Novel multifunctional dopamine D2/D3receptors agonists with potential neuroprotection and anti-alpha synuclein protein aggregation properties

Article abstract of DOI:10.1016/j.bmc.2016.08.021

Our ongoing drug development endeavor to design compounds for symptomatic and neuroprotective treatment of Parkinson's disease (PD) led us to carry out a structure activity relationship study based on dopamine agonists pramipexole and 5-OHDPAT. Our goal was to incorporate structural elements in these agonists in a way to preserve their agonist activity while producing inhibitory activity against aggregation of α-synuclein protein. In our design we appended various catechol and related phenol derivatives to the parent agonists via different linker lengths. Structural optimization led to development of several potent agonists among which (?)-8a, (?)-14 and (?)-20 exhibited potent neuroprotective properties in a cellular PD model involving neurotoxin 6-OHDA. The lead compounds (?)-8a and (?)-14 were able to modulate aggregation of α-synuclein protein efficiently. Finally, in an in vivo PD animal model, compound (?)-8a exhibited efficacious anti-parkinsonian effect.

Full text of DOI:10.1016/j.bmc.2016.08.021

Accepted Manuscript
Novel multifunctional dopamine D2/D3 receptors agonists with potential neu-
roprotection and anti-alpha synuclein protein aggregation properties
Dan Luo, Horrick Sharma, Deepthi Yedlapudi, Tamara Antonio, Maarten E.A.
Reith, Aloke K. Dutta
PII:
S0968-0896(16)30628-9
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.021
BMC 13204
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
1 June 2016
15 August 2016
18 August 2016
Please cite this article as: Luo, D., Sharma, H., Yedlapudi, D., Antonio, T., Reith, M.E.A., Dutta, A.K., Novel
multifunctional dopamine D2/D3 receptors agonists with potential neuroprotection and anti-alpha synuclein protein
aggregation properties, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.
2016.08.021
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Novel multifunctional dopamine D2/D3 receptors agonists with potential
neuroprotection and anti-alpha synuclein protein aggregation properties
Dan Luo1, Horrick Sharma1, Deepthi Yedlapudi1, Tamara Antonio2, Maarten E.A. Reith2,
Aloke K. Dutta1*
1. Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202
2. Department of Psychiatry, New York University, New York, N.Y. 10016
Correspondence Address:
Aloke K. Dutta, Ph.D.
Department of Pharmaceutical Sciences
Eugene Applebaum College of Pharmacy & Health Sciences
Wayne State University
Detroit, MI 48202
Tel: 1-313-577-1064, Fax: 1-313-577-2033, e-mail: adutta@wayne.edu
1

Abstract
Our ongoing drug development endeavor to design compounds for symptomatic and
neuroprotective treatment of Parkinson’s disease (PD) led us to carry out a structure
activity relationship study based on dopamine agonists pramipexole and 5-OHDPAT. Our
goal was to incorporate structural elements in these agonists in a way to preserve their
agonist activity while producing inhibitory activity against aggregation of -synuclein
protein. In our design we appended various catechol and related phenol derivatives to the
parent agonists via different linker lengths. Structural optimization led to development of
several potent agonists among which (-)-8a, (-)-14 and (-)-20 exhibited potent
neuroprotective properties in a cellular PD model involving neurotoxin 6-OHDA. The lead
compounds (-)-8a and (-)-14 were able to modulate aggregation of -synuclein protein
efficiently. Finally, in an in vivo PD animal model, compound (-)-8a exhibited efficacious
anti-parkinsonian effect.
Key Words: Parkinson’s disease, Dopamine agonist, Multifunctional drug,
Neuroprotection, Alpha Synuclein, Structure activity relationship study
1. Introduction
Parkinson’s disease (PD), affecting approximately 1% of population over age 65, is the
second most common neurodegenerative disorder worldwide.1 Two pathologic hallmarks
of this disease have been described as involving a progressive loss of dopaminergic
neurons in the substantia nigra pars compacta (SNpc) and the presence of cytoplasmic
"Lewy bodies (LBs)" in the brain.2 Symptoms of PD are broad and are usually classified
into motor and non-motor features. Bradykinesia, resting tremor, rigidity, and postural
instability are the key clinical features.3 Studies have revealed complexity in the
pathogenesis of PD. Multiple pathogenic factors including oxidative stress, -synuclein
(SN) protein aggregation, mitochondrial dysfunction, and genetic/environmental aspects
2

are strongly implicated in the PD progression.1,4-7
The current treatments available for PD generally fall into four major categories, namely
levodopa, catechol-O-methyl transferase (COMT) inhibitors, monoamine oxidase B (MAO-
B) inhibitors, and dopamine agonists.8 Levodopa, an orally active precursor of dopamine,
remains the gold standard of PD treatment.9 Dopamine agonists, which mimic dopamine,
can directly activate dopaminergic receptors to improve PD motor symptoms.10 However,
the benefits provided by these treatments are mostly symptom-relieving. Furthermore,
those anti-parkinsonian agents suffer from loss of efficacy as the disease progresses, and
the long-term use of levodopa induces motor complications such as motor fluctuations and
dyskinesia.11
Based on the multifactorial nature of PD pathogenesis, treatments targeting multiple
pathogenic factors could be promising in terms of symptom-relieving and disease-
modifying treatment for PD. In our previous study on development of symptomatic and
neuroprotective agents for PD, we have established a hybrid molecular template to
develop multifunctional dopamine D2/D3 agonists. Our focus on development of potent
D2/D3 agonists is directed to address the symptomatic aspect of the disease process while
incorporating relevant neuroprotective properties to produce disease modifying effects.
The dopamine D2/D3 receptors, which share a high degree of homology12, are
predominantly expressed in the striatum of the brain and play a principle role in motor
function.13 In this regard, the dopamine receptors are classified into two subtypes (D1-type
and D2-type), which is based on distinct physiological and pharmacological properties of
these two receptors as stimulation of the D1-type receptors (D1 and D5) lead to activation of
adenylate cyclase, which promotes synthesis of cAMP (cyclic adenosine monophosphate),
whereas D2-type receptor (D2, D3 and D4) activation leads to inhibition of adenylate cyclase
activity.14-17
Some of our lead compounds, such as D-520 and D-512 (Figure 1), exhibited high agonist
potency at D2/D3 receptors and efficacious activity in PD animal models with additional
3

neuroprotective activity.18-21 These lead compounds possess a piperazine linker that
connects the head group, aminotetralin or tetrahydrobenzo 2-aminothiazole moiety, to an
indole or a biphenyl accessory binding site fragment.18-20 In our present study, we have
investigated the effect of replacing the piperazine ring in the linker region by alkyl linkers.
The effects of saturated and unsaturated linker moiety and the linker length on D2/D3
agonistic activities have been evaluated.
Figure 1. Molecular structures of D2/D3 agonists
Additionally, in our design we have briefly explored the accessory binding site moiety with
a goal to produce neuroprotective properties in these molecules as well as potentially
targeting SN for inhibition of aggregation, which has been one of the major targets for the
development of PD therapeutics.22-24 Several molecules containing catechol moiety or its
derivatives, such as rifampicin, curcumin, and flavonoids, have been reported to exert
inhibitory effect on SN aggregation, and it has been speculated that the polyphenol
structure interact with SN to alter the aggregation pathway.25-31 Thus, we have
incorporated the catechol moiety in our lead compound D-520 located distally from the
agonist binding moiety in the molecule and briefly explored its related derivatives.
Therefore, our drug developmental strategy incorporates structural features that can
provide neuroprotective property while maintaining the potent D2/D3 agonistic activity
based on the modified hybrid template (Figure 2). Compounds were characterized both in
4

the in vitro binding assay and GTPγS functional assay. Selected lead compounds were
studied in vitro to evaluate their neuroprotective activity against 6-hydroxydopamine (6-
OHDA) induced toxicity. Their ability on modulating αSN aggregation has been
investigated briefly through a cell-free assay procedure. In vivo studies of the lead
compound were also conducted in a PD animal model for evaluating their potency to
reverse the reserpine induced hypolocomotion.
Figure 2. Molecular template for novel D2/D3 multifunctional agonists with potential SN
aggregation modulation property
2. Chemistry
5

