Design, Synthesis, and Pharmacological Characterization of Carbazole Based Dopamine Agonists as Potential Symptomatic and Neuroprotective Therapeutic Agents for Parkinson's Disease

Article abstract of DOI:10.1021/acschemneuro.8b00291

We have developed a series of carbazole-derived compounds based on our hybrid D₂/D₃ agonist template to design multifunctional compounds for the symptomatic and disease-modifying treatment of Parkinson's disease (PD). The lead molecules (-)-11b (D-636), (-)-15a (D-653), and (-)-15c (D-656) exhibited high affinity for both D₂ and D₃ receptors and in GTPγS functional assay, the compounds showed potent agonist activity at both D₂ and D₃ receptors (EC₅₀ (GTPγS); D₂ = 48.7 nM, D₃ = 0.96 nM for 11b, D₂ = 0.87 nM, D₃ = 0.23 nM for 15a and D₂ = 2.29 nM, D₃ = 0.22 nM for 15c). In an animal model of PD, the test compounds exhibited potent in vivo activity in reversing hypolocomotion in reserpinized rats with a long duration of action compared to the reference drug ropinirole. In a cellular antioxidant assay, compounds (-)-11b, (-)-15a, and (-)-15c exhibited potent activity in reducing oxidative stress induced by neurotoxin 6-hydroxydopamine (6-OHDA). Also, in a cell-based PD neuroprotection model, these lead compounds significantly increased cell survival from toxicity of 6-OHDA, thereby producing a neuroprotective effect. Additionally, compounds (-)-11b and (-)-15a inhibited aggregation and reduced toxicity of recombinant alpha synuclein protein in a cell based in vitro assay. These observations suggest that the lead carbazole-based dopamine agonists may be promising multifunctional molecules for a viable symptomatic and disease-modifying therapy of PD and should be further investigated.

Full text of DOI:10.1021/acschemneuro.8b00291

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Article
Design, Synthesis and Pharmacological Characterization of
Carbazole Based Dopamine Agonists as Potential Symptomatic
and Neuroprotective Therapeutic Agents for Parkinson’s Disease
Asma Elmabruk, Banibrata Das, DEEPTHI YEDLAPUDI, Liping
Xu, Tamara Antonio, Maarten E.A. Reith, and Aloke K. Dutta
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00291 • Publication Date (Web): 09 Oct 2018
Downloaded from http://pubs.acs.org on October 17, 2018
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Design, Synthesis and Pharmacological Characterization of Carbazole Based
Dopamine Agonists as Potential Symptomatic and Neuroprotective Therapeutic
Agents for Parkinson’s Disease
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Asma Elmabruk, Banibrata Das, Deepthi Yedlapudi, Liping Xu, Tamara Antonio,
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Maarten E.A. Reith, Aloke K. Dutta
1. Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48202
. Department of Psychiatry, New York University, New York, NY 10016
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§Both authors contributed equally to this work
*Corresponding Author:
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
Keywords: Parkinson’s disease, D /D
agonist, carbazole, neuroprotection,
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multifunctional drug, alpha synuclein
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Abstract
We have developed a series of carbazole-derived compounds based on our hybrid
D /D agonist template to design multifunctional compounds for the symptomatic and
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disease-modifying treatment of Parkinson’s disease (PD). The lead molecules (-)-11b (D-
36), (-)-15a (D-653) and (-)-15c (D-656) exhibited high affinity for both D and D
6
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3
receptors and in GTPγS functional assay, the compounds showed potent agonist activity
at both D and D receptors (EC (GTPγS); D = 48.7 nM, D = 0.96 nM for 11b, D =
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0.87 nM, D = 0.23 nM for 15a and D = 2.29 nM, D = 0.22 nM for 15c). In an animal
3 2 3
model of PD, the test compounds exhibited potent in vivo activity in reversing
hypolocomotion in reserpinized rats with a long duration of action compared to the
reference drug ropinirole. In a cellular antioxidant assay, compounds (-)-11b, (-)-15a and
(-)-15c exhibited potent activity in reducing oxidative stress induced by neurotoxin 6-
hydroxydopamine (6-OHDA). Also, in a cell-based PD neuroprotection model, these lead
compounds significantly increased cell survival from toxicity of 6-OHDA, thereby,
producing a neuroprotective effect. Additionally, compounds (-)-11b and (-)-15a inhibited
aggregation and reduced toxicity of recombinant alpha synuclein protein in a cell based
in vitro assay. These observations suggest that the lead carbazole-based dopamine
agonists may be promising multifunctional molecules for a viable symptomatic and
disease modifying therapy of PD and should be further investigated.
Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disease that develops
from gradual depletion of dopamine (DA) in the basal ganglia with extensive loss of
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dopaminergic neurons in the substantia nigra pars compacta (SNc) of the midbrain
accompanied by accumulation of presynaptic protein α-synuclein (α-syn) known as Lewy
bodies.1-3 PD has been identified as the second most common neurodegenerative
disorder after Alzheimer’s disease and it affects 2-3% of the population ≥65 years of age.
The four cardinal symptoms associated with PD are tremor, rigidity, bradykinesia, and
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postural instability. Although progression and development of PD increase with age, the
exact pathogenesis and etiology remain unclear. Monogenic mutations account for less
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than 10% of PD cases. On the other hand environmental factors and genetic
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susceptibility are thought to contribute to development of sporadic PD cases. Several
factors have been implicated in the pathogenesis of PD including protein aggregation,
oxidative stress, mitochondrial dysfunction, metal toxicity, and exposure to pesticides.4
Compromised antioxidant defense mechanism could lead to cell death because
increased oxidative stress leads to misfolding of proteins such as α-syn and parkin in the
substantia nigra.4,6
The current treatment of Parkinson’s disease can be classified into four categories:
levodopa, DA agonists, monoamine oxidase inhibitors (MAO-Is), and catechol-o-methyl
transferase inhibitors (COMT-Is).7,8 DA agonists activate mostly the postsynaptic DA
receptors. These receptors are divided into D -like (D and D ) which activate the
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adenylate cyclase and D -like (D , D , and D ) which inhibit the adenylate cyclase.
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COMT-Is are also used in combination with levodopa to treat the wearing off symptoms
of PD, which occur after prolonged therapy with levodopa. Even though several
medications are available on the market to treat PD, there is still no cure of the disease.4,10
Moreover, the current Parkinsonism treatments act by reducing the symptoms only but
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they do not slow or stop the progression of the disease process, and they also produce
severe side effects such as dyskinesia and motor fluctuation with long-term therapy.6,11
New strategies have been adopted to overcome the current concerns with PD therapy by
developing multifunctional agents to target multiple aspects of the pathogenesis of the
disease. The rationale behind this strategy was built upon the fact that PD has been
identified as multifactorial in nature, thereby, development of a multifunctional agent could
be beneficial to treat patients with PD; however, no such agents are available in the
clinic.12,13
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Our efforts to design such multifunctional compounds to address multiple pathogenic
factors of PD as well as to address the symptomatic aspect led us to embark upon a drug
discovery approach focused on development of novel multifunctional dopamine D2/D3
agonist molecules. Specifically, as reported by us earlier, our hybrid structure strategy
which combines D /D agonist head groups to other moieties via piperazine linker,
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produced potent agonists for D2/D3 receptors.14,15 It seems the piperazine moiety not
only assists in producing D2/D3 potency but also provides suitable pharmacokinetic
properties. Thus, such hybrid molecules containing suitable moieties to modulate the
pathogenic pathway of PD, led to development of molecules to validate our proof of
concept.16-21 We have shown in our recent studies that lead molecule like D-512 and D-
607 not only have the potential to provide symptomatic effects but also produced potent
neuroprotective effects in various in vitro and in vivo experiments.16,21-23 In addition, we
have demonstrated that the compound D-512 (Figure 1) exhibits superior
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antiparkinsonian effects in vivo over a clinically approved drug, ropinirole. In our current
study, we carried out structure activity relationship (SAR) studies on carbazole-based
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molecules that have previously been identified to have neuroprotective properties.25-28
Previous studies have shown that carbazole-containing compounds exert neuroprotective
properties by enhancing the formation of neurons in the subgranular zone of the dental
gyrus.29,30 Moreover, it has been reported that carbazole analogues exhibit the ability to
regenerate neurons in the substantia nigra in neurodegenerative disease.27,30,31 Thus,
aminopropyl carbazole P7C3 and its analogue P7C3A20 (Figure 1) have shown
proneurogenic and neuroprotective properties in aged rats, stabilized mitochondrial
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membrane potential and inhibited newborn hippocampal neuron apoptosis. In addition,
tricyclic neuroleptic phenothiazines and tricyclic antidepressants like nortriptyline have
shown properties to interact with α-syn.32-34 Carbazole moiety bearing a close
resemblance to such tricyclic structures may also produce an interaction with α-syn
protein. Based on these findings, we designed and developed a number of multifunctional
molecules by covalently attaching D /D agonist head groups such as pramipexole and
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5-OH-MPAT to various carbazole moieties through a piperazine linker (Schemes 1-3).
