In our overall goal to develop multifunctional dopamine D2/D3 agonist drugs for the treatment of Parkinson's disease (PD), we previously synthesized potent D3 preferring agonist D-264 (1a), which exhibited neuroprotective properties in two animal models of PD. To enhance the in vivo efficacy of 1a, a structure-activity relationship study was carried out. Competitive binding and [(35)S]GTPγS functional assays identified compound (-)-9b as one of the lead molecules with preferential D3 agonist activity (EC50(GTPγS); D3 = 0.10 nM; D2/D3 (EC50): 159). Compounds (-)-9b and (-)-8b exhibited high in vivo activity in two PD animal models, reserpinized and 6-hydroxydopamine (OHDA)-induced unilateral lesioned rats. On the other hand, 1a failed to show any in vivo activity in these models unless the compound was dissolved in 5-10% beta-hydroxy propyl cyclodextrin solution. Lead compounds exhibited appreciable radical scavenging activity. In vitro experiments with dopaminergic MN9D cells indicated neuroprotection by both 1a and (-)-9b from toxicity of MPP+.
In our overall goal to develop multifunctional dopamineD2/D3 agonist drugs for the treatment of Parkinson's disease (PD), we previously synthesized potent D3 preferring agonist D-264 (1a), which exhibited neuroprotective properties in two animal models of PD. To enhance the in vivo efficacy of 1a, a structure-activity relationship study was carried out. Competitive binding and [(35)S]GTPγS functional assays identified compound (-)-9b as one of the lead molecules with preferential D3 agonist activity (EC50(GTPγS); D3 = 0.10 nM; D2/D3 (EC50): 159). Compounds (-)-9b and (-)-8b exhibited high in vivo activity in two PD animal models, reserpinized and 6-hydroxydopamine (OHDA)-induced unilateral lesioned rats. On the other hand, 1a failed to show any in vivo activity in these models unless the compound was dissolved in 5-10% beta-hydroxy propyl cyclodextrin solution. Lead compounds exhibited appreciable radical scavenging activity. In vitro experiments with dopaminergicMN9Dcells indicated neuroprotection by both 1a and (-)-9b from toxicity of MPP+.
Parkinson’s disease (PD) is a progressive
age-related neurodegenerative disorder of the central nervous system
that is characterized by gradual loss of dopaminergic neurons in the
substantia nigra region of the brain.[1] It
is estimated that PD affects 1–2% of the people older than
65 years of age. According to a statistical analysis published by
the Parkinson’s Disease foundation, approximately 60 000
Americans are diagnosed with PD each year, and an estimated 7–10
million people worldwide are living with PD. Common symptoms associated
with PD include rigidity, bradykinesia, resting tremors, postural
instability, and cognitive psychiatric complications.[2−4] The etiology of PD is not clear yet, but it has been shown that
both mitochondrial dysfunction and oxidative stress are interdependent,
which is thought to play a central role in the pathogenesis of the
disease process.[5,6] Oxidative stress and excessive
amounts of metals especially ironcan lead to the formation of reactive
oxygen species (ROS). These mitochondria-derived ROS inhibit mitochondrial
respiration and promote the aggregation of alpha synuclein protein
(αSN), which ultimately forms Lewy bodies (LBs) and Lewy neuritis
(LN).[7] LBs and LN are neuropathological
hallmarks of PD and toxic toward dopaminergic neurons. Levodopa (L-DOPA)
became available in 1960 for the treatment of PD and is still being
considered a main stream therapy.[8] However,
prolonged use of L-DOPA gives rise to “on” and “off”
episodes along with motor fluctuations, and eventual oxidation of
dopamine (DA) derived from L-DOPA further facilitatesneurodegeneration.[9] One of the current strategies of PD therapy is
to delay the initiation of L-DOPA therapy, by using various combinations
of other therapeutic agents including, but not limited to, DA agonists,
inhibitors of DA metabolism.[10] However,
none of these strategies address the limitations of L-DOPA. Therefore,
the need for therapeutic agents with disease-modifying effects is
of paramount importance.The DA receptors, belonging to a class
of G-protein-coupled receptor (GPCR) family, are mainly found in the
central nervous system (CNS) (controlling neuronal signaling thereby
modulating many important behaviors) and in the periphery (to affect
cardiovascular and renal functions).[10] The
D1-like receptors (D1 and D5 subtypes)
and the D2-like receptors (D2, D3, and D4 subtypes) transduce signals via adenylate cyclase,
an effectors molecule. Upon receptor activation, D1-like
receptors activate adenylate cyclase, whereas D2-like receptors
inhibit it. Interestingly, the DA D3 receptor has a different
distribution in the brain compared to the D2 receptor.[11,12] The D3 receptor is found to be densest in the limbic
region of the brain, whereas the highest level of D2 expression
is in the striatum of the midbrain.[13] It
is important to mention that D2 and D3 receptor
subtypes exhibit 50% homology in their amino acid sequence, which
increases to 75–80% in the helical transmembrane spanning domains,
where agonist binding sites are believed to be located.[14,15] This makes the task of developing D3-selective ligands
challenging. Interestingly, DA D3 preferring agonists were
shown to provide an additional neuroprotective effect compared to
the DA D2 receptor agonist, probably via the production
of neurotrophic factor.[16,17] An enormous amount
of work has been done to develop D3 selective agonists
and to identify key pharmacophoric features responsible for selectivity
for D3 receptor over D2.[18−27] It is important to mention that D3 receptor bound to
an antagonist was recently crystallized to provide a detailed molecular
structure.[28]The research from the
past two decades in the PD area has provided more insights into the
basic pathogenetic factors of PD such as roles of oxidative stress,
aggregation of αSN proteins in the form of soluble toxic aggregates
and fibrils, and increased concentration of iron in the PD brain.[29−31] αSN is a component of Lewy bodies, a pathological hallmark
of PD. αSN along with oxidized DA (DA-quinone) could have a
synergistic effect in terms of disease susceptibility and progression.[32−35]It is increasingly evident that drugs aimed at a single target
may be inadequate for the treatment of complex diseases such as PD,
which is multifactorial in nature. Thus, it is hypothesized that multifunctional
drugs exhibiting multiple pharmacological activities addressing underlying
pathogenic factors of PD will be effective as disease modifying agents.[36] With this in mind, we initiated our drug discovery
approach aimed at identifying novel multifunctional agents possessing
D2/D3 agonist or D3 preferring agonist
activity along with antioxidant, ironchelating, and modulation of
αSN aggregation activities. In this regard, we have designed
and explored a novel, hybrid molecular template by combining known
D2/D3 agonists with D2/D3 antagonist fragments, which led to the development of a number of
potent D3 preferring and D2/D3 agonists
and lead molecules exhibiting potent in vivo activity in PD animal
models.[37−44] One such lead compound, D-264 (1a, Figure 1), exhibited potent in vivo activity in PD animal
models and also exhibited neuroprotective properties in two different
PD animal neuroprotection models.[45]
Figure 1
Molecular structures
of D2/D3 agonists.
Molecular structures
of D2/D3 agonists.Although its neuroprotective action is an important feature, 1a suffers from poor in vivo efficacy probably due to lack
of sufficient brain penetration, although additionally high plasma
protein binding and possibly binding to adipose tissue can also potentially
contribute to less efficacy. In vivo activity of 1a was
enhanced significantly when 1a was solubilized in 5–10%
β-hydroxy-propyl-cyclodextrin (BHPC) solution presumably by
encapsulating the molecule leading to enhanced blood brain barrier
penetration of 1a. The present structure–activity
study with 1a-related compounds has been designed to
enhance the in vivo efficacy without compromising their multifunctional
agonist and neuroprotective properties. Introduction of different
polar hydroxyl group(s) will contribute toward reducing the lipophilicity
of the parent 1a, which will help bring the compounds,
for example, (−)-9b, more in compliance with Lipinski’s
rule of five compared to the parent 1a.[46] Consequently, this should contribute toward a higher in
vivo efficacy.
Chemistry
Scheme 1 describes the synthesis of final compounds (±)-8a, (±)-8b, (±)-8c, (±)-9a, (±)-9b, (±)-9c, (±)-9d, (−)-11 and their enantiomers. Iodination
of phenyl piperazine was done following the literature procedure.