Scheme 1. Reaction and Conditions: (a) TPP, toluene, reflux, 16 h; (b) NaHMDS, THF, -
78 °C to rt, 48 h; (c) 10% Pd/C, H2, EtOH, rt, 2 h; (d) DIBALH, toluene, -78 °C, 2 h; (e) (±),
(−)-pramipexole or (−)-5-methoxy-N-propyl-2-aminotetralin, NaBH(OAc)3, CH2Cl2, rt, 36 h;
(f) BBr3, CH2Cl2, -78 °C to rt, 6 h; (g) TBDMSCl, imidazole, DMF, rt, 2 h; (h) DIBALH, THF,
-10 °C to rt, 6 h; (i) PCC, CH2Cl2, 0 °C to rt, 9h; (j) TBAF, THF, 0 °C, 1.5 h; (k) 48% HBr,
reflux, 6 h.
Scheme 1 describes the synthesis of a series of compounds where the 2-aminothiazole
head group is attached to various diphenol- or methoxyphenol fragments via five-carbon
alkyl linkers. One aminotetralin analog is also included in this scheme. Commercially
available ethyl 4-bromobutyrate (1) was reacted with triphenylphosphine to give
6

phosphonium bromide 2, which was subjected to Wittig olefination with aldehydes 3a or 3b
using sodium hexamethyldisilazide (NaHMDS) to obtain preferentially (Z:E > 20:1) (Z)-
alkene products 4a and 4b, respectively. Their stereochemistry was confirmed by a smaller
coupling constant (J = 11.2 Hz) observed for the Z-isomers as opposed to a larger vicinal
coupling (J = 16 Hz) obtained for the corresponding (E)-isomers. Pd/C-catalyzed
hydrogenation of 4a and 4b yielded corresponding saturated intermediates 5a and 5b.
Then these esters were reduced to aldehydes 6a and 6b by using diisobutylaluminium
hydride (DIBALH). Reductive amination with sodium triacetoxyborohydride was used to
couple 6a and 6b with (±)- or (−)-pramipexole to give (±)-7a, (−)-7a, (±)-7b, and (−)-7b,
respectively, which were then reacted with boron tribromide to yield corresponding
dihydroxy-compounds (±)-8a, (−)-8a, (±)-8b, and (−)-8b. The (Z)-isomer of 4a was
separated by normal phase silica gel chromatography and used to synthesize target
compound (±)-12. Tert-butyldimethylsilyl (TBS) protection of 4a yielded intermediate 9,
which was reduced to alcohol 10 by DIBALH and then oxidized to aldehyde intermediate
11 by PCC oxidation. Deprotection of the TBS group and subsequent reductive amination
gave the final compound (±)-12. Compound (−)-14, the aminotetralin analog of (−)-8a, was
synthesized by coupling aldehyde 6a with (−)-5-methoxy-N-propyl-2-aminotetralin through
reductive amination followed by demethylation using hydrobromic acid.
Scheme 2. Reaction and Conditions: (a) TPP, toluene, reflux, 16 h; (b) HMDS, n-BuLi, THF,
7

0 °C, 15 min; (c) vanillin, LiHMDS, THF, rt, 24 h; (d) CH3COCl, MeOH, 0 °C to rt, 4 h; (e)
DIBALH, toluene, -78 °C, 2 h; (f) (±) or (−)-pramipexole, NaBH(OAc)3, CH2Cl2, rt, 36-48 h.
Scheme 2 depicts the synthesis of additional five-carbon linker containing compounds, (±)-
20 and (−)-20, where the unsaturated linker fragment is in the (E)-conformation.
Phosphonium salt 16 was first prepared by reacting 4-bromobutyric acid (15) with
triphenylphosphine. Then Wittig olefination of 16 with vanillin (3a) using freshly prepared
lithium hexamethyldisilazide (LiHMDS) gave preferentially (E:Z > 10:1) (E)-alkene
intermediate 17. 17 was converted to its corresponding methyl ester 18 and its (E)-isomer
(J = 15.6 Hz) was separated by column chromatography. Reduction to aldehyde followed
by reductive amination with (±)- or (−)-pramipexole gave target compounds (±)-20 and (−)-
20.
Scheme 3. Reaction and Conditions: (a) HMDS, n-BuLi, THF, 0 °C, 15 min; (b) vanillin,
LiHMDS, THF, rt, 24 h; (c) 10% Pd/C, H2, EtOH, rt, 2 h; (d) CH3COCl, MeOH, 0 °C to rt, 4
h; (e) DIBALH, toluene, -78 °C, 2 h; (f) (±)-pramipexole, NaBH(OAc)3, CH2Cl2, rt, 48 h.
Scheme 3 describes the synthesis of target compound (±)-26, which contains a saturated
six-carbon linker. Commercially available phosphonium salt 21 was reacted with vanillin
(3a) in the presence of freshly prepared LiHMDS resulting preferentially in (E)-alkene
product 22. This unsaturated carboxylic acid precursor was reduced through Pd/C-
8

catalyzed hydrogenation and converted to corresponding methyl ester 24. Final compound
(±)-26 was obtained by reduction of ester intermediate 24 to aldehyde 25 followed by
reductive amination of 25 with (±)-pramipexole.
Scheme 4. Reaction and Conditions: (a) TPP, toluene, reflux, 16 h; (b) 1 M NaOH aq., 15
min; (c) vanillin, CHCl3, reflux, 5 h; (d) 10% Pd/C, H2, EtOH, rt, 2 h; (e) DIBALH, toluene, -
78 °C, 2 h; (f) (±)-pramipexole, NaBH(OAc)3, CH2Cl2, rt, 36-48 h; (g) BBr3, CH2Cl2, -78 °C
to rt, 6 h; (h) TBDMSCl, imidazole, DMF, rt, 2 h; (i) DIBALH, THF, -10 °C to rt, 6 h; (j) MnO2,
CH2Cl2, rt, 24h; (k) TBAF, THF, 0 °C, 1 h.
Scheme 4 shows the synthesis of another series of compounds with three-carbon linkers
connecting the tetrahydrobenzo 2-aminothiazole moiety head group to a diphenol- or
methoxyphenol fragment. (E)-alkene intermediate 29 was prepared via the Wittig reaction.
Pd/C-catalyzed hydrogenation was used to generate saturated ester precursor 30, which
was then converted to aldehyde 31 by DIBALH. Target compound (±)-32 was prepared by
9

reductive amination of aldehyde 31 with (±)-pramipexole. Demethylation was then
achieved to obtain target compound (±)-33. To prepare compound (±)-38, intermediate 29
was first protected and reduced to alcohol 35. Then aldehyde 37 was synthesized by
oxidation of alcohol intermediate 35 using manganese dioxide followed by deprotection of
TBS group in intermediate 36. The aldehyde intermediate 37 was then reacted with (±)-
pramipexole to yield final compound (±)-38.
Scheme 5. Reaction and Conditions: (a) BBr3, CH2Cl2, -78 °C to rt, 6 h; (b) TBDMSCl,
imidazole, DMF, rt, 2 h; (c) phosphonium bromide 2, NaHMDS, THF, -78 °C to rt, 48 h; (d)
DIBALH, toluene, -78 °C, 2 h; (e) (±)-pramipexole, NaBH(OAc)3, CH2Cl2, rt, 36 h; (f) TBAF,
THF, 0 °C, 1.5 h.
Scheme 5 outlines synthesis of D-592, a mixture of compounds (±)-44, 45 as deduced
from NMR and mass spectroscopy (m/z [M + H]+: major 388.46, minor 386.44). Air
oxidation of the catechol to the corresponding dione could be a result of stabilization from
an extended conjugation in the dione state. Prior to coupling to the phosphonium salt
through Wittig reaction, aldehyde 3a was first demethylated and TBS-protected by tert-
butyldimethylsilyl chloride (TBDMSCl) to give intermediate 40. After the Wittig olefination
with phosphonium bromide 2, reduction to aldehyde intermediate 42 was achieved by
10