In our current work, a series of compounds were synthesized, and the selected
compounds were characterized by both in vitro and in vivo assays. The lead molecules
-)-11b, (-)-15a and (-)-15c exhibited high affinity and functional potency at both D and
(
2
D receptors. The target compounds also exhibited potent in vivo activity in reserpinized
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rats and demonstrated significant dose-dependent neuroprotection effect in 6-
hydroxydopamine treated cells. This study will, therefore, shed additional light on the
importance of a carbazole moiety 25 as a potential molecular component in hybrid drug
design for developing disease-modifying therapeutics for PD.
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Results and Discussion
In this study, a series of compounds were synthesized by incorporating the
aminotetralin or bioisosteric equivalent agonist head group with carbazole functionality
via ethylpiperazine linker (scheme 1, 2 and 3). Scheme 1 describes the syntheses of final
compounds (±)-10a, (±)-10b, (±)-10c and (-)-11a, (-)-11b, (-)-11c. A palladium catalyzed
coupling of (4-bromophenyl)boronic acid and 1-bromo-2-nitrobenzene afforded 4'-bromo-
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2-nitro-1,1'-biphenyl (2a). This compound was then cyclized in the presence of PPh to
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produce 2-bromo-9H-carbazole (3a) which then underwent N-protection using di-tert-
butyl dicarbonate in the presence of 4-dimethylaminopyridine (4-DMAP) to yield 4a.
Palladium-catalyzed mediated cross coupling of 4a with 1-(2-((tert-butyldimethylsilyl)oxy)-
ethyl)piperazine18 under refluxing conditions in the presence of Cs CO and BINAP in
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toluene produced intermediate 5a. Treatment with n-Bu NF (TBAF) in THF removed the
4
silyl protecting group of compound 5a to produce the alcohol 6a, which in the presence
of pyridine-sulfur trioxide was oxidized to yield the corresponding aldehyde 7a. Reductive
amination of the aldehyde with either (±) or (-)-pramipexole in the presence of
NaBH(OAc) afforded compounds 8a and 9a, respectively. In the final step, the amine
3
protecting t-Boc groups were removed by treatment with trifluoroacetic acid to produce
the final compounds (±)-10a and (-)-11a as TFA salts. The other final compounds (±)-
10b, (±)-10c, (-)-11b and (-)-11c were also synthesized in the similar fashion as described
above, where 3-Bromo-9H-carbazole and 4-Bromo-9H-carbazole were used as the
starting materials, respectively.
The syntheses of the final compounds (±)-14a, (±)-14b, (±)-14c and (-)-15a, (-)-15b,
(-)-15c is shown in Scheme 2. To prepare these compounds, we employed (±) and (-)-(5-
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methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine and reductively alkylated with
intermediate aldehydes 7a-7c in the presence of NaBH(OAc) to produce compounds (±)-
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12a, (±)-12b, (±)-12c, (-)-13a, (-)-13b, and (-)-13c. Finally, demethylation and removal of
the protecting t-Boc groups were carried out in one step by refluxing with aq. HBr to give
the final compounds (±)-14a, (±)-14b, (±)-14c, (-)-15a, (-)-15b, and (-)-15c as HBr salts.
The syntheses of two more target compounds (±)-20 and (-)-21 are illustrated in
scheme 3. N-alkylation was first performed by refluxing carbazole with dibromoethane in
presence of a mixture of K CO , KOH and TBAB to afford 9-(2-bromoethyl)-9H-carbazole
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16. Base-catalyzed condensation of 1-(2-((tert-butyldimethylsilyl)-oxy)ethyl)piperazine
with intermediate 16 yielded compound 17, which on TBDMS deprotection in the
presence of TBAF in THF afforded alcohol 18 in excellent yield. Pyridine-sulfur trioxide
was used to oxidize alcohol 18 next to yield the corresponding aldehyde 19, which was
then underwent reductive amination with either (±) or (-)- pramipexole in the presence of
NaBH(OAc) to produce the final compounds (±)-20 and (-)-21. These two molecules were
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converted to their corresponding HCl salts by treatment with ethereal HCl. All the final
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compounds were characterized by H and C NMR as well as elemental analysis.
A well-established radioligand competition assay was carried out to evaluate binding
affinity of the test compounds and were compared with that of the reference agent (S)-5-
OH-DPAT (Table 1). Binding affinity to rat DA D and D receptors expressed in HEK-293
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17
cells was determined as described by us previously. Table 1 lists the binding data of
new compounds. Compounds (±)-10a-c, which incorporate racemic 2-aminothiazole
head group and a piperazine ring connected to the different positions of the carbazole
ring, exhibited high affinity for D and low to moderate affinity for D receptors. When the
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positions of attachment are at carbon 2 and 3 of the carbazole moiety for compounds 10a
and 10b, respectively, both the compounds displayed low affinity for D and high affinity
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for the D receptors with high selectivity (K, D = 902 nM, D = 6.18 nM, D /D = 146 and
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D = 612 nM, D = 3.12 nM, D /D = 196 for 10a and 10b, respectively). Interestingly,
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covalent attachment at position 4 of the carbazole ring dramatically improved the affinity
for D while that for D receptor remained the same (K, D = 76.9 nM, D = 7.8 nM, D /D
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9.86 for 10c). This indicated highest tolerance of the 4-substituted carbazole derivative
for interaction with the D2 and D3 receptors. As expected, we observed a 2-4-fold
improvement in binding affinity when enantiomerically pure aminothiazole moiety was
attached to the carbazole as in (-)-11a and (-)-11b compared to their racemic counterparts
(
K, D = 504 nM, D = 3.94 nM, D /D = 128 and D = 135 nM, D = 3.80 nM, D /D = 35
i 2 3 2 3 2 3 2 3
for (-)-11a and (-)-11b respectively). However, for (-)-11c we did not observe much
difference from its racemic version.
Next, the effect of bioisosteric replacement of the aminothiazole moiety with
aminotetraline functionality on the receptor binding of target compounds was evaluated.
In corroboration with our previous results,16,38 aminotetraline substituted compounds (±)-
1
4a-c and (-)-15a-c exhibited high affinity at both D and D receptors. For instance, the
2 3
aminotetraline analogue (-)-15a has been found to have very high affinity for D while
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displaying subnanomolar affinity for D receptor compared to the corresponding
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thiazolidium counterpart (-)-11a (K, D = 71.2 nM, D = 0.40 nM, D /D = 177 for (-)-15a
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vs D = 504 nM, D = 3.94 nM, D /D = 128 for (-)-11a). Among the three enantiomerically
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pure isomers (-)-15a-c, which differ only in the substitution positions at the carbazole
moiety, positions 2, 3 and 4, showed variable binding affinity at both D and D receptors
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(
K, D = 71.2 nM, D = 0.40 nM for (-)-15a (; D = 61.6 nM, D = 1.94 nM for (-)-15b and
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D = 16.9 nM, D = 0.36 nM for (-)-15c). As discussed before, substitution at the 4-position
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of the carbazole aromatic ring resulted in compounds 10c, (-)-11c, 14c and (-)-15c) with
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better D /D binding affinities in comparison to other isomeric analogues with compound
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(-)-15c exhibiting the highest affinity among all the molecules, underscoring the
importance of positional attachment to the carbazole ring. Finally, the binding affinities
were evaluated for another series of compounds in which the piperazine ring of the
agonist fragment was appended directly to the carbazole nitrogen atom through a
methylene linker. As shown in Table 1, enantiomeric compound (-)-21 displayed relatively
higher binding affinity at D and comparable affinity at D receptor with moderate
2
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selectivity compared to the racemic compound ()-20 (K, D = 435 nM, D = 6.60 nM,
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D /D = 65.9 and D = 82.6 nM, D = 7.18 nM, D /D = 12 for 20 and (-)-21, respectively).