The 1-(4-iodophenyl) piperazine was treated with t-Boc-anhydride to synthesize the t-Boc protected
intermediate (1). The t-Boc protected
intermediate was then subjected to Suzuki coupling reaction[47,48] with various commercially available substituted benzene boronic
acids. The amine protecting t-Boc group was removed
by using trifluoroacetic acid. The free amines (4a–d) were subjected to N-alkylation reaction with TBDMS protected
bromoalcohol to get intermediates (5a–e) which further underwent TBDMS elimination using tetrabutyl ammonium
fluoride (TBAF) solution to get the alcohol intermediate (6a–e). These alcohol intermediates (6a–e) were oxidized under Swern oxidation conditions
to get the arylpiperazine aldehydes (7a–e), which were further condensed with (±)-, S-(−), or R-(+)-pramipexole under reductive
amination conditions to give four final compounds (±)-8a,(±)-8b,(±)-8c,(−)-8b and the four carbon
linker intermediate (±)-8d. The demethylation of
these intermediates with either boron tribromide or with freshly distilled
aqueous hydrobromic acid (48%) yielded the four final compounds (9a–d) and their enantiomers. One of the
intermediates described in Scheme 1, the arylpiperazinealdehyde, 7b, was subjected under reductive amination
conditions to react with (S)-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine
to get corresponding mehtoxy intermediates (−)-10 and subsequently was treated with aqueous hydrobromic acid (48%)
to furnish the final compound (−)-11.
Scheme 1
Scheme 2 depicts the synthesis of final target compounds
(±)-22. 2-Methoxyaniline (12) was subjected
to cyclization by following the literature procedure[49] to produce the intermediate 13. Further, bromination
of the intermediate, 13, yielded bromo derivative, 14. This amine intermediate, 14, was converted
into t-Boc protected compound, 15, followed
by their Suzuki coupling reaction with commercially available benzeneboronic acids, and subsequently t-Boc group was removed
by using TFA to yield 17. The free amine intermediate 17 was N-alkylated with (2-bromo ethoxy)-tert-butyldimethylsilane to get compound 18, which on TBDMS
elimination yielded alcohol, 19. Compound 19 was converted into aldehyde derivatives 20 under Swern
oxidation conditions followed by condensation with (±)-pramipexole
under reductive amination conditions and subsequently treated with
aqueous hydrobromic acid (48%) to yield the final compound 22.
Scheme 2
In Scheme 3, we describe the
synthesis of bioisosteric analogues of 2-aminothiazole agonist pharmacophoic
headgroup using the quinazoline moiety. The quanazoline derivatives
were synthesized as reported in our earlier publication. Briefly,
1,4-cyclohexanedionemonoethyleneketal, on treatment with n-propylamine under reductive amination conditions, yielded intermediate 23. This intermediate 23 was coupled with aldehyde 7d and 7b under reductive amination conditions
to afford 24a and 24b. Removal of the ketal
group by dilute HCl in THF followed by ring formation in a two-step
synthesis afforded the final compound 26a and the intermediate 26b. Final target 27 was produced by demethylation
of methoxy group of 26b, using 48% aqueous HBr.
Scheme 3
The
synthesis of the final compound 35 is shown in Scheme 4. Mono-t-Boc protected amine 28 was reacted with commercially available biphenyl carbonyl
chloride 29 at room temperature in THF in the presence
of diisopropylethylamine as base to provide 30. The t-Boc group was removed using TFA followed by N-alkylation
with TBDMS protected bromoethanol, and subsequently the TBDMS group
was eliminated using TBAF to yield the corresponding alcohol 33. Alcohol 33 was converted, under Swern oxidation
conditions, into its aldehyde derivative 34 followed
by reductive amination with (±)-pramipexole to afford the final
compound 35.
Scheme 4
Results and Discussion
Potency
and Agonism at DA D2 and D3 Receptors
Our first-generation hybrid compound 1a, a potent and
D3 preferring agonist with multifunctional properties for
potential PD treatment, was the starting point for designing compounds
with enhanced in vivo efficacy without compromising agonist potency.
The structural modifications are mainly centered on the introduction
of methoxy and hydroxyl groups at various positions on the biphenyl
moiety of this hybrid molecule. Methoxy and hydroxyl substitutions
also should help us to examine the possible contribution of any hydrogen-bonding
interaction originating from this region of the molecule with D2 and D3 receptors. Apart from these modifications,
other molecular alterations involving bioisosteric replacement of
the thiazolidium moiety by aminotetraline or quanazoline rings, change
of ethylene linker length, and incorporation of amide bond at the
piperazinenitrogen atom distal to the agonist headgroup have also
been incorporated.First, the influence of methoxy and hydroxyl
substitutions on the biphenyl ring of 1a was tested in
binding assays with rat DA D2 and D3 (rD2 and rD3) receptors expressed in HEK-293cells.
To this end, racemic derivatives 8a–c, 9a–c, and 22 were
synthesized and characterized. It is evident from Table 1 that most of these compounds displayed high affinity for
D3 and moderate affinity for D2 receptors. Among
this series of analogues, compound 9b with monohydroxyl
substitution on the meta position of the phenyl ring distal to the
piperazine was found to be the most potent and selective for D3 tested at this point (Ki, D2 = 347, D3 = 1.20 nM, D2/D3 = 289). On the other hand, 22 with the hydroxyl group
at the ortho position of the phenyl ring proximal to the piperazine
ring proved to be the most potent of the compounds tested at this
point for D2 (Ki, D2 = 70.6, D3 = 2.35 nM, D2/D3 = 30).
Compound 8b, a methoxy analogue of 9b, exhibited
a somewhat lower binding affinity at D2 receptor compared
to 9b, while D3 affinity decreased approximately
2-fold in comparison to 9b (Ki, D2 = 464, D3 = 2.11 nM, D2/D3 = 220 for 8b). These results indicated that
introduction of monomethoxy and monohydroxyl groups is well tolerated
on the distal phenyl ring of 1a and actually increases
selectivity for the D3 receptor.
Table 1
Inhibition
of [3H]Spiroperidol Binding to rD2L and rD3 Receptors Expressed in HEK-293 Cellsa
compound
Ki (nM), rD2L [3H]spiroperidol
Ki (nM), rD3 [3H]spiroperidol
D2L/D3
(−)-5-OH-DPATb
58.8 ± 11.0
1.36 ± 0.28
43.2
1ab
186 ± 34
2.10 ± 0.34
86
1bb
1,073 ± 92
1.84 ± 0.51
583
8a
213 ± 12
1.41 ± 0.12
151
8b
464 ± 93
2.11 ± 0.34
220
(−)-8b
343 ± 65
2.33 ± 0.26
147
8c
274 ± 45
3.57 ± 0.44
78
9a
230 ± 50
1.17 ± 0.37
196
9b
347 ± 54
1.20 ± 0.14
289
(−)-9b
369 ± 39
1.73 ± 0.14
213
(+)-9b
1507 ± 312
19.7 ± 2.1
76
9c
208 ± 15
1.80 ± 0.38
115
9d
567 ± 83
9.43 ± 1.14
60
(−)-11
27.8 ± 1.8
0.77 ± 0.030
36
22
70.6 ± 10.2
2.35 ± 0.13
30
26a
735 ± 198
3.65 ± 0.64
201
27
13,121 ± 4539
67 ± 7.8
196
35
1666 ± 282
9.58 ± 1.18
174
Results are the means ± SEM for 3–6 experiments each
performed in triplicate.
From previous ref (44).
