to survive exposure to the
lumen of the stomach and small intestine, whereas in the serum
L
3. Discussion
the diacetyl derivative (11b) is rapidly converted to
the exception of 11b, none of the other diacetyl prodrugs investi-
gated in this study led to appreciable blood levels of -dopa after
oral administration. This confirms the suggestion that the N-alky-
lated amide prodrugs are not cleaved in plasma, even though they
are predicted to be absorbed from the gut.
L-dopa. With
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
brain’s capacity to recover from or compensate for specific neuro-
chemical depletions, and to investigate the promotive effects of
experimental and clinical approaches which are relevant for the
L
O
O
O
O
O
O
HO
HO
BnO
BnO
NRR'
NRR'
Cl
b
NRR'
Cbz
a
NH3
Cl
NH3
HN
10b: R = R' = H
6b-6g
O
10c: R = H, R' = Me
10d: R = H, R' = Et
10e: R = H, R' = i-Pr
10f: R = R' = Me
11b: R = R' = H
11c: R = H, R' = Me
11d: R = H, R' = Et
11e: R = H, R' = i-Pr
11f: R = R' = Me
10g: R = H, R' = CH2CH2Ph
Scheme 3. Reagents and conditions: (a) 30 psi H2, Pd/C, HCl, EtOAc/MeOH; (b) HOAc-HCl, AcCl.

4038
T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
Table 1
PD analysis.
Prodrugs
Route of
administration
Dose
AUC activity
(rot/test period)
Maximum activity
(rot/10 min)
Duration of activity
(min)
clogP
(mg/kg)
(mmol/kg)
L
-Dopa
-Dopa
p.o.
i.p.
p.o.
i.p.
p.o.
i.p.
p.o.
i.p.
p.o.
i.p.
p.o.
i.p.
p.o.
p.o.
p.o.
p.o.
p.o.
12.5
12.5
63.39
63.39
63.35
63.35
63.66
63.66
63.65
63.65
63.58
63.58
63.46
63.46
63.40
63.40
63.40
63.26
63.40
522
1021
612
1186
29
71
104
105
144
5
119
160
103
155
10
0
39
18
30
124
147
140
0
ꢀ2.20
ꢀ0.70
ꢀ0.88
ꢀ0.51
0.92
L
10b
10b
10c
10c
10d
10d
10g
10g
11b
11b
11c
11d
11e
11f
9e
14.74
14.74
15.64
15.64
16.53
16.53
21.35
21.35
20.1
16
2
324
117
94
825
1085
838
26
20
26
26
579
39
40
17
124
107
69
1
6
2
1
90
ꢀ1.09
20.1
20.97
21.86
22.75
21.81
23.89
ꢀ1.28
ꢀ0.90
ꢀ0.60
ꢀ1.47
0.37
3
0
0
153
Vehicle: saline; test period: 240 min.
It is assumed on the basis of PD analysis that 10g did not
generate high levels of blood -dopa when administered orally but
did so when administered by the ip route. The reason for the poor
were obtained by infusing samples into an LCQ Deca XP ion trap
instrument. High resolution mass spectra (HRMS) were obtained on
a QTOF Micro by direct infusing samples into the EI source.
L
absorption from the intestine is not clear, particularly as 10g has
a relatively high logP value. Generation of high levels of
L
-dopa of
4.1.1. Preparation of (S)-3-(3,4-dihydroxyphenyl)-1-methoxy-1-
oxopropan-2-aminium chloride (2)
this molecule, when administered via the ip route, indicates that
there is a serum peptidase capable of cleaving the aryl substituted
amide function.
To methanol (50 mL) cooled to e5 ꢁC was added thionyl chloride
(5 mL) slowly and the temperature was kept under 10 ꢁC.
L-dopa
Although the dicarbonate (9e) results in high
not as efficient as the corresponding diacetyl derivative (11b).
Introduction of a more hydrophobic ester group on the catechol
L
-dopa levels, it is
(10 g, 50.7 mmol) was then added in portions. The solution was
slowly warmed to room temperature and then stirred at 50 ꢁC
overnight. After removal of the solvent, the residue was redissolved
in methanol, and titratated with diethyl ether, and stored at 4 ꢁC.
The product 2 was obtained as a white solid by filtration, 11.6 g (92%
function reduces the rate of conversion to
The most efficient prodrug of the present series for producing
more prolonged blood levels of -dopa, when administered via the
oral route, is the diacetyl derivative of the simple -dopa amide
L-dopa in the serum.
L
yield). 1H NMR (DMSO-d6)
d 2.90e3.03 (m, 2H), 3.64 (s, 3H), 4.10
L
(m, 1H), 6.45 (m, 1H), 6.61 (m, 1H), 6.69 (m, 1H), 8.61 (br, 3H), 8.97
(11b). After oral administration, this molecule resulted in a rota-
(s, 1H), 9.00 (s, 1H). ESI-MS: m/z 212 ([MeCl]þ).
tional response that was double that seen after administration of
L
-dopa with a greater maximal effect and a longer duration of
action. The characteristics of the compound are favourable when
compared to the absorption of orally administered -dopa, which in
the same experimental system is about 50% of that corresponding
to 11b (Table 1). The resulting levels of -dopa in plasma at Cmax are
4.1.2. Preparation of (S)-methyl 2-(tert-butoxycarbonyl)-3-(3,4-
dihydroxyphenyl)propanoate (3a)
L
To a solution of 2 (11 g, 44.4 mmol) in THF (80 mL) was added
saturated aqueous NaHCO3 (80 mL). The resulting solution was
cooled with ice-bath and di-tert-butyl dicarbonate (10.66 g in
50 mL THF, 48.84 mmol) was added dropwise. The reactant was
allowed to warm to room temperature and stirred for 1 h. The
organic solvent was removed and the aqueous layer was extracted
with dichloromethane (2 ꢂ 60 mL). The combined organic layers
were washed subsequently with water, aqueous KHSO4, brine, and
dried over Na2SO4. After removal of the solvent, the residue was
purified by silica gel chromatography using ethyl acetate/hexane
(1:1) as an eluent to afford product 3a as a white solid (13.0 g, 94%).
L
in the therapeutic range known to result in an antiparkinsonian
response in man.
