Structure-activity-relationship study of N6-(2(4-(1H-indol-5-yl)piperazin-1-yl)ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine analogues: Development of highly selective D3 dopamine receptor agonists along with a highly potent D2/D3 agonist and their pharmacological characterization

Mark Johnson; Tamara Antonio; Maarten E A Reith; Aloke K Dutta

doi:10.1021/jm300268s

. Author manuscript; available in PMC: 2013 Jun 28.

Published in final edited form as: J Med Chem. 2012 Jun 13;55(12):5826–5840. doi: 10.1021/jm300268s

Structure-activity-relationship study of N⁶-(2(4-(1H-indol-5-yl)piperazin-1-yl)ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine analogues: Development of highly selective D3 dopamine receptor agonists along with a highly potent D2/D3 agonist and their pharmacological characterization

Mark Johnson ¹, Tamara Antonio ², Maarten E A Reith ^2,³, Aloke K Dutta ^1,^*

PMCID: PMC3386472 NIHMSID: NIHMS381896 PMID: 22642365

Abstract

In our effort to develop multifunctional drugs against Parkinson’s disease, a structure-activity-relationship study was carried out based on our hybrid molecular template targeting D2/D3 receptors. Competitive binding with [³H]spiroperidol was used to evaluate affinity (K_i) of test compounds. Functional activity of selected compounds in stimulating [³⁵S]GTPγS binding was assessed in CHO-cells expressing either human D2 or D3 receptors. Our results demonstrated development of highly selective compounds for D3 receptor ((−)-40; K_i D3 = 1.84 nM, D2/D3 = 583.2, (−)-45; K_i D3 = 1.09 nM, D2/D3 = 827.5). Functional data identified (−)-40 (EC₅₀ D2 = 114 nM and D3 = 0.26 nM, D2/D3 = 438) as one of the highest D3 selective agonists known to date. In addition, high affinity, non-selective, D3 agonist, (−)-19 (EC₅₀ D2 = 2.96 nM and D3 = 1.26 nM), was also developed. Lead compounds with antioxidant activity were evaluated using an in vivo PD animal model.

Introduction

Dopaminergic receptor systems have been targeted for the development of pharmacotherapeutic agents for a number of CNS related disorders, including drug addiction, schizophrenia, depression and Parkinson’s disease (PD). Dopamine (DA) receptor agonists have been employed more extensively in the treatment of Parkinson’s disease than any other type of pharmacotherapy. Levodopa (L-dopa), the immediate precursor to endogenous DA, is the current gold-standard treatment option for PD. DA receptors belong to the family of transmembrane proteins known as G-protein coupled receptors (GPCRs). DA receptors are widely distributed in the CNS, are also present in the periphery, and are divided into five subtypes. Based on the stimulatory action on adenylyl cyclase, D1 and D5 are grouped together as D1-type. D2–D4 receptors are classified as D2-type due to their inhibitory action on adenylyl cyclase activity.^1–7 Interestingly, the D3 receptor was found to have a distribution in the brain that is somewhat different than that of the D2 receptor. The highest levels of D3 receptor expression were found to be in the limbic region of the brain, while D2 receptor expression is most dense in the striatum of the midbrain.⁸ D2 and D3 receptor subtypes occur post- as well as pre-synaptically; in the latter location they function as auto-receptors that regulate DA synthesis, metabolism and release.⁹ It is noteworthy that D2 and D3 receptor subtypes share 50% overall amino acid sequence homology and 75–80% in their agonist binding sites. As a result, development of ligands selective for either subtype is a challenging task.^{10, 11}

Parkinson’s disease (PD) is a progressive, neurodegenerative disorder that results from the death of DA-producing cells in the substantia nigra region of the midbrain. Common symptoms include: resting tremor, muscular rigidity, bradykinesia, along with postural instability and cognitive psychiatric complications.^12–14 Although the etiology of PD is not yet clear and may be multifactorial, oxidative stress and mitochondrial dysfunction are thought to play a central role in the pathology of the disease. Recent studies on various genetic mutations have provided new insights into the disease process.^15–17 Oxidative stress has been strongly implicated in midbrain dopaminergic cell death.¹⁵ Toxicity from endogenous and exogenous origins, caused by oxidative mechanisms, has been implicated as a fundamental process in progressive nigral cell loss.¹⁸ Along with motor fluctuations and wearing off after long-term treatment, side effects associated with L-dopa treatment and the eventual oxidation of DA derived from L-dopa have been speculated to produce further oxidative stress.¹⁹

In addition, α-synuclein, a presynaptic protein involved in fibrilization, has been implicated in the pathogenesis of PD.^{20, 21} A recent report demonstrated in cultured, human dopaminergic neurons that accumulation of α-synuclein induces apoptosis in the presence of DA and reactive oxygen species.²² Furthermore, an interaction between calcium, cytosolic DA and α-synuclein has been implicated in the loss of DA neurons in the substantia nigra.²³ In this case, DA-dependent neurotoxicity is mediated by a soluble protein complex containing α-synuclein.²⁴ Therefore, α-synuclein, together with oxidized DA, could have synergistic effects in terms of disease susceptibility and progression.

It is increasingly evident that for a complex disease such as PD, a drug aimed at one target site will only partially address the therapeutic need of the disease. Thus, it is hypothesized that multi-functional drugs, having multiple pharmacological activities, will be more effective in the case of PD.²⁵ Our approach in developing such agents involves alleviating symptoms of the disease, along with preventing or halting the neurodegeneration process. Our drug development approach encompasses and incorporates some of the critical pathogenic factors implicated in PD.^{26, 27} We have designed 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 D3-preferring agonists.^28–33 One such compound, D-264, was shown to be neuroprotective in two PD animal models.³⁴ Furthermore, we demonstrated that our D3-preferring agonist, D-264, significantly improved behavioral syndromes in both acute MPTP and progressive lactacystin mouse models of PD.³⁴ Moreover, D-264 exhibited pronounced neuroprotection in both MPTP- and lactacystin-lesioned animal models, in which degeneration of dopaminergic pathways is known to occur. The neuroprotective effect of D-264 was attributed, in part, to activation of the D3 receptor. Structural flexibility in the D2/D3 antagonist fragment of our hybrid, molecular template has allowed us to design and develop new DA agonists with the capacity to bind iron. Iron has been implicated in the pathogenesis of PD, likely via increasing oxidative stress levels.³¹ In our recent study, we have demonstrated development of brain penetrant, multifunctional compounds with agonist activity at D2/D3 receptors along with capacity to chelate iron. One of our lead compounds in this series exhibited in vivo neuroprotection in a mouse MPTP model.^{31, 35}

As mentioned above, we have previously reported a hybrid structure approach as part of our ongoing effort to design and develop selective agonists for the DA D3 receptor. Our hybrid approach combines, via suitable linker length, an agonist binding moiety (aminotetralin or bioisosteric equivalent) with aryl-piperazine fragments. This approach has yielded molecules that retain agonist activity, while exhibiting varying selectivity for the D3 receptor (Figure 1). Our current structure-activity-relationship study is focused on introducing several indole derivatives on the piperazine ring. Our goal is to assess indole substitution, in terms of its effect on selectivity and affinity for the D3 receptor. It is important to mention that indole derivatives are well known for their potent antioxidant activity.^{36, 37} Another goal is to produce synergistic, antioxidant activity by combining the 2-aminothiazole moiety with an indole functionality.³⁸

Molecular structure of dopamine D3-receptor-preferring agonists and antagonists.

Chemistry

Scheme 1 outlines the synthesis of 9a, 9b and 9c. 5-methoxy-2-tetralone was first condensed with n-propyl amine, in the presence of sodium cyanoborohydride, under reductive amination conditions, to yield secondary amine 2. In preparation of 7, amine 4 was N-alkylated using 2-bromoethanol to provide alcohol 5. Alcohol 5 was converted, under Swern oxidation conditions, to its aldehyde derivative. Condensation of aldehyde 6 with amine 2, using sodium triacetoxyborohydride as reducing agent, yielded compound 7. Treatment of intermediate 7 with aq. HBr (48%) provided amine 8. Finally, condensation of secondary amine 8 with properly substituted, indole-carbaldehydes, afforded final compounds 9a–c.

Scheme 2 describes the synthesis of final compounds (±)-19, (+)-19, (−)-19 and 21. Commercially available, 5-bromoindole was N-protected using triisopropylsilyl chloride in the presence of NaH to give intermediate 11. Palladium-catalyzed cross-coupling of 11 with amine 4, using PdCl₂[P(o-tol)₃]₂ and NaOtBu in xylenes at reflux yielded intermediate 12. Successive deprotection of TIPS and Boc groups with trifluoroacetic acid gave amine 13, which on selective, N-alkylation with (2-bromo-ethoxy)-tert-butyl-dimethyl-silane yielded compound 14. The indole moiety of intermediate 14 was N-protected giving compound 15, which on TBDMS deprotection yielded alcohol 16. Compound 16 was converted to its aldehyde derivative 17, under Swern oxidation conditions. Aldehyde 17 was subsequently condensed with (±), S-(−) or R-(+)-pramipexole to yield intermediates (±)-18, S-(−)-18 and R-(+)-18, which were each treated with trifluoroacetic acid to afford final compounds (±)-19, S-(−)-19 and R-(+)-19. Aldehyde 17 was further condensed with 5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-propyl-amine under reductive amination conditions and subsequently treated with aq. HBr (48%) to furnish final compound 21.