Results are the means ± SEM for 3–6 experiments each
performed in triplicate.From previous ref (44).Among the racemiccompounds
with D3 affinity in the nanomolar range, 9b exhibited the highest selectivity for D3; we therefore
chose this racemiccompound for synthesizing both the (−)-
and (+)-enantiomer to evaluate the differential potency and selectivity
of the enantiomers at DA receptors. In agreement with our earlier
results on stereoselectivity in this type of compounds, (−)-9b (Ki, D2 = 369 nM,
D3 = 1.73 nM, D2/D3 = 213) exhibited
higher potency at both D2 and D3 receptors compared
to (+)-9b (Ki, D2 = 1507 nM, Ki D3 = 19.7 nM,
D2/D3 = 76). In compound (−)-9b, an additional hydroxyl functionality is present compared with the
parent compound 1a, resulting in retention of high binding
affinity at D3 with slightly reduced affinity for D2, that is, overall higher selectivity of (−)-9b for D3compared to 1a (Ki; D2/D3 = 213 vs D2/D3 = 86 for (−)-9b and 1a,
respectively). The (−)-isomer of 8b was made to
evaluate whether the free hydroxyl group in (−)-9b is critical for activity (Table 1). Compound
(−)-8b, which is a methoxy analogue maintained
D2 receptor affinity similar to (−)-9b (Ki; D2 = 343 nM vs D2 = 369 nM for (−)-8b and (−) 9b, respectively), while the binding affinity toward D3 dropped slightly (Ki; D3 = 2.33 nM vs D3 = 1.73 nM for (−)-8b and (−)-9b, respectively); this resulted in
a somewhat lower selectivity for D3 over D2 receptors
(D2/D3 = 147 vs D2/D3 =
213 for (−)-8b and (−)-9b,
respectively). All compounds in this series showed nanomolar binding
potency for the D3 receptor.In our next series of
compounds, aminotetraline and amino pyrimidine moieties were incorporated
as bioisosteric replacement of the thiazolidium moiety of pramipexole
in 9b or 1a, resulting in (−)-11, 26a, and 27.[50] It was hypothesized that in both cases H-bonding interaction
of the parent amino group with serine-192 at the DA receptor should
be maintained.[51] Specifically, the (−)
isomer of 5-hydroxy aminotetraline was synthesized, as we have shown
in our previous reports that the (−)-enantiomer exhibits the
highest affinity compared to the (+)-isomer for both D2 and D3 receptors. As expected, the 5-hydroxy aminotetraline
analogue (−)-11 exhibited higher affinity, compared
to (−)-9b, for both D2 and D3 receptors with overall less selectivity for D3 receptor
(Ki, D2 = 27.8, D3 = 0.77 nM, D2/D3 = 36). In our previous report,
the phenolic moiety of 5-hydroxy aminotetraline was replaced by an
amino pyrimidine moiety, which is a known bioisostere of a phenolic
group. Here we wanted to explore this further with linearly fused
biphenyl rings at the other side of the molecule. Incorporation of
amino pyrimidine to this moiety in compound 26a resulted
in reduced potency for both D2/D3 receptors
(Ki, D2 = 735 nM, D3 3.65 nM) with decreased in selectivity (D2/D3 = 201) compared to 9b. Next, in compound 27 we introduced a hydroxyl group on the biphenyl ring of 26a (targeting the accessory binding domain of the receptor). Compound 27, which is a bioisosteric analogue compound 9b, exhibited significantly decreased binding affinity, compared to 9b, at both D2 and D3 receptors (Ki, D2 = 13121 nM, D3 =
67 nM, vs D2 = 235 nM, D3 0.70 nM for 27 vs 9b, respectively). This suggests the combination
of either 2-aminothiazole or hydroxy-tetralin, and a linearly fused
biphenyl moiety gives rise to D2 and D3 potency
and D2/D3 selectivity. Next, we increased the
length of the two-carbon linker in 9b to four carbons
in compound 9d. In agreement with our previous results
with a four-methylene linker at this position, compound 9d (Ki, D2 = 567 nM, D3 = 9.43 nM) displayed lower potency at both D2 and D3 receptors compared to 9b.[38] The reason behind characterizing bio-isosteric ((−)-11, 26a, and 27) and higher chain
length (9d) compound is to expand the SAR study to better
understand molecular interaction of our hybrid molecules with D2/D3 receptors.Finally, in an earlier publication,
we reported compound (S)-(4-{2-[(2-amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)-propylamino]ethyl}piperazin-1-yl)-(1H-indol-2-yl)methanone
(D-440) as one of the most potent and selective agonists
for D3 receptor known to date, and this compound contains
a carbonyl bond between the piperazinenitrogen atom and 5-position
of indole, distal to the agonist headgroup.[44] So, to probe the impact of introduction of a carbonyl group on D3 receptor selectivity in our first generation hybrid compound 1a, we incorporated a carbonyl bond between the piperazinenitrogen and the accessory binding biphenyl ring of 1a. This modification generated compound 35, Scheme 4, which exhibited lower binding affinity for D2/D3 receptors (Ki,
D2 = 1666 nM, D3 = 9.58 nM), and its selectivity
was increased (D2/D3 = 174) compared to parent
compound 1a. Thus, introduction of a carbonyl group between
the piperazinenitrogen and the biphenyl ring in this series of compounds
impacted D3 affinity and selectivity in opposite fashion.On the basis of the binding results, selected compounds (−)-8b and (−)-9b were subjected to the GTPγS
binding functional assay for D2 and D3 receptors
and compared with endogenous ligand DA and the parent compound 1a. The functional assay measures quantitatively the ability
of the compound to stimulate the receptor as an agonist. Comparison
with the maximum stimulation (Emax), produced
by the full agonist DA, indicates whether the compound is a full agonist,
a partial agonist, or an antagonist. The assays were carried out with
cloned humanD2 and D3 receptors expressed in
CHO cells. Compound (−)-9b displayed higher functional
potency for D2/D3 and selectivity for D3 receptor in comparison to 1a and dopamine (Table 2). (−)-9b displayed a 15-fold
increase in D3 functional potency in comparison to 1a (EC50 = 15.9 nM vs 33.1 nM for D2 and 0.1 nM vs 1.51 nM for D3, for (−)-9b vs 1a, respectively) and a 7-fold increase in functional
selectivity (D2/D3 = 159 vs 22.1 for (−)-9b
vs 1a). Compounds (−)-9b and 1a exhibited full agonist activity at D2 and D3 receptors, while their selectivity for D3 receptor
dropped significantly when compared to the binding data (Table 1). On the other hand, compound (−)-8b turned out to be functionally 2-fold less potent at D3 receptor (EC50 = 3.42 nM) in comparison to 1a and was a partial agonist at D3 but full agonist
at D2 receptor. The functional potency of compound (−)-8b for D2 was comparable to 1a (EC50 = 36.8 nM vs 33.1 nM for (−)-8b vs 1a, respectively).
Table 2
Stimulation of [35S]GTPγS Binding to hD2 and hD3 Receptors Expressed in CHO Cells
CHO–D2
CHO–D3
compd
EC50 (nM)a [35S]GTPγS
%Emax
EC50 (nM)a [35S]GTPγS
%Emax
D2/D3
dopamine
218 ± 12
100
10.6 ± 2.1
100
26.5
1ab
33.1 ± 6.6
104 ± 5
1.51 ± 0.02
90 ± 4.3
22.1
(−)-8b
36.8 ± 7.2
105 ± 6
3.42 ± 1.01
67.3 ± 5.6
10.8
(−)-9b
15.9 ± 1.8
116 ± 10
0.10 ± 0.02
95.8 ± 3.7
159
EC50 is
the concentration producing half-maximal stimulation; for each compound,
maximal stimulation (Emax) is expressed
as percent of the Emax observed with 1
mM (D2) or 100 uM (D3) of the full agonist DA
(%Emax). Results are the means ±
SEM for 3–6 experiments each performed in triplicate.
From previous ref (43).
EC50 is
the concentration producing half-maximal stimulation; for each compound,
maximal stimulation (Emax) is expressed
as percent of the Emax observed with 1
mM (D2) or 100 uM (D3) of the full agonist DA
(%Emax). Results are the means ±
SEM for 3–6 experiments each performed in triplicate.From previous ref (43).
Evaluation of Free Radical Scavenging Activity
Scavenging
of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical by 1a, (−)-9b, (−)-8b, (−)-11, ropinirole, and ascorbic acid was monitored (Figure 2). As shown in Figure 2,
all compounds inhibited DPPH radical activity dose dependently. Overall,
all of the compounds exhibited similar antioxidant efficacy as ascorbic
acid except ropinirole which exhibited poor activity in this assay.
Interestingly, (−)-11 exhibited less antioxidant
activity in this assay than (−)-9b, indicating
a more efficacious antioxidant activity of the thiazolidum moiety
compared to aminotetraline.
Figure 2
DPPH radical scavenging activity by 1a, (−)-9b, (−)-8b, (−)-11, ropinirole, and ascorbic acid.
DPPH radical scavenging activity by 1a, (−)-9b, (−)-8b, (−)-11, ropinirole, and ascorbic acid.
Reversal of Reserpine-Induced Hypolocomotion in Rats by 1a, (−)-8b, (−)-9b,and Ropinirole
Reserpine induces depletion of catecholamine
in nerve terminals, resulting in a cataleptic condition in rats, which
is a well established animal model for PD.[52,53] Significant inhibition of locomotion of rats was observed 18 h after
the administration of reserpine (5 mg/kg, s.c.) which indicated the
development of akinesia. Compounds (−)-8b and
(−)-9b at a dose of 5 μmol/kg, i.p., in
DI water, were highly efficacious in reversing akinesia (Figure 3), while 1a at the same dose (5 μmol/kg,
i.p., in DI water) failed to produce any significant effect in reversing
akinesia in reserpine-treated rats. However, 1a was more
effective when it was dissolved in 10% beta-hydroxy cyclodextrin solution
(Figure 3). The reference drug Ropinirole exhibited
much shorter duration of action compared to the test compounds (Figure 3). The locomotor activity of (−)-8b at the end of 6h remained high compared to (−)-9b. It is evident from the result that compounds (−)-8b and (−)-9b were more efficacious in producing
reversal of akinesia than 1a ((−)-8b > (−)-9b > 1a).