4. Experimental
4.1. General
All chemicals were purchased from Aldrich or Fluka and were used
withoutfurtherpurification.1H NMR Spectrawere recorded on Bruker
Avance 400 spectrometer using CDCl3 or DMSO-d6 as the solvent with
TMSasaninternalstandard.Electrosprayionization(ESI)massspectra
1H NMR (DMSO-d6)
d 1.34 (s, 9H), 2.63e2.80 (m, 2H), 3.59(s, 3H),
4.04 (m, 1H), 6.45 (m, 1H), 6.60 (m, 2H), 7.18 (d, J ¼ 8.0 Hz, 1H, NH),
8.75 (br, 2H, OH). ESI-MS: m/z 312 (MHþ).
Table 2
Pharmacokinetic analysis result.
Compound
Route of
admin.
Dose
(mg/kg)
Dose
mol/kg)
L
-dopa Cmax
L
-dopa t1/2 apparent
L
-dopa AUC (0e4 h)
(
m
(ng/ml)
(min)
(mg.min/ml)
L
-dopa
p.o.
p.o.
p.o.
p.o.
p.o.
p.o.
12.5
14.8
20.1
42.5
25.4
23.9
63.5
63.5
63.5
106
63.5
63.5
4088
2203
1746
415
860
690
75
52
97
51
78
ND
434
256
245
44
98
95
10b
11b
9a
9b
9e

T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
4039
4.1.3. Preparation of (S)-methyl 2-(benzyloxycarbonyl)-3-(3,4-
dihydroxyphenyl)propanoate (3b)
4.1.6. General preparation of dibenzyl protected
derivatives 6ae6g
L-dopa amide
To a solution of 2 (15.6 g, 63.0 mmol) in anhydrous DMF
(100 mL) was added Et3N (14.0 g, 138.6 mmol). The solution was
cooled on an ice-bath and benzyl chloroformate (11.8 g, 69.3 mmol)
was added in three portions, stirring was continued at 0 ꢁC for 3 h.
Cold aqueous HCl (500 mL,1.5 M) was added to the reactant and the
resulting solution was extracted with diethyl ether (2 ꢂ 400 mL).
The combined organic layer was washed with brine and dried over
Na2SO4. After removal of the solvent, the crude product 3b was
obtained as a colorless oil (21.2 g, 97%) which was used in next
A mixture of 5a or 5b (10.26 mmol), NHS (1.42g, 12.32 mmol),
EDC (2.36g, 12.32 mmol) and CH2Cl2/DMF (3:1, 140 mL) was stirred
at room temperature overnight. NH3 solution (0.5 M in dioxane,
24.65 mL) or other amine (1.2 equiv) was then added with
continued stirring for 4 h. The reactant was diluted with CHCl3, and
washed with saturated NaHCO3, brine, and dried over Na2SO4. After
removal of the solvent, the residue was washed with cold meth-
anol/diethyl ether to afford product as a white solid.
reaction without further purification. 1H NMR (CDCl3)
d
2.96 (m, 2H,
4.1.6.1. (S)-tert-butyl 3-(3,4-bis(benzyloxy)phenyl)-1-amino-1-oxo-
CH2), 3.71 (s, 3H, OCH3), 4.60 (m, 1H, CH), 5.09 (s, 2H, CH2), 5.27
(m,1H, NH), 6.49 (d, J ¼ 8.0,1H, 6-ArH), 5.93 (br, 2H, OH), 6.59(s,1H,
2-ArH), 6.72 (d, J ¼ 8.0 Hz, 1H, 5-ArH), 7.29e7.42 (m, 5H, PhH).
ESI-MS: m/z 346 (MHþ).
propan-2-ylcarbamate (6a). Yield 96%; 1H NMR (CDCl3)
d 1.42 (s,
9H, Boc-CH3), 2.84e2.89 and 3.01e3.06 (m, 2H, CH2), 4.23 (m, 1H),
5.08 (m, 2H, NH2), 5.14 and 5.16 (s, 4H, CH2), 5.48 (br, 1H, NH), 6.72
(d, J ¼ 8.1, 1H, 6-ArH), 6.81 (s, 1H, 2-ArH), 6.86 (d, J ¼ 8.1 Hz, 1H,
5-ArH), 7.28e7.45 (m, 10H, PhH). ESI-MS: m/z 477 (MHþ).
4.1.4. General procedure for dibenzylation of catechol hydroxyl
groups
4.1.6.2. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-amino-1-oxopro-
A mixture of 3a or 3b (27 mmol), K2CO3 (11.2 g, 81 mmol) and
benzyl bromide (13.85 g, 81 mmol) in acetone (200 mL) was
refluxed under nitrogen for 3 days, cooled and filtered. The filtrate
was concentrated and the residue was then dissolved in dichloro-
methane (100 mL). The solution was washed with water
(2 ꢂ 100 mL), brine (100 mL), dried over Na2SO4, filtered and
concentrated. The residue was washed with cold methanol to
afford 4a or 4b as white solids.
pan-2-ylcarbamate (6b). Yield 93%; 1H NMR (CDCl3)
d 2.82e2.88
and 3.05e3.09 (m, 2H, CH2), 4.28 (m, 1H, CH), 5.08 (br, 1H, NH), 5.09
and 5.14 (s, 6H, CH2), 5.32 (m, 2H, NH2), 6.70 (d, J ¼ 8.0 Hz, 1H,
6-ArH), 6.79(s, 1H, 2-ArH), 6.85 (d, J ¼ 8.1 Hz, 1H, 5-ArH), 7.29e7.45
(m, 15H, PhH). ESI-MS: m/z 511 ([MþH]þ), 533 ([MþNa]þ).
4.1.6.3. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-(methylamino)-1-
oxopropan-2-ylcarbamate (6c). Yield 93%; 1H NMR (CDCl3)
d 2.60
(d, J ¼ 4.9 Hz, 3H, CH3), 2.82e2.88 and 3.02e3.07 (m, 2H, CH2), 4.22
(m, 1H, CH), 5.08 and 5.13 (s, 6H, CH2), 5.32 (m, 1H, NH), 5.40(m, 1H,
NH), 6.65 (d, J ¼ 7.9 Hz, 1H, 6-ArH), 6.79(s, 1H, 2-ArH), 6.83 (d,
J ¼ 8.1 Hz, 1H, 5-ArH), 7.28e7.44 (m, 15H, PhH). ESI-MS: m/z 525
([MþH]þ), 547 ([MþNa]þ).