chemistry described above. Subsequent reductive amination of 42 with (±)-pramipexole
resulted in intermediate (±)-43, which was deprotected to give D-592 as a mixture of
compounds (±)-44 and 45.
3. Results and Discussion
3.1 Affinity and Efficacy to D2/D3 Receptors
In our current SAR study to explore the effect of structural variations in the linker region
that connects the D2/D3 agonistic head group with accessory binding site moiety, we have
replaced the piperazine ring in our lead compounds as in D-520 with saturated and
unsaturated alkyl linkers. We varied the alkyl chain length resulting in compounds with
three to six carbon linkers. The effect of linker length on the D2/D3 binding affinity can be
inferred from compounds in the saturated series, which includes (±)-32, (±)-7a, and (±)-26.
Among them, compound (±)-26 with a six-carbon linker exhibited the highest affinity for
both D2/D3 receptors (Ki, D2=58.5 nM, D3=0.675 nM, Table 1). Compound (±)-7a, which
has a five-carbon linker, also displayed potent binding affinity at D3 receptor (Ki, D3=3.62
nM) and moderate affinity for D2 receptor (Ki, D2=175 nM). While the three-carbon linker
compound (±)-32 gave much weaker binding, especially for D2 receptor (Ki, D2=2,404 nM,
D3=15.8 nM). These results suggest that linker length has a considerable effect on the
binding affinity of these compounds: lengthening the linker chain from three-carbon to six-
carbon greatly improves the affinity for both D2 receptor (by 41-fold) and D3 receptor (by
23-fold). The high activity of the six-carbon linker compound (±)-26 could be because of
better accessibility and/or interactions of the tetrahydrobenzo 2-aminothiazole head group
moiety at the D2/D3 receptors binding sites. The binding data of (±)-32 also suggest that a
greater selectivity for D3 receptor could be achieved by connecting the head group and the
accessory binding fragment through a linker with appropriate length.
The increasing trend of receptor binding affinity may indicate lesser steric hindrance
occurred between the phenyl ring in the designed molecules and the binding pocket of
11

receptors when extended linkers were incorporated. It may also be reasoned that a six- or
a five-carbon linker could confer more flexibility; thus, (±)-26 and (±)-7a may adopt different
conformations than the corresponding three-carbon linker compound (±)-32. Towards this
end, conformational restrictions were introduced in the linker portion through insertion of a
double bond. Further, with an aim to impart potential neuroprotective property, a
conjugated double bond, as in curcumin, was particularly envisaged resulting in substituted
styrene accessory binding fragments. The effect of introducing a double bond in the linker
portion is more complicated. For compounds containing three-carbon linkers, incorporation
of an (E)-double bond in compound (±)-38 greatly reduces the binding affinity for both D2
and D3 receptors in comparison to its corresponding saturated linker analog (±)-32 (Ki, D2=
4,634 vs. 2,404 nM, D3= 39.1 vs. 15.8 nM, Table 1). For a five-carbon linker saturated
analog (±)-7a (Table 1), introduction of an (E)-double bond as in (±)-20 improved binding
affinity at D2 receptor (Ki, D2=150 nM) and was also tolerated at D3 receptor (Ki, D3=4.24
nM). To determine the effect of stereochemistry, corresponding (Z)-isomer analog,
compound (±)-12, was also synthesized. As predicted, stereochemistry did influence the
binding affinity as the (Z)-isomer analog (±)-12 displayed reduced affinity at both D2 and D3
receptors (Ki, D2=233 nM, D3=23.3 nM) when compared to its saturated (±)-7a and the (E)-
isomer counterpart (±)-20. These results suggest that the length along with the
stereochemistry of linkers influences binding of the head group moiety at both D2 and D3
receptors. Overall, our SAR study demonstrates that the piperazine ring present in our
lead compounds could be replaced by an appropriate alkyl linker in our present series of
compounds while maintaining affinity and potency.
Next, we focused our efforts on the terminal accessory binding fragment that is designed
to impart multifunctional properties to our compounds for combating neurodegeneration in
PD. Structural motifs present in naturally-occurring compounds including polyphenols and
curcumin, were substituted as the accessory binding fragments. Effects of various hydroxyl
or methoxy substitutions on the phenyl ring were investigated through compounds (±)-7a,
12

(±)-8a, (±)-32, and (±)-33. We explored compounds with either an ortho- or a para-
substitution on the aromatic ring, as such substitution has a potential of forming a
quinone/semiquinone moiety for SN aggregation. Compound (±)-7a, with a hydroxyl at
the p-position and a methoxy at the m-position, exhibited potent binding affinity for D3
receptor (Ki, D3=3.62 nM) and moderate affinity for the D2 receptor (Ki, D2=175 nM).
Replacement of m-methoxy group in (±)-7a with a hydroxyl group resulting in (±)-8a
exhibited a moderate improvement for both D2 and D3 binding (Ki, D2=116 nM, D3=2.59 nM,
Table 1). Among the three-carbon linker analogs, the dihydroxyl analog (±)-33 showed a
significant increase in affinity for D2 receptor (Ki, D2=499 nM) as compared to the methoxy-
substituted analog (±)-32 (Ki, D2= 2,404 nM). However, for D3 binding, (±)-33 (Ki, D3=11.9
nM) showed little or no enhancement over (±)-32 (Ki, D3=15.8 nM). These results
demonstrate that the dihydroxy-compounds (±)-8a and (±)-32 exhibited better binding
profiles. Next, to briefly assess the effect of substituent position, compound (±)-8b, having
hydroxyls at the 2′- and 5′-positions, was synthesized. Compound (±)-8b exhibited
comparable binding affinity for D2 receptor and a slightly improved binding for D3 receptor
compared to its counterpart (±)-8a (Ki, D2= 117 vs. 116 nM, D3= 1.01 vs. 2.59 nM).
Thereafter, to investigate the effect of a dihydroxyl substituent in the presence of an
unsaturated linker we pursued synthesis of compound (±)-44. However, probably as a
result of air oxidation, the resulting compound was obtained as an inseparable mixture of
(±)-44 and dione (±)-45 which showed moderate affinity for both D2 and D3 receptors (Ki,
D2=121 nM, D3=25.0 nM).
Our previous studies have demonstrated that compounds with (−)-(S)-isomer of
pramipexole as head group generally resulted in greater binding affinity than their (+)-(R)-
isomer counterparts.32 Thus, (−)-isomers of selected compounds 7a, 8a, 8b, 20 were
synthesized. Compounds (−)-7a (Ki, D2=68.3 nM, D3=1.57 nM) and (−)-8a (Ki, D2=65.7 nM,
D3=1.42 nM) demonstrated improved binding profile, particularly for the D2 receptor, in
comparison to their racemic counterparts (±)-7a and (±)-8a, respectively. Compound (−)-
13