Thus, the results indicate that compounds (−)-8b and (−)-9b exhibited higher in vivo efficacy
which might be due to efficient crossing of the blood brain barrier.
Compound 1a was able to produce in vivo activity only
if dissolved in 10% beta-hydroxy propyl cyclodextrin solution, indicating
limitations in brain uptake for this compound when administered by
itself. Interestingly, all three compounds displayed a long duration
of action (Figure 3).
Figure 3
Effect of different drugs
upon reserpine (5.0 mg/kg, s.c.)-induced hypolocomotion in rats. Data
are means ± SEM, n = 4 per value. Horizontal
activity was measured as described under materials and methods. The
plots are the representation of horizontal locomotor activity at discrete
30-min intervals after the administration of (−)-9b (i.p.), (−)-8b (i.p.), ropinirole (s.c) and 1a (i.p.) at the dose of 5 μmol/kg compared to control
reserpine treated rats in 18 h post reserpine treatment. One way ANOVA
analysis demonstrates significant effect among treatments F (5,95)
= 14.16 (P < 0.0001). Dunnett’s analysis
following ANOVA showed that the effects of (−)9b (P < 0.01), (−)-8b (P < 0.01), and ropinirole (P < 0.01)
were significantly different compared to reserpine control.
Effect of different drugs
upon reserpine (5.0 mg/kg, s.c.)-induced hypolocomotion in rats. Data
are means ± SEM, n = 4 per value. Horizontal
activity was measured as described under materials and methods. The
plots are the representation of horizontal locomotor activity at discrete
30-min intervals after the administration of (−)-9b (i.p.), (−)-8b (i.p.), ropinirole (s.c) and 1a (i.p.) at the dose of 5 μmol/kg compared to control
reserpine treated rats in 18 h post reserpine treatment. One way ANOVA
analysis demonstrates significant effect among treatments F (5,95)
= 14.16 (P < 0.0001). Dunnett’s analysis
following ANOVA showed that the effects of (−)9b (P < 0.01), (−)-8b (P < 0.01), and ropinirole (P < 0.01)
were significantly different compared to reserpinecontrol.
In Vivo Pharmacology in
6-OHDA Lesioned Rats
On the basis of the above locomotor
data, compounds (−)-8b and (−)-9b as well as the reference Ropinirole were selected for in vivo evaluation
in ratscarrying an unilateral lesion in the medial forebrain bundle;
the lesion was induced by application of the neurotoxin 6-hydroxydopamine
(6-OHDA), resulting in the production of supersensitized DA receptors
on the lesioned side. Such rats, when challenged with direct acting
DA agonists, respond with contralateral rotations away from the lesioned
side. This rat model is considered to be one of the standard models
for preclinical screening of drugs for possible antiparkinsonian activity.[54] Both compounds (−)-8b and
(−)-9b produced potent rotational activity in
a dose-dependent manner when administered intraperitoneally (i.p).
At a 10 μmol/kg dose, both (−)-8b (6.56
mg/kg) and (−)-9b (9.28 mg/kg) produced potent
rotation that lasted for more than 10 h (Figure 4). Compound (−)-8b was more efficacious in producing
rotation compared to (−)-9b (total of 5866 vs
2653 rotations for (−)-8b vs (−)-9b, respectively). Peak effect of both compounds was reached
at 7.5 h. This is an indication of long duration of action of both
compounds in producing contralateral rotation. When tested at a lower
doses (5 μmol/kg), both compounds, (−)-8b (3.28 mg/kg) and (−)-9b (4.64 mg/kg), produced
a lower total number of rotations (3333 and 1839 for (−)-8b and (−)-9b, respectively) than at the
10 μmol/kg dose. The rotation in this case lasted for more than
7 h (Figure 4). Interestingly, both compounds
produced initial increase of rotational activity followed by a brief
decrease of activity before exhibiting a steady increase of rotational
activity. At present, the reason for such biphasic activity is unknown.
At both tested doses (−)-8b was more efficacious
than (−)-9b in the rotation test, just as in the
locomotor activity study with reserpinized rats (Figure 3). The reference drug Ropinirole at a higher dose (10 μmol/kg)
exhibited much shorter duration of action. As we have reported in
the Supporting Information section, pretreatment
studies with the potent DA receptor antagonist haloperidol demonstrated
block of the production of rotation by our hybrid D2/D3 agonist, indicating site-specific interaction at the target
D2/D3 receptor sites.
Figure 4
Effect on turning behavior
of two different doses of (−)-9b (i.p.), (−)-8b (i.p.), ropinirole and vehicle in lesioned rats studied
for maximum 12 h. Each point is the mean ± SEM of 3–4
rats. The drugs were administered i.p. One way ANOVA analysis demonstrates
significant effect among treatments: F (5, 95) = 29.70 (P < 0.0001). Dunnett’s analysis showed that the effect of
(−)-9b, (−)-8b and ropinirole
on rotations at two doses was significantly different compared to
vehicle (P < 0.01).
Effect on turning behavior
of two different doses of (−)-9b (i.p.), (−)-8b (i.p.), ropinirole and vehicle in lesioned rats studied
for maximum 12 h. Each point is the mean ± SEM of 3–4
rats. The drugs were administered i.p. One way ANOVA analysis demonstrates
significant effect among treatments: F (5, 95) = 29.70 (P < 0.0001). Dunnett’s analysis showed that the effect of
(−)-9b, (−)-8b and ropinirole
on rotations at two doses was significantly different compared to
vehicle (P < 0.01).
Neuroprotection against MPP+ Toxicity
The dose-dependent
effect of treatment of 1a and (−)-9b in reversing
the toxicity of MPP+ to dopaminergicMN9Dcells is demonstrated in
Figure 5. From our previous dose–effect
experiment with MPP+, we chose 100 μM of MPP+ which can induce
50–60% cell death, for our study.[55] To test whether 1a and (−)-9b can
protect dopaminergicMN9Dcells from MPP+ induced toxicity, the cells
were pretreated with various concentrations of (20, 10, 5, 1, 0.1,
0.01, and 0.001 μM) of either 1a or (−)-9b for 1 h and then cotreated with 100 μM MPP+ for an
additional 24 h. The data from the MTT assay indicated that both 1a and (−)-9b are able to protect the
MN9Dcells in a dose-dependent manner. For 1a, significant
protection from toxicity of MPP+ was conferred by 1, 5, 10, and 20
μM doses, and this result correlates well with in vivo neuroprotection
result that we published earlier.[45] For
(−)-9b, significant neuroprotection was conferred
at 5 and 10 μM doses. It seems 1a is relatively more potent
and efficacious than (−)-9b in this neuroprotection
assay. Interestingly, (−)-8b did not show any
neuroprotection when the assay was carried out under identical condition
(see Supporting Information).
Figure 5
Dose-dependent
effect of combination of pretreatment followed by cotreatment of 1a and (−)-9b with 100 μM MPP+ on
cell viability of MN9D cells from toxicity of 100 μM MPP+. (A,
B) MN9D cells were pretreated with different doses of 1a and (−)-9b for 1 h followed by cotreatment with
100 μM MPP+ for 24 h. The values shown are means ± SDs
of three independent experiments performed in 4–6 replicates.
One-way ANOVA analysis followed by Tukey’s multiple comparison
post hoc test were performed (**p < 0.01 compared
to the MPP+ group. ##p < 0.001 compared to the
control group).
Dose-dependent
effect of combination of pretreatment followed by cotreatment of 1a and (−)-9b with 100 μM MPP+ on
cell viability of MN9Dcells from toxicity of 100 μM MPP+. (A,
B) MN9Dcells were pretreated with different doses of 1a and (−)-9b for 1 h followed by cotreatment with
100 μM MPP+ for 24 h. The values shown are means ± SDs
of three independent experiments performed in 4–6 replicates.
One-way ANOVA analysis followed by Tukey’s multiple comparison
post hoc test were performed (**p < 0.01 compared
to the MPP+ group. ##p < 0.001 compared to the
control group).