4.1.4.1. (S)-methyl 3-(3,4-bis(benzyloxy)phenyl)-2-(tert-butoxycarbonyl)
propanoate (4a). Yield 92%; 1H NMR (CDCl3)
d 1.42 (s, 9H, Boc-CH3),
2.98 (m, 2H, CH2), 3.64 (s, 3H, OCH3), 4.52 (m, 1H, CH), 4.94 (d,
J ¼ 8.2 Hz, 1H, NH), 5.121 and 5.125 (s, 4H, CH2), 6.64 (d, J ¼ 8.1 Hz,
1H, 6-ArH), 6.73(d, J ¼ 1.8 Hz, 1H, 2-ArH), 6.85(d, J ¼ 8.1 Hz, 1H,
5-ArH), 7.29e7.45 (m, 10H, PhH). ESI-MS: m/z 492 (MHþ).
4.1.6.4. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-(ethylamino)-1-oxo-
propan-2-ylcarbamate (6d). Yield 90%; 1H NMR (CDCl3)
d 0.89 (t,
4.1.4.2. (S)-methyl 3-(3,4-bis(benzyloxy)phenyl)-2-(benzyloxycarbonyl)
J ¼ 7.2 Hz, 3H, CH3), 2.79e2.85 (m, 1H, DOPA-CH2), 3.03e3.16 (m,
3H, 2H from CH2 and 1H from DOPA-CH2), 4.18 (m,1H, CH), 5.10 and
5.13 (s, 6H, CH2), 5.24 (m, 1H, NH), 5.34(m, 1H, NH), 6.66 (d,
J ¼ 8.2 Hz,1H, 6-ArH), 6.83 (m, 2H, 2-ArH and 5-ArH), 7.28e7.44 (m,
15H, PhH). ESI-MS: m/z 539 ([MþH]þ), 561([MþNa]þ).
propanoate (4b). Yield 87%; 1H NMR (CDCl3)
d 3.00 (m, 2H, CH2),
3.64 (s, 3H, OCH3), 4.60 (m, 1H, CH), 5.08, 5.10 and 5.12 (s, 6H, CH2),
5.16 (d, J ¼ 8.2 Hz, 1H, NH), 6.60 (dd, J ¼ 8.2 and 1.9 Hz, 1H, 6-ArH),
6.69(s, 1H, 2-ArH), 6.83(d, J ¼ 8.2 Hz, 1H, 5-ArH), 7.28e7.44 (m, 15H,
PhH). ESI-MS: m/z 526 (MHþ).
4.1.6.5. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-(isopropylamino)-
4.1.5. General procedure for hydrolysis of 4a and 4b
1-oxopropan-2-ylcarbamate (6e). Yield 85%; 1H NMR (CDCl3)
d 0.84
To a solution of 4a or 4b (23.76 mmol) in THF/MeOH (3:1,
160 mL) was added aqueous LiOH (2 M, 35.6 mL). The solution was
stirred at room temperature for 3 h and then water (300 mL) was
added. The resulting solution was acidified to pH 2e3 with aqueous
KHSO4 and extracted with CH2Cl2 (3 ꢂ 300 mL). The combined
organic extracts were washed with water, brine, dried over Na2SO4.
After removal of the solvent, the product was obtained as a white
solid.
(d, J ¼ 6.5 Hz, 3H, CH3), 0.98 (d, J ¼ 6.5 Hz, 3H, CH3), 2.77e2.83 and
3.03e3.09 (m, 2H, CH2), 3.84e3.93(m,1H, CH), 4.15 (m,1H, CH), 5.06
(d, J ¼ 8.0 Hz,1H, NH), 5.10 and 5.13 (s, 6H, CH2), 5.39(m,1H, NH), 6.66
(d, J ¼ 7.9 Hz, 1H, 6-ArH), 6.83 (d, J ¼ 8.1 Hz, 2H, 2-ArH and 5-ArH),
7.27e7.43 (m,15H, PhH). ESI-MS: m/z 553 ([MþH]þ), 575([MþNa]þ).
4.1.6.6. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-(dimethylamino)-
1-oxopropan-2-ylcarbamate (6f). Yield 94%; 1H NMR (CDCl3)
d 2.43
(s, 3H, CH3), 2.76(s, 3H, CH3), 2.79-2.85 and 2.91-2.96 (m, 2H, CH2),
4.77 (m, 1H, CH), 5.08 (d, J ¼ 2.1 Hz, 2H, CH2), 5.10 and 5.13 (s, 4H,
CH2), 5.66 (d, J ¼ 8.6 Hz, 1H, NH), 6.67 (dd, J ¼ 8.1 and 1.8 Hz, 1H,
6-ArH), 6.80 (d, J ¼ 1.8 Hz,1H, 2-ArH), 6.83 (d, J ¼ 8.1 Hz,1H, 5-ArH),
7.29e7.46 (m, 15H, PhH). ESI-MS: m/z 539 ([MþH]þ), 561
([MþNa]þ).
4.1.5.1. (S)-3-(3,4-bis(benzyloxy)phenyl)-2-(tert-butoxycarbonyl)
propanoic acid (5a). Yield 97%; 1H NMR (CDCl3)
d 1.42 (s, 9H, Boc-
CH3), 2.97-3.05 (m, 2H, CH2), 4.52 (m,1H, CH), 4.90 (d, J ¼ 7.3 Hz,1H,
NH), 5.11 (s, 4H, CH2), 6.68 (d, J ¼ 8.1 Hz, 1H, 6-ArH), 6.78(s, 1H,
2-ArH), 6.85(d, J ¼ 8.1 Hz, 1H, 5-ArH), 7.29e7.44 (m, 10H, PhH).
ESI-MS: m/z 478 (MHþ).
4.1.6.7. (S)-benzyl 3-(3,4-bis(benzyloxy)phenyl)-1-oxo-1-(phenethyl-
4.1.5.2. (S)-3-(3,4-bis(benzyloxy)phenyl)-2-(benzyloxycarbonyl)
amino)propan-2-ylcarbamate (6g). Yield 91%; 1H NMR (CDCl3)
d 2.49e2.67 (m, 2H, CH2), 2.84 and 3.01 (m, 2H, DOPA-CH2),
propanoic acid (5b). Yield 98%; 1H NMR (CDCl3)
d
2.95-3.09 (m, 2H,
CH2), 4.60 (m, 1H, CH), 5.05 (1H, NH, buried), 5.08 and 5.10 (s, 6H,
CH2), 6.63 (dd, J ¼ 8.1 and 1.8 Hz,1H, 6-ArH), 6.72(s,1H, 2-ArH), 6.81
(d, J ¼ 8.1 Hz, 1H, 5-ArH), 7.29e7.42 (m, 15H, PhH). ESI-MS: m/z 512
(MHþ).