Scheme 3 depicts the synthesis of final compounds (±)-40, R-(+)-40, S-(−)-40, 41 and 42. Indole-2-carboxylic acid was reacted with amine 4, under amide coupling conditions, to yield intermediate 23. Deprotection, followed by selective, N-alkylation with appropriately substituted TBDMS-protected alkyl halides afforded intermediates 25-27. Protection of the indole moiety, followed by selective deprotection of the hydroxyl functionality, gave alcohols 31–33. Alcohols 31–33 were subsequently converted to their aldehyde derivative, under Swern oxidation conditions, to yield 34–36. Aldehyde 34 was coupled with (±), R-(+) and S-(−)-pramipexole, under reductive amination conditions, to give condensed products (±)-37, R-(+)-37 and S-(−)-37. Deprotection of the indole moiety afforded final compounds (±)-40, R-(+)-40 and S-(−)-40. Aldehydes 35 and 36 were condensed with (±)-pramipexole to yield intermediates 38 and 39, which were deprotected under acidic conditions to furnish final compounds 41 and 42.

Scheme 4 outlines the synthesis of final compounds (±)-45, R-(+)-45, S-(−)-45, (±)-46, R-(+)-46, S-(−)-46, 48 and 49. Aldehyde 6 was coupled with (±), (+) or (−)-pramipexole to yield condensed products (±)-43, R-(+)-43 and S-(−)-43. Subsequent treatment with trifluoroacetic acid yielded deprotected intermediates (±)-44, R-(+)-44 and S-(−)-44. Separately, under amide coupling conditions, (±)-44, R-(+)-44 and S-(−)-44 were reacted with either indole-3-carboxylic acid or indole-5-carboxylic acid to yield final compounds (±)-45, R-(+)-45, S-(−)-45, (±)-46, R-(+)-46 and S-(−)-46. Reaction of (±)-44 with 5-methoxy-1H-indole-3-carboxylic acid, under amide coupling conditions, or 1H-indole-5-carbaldehyde under reductive amination conditions, afforded intermediate 47 and final compound 49. Finally, demethylation of 47 with boron tribromide furnished final compound 48.

Results and Discussion

Our current study is aimed at investigating the molecular and chemical flexibility, along with basicity, of the aryl-piperazine fragment of our hybrid template as it relates to D2/D3 receptor binding and functional activity. The present series of compounds are comprised of various indole derivatives, as our previous studies have indicated that an indole substituent in the aryl-piperazine region is well tolerated, producing molecules with high D2/D3 affinity and preference for D3 receptor.³² We have carried out our binding studies with rat dopamine D2 and D3 (rD2 and rD3) receptors expressed in HEK-293 cells and functional characterization with human D2 and D3 receptors (hD2 and hD3) expressed in CHO cells. In our own findings, we did not see any significant difference of affinity of compounds interacting with either rat or human dopamine D2 and D3 receptors. An overall high degree of homology exists between the two species, 95% for D2 and greater than 78% for D3 with somewhat shorter 3^rd intracellular loop in the human version of the D3 receptor. ^{2, 39}

Table 1 summarizes the binding data for analogues that were synthesized. Compounds 9a–c, which incorporates the 5-hydroxy-aminotetralin head group and a methylene unit connecting piperazine to indole at various positions, displayed high affinity for D3 and moderate affinity for D2 receptors. Amongst this series of analogues, the 5-substituted indole derivative, 9a, displayed the highest selectivity for D3 (K_i D2 = 269 nM, D3 = 4.17 nM, D2/D3 = 64.5), while 9c (K_i D2 = 82.1 nM, D3 = 3.20 nM, D2/D3 = 25.6) proved to be the most potent and least selective. Analogues 9a–c exhibited lower affinity for D2 and, in the case of 9a, higher selectivity for D3 compared to parent compound D–237 (9a; K_i D2 = 269 nM, D2/D3 = 64.5 vs D-237; K_i D2 = 26.0 nM, D2/D3 = 31.5). A similar 5-substituted, indole derivative, 21, lacks a linking carbon between piperazine and indole and displayed low selectivity and lost some potency (K_i D2 = 76.4 nM, D3 = 10.4 nM, D2/D3 = 7.3) for D3 receptor compared to counterparts 9a-c. Compound 49, analogous to 9a–c, with 2-aminothiazole substitution in the agonist head group, maintained D2 receptor affinity within the range displayed by 9a–c, while D3 affinity decreased by approximately 2-fold (K_i D2 = 132 nM, D3 = 8.07 nM, D2/D3 = 16.4).

Table 1.

Inhibition constants for competition with [³H]spiroperidol binding to cloned rat D2L and D3 receptors expressed in HEK-293 cells.

Compound	K_i (nM), rD2L [³H]spiroperidol	K_i (nM), rD3 [³H]spiroperidol	D2L/D3
(−)-5-OH-DPAT	58.8 ± 11.0	1.36 ± 0.28	43.2
D-237	26.0 ± 7.5	0.83 ± 0.13	31.5
D-301	269 ± 16	2.23 ± 0.60	121
D-264	264 ± 40	0.92 ± 0.23	253
9a	269 ± 183	4.17 ± 0.36	64.5
9b	183 ± 26	5.48 ± 0.86	33.4
9c	82.1 ± 7.1	3.20 ± 0.32	25.6
(±)-19	46.7 ± 6.6	1.92 ± 0.38	24.3
(−)-19	39 ± 5	2.19 ± 0.39	17.8
(+)-19	134 ± 12	15.9 ± 3.6	8.46
21	76.4 ± 2.4	10.4 ± 1.6	7.3
(±)-40	852 ± 209	4.59 ± 0.15	185.6
(−)-40	1,073 ± 92	1.84 ± 0.51	583
(+)-40	2,558 ± 112	54.1 ± 4.2	47.3
41	928 ± 152	2.78 ± 0.25	334
42	531 ± 119	1.74 ± 0.25	305
(±)-45	1,503 ± 67	4.17 ± 0.30	360
(−)-45	902 ± 130	1.09 ± 0.14	828
(+)-45	1,316 ± 244	48.2 ± 8.6	27.3
(±)-46	1,243 ± 130	4.10 ± 0.57	303
(−)-46	1,031 ± 182	1.40 ± 0.29	736
(+)-46	2,626 ± 229	52.8 ± 8.3	49.7
48	1,079 ± 139	16.8 ± 0.6	64.2
49	132 ± 22	8.07 ± 0.93	16.4

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Results are means ± SEM for 3–6 independent experiments, each performed in triplicate.

Previous and current results consistently demonstrate that in the 2-aminothiazole series of hybrid compounds, the (−)-isomeric version exhibits the highest affinity for the D3 receptor compared to the (+)-isomer. Bioisosteric equivalent molecules were synthesized with the aminotetralin head group replaced with 2-aminothiazole. The first of these analogues incorporated a bond that linked the piperazine moiety directly to the indole group. The most active, optical isomer of this molecule, (−)-19, displayed the highest affinity for the D2 receptor among the compounds in our study, along with low selectivity (K_i D2 = 39 nM, D3 = 2.19 nM, D2/D3 = 17.8). In contrast, one of the parent compounds, D-301, displayed lower D2 affinity and higher D3 selectivity (K_i D2 = 269 nM, D3 = 2.23 nM, D2/D3 = 121) compared to (−)-19. This suggests that the indole moiety alone does not give rise to selectivity for either receptor subtype. In order to investigate the effect of lowering the basicity of the piperazine ring, we synthesized a number of analogues that incorporate an amide bond at the piperazine nitrogen atom, distal to the agonist head group. Interestingly, in these molecules, D3 receptor affinity was maintained in the low nanomolar range, while D2 affinity dropped significantly to the low micromolar range. The 2-aminothiazole derived, 3-substituted, indole-acyl derivative (−)-45, displayed the highest selectivity in binding (K_i D2 = 902 nM, D3 = 1.09 nM, D2/D3 = 828) to D3 receptor in our current series of molecules. A more than 3-fold increase in D3 selectivity was observed for (−)-45 when compared to previous lead and parent compound D-264 (D2/D3; 828 vs 253 for (−)-45 vs D-264). Compound (−)-45 was also more selective than another lead compound, D-301 (D2/D3; 828 vs. 121 for (−)-45 vs D-301). Our next goal was to synthesize isomeric, 2-substituted, indole-acyl derivative, (±)- 40, connected to the piperazine ring via amide linkage. The most active enantiomer, (−)-40, exhibited high affinity and selectivity for D3 receptor (K_i D3 = 1.84 nM, D2/D3 = 583). Next, the isomeric, 5-substituted, indole-acyl derivative (±)-46, was prepared. The most active enantiomer, (−)-46, produced a similar, high affinity and selectivity profile for D3 receptor (K_i D3 = 1.40 nM, D2/D3 = 736). Thus, affinity and selectivity for D3 receptor were similar in isomeric compounds (−)-40, (−)-45 and (−)-46.