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This structural modification suggests no significant differences in DA receptor interaction
between compounds where the carbazole moiety is attached either at the 2/3 positions
of the aromatic ring or through the nitrogen atom; however, a prominent difference exists
for compounds where the carbazole nitrogen is sterically free to probably participate in
additional receptor interaction (e.g. (-)-15c vs (-)-15b and (-)-21).
Based on binding data, functional activities of the selected optically active lead
compounds for human DA D and D receptors expressed in CHO cells were measured
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17
by using [ S]GTPγS binding assay. Agonist or partial agonist activity was judged by
comparing with the maximum stimulation (Emax), produced by the full agonist DA. As
shown in Table 2, aminothiazole containing compounds (-)-11b and (-)-11c demonstrated
moderate potency at both D and D receptors (EC (GTPγS); D = 48.7, D = 0.96 nM
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and D = 22.2, D = 1.67 nM, respectively), correlating well with binding data. While (-)-
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1b showed full agonist activity at both D and D receptors (E
= 87-93%), compound
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(-)-11c revealed partial agonist activity at D and full agonism at D receptor (E = 57%
max
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vs 82%, respectively for D and D ). On the other hand, aminotetraline compound (-)-15a
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displayed very high functional potency (EC (GTPγS); D = 0.87 and D = 0.23 nM) and
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full agonism (Emax = 85-92%) at both the receptors. Compound (-)-15c was also found to
be highly potent and efficacious in stimulating both receptors (EC (GTPγS); D = 2.29
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and D = 0.22 nM; E
= 74-88%). Neither compounds exhibited appreciable selectivity
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for D over D (Table 2) and their selectivity for D receptor was much less when
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compared to the binding data (Table 1). The values for the ClogP and tPSA for all the
compounds were calculated as shown in Table 1. In general, these values predict that
these compounds should cross the blood brain barrier to produce in vivo CNS efficacy
which we observed in case of (-)-11b, (-)-15a and (-)-15c (see below). Therefore, our
SAR results of a series of carbazole compounds show that the affinity and selectivity for
the D /D receptors are governed by the nature of covalent attachment to the carbazole
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moiety and the structure of agonist binding head group in the hybrid molecule.
In vivo evaluation of the compounds (-)-11b, (-)-15a and (-)-15c in a reserpine-based
PD animal model was performed next. In this well-established animal model for PD,
depletion of catecholamines by reserpine results in catalepsy in rats.39,40 Reserpine
produced a significant inhibition of locomotion in rats 18 h after the administration of the
drug (5 mg/kg, sc), which indicated the development of akinesia (Figure 2). Compounds
(-)-11b, (-)-15a and (-)-15c at the dose of 10 μMol/kg, ip, significantly reversed akinesia
in rats compared to reserpine treatment alone. The compounds also demonstrated
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significant enhancement of locomotion for the duration of the study of 6 h. The total
average number of horizontal locomotor counts generated by (-)-15a, (-)-15c, (-)-11b and
Ropinirole were 3718, 2680, 2408 and 1528, respectively. Thus, among the molecules
tested, (-)-15a was found to have the highest in vivo activity and this finding correlates
with in vitro functional assay where the compound exhibited subnanomolar potency for
stimulation of D /D receptors along with full agonist property. On the other hand, the
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reference drug ropinirole at 10 μMol/kg, ip, produced a fast onset of activation of
locomotion compared to the control but had a shorter duration of action compared to the
test compounds. It is also important to note that pharmacokinetic properties of these
compounds should also play an important role in efficacy. It has been shown that the
locomotor stimulation in this reserpine model is mediated by postsynaptic D /D receptor
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activation by the compounds.
Antioxidant Activity
Experimental detection of reactive oxygen species (ROS) produced by 6-OHDA was
carried out by DCFDA assay. 6-OHDA, a well known neurotoxin, produces oxidative
stress similar to what is observed in PD and the mechanism of neurotoxicity of 6-OHDA
takes place via its auto-oxidation and subsequent generation of hydrogen peroxide.41,42
DCFDA is a non-fluorogenic dye but in the presence of ROS, it is oxidized to produce
DCF which produces florescence. We have previously shown that 6-OHDA causes cell
43
death in a dose-dependent manner. We chose 75 M 6-OHDA as we have shown that
this dose induces 40-50% cell death. To examine whether our compounds (-)-11b, (-)-
15a and (-)-15c can protect PC12 cells from the ROS produced by the exposure to 75
M 6-OHDA, the PC12 cells were treated with 6-OHDA after pretreatment with various
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concentrations of drugs (5, 10, 20 M) for 24 h, and compared with 6-OHDA alone treated
cells. As shown in Figure 3, a well over two-fold increase in ROS was observed in cells
treated with 6-OHDA (75 M) alone compared to the control untreated cells. However,
the test compounds could dose dependently decrease, albeit without any significant
differences, the production of ROS induced by 6-OHDA (75 M) in PC12 cells. In this
regard, highest dose of all the three compounds was the most efficacious in producing
significant antioxidant effect. Thus at 20 M, a reduction in 76%, 93% and 36% ROS were
induced by (-)-11b, (-)-15a and (-)-15c, respectively (Figure 3A-C).
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Cellular Neuroprotection Activity
To investigate the neuroprotection property of the selected target molecules (-)-11b,
(-)-15a and (-)-15c against 6-OHDA-induced cytotoxicity, we used dopaminergic rat
adrenal Pheochromocytoma PC12 cells. As reported above, treatment of PC12 cells with
6-OHDA at 75 µM for 24 h produced ~ 50% loss in cells viability and this concentration
22
was used in subsequent experiments. In contrast, it was observed that the cells treated
with increasing concentrations of either (-)-11b or (-)-15c alone (0.01–30 µM) showed no
cells loss compared to untreated controls (Figure 4A and 4C, respectively), indicating no
toxicity of the compounds at the doses tested. However, (-)-15a produced some
incremental toxicity starting at 20 µM dose (Figure 4B). The potential neuroprotective
effects of (-)-11b, (-)-15a and (-)-15c against 6-OHDA-induced toxicity were evaluated
next. Thus, pre-treatment of the cells with the test compounds for 24 h followed by
exposure to 6-OHDA 75 µM for another 24 h produced dose-dependent protection of the
cells from the neurotoxic insult and the greatest protective effect was observed at 5 µM
for (-)-11b and 20 µM for (-)-15c. At those concentration both drugs increased cell survival
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by ~20% compared to 6-OHDA (75 µM) treated alone (Figure 4D and 4F), indicating
neuroprotective effect of these drugs. Compound (-)-15a also revealed dose-dependent
neuroprotection and the highest effect was observed at the dose of 10 µM (15%) but the
effect was not observed at the higher doses (Figure 4E). This could be due to the fact
that the compound by itself produced toxicity to the cells at doses ≥20 µM and the
presence of 6-OHDA enhanced the toxicity (Figure 4B).
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Effect of (-)-11b and (-)-15a on the aggregation of α-syn with 0.5% seeding
The α-syn assay was carried out by following the procedures developed by us
earlier.37 The compounds (-)-11b and (-)-15a were incubated with α-syn seeded with
0.5% PFFs for a period of 30D at 37 °C without agitation. Seeding of monomeric α-syn
with 0.5% PFFs leads to a significant increase in aggregation characterized by a 15-fold
increase in ThT fluorescence at 30D. Both (-)-11b and (-)-15a lead to a significant
decrease in the ThT fluorescence (p≤0.0001) when compared to α-syn seeded with 0.5%
PFFs alone at 30D (Fig. 5A). (-)-11b and (-)-15a showed a 16-fold and 3-fold decrease in
ThT fluorescence respectively, when compared to seeded α-syn at 30D.
When PC12 cells were treated with the aggregates formed by 0.5% PFFs seeding at
30D, it was observed that the viability went down to 37% when compared to control.
However, the seeded samples formed in the presence of (-)-11b and (-)-15a showed
significant less toxicity when compared to seeded α-syn without compounds. The viability
values of PC12 cells when treated with α-syn seeded samples in the presence of (-)-11b
and (-)-15a were 71% and 72% at 30D when compared to control (Figure 5B). We have
previously shown that the parent compounds to our hybrid molecules, Pramipexole and
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-OHDPAT were far less effective in decreasing ThT fluorescence and reducing toxicities
when compared to our multifunctional hybrid agonist.37
In conclusion, the present study represents the development of a novel series of
carbazole-based multifunctional DA D /D receptor agonists. Compounds (-)-11b, (-)-15a
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and (-)-15c exhibited high binding affinity and full agonist activity at the both D and D3
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receptors. The lead molecules were highly efficacious in a PD animal model by reversing
hypo-locomotion activity with a long duration of action, indicating their potential in relieving
motor dysfunction in PD. In a cellular model, the lead compounds exhibited potent anti-
oxidant activity produced by neurotoxin 6-OHDA. To gain further insight into their possible
multifunctional property, the data presented here also demonstrates that all three
compounds (-)-11b, (-)-15a and (-)-15c exhibited neuroprotective property in an in vitro
cellular model of PD by protecting neuronal PC12 cells from neurotoxin 6-OHDA.