3.26e3.41 (m, 2H, CH2), 4.18 (m, 1H, DOPA-CH), 5.08, 5.11 and 5.12
(s, 6H, CH2), 5.28 (m, 1H, NH), 5.46 (t, J ¼ 5.8 Hz, 1H, NH), 6.64 (d,
J ¼ 7.8 Hz, 1H, 6-ArH), 6.81 (m, 2H, 2-ArH and 5-ArH), 7.02 (m, 2H,
PhH), 7.17e7.43 (m, 18H, PhH). ESI-MS: m/z 615 (MHþ).

4040
T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
4.1.7. Preparation of (S)-tert-butyl 1-amino-3-(3,4-
dihydroxyphenyl)-1-oxopropan-2-ylcarbamate (7)
CH), 7.17 (s, 1H, ArH), 7.20 (s, 2H, ArH), 7.58 and 7.99 (s, 2H, NH2),
8.30 (br, 3H, NHþ3 ). ESI-MS: m/z 365 ([MeCl]þ). HRMS: Calcd for
C19H29N2O5: 305.2076 ([MeHClþH]þ), found: 305.2067.
To a suspension of 6a (2.38 g, 5 mmol) in MeOH/EtOAc (1:1,
40 mL) was added 5% Pd/C (0.24 g). Hydrogenation was carried out
at 30 psi H2 for 3 h. After filtration to remove the catalyst, the
filtrate was concentrated and 7 was obtained as a white solid
4.1.9.2. (S)-3-(3,4-bis(3-methylbutanoyloxy)phenyl)-1-amino-1-
oxopropan-2-ammonium chloride (9b). Quantitative yield; 1H
(1.48 g, 100%). 1H NMR (DMSO-d6)
d
1.33 (s, 9H, CH3), 2.55 and 2.76
NMR (DMSO-d6) d 0.99 (m, 12H, CH3), 2.06 (m, 2H, CH), 2.44 (m,
(m, 2H, DOPA-CH2), 3.98 (m, 1H, DOPA-CH), 6.49 (d, J ¼ 6.5, 1H,
6-ArH), 6.62 (m, 2H, 2-ArH and 5-ArH), 6.95 and 7.29(s, 2H, NH2),
8.66 (d, J ¼ 6.8, 1H, NH). ESI-MS: m/z 297 (MHþ).
4H, CH2), 3.02 and 3.13 (m, 2H, CH2), 3.97 (m, 1H, CH), 7.18 (s,
1H, ArH), 7.22 (s, 2H, ArH), 7.60 and 8.00 (s, 2H, NH2), 8.26 (br,
3H, NHþ3 ). ESI MS: 365 ([MeCl]þ), 387 ([MeHClþNa]þ). HRMS:
Calcd for C19H29N2O5: 305.2076 ([MeHClþH]þ), found:
305.2068.
4.1.8. General preparation of diacyl Boc-protected L-dopa amide
derivatives (8ae8e)
To a suspension of 7 (0.5 g, 1.69 mmol) in CH2Cl2 (20 mL) cooled
on an ice-bath was added Et3N (0.41 g, 4.06 mmol), followed by the
dropwise addition of a variety of acyl chlorides (4.056 mmol). The
mixture was stirred for 3 h and then washed with aqueous NaHCO3,
brine, and dried over Na2SO4. After removal of the solvent, the
residue was purified by silica gel chromatography (EtOAc/Hexane
1:1) to obtain the products as white solids.
4.1.9.3. (S)-3-(3,4-bis(2,2-dimethylbutanoyloxy)phenyl)-1-amino-1-
oxopropan-2-ammonium chloride (9c). Quantitative yield; 1H NMR
(DMSO-d6) d 0.92 (m, 6H, CH3), 1.24 and 1.25 (s, 12H, CH3), 1.66 (m,
4H, CH2), 3.02 and 3.14 (m, 2H, CH2), 3.99 (t, J ¼ 6.5 Hz, 1H, CH),
7.15e7.22 (m, 3H, ArH), 7.60 and 7.99 (s, 2H, NH2), 8.25(br, 3H, NHþ3 ).
ESI MS: 393 ([MeCl]þ), 410 ([MeHClþNH4]þ), 415 ([MeHClþNa]þ).
HRMS: Calcd for C21H33N2O5: 393.2389 ([MeHClþH]þ), found:
393.2400.
4.1.8.1. (S)-tert-butyl 1-amino-3-(3,4-bis(pivaloyloxy)phenyl)-1-oxo-
propan-2-ylcarbamate (8a). Yield 62%; 1H NMR (CDCl3)
18H, CH3), 1.44 (s, 9H, CH3), 2.95 and 3.14 (m, 2H, CH2), 4.32 (m, 1H,
CH), 5.22 (m, 1H, NH), 5.41 and 5.87 (br, 2H, NH2), 7.00e7.12 (m, 3H,
ArH). ESI-MS: m/z 465 (MHþ).
d
1.33 (s,
4.1.9.4. (S)-3-(3,4-bis(3,3-dimethylbutanoyloxy)phenyl)-1-amino-1-
oxopropan-2-ammonium chloride (9d). Quantitative yield; 1H NMR
(DMSO-d6)
d 1.07 and 1.08 (s, 18H, CH3), 2.44 and 2.45 (s, 4H, CH2),
3.06 and 3.14 (m, 2H, CH2), 4.00 (m,1H, CH), 7.18 (s,1H, ArH), 7.22 (s,
2H, ArH), 7.60 and 8.03 (br, 2H, NH2), 8.28 (br, 3H, NHþ3 ). ESI MS:
393 ([MeCl]þ), 415 ([MeHClþNa]þ). HRMS: Calcd for C21H33N2O5:
393.2389 ([MeHClþH]þ), found: 393.2386.