Next, to determine the impact of the linker length on D2/D3 receptor binding, we varied the length of the two-carbon tether between the agonist head group and the aryl piperazine fragment. The two-carbon linker of (±)-40 was increased to three and four. Compound 41 (K_i D2 = 928 nM, D3 = 2.78 nM, D2/D3 = 333.8) contained a three-carbon linker and displayed an almost 2-fold increase in D3 selectivity compared to the parent compound, (±)-40 (K_i D2 = 852 nM, D3 = 4.59 nM, D2/D3 = 186). Compound 42 contained a four-carbon linker, which increased affinity for both D2 and D3 receptor (K_i D2 = 531 nM, D3 = 1.74 nM, D2/D3 = 305) compared to (±)-40 and 41. Finally, we modified (±)-45 to incorporate a hydroxyl substituent at the 5-position of the indole moiety. Hydroxyl substitution helped us to study the electronic effects of an electron-releasing group on the indole nucleus and a possible contribution of hydrogen-bond interactions in this region of D2 and D3 receptors. This modification produced compound 48, which maintained micromolar D2 affinity of the parent compound, while D3 affinity decreased 4-fold (K_i D2 = 1,079 nM, D3 = 16.8 nM, D2/D3 = 64.2). This result indicated that introduction of a 5-hydroxyl group on the indole nucleus did not have a significant effect on D2 affinity, while D3 affinity and selectivity were impacted unfavorably.

The next goal of our study was to investigate the functional activity of selected compounds at D2/D3 receptors. The most selective D3 ligands, based on binding results, were selected for functional activity evaluation. Optically active, lead compounds (−)-40, (−)-45 and (−)-46 were tested in the [³⁵S]GTPγS functional assay to characterize their ability to stimulate D2/D3 receptors in comparison to the endogenous ligand DA, and to the parent compounds D-264 and D-301. Each of the three compounds tested displayed higher functional selectivity and potency for D3 receptor in comparison to D-264 and DA (Table 2). In particular, (−)-40 maintained high functional selectivity for D3 receptor (EC₅₀ D2 = 114 nM, D3 = 0.26 nM, D2/D3 = 438), correlating well with binding data and exhibited full agonist activity at both D2 and D3 receptors (% E_max close to 100%). Compound (−)-40 demonstrated a 5-fold increase in functional potency (EC₅₀ D3 = 1.51 vs 0.26 for D-264 vs (−)-40) and an almost 20-fold increase in functional selectivity (D2/D3; 22.1 vs 438 nM for D-264 vs (−)-40) for the D3 receptor when compared to D-264. On the other hand, in comparison to D-301, the selectivity was 3-fold higher (EC₅₀ D2/D3 = 438 vs. 141). Compounds (−)-45 (EC₅₀ D2 = 86.4, D3 = 0.87 nM, D2/D3 = 99.3) and (−)-46 (EC₅₀ D2 = 70.7 nM, D3 = 0.56 nM, D2/D3 = 126) each exhibited full agonist activity (% E_max not significantly different from 100) at D2 and D3 receptors, while their selectivity for D3 receptor dropped considerably when compared to binding data. In contrast to the above mentioned, indole-acyl derivatives, compound (−)-19 was exceptionally potent at D2 receptor (EC₅₀ D2 = 2.96 nM and D3 = 1.26 nM), while also exhibiting high potency for D3 receptor. Thus, (−)-19 was indiscriminate in its binding to D2 and D3 receptors.

Table 2.

Stimulation of [³⁵S]GTPγS Binding to Cloned Human D2 Receptor and D3 receptor expressed in CHO cells.

Compound	hCHO-D2		hCHO-D3
	EC₅₀ (nM) [³⁵S]GTPγS	% E_max	EC₅₀ (nM) [³⁵S]GTPγS	% E_max	D2/D3
DA	227 ± 11	100	8.57	100	26.5
D-301	116 ± 16	88.4 ± 3.9	0.82 ± 0.20	102 ± 2	141
D-264	33.1 ± 6.6	104 ± 5	1.51 ± 0.22	90.0 ± 4.3	22.1
(−)-40	114 ± 12	101 ± 5	0.26 ± 0.07	103 ± 10	438
(−)-45	86.4 ± 6.2	91.6 ± 5.9	0.87 ± 0.11	100 ± 6	99.3
(−)-46	70.7 ± 14.9	107 ± 4	0.56 ± 0.14	98.1 ± 3.3	126
(−)-19	2.96 ± 0.3	107 ± 3	1.26 ± 0.2	93.1 ± 4.4	2.35

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EC₅₀ is the concentration producing half-maximal stimulation. For each compound, maximal stimulation (E_max) is expressed as a percent of the E_max observed with 1 mM (D2) or 100 µM (D3) of the full agonist DA (% E_max). Results are means ± SEM for 3–6 independent experiments, each performed in triplicate.

Evaluation of Free Radical Scavenging Activity

Scavenging of the DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical by (±)-40, (±)-45, (±)-46, (±)-19 and ascorbic acid is shown in Figure 2.⁴⁰ The scavenging effect is expressed as percent of control. As shown in Figure 2, each compound inhibited DPPH radical activity dose dependently. The standard compound, ascorbic acid, had an IC₅₀ of 24.9 µM in this assay procedure, whereas the IC₅₀ value for (±)-19 was 14.5 µM (n=3–4 for all compounds tested). All other compounds exhibited potencies comparable to that of ascorbic acid. It is clear from the data that compound (±)-19 is nearly twice as potent as ascorbic acid in quenching the DPPH radical.

DPPH radical scavenging activity by (±)-19, (±)-40, (±)-45, (±)-46 and ascorbic acid.

Reversal of Reserpine-Induced Hypolocomotion in Rats by (−)-19, (−)-46, (−)-40 and Ropinirole

Reserpine induces depletion of catecholamines in nerve terminals, resulting in a cataleptic condition in rats, which is a well established animal model for PD.⁴¹ Significant reduction of locomotion of the rats was observed 18 h after the administration of reserpine (5 mg/kg, sc), which indicated the development of akinesia in rats. Compound (−)-19 (5 µmol/kg, sc) was efficacious in significantly reversing akinesia in rats, compared to reserprine treatment alone, over a 6-h period. Similarly, treatment with the reference drug, ropinirole (5 µmol/kg, sc), produced a significant locomotor activation compared to control, reaching higher levels of locomotion but with a much shorter duration of action compared with (−)-19. On the other hand, compounds (−)-46 and (−)-40 (10 µmol/kg, sc) failed to produce any appreciable effect in reversing akinesia in reserpine-treated rats

Conclusion

Results from binding and functional activity studies indicate that we have developed a novel class of D3-selective agonists, based on our hybrid template. Our current series of compounds include those with low, moderate and high selectivity for D3 receptor. D3 selectivity, according to our SAR results, is influenced by three factors: linker length, molecular structure and basicity of the aryl-piperazine fragment. Our results indicate a correlation between basicity of the nitrogen atom that connects piperazine to the indole ring, with D2/D3 selectivity and potency. In the case of compound 49, piperazine and indole are connected via a methylene unit, giving rise to high basicity of the piperazine nitrogen atom and low selectivity for D3 receptor (D2/D3 = 16.4 for 49). On the other hand, when the connecting carbon is a planar sp² hybridized carbonyl group, the basicity of the nitrogen atom is low and much higher selectivity is observed for the D3 receptor (D2/D3 = 583, 828 and 736 for (−)-40, (−)-45 and (−)-46, respectively). It is also possible that an electronic effect of carbonyl group on the indole ring might also additionally play an important role in selectivity for D3 receptor. In the GTPγS functional assay, compound (−)-40 exhibited high selectivity for the D3 receptor and is one of the most selective, D3 agonists known to date (D2/D3 = 438). These results indicate that amide linkage, connecting the piperazine ring to the indole moiety, is unfavorable, in terms of binding to the D2 receptor, while affinity for the D3 receptor remains high. In this regard, in a recent publication, the importance of the presence of amide linkage has been shown in highly selective D3 antagonist compounds.⁴² The length of the tether between the agonist head group and the aryl-piperazine fragment affects the affinity and selectivity for D2 and D3 receptors. In the series (±)-40, 41 and 42, in which the linker length is increased from n = 2 to n=4, an increase in D3 affinity and selectivity occurs.

Furthermore, radical quenching study indicates that compound (±)-19 has potent antioxidant activity. Antioxidant activity is highly relevant, as there has been a strong implication of oxidative stress in the pathogenesis of PD. Further evaluation of the antioxidant activity of these compounds is underway. In the present in vivo PD animal model, compound (−)-19 produced a significant, long-lasting reversal of hypolocomotion in reserpinized rats. Our ongoing studies with (−)-19 are directed at evaluating the full potential of this compound as a multifunctional, neuroprotective agent against PD.

Experimental Section

Reagents and solvents were purchased from commercial suppliers and used as received, unless otherwise noted. Dry solvent was obtained following the standard procedure. All reactions were performed under N₂ atmosphere, unless otherwise 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), Dragendorff’s reagent or ninhydrin. Whatman Purasil® 60A silica gel 230–400 mesh was used for flash column, chromatographic purifications. Proton nuclear magnetic resonance (¹H NMR) spectra were measured on Varian 400 MHz FT NMR spectrometer, using tetramethylsilane (TMS) as an internal standard. The NMR solvent used was CDCl₃, unless otherwise indicated. Optical rotations were recorded on Perkin-Elmer 241 polarimeter. Mass spectra were recorded on Micromass QuattroLC triple quadrupole mass spectrometer. 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. and were within ± 0.4% of the theoretical value.