8b exhibited a binding Ki, for D3 (0.78 nM) that was among the higher affinity values
observed in this series of compounds. In general, (S)-isomers displayed somewhat higher
binding affinities for both D2/D3 receptors than their racemic counterparts except for (−)-20
(Ki, D2=123 nM, D3=22.3 nM) where a decrease of D3 binding and little or no change in D2
binding was observed.
A bioisosteric replacement of the 2-aminothiazole head group in compound (−)-8a with
aminotetralin moiety to give (−)-14 was performed. In consistence with our previous data,
the aminotetralin analog (−)-14 (Ki, D2=4.67 nM, D3=0.493 nM) has shown much better
binding affinity at both D2 and D3 receptors compared to the 2-aminothiazole derivative (−)-
8a (Ki, D2=65.7 nM, D3=1.42 nM).
These optically active lead compounds 7a, 8a, 8b, 14 and 20 were also assessed by
measuring stimulation of [35S]GTPγS binding in comparison to dopamine, the full agonist,
to evaluate their ability of activating human dopamine hD2 and hD3 receptors expressed in
CHO cells. Compounds (−)-7a, (−)-8a, (−)-8b, (−)-14, (−)-20 exerted high functional
potency and efficacy at both D2/D3 receptors (Table 2). In contrast to the more selective
binding affinity observed for D3 receptor, all tested compounds turned out to be comparably
potent and full agonist at both D2 and D3 receptors. Compound (−)-7a was the most potent
among the 2-aminothiazole derivatives with a very high functional potency (EC50 (GTPγS);
D2 =2.09 nM and D3 =0.78 nM) and full agonist activity (%Emax, D2=96.2, D3=94.4) at both
D2 and D3 receptors. Similarly, compound (−)-8b also turned out to be an agonist with
comparable potency (EC50 (GTPγS); D2=9.3 nM and D3=1.22 nM). On the other hand, the
aminotetralin analog (−)-14, showed a non-selective profile (D2/D3=0.82). When compared
to its 2-aminothiazole derivative counterpart (−)-8a, a great improvement in potency at
both D2/D3 receptors (EC50 (GTPγS); D2 =0.27 vs. 22.9 nM and D3 =0.33 vs. 2.62 nM for
(−)-14 and (−)-8a, respectively) was observed. As for the efficacy, a decrease at D2
receptor and an increase at the D3 receptor were observed when comparing compound
(−)-14 to (−)-8a (%Emax, D2= 88.8 vs. 97.4, D3=90.9 vs. 83.4).
14

Table 1. Ki values (nM) for inhibition of [3H] spiroperidol binding (HEK - D2, 3 cells)a (cLogP
and tPSA values are calculated using ChemDraw)
Ki (nM), [3H]spiperone
Compound
D2L/D3 cLogP tPSA
D2L
D3
dopamineb
(−)-5-OH-DPAT
(±)-7a, D-548
(−)-7a, D-591
(±)-8a, D-575
(−)-8a, D-593
(±)-8b, D-584
(−)-8b, D-601
(±)-12, D-567
(−)-14, D-644
(±)-20, D-604
(−)-20, D-618
(±)-26, D-606
(±)-32, D-547
(±)-33, D-573
(±)-38, D-570
(±)-44&45, D-592
990 ± 198
101 ± 14.9
10
74
153 ± 32
175 ± 8
2.07 ± 0.38
3.62 ± 0.78
1.57 ± 0.41
2.59 ± 0.31
1.42 ± 0.14
1.01 ± 0.28
0.78 ± 0.20
23.3 ± 4.0
48
43
45
46
116
143
10
9
4.42 71.08
4.42 71.08
3.97 82.08
3.97 82.08
3.85 82.08
3.85 82.08
4.12 71.08
5.22 63.93
4.12 71.08
4.12 71.08
4.95 71.08
3.36 71.08
2.92 80.08
3.26 71.08
68.3 ± 12.0
116 ± 8
65.7 ± 11.7
117 ± 17
112 ± 8
233 ± 8
4.67 ± 0.65 0.493 ± 0.067
150 ± 20
123 ± 18
4.24 ± 0.74
22.3 ± 2.2
35
6
58.5 ± 7.3 0.675 ± 0.178
87
153
42
119
5
2,404 ± 29
499 ± 16
15.8 ± 2.0
11.9 ± 1.9
39.1 ± 5.4
25.0 ± 1.96
4,634 ± 325
121 ± 21
15

a Results are expressed as means ± SEM for three to four experiments, each performed in
triplicate.
b Data was obtained under almost identical assay conditions.33
Table 2. Stimulation of [35S]GTPγS Binding to hD2 and hD3 receptors expressed in CHO
cellsa
CHO-D2
[35S]GTPγS
CHO-D3
[35S]GTPγS
Compound
D2/D3
%Emax
%Emax
EC50 (nM)
EC50 (nM)
Dopamine
(−)-7a, D-591
(−)-8a, D-593
(−)-8b, D-601
(−)-14, D-644
(−)-20, D-618
376 ± 39
100
7.26 ± 0.21
100
51.79
2.09 ± 0.22 96.2 ± 2.3 0.78 ± 0.10 94.4 ± 4.2 2.68
22.9 ± 1.6 97.4 ± 3.6 2.62 ± 0.49 83.4 ± 8.2 8.74
9.3 ± 2.49 88.0 ± 3.6 1.22 ± 0.30 90.9 ± 5.5 7.62
0.27 ± 0.62 88.8 ± 3.5 0.33 ± 0.09 90.9 ± 2.6 0.82
37.7 ± 5.6 84.3 ± 3.6 20.4 ± 5.8 87.5 ± 6.2 1.85
a Results are expressed as means ± SEM for three to five experiments, each performed in
triplicate.
3.2 Reversal of Reserpine-Induced Hypolocomotion in Rats
Catalepsy was induced in rats by administrating reserpine, which can block the vesicular
monoamine transporter (VMAT) and subsequently lead to depletion of monoamines in the
synapse of the peripheral sympathetic nerve terminals.34-36 In this well-established PD
model, significant inhibition of locomotion of rats, which indicates the development of
akinesia, was observed 18 h after administrating reserpine (5 mg/kg, s.c.) (Figure 3). The
ability of compound (−)-8a to reverse the reserpine-induced hypolocomotion was
investigated in this assay because of its high affinity and efficacy at D2 and D3 receptors in
the receptor assays. At the dose of 10 µmol/kg i.p., compound (−)-8a has effectively
reversed akinesia induced by reserpine treatment and displayed a steady continuous
16

effect in comparison to the peak effect from reference drug ropinirole (Figure 3). This
significant locomotor stimulation is likely to be mediated by postsynaptic D2/D3 receptor
activation, which suggested that compound (−)-8a can cross the blood-brain barrier
effectively and function as a potent agonist.
Figure 3. Effect of compound (−)-8a and ropinirole on reserpine-induced (5.0 mg/kg, s.c.)
hypolocomotion in rats. Results are expressed as Mean ± SEM; n=3 rats per value.
Horizontal activity (HACTV), which is the total number of beam interruptions that occurred
in the horizontal sensor during a given sample period, was measured as described in the
Experimental Section. Locomotor data during each 30-min interval after the administration
of compound (−)-8a (i.p.) or ropinirole (i.p.) at the dose of 10 µmol/kg compared to control
reserpine treated rats in 18 h post reserpine treatment was plotted. Differences among
treatments were significant by one-way ANOVA analysis (F (2, 36) = 41.24; p< 0.0001).
Dunnett’s analysis following ANOVA revealed that the effect of compound (−)-8a (p<
0.0001) was significantly different when compared to reserpine control.
3.3 Neuroprotection against 6-OHDA-inudced Toxicity
6-OHDA is a dopaminergic neurotoxin that is widely used to study PD as it is known to
cause cell death via production of reactive oxygen species in a dose dependent manner.
17