4.1.8.2. (S)-tert-butyl 1-amino-3-(3,4-bis(3-methylbutanoyloxy)phenyl)-
1-oxopropan-2-ylcarbamate (8b). Yield 61%; 1H NMR (CDCl3)
d 1.05
(d, J ¼ 6.5 Hz, 12H, CH3), 1.44 (s, 9H, CH3), 2.20 (m, 2H, CH), 2.40 (d,
J ¼ 7.1 Hz, 4H, CH2), 2.97 and 3.15 (m, 2H, CH2), 4.33 (m, 1H, CH),
5.14 (m, 1H, NH), 5.31 and 5.81 (br, 2H, NH2), 7.05 (s, 1H, 6-ArH), 7.11
(s, 2H, 2-ArH and 5-ArH). ESI-MS: m/z 465 (MHþ).
4.1.9.5. (S)-3-(3,4-bis(ethoxycarbonyloxy)phenyl)-1-amino-1-oxo-
propan-2-ammonium chloride 9e. Quantitative yield; 1H NMR
(DMSO-d6)
d
1H NMR (DMSO-d6)
d
1.29 (t, J ¼ 3.5 Hz, 6H, CH3), 3.06
and 3.15 (m, 2H, CH2), 4.00 (m, 1H, CH), 4.25 (q, J ¼ 4.0 Hz, 4H, CH2),
7.27 (s, 1H, ArH), 7.32 (d, J ¼ 2.0 Hz, 1H, ArH), 7.38 (d, J ¼ 8.5 Hz, 1H,
ArH), 7.60 and 8.01 (s, 2H, NH2), 8.24 (br, 3H, NHþ3 ). ESI MS: 341
([MeCl]þ), 363 ([MeHClþNa]þ). Calcd for C15H21N2O7: 341.1348
([MeHClþH]þ), found: 341.1339.
4.1.8.3. (S)-tert-butyl 1-amino-3-(3,4-bis(2,2-dimethylbutanoyloxy)
phenyl)-1-oxopropan-2-ylcarbamate (8c). Yield 68%; 1H NMR
(CDCl3)
d
0.95 (t, J ¼ 7.4 Hz, 6H, CH3), 1.28 (s, 12H, CH3), 1.44 (s, 9H,
CH3), 1.70 (q, J ¼ 7.4 Hz, 4H, CH2), 2.94 and 3.16 (m, 2H, CH2), 4.32
(m,1H, CH), 5.15 (m,1H, NH), 5.31 and 5.74 (br, 2H, NH2), 6.98 (s,1H,
6-ArH), 7.07 (m, 2H, 2-ArH and 5-ArH). ESI-MS: m/z 493 (MHþ).
4.1.10. General preparation of the hydrochlorides of L-dopa amide
derivatives (10be10g)
4.1.8.4. (S)-tert-butyl 1-amino-3-(3,4-bis(3,3-dimethylbutanoyloxy)
To a suspension of 6 (9.60 mmol) in MeOH/EtOAc (1:1,
120 mL) was added 5% Pd/C (20% weight of 6), followed by the
addition of HCl solution (12 mL, 1.25 M in MeOH). Hydrogenation
was carried out under 30 psi H2 at room temperature for 3 h.
After filtration to remove the catalyst, the filtrate was concen-
trated. The residue was redissolved in MeOH (6 mL) and then
diethyl ether was added to precipitate the product, which was
collected as a white solid.).
phenyl)-1-oxopropan-2-ylcarbamate (8d). Yield 75%; 1H NMR
(CDCl3)
d 1.12 (s, 18H, CH3), 1.43 (s, 9H, CH3), 2.41 (s, 4H, CH2), 2.98
and 3.13 (m, 2H, CH2), 4.34 (m, 1H, CH), 5.14 (m, 1H, NH), 5.37 and
5.83 (br, 2H, NH2), 7.04 (s, 1H, 6-ArH), 7.11 (s, 2H, 2-ArH and 5-ArH).
ESI-MS: m/z 493 (MHþ).
4.1.8.5. (S)-tert-butyl 1-amino-3-(3,4-bis(ethoxycarbonyloxy)phenyl)-
1-oxopropan-2-ylcarbamate (8e). Yield 65%; 1H NMR (CDCl3)
d 1.3 (t,
6H, CH3), 1.43 (s, 9H, CH3), 2.96 and 3.15 (m, 2H, CH2), 4.20 (q, 4H,
CH2), 4.33 (m, 1H, CH), 5.14 (m, 1H, NH), 5.35 and 5.80 (br, 2H, NH2),
7.04 (s, 1H, 6-ArH), 7.10 (s, 2H, 2-ArH and 5-ArH). ESI-MS: m/z 441
(MHþ).
4.1.10.1. (S)-1-amino-3-(3,4-dihydroxyphenyl)-1-oxopropan-2-
ammonium chloride (10b). Quantitative yield; 1H NMR (DMSO-d6)
d
2.75-2.81 and 2.88-2.94 (m, 2H, CH2), 4.15 (m, 1H, CH), 6.51 (dd,
J ¼ 8.0 and 1.9 Hz, 1H, 6-ArH), 6.66 (m, 2H, 2-ArH and 5-ArH), 7.50
and 7.90 (s, 2H, NH2), 8.01 (br, 3H, NHþ3 ), 8.90 and 8.94 (s, 2H, OH).
ESI MS: 197 ([MeCl]þ).
4.1.9. General preparation of the hydrochlorides of diacyl L-dopa
amide derivatives (9ae9e)
To a solution of 8 (1 mmol) in CH2Cl2 (6 mL) cooled on an ice-
bath was added HCl solution (2 mL, 4 M in dioxane). The solution
was stirred for 2 h. After removal of the solvent, product 9 was
obtained as a white solid.
4.1.10.2. (S)-3-(3,4-dihydroxyphenyl)-1-(methylamino)-1-oxopro-
pan-2-ammonium chloride (10c). Quantitative yield; 1H NMR
(DMSO-d6)
d
2.59 (d, J ¼ 4.5 Hz, 3H, CH3), 2.73-2.89 (m, 2H, CH2),
3.78 (m, 1H, CH), 6.45 (dd, J ¼ 8.0 and 1.9 Hz, 1H, 6-ArH), 6.60 (d,
J ¼ 1.9 Hz, 1H, 2-ArH), 6.66 (d, J ¼ 8.0 Hz, 1H, 5-ArH), 8.21 (br, 3H,
NHþ3 ), 8.43 (m, 1H, NH), 8.92 (br, 2H, OH). ESI MS: 211 ([MeCl]þ).