5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (2)

Into a stirring solution of n-propylamine (14.9 mL, 181.6 mmol) and ketone 1 (12.8 g, 72.6 mmol), in CH₂Cl₂ (70 mL), was added glacial acetic acid (17.3 mL, 290.5 mmol). After stirring for 0.5 h, NaCNBH₃ (11.4 g, 181.6 mmol) was added portion wise at 0 °C, followed by methanol (20 mL). The mixture was allowed to reach room temperature and stirred overnight. The reaction mixture was quenched with a saturated NaHCO₃ solution at 0 °C and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was washed with water, brine and dried over Na₂SO₄. Solvent was removed under reduced pressure. Crude product was purified by column chromatography (EtOAc/MeOH, 9:1) to give compound 2 (11.1 g, 70%). ¹H NMR (CDCl₃, 400 MHz): δ 0.96 (t, J = 7.2 Hz, 3H), 1.54–1.65 (m, 3H), 2.09–2.16 (m, 1H), 2.52–2.74 (m, 4H), 2.88–3.07 (m, 3H), 3.81 (s, 3H), 6.66 (d, J = 8.0 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 8.4 Hz, 1H).

4-(2-Hydroxy-ethyl)piperazine-1-carboxylic acid tert-butyl ester (5)

A mixture of compound 4 (10.0 g, 53.7 mmol), 2-bromoethanol (10.1 g, 80.6 mmol) and K₂CO₃ (22.3 g, 161.1 mmol) in CH₃CN (100 mL) was refluxed for 14 h according to procedure D. The crude material was purified by silica gel column chromatography (EtOAc/MeOH, 20:1) to give compound 5 (7.12 g, 58%). ¹H NMR (CDCl₃, 400 MHz): δ 1.40 (s, 9H), 2.40 (t, J = 4.8 Hz, 4H), 2.50 (t, J = 5.2, 2H), 3.38 (t, J = 4.8 Hz, 4H), 3.58 (t, J = 5.2 Hz, 2H).

Procedure A. 4-(2-Oxo-ethyl)piperazine-1-carboxylic acid tert-butyl ester (6)

Into a stirring solution of oxalyl chloride (5.5 g, 43.3 mmol) in CH₂Cl₂ (80 mL) at −78 °C, DMSO (6.2 mL, 78.7 mmol) was added. The reaction mixture was stirred for 0.5 h, followed by addition of compound 5 (5.0 g, 21.7 mmol, solution in 20 mL of CH₂Cl₂). The reaction mixture was stirred at the same temperature for 0.5 h, followed by addition of Et₃N (18.2 mL, 179.4 mmol) and stirring was continued for 1.5 h, while allowing the reaction mixture to reach room temperature. The reaction mixture was quenched by addition of a saturated solution of NaHCO₃ and extracted with CH₂Cl₂ (3 × 100 mL). The combined organic layer was dried using Na₂SO₄ and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 20:1) to give compound 6 (4.96 g, ~100%).

Procedure B. 4-{2-[(5-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)propyl amino]ethyl}piperazine-1-carboxylic acid tert-butyl ester (7)

Into a stirring solution of amine 2 (4.76 g, 21.7 mmol) in CH₂Cl₂ (50 mL), aldehyde 6 (4.96 g, 21.7 mmol) was added. After stirring for 1 h, NaBH(OAc)₃ (8.29 g, 39.1 mmol) was added portion wise and the mixture was stirred for 48 h at room temperature. The reaction mixture was quenched with a saturated solution of NaHCO₃ at 0 °C and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was dried over Na₂SO₄ and the solvent was removed under reduced pressure. Crude product was purified by column chromatography (EtOAc/MeOH, 20:1) to give compound 7 (6.46 g, 69%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 7.6 Hz, 3H), 1.20 (s, 9H), 1.30–1.52 (m, 3H), 1.90–2.12 (m, 1H), 2.20–3.06 (m, 13H), 3.20–3.60 (m, 6H), 3.77 (s, 3H), 6.61 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 7.05 (t, J = 7.6 Hz, 1H).

6-[(2-piperazin-1-yl-ethyl)-propyl amino]-5,6,7,8-tetrahydronaphthalen-1-ol (8)

A mixture of compound 7 (6.46 g, 14.9 mmol) and 48% aq. HBr (40 ml) was refluxed at 125 °C for 12 h. The reaction mixture was evaporated to dryness and a saturated solution of NaHCO₃ was added into it at 0 °C. The reaction mixture was then extracted with ethyl acetate (3 × 100 mL). The combined organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield compound 8 (3.80 g, 80%). ¹H NMR (CD₃OD, 400 MHz): δ 1.06 (t, J = 7.2 Hz, 3H), 1.80–2.02 (m, 3H), 2.36–2.48 (m, 1H), 2.60–2.80 (m, 1H), 2.96–4.02 (m, 18H), 6.61 (d, J = 8 Hz, 1H), 6.66 (d, J = 8 Hz, 1H), 6.96 (t, J = 8 Hz, 1H).

6-({2-[4-(1H-indol-5-ylmethyl)piperazin-1-yl]ethyl}propyl amino)-5,6,7,8-tetrahydro naphthalen-1-ol (9a)

Amine 8 (200 mg, 0.63 mmol) was reacted with 1H-indole-5-carbaldehyde (91 mg, 0.63 mmol) and NaBH(OAc)₃ (240 mg, 1.13 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography (MeOH/EtOAc, 1:6) to afford compound 9a (220 mg, 79%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 7.2 Hz, 3H), 1.30–1.52 (m, 3H), 1.90–2.10 (m, 1H), 2.46–3.02 (m, 19H), 3.76 (s, 2H), 6.51 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 7.06–7.25 (m, 3H), 7.36 (d, J = 8 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 8.26 (bs, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 22.3, 24.0, 25.7, 29.9, 32.4, 47.9, 52.8, 53.5, 53.6, 53.8, 57.4, 58.7, 111.2, 112.2, 119.6, 121.4, 122.1, 123.6, 124.2, 126.3, 128.2, 136.5, 138.5, 154.2, 171.4. The free base was converted to its hydrochloride salt. M.p. 165–169 °C. Anal. calculated for C₃₀H_46.5Cl_3.5N₄O: C, H, N.

6-({2-[4-(1H-indol-2-ylmethyl)piperazin-1-yl]ethyl}propyl amino)-5,6,7,8-tetrahydro naphthalen-1-ol (9b)

Amine 8 (200 mg, 0.63 mmol) was reacted with 1H-Indole-2-carbaldehyde (91 mg, 0.63 mmol) and NaBH(OAc)₃ (240 mg, 1.13 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography (MeOH/EtOAc, 1:6) to afford compound 9b (225 mg, 81%). ¹H NMR (CDCl₃, 400 MHz): δ 0.90 (t, J = 7.2 Hz, 3H), 1.40–1.71 (m, 3H), 1.96–2.10 (m, 1H), 2.36–3.08 (m, 19H), 3.77 (s, 2H), 5.36 (bs, 2H), 6.39 (s, 1H), 6.60 (dd, J = 7.2 Hz, 1H), 6.95 (t, J = 7.6 Hz, 1H), 7.04–7.12 (m, 2H), 7.17 (t, J = 7.6 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.56 (d, J = 8.4 Hz, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.1, 21.6, 24.0, 25.4, 28.4, 30.0, 31.8, 47.5, 52.5, 52.8, 53.2, 53.6, 55.5, 57.2, 57.8, 103.0, 111.3, 119.9, 120.5 122.0, 123.7, 126.0, 128.3, 133.8, 136.7, 137.6, 154.6, 178.0. The free base was converted to its hydrochloride salt. M.p. 180–185 °C. Anal. calculated for C₂₈H₄₇Cl₄N₄O_3.5: C, H, N.

6-({2-[4-(1H-indol-3-ylmethyl)piperazin-1-yl]ethyl}propyl amino)-5,6,7,8-tetrahydro naphthalen-1-ol (9c)

Amine 8 (200 mg, 0.63 mmol) was reacted with 1H-indole-3-carbaldehyde (91 mg, 0.63 mmol) and NaBH(OAc)₃ (240 mg, 1.13 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography (MeOH/EtOAc, 1:6) to afford compound 9c (213 mg, 77%). ¹H NMR (CDCl₃, 400 MHz): δ 0.85 (t, J = 7.2 Hz, 3H), 1.20–1.56 (m, 2H), 1.80–2.04 (m, 1H), 2.20–3.00 (m, 18H), 3.20–3.45 (m, 2H), 3.74 (s, 2H), 6.48 (d, J = 7.6 Hz, 1H), 6.57 (d, J = 7.6 Hz, 1H), 6.93 (t, J = 7.2 Hz, 1H), 7.02–7.25 (m, 3H), 7.35 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 7.6, 1H), 8.24 (s, 1H). ¹³C (CDCl₃, 100 MHz): 12.0, 22.5, 25.4, 26.0, 26.7, 29.9, 48.4, 52.9, 53.7, 58.3, 58.6, 63.6, 102.6, 111.0, 117.5, 121.7, 124.0, 124.7, 128.1, 128.8, 135.5, 145.2, 165.9. The free base was converted to its hydrochloride salt. M.p. 180–185 °C. Anal. calculated for C₂₈H₄₇Cl₄N₄O_3.5: C, H, N.