Conclusion
In this paper, we describe an SAR study based on our earlier lead
molecule 1a, with some highly potent agonist molecules
for D2 and D3 receptors with enhanced blood
brain barrier crossing ability compared to the parent molecule 1a. SAR results have demonstrated that hydroxyl derivatives
of 1a have higher affinity for the D3 receptor.
In both binding and functional assays, compound (−)-9b exhibited the highest selectivity for D3 over D2 receptors. Lead molecules also exhibited potent free radical quenching
property, indicating their antioxidant property. Furthermore, lead
molecules were tested in two PD animal models and compared with parent
molecule 1a. Compounds (−)-9b and
(−)-8b exhibited significant, long-lasting reversal
of hypolocomotion in reserpinized rats; on the other hand, 1a was efficacious in this model only if dissolved in 10% BHCD solution.
Similarly, in 6-OHDA animal model studies, compounds (−)-8b and (−)-9b produced extensive rotational
activity with long duration of action. In vitro neuroprotection experiments
with dopaminergicMN9Dcells treated with 1a and (−)-9b indicated protection from toxicity of MPP+.
Experimental Section
Reagents and solvents were purchased
from commercial suppliers and used as received unless otherwise indicated.
Dry solvent was obtained according to the standard procedure. All
reactions were performed under inert atmosphere (N2) unless
otherwise noted. 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 purifications
were performed using Whatman Purasil 60A silica gel 230–400
mesh. The proton nuclear magnetic resonance (1H NMR) spectra
were measured on a Varian 400 MHz FT NMR spectrometer using tetramethylsilane
(TMS) as an internal standard. The NMR solvent used was CDCl3 or CD3OD as indicated. Optical rotations were recorded
on Perkin-Elmer 241 polarimeter. Melting points were recorded using
MEL-TEMP II (Laboratory Devices Inc., USA) capillary melting point
apparatus and were uncorrected. Elemental analyses were performed
by Atlantic Microlab, Inc.
Into a stirring solution of 1-phenylpiperazine
(21.8 g, 134.0 mmol) in acetic acid/water (3:1, 42 mL), a suspension
of iodine monochloride (24.0 g, 148.0 mmol) in acetic acid/water (3:1,
42 mL) was added at 55 °C. The reaction was stirred at 55 °C
for 1 h and then at room temperature for another 1 h. The solution
was poured into 400 mL of crushed ice, and the pH was adjusted to
13 with 4 N NaOH. The product was then extracted with dichloromethane
(3 × 100 mL). The combined organic layer was dried over Na2SO4, filtered, and evaporated in vacuo to provide
the free amine of compound 1 as a pale yellow solid (28.69
g, 74%) which was converted to t-Boc derivative without
further purification.Into a stirring solution of this amine
(28.0 g, 97.17 mmol) in dichloromethane (80 mL), (Boc)2O (25.44 g, 116.60 mmol) and Et3N (35.26 mL, 252.64 mmol)
were added at room temperature. The reaction mixture was stirred at
the same temperature for 12 h and was extracted with CH2Cl2 (3 × 100 mL), washed with water, dried over Na2SO4, filtered, and concentrated in vacuo. The crude
material was purified by column chromatography over silica gel (hexane/EtOAc,
9.0:1.0) to give compound 1 (34.70 g, 92%). 1H (CDCl3, 400 MHz): δ1.48 (s, 9 H), 3.10 (t, J = 4.8 Hz, 4H), 3.56 (t, J = 4.8 Hz, 4H),
6.68 (d, J = 8.8 Hz, 2H), 7.53 (d, J = 9.2 Hz, 2H).
Procedure A. t-Butyl 4-(4′-methoxybiphenyl-4-yl)piperazine-1-carboxylate
(3a)
A suspension of (4-methoxyphenyl)boronic
acid 2a (2.34 g, 15.49 mmol), iodo compound 1 (6.01 g, 15.49 mmol), Na2CO3 (3.28 g, 30.98
mmol, 2 M solution in water), and Pd(PPh3)4 (875
mg, 0.75 mmol) in dimethoxy ethane/ethanol (1:1) was refluxed for
1 h. The solvents were removed in vacuo, and the crude product was
purified by flash chromatography using the solvent system hexane/ethyl
acetate (4.0:1.0) to yield compound 3a (3.82 g, 67%). 1H NMR (CDCl3, 400 MHz): δ 1.49 (s, 9H), 3.17
(t, J = 4.8 Hz, 4H), 3.61 (t, J =
4.8 Hz, 4H), 3.85 (s, 3H), 6.96 (d, J = 8.8 Hz, 2H),
6.98 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H).
Commercially available benzeneboronic acid, 2d (2.5 g, 20.48 mmol), was reacted with iodo compound 1 (7.95 g, 20.48 mmol), Na2CO3 (4.34
g, 40.96 mmol, 2 M solution in water), and Pd(PPh3)4 (1.18 g, 1.02 mmol) in dimethoxy ethane/ethanol (25 mL/25
mL) by following procedure A to yield compound 3d (1.74 g, 80%). 1H NMR (CDCl3, 400
MHz): δ 1.49 (s, 9H), 3.07 (bs, 4H), 3.61 (t, J = 4.8 Hz, 4H), 6.96 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 7.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 2H),
7.49 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 7.2 Hz, 2H).
Procedure B. 1-(4′-Methoxy-biphenyl-4-yl)piperazine
(4a)
Into a stirring solution of compound 3a (3.4 g, 9.23 mmol) in CH2Cl2 (30
mL), TFA (20 mL) was added slowly at room temperature, and the reaction
mixture was stirred for 4 h. Unreacted TFA and solvent CH2Cl2 were removed in vacuo, and the salt formed was washed
with diethyl ether. Saturated solution of sodium bicarbonate was added
to the salt, and it was extracted with dichloromethane (50 ×
3 mL). The combined organic layer was dried over Na2SO4, filtered, and evaporated in vacuo to provide the compound 4a (2.22 g, 90%). 1H NMR (CDCl3, 400
MHz): δ 1.63 (bs, 1H); 3.06 (t, J = 4.4 Hz,
4H); 3.19 (t, J = 4.6 Hz, 4H), 3.84 (s, 3H); 6.95
(d, J = 8.4 Hz, 2H), 6.98(d, J =
8.8 Hz, 2H), 7.47 (d, J = 8.0 Hz, 2H), 7.49 (d, J = 8.0 Hz, 2H).
Procedure D. 2-(4-(4′-Methoxybiphenyl-4-yl)piperazin-1-yl)ethanol
(6a)
Into a stirring solution of compound 5a (1.5 g, 3.52 mmol) in anhydrous THF (30 mL), n-tetrabutylammonium fluoride (0.92 g, 3.52 mmol, 1.0 M solution in
THF) was added at 0 °C. The reaction mixture was then stirred
at room temperature for 1.5 h. THF was evaporated in vacuo, and the
residue was diluted with CH2Cl2 (50 mL) and
washed with water. The water layer was extracted with CH2Cl2 (3 × 75 mL). The combined organic layer was washed
with brine, dried over Na2SO4, and evaporated
in vacuo. The crude product was purified by silica gelcolumn chromatography
(EtOAc) to yield compound 6a (1.04 g, 95%). 1H NMR (CDCl3, 400 MHz): δ 2.62 (t, J = 5.2 Hz, 2H), 2.70 (t, J = 4.8 Hz, 4H), 3.25 (t, J = 4.8 Hz, 4H), 3.67 (t, J = 5.4 Hz, 2H),
3.83 (s, 3H), 6.95 (d, J = 9.2 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H), 7.46 (d, J = 8.0 Hz, 2H),
7.48 (d, J = 9.2 Hz, 2H).
Compound 5e (2.6 g, 5.72 mmol)
was reacted with n-tetrabutylammonium fluoride (1.50
g, 5.72 mmol, 1.0 M solution in THF) in THF (40 mL) by following procedure D to yield compound 6e (1.40 g, 72%). H NMR (CDCl3, 400 MHz): 1.62 (t, J = 8.0 Hz, 4H), 2.41 (t, J = 7.2 Hz, 2H),
2.62 (t, J = 7.6 Hz, 4H), 3.27 (t, J = 6.8 Hz, 4H), 3.64 (t, J = 7.2 Hz, 2H), 3.85 (s,
3H), 6.85 (dd, J = 1.6 Hz, 8.0 Hz, 1H), 6.99 (d, J = 8.8 Hz, 2H), 7.09 (bs, 1H), 7.15 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 2H).