HRMS: Calcd for C10H15N2O3: 211.1082 ([MeHClþH]þ), found:
211.1081.
4.1.9.1. (S)-3-(3,4-bis(pivaloyloxy)phenyl)-1-amino-1-oxopropan-2-
ammonium chloride (9a). Quantitative yield; 1H NMR (DMSO-d6)
d
1.27 and 1.28 (s, 18H, CH3), 3.00e3.15 (m, 2H, CH2), 3.99 (m, 1H,

T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
4041
4.1.10.3. (S)-3-(3,4-dihydroxyphenyl)-1-(ethylamino)-1-oxopropan-
2-ammonium chloride (10d). Yield 96%; 1H NMR (DMSO-d6)
0.96
3H, ArH), 8.44 (br, 3H, NHþ3 ), 8.58 (t, J ¼ 5.4 Hz, 1H, NH). ESI MS: 309
([MeCl]þ), 331 ([MeHClþNa]þ). HRMS: Calcd for C15H21N2O5:
309.1450 ([MeHClþH]þ), found: 309.1445.
d
(t, J ¼ 7.2 Hz, 3H, CH3), 2.70e2.85 (m, 2H, DOPA-CH2), 2.99e3.15 (m,
2H, CH2), 3.67 (t, J ¼ 7.0 Hz, 1H, DOPA-CH), 6.45 (dd, J ¼ 8.0 and
1.9 Hz, 1H, 6-ArH), 6.60 (d, J ¼ 1.9 Hz, 1H, 2-ArH), 6.65 (d, J ¼ 8.0 Hz,
1H, 5-ArH), 7.56 (br, 3H, NHþ3 ), 8.29 (t, J ¼ 5.3 Hz, 1H, NH), 8.88 (br,
2H, OH). ESI MS: 225 ([MeCl]þ). HRMS: Calcd for C11H17N2O3:
225.1239 ([MeHClþH]þ), found: 225.1224.
4.1.11.4. (S)-3-(3,4-diacetoxyphenyl)-1-(isopropylamino)-1-oxopro-
pan-2-ammonium chloride (11e). Yield 75%; 1H NMR (DMSO-d6)
d
0.88 (d, J ¼ 6.5 Hz, 3H, CH3), 1.04 (d, J ¼ 6.5 Hz, 3H, CH3), 2.25 (s,
3H, CH3), 2.27 (s, 3H, CH3), 3.00 and 3.12 (m, 2H, DOPA-CH2), 3.76
(m, 1H, CH), 3.95(m, 1H, DOPA-CH), 7.13-7.25 (m, 3H, ArH), 8.42 (d,
J ¼ 7.6 Hz, 1H, NH), 8.47 (br, 3H, NHþ3 ). ESI MS: 323 ([MeCl]þ), 345
([MeHClþNa]þ). HRMS: Calcd for C16H23N2O5: 323.1607
([MeHClþH]þ), found: 323.1601.
4.1.10.4. (S)-3-(3,4-dihydroxyphenyl)-1-(isopropylamino)-1-oxopro-
pan-2-ammonium chloride (10e). Quantitative yield; 1H NMR
(DMSO-d6)
d
0.94 (d, J ¼ 6.6 Hz, 3H, CH3), 1.05 (d, J ¼ 6.6 Hz, 3H,
CH3), 2.79 (m, 2H, DOPA-CH2), 3.70 (t, J ¼ 7.0 Hz, 1H, DOPA-CH),
3.81 (m, 1H, CH), 6.45 (dd, J ¼ 8.0 and 1.9 Hz, 1H, 6-ArH), 6.60 (d,
J ¼ 1.9 Hz, 1H, 2-ArH), 6.65 (d, J ¼ 8.0 Hz, 1H, 5-ArH), 7.95 (br, 3H,
NHþ3 ), 8.18 (d, J ¼ 7.6 Hz, 1H, NH), 8.87 and 8.90 (br, 2H, OH). ESI MS:
239 ([MeCl]þ). HRMS: Calcd for C12H19N2O3: 239.1396
([MeHClþH]þ), found: 239.1400.
4.1.11.5. (S)-3-(3,4-diacetoxyphenyl)-1-(dimethylamino)-1-oxopro-
pan-2-ammonium chloride (11f). Yield 92%; 1H NMR (DMSO-d6)
d
2.26 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.61 (s, 3H, CH3), 2.76 (s, 3H,
CH3), 2.94 and 3.16 (m, 2H, DOPA-CH2), 4.54 (m, 1H, DOPA-CH),
7.11e7.23 (m, 3H, ArH), 8.50 (br, 3H, NHþ3 ). ESI MS: 309 ([MeCl]þ),
331 ([MeHClþNa]þ). HRMS: Calcd for C15H21N2O5: 309.1450
([MeHClþH]þ), found: 309.1445.
4.1.10.5. (S)-3-(3,4-dihydroxyphenyl)-1-(dimethylamino)-1-oxopro-
pan-2-ammonium chloride (10f). Quantitative yield; 1H NMR
(DMSO-d6)
d
2.61 (s, 3H, CH3), 2.74 (m, 1H, DOPA-CH2), 2.80 (s, 3H,
4.2. Pharmacological evaluation
CH3), 2.89 (m, 1H, DOPA-CH2), 4.39 (m, 1H, DOPA-CH), 6.43 (dd,
J ¼ 8.0 and 1.9 Hz,1H, 6-ArH), 6.59 (d, J ¼ 1.9 Hz, 1H, 2-ArH), 6.68 (d,
J ¼ 8.0 Hz, 1H, 5-ArH), 8.23 (br, 3H, NHþ3 ), 8.96 (br, 2H, OH). ESI MS:
225 ([MeCl]þ).
4.2.1. Animals
Male Wistar rats (250e500g, Harlan Ltd.). Animals were housed
in groups of 4 on a 12-h light-dark cycle with an environment of
50% humidity and a temperature of 21 ꢃ 2 ꢁC in accordance with
Animals (Scientific Procedures) Act 1996 Home Office regulations.
Rats had access to food and water ad libitum. All animals used in
this study were treated in accordance with the UK 1986 Animals
(Scientific Procedures Act).