5-Bromo-1-(triisopropylsilyl)-1H-indole (11)

Into a stirring solution of NaH (4.03 g, 170.0 mmol) in dry THF (150 mL), compound 10 (16.44 g, 83.9 mmol) was added portion wise at 0 °C. The reaction mixture was allowed to stir at room temperature for 1 h, followed by dropwise addition of triisopropylsilyl chloride (20 g, 103.7 mmol). The reaction mixture was stirred for 12 h and then filtered through celite. The crude residue was purified by column chromatography using hexane as solvent to afford compound 11 (22 g, 75%). ¹H NMR (CDCl₃, 400 MHz): δ 1.23 (s, 18H), 1.74 (heptet, J = 7.6 Hz, 3H), 6.64 (d, J = 3.2 Hz, 1H), 7.07 (d, J = 6 Hz, 1H), 7.14 (s, 1H), 7.31 (d, J = 3.2 Hz, 1H), 7.45 (d, J = 8.8, 1H).

tert-Butyl 4-(1-(triisopropylsilyl)-1H-indol-5-yl)piperazine-1-carboxylate (12)

A mixture of compound 11 (22.0 g, 63.0 mmol), 4 (11.71 g, 63.0 mmol), PdCl₂[P(O-tol)₃]₂ (2.47 g, 3.1 mmol) and NaOtBu (9.08 g, 94.4 mmol) in xylenes (175 mL) was heated at 110 °C for 12 h. The reaction mixture was filtered through celite and concentrated in vacuo. The crude residue was purified by column chromatography (EtOAc/hexane, 1:20) to afford compound 12 (13.22 g, 46%). ¹H NMR (CDCl₃, 400 MHz): δ 1.19 (s, 18H), 1.55 (s, 9H), 1.74 (heptet, J = 6.8 Hz, 3H), 3.14 (bs, 4H), 3.67 (bs, 4H), 6.60 (t, J = 6 Hz, 1H), 6.94 (d, J = 8.8, 1H), 7.19 (s, 1H), 7.26 (t, J = 2.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H).

Procedure C. 5-Piperazin-1-yl-1H-indole (13)

To a stirring solution of compound 12 (7.70 g, 16.8 mmol) in CH₂Cl₂ (15 mL), TFA (15 mL) was added slowly at room temperature and the reaction mixture was stirred for 2 h. Unreacted TFA and solvent were removed under reduced pressure and the salt was washed with diethylether. A saturated solution of NaHCO₃ was added to the salt, followed by extraction with CH₂Cl₂ (3 × 50 mL). The combined organic layer was dried over Na₂SO₄, filtered and evaporated in vacuo to provide compound 13 (2.88 g, 85%). ¹H NMR (CDCl₃, 400 MHz): δ 1.85 (bs, 1H), 2.80–3.28 (m, 8H), 6.85–7.10 (m, 1H), 7.02–7.40 (m, 4H), 8.31 (bs, 1H).

Procedure D. 5-{4-[2-(tert-Butyl-dimethyl-silanyloxy)ethyl]piperazin-1-yl}-1H-indole (14)

A mixture of compound 13 (2.88 g, 14.3 mmol), (2-bromoethoxy)-tert-butyl-dimethylsilane (3.42 g, 14.3 mmol) and K₂CO₃ (5.93 g, 42.9 mmol) in CH₃CN (50 mL) was refluxed for 14 hours. After filtration, acetonitrile was evaporated under reduced pressure and the crude material was purified by silica gel column chromatography (EtOAc/hexane, 3:1) to give compound 14 (4.01 g, 78%).¹H NMR (CDCl₃, 400 MHz): δ 0.02 (s, 6H), 0.83 (s, 9H), 2.30–2.80 (m, 6H), 2.82–3.30 (m, 4H), 3.52–3.82 (m, 2H), 6.25–6.48 (m, 1H), 6.75–7.30 (m, 4H), 8.09 (s, 1H).

5-{4-[2-(tert-Butyl-dimethyl-silanyloxy)ethyl]piperazin-1-yl}indole-1-carboxylic acid tert-butyl ester (15)

Amine 14 (4.0 g, 11.1 mmol) was reacted with (Boc)₂O (2.68 g, 12.2 mmol) and DMAP (1.49 g, 12.2 mmol) in THF (50 mL) at room temperature using procedure G. The crude material was purified by column chromatography over silica gel (EtOAc/hexane, 1:1) to give compound 15 (5.2 g, ~100%). ¹H NMR (CDCl₃, 400 MHz): δ 0.08 (s, 6H), 0.95 (s, 9H), 1.65 (s, 9H), 2.61 (t, J = 6.4 Hz, 2H), 2.73 (t, J = 4.8 Hz, 4H), 3.19 (t, J = 4.8 Hz, 4H), 3.81 (t, J = 6.4 Hz, 2H), 6.47 (d, J = 3.6 Hz, 1H), 7.01 (dd, J = 6.4, 2.4 Hz, 1H), 7.06 (dd, J = 6.4, 2.4 Hz, 1H), 7.53 (s, 1H), 8.00 (s, 1H).

Procedure E. 5-[4-(2-Hydroxy-ethyl)piperazin-1-yl]indole-1-carboxylic acid tert-butyl ester (16)

Into a stirring solution of compound 15 (2.0 g, 4.3 mmol) in THF (30 mL), n-tetrabutylammonium fluoride (1.14 g, 4.3 mmol, 1.0 M solution in THF) was added at 0 °C. The reaction mixture was then stirred at room temperature for 1 h. THF was evaporated in vacuo, 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. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 20:1) to yield compound 16 (1.49 g, 99%). ¹H NMR (CDCl₃, 400 MHz): δ 1.65 (s, 9 H), 2.61 (t, J = 5.2 Hz, 2H), 2.70 (t, J = 4.8 Hz, 4H), 3.19 (t, J = 4.8 Hz, 4H), 3.67 (t, J = 5.2 Hz, 2H), 6.47 (d, J = 3.6 Hz, 1H), 7.01 (dd, J = 6.8, 2 Hz, 1H), 7.06 (d, J = 2 Hz, 1H), 7.53 (s, 1H), 8.00 (s, 1H).

5-[4-(2-Oxo-ethyl)piperazin-1-yl]indole-1-carboxylic acid tert-butyl ester (17)

Compound 16 (1.49 g, 4.3 mmol) was reacted with oxalyl chloride (0.75 mL, 8.6 mmol), DMSO (1.23 mL, 17.3 mmol) and Et₃N (3.6 mL, 25.8 mmol) in CH₂Cl₂ (40 mL) using procedure A. The crude residue was purified by column chromatography using ethyl acetate as solvent to afford compound 17 (1.23 g, 83%).

5-(4-{2-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl)propyl amino]ethyl}piperazin-1-yl)indole-1-carboxylic acid tert-butyl ester [(±)-18]

Compound 17 (175 mg, 0.51 mmol) was reacted with (±)-pramipexole (108 mg, 0.51 mmol) and NaBH(OAc)₃ (194 mg, 0.92 mmol) in CH₂Cl₂ (15 mL) according to procedure B. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 20:1) to yield compound (±)-18 (150 mg, 55%). ¹H NMR (CDCl₃, 400 MHz): δ 0.91 (t, J = 6.8 Hz, 1H), 1.35–1.60 (m, 2H), 1.67 (s, 9H), 1.89–2.10 (m, 1H), 2.30–3.30 (m, 20H), 4.94 (bs, 2H), 6.67 (t, J = 3.2 Hz, 1H), 7.09 (dd, J = 8.8, 2.8 Hz, 1H), 7.28 (dd, J = 3.2 Hz, 1H), 7.59 (s, 1H), 7.97 (d, J = 6.4 Hz, 1H).

(S)-5-(4-{2-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl)propyl amino]ethyl} piperazin-1-yl)indole-1-carboxylic acid tert-butyl ester [(−)-18]

Compound 17 (175 mg, 0.51 mmol) was reacted with S-(−)-pramipexole (108 mg, 0.51 mmol) and NaBH(OAc)₃ (194 mg, 0.92 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography (EtOAc/MeOH, 20:1) to afford compound S-(−)-18 (161 mg, 59%). ¹H NMR (CDCl₃, 400 MHz): δ 0.93 (t, J = 6.8 Hz, 1H), 1.35–1.60 (m, 2H), 1.68 (s, 9H), 1.89–2.10 (m, 1H), 2.30–3.30 (m, 20H), 4.94 (bs, 2H), 6.67 (t, J = 3.2 Hz, 1H), 7.09 (dd, J = 8.8, 2.8 Hz, 1H), 7.28 (dd, J = 3.2 Hz, 1H), 7.60 (s, 1H), 7.97 (d, J = 6.4 Hz, 1H).

(R)-5-(4-{2-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl)propyl amino]ethyl}piperazin-1-yl)indole-1-carboxylic acid tert-butyl ester [(+)-18]

Compound 17 (175 mg, 0.51 mmol) was reacted with R-(+)-pramipexole (108 mg, 0.51 mmol) and NaBH(OAc)₃ (194 mg, 0.92 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography using (EtOAc/MeOH, 20:1) to afford compound R-(+)-18 (164 mg, 60%). ¹H NMR (CDCl₃, 400 MHz): δ 0.91 (t, J = 6.8 Hz, 1H), 1.37–1.60 (m, 2H), 1.67 (s, 9H), 1.89–2.10 (m, 1H), 2.30–3.30 (m, 20H), 4.94 (bs, 2H), 6.67 (t, J = 3.2 Hz, 1H), 7.10 (dd, J = 8.8, 2.8 Hz, 1H), 7.28 (dd, J = 3.2 Hz, 1H), 7.59 (s, 1H), 7.98 (d, J = 6.4 Hz, 1H).