Furthermore, in vitro assays with recombinant α-syn protein demonstrated potent
modulatory effect of (-)-11b and (-)-15a on aggregation and toxicity of α-syn protein. In
summary, we are able to demonstrate that multifunctional drugs like (-)-11b, (-)-15a and
(-)-15c have the potential not only to alleviate motor dysfunction in PD patients, but also
to modify disease progression. More in depth future studies to establish the disease-
modifying effects of the compounds will shed more light into their potential therapeutic
application.
Experimental Section
Reagents and solvents were obtained from commercial suppliers and used as
received unless otherwise indicated. Dry solvent was obtained according to the standard
procedure. All reactions were performed under N atmosphere unless otherwise
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indicated. Analytical silica gel 60 F254-coated TLC plates were purchased from EMD
Chemicals, Inc. and were visualized with UV light or by treatment with phosphomolybdic
acid (PMA), or ninhydrin. Flash column chromatographic purification was done using
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Whatman Purasil 60A silica gel 230−400 mesh. Proton nuclear magnetic resonance ( H
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NMR) spectra and carbon nuclear magnetic resonance ( C NMR) spectra were
measured on Varian 400 and 600 MHz NMR spectrometer with tetramethylsilane (TMS)
as an internal standard. The NMR solvent used was either CDCl or CD OD unless
3
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otherwise indicated. Autopol III automatic polarimeter (Rudolph Research Analytical) was
used to record the optical rotations. MEL-TEMP II (Laboratory Devices Inc., U.S.) capillary
melting point apparatus was used to record the melting points. Purity of the compounds
was determined by elemental analysis and was within ±0.4% of the theoretical value
(≥95% purity). Elemental analyses were performed by Atlantic Microlab, Inc., GA, USA.
Procedure A. 4'-Bromo-2-nitro-1,1'-biphenyl (2a). To a stirring solution of 1-bromo-2-
nitrobenzene (2.0 g, 9.9 mmol) and (4-bromophenyl)boronic acid (2.19 g, 10.89 mmol) in
THF (25 mL) were added Pd(PPh ) (0.572 g, 0.50 mmol) followed by 2M K CO (5.53 g
3
4
2
3
in 20 mL water) at room temperature. The reaction mixture was stirred at 90 °C for 12 h
after which it was cooled and extracted with CH Cl . The combined organic layer was
2
2
dried over Na SO4, filtered, and concentrated in vacuo. The crude material was purified
2
by column chromatography over silica gel using hexane:ethyl acetate (9:1) as solvent to
1
give compound 2a (2.7 g, 98%). H NMR (600 MHz, CDCl ): δ 7.88 (d, J = 8.4 Hz, 1H),
3
7
.64-7.61 (m, 1H), 7.59-7.55 (m, 2H), 7.53-7.50 (m, 1H), 7.42-7.40 (m, 1H), 7.20-7.18 (m,
H).
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-Bromo-2'-nitro-1, 1’-biphenyl (2c). To a stirring solution of 1-bromo-2-nitrobenzene
(2.50 g, 12.37 mmol) and (2-bromophenyl)boronic acid (2.73 g, 13.61 mmol) in THF (30
mL) were added Pd(PPh ) (0.715 g, 0.61 mmol) followed by 2M K CO (5.53 g in 20 mL
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water) according to procedure A to give compound 2c (2.18 g, 94%). H NMR (600 MHz,
CDCl ): δ 7.87 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.40 (d, J = 9 Hz, 1H), 7.30
3
(d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.2 Hz, 1H), 7.10 (d, J = 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz,
2H).
Procedure B. 2-Bromo-9H-carbazole (3a). Compound 2a and PPh were dissolved in
3
1,2-dichlorobenzene and the resulting solution was stirred at 170 °C for 12 h after which
it was cooled and extracted with CH Cl /H O. The combined organic layer was dried over
2
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2
Na SO , filtered, and concentrated in vacuo. The crude material was purified by column
2
4
chromatography over silica gel using hexane:ethyl acetate (9:1) to yield compound 3a
1
(1.57 g, 89%). H NMR (600 MHz, CDCl ): δ 8.04 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 8.4 Hz,
3
1H), 7.62-7.58 (m, 2H), 7.52-7.47 (m, 2H), 7.39 (d, J = 8.4 Hz, 1H).
4-Bromo-9H-carbazole (3c). Compound 2c and PPh3 were reacted in 1,2-
1
dichlorobenzene according to procedure B to yield compound 3c (1.25 g, 87%). H NMR
(600 MHz, CDCl ): δ 8.81 (d, J = 7.8 Hz, 1H), 7.97 (s, 1H), 7.50 (t, J = 7.2 Hz, 1H), 7.44
3
(d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 7.30 (d, J = 7.8 Hz, 1H), 7.25-7.20 (m, 1H).
Procedure C. tert-Butyl 2-bromo-9H-carbazole-9-carboxylate (4a). To a stirring
solution of compound 3a (1.5 g, 6.09 mmol) in THF (20 mL) were added (Boc) O (1.46
2
g, 6.7 mmol) and DMAP (0.819 g, 6.7 mmol) in THF (20 mL) at room temperature. The
reaction mixture was stirred at the same temperature for 12 h. The crude mixture was
evaporated under reduced pressure, followed by extraction with EtOAc (3 × 20 mL) in
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water. The combined organic layer was dried over Na SO , filtered, and concentrated in
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vacuo. The crude material was purified by silica gel column chromatography
1
(hexane:EtOAc = 19:1) to yield compound 4a (1.78 g, 84%). H NMR (600 MHz, CDCl ):
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δ 8.48 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.18 (d, J =
7.8 Hz, 1H), 7.11 (t, J = 7.2 Hz, 1H), 6.97 (t, J = 8.10 Hz, 1H), 1.53 (s, 9H).
tert-Butyl 3-bromo-9H-carbazole-9-carboxylate (4b). Commercially available 3-
Bromo-9H-carbazole (3b) (2.0 g, 8.13 mmol) was reacted with (Boc) O (1.95 g, 8.94
2
mmol) and DMAP (1.09 g, 8.94 mmol) in THF (20 mL) according to procedure C. The
crude material was purified by column chromatography over silica gel using hexane:ethyl
1
acetate (19:1) as solvent to give compound 4b (2.8 g, ∼100%). H NMR (600 MHz,
CDCl ): δ 8.27 (d, J = 8.4 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 2.4 Hz, 1H), 7.92
3
(qd, J = 7.8, 0.6 Hz, 1H), 7.55 (dd, J = 6.6, 2.4 Hz, 1H), 7.49 (td, J = 8.4, 1.2 Hz, 1H), 7.36
(td, J = 8.4, 1.2 Hz, 1H), 1.76 (s, 9H).
tert-Butyl 4-bromo-9H-carbazole-9-carboxylate (4c). Compound 3c (1.5 g, 6.09
mmol) was reacted with (Boc) O (1.46 g, 6.7 mmol) and DMAP (0.819 g, 6.7 mmol) in
2
THF (20 mL) according to procedure C. The crude material was purified by silica gel
1
column chromatography (hexane:EtOAc = 19:1) to yield compound 4c (1.78 g, 84%). H
NMR (600 MHz, CDCl ): δ 8.53 (s, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H),
3
7.82 (d, J = 8.4 Hz, 1H), 7.49-7.46 (m, 2H), 7.36 (t, J = 7.2 Hz, 1H), 1.77 (s, 9H).