37,38
From our previous study, treatment of PC12 cells with 75 µM 6-OHDA for 24 h
resulted in a significant decrease of cell viability.32 We selected three representative
compounds (−)-8a, (−)-14 and (−)-20 to evaluate their dose dependent effect in reversing
toxicity of 6-OHDA. These three compounds were chosen based on their structural
diversity on terminal aromatic functionalization and the agonist head groups. To evaluate
the protective effect of selected compounds against 6-OHDA-induced toxicity, PC12 cells
were first pretreated 24 h with increasing doses of compound (−)-8a (0.1-30 µM), (−)-14
(0.1-30 µM), or (−)-20 (1-50 µM), followed by single treatment of 75 µM 6-OHDA for
additional 24 h. Cell viability was assessed by MTT assay. As shown in Figure 4,
compound (−)-8a, (−)-14, and (−)-20 are able to protect cells against 6-OHDA-induced
toxicity in a dose-dependent manner. The greatest protection (18-25%) was seen at 30 µM
dose point for all the three compounds.
Figure 4. Dose-dependent effect on the viability of PC12 cells by 24 h pretreatment of
compounds (−)-8a (a), (−)-20 (b), and (−)-14 (c) at various concentrations followed by
single treatment of 75 µM 6-OHDA. The results shown are mean ± SEM of three
independent experiments performed in seven to eight replicates. One-way ANOVA
followed by Tukey’s multiple comparison post hoc test was performed (#### p ≤ 0.0001
compared to the Ctrl; * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001 compared to the 6-OHDA
control).
3.4 Evaluation of Antifibrillar Activity of (−)-8a, (−)-14 on SN Aggregation
°
In this experiment, SN was shaken at 37 C for six days to form aggregates to yield β-
18

sheet-positive fibrillar structures, which were detected by thioflavin T (ThT). Next, we
wanted to observe the effect of our lead compounds, (−)-8a (D-593) and (−)-14 (D-644),
and the reference control dopamine agonists, (−)-pramipexole and (−)-5-OHDPAT, on
aggregation of SN by monitoring the ThT activity. We wanted to test the control dopamine
agonists since our lead agonists (−)-8a and (−)-14 were derived from them. Figure 5
displays the activity of ThT when SN was shaken alone as well as in the presence of the
drugs. A significant inhibition of ThT activity was observed at day 6 in the presence of lead
compounds (−)-8a and (−)-14 compared to SN alone, indicating alteration of the
aggregation process in the presence of drugs. Interestingly, dopamine agonists
pramipexole and 5-OHDPAT slightly enhanced aggregation activity of SN over the six
days shaking. The results indicate distinct capacity of lead hybrid drugs to inhibit
aggregation whereas their dopamine agonists precursors ((−)-pramipexole and (−)-5-
OHDPAT) enhanced the aggregation.
19

Figure 5: Modulation of SN fibrillization by (−)-8a (D-593) and (−)-14 (D-644) as well as
by the control drugs (−)-pramipexole and (−)-5-OHDPAT. The effect on ThT activity from
shaking of SN alone for six days (SN d6) is normalized with respect to other treatments.
The drugs were incubated with SN over a period of 6 days, and the fibrillization was
measured by ThT fluorescence assay. The drugs were also tested alone to show any
effect of drugs in ThT fluorescence activity. Data values shown are means ± SEM of three
independent experiments. One-way ANOVA followed by Tukey’s multiple comparison post
hoc test was performed (** p ≤ 0.01, * p ≤ 0.05 compared to the SN d6).
4. Conclusion
Based on our modified hybrid molecular template for multifunctional D2/D3 agonists, we
have carried out an initial SAR study on a series of catechol compounds and related
analogs as novel multifunctional agents for the treatment of PD. The current study
demonstrated the effect of variations in the linker and the accessory binding site fragments
on D2/D3 receptor activity. In general, five or higher carbon linker lengths were tolerated the
best for receptor interaction. Compounds (−)-7a, (−)-8a, (−)-8b, (−)-14 and (−)-20 have
exhibited high agonist potency at both D2/D3 receptor, and (−)-8a was tested in vivo and
displayed efficacious activity in reserpine-induced hypolocomotion rat model. We have
shown that compounds (−)-8a, (−)-14, and (−)-20 are neuroprotective in an in vitro
neuroprotection PD model involving dopaminergic PC12 cells treated with neurotoxin 6-
OHDA. In addition, the lead compounds (−)-8a and (−)-14 can greatly inhibit SN
aggregation as shown by ThT assay. Thus, the lead compound (−)-8a seems to be a
promising D2/D3 agonist with neuroprotective activity, and should have the potential to
provide symptom-relieving and disease-modifying effect for the treatment of PD.
5. Experimental Section
All reagents and solvents were purchased from commercial suppliers and used as
20

received unless otherwise stated. Dry solvent was obtained according to the standard
procedure. All reactions were performed under inert atmosphere (N2) unless otherwise
indicated. Analytical silica gel 60 F254-coated TLC plates were obtained from EMD
Chemicals, Inc. and were visualized with UV light or by treatment with phosphomolybdic
acid (PMA), Dragendorff’s reagent, or ninhydrin. Flash column chromatographic
1
purifications were performed using Whatman Purasil 60A silica gel 230−400 mesh. H
NMR spectra were acquired on a Varian 400 and 600 MHz FT NMR spectrometer using
tetramethylsilane (TMS) as the internal standard. The NMR solvent used was CDCl3 or
CD3OD as indicated. Optical rotations were recorded on PerkinElmer 241 polarimeter.
Melting points were recorded using a MEL-TEMP II (Laboratory Devices Inc., Placerville,
CA) capillary melting point apparatus and were uncorrected. Elemental analyses were
performed by Atlantic Microlab, Inc.
5.1. Procedure A: (4-Ethoxy-4-oxobutyl)triphenylphosphonium bromide (2)
Triphenylphosphine (3.76 g, 14.34 mmol) and ethyl 4-bromobutyrate (2.0 g, 10.25 mmol)
were added to a dried round bottom flask under argon. The reaction mixture was heated to
120 °C under condenser for 16 h after which the reaction was allowed to come to room
temperature. CH2Cl2 (10 mL) was added, followed by diethyl ether until no further
precipitation of product was observed. The precipitate was further washed with ether (100
mL) and dried in vacuo to give pure compound 2 as a white solid (4.64 g) in quantitative
yield. 1H NMR (400 MHz, CDCl3): δ ppm 7.67-7.90 (m, 15 H), 3.98-4.12 (m, 4H), 2.86-2.89
(m, 2H), 1.88-1.94 (m, 2H), 1.22 (t, J = 7.2 Hz, 3H).
5.2. Procedure B: Ethyl 5-(4-hydroxy-3-methoxyphenyl)pent-4-enoate (4a)
Starting material 2 (6.61 g, 14.45 mmol) was added to dry THF (15 mL) in an oven-dried
round bottom flask. The resulting suspension was cooled to -78 °C. Then a solution of
NaHMDS (1M in THF, 15.77 mL, 15.77 mmol) was added dropwise, and the reaction
mixture was stirred at -78 °C for 1 h. Thereafter, a solution of vanillin 3a (2.0 g, 13.14 mmol)
in dry THF (5 mL) was added dropwise, and reaction stirred at -78 °C for 2 h. Then it was
21

warmed to room temperature and stirred for 48 h. The reaction mixture was then extracted
with ethyl acetate (2 ×100 mL) and washed with brine. The combined organic layer was
dried over Na2SO4 and concentrated in vacuo. The crude thus obtained was purified by
column chromatography using 10 % ethyl acetate in hexanes to yield compound 4a (1.17 g,
36%) as colorless oil with a preferential Z:E ratio of > 20:1. 1H NMR (400 MHz, CDCl3): δ
ppm 6.88 (d, J = 8.2 Hz, 1H), 6.79-6.82 (m, 2H), 6.37 (d, J = 11.2 Hz, 1H), 5.64 (s, 1H),
5.49-5.55 (m, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.88 (s, 3H), 2.63-2.69 (m, 2H), 2.45 (t, J = 8.0
Hz, 2H), 1.24 (t, J = 7.2 Hz, 3H).
5.3. Ethyl 5-(2,5-dimethoxyphenyl)pent-4-enoate (4b)
Starting material 2 (7.56 g, 16.53 mmol) was reacted with 2,5-dimethoxy benzaldehyde 3b
(2.5 g, 15.04 mmol) in the presence of NaHMDS 1M in THF (18.05 mmol, 18.05 mL)
according to procedure B. The crude was purified by column chromatography using 7-10%
1
ethyl acetate in hexanes to give compound 4b (1.71 g, 43%). H NMR (400 MHz, CDCl3):
δ ppm 6.82 (d, J = 2.4 Hz, 1H), 6.74-6.79 (m, 2H), 6.52 (d, J = 11.2 Hz, 1H), 5.65-5.72 (m,
1H), 4.12 (q, J = 7.2 Hz, 2H), 3.78 (s, 3H), 3.77 (s, 3H), 2.55-2.60 (m, 2H), 2.41 (t, J = 7.2
Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H).
5.4. Procedure C: Ethyl 5-(4-hydroxy-3-methoxyphenyl)pentanoate (5a)
Intermediate 4a (1.0 g, 4.00 mmol) was dissolved in ethanol (10 mL) in a round bottom
flask, and 10% Pd/C (0.10 g, 10 wt%) was added to it. The reaction flask was degassed,
and then the reaction mixture was stirred under hydrogen atmosphere for 2 h at room
temperature. After the completion of reaction, the mixture was diluted with ethanol (20 mL)
and passed through a short bed of celite. The organic layer was concentrated to afford
1
compound 5a (0.98 g) in quantitative yield, which was pure enough for the next step. H
NMR (400 MHz, CDCl3): δ ppm 6.82 (d, J = 8.0 Hz, 1H), 6.65-6.67 (m, 2H), 4.12 (q, J = 7.2
Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 3.87 (s, 3H), 2.31 (t, J = 7.2 Hz, 2H), 1.58-1.70 (m,4H),
1.24 (t, J = 7.2 Hz, 3H).
5.5. Ethyl 5-(2,5-dimethoxyphenyl)pentanoate (5b)
22