N⁶-{2-[4-(1H-Indol-5-yl)piperazin-1-yl]ethyl}-N⁶-propyl-4,5,6,7-tetrahydro benzothiazole-2,6-diamine [(±)-19]

Compound (±)-18 (150 mg, 0.28 mmol) was reacted with TFA (10 mL) in CH₂Cl₂ (10 mL) using procedure C. Unreacted TFA and solvent were removed in vacuo and the salt was washed with diethylether and recrystallized from ethanol to afford compound (±)-19 (106 mg, 38%). ¹H NMR (CD₃OD, 400MHz): δ 0.99 (t, J = 7.2 Hz, 3H), 1.52–1.74 (m, 2H), 1.76–2.04 (m, 1H), 2.19 (d, J = 9.2 Hz, 1H), 2.52–2.84 (m, 6H), 3.10–3.58 (m, 13H), 6.50 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H). ¹³C (CD₃OD, 100 MHz): δ 12.0, 22.2, 24.0, 25.1, 47.0, 51.1, 54.4, 54.8, 59.2, 101.4, 101.5, 111.8, 115.3, 116.1, 127.8, 129.7, 135.1, 136.0, 140.7, 171.0. M.p. 110–115 °C. Anal. calculated for C₃₀H₄₀F₉N₆O_7.5S: C, H, N.

(S)-N⁶-{2-[4-(1H-Indol-5-yl)piperazin-1-yl]ethyl}-N⁶-propyl-4,5,6,7-tetrahydro benzothiazole-2,6-diamine [(−)-19]

Compound (−)-18 (150 mg, 0.28 mmol) was reacted with TFA (10 mL) in CH₂Cl₂ (10 mL) using procedure C. Unreacted TFA and solvent were removed in vacuo and the salt was washed with diethylether and recrystallized from ethanol to afford compound S-(−)-19 (120 mg, 43%). ¹H NMR (CD₃OD, 400MHz): δ 0.98 (t, J = 7.2 Hz, 3H), 1.54–1.74 (m, 2H), 1.76–2.04 (m, 1H), 2.19 (d, J = 9.2 Hz, 1H), 2.52–2.84 (m, 6H), 3.10–3.58 (m, 13H), 6.51 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H). ¹³C (CD₃OD, 100 MHz): δ 12.0, 22.3, 24.0, 25.1, 47.0, 51.1, 54.5, 54.8, 59.2, 101.4, 101.6, 111.8, 115.3, 116.1, 127.8, 129.7, 135.1, 136.0, 140.8, 171.0. [α]²⁵_D = −11.0° (c = 1.0, CH₃OH). M.p. 115–120 °C. Anal. calculated for C₃₁H_37.5F_10.5N₆O₇S: C, H, N.

(R)-N⁶-{2-[4-(1H-Indol-5-yl)-piperazin-1-yl]ethyl}-N⁶-propyl-4,5,6,7-tetrahydro benzothiazole-2,6-diamine [(+)-19]

Compound (+)-18 (150 mg, 0.28 mmol) was reacted with TFA (10 mL) in CH₂Cl₂ (10 mL) using procedure C. Unreacted TFA and solvent were removed in vacuo and the salt was washed with diethylether and recrystalized from ethanol to afford compound R-(+)-19 (140 mg, 50%). ¹H NMR (CD₃OD, 400MHz): δ 0.98 (t, J = 7.2 Hz, 3H), 1.54–1.78 (m, 2H), 1.76–2.04 (m, 1H), 2.20 (d, J = 9.2 Hz, 1H), 2.52–2.84 (m, 6H), 3.10–3.58 (m, 13H), 6.51 (d, J = 3.2 Hz, 1H), 7.05 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 3.2 Hz, 1H), 7.34 (d, J = 8.8 Hz, 2H). ¹³C (CD₃OD, 100 MHz): δ 12.2, 22.3, 24.0, 25.1, 47.0, 51.2, 54.5, 54.8, 59.2, 101.4, 101.6, 111.8, 115.3, 116.1, 127.8, 129.7, 135.1, 136.2, 140.8, 171.0. [α]²⁵_D = −15.5° (c = 1.0, CH₃OH). M.p. 115–120 °C. Anal. calculated for C₃₁H_37.9F_10.5N₆O_7.2S: C, H, N.

tert-Butyl 5-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl) piperazin-1-yl)-1H-indole-1-carboxylate (20)

Aldehyde 17 (320 mg, 0.93 mmol) was reacted with amine 2 (204 mg, 0.93 mmol) and NaBH(OAc)₃ (355 mg, 1.68 mmol) in CH₂Cl₂ (20 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/hexane, 1:1) to give compound 20 (190 mg, 38%). ¹H NMR (CDCl₃, 400 MHz): δ 0.91 (t, J = 7.2 Hz, 3H), 1.33–1.75 (m, 13H), 1.95–2.13 (m, 1H), 2.35–3.23 (m, 18H), 3.81 (s, 3H), 6.55–6.77 (m, 3H), 7.03–7.15 (m, 3H), 7.58 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 6.8 Hz, 1H).

6-({2-[4-(1H-Indol-5-yl)piperazin-1-yl]-ethyl}propyl amino)-5,6,7,8-tetrahydro naphthalen-1-ol (21)

A mixture of compound 20 (60 mg, 0.11 mmol) and 48% aq. HBr (10 ml) was refluxed for 5 h. The reaction mixture was evaporated to dryness and the residue was washed with diethylether. Finally, the HBr salt was recrystalized from ethanol to furnish compound 21 (50 mg, 60%). ¹H NMR (CD₃OD, 400 MHz): δ 0.95 (t, J = 7.2 Hz, 3H), 1.41–1.57 (m, 3H), 2.00–2.22 (m, 1H), 2.58–3.18 (m, 19H), 6.61 (d, J = 8 Hz, 1H), 6.75 (d, J = 8 Hz, 1H), 7.08 (t, J = 7.6 Hz, 1H), 7.19–7.38 (m, 3H), 7.51 (d, J = 8 Hz, 1H), 7.90 (d, J = 8.2 Hz, 1H), 8.50 (bs, 1H). M.p. 250–260 °C. Anal. calculated for C₂₇H_41.4Br₄N₄O_1.7: C, H, N.

Procedure F. 4-(1H-Indole-2-carbonyl)piperazine-1-carboxylic acid tert-butyl ester (23)

To a stirring solution of EDCI.HCl (15.45 g, 80.6 mmol) in CH₂Cl₂ (150 mL), acid derivative 22 (10.39 g, 64.5 mmol) was added at room temperature. The reaction mixture was stirred for 0.5 h, followed by addition of amine 4 (10.0 g, 53.7 mmol), HOBt (10.89 g, 80.6 mmol) and Et₃N (22.46 g, 161.2 mmol). The reaction mixture was stirred for 3 h, followed by addition of a saturated solution of NaHCO₃. The aqueous layer was extracted with CH₂Cl₂ (3 × 150 mL). The combined organic layer was dried over Na₂SO₄, evaporated under reduced pressure and the crude product was purified by flash column chromatography (CH₂Cl₂/MeOH, 50:1) to afford compound 23 (15.6 g, 88%). ¹H NMR (CDCl₃, 400 MHz): δ 1.49 (s, 9H), 3.56 (t, J = 5.6 Hz, 4H), 3.74–4.04 (m, 4H), 6.78 (s, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 9.14 (bs, 1H).

(1H-Indol-2-yl)-piperazin-1-yl-methanone (24)

Compound 23 (12 g, 36.4 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C to provide compound 24 (7.85 g, 94%). ¹H NMR (CDCl₃, 400 MHz): δ 2.52–3.04 (m, 4H), 3.60–4.06 (m, 4H), 6.77 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.27 (d, J = 6.8 Hz, 1H), 7.43 (d, J = 6.8 Hz, 1H), 7.65 (d, J = 6.8 Hz, 1H), 9.50 (bs, 1H).

{4-[2-(tert-Butyldimethylsilanyloxy)ethyl]piperazin-1-yl}-(1H-indol-2-yl)methanone (25)

Compound 24 (2.50 g, 10.9 mmol) was reacted with (2-bromo-ethoxy)-tert-butyl-dimethylsilane (3.13 g, 13.1 mmol) and K₂CO₃ (4.52 g, 32.7 mmol) in CH₃CN (50 mL) using procedure D. The crude material was purified by silica gel column chromatography using ethyl acetate as solvent to give compound 25 (3.55 g, 84%). ¹H NMR (CDCl₃, 400 MHz): δ 0.09 (s, 6H), 0.92 (s, 9H), 2.02–2.80 (m, 6H), 3.01–4.20 (m, 6H), 6.77 (s, 1H), 7.11 (t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8 Hz, 1H), 10.4 (bs, 1H).

{4-[3-(tert-Butyldimethylsilanyloxy)propyl]piperazin-1-yl}-(1H-indol-2-yl)methanone (26)

Compound 24 (2.50 g, 10.9 mmol) was reacted with (3-bromo-propoxy)-tert-butyl-dimethylsilane (3.31 g, 13.1 mmol) and K₂CO₃ (4.52 g, 32.7 mmol) in CH₃CN (50 mL) using procedure D. The crude material was purified by silica gel column chromatography (EtOAc/hexane, 1:1) to give compound 26 (3.33 g, 76%). ¹H NMR (CDCl₃, 400 MHz): δ 0.06 (s, 6H), 0.90 (s, 9H), 1.52–1.86 (m, 2H), 2.45–2.70 (m, 6H), 3.68 (t, J = 12 Hz, 2H), 3.72–4.20 (m, 4H), 6.78 (s, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.20–7.32 (m, 2H), 7.43 (d, J = 7.6 Hz, 1H), 7.64 (d, J = 7.6 Hz, 1H), 9.35 (s, 1H).