Procedure E. 2-(4-(4′-Methoxybiphenyl-4-yl)piperazin-1-yl)acetaldehyde
(7a)
Into a stirred solution of oxalyl chloride
(0.324 mL, 2.56 mmol) in CH2Cl2 (40 mL) at −78
°C, DMSO (0.40 mL, 5.12 mmol) was added. The reaction mixture
was stirred for 10 min followed by addition of compound 6a (400 mg, 1.28 mmol, dissolved in 5 mL of CH2Cl2). The reaction mixture was stirred at the same temperature for 15
min. Then Et3N (0.78 mL, 7.68 mmol) was added next, and
stirring was continued for another 1 h and 20 min while allowing the
reaction mixture to reach at room temperature. The reaction mixture
was quenched by addition of water and extracted with CH2Cl2 (3 × 25 mL). The combined organic layer was washed
with brine and concentrated to yield the compound 7a (321
mg, 81%), which was used without purification in the next step.
Compound 6e (1.2 g, 3.52 mmol)
was reacted with oxalyl chloride (0.60 mL, 7.05 mmol), DMSO (1.00
mL, 14.08 mmol), and Et3N (2.92 mL, 21.12 mmol) in dichloromethane
(30 mL) by following procedure E to yield compound 7e (0.89 g, 75%).
Procedure F. N6-(2-(4-(4′-Methoxybiphenyl-4-yl)piperazin-1-yl)ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (±)-(8a)
Into a
stirring solution of compound 7a (321 mg, 1.03 mmol)
in CH2Cl2 (10 mL), (±)-pramipexole (219
mg, 1.03 mmol) was added at room temperature. The reaction mixture
was stirred for 1 h, and then NaBH(OAc)3 (393 mg, 1.85
mmol) was added into the reaction mixture. After the reaction was
stirred for 48 h, a saturated solution of NaHCO3 was added
into the reaction mixture, and it was extracted with CH2Cl2 (3 × 50 mL). The combined organic layer was washed
with brine and finally purified by silica gelcolumn chromatography
(EtOAc/MeOH, 9:1) to yield compound (±)-8a (313
mg, 60%). 1H NMR (CDCl3, 400 MHz): δ 0.91
(t, J = 7.2 Hz, 3H), 1.52–1.56 (m, 2H), 1.76–1.79
(m, 1H), 2.06 (d, J = 8.8 Hz, 1H), 2.59–2.80
(m, 13H), 3.17–3.26 (m, 6H), 3.84 (s, 3H), 6.91–7.01(m,
4H), 7.42–7.49 (m, 4H). The product was converted into corresponding
hydrochloride salt, m.p. 268 °C. Anal. (C29H39N5OS·4.0HCl·2.0H2O): C, H, N.
Compound 7b (500 mg, 1.61 mmol) was reacted with (−)pramipexole
(340.24 mg, 1.61 mmol) and NaBH(OAc)3 (612.50 mg, 2.89
mmol) in dichloromethane (100 mL) by following procedure F to yield compound (−)-8b (526 mg, 65%). [α]25D = −34.6 (c = 1, CH3OH). Spectral data matching with compound (±)-8b. The product was converted into corresponding hydrochloride salt,
m.p. 245 °C. Anal. (C29H39N5OS·4.0 HCl·1.0H2O): C, H, N.
Compound 7b (100 mg, 0.322 mmol) was reacted with (+)-pramipexole (68.04
mg, 0.322 mmol) and NaBH(OAc)3 (122.84 mg, 0.579 mmol)
in dichloromethane (20 mL) by following procedure F to
yield compound (+)-8b (105 mg, 65%). Spectral data matching
with compound (±)-8b.
Procedure G. 4′-(4-(2-((2-Amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)ethyl)piperazin-1-yl)-[1,1′-biphenyl]-4-ol
((±)-9a)
Into a stirring solution of compound
(±)-8a (60 mg, 0.11 mmol) in anhydrous CH2Cl2 (10 mL) at −78 °C, boron tribromide (1.1
mL, 1.1 mmol, 1 M solution in CH2Cl2) was added.
The reaction mixture was allowed to come to room temperature and was
stirred for 48 h. The reaction was quenched by addition of saturated
NaHCO3 solution, and the mixture was extracted with CH2Cl2. The combined organic layer was dried over
Na2SO4 and evaporated under a vacuum, and the
crude product was purified by flash chromatography (CH2Cl2/MeOH = 9:1) to afford compound (±)-9a (0.029 g, 50%). 1H NMR (CDCl3, 400 MHz): δ
0.96 (t, J = 7.2 Hz, 3H), 1.60–1.64 (m, 2H),
1.81–1.85 (m, 1H), 2.08 (d, J = 7.2 Hz, 1H),
2.52–3.04 (m, 13H), 3.11–3.24 (m, 6H), 6.81(d, J = 8.4 Hz, 2H), 7.00 (d, J = 8.8 Hz, 2H),
7.38 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H). The product was converted into corresponding hydrochloride
salt, m.p. 272 °C. Anal. (C28H37N5OS·4.0 HCl·1.0H2O): C, H, N.
Compound (+)-8b (100 mg,
0.20 mmol) and 48% aqueous HBr (10 mL) was refluxed for 12 h by following
procedure H to afford compound (+)-9b (105
mg, 60%, recrystallized from ethanol). Spectral data matching with
compound (−)-9b. [α]25D = +16.0 (c = 0.5, CH3OH). Hydrobromide
salt, m.p. 270 °C. Anal. (C28H37N5OS·5.0 HBr·1.0H2O): C, H, N. m/z calculated for C28HN5OS [M + H+]: calculated 491.27; found
492.27.
Compound (−)-10 (300 mg, 0.58 mmol) and 48% aqueous HBr (15 mL) was refluxed for
10 h by following procedure H to afford compound (−)-11 (296 mg, 70%, recrystallized from ethanol). 1H NMR of HBrsalt (400 MHz, CD3OD): δ 1.04 (t, J = 7.2 Hz, 3H), 1.73–1.93 (m, 3H), 2.30–2.69
(m, 2H), 3.07–3.83 (m, 18 H), 6.60 (d, J =
5.2 Hz, 1H), 6.69 (d, J = 6.4 Hz, 1H), 6.73 (d, J = 7.6 Hz, 1H), 6.96–7.04 (m, 3H), 7.19–7.23
(m, 3H), 7.56 (d, J = 7.2 Hz, 2H). [α]25D = −41.0 (c = 1.0, CH3OH). Hydrobromide salt, m.p. 290 °C. Anal. (C31H39N3O2S·3.0 HBr·2.0 H2O): C, H, N.
Procedure I. 1-(2-Methoxyphenyl)piperazine
(13)
A stirring solution of 2-methoxyaniline 12 (31.60 g, 256.91 mmol) and bis-(2-chloroethyl)amine (45.85
g, 256.91 mmol) was heated at 150 °C in diethylene glycol monomethyl
ether (100 mL) for 6 h. After being cooled to room temperature, the
mixture was dissolved in MeOH (4 mL) followed by addition of Et2O (300 mL). The precipitate was filtered off and washed with
Et2O to provide HCl salt. The HCl salt was further converted
to free amine by treatment with Na2CO3 solution
and extracted with EtOAc (2 × 100 mL). The combined organic layers
were dried over Na2SO4, and concentrated in
vacuo to provide the pure free amine product 13 (34.34
g, 70%). 1H NMR (CDCl3, 400 MHz): δ 3.12
(t, J = 7.6 Hz, 4H), 3.37 (t, J =
6.4 Hz, 4H), 3.79 (s, 3H), 6.86 (t, J = 7.6 Hz, 1.6
Hz, 1H), 6.93 (t, J = 4.8 Hz, 2H), 6.94–7.07
(m, 1H).
Procedure J. 1-(4-Bromo-2-methoxyphenyl)piperazine
(14)
Amine 13 (15.0 g, 78.07 mmol)
was dissolved in CH2Cl2 (200 mL) and cooled
to 0 °C. Bromine (4.02 mL, 78.07 mmol) was added dropwise into
the above solution. After 2 h, reaction mixture was washed with 1
N sodium hydroxide, and the organic layer was separated, dried over
Na2SO4, and concentrated in vacuo to yield 14 (16.86 g, 80% yield). The crude product 14 thus obtained was converted into its t-Boc derivative
without further purification.