Procedure D. tert-Butyl 2-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperazin-1-yl)-
9H-carbazole-9-carboxylate (5a). To a mixture of compounds 4a (0.8 g, 2.31 mmol), 1-
(
2-((tert-butyldimethylsilyl)oxy)-ethyl)piperazine18 (1.13 g, 4.62 mmol), BINAP (0.144 g,
0.23 mmol) and Cs CO (2.26 g, 6.93 mmol), toluene (15 mL) was added under N
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atmosphere. The reaction mixture was degassed by bubbling N for 5 min and then
2
Pd(OAc) (0.039 g, 0.17 mmol) was added quickly. The system was degassed again and
2
refluxed for 24 h under inert condition. The reaction mixture was cooled to room
temperature, filtered through a pad of celite, washed with dichloromethane and
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concentrated in vacuum. The crude residue was purified by column chromatography
1
(hexane:EtOAc = 4:1) to afford compound 5a (0.97 g, 82%). H NMR (600 MHz, CDCl ):
3
δ 8.23 (d, J = 7.8 Hz, 1H), 7.92 (s, 1H), 7.84 (d, J = 7.2 Hz, 1H), 7.81 (d, J = 9.0 Hz, 1H),
7
.35 (t, J = 7.2 Hz, 1H), 7.29 (t, J = 7.2 Hz, 1H), 7.00 (dd, J = 9.0, 1.8 Hz, 1H), 3.82 (t, J
6.6 Hz, 2H), 3.32 (t, J = 4.8 Hz, 4H), 2.74 (t, J = 4.8 Hz, 4H), 2.62 (t, J = 6.6 Hz, 2H),
=
1.75 (s, 9H), 0.91 (s, 9H), 0.09 (s, 6H).
tert-Butyl 3-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperazin-1-yl)-9H-carbazole-9-
carboxylate (5b). A mixture of compound 4b (1.2 g, 3.47 mmol), 1-(2-((tert-
butyldimethylsilyl)oxy)ethyl)piperazine (1.27 g, 5.20 mmol), Pd(OAc) (0.058 g, 0.26
2
mmol), BINAP (0.216 g, 0.35 mmol) and Cs CO (3.39 g, 10.4 mmol) in toluene (25 mL)
2
3
o
was heated at 110 C for 24 h according to procedure D. The crude material was purified
by silica gel column chromatography (hexane:EtOAc = 4:1) to give compound 5b (1.4 g,
1
7
9%). H NMR (600 MHz, CDCl ): δ 8.27 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H),
3
7.93 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.32 (t, J = 7.8
Hz, 1H), 7.13 (dd, J = 6.6, 2.4 Hz, 1H), 3.83 (t, J = 6.6 Hz, 2H), 3.28 (t, J = 4.8 Hz, 4H),
2.77 (t, J = 4.8 Hz, 4H), 2.63 (t, J = 6.6 Hz, 2H), 1.75 (s, 9H), 0.92 (s, 9H), 0.09 (s, 6H).
tert-Butyl 4-(4-(2-((tert-butyldimethylsilyl)oxy)ethyl)piperazin-1-yl)-9H-carbazole-9-
carboxylate (5c). A mixture of 4c (2.70 g, 7.80 mmol), 1-(2-((tert-butyldimethylsilyl)oxy)-
ethyl)piperazine (3.24 g, 13.25 mmol), Pd(OAc) (0.0.13 g, 0.59 mmol), BINAP (0.49 g,
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.78 mmol) and Cs CO (7.62 g, 23.4 mmol) in toluene (30 mL) was heated at 110°C for
2
3
24 h according to procedure D. The crude residue was purified by column
1
chromatography (hexane:EtOAc = 4:1) to afford compound 5c (3.24 g, 82%). H NMR
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9
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8
9
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(600 MHz, CDCl ): δ 8.34 (dd, J = 8.1,4.8 Hz, 2H), 8.08 (d, J = 8.4 Hz, 1H), 7.45 (t, J =
3
7
.2 Hz, 1H), 7.41-7.36 (m, 2H), 7.04 (d, J = 8.4 Hz, 1H), 3.85 (t, J = 6.6 Hz, 2H), 3.33 (d,
J = 5.4 Hz, 2H), 2.99 (t, J = 5.4 Hz, 2H), 2.71 (t, J = 6.6 Hz, 4H), 1.75 (s, 9H), 0.91 (s,
H), 0.10 (s, 6H).
9
Procedure E. tert-Butyl 2-(4-(2-hydroxyethyl)piperazin-1-yl)-9H-carbazole-9-
carboxylate (6a). Into a stirring solution of compound 5a (0.95 g, 1.86 mmol) in THF (10
mL) was added n-tetrabutylammonium fluoride (2.8 mL, 2.8 mmol, 1.0 M solution in THF)
at 0 °C. The reaction mixture was then stirred at room temperature for 2 h. THF was
evaporated in vacuo, and the residue was diluted with CH Cl (25 mL) and washed with
2
2
a saturated solution of NaHCO . The water layer was extracted with CH Cl (3 × 50 mL).
3
2
2
The combined organic layer was washed with brine, dried over Na SO , and evaporated
2
4
under reduced pressure. The crude product was purified by silica gel column
1
chromatography (EtOAc:MeOH = 9:1) to give compound 6a (0.595 g, 81%). H NMR (600
MHz, CDCl ): δ 8.23 (d, J = 7.8 Hz, 1H), 7.94 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.82 (d, J
3
=
9.0 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.2 Hz, 1H), 7.00 (dd, J = 6.6, 1.8 Hz,
H), 3.68 (t, J = 5.4 Hz, 2H), 3.34 (t, J = 4.8 Hz, 4H), 2.73 (t, J = 4.8 Hz, 4H), 2.64 (t, J =
1
5.4 Hz, 2H), 1.76 (s, 9H).
tert-Butyl 3-(4-(2-hydroxyethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (6b).
Compound 5b (1.2 g, 2.35 mmol) in THF (15 mL) was reacted with n-tetrabutylammonium
fluoride (4.71 mL, 4.71 mmol, 1.0 M solution in THF) according to procedure E. The crude
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product was purified by silica gel column chromatography (EtOAc:MeOH = 9:1) to yield
1
compound 6b (0.78 g, 84%). H NMR (600 MHz, CDCl ): δ 8.28 (d, J = 8.4 Hz, 1H), 8.18
3
(d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 7.8 Hz,
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1H), 7.33 (t, J = 7.8 Hz, 1H), 7.13 (dd, J = 6.6, 2.4 Hz, 1H), 3.69 (t, J = 5.4 Hz, 2H), 3.29
(t, J = 4.8 Hz, 4H), 2.76 (t, J = 4.8 Hz, 4H), 2.66 (t, J = 5.4 Hz, 2H), 1.75 (s, 9H).
tert-Butyl 4-(4-(2-hydroxyethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (6c).
Compound 5c (3.30 g, 6.47 mmol) was reacted with n-tetrabutylammonium fluoride (9.70
mL, 9.70 mmol, 1.0 M solution in THF) in THF (30 mL) according to procedure E. The
crude product was purified by silica gel column chromatography (EtOAc:MeOH = 9:1) to
1
give compound 6c (2.01 g, 80%). H NMR (600 MHz, CDCl ): δ 8.27 (d, J = 6.6 Hz, 1H),
3
8
.17 (d, J = 7.8 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.47 (s, 1H), 7.43 (t, J = 7.2 Hz, 1H),
.31 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 9.0 Hz, 1H), 3.69 (t, J = 5.4 Hz, 2H), 3.26 (s, 4H),
7
2.76 (s, 4H), 2.62 (t, J = 5.4 Hz, 2H), 1.73 (s, 9H).
Procedure F. tert-Butyl 2-(4-(2-oxoethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate
(
7a). Into a stirring solution of compound 6a (0.30 g, 0.76 mmol) in CH Cl (6 mL) and
2 2
DMSO (3 mL), was added Et N (0.74 mL, 5.31 mmol) at 0 °C. The reaction mixture was
3
stirred for 5 min followed by addition of SO .py complex (0.604 g, 3.79 mmol) at 0 °C. Ice
3
bath was removed and the reaction mixture was stirred at room temperature for 2 h. The
reaction mixture was quenched by addition of water and extracted with CH Cl (3 × 30
2
2
mL). The combined organic layer was dried using Na SO , and the solvent was removed
2
4
under reduced pressure. The crude product was purified by silica gel column
chromatography (hexane:EtOAc = 3:7) to give aldehyde 7a (0.25 g, 84%). The purified
1
aldehyde was used immediately for next step. H NMR (600 MHz, CDCl ): δ 9.74 (s, 1H),
3
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.23 (d, J = 8.4 Hz, 1H), 7.94 (s, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H),
.36 (td, J = 7.2, 1.2 Hz, 1H), 7.29 (td, J = 7.2, 1.2 Hz, 1H), 6.97 (dd, J = 6.6, 1.8 Hz, 1H),
7
3.36 (t, J = 4.8 Hz, 4H), 3.24 (t, J = 1.2 Hz, 2H), 2.73 (t, J = 4.8 Hz, 4H), 1.75 (s, 9H).
tert-Butyl 3-(4-(2-oxoethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (7b).