{4-[4-(tert-Butyldimethylsilanyloxy)butyl]piperazin-1-yl}-(1H-indol-2-yl)methanone (27)

Compound 24 (2.50 g, 10.9 mmol) was reacted with (4-bromo-butoxy)-tert-butyl-dimethylsilane (3.50 g, 13.1 mmol) and K₂CO₃ (4.52 g, 32.7 mmol) in CH₃CN (50 mL) using procedure D. The crude material was purified by silica gel column chromatography (EtOAc/hexane, 2:3) to give compound 27 (4.31 g, 95%). ¹H NMR (CDCl₃, 400 MHz): δ 0.05 (s, 6H), 0.90 (s, 9H), 1.47–1.75 (m, 4H), 2.40 (t, J = 6.4 Hz, 2H), 2.53 (t, J = 4.4 Hz, 4H), 3.64 (t, J = 5.6 Hz, 2H), 3.80–4.05 (m, 4H), 6.77 (s, 1H), 7.12 (t, J = 6.8 Hz, 1H), 7.26 (t, J = 7.2 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8 Hz, 1H), 10.00 (s, 1H).

Procedure G. 2-{4-[3-(tert-Butyldimethylsilanyloxy)ethyl]piperazine-1-carbonyl}indole-1-carboxylic acid tert-butyl ester (28)

To a stirring solution of amine 25 (3.55 g, 9.2 mmol) in THF (50 mL), (Boc)₂O (2.21 g, 10.1 mmol) and DMAP (1.23 g, 10.1 mmol) were added 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 CH₂Cl₂ (3 × 100 mL) in water. The combined organic layer was dried over Na₂SO₄, filtered, and concentrated in vacuo. The crude material was purified by column chromatography over silica gel using ethyl acetate as solvent to give compound 28 (4.46 g, ~100%). ¹H NMR (CDCl₃, 400 MHz): δ 0.05 (s, 6H), 0.09 (s, 9H), 1.62 (s, 9H), 2.48 (t, J = 4 Hz, 2H), 2.56 (t, J = 6 Hz, 2H), 2.63 (t, J = 4 Hz, 2H), 3.38 (t, J = 4 Hz, 2H), 3.76 (t, J = 6 Hz, 4H), 6.60 (s, 1H), 7.20–7.28 (m, 1H), 7.34 (t, J = 7.2 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H).

2-{4-[3-(tert-Butyldimethylsilanyloxy)propyl]piperazine-1-carbonyl}indole-1-carboxylic acid tert-butyl ester (29)

Amine 26 (3.33 g, 8.3 mmol) was reacted with (Boc)₂O (2.00 g, 9.1 mmol) and DMAP (1.11 g, 9.1 mmol) in THF (50 mL) using procedure G. The crude material was purified by column chromatography over silica gel (EtOAc/hexane, 1:1) to give compound 29 (4.04 g, 97%). ¹H NMR (CDCl₃, 400 MHz): δ 0.002 (s, 6H), 0.84 (s, 9H), 1.57 (s, 9H), 1.50–1.80 (m, 2H), 2.20–2.70 (m, 6H), 3.30–3.45 (m, 2H), 3.62 (t, J = 12 Hz, 2H), 3.66–3.80 (m, 2H), 6.55 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H).

2-{4-[4-(tert-Butyldimethylsilanyloxy)butyl]piperazine-1-carbonyl}-indole-1-carboxylic acid tert-butyl ester (30)

Amine 27 (4.31 g, 10.4 mmol) was reacted with (Boc)₂O (2.50 g, 11.4 mmol) and DMAP (1.39 g, 11.4 mmol) in THF (50 mL) using procedure G. The crude material was purified by column chromatography over silica gel (EtOAc/hexane, 1:3) to give compound 30 (4.92 g, 92%). ¹H NMR (CDCl₃, 400 MHz): δ 0.001 (s, 6H), 0.84 (s, 9H), 1.25–1.70 (m, 13H), 2.15–2.40 (m, 4H), 2.42–2.55 (m, 2H), 3.25–3.45 (m, 2H), 3.47–3.65 (m, 2H), 3.67–3.85 (m, 2H), 6.54 (s, 1H), 7.18 (t, J = 7.2 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H).

2-[4-(2-Hydroxyethyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (31)

Compound 28 (4.46 g, 9.2 mmol) was reacted with n-tetrabutylammonium fluoride (2.39 g, 9.2 mmol, 1.0 M solution in THF) in THF (30 mL) using procedure E. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 9:1) to yield compound 31 (3.08 g, 90%). ¹H NMR (CDCl₃, 400 MHz): δ 1.62 (s, 9H), 2.46 (t, J = 4.8 Hz, 2H), 2.54–2.68 (m, 4H), 3.41 (t, J = 5.2 Hz, 2H), 3.64 (t, J = 5.2 Hz, 2H), 3.72–3.78 (m, 2H), 6.60 (s, 1H), 7.21–7.28 (m, 1H), 7.35 (t, J = 8.4 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 8.14 (d, J = 7.6 Hz, 1H).

2-[4-(3-Hydroxypropyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (32)

Compound 29 (4.04 g, 8.1 mmol) was reacted with n-tetrabutylammonium fluoride (2.11 g, 8.1 mmol, 1.0 M solution in THF) in THF (30 mL) using procedure E. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 9:1) to yield compound 32 (2.84 g, 91%). ¹H NMR (CDCl₃, 400 MHz): δ 1.30–1.75 (m, 11H), 2.33 (m, 2H), 2.47 (t, J = 4.4 Hz, 4H), 3.29 (m, 2H), 3.63 (t, J = 4.8 Hz, 4H), 6.50 (s, 1H), 7.13 (t, J = 7.6 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 8.05 (d, J = 8.4 Hz, 1H).

2-[4-(4-Hydroxybutyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (33)

Compound 30 (4.92 g, 9.5 mmol) was reacted with n-tetrabutylammonium fluoride (2.50 g, 9.5 mmol, 1.0 M solution in THF) in THF (30 mL) using procedure E. The crude product was purified by silica gel column chromatography (EtOAc/MeOH, 9:1) to yield compound 33 (3.72 g, 97%). ¹H NMR (CDCl₃, 400 MHz): δ 0.95–1.70 (m, 13H), 2.30 (t, J = 6 Hz, 4H), 2.37–2.65 (m, 2H), 3.15–3.36 (m, 2H), 3.37–3.55 (m, 2H), 3.55–3.82 (m, 2H), 6.48 (s, 1H), 7.12 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 8.05 (d, J = 8 Hz, 1H).

2-[4-(2-Oxoethyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (34)

Compound 31 (1.0 g, 2.7 mmol) was reacted with oxalyl chloride (0.47 mL, 5.4 mmol), DMSO (0.76 mL, 10.7 mmol) and Et₃N (2.2 mL, 16.1 mmol) in CH₂Cl₂ (40 mL) using procedure A. The crude residue was purified by column chromatography (EtOAc/MeOH, 20:1) to afford compound 34 (0.81 g, 81%).

2-[4-(3-Oxopropyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (35)

Compound 32 (0.8 g, 2.1 mmol) was reacted with oxalyl chloride (0.36 mL, 4.1 mmol), DMSO (0.59 mL, 8.3 mmol) and Et₃N (1.73 mL, 12.4 mmol) in CH₂Cl₂ (40 mL) using procedure A. The crude residue was purified by column chromatography (EtOAc/MeOH, 20:1) to afford compound 35 (0.62 g, 78%).

2-[4-(4-Oxobutyl)piperazine-1-carbonyl]indole-1-carboxylic acid tert-butyl ester (36)

Compound 33 (1.0 g, 2.5 mmol) was reacted with oxalyl chloride (0.43 mL, 5.0 mmol), DMSO (0.71 mL, 10.0 mmol) and Et₃N (2.08 mL, 15.0 mmol) in CH₂Cl₂ (40 mL) using procedure A. The crude residue was purified by column chromatography (EtOAc/MeOH, 20:1) to afford compound 36 (0.75 g, 75%).

2-(4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl)propyl amino]ethyl}piperazine-1-carbonyl)indole-1-carboxylic acid tert-butyl ester [(±)-37]

Aldehyde 34 (480 mg, 1.3 mmol) was reacted with (±)-pramipexole (273 mg, 1.3 mmol) and NaBH(OAc)₃ (451 mg, 2.3 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 9:1) to give compound (±)-37 (0.52 g, 71%). ¹H NMR (CDCl₃, 400 MHz): δ 0.85 (t, J = 3.6 Hz, 3H), 1.30–1.65 (m, 2H), 1.60 (s, 9H), 1.95 (d, J = 10.8 Hz, 1H), 2.20–3.10 (m, 16H), 3.15–3.82 (m, 4H), 4.94 (bs, 2H), 6.58 (s, 1H), 7.24 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.53 (d, J = 6 Hz, 1H), 8.13 (d, J = 6 Hz, 1H).

(R)-2-(4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl)propyl amino]ethyl} piperazine-1-carbonyl)indole-1-carboxylic acid tert-butyl ester [(+)-37]

Aldehyde 34 (200 mg, 0.54 mmol) was reacted with R-(+)-pramipexole (114 mg, 0.54 mmol) and NaBH(OAc)₃ (205 mg, 0.97 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 9:1) to yield compound R-(+)-37 (186 mg, 61%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 3.6 Hz, 3H), 1.30–1.65 (m, 2H), 1.61 (s, 9H), 1.95 (d, J = 10.8 Hz, 1H), 2.20–3.10 (m, 16H), 3.15–3.82 (m, 4H), 4.94 (bs, 2H), 6.58 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.53 (d, J = 6 Hz, 1H), 8.13 (d, J = 6 Hz, 1H).