Procedure K. t-Butyl 4-(4-bromo-2-methoxyphenyl)piperazine-1-carboxylate
(15)
Into a stirring solution of amine 14 (14.0 g, 51.84 mmol) in dichloromethane (40 mL), (Boc)2O (11.31 g, 51.84 mmol) and Et3N (21.55 mL, 155.52
mmol) were added at room temperature. The reaction mixture was stirred
at the same temperature for 12 h and was extracted with CH2Cl2 (3 × 100 mL), washed with water, dried over Na2SO4, filtered, and concentrated. The crude material
was purified by column chromatography over silica gel (hexane/EtOAc,
8:2) to give compound (15) (16.30 g, 85%). 1H NMR (CDCl3, 400 MHz): δ 1.49 (s, 9H), 2.95 (t, J = 4.4 Hz, 4H), 3.58 (t, J = 5.2 Hz, 4H),
3.86 (s, 3H), 6.75 (d, J = 8.4 Hz, 1H), 6.97 (d, J = 2.0 Hz, 1H), 7.58 (dd, J = 8.4 Hz,
2.0 Hz, 1H).
Compound 19 (500 mg, 1.60
mmol) was reacted with oxalyl chloride (0.28 mL, 3.20 mmol), DMSO
(0.45 mL, 6.40 mmol) and Et3N (1.33 mL, 9.6 mmol) in dry
dichloromethane (30 mL) by following procedure E. The
crude product was purified by silica gelcolumn chromatography (EtOAc/MeOH,
9.5:0.5) to yield compound 20 (397 mg, 80%).
A solution of ketal 24b (700
mg, 1.41 mmol) in THF (50 mL) and 1 N HCl (10 mL) was stirred at 80
°C under N2 for 2 h followed by procedure M to yield (540 mg, 85%) of compound 25b. 1H NMR (CDCl3, 400 MHz): δ 0.89 (t, J = 7.2 Hz, 3H), 1.45–1.52 (m, 2H), 1.82–1.91 (m, 2H),
2.04–2.12 (m, 2H), 2.30–2.52 (m, 8H), 2.66–2.70
(m, 6H), 3.15 (t, J = 7.2 Hz, 1H), 3.67 (t, J = 6.0 Hz, 4H), 3.85 (s, 3H), 6.83 (m, 1H), 6.98 (d, J = 8.8 Hz, 2H), 7.14 (bs, 1H), 7.13–7.15 (m, 1H),
7.31 (t, J = 8.0 Hz, 1H), 7.50 (d, J = 8.4 Hz, 2H).
Procedure N. N6-(2-(4-([1,1′-Biphenyl]-4-yl)piperazin-1-yl)ethyl)-N6-propyl-5,6,7,8-tetrahydroquinazoline-2,6-diamine (26a)
Into a solution of ketone 25a (450
mg, 1.07 mmol) in dry toluene (20 mL), tris(dimethylamino)methane
(780 mg, 5.36 mmol) was added, and the mixture was stirred under nitrogen
at 90 °C for 4 h. The solvent was removed under vacuo, and the
residue was dissolved in EtOH (50 mL). Guandine carbonate (460 mg,
2.55 mmol) was added next. The mixture was then refluxed for 17 h.
The solvent was evaporated in vacuo, and the residue was diluted with
CH2Cl2 and washed with brine. The organic layer
was dried over Na2SO4 and evaporated to yield
crude product, which was purified by purified by silica gelcolumn
chromatography (EtOAc/MeOH, 7:3) to yield (378 mg, 75%) of compound 26a. 1H NMR (CDCl3, 400 MHz): δ
0.87 (t, J = 6.4 Hz, 3H), 1.57–1.76 (m, 3H),
2.05–2.13 (m, 1H), 2.64–2.98 (m, 15H), 3.27 (t, J = 4.4 Hz, 4H), 4.93 (s, 2H), 6.97 (d, J = 8.8 Hz, 2H), 7.28 (d, J = 7.6 Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H),
7.54 (d, J = 7.2 Hz, 2H), 8.07 (s, 1H). The product
was converted into corresponding hydrochloride salt, m.p. 232 °C.
Anal. (C29H38N6·4.0HCl·1.0CH3COOCH2CH3): C, H, N. MS (ES+): m/z calculated for C29H38N6 [M + H+]: calculated 470.32; found
471.52.
Compound 26b (100 mg, 0.59
mmol) and 48% aqueous HBr (10 mL) was refluxed for 8 h using procedure H to afford compound 27 (118 mg, 65%, recrystallized
from ether). 1H NMR of HBrsalt (CD3OD, 400
MHz): δ 1.08 (t, J = 7.2 Hz, 3H), 1.95–2.01
(m, 2H), 2.27–2.29 (m, 1H), 2.58–2.64 (m, 1H), 3.01–3.39
(m, 6 H), 3.48 (bs, 8H), 3.91–4.01 (m, 4H), 4.14 (bs, 1H),
6.71 (d, J = 8.0 Hz, 1H), 6.99 (bs, 1H), 7.03 (d, J = 7.6 Hz, 2H), 7.19–7.26 (m, 3H), 7.56 (d, J = 8.4 Hz, 2H), 8.67 (bs, 1H). Hydrobromide salt, m.p.
255 °C. Anal. (C29H38N6O·6.0
HBr·3.0H2O): C, H, N.
Procedure O. t-Butyl 4-(biphenylcarbonyl)piperazine-1-carboxylate
(30)
To a stirring solution of t-Boc-piperazine, 28 (1.5 g, 8.05 mmol) in THF (25 mL),
4-biphenyl carbonyl chloride, 29 (1.6 g, 7.24 mmol) and
diisopropylethylamine (2.53 mL, 14.49 mmol) were added at room temperature.
The reaction mixture was stirred at the same temperature for overnight
and partitioned between brine and ethyacetate. The organic layer was
separated and washed with brine, dried over Na2SO4, and concentrated. The crude material was purified by column chromatography
over silica gel (hexane/EtOAc, 8.0:2.0) to give compound 30 (2.16 g, 80%). 1H (CDCl3, 400 MHz): δ1.47
(s, 9 H), 3.47 (bs, 4H), 3.74 (bs, 4H), 7.37 (t, J = 7.2 Hz, 1H), 7.43–7.49 (m, 4H), 7.59 (d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H).
Biphenyl-4-yl(piperazin-1-yl)methanone
(31)
Compound 30 (2.1 g, 5.73 mmol)
was reacted with TFA (20 mL) in CH2Cl2 (30 mL)
by following procedure B to give compound 31 (1.44 g, 95%). 1H (CDCl3, 400 MHz): 3.47 (bs,
4H), 3.74 (bs, 4H), 7.38 (t, J = 7.2 Hz, 1H), 7.44–7.49
(m, 4H), 7.59 (d, J = 7.2 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H).
Compound 34 (400 mg, 1.29 mmol)
was reacted with (±)-pramipexole (275 mg, 1.29 mmol) and NaBH(OAc)3 (492.12 mg, 2.32 mmol) in dichloromethane (20 mL) using procedure F to yield compound (±)-35 (420 mg, 70%). 1H NMR (CDCl3, 400 MHz): δ 0.86 (t, J = 7.2 Hz, 3H), 1.41–1.46 (m, 2H), 1.57–1.70
(m, 1H), 1.95 (d, J = 11.6 Hz, 1H), 2.40–2.70
(m, 13H), 3.01–3.06 (m, 1H), 3.48 (bs, 2H), 3.78 (bs, 2H),
4.02–4.12 (m, 1H), 4.96 (bs, 2H), 7.35 (t, J = 7.2 Hz, 1H), 7.41–7.47 (m, 4H), 7.56–7.61 (m, 4H).
The product was converted into corresponding hydrochloride salt, m.p.
255 °C. Anal. (C29H37N5OS·5.0
HCl·1.0 C2 H5OC2H5): C, H, N.
Evaluation of Antioxidant Activity. DPPH
Radical Scavenging Assay
To a 96-well plate, an amount of
100 μL of drug solutions (dissolved in methanol) ranging from
20 to 250 μM was added. Next 100 μL of 200 μM methanolic
solution of 1,1-DPPH was added, and the plate was shaken vigorously
at 30 °C for 25 min. Control wells received 100 μL of methanol
and 100 μL of 200 μM methanolicDPPH solution. Wells containing
only 200 μL of methanol served as a background correction. The
change in absorbance of all samples and standard (ascorbic acid) was
measured at 517 nm. Radical scavenging activity was expressed as inhibition
percentage and was calculated using the following formula: % scavenging
activity = (absorbance of control – absorbance of sample)/
(absorbance of control)] × 100.
Animal Experiments. Drugs
and Chemicals
The following commercially available drugs
were used in the experiment: reserpine hydrochloride (Alfa Aesar),
Ropinirole (Sigma Aldrich). The hydrochloride salts of (−)-8b and hydrobromide salt of (−)-9b were
dissolved in water for both locomotor and 6-OH-DA rotational experiments.