Compound 6b (0.45 g, 1.14 mmol) in CH Cl (10 mL) and DMSO (5 mL), was oxidized
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2
using SO .py complex (0.905 g, 5.69 mmol) and Et N (1.11 mL, 7.96 mmol) according to
3
3
procedure F. The crude product was purified by silica gel column chromatography
1
(EtOAc) to yield compound 7b (0.35 g, 78%). H NMR (600 MHz, CDCl ): δ 9.76 (s, 1H),
3
8
.28 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 2.4
Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.12 (dd, J = 6.6, 2.4 Hz, 1H),
.31 (t, J = 4.2 Hz, 4H), 3.25 (t, J = 1.2 Hz, 2H), 2.74 (t, J = 4.2 Hz, 4H), 1.74 (s, 9H).
3
tert-Butyl 4-(4-(2-oxoethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (7c). Alcohol
6c (0.35 g, 0.88 mmol) was oxidized using SO .py complex (0.704 g, 4.425 mmol), DMSO
3
(9 mL) and Et N (0.86 mL, 6.19 mmol) in CH Cl (6 mL) according to procedure F. The
3 2 2
crude product was purified by silica gel column chromatography (hexane:EtOAc = 3:7) to
give aldehyde 7c (0.31 g, 89%). The purified aldehyde was used immediately for next
1
step. H NMR (600 MHz, CDCl ): δ 9.78 (s, 1H), 8.33 (d, J = 7.8 Hz, 1H), 8.27 (d, J = 7.8
3
Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.44-7.40 (m, 2H), 7.35 (t, J =
7.2 Hz, 1H), 7.06 (dd, J = 7.8, 1.8 Hz, 1H), 3.36-3.34 (m, 4H), 3.06 (t, J = 11.1 Hz, 2H),
2.73 (d, J = 11.4 Hz, 2H), 2.71 (d, J = 10.2 Hz, 2H), 1.75 (s, 9H).
Procedure G. tert-Butyl 2-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-
yl)(propyl)amino)-ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (8a). Into
a
6
stirring solution of racemic N -propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine
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6
(
0.058 g, 0.27 mmol) in CH Cl (7 mL) was added aldehyde 7a (0.12 g, 0.31 mmol). After
2
2
the mixture was stirred for 1.5 h, NaBH(OAc) (0.13 g, 0.61 mmol) was added and the
3
mixture was stirred for another 46 h at room temperature. The reaction mixture was
quenched with a saturated solution of NaHCO at 0 °C and extracted with CH Cl (3 × 50
0
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9
0
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2
2
mL). The combined organic layer was dried over Na SO , and the solvent was removed
2
4
under reduced pressure. The crude product was purified by silica gel column
1
chromatography (CH Cl :MeOH = 19:1) to afford compound 8a (0.06 g, 38%). H NMR
2
2
(600 MHz, CDCl ): δ 8.23 (d, J = 8.4 Hz, 1H), 7.92 (s, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.81
3
(d, J = 8.4 Hz, 1H), 7.35 (td, J = 7.2, 1.2 Hz, 1H), 7.29 (td, J = 7.2, 1.2 Hz, 1H), 7.00 (dd,
J = 6.0, 2.4 Hz, 1H), 4.77 (bs, 2H), 3.33 (t, J = 4.8 Hz, 4H), 3.07 (m, 1H), 2.78‒2.68 (m,
8
H), 2.60‒2.47 (m, 6H), 2.02‒2.00 (m, 1H), 1.75 (s, 10H), 1.51‒1.47 (m, 2H), 0.90 (t, J =
7
.2 Hz, 3H).
tert-Butyl 3-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)
ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (8b). Aldehyde 7b (0.15 g, 0.38
6
mmol) in CH Cl
(10 mL) was reacted with racemic N -propyl-4,5,6,7-
2
2
tetrahydrobenzo[d]thiazole-2,6-diamine (0.073 g, 0.34 mmol) and NaBH(OAc) (0.162 g,
3
0
.76 mmol) according to procedure G. Crude product was purified by column
1
chromatography (EtOAc:MeOH = 9:1) to afford compound 8b (0.065 g, 32%). H NMR
(400 MHz, CDCl ): δ 8.27 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 9.2 Hz, 1H), 7.93 (d, J = 7.6
3
Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.44 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.12
dd, J = 7.2, 2.4 Hz, 1H), 5.12 (bs, 2H), 3.28 (t, J = 4.8 Hz, 4H), 3.07‒3.02 (m, 1H),
.78‒2.67 (m, 8H), 2.58‒2.45 (m, 6H), 2.00‒1.98 (m, 1H), 1.74 (s, 10H), 1.52‒1.46 (m,
2H), 0.90 (t, J = 7.2 Hz, 3H).
(
2
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tert-Butyl 4-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)
ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (8c). Aldehyde 7c (0.15 g, 0.38
6
mmol) was reacted with racemic N -propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-
1
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0
diamine (0.073 g, 0.34 mmol) and NaBH(OAc) (0.162 g, 0.76 mmol) in CH Cl (10 mL)
3
2
2
according to procedure G. The crude product was purified by silica gel column
1
chromatography (CH Cl :MeOH = 19:1) to afford compound 8c (0.91 g, 41%). H NMR
2
2
(
600 MHz, CDCl ): δ 8.23 (d, J = 6.6 Hz, 1H), 8.28 (d, J = 6.6 Hz, 1H), 8.06 (d, J = 6.6
3
Hz, 1H), 7.43 (d, J = 5.4 Hz, 1H), 7.37 (d, J = 5.4 Hz, 2H), 7.02 (d, J = 6.0 Hz, 1H), 5.03
s, 2H), 3.10-3.05 (m, 3H), 3.07-2.98 (m, 2H), 2.76‒2.69 (m, 4H), 2.62‒2.60 (m, 8H),
2.01‒2.00 (m, 1H), 1.73 (s, 10H), 1.50‒1.46 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H).
S)-tert-Butyl 2-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)
(
(
amino)ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (9a). Aldehyde 7a (0.125 g,
0.32 mmol) was reacted with (-)-pramipexole (0.06 g, 0.29 mmol) and NaBH(OAc) (0.135
3
g, 0.64 mmol) in CH Cl (8 mL) according to procedure G. The crude product was purified
2
2
by silica gel column chromatography (CH Cl :MeOH = 19:1) to afford compound 9a
2
2
1
(
0.067 g, 40%). H NMR (600 MHz, CDCl ): δ 8.23 (d, J = 8.4 Hz, 1H), 7.92 (s, 1H), 7.85
3
(d, J = 7.2 Hz, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.35 (td, J = 7.2, 1.2 Hz, 1H), 7.29 (td, J =
7
.2, 1.2 Hz, 1H), 7.00 (dd, J = 6.6, 2.4 Hz, 1H), 4.76 (bs, 2H), 3.33 (t, J = 4.8 Hz, 4H),
.07 (m, 1H), 2.79‒2.68 (m, 8H), 2.61‒2.48 (m, 6H), 2.02‒2.00 (m, 1H), 1.75 (s, 10H),
3
1.51‒1.48 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H); [α]D25= -44.0 (c=1.0 in CH Cl ).
2
2
(S)-tert-Butyl
3-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)
amino)ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (9b). Aldehyde 7b (0.30 g,
0.76 mmol) was reacted with (-)-pramipexole (0.145 g, 0.69 mmol) and NaBH(OAc)3
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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5
5
5
6
(
0.323 g, 1.52 mmol) in CH Cl (15 mL) according to procedure G. The crude product was
2
2
purified by silica gel column chromatography (EtOAc:MeOH = 9:1) to afford compound
1
9b (0.158 g, 39%). H NMR (600 MHz, CDCl ): δ 8.27 (d, J = 7.2 Hz, 1H), 8.16 (d, J = 8.4
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 2.4 Hz, 1H), 7.43 (t, J = 7.2 Hz, 1H), 7.32
(
t, J = 7.2 Hz, 1H), 7.12 (dd, J = 6.6, 2.4 Hz, 1H), 5.21 (bs, 2H), 3.28 (t, J = 4.8 Hz, 4H),
3.07‒3.02 (m, 1H), 2.78‒2.66 (m, 8H), 2.59‒2.46 (m, 6H), 2.01‒1.99 (m, 1H), 1.74 (s,
0H), 1.52‒1.46 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H).