4.1.10.6. (S)-3-(3,4-dihydroxyphenyl)-1-(phenethylamino)-1-oxo-
propan-2-ammonium chloride (10g). Yield 91%; 1H NMR (DMSO-d6)
d
2.58e2.70 (m, 2H, CH2), 2.72e2.89 (m, 2H, DOPA-CH2), 3.18 (m,
1H, CH2, anther H was buried by water peak), 3.79 (t, J ¼ 6.9 Hz, 1H,
DOPA-CH), 6.45 (d, J ¼ 7.9 Hz, 1H, 6-ArH), 6.63 (s, 1H, 2-ArH), 6.67
(d, J ¼ 8.0 Hz, 1H, 5-ArH), 7.16e7.22 (m, 3H, PhH), 7.26e7.31 (m, 2H,
PhH), 8.14 (br, 3H, NHþ3 ), 8.58 (m, 1H, NH), 8.93 (br, 2H, OH). ESI MS:
301 ([MeCl]þ). HRMS: Calcd for C17H21N2O3: 301.1552
([MeHClþH]þ), found: 301.1546.
4.2.2. Pharmacological and pharmacokinetic evaluation
4.2.2.1. Surgical procedures. 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. Rats were
anaesthetized in an induction chamber using isofluorane (1e2% in
95% O2, 5% CO2 carrier gas), placed in a Kopf stereotaxic frame and
anaesthesia maintained with 0.5e1.0% isofluorane. An incision was
made in the scalp and a 0.8-mm-diameter hole made in the skull at
coordinates AP: ꢀ0.26mm L: þ2.0 mm (all co-ordinated measured
from bregma). The neurotoxin 6-hydroxydopamine hydrobromide
4.1.11. General preparation of the hydrochlorides of the diacetyl L-
dopa amide derivatives (11be11f)
To a solution of 10 (9 mmol) in acetic acid (20 mL) was bubbled
HCl gas at 40 ꢁC, followed by the dropwise addition of acetyl
chloride (12 mL). The solution was allowed to cool to room
temperature and was stirred overnight. Addition of diethyl ether
caused to precipitation of the product 11, which after filtration was
obtained as a white solid.
(6-OHDA) (8
ascorbic acid) was injected into the left median forebrain bundle at
a constant rate over 4 min (1 L/min) using a 10- L Hamilton
mg free base in 4 mL of 0.9% saline containing 0.05%
m
m
syringe lowered to ꢀ8 mm below the dura. The needle remained in
place for a further 4 min before being removed, and the wound
cleaned and sutured. Flunixin hydrochloride (2.5 mg/kg, Dunlop’s
Veterinary Supplies, Dumfries, UK) was administered for pain relief
and a rehydration treatment of 5% glucose in 0.9% saline (up to 5 mL
ip) was given prior to recovery from the anaesthetic.
4.1.11.1. (S)-1-amino-3-(3,4-diacetoxyphenyl)-1-oxopropan-2-ammo-
nium chloride (11b). Yield 84%; 1H NMR (DMSO-d6)
d 2.27 (s, 3H,
CH3), 2.28 (s, 3H, CH3), 3.04 and 3.14 (m, 2H, DOPA-CH2), 3.99 (m,1H,
CH), 7.19e7.25 (m, 3H, ArH), 7.61 and 8.04 (s, 2H, NH2), 8.30 (br, 3H,
NHþ3 ). ESI MS: 281 ([MeCl]þ), 303 ([MeHClþNa]þ). HRMS: Calcd for
C13H17N2O5: 281.1137 ([MeHClþH]þ), found: 281.1140.
4.2.2.2. Confirmation of lesion of the nigro-striatal tract. At least 2
weeks following surgery, animals were assessed for rotational
behaviour (see below) in response to the administration of
apomorphine hydrochloride (0.5 mg/kg s.c. in 0.9% saline contain-
ing 0.05% ascorbic acid) to evaluate the extent of the lesion. Only
those rats exhibiting >6 turns/min at peak activity were used in
subsequent studies.
4.1.11.2. (S)-3-(3,4-diacetoxyphenyl)-1-(methylamino)-1-oxopro-
pan-2-ammonium chloride (11c). 82% yield. 1H NMR (DMSO-d6)
d
2.26 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.58 (d, J ¼ 4.3 Hz, 3H, CH3),
3.06 (d, J ¼ 6.9 Hz, 2H, DOPA-CH2), 3.98 (m, 1H, DOPA-CH),
7.13e7.23 (m, 3H, ArH), 8.43 (br, 3H, NHþ3 ), 8.60 (m, 1H, NH). ESI MS:
295 ([MeCl]þ). HRMS: Calcd for C14H19N2O5: 295.1294
([MeHClþH]þ), found: 295.1272.
4.2.2.3. Assessment of the induction of rotational activity by test
compounds. At least 1 week after apomorphine administration, rats
(n¼4e8 per treatment) were assessed for rotational activity with
4.1.11.3. (S)-3-(3,4-diacetoxyphenyl)-1-(ethylamino)-1-oxopropan-
2-ammonium chloride (11d). Yield 90%; 1H NMR (DMSO-d6)
d 0.93
(t, J ¼ 7.2 Hz, 3H, CH3), 2.26 (s, 3H, CH3), 2.27 (s, 3H, CH3), 2.94-3.16
either L-dopa or a test drug. Compounds were administered either
(m, 4H, DOPA-CH2 and NCH2), 3.95 (m, 1H, DOPA-CH), 7.13-7.22 (m,
via the intraperitoneal (ip) route or orally by gavage (po). Animals

4042
T. Zhou et al. / European Journal of Medicinal Chemistry 45 (2010) 4035e4042
were treated with the peripheral decarboxylase inhibitor benser-
azide (10 mg/kg) to prevent degradation of -dopa to dopamine in
The supernatant was analysed by LC-MS/MS method which is
described in detail below.