Reserpine was dissolved in 10–25 μL of glacial acetic
acid and further diluted with 5.5% glucose solution. All compounds
for this study were administered in a volume of 0.1–0.2 mL
for subcutaneous administration and 0.5–0.7 mL for intraperitoneal
administration into each rat.
Animals
In rodent
studies, animals were male Sprague–Dawley rats from Harlan
(Indianapolis, IN) weighing 220–225 g unless otherwise specified.
The lesioned rats (290–320 g) were purchased from Charles River
(Rensselaer, NY), and their unilateral lesion was checked twice by
apomorphinechallenge following the surgery. Animals were maintained
in sawdust-lined cages in a temperature and humidity controlled environment
at 22 ± 1 °C and 60 ± 5%, respectively, with a 12 h
light/dark cycle, with lights on from 6:00 a.m. to 6:00 p.m. They
were group-housed with unrestricted access to food and water. All
experiments were performed during the light component. All animal
use procedures were in compliance with the Wayne State University
Animal Investigation Committee consistent with AALAC guidelines.
Reversal of Reserpine-Induced Hypolocomotion in Rats
Administration
of reserpine inducescatalepsy in rodents primarily by blocking the
vesicular monoamine transporter (VMAT) which helps in the internalization
of monoamines into vesicles, resulting in metabolism of unprotected
monoamines in the cytosol that ultimately causes depletion of monoamines
in the synapse of the peripheral sympathetic nerve terminals. The
ability of the compound (−)-8b, (−)-9b, 1a and ropinirole to reverse the reserpine
induced hypolocomotion was investigated. Prior to administration of
reserpine animals were anaesthetized using isoflurane. Reserpine (5.0
mg/kg, sc) or saline (sc) was administered 18 h before the injection
of drug or vehicle (ip). The rats were placed individually in chambers
for 1 h for acclimatization purposes before the administration of
the test drug, standard drug, or vehicle. Immediately after administration
of drug or vehicle, animals were individually placed in VersaMax animal
activity monitor chamber (45 cm 30 cm 20 cm) (AccuScan Instruments,
Inc. Columbus, OH) to start measuring locomotor activity. Locomotion
was monitored for 6 h. Consecutive interruption of two infrared beams
situated 24 cm apart and 4 cm above the cage floor in the monitor
chamber recorded movement. The data were presented as horizontal counts
(HACTV). The effect of the individual doses of drugs on locomotor
activity was compared with respect to saline treated controls (mean ±
SEM). The data were analyzed by one way analysis of variance (ANOVA)
followed by Dunnett’s post hoc test. The effect was considered
significant if the difference from control group was observed at p < 0.05.
In Vivo Rotational Experiment with 6-OH-DA
Lesioned Rats
In the first 14 days post lesion challenge
with apomorphine was done with lesioned animals to observe a complete
rotation session post administration. In the second challenge with
apomorphine (0.05 mg/kg) 21 days postlesion, contralateral rotations
were recorded for 30 min; apomorphine produced rotations in all four
rats (average rotation of >250) indicating successful unilateral
lesion. In these rats, lesion was performed on the left side of the
medial forebrain bundle in the brain, and the coordinates used from
Bregma are the following: AP, −4.3; ML, p1.2; DV, −8.3. The rotations produced upon agonist challenge were clockwise. In
this study, apomorphine was also used as a reference compound. The
test drugs were dissolved in saline. The drugs (−)-8b, 1a, and (−)-9b were administered
i.p. The rotations were measured over 7–12 h. For control,
vehicle was administered alone. Rotations were measured in the Rotomax
rotometry system (AccuScan Instruments, Inc. Columbus, OH) equipped
with Rotomax analyzer, high resolution sensor, and animal chambers
with harnesses. Data were analyzed with Rotomax Windows software program.
The rotations were measured in a rotational chamber immediately after
administration of drugs. The data were collected at every 30 min.
Data were analyzed by the GraphPad (version 4, San Diego, CA) program.
All drugs produced contralateral rotations in all lesioned rats, which
lasted over 3–10 h.
Cell Culture and Treatments
The
hybridoma dopaminergicMN9Dcells are derived from the somatic infusion
of rostral mesencephalic neurons from embryonicC57BL/6J (E14) mice
with N18TG2 mousecells. They were cultured in T-75 flask (Greiner
Bio One, Frickenhausen, Germany) coated with 1 mg/mLpoly-l-lysine and maintained in DMEM (high glucose with phenol red) supplemented
with 10% Fecal Clone III serum, penicillin (50 units/mL), and streptomycin
(50 μg/mL) at 37 °C under 5% CO2 atmosphere.
Stock solution of 1a and (−)-9b were
prepared in DMSO and stored at −20 °C for the period of
experiments. MN9Dcells were pretreated with various concentrations
of drugs for 1 h and then cotreated with 100 μM MPP+ (prepared
freshly before addition from a stock solution in DMSO stored at −20
°C) for 24 h. The control cells were treated with the above medium
having 0.01% DMSO only.
Assessment of Cell Viability
To
evaluate the neuroprotection ability of the test compounds in the
presence of the neurotoxins MPP+, the quantitative and colorimetricMTT (3–4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide)
tetrazolium salt assay was used to assess cell viability. MN9Dcells
were seeded into poly-l-lysinecoated 96-well plates at 1
× 104 cells/well in 100 μL medium. After the
plate was equilibrated for 40 h, old medium was taken out from each
well, and 160 μL of fresh medium (containing 0.01% DMSO) was
added to control wells and wells which were to be treated with MPP+.
A solution of 160 μL of 1a or (−)-9b in the above medium without DMSO in 20, 10, 5, 1, 0.1,
0.01, 0.001 μM were added to wells which would be cotreated
with MPP+. The plate was incubated for 1 h at 37 °C under 5%
CO2 atmosphere. At the end of incubation, required amount
of MPP+ was added to each well (except the control wells) to maintain
a final concentration of 100 μM. The plate was then incubated
for 24 h at 37 °C under 5% CO2 atmosphere. Next, 20
μL of MTT stock solution (prepared in Dulbecco’s phosphate-buffered
saline) was added to each well to maintain a final concentration of
0.5 mg/mL, and the plate was incubated for another 3 h at 37 °C
under 5% CO2 atmosphere. Next, the plate was centrifuged
at 1500 rpm for 10 min, and the supernatants were removed carefully.
The formazancrystals were dissolved in 100 μL of a 1:1 mixture
of DMSO/methanol solution by shaking gently at 400 rpm for 30 min
at room temperature on a Thermomix R shaker (Eppendorf, Hamburg, Germany).
Then, the absorbance was measured at 570 nM and 690 nM using an Epoch
microplate reader (BioTek, Winooski, VT, USA). Background corrected
values (570–690 nM) were used to plot the graph. Data from
at least three experiments were analyzed using GraphPad software (Version
4, San Diego, USA).
DA D2 and D3 Receptor Assays
Binding potency was monitored by
inhibition of [3H]spiroperidol (16.2 Ci/mmol, Perkin-Elmer)
binding to dopaminerD2 and rD3 receptors expressed
in HEK-293cells, in a buffer containing 0.9% NaCl under conditions
corresponding to our “high [radioligand] protocol” as
described by us previously.[40,56] Observed IC50 values were converted to inhibition constants (Ki) by the Cheng–Prusoff equation (see ref (39)).[39] Functional activity of test compounds in activating dopaminehD2 and hD3 receptors expressed in CHO cells was measured
by stimulation of [35S]GTPγS (1250 Ci/mmol, Perkin-Elmer)
binding in comparison to stimulation by the full agonist dopamine
as described by us previously.[40]
Authors: Frank Boeckler; Ursula Ohnmacht; Thomas Lehmann; Wolfgang Utz; Harald Hübner; Peter Gmeiner Journal: J Med Chem Date: 2005-04-07 Impact factor: 7.446
Authors: Luana L Skalisz; Vanessa Beijamini; Samia L Joca; Maria A B F Vital; Claudio Da Cunha; Roberto Andreatini Journal: Prog Neuropsychopharmacol Biol Psychiatry Date: 2002-06 Impact factor: 5.067
Authors: Banibrata Das; Seenuvasan Vedachalam; Dan Luo; Tamara Antonio; Maarten E A Reith; Aloke K Dutta Journal: J Med Chem Date: 2015-11-30 Impact factor: 7.446
Figure 1
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Figure 2
Figure 3
Figure 4
Figure 5