Intermediate 4b (1.0 g, 3.78 mmol) was dissolved in ethanol (10 mL) and reacted with 10%
Pd/C (0.1 g, 10 wt%) according to procedure C to afford compound 5b (0.96 g, 95%),
which was used without further purification. 1H NMR (400 MHz, CDCl3): δ ppm 6.75 (d, J =
8.8 Hz, 1H), 6.72 (d, J = 3.2 Hz, 1H), 6.68 (dd, J = 8.4, 2.4 Hz, 1H), 4.12 (q, J = 7.2 Hz,
2H), 3.76 (s, 3H), 3.75 (s, 3H), 2.60 (t, J = 7.4 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.56-1.70
(m, 4H), 1.24 (t, J = 7.2 Hz, 3H).
5.6. Procedure D: 5-(4-Hydroxy-3-methoxyphenyl)pentanal (6a)
To a solution of compound 5a (0.66 g, 2.62 mmol) in dry toluene (10 mL) under argon was
added DIBALH solution (1M in hexane, 2.87 mL, 2.87 mmol) at -78 °C. The reaction
mixture was stirred at -78 °C for 2 h. MeOH (0.20 mL) was added to the reaction mixture,
and the reaction mixture was allowed to come to 0 °C. It was then added to a separating
funnel containing HCl (1N, 10 mL) and extracted with ethyl acetate (3 × 30 mL). The
combined organic layer was washed with brine, dried over Na2SO4, and evaporated under
reduced pressure. The crude was purified by column chromatography using 11-15% ethyl
acetate in hexanes to yield compound 6a (0.23 g, 42%) as colorless oil.1H NMR (400 MHz,
CDCl3): δ ppm 9.75 (t, J = 1.6 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.65 (m, 2H), 3.87 (s, 3H),
2.56 (t, J = 7.2 Hz, 2H), 2.44 (td, J1 = 6.40 Hz, J2 = 1.60 Hz 2H), 1.59-1.68 (m, 4H).
5.7. 5-(2,5-Dimethoxyphenyl)pentanal (6b)
Intermediate 5b (0.6 g, 2.25 mmol) was reduced with DIBALH solution (1M in hexane, 2.47
mL, 2.47 mmol) in dry toluene (10 mL) using procedure D. The crude was purified by
column chromatography using 11-15% ethyl acetate in hexanes to give compound 6b (0.4
g, 80 %) as yellow oil. 1H NMR (400 MHz, CDCl3): δ ppm 9.74 (d, J = 1.6 Hz, 1H), 6.76 (d,
J = 8.8 Hz, 1H), 6.67-6.71 (m, 2H), 3.76 (s, 3H), 3.75 (s, 3H), 2.60 (t, J = 6.4 Hz, 2H), 2.44
(t, J = 6.8 Hz, 2H), 1.58-1.37(m, 4H).
5.8. Procedure E: 4-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pentyl)-2-methoxyphenol ((±)-7a) (D-548).
Into a stirring solution of (±)-pramipexole (0.15 g, 0.71mmol) in CH2Cl2 (8 mL) was added
23

aldehyde 6a (0.148 g, 0.71 mmol), and the mixture stirred for 1 h. NaBH(OAc)3 (0.27 g,
1.27 mmol) was then added portion-wise followed by MeOH (0.8 mL). The reaction was
stirred for 36 h at room temperature. The reaction mixture was quenched with a sat.
NaHCO3 solution at 0 °C and extracted with CH2Cl2 (3 × 25 mL). The combined organic
layer was dried over Na2SO4, and solvent was removed under reduced pressure. The
crude product was purified by column chromatography using 5-7% MeOH in CH2Cl2 to
give compound (±)-7a (0.2 g, 70%).1H NMR (400 MHz, DMSO-d6): δ ppm 6.64-6.72 (m,
2H), 6.58 (d, J = 8.4 Hz, 1H), 3.82 (s, 3H), 3.05 (bs, 1H), 2.50-2.67 (m, 10H), 1.59-1.70 (m,
3H), 1.48-1.53 (m, 4H), 1.28-1.36 (m, 3H), 0.89 (t, J = 7.2 Hz, 3H). The free base was
converted into its corresponding hydrochloride salt. Mp 224-226 °C. Anal. (C26H43Cl2N3O4S)
C, H, N.
5.9. (S)-4-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)pentyl)-
2-methoxyphenol ((−)-7a) (D-591)
Aldehyde 6a (0.148 g, 0.71 mmol), (−)-pramipexole (0.15 g, 0.71mmol), and NaBH(OAc)3
(0.27 g, 1.27 mmol) in CH2Cl2 (8 mL) and MeOH (0.8 mL) were reacted using procedure E,
and the resulting crude was purified by column chromatography using 5-7% MeOH in
CH2Cl2 to give compound (−)-7a (0.19 g, 66%). 1H NMR (600 MHz, CD3OD): δ ppm 6.64-
6.72 (m, 2H), 6.58 (d, J = 8.4 Hz, 1H), 3.82 (s, 3H), 3.05 (bs, 1H), 2.65-2.67 (m, 1H), 2.50-
2.65 (m, 9H), 1.59-1.70 (m, 3H), 1.48-1.53 (m, 4H), 1.28-1.36 (m, 3H), 0.90 (t, J = 7.2 Hz,
3H). 13C NMR (150 MHz, CD3OD): δ ppm 170.18, 147.44, 145.60, 144.20, 133.48, 133.15,
120.41, 114.63, 111.74, 58.87, 54.95, 53.12, 51.70, 34.73, 30.69, 25.76, 24.86, 22.57,
22.04, 21.57, 18.46, 9.82. The free base was converted into its corresponding
25
hydrochloride salt. [α]D (salt) = −46.8 (c = 0.5 in CH3OH). Mp 196-198 °C. Anal.
(C22H37Cl2N3O3S) C, H, N.
5.10. N6-(5-(2,5-Dimethoxyphenyl)pentyl)-N6-propyl-4,5,6,7-
tetrahydrobenzo[d]thiazole-2,6-diamine ((±)-7b)
Aldehyde 6b (0.157 g, 0.71 mmol), (±)-pramipexole (0.15 g, 0.71 mmol), and NaBH(OAc)3
24