L
peripheral tissues and placed in rotometers (Med Associates) for up
to 30 min to measure basal rotational activity. They were then
4.2.3.3. LC-MS/MS analysis. The LC-MS/MS system consisted of an
Agilent 1100 series gradient HPLC pump (Agilent Technologies, Palo
Alto, CA), a CTC HTS PAL Autosampler (CTC Analytics, Zwingen,
Switzerland) and an Applied Biosystems/MDS Sciex API 3000 triple
quadrupole mass spectrometer (Applied Biosystems, Foster City,
CA) equipped with a turbo ionspray interface and operated in
positive electrospray mode. Analytes in incubation mixtures were
separated by reverse phase HPLC using a Phenomenex Sphereclone
treated with
equivalent dose (63.4
assessed for up to 4 hours after test drug/
L
-dopa or a test compound at a single fixed molar
mole/kg ip or po). Rotational behaviour was
-dopa administration.
m
L
Animals were typically treated with a series of compounds for
comparative purposes with each treatment being administered at
least 1 day apart.
4.2.2.4. Data analysis. The number of rotations measured per
10 min over the 4-hour period was determined. Animals were
considered active if they turned >10 turns per 10 min. From this
data the following parameters were measured:
ODS 2 column (150 ꢂ 4.6 mm, 3
mm, Phenomenex, Torrance, CA).
A gradient elution program was used at a flow rate of 1 ml/min
with a mobile phase consisting of acetonitrile/0.1% formic acid
(5% v/v) in water/0.1% formic acid, delivered for 1.5 min, after which
time the acetonitrile concentration was increased to 95% over
0.5 min and held at 95% for four minutes before restoring it back to
A: Total activity (AUC activity, where AUC ¼ area under the
locomotor activity/time curve)
B: Peak activity
5% for the remaining two minutes. The injection volume was 20 ml.
Approximately 10% of the eluent was introduced into the mass
spectrometer source. The source temperature of the mass
spectrometer was maintained at 450 ꢁC, and other source param-
eters (e.g., collision energy, declustering potential, curtain gas
pressure etc.) were optimised on the day of analysis to achieve
C: Duration of activity
Values are quoted as % of
L-dopa induced effects.
4.2.3. Pharmacokinetic analysis
maximum sensitivity. Quantification of L-dopa and warfarin was
4.2.3.1. Pharmacokinetics dosing protocol. Naïve male Wistar rats
(250e500 g) were used for the pharmacokinetic studies. Animals
were fasted overnight. Test compounds were dissolved in 0.9%
saline and co-dosed with benserazide (10 mg/kg) at a molecular
achieved by monitoring the transitions of m/z ¼ 198.075/152.1 and
m/z ¼ 369.069/163, respectively.
weight equivalent dose to 12.5 mg/kg L-dopa. Blood samples were
References
taken via a butterfly needle located into the lateral tail vein and
collected into sample tubes containing heparin as the anti-coagu-
lant. Blood samples were centrifuged at 5000 rpm for 10 min; the
supernatant plasma was removed and stored at ꢀ80 ꢁC.
[1] P.A. Serra, G. Esposito, P. Enrico, M.A. Mura, R. Migheli, M.R. Delogu, et al., Br. J.
Pharmacol. 130 (2000) 937e945.
[2] G.K. Tofaris, M.G. Spillantini, Mov. Disord. 20 (2005) S37e44.
[3] J. Weber, G.M. Keating, CNS Drugs 23 (2009) 81e89.
[4] A. Antonini, P. Barone, Neurol. Sci. 29 (2008) S371eS374.
[5] H.P. Wang, J.S. Lee, M.C. Tsai, H.H. Lu, W.L. Hsu, Bioorg. Med. Chem. Lett. 5
(1995) 2195e2198.
[6] A. Di Stefano, B. Mosciatti, G.M. Cingolati, G. Giorgioni, M. Riccitutelli,
I. Cacciatore, et al., Bioorg. Med. Chem. Lett. 11 (2001) 1085e1088.
[7] E. Santini, E. Valjent, G. Fisone, FEBS J. 275 (2008) 1392e1399.
[8] A.D. Stefano, P. Sozio, S.L. Cerasa, Molecules 13 (2008) 46e68.
[9] F. Pinnen, I. Cacciatore, C. Cornacchia, P. Sozio, A. Iannitelli, M. Costa, et al.,
J. Med. Chem. 50 (2007) 2506e2515.
[10] F. Pinnen, I. Cacciatore, C. Cornacchia, P. Sozio, S.L. Cerasa, A. Iannitelli, et al.,
J. Med. Chem. 52 (2009) 559e563.
[11] D. Atlas, L-Dopa amide derivatives and uses thereof. US2006025385(A1).
[12] R.K.W. Schwarting, J.P. Huston, Prog. Neurobiol. 50 (1996) 275e331.
[13] M. Ihara, Y. Tsuchiya, Y. Sawasaki, A. Hisaka, H. Takehana, K. Tomimoto, et al.,
J. Pharm. Sci. 78 (1989) 525.
4.2.3.2. Preparation of sample and standard solutions. Stock solu-
tions of 10 mM L-dopa and warfarin were prepared in 20% TFA,
10 mM sodium meta bisulphite and DMSO, respectively.
Standard curves and Quality control (QC) samples were
prepared by spiking control rat plasma with
initial concentration of 50 M. Serial dilutions of this solution were
performed in rat plasma to result in solutions with 25, 6.25, 3.125,
1.56, 0.78 and 0.39 -dopa.
L-dopa to achieve an
m
mM L
One volume of sample plasma, standard and QC sample plasma
was transferred from each sample vial to a 96 well plate.
Compounds were extracted from plasma by addition of one volume
of 20% TFA in 10 mM sodium metabisulphite containing the internal
[14] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliver. Rev. 23
(1997) 3e25.
[15] http://www.molinspiration.com/cgi-bin/properties.
[16] U. Ungerstedt, Acta Physiol. Scand. 367 (Suppl.) (1971) 69e93.
[17] F.M. Otis, L. Levesque, F. Marceau, J. Lacroix, R.C. Gaudreault, Inflammo-
pharmacology 1 (1992) 201e212.
standard, warfarin, at 0.5 mM. The samples were vortex mixed and
centrifuged at 4500 rpm for 4 min to precipitate plasma proteins.
One volume of water was added to each well and the protein pellet
was re-suspended. The samples were again vortex mixed and
centrifuged at 4500 rpm for 9 min to precipitate plasma proteins.
[18] L.E. Piatnitski-Chekler, A.J. Butera, D. Li, E.R. Swillo, A.G. Morgan, I.E. Rossman,
et al., Biorg. Med. Chem. Lett. 19 (2009) 4551e4554.