S)-tert-Butyl 4-(4-(2-((2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)
1
(
amino)ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (9c). Aldehyde 7c (0.150 g,
0.38 mmol) was reacted with (-)-pramipexole (0.73 g, 0.54 mmol) and NaBH(OAc) (0.162
3
g, 0.76 mmol) in CH Cl (10 mL) according to procedure G. The crude product was
2
2
purified by silica gel column chromatography (CH Cl :MeOH = 19:1) to afford compound
2
2
1
9c (0.097 g, 43%). H NMR (600 MHz, CDCl ): δ 8.32 (d, J = 7.2 Hz, 1H), 8.28 (d, J = 7.8
3
Hz, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.43 (d, J = 7.2 Hz, 1H), 7.40-7.35 (m, 2H), 7.30 (d, J =
7
.2 Hz, 1H), 4.83 (bs, 2H), 3.33 (d, J = 8.4 Hz, 2H), 3.06 (m, 3H), 2.99 (m, 2H), 2.76‒2.70
(
m, 4H), 2.60 (m, 7H), 1.74 (s, 10H), 1.51‒1.48 (m, 2H), 0.90 (t, J = 6.6 Hz, 3H); [α]D25= -
2
7.20 (c=1.0 in CH Cl ).
2 2
6
6
Procedure H. N -(2-(4-(9H-carbazol-2-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-
tetrahydrobenzo-[d]thiazole-2,6-diamine (10a) (D-652). To a stirred solution of 8a
o
(0.055 g, 0.09 mmol) in CH Cl (3 mL) at 0 C, trifluoroacetic acid (3 mL) was added
2
2
slowly and the reaction mixture was stirred for 3 h at room temperature. Unreacted TFA
and solvent were removed under reduced pressure and the obtained TFA salt was
1
washed with ether for several times followed by drying to yield 10a (0.079 g, 90%). H
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NMR (600 MHz, CD OD): δ 7.99 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.42 (d, J
3
=
7.8 Hz, 1H), 7.32 (t, J = 7.2 Hz, 1H), 7.25 (s, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.03 (d, J =
8.4 Hz, 1H), 3.84 (m, 1H), 3.64‒3.55 (m, 5H), 3.48-3.42 (m, 3H), 3.36 (s, 4H), 3.23-3.14
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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7
8
9
0
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9
0
(m, 2H), 3.01-2.99 (m, 1H), 2.84-2.79 (m, 1H), 2.71‒2.65 (m, 2H), 2.32-2.31 (m, 1H),
2
.02-1.95 (m, 1H), 1.83‒1.79 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz,
CD OD): δ 170.28, 160.68, 145.32, 140.57, 132.69, 125.56, 124.53, 122.50, 121.20,
3
1
20.14, 119.84, 119.33, 118.79, 118.29, 117.08, 115.15, 111.37, 110.77, 109.73, 59.75,
2.49, 51.56, 50.08, 22.29, 21.88, 21.39, 18.01, 10.19; Anal. Calcd for
5
C H N S.4CF COOH: C, 45.77; H, 4.27; N, 8.90. Found: C, 45.71; H, 4.64; N, 8.87.
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3
6
6
N -(2-(4-(9H-carbazol-3-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-tetrahydrobenzo
[
d]thiazole-2,6-diamine (10b) (D-627). Compound 8b (0.05 g, 0.09 mmol) in CH Cl (2
2 2
o
mL) at 0 C, was treated with trifluoroacetic acid (2 mL) according to procedure H to obtain
1
the TFA salt of compound 10b (0.052 g, 84%). H NMR (400 MHz, CD OD): δ 8.01 (d, J
3
= 8.0 Hz, 1H), 7.66 (d, J = 1.6 Hz, 1H), 7.41-7.36 (m, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.16-
7.14 (m, 1H), 7.11 (t, J = 7.2 Hz, 1H), 3.34 (s, 1H), 3.19 (s, 4H), 2.98‒2.91 (m, 2H), 2.80-
2.51 (m, 12H), 2.07‒2.04 (m, 1H), 1.81-1.77 (m, 1H), 1.63‒1.53 (m, 2H), 0.95 (t, J = 7.2
13
Hz, 3H); C NMR (100 MHz, CD OD): δ 168.63, 144.25, 143.69, 143.37, 140.69, 135.84,
3
1
25.07, 123.21, 122.92, 119.50, 118.21, 118.00, 113.45, 110.87, 110.41, 107.90, 58.85,
5.41, 53.24, 53.12, 51.07, 25.37, 24.70, 23.79, 20.35, 10.43; Anal. Calcd for
5
C H N S.2CF COOH: C, 53.62; H, 5.34; N, 11.73. Found: C, 53.64; H, 5.83; N, 11.34.
28
36
6
3
6
6
N -(2-(4-(9H-carbazol-4-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-tetrahydrobenzo
[d]thiazole-2,6-diamine (10c) (D-658). Compound 8c (0.082 g, 0.14 mmol) was treated
with trifluoroacetic acid (3 mL) in CH Cl (3 mL) according to procedure H to furnish the
2
2
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TFA salt of 10c (0.091g, 90%). H NMR (600 MHz, CD OD): δ 8.08 (d, J = 7.8 Hz, 1H),
3
7
.45 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.22 (d, J = 7.8
Hz, 1H), 7.19 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 7.8 Hz, 1H), 3.94‒3.89 (m, 1H), 3.83 (d, J =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
.2 Hz, 2H), 3.77-3.59 (m, 8H), 3.29 (d, J = 1.2 Hz, 1H), 3.24-3.20 (m, 3H), 3.06-3.04 (m,
H), 2.92-2.88 (m, 1H), 2.78‒2.71 (m, 2H), 2.37-2.36 (m, 1H), 2.11-2.04 (m, 1H),
1
1.86‒1.82 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H); 13C NMR (150 MHz, CD OD): δ 170.32,
3
1
1
2
61.19, 146.42, 141.50, 139.78, 132.81, 125.91, 124.74, 121.78, 121.34, 118.62, 115.64,
11.66, 110.23, 107.39, 59.17, 53.36, 53.25, 52.80, 50.81, 48.60, 48.01, 44.95, 22.51,
2.03, 21.49, 18.34, 9.79; Anal. Calcd for C H N S, 3CF COOH, CH OH, H O
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6
3
3
2
: C, 47.73; H, 5.15; N, 9.54. Found: C, 47.63; H, 4.67; N, 9.23.
6
6
(
S)-N -(2-(4-(9H-carbazol-2-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-tetrahydro-
benzo[d]thiazole-2,6-diamine (11a) (D-651). Compound 9a (0.065 g, 0.11 mmol) was
treated with trifluoroacetic acid (3 mL) in CH Cl (3 mL) according to procedure H to
2
2
1
furnish the TFA salt of 11a (0.085 g, 93%). H NMR (600 MHz, CD OD): δ 7.99 (d, J =
3
8
.4 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 7.2 Hz, 1H),
.26 (s, 1H), 7.13 (t, J = 7.2 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 3.86 (m, 1H), 3.65‒3.56
7
(
m, 5H), 3.49-3.42 (m, 3H), 3.37 (s, 4H), 3.25-3.16 (m, 2H), 3.02-3.00 (m, 1H), 2.85-2.81
(
m, 1H), 2.73‒2.66 (m, 2H), 2.34-2.32 (m, 1H), 2.04-1.97 (m, 1H), 1.83‒1.80 (m, 2H),
13
1
.03 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, CD OD): δ 170.29, 160.41, 145.25, 140.58,
3
132.67, 125.55, 124.55, 122.49, 121.21, 120.15, 119.82, 119.32, 118.82, 118.24, 116.97,
1
15.06, 111.39, 110.76, 109.70, 59.75, 52.50, 51.55, 50.17, 22.31, 21.89, 21.40, 17.98,
1
0.18; [α]D25= -24.8 (c=1.0 in CH OH); Anal. Calcd for C H N S.3CF COOH.2H O: C,
3
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3
2
47.11; H, 5.00; N, 9.70. Found: C, 47.63; H, 4.67; N, 9.23.