(S)-2-(4-{2-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl)propyl amino]ethyl} piperazine-1-carbonyl)indole-1-carboxylic acid tert-butyl ester [(−)-37]

Aldehyde 34 (300 mg, 0.81 mmol) was reacted with S-(−)-pramipexole (170 mg, 0.81 mmol) and NaBH(OAc)₃ (308 mg, 1.45 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 9:1) to yield compound S-(−)-37 (284 mg, 62%). ¹H NMR (CDCl₃, 400 MHz): δ 0.83 (t, J = 3.6 Hz, 3H), 1.30–1.65 (m, 2H), 1.61 (s, 9H), 1.95 (d, J = 10.8 Hz, 1H), 2.20–3.10 (m, 16H), 3.15–3.82 (m, 4H), 4.94 (bs, 2H), 6.58 (s, 1H), 7.25 (d, J = 8.4 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.53 (d, J = 6 Hz, 1H), 8.14 (d, J = 6 Hz, 1H).

2-(4-{3-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl) propyl amino] propyl} piperazine-1-carbonyl) indole-1-carboxylic acid tert-butyl ester (38)

Aldehyde 35 (220 mg, 0.57 mmol) was reacted with (±)-pramipexole (121 mg, 0.57 mmol) and NaBH(OAc)₃ (218 mg, 1.03 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 9:1) to give compound 38 (215 mg, 65%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 7.2 Hz, 3H), 1.15–1.85 (m, 13H), 1.96 (d, J = 7.2 Hz, 1H), 2.10–2.85 (m, 15H), 2.90–3.15 (m, 1H), 3.20–3.50 (m, 2H), 3.55–3.95 (m, 2H), 6.58 (s, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.52 (d, J = 7.6 Hz, 1H), 8.13 (d, J = 8 Hz, 1H).

2-(4-{4-[(2-Amino-4,5,6,7-tetrahydro benzothiazol-6-yl) propyl amino] butyl}piperazine 1-carbonyl) indole-1-carboxylic acid tert-butyl ester (39)

Aldehyde 36 (233 mg, 0.58 mmol) was reacted with (±)-pramipexole (123 mg, 0.58 mmol) and NaBH(OAc)₃ (222 mg, 1.05 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 20:1) to yield compound 39 (190 mg, 55%). ¹H NMR (CDCl₃, 400 MHz): δ 0.87 (t, J = 7.2 Hz, 3H), 1.10–1.78 (m, 15H), 1.96 (d, J = 11.2 Hz, 1H), 2.12–2.85 (m, 15H), 2.90–3.15 (m, 1H), 3.25–3.50 (m, 2H), 3.65–3.92 (m, 2H), 4.78 (s, 2H), 6.60 (s, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.54 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 8.4 Hz, 1H).

(4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl) propyl amino] ethyl} piperazin-1-yl)-(1H-indol-2-yl) methanone [(±)-40]

Compound (±)-37 (200 mg, 0.35 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C. Unreacted TFA and solvent were removed under reduced pressure. The TFA salt was converted to the free base by extraction using CH₂Cl₂ (3 × 100 mL) and a saturated solution of NaHCO₃. The crude material was purified by column chromatography over silica gel to afford compound (±)-40 (228 mg, 80%). ¹H NMR (CD₃OD, 400 MHz): δ 1.04 (t, J = 7.2 Hz, 3H), 1.70–1.88 (m, 2H), 2.01–2.18 (m, 1H), 2.32 (d, J = 10.8 Hz, 1H), 2.60–3.60 (m, 13H), 3.72–4.20 (m, 6H), 6.85 (s, 1H), 7.07 (t, J = 8 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.62 (d, J = 8 Hz, 1H). ¹³C (CD₃OD, 100 MHz): δ 14.1, 23.0, 26.0, 26.7, 27.0, 56.6, 57.0, 57.8, 63.1, 109.2, 115.7, 116.3, 124.1, 125.4, 127.9, 131.3, 132.7, 137.4, 137.5, 167.8, 174.4. The free base was converted to its hydrochloride salt. M.p. 270–275 °C. Anal. calculated for C₂₅H₄₀Cl₄N₆O₂S: C, H, N.

(R)-(4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl) propyl amino] ethyl} piperazin-1-yl)-(1H-indol-2-yl) methanone [(+)-40]

Compound (+)-37 (150 mg, 0.26 mmol) was reacted with TFA (15 mL) in CH₂Cl₂ (15 mL) using procedure C. Unreacted TFA and solvent were removed in vacuo and the salt was washed with diethylether and recrystallized from ethanol to afford compound R-(+)-40 (160 mg, 75%). ¹H NMR (CD₃OD, 400 MHz): δ 1.05 (t, J = 7.2 Hz, 3H), 1.70–1.88 (m, 2H), 2.01–2.18 (m, 1H), 2.34 (d, J = 10.8 Hz, 1H), 2.60–3.60 (m, 13H), 3.72–4.20 (m, 6H), 6.85 (s, 1H), 7.07 (t, J = 8 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.63 (d, J = 8 Hz, 1H). ¹³C (CD₃OD, 100 MHz): δ 14.1, 23.0, 26.2, 26.7, 27.0, 56.6, 57.0, 57.8, 63.1, 109.3, 115.7, 116.3, 124.1, 125.4, 127.9, 131.3, 132.7, 137.4, 137.6, 167.8, 174.4. [α]²⁵_D = +25.2° (c = 1.0, CH₃OH). M.p. 100–110 °C. Anal. calculated for C₃₁H_38.₂F₉N₆O_7.6S: C, H, N.

(S)-(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl) propyl amino]ethyl}piperazin-1-yl)-(1H-indol-2-yl)methanone [(−)-40]

Compound (−)-37 (200 mg, 0.35 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C. Unreacted TFA and solvent were removed in vacuo and the salt was washed with diethylether and recrystallized from ethanol to afford compound S-(−)-40 (237 mg, 83%). ¹H NMR (CD₃OD, 400 MHz): δ 1.02 (t, J = 7.2 Hz, 3H), 1.73–1.88 (m, 2H), 2.01–2.18 (m, 1H), 2.32 (d, J = 10.8 Hz, 1H), 2.60–3.60 (m, 13H), 3.72–4.20 (m, 6H), 6.85 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.44 (d, J = 8 Hz, 1H), 7.62 (d, J = 8 Hz, 1H). ¹³C (CD₃OD, 100 MHz): δ 14.3, 23.0, 26.2, 26.7, 27.0, 56.6, 57.0, 57.8, 63.1, 109.3, 115.7, 116.3, 124.1, 125.4, 127.9, 131.3, 132.7, 137.4, 137.5, 167.8, 174.4. [α]²⁵_D = −31.2° (c = 1.0, CH₃OH). M.p. 110–115 °C. Anal. calculated for C₃₁H₄₀F₉N₆O_8.5S: C, H, N.

(4-{3-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]propyl}piperazin-1-yl)-(1H-indol-2-yl)methanone (41)

Compound 38 (200 mg, 0.34 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C. Unreacted TFA and solvent were removed under reduced pressure, followed by washing of the salt with diethylether and recrystallization from ethanol to afford compound 41 (266 mg, 94%). ¹H NMR (CD₃OD, 400 MHz): δ 1.04 (t, J = 7.6 Hz, 3H), 1.65–1.90 (m, 2H), 1.95–2.18 (m, 1H), 2.20–2.48 (m, 3H), 2.56–3.70 (m, 12H), 3.78–4.40 (m, 5H), 6.90 (s, 1H), 7.08 (t, J = 8 Hz, 1H), 7.23 (t, J = 8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H). ¹³C (CD₃OD, 100 MHz): δ 10.0, 18.4, 20.1, 21.8, 22.3, 22.7, 51.8, 53.1, 53.6, 59.2, 104.4, 105.7, 111.8, 120.2, 121.6, 124.2, 127.4, 128.2, 133.7, 136.9, 163.8, 170.5. M.p. 115–120 °C. Anal. calculated for C₃₂H₄₂F₉N₆O_8.5S: C, H, N.

(4-{4-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]butyl}piperazin-1-yl)-(1H-indol-2-yl)methanone (42)

Compound 39 (175 mg, 0.29 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C. Unreacted TFA and solvent were removed under reduced pressure, followed by washing of the salt with diethylether and recrystallization from ethanol to afford compound 42 (175 mg, 71%). ¹H NMR (CD₃OD, 400 MHz): δ 1.03 (t, J = 7.2 Hz, 3H), 1.50–2.02 (m, 6H), 2.03–2.20 (m, 1H), 2.35 (d, J = 10.8 Hz, 1H), 2.54–3.70 (m, 18H), 3.74–4.01 (m, 1H), 6.90 (s, 1H), 7.08 (t, J = 7.6 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H). ¹³C (CD₃OD, 100 MHz): δ 10.0, 18.4, 21.0, 21.7, 22.0, 22.2, 22.7, 51.6, 56.0, 59.1, 105.7, 105.7, 111.8, 120.3, 121.6, 124.2, 127.4, 128.2, 133.2, 136.9, 163.8, 170.6. M.p. 100–105 °C. Anal. calculated for C₃₅H_43.6F₁₂N₆O_9