(0.27 g, 1.27 mmol) in CH2Cl2 (9 mL) and MeOH (0.8 mL) were reacted using procedure E,
and the resulting crude was purified by column chromatography using 5-7% MeOH in
CH2Cl2 to give compound (±)-7b (0.17 g, 58%). 1H NMR (400 MHz, CDCl3): δ ppm 6.75 (d,
J = 8.8 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.66 (dd, J = 8.8, 2.1 Hz, 1H), 3.75 (s, 3H), 3.74
(s, 3H), 3.07 (bs, 1H), 2.69 (dd, J = 12.0, 4.04 Hz, 2H), 2.47-2.58 (m, 8H), 1.99-2.03(m,
1H), 1.68-1.78 (m, 1H), 1.40-1.61 (m, 6H), 1.31-1.37 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).
5.11. (S)-N6-(5-(2,5-Dimethoxyphenyl)pentyl)-N6-propyl-4,5,6,7-
tetrahydrobenzo[d]thiazole-2,6-diamine ((−)-7b)
Aldehyde 6b (0.105 g, 0. 47 mmol), (−)-pramipexole (0.10 g, 0.47 mmol), and NaBH(OAc)3
(0.18 g, 0.85 mmol) in CH2Cl2 (7 mL) and MeOH (0.6 mL) were reacted using procedure E,
and the resulting crude was purified by column chromatography using 5-7% MeOH in
1
CH2Cl2 to give compound (−)-7b (0.105 g, 53%). H NMR (600 MHz, CDCl3): δ ppm 6.76
(d, J = 8.4 Hz, 1H), 6.71 (d, J = 1.8 Hz, 1H), 6.67 (dd, J = 8.4, 3.0 Hz, 1H), 4.71 (bs, 2H),
3.77 (s, 3H), 3.76 (s, 3H), 3.04 (bs, 1H), 2.69 (d, J = 7.2 Hz, 2H), 2.4-2.6 (m, 8H), 2.0 (bs,
1H), 1.62-1.72 (m, 2H), 1.55-1.61 (m, 2H), 1.41-1.52 (m, 3H), 1.33-1.37 (m, 2H), 0.88 (t, J
= 7.2 Hz, 3H).
5.12. Procedure F: 4-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pentyl)benzene-1,2-diol ((±)-8a) (D-575)
BBr3 1M in CH2Cl2 (2.66 mL, 2.66 mmol) was added to a solution of compound (±)-7a (0.2
g, 0.50 mmol) in CH2Cl2 (20 mL) at -78 °C and stirred for 2 h. The reaction mixture was
allowed to come to room temperature and stirred for another 4 h. The reaction was then
quenched by MeOH (20 mL) at 0° C, and the solvent was concentrated in vacuo. MeOH
(20 mL) was added to the residue and again evaporated. This process was repeated three
times. The residue obtained was purified by column chromatography using 10-15% MeOH
1
in CH2Cl2 to give pure compound (±)-8a (0.123 g, 64 %). H NMR (400 MHz, CD3OD): δ
ppm 6.64 (d, J = 8.0 Hz, 1H), 6.60 (d, J = 2.4Hz, 1H), 6.47 (dd, J = 8.0, 2.4 Hz, 1H), 3.35-
3.40 (m, 1H), 2.75-2.85 (m, 5H), 2.62-2.71 (m, 2H), 2.55-2.57 (m, 1H), 2.47 (t, J = 7.2 Hz,
25

2H), 2.09-2.14 (m, 1H), 1.79-1.89 (m, 1H), 1.57-1.64 (m, 6H), 1.31-1.38 (m, 2H), 0.95 (t, J
= 7.6 Hz, 3H). 13C NMR (100 MHz, CD3OD): δ ppm 168.79, 144.70, 143.39, 142.80,
133.78, 119.23, 115.11,114.79, 113.73, 58.94, 52.66, 50.93, 34.65, 30.97, 26.37, 26.17,
25.14, 24.33, 23.50, 19.79, 10.29. The free base was converted into its corresponding
hydrochloride salt. Mp 196-198 °C. Anal. (C21H34Cl3N3O2S) C, H, N.
5.13. (S)-4-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pentyl)benzene-1,2-diol ((−)-8a) (D-593)
BBr3 1M in CH2Cl2 (1.61mL, 1.61 mmol) was reacted with compound (−)-7a (0.132 g, 0.33
mmol) in CH2Cl2 (10 mL) according to Procedure F to afford crude, which was purified by
column chromatography using 10-15% MeOH in CH2Cl2 to give compound (−)-8a (90 mg,
1
71%). H NMR (600 MHz, CD3OD): δ ppm 6.63 (d, J = 7.8 Hz, 1H), 6.58 (d, J = 1.8 Hz,
1H), 6.45 (dd, J = 7.8, 1.8 Hz, 1H), 2.97-3.02 (m, 1H), 2.42-2.62 (m, 10H), 1.95-1.97 (m,
1H), 1.60-1.73 (m, 1H), 1.53-1.58 (quintet, J = 7.8Hz, 2H), 1.42-1.48 (septet, J = 7.2 Hz,
4H), 1.26-1.31 (quintet, J = 7.2 Hz, 2H), 0.88 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz,
CD3OD): δ ppm 168.35, 144.65, 143.49, 142.71, 134.05, 119.21, 115.10, 114.77, 114.64,
25
57.64, 52.52, 50.53, 34.82, 31.33, 27.97, 26.66, 25.74, 25.21, 24.07, 21.22, 10.78. [α]D
(free base) = − 51.8 (c = 1.0 in CH3OH); the free base was converted into its
corresponding hydrochloride salt. Mp 211-212 °C. Anal. (C21H35Cl2N3O3S) C, H, N.
5.14. 2-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pentyl)benzene-1,4-diol ((±)-8b) (D-584)
BBr3 1M in CH2Cl2 (2.27 mL, 2.27 mmol) was reacted with compound (±)-7b (0.16 g, 0.38
mmol) according to Procedure F to afford crude, which was purified by column
chromatography using 10-15% MeOH in CH2Cl2 to afford compound (±)-8b (0.12 g, 80%).
1H NMR (600 MHz, CD3OD): δ ppm 6.55 (d, J = 8.4 Hz, 1H), 6.51 (d, J = 3.0 Hz, 1H), 6.41
(dd, J = 8.4, 3.0 Hz, 1H), 3.0 (m, 1H), 2.44-2.63 61 (m, 10H), 1.98 (d, J = 10.2 Hz, 1H),
1.65-1.68 (m, 1H), 1.56-1.61 (m, 2H), 1.44-1.52 (m, 4H), 1.32-1.36 (m, 2H), 0.88 (t, J = 7.2
Hz, 3H). 13C NMR (150 MHz, CD3OD): δ ppm 168.33, 149.56, 147.69, 143.48, 129.72,
26

116.27, 115.16, 114.71, 112.46, 57.62, 52.54, 50.50, 29.79, 29.44, 27.94, 26.88, 25.74,
25.19, 24.10, 21.22, 10.89. The free base was converted into its corresponding
hydrochloride salt. Mp 217-219 °C. Anal. (C22H38Cl3N3O3S) C, H, N.
5.15. (S)-2-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pentyl)benzene-1,4-diol ((−)-8b) (D-601)
BBr3 1M in CH2Cl2 (1.33 mL, 1.33 mmol) was reacted with compound (−)-7b (93 mg, 0.22
mmol) in CH2Cl2 (6 mL) according to Procedure F to afford crude, which was purified by
column chromatography using 10-15% MeOH in CH2Cl2 to give compound (−)-8b (62 mg,
1
71%). H NMR (600 MHz, CD3OD): δ ppm 6.56 (dd, J = 8.4, 1.8 Hz, 1H), 6.52 (d, J = 2.4
Hz, 1H), 6.43 (dt, J = 8.4, 1.80 Hz, 1H), 2.98-3.03 (m, 1H), 2.44-2.64 (m, 10H), 1.99-2.00
(m, 1H), 1.63-1.71 (m, 1H), 1.57-1.62 (m, 2H), 1.46-1.52 (m, 4H), 1.32-1.37 (m, 2H), 0.90
13
(t, J = 7.2 Hz, 3H). C NMR (150 MHz, CD3OD): δ ppm 168.31, 149.56, 147.69, 143.49,
129.73, 116.22, 115.20, 114.79, 112.49, 57.58, 52.54, 50.49, 29.79, 29.44, 28.00, 26.89,
25.76, 25.24, 24.11, 21.27, 10.78.. The free base was converted into its corresponding
25
hydrochloride salt. [α]D (salt) = −18.4 (c = 0.5 in CH3OH); Mp 222-224 °C. Anal.
(C21H34Cl3N3O2S) C, H, N.
5.16. Procedure G: (Z)-Ethyl 5-(4-((tert-butyldimethylsilyl)oxy)-3-
methoxyphenyl)pent-4-enoate (9)
The (Z)-isomer of 4a was obtained by further purification of the mixture of (E)-, (Z)-isomers
through column chromatography using 10% ethyl acetate in hexanes. Imidazole (0.35 g,
5.14 mmol) was added to a solution of intermediate 4a (0.52 g, 2.08 mmol) and tert-
butyldimethylsilyl chloride (0.37 g, 2.45 mmol) in DMF (5 mL). The solution was stirred at
room temperature for 2 h. After the completion of reaction, sat. NaHCO3 (10 mL) was
added and stirred for another 30 min. The reaction mixture was extracted with CH2Cl2 (2 x
50 mL). The combined organic layer was dried over Na2SO4 and concentrated in vacuo.
The crude thus obtained was purified by column chromatography using 5% ethyl acetate in
hexanes to afford compound 9 (0.75 g) in quantitative yield. 1H NMR (400 MHz, CD3OD): δ
27

ppm 6.85 (s, 1H), 6.79 (d, J = 8.0 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 6.37 (d, J = 11.2 Hz,
1H), 5.47-5.55 (m, 1H), 4.10 (m, J = 7.2 Hz, 2H), 3.78 (s, 3H), 2.63 (q, J = 7.2 Hz, 2H),
2.44 (t, J = 7.2 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H), 0.99 (s, 9H), 0.13 (s, 6H).
5.17. Procedure H: (Z)-5-(4-((tert-Butyldimethylsilyl)oxy)-3-methoxyphenyl)pent-4-en-
1-ol (10)
Into a stirring solution of compound 9 (0.71 g, 1.95 mmol) in anhydrous THF (15 mL) was
added DIBAL-H solution 1 M in THF (9.73 mL, 9.73 mmol) dropwise at -10 °C. The
reaction mixture was stirred at room temperature for 6 h and was quenched with MeOH (1
mL). The mixture was neutralized with 1N HCl (15 mL) solution. The organic layer was
separated, and the aqueous layer was extracted with ethyl acetate (2 x 100 mL). The
combined organic layer was dried over Na2SO4 and concentrated in vacuo. The crude
product was purified by column chromatography using 11-15% ethyl acetate in hexanes to
1
give compound 10 (0.55 g, 88%). H NMR (CDCl3, 400 MHz): δ ppm 6.74-6.85 (m, 3H),
6.37 (d, J = 11.2 Hz, 1H), 5.48-5.53 (m, 1H), 3.80 (s, 3H), 2.67 (q, J = 7.2 Hz, 2H), 2.43 (t,
J = 7.2 Hz, 2H), 1.22 (t, J = 7.2 Hz, 2H), 1.06 (s, 9H), 0.15 (s, 6H).
5.18. Procedure I: (Z)-5-(4-((tert-Butyldimethylsilyl)oxy)-3-methoxyphenyl)pent-4-enal
(11)
Alcohol 10 (0.9 g, 2.79 mmol) was slowly added to an ice cooled suspension of PCC (0.80
g, 3.71 mmol) in CH2Cl2 (90 mL), and the mixture was stirred at rt for 9 h. The reaction
mixture was then filtered over celite, and solvent was evaporated to get residue, which was
purified by column chromatography using 10-15% of ethyl acetate in hexanes to give
compound 11 (0.36 g, 40%). 1H NMR (CDCl3, 400 MHz): δ ppm 9.78 (s, 1H), 6.74-6.85 (m,
3H), 6.37 (d, J = 11.2 Hz, 1H), 5.49-5.52 (m, 1H), 3.80 (s, 3H), 2.56-2.68 (m, 4H), 0.99 (s,
9H), 0.16 (s, 6H).
5.19. Procedure J: (Z)-4-(5-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)pent-1-en-1-yl)-2-methoxyphenol ((±)-12) (D-567)
Compound 11 (0.315 g, 0.98 mmol) was dissolved in THF (10 mL) and cooled to 0 °C.
28

TBAF 1M in THF (1.02 mL, 1.02 mmol) was added, and reaction stirred in ice for 1.5 h.
After reaction was complete, 10% NaHCO3 solution (10 mL) was added, and reaction
mixture was extracted with CH2Cl2 (3 x 50 mL) to obtain aldehyde, which was immediately
taken to the next step without further purification. Aldehyde (0.195 g, 0.95 mmol), (±)-
pramipexole (0.20 g, 0.95 mmol), and NaBH(OAC)3 (0.36 g, 1.70 mmol) in CH2Cl2 (7 mL)
and MeOH (1.0 mL) were reacted using procedure E, and the resulting crude was purified
by column chromatography using 5-7% MeOH in CH2Cl2 to give compound (±)-12 (0.148 g,
1
39%). H NMR (400 MHz, CD3OD): δ ppm 6.86 (d, J = 8.0 Hz, 1H), 6.76-6.82 (m, 2H),
6.35 (d, J = 12.4 Hz, 1H), 5.49-5.56 (m, 1H), 4.98 (bs, 1H), 3.86 (s, 3H), 3.1 (bs, 1H), 2.50-
2.71 (m, 8H), 2.37 (q, J = 7.2 Hz, 2H), 2.03 (bs, 1H), 1.44-1.70 (m, 5H), 0.88 (t, J = 7.2 Hz,
13
3H). C NMR ppm (100 MHz, CD3OD): δ ppm 165.63, 147.19, 145.15, 143.41, 129.50,
129.42, 121.48, 114.57, 114.28, 112.11, 58.18, 54.96, 52.60, 50.10, 27.88, 25.84, 24.76,
23.91, 23.35, 20.72, 10.56.. The free base was converted into its corresponding
hydrochloride salt. Mp 212-214 °C. Anal. (C22.5H36Cl3N3O2.5S) C, H, N.
5.20. (S)-2-Methoxy-4-(5-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-
yl)(propyl)amino)pentyl)phenol ((−)-13)
Aldehyde 6a (0.12 g, 0.58 mmol), (−)-5-methoxy-N-propyl-2-aminotetralin (0.11 g, 0.50
mmol), and NaBH(OAc)3 (0.24 g, 1.13 mmol) in CH2Cl2 (8 mL) were reacted using
procedure E, and the resulting crude was purified by column chromatography using 5%
1
MeOH in CH2Cl2 to give compound (±)-13 (0.17 g, 82%). H NMR (600 MHz, CDCl3): δ
ppm 7.08 (t, J = 7.8 Hz, 1H), 6.81 (d, J = 8.4Hz, 1H), 6.70 (d, J = 7.8Hz, 1H), 6.67-6.63 (m,
3H), 3.86 (s, 3H), 3.80 (s, 3H), 3.00-2.97 (m, 1H), 2.94-2.90 (m, 1H), 2.84-2.71 (m, 2H),
2.55-2.45 (m, 7H), 2.07-2.01 (m, 1H), 1.62-1.51 (m, 3H), 1.51-1.44 (m, 4H), 1.43-1.32 (m,
2H), 0.88 (t, J = 7.2 Hz, 3H).
5.21. (S)-4-(5-((5-Hydroxy-1,2,3,4-tetrahydronaphthalen-2-
yl)(propyl)amino)pentyl)benzene-1,2-diol ((−)-14) (D-644)
Compound (−)-13 (95 mg, 0.23 mmol) was treated with 48% hydrobromic acid (9.5 mL)
29