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(
S)-N -(2-(4-(9H-carbazol-3-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-tetrahydro-
benzo[d]thiazole-2,6-diamine (11b) (D-636). Compound 9b (0.15 g, 0.25 mmol) was
treated with trifluoroacetic acid (8 mL) in CH Cl (8 mL) according to procedure H to
2
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
furnish the TFA salt of 11b (0.20 g, 96%). H NMR (600 MHz, CD OD): δ 8.27 (s, 1H),
3
8
.11 (d, J = 7.2 Hz, 1H), 7.59-7.54 (m, 2H), 7.49 (d, J = 8.4 Hz, 1H), 7.44 (t, J = 7.2 Hz,
1H), 7.21 (t, J = 7.2 Hz, 1H), 3.96 (m, 1H), 3.80 (s, 4H), 3.57‒3.45 (m, 2H), 3.34-3.27 (m,
2
H), 3.20-3.10 (m, 7H), 2.97-2.92 (m, 1H), 2.83-2.75 (m, 2H), 2.44‒2.42 (m, 1H), 2.17-
13
2
.10 (m, 1H), 1.90‒1.84 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, CD OD):
3
δ 170.35, 160.54, 141.04, 139.17, 135.08, 132.80, 126.39, 123.44, 122.29, 119.95,
119.05, 117.60, 111.67, 111.55, 111.45, 110.90, 59.31, 54.18, 53.61, 51.21, 50.61, 22.42,
2
2.09, 21.50, 17.98, 9.82; [α]D25= -17.4 (c=1.0 in CH OH); Anal. Calcd for
3
C H N S.3CF COOH.H O: C, 48.11; H, 4.87; N, 9.90. Found: C, 48.25; H, 4.99; N, 9.58.
28
36
6
3
2
6
6
(S)-N -(2-(4-(9H-carbazol-4-yl)piperazin-1-yl)ethyl)-N -propyl-4,5,6,7-tetrahydro-
benzo[d]thiazole-2,6-diamine (11c) (D-657). Compound 9c (0.83 g, 0.14 mmol) was
treated with trifluoroacetic acid (7 mL) in CH Cl (7 mL) according to procedure H to
2
2
1
furnish the TFA salt of 11c (0.085 g, 93%). H NMR (600 MHz, CD OD): δ 8.07 (d, J =
3
7
7
3
.2 Hz, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H),
.22 (d, J = 7.8 Hz,, 1H), 7.19 (t, J = 7.2 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 3.90 (s, 1H)
.84 (d, J = 6.6 Hz, 2H), 3.78‒3.71 (m, 4H), 3.56-3.43 (m, 4H), 3.29 (s, 2H), 3.26-3.21
(m, 3H), 3.06-3.04 (m, 1H), 2.92-2.88 (m, 1H), 2.78‒2.71 (m, 2H), 2.36 (bs, 1H), 2.09-
13
2
.04 (m, 1H), 1.86‒1.82 (m, 2H), 1.04 (t, J = 7.2 Hz, 3H); C NMR (150 MHz, CD OD):
3
δ 170.32, 161.06, 146.42, 139.77, 132.80, 126.38, 124.29, 122.26, 121.30, 119.075,
118.18, 115.62, 111.61, 110.74, 109.72, 59.72, 58.72, 53.27, 52.76, 50.70, 44.91, 22.99,
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2
2.34, 21.44, 18.30, 10.07, 9.46; [α]D25= -25.0 (c=1.0 in CH OH); Anal. Calcd for
3
C H N S.3CF COOH: C, 49.16; H, 4.73; N, 10.12. Found: C, 48.93; H, 4.84; N, 9.77.
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tert-Butyl
2-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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8
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0
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4
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7
8
9
0
ethyl)-piperazin-1-yl)-9H-carbazole-9-carboxylate (12a). Compound 7a (0.10 g, 0.25
mmol) was reacted with (±)-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine
(0.056 g, 0.25 mmol) and NaBH(OAc) (0.108 g, 0.51 mmol) in CH Cl (7 mL) according
3
2
2
to procedure G. The crude product was purified by silica gel column chromatography
1
(hexane:EtOAc = 1:3) to afford compound 12a (0.115 g, 77%). H NMR (600 MHz,
CDCl ): δ 8.23 (d, J = 7.8 Hz, 1H), 7.91 (s,1H), 7.83 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.4
3
Hz, 1H), 7.34 (t, J = 7.2, Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 6.98
(
dd, J = 6.6, 1.8 Hz, 1H), 6.72 (d, J = 7.2, Hz, 1H), 6.64 (d, J = 7.8, Hz, 1H), 3.79 (s, 3H),
.32 (t, J = 4.8 Hz, 4H), 3.02‒2.93 (m, 2H), 2.88-2.85 (m, 1H), 2.79‒2.75 (m, 3H), 2.70 (t,
J = 4.8 Hz, 4H), 2.56‒2.50 (m, 5H), 2.09‒2.06 (m, 1H), 1.74 (s, 9H), 1.62‒1.55 (m, 1H),
.53-1.47 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
tert-Butyl 3-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)
3
1
ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (12b). Compound 7b (0.170 g, 0.43
mmol) was reacted with (±)-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine
(0.095 g, 0.43 mmol) and NaBH(OAc) (0.183 g, 0.86 mmol) in CH Cl (10 mL) according
3 2 2
to procedure G. The crude product was purified by silica gel column chromatography with
1
EtOAc to afford compound 12b (0.11 g, 43%). H NMR (600 MHz, CDCl ): δ 8.27 (d, J =
3
7
.8 Hz, 1H), 8.16 (d, J = 8.4 Hz, 1H), 7.92 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 2.4 Hz, 1H),
.43 (t, J = 7.2, Hz, 1H), 7.31 (t, J = 7.2 Hz, 1H), 7.12 (dd, J = 6.6, 2.4 Hz, 1H), 7.08 (t, J
7
= 8.4 Hz, 1H), 6.72 (d, J = 7.2, Hz, 1H), 6.64 (d, J = 8.4 Hz, 1H), 3.79 (s, 3H), 3.27 (t, J =
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.8 Hz, 4H), 3.03‒2.98 (m, 2H), 2.90-2.87 (m, 1H), 2.81‒2.77 (m, 3H), 2.73 (t, J = 4.8 Hz,
H), 2.59‒2.51 (m, 5H), 2.11‒2.08 (m, 1H), 1.74 (s, 9H), 1.63‒1.57 (m, 1H), 1.55-1.49
4
(m, 2H), 0.91 (t, J = 7.2 Hz, 3H).
1
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0
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8
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0
1
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8
9
0
tert-Butyl
4-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)
ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (12c). Compound 7c (0.10 g, 0.25
mmol) was reacted with (±)-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine
(0.055 g, 0.25 mmol) and NaBH(OAc) (0.108 g, 0.50 mmol) in CH Cl (8 mL) according
3 2 2
to procedure G. The crude product was purified by silica gel column chromatography
1
(hexane:EtOAc = 1:3) to afford compound 12c (0.112 g, 74%). H NMR (600 MHz,
CDCl ): δ 8.23 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 8.4, Hz, 1H), 8.09 (d, J = 7.2 Hz, 1H), 7.47
3
(
d, J = 7.8 Hz, 1H), 7.39 (m, 2H), 7.13 (t, J = 7.8 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.70
q, J = 7.8 Hz, 2H), 3.92 (bs, 2H), 3.79 (s, 3H), 3.58‒3.48 (m, 4H), 2.29 (m, 2H), 3.06-
(
3.04 (m, 5H), 2.62, (m, 1H), 2.37 (bs, 1H), 2.09‒2.06 (m, 1H), 1.74 (s, 9H), 1.62‒1.55 (m,
H), 1.53-1.47 (m, 2H), 0.92 (t, J = 7.2 Hz, 3H).
S)-tert-Butyl 2-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)
amino)ethyl)piperazin-1-yl)-9H-carbazole-9-carboxylate (13a). Compound 7a (0.10 g,
.25 mmol) was reacted with (-)-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-
amine (0.056 g, 0.25 mmol) and NaBH(OAc) (0.108 g, 0.51 mmol) in CH Cl (7 mL)
1
(
0
3
2
2
according to procedure G. The crude product was purified by silica gel column
1
chromatography (hexane:EtOAc = 1:3) to afford compound 13a (0.11 g, 74%). H NMR
(600 MHz, CDCl ): δ 8.23 (d, J = 8.4 Hz, 1H), 7.91 (s,1H), 7.82 (d, J = 7.8 Hz, 1H), 7.78
3
(d, J = 8.4 Hz, 1H), 7.34 (t, J = 7.2, Hz, 1H), 7.28 (t, J = 7.2 Hz, 1H), 7.08 (t, J = 7.8 Hz,
1H), 6.97 (dd, J = 6.6, 1.8 Hz, 1H), 6.71 (d, J = 7.2, Hz, 1H), 6.63 (d, J = 7.8, Hz, 1H),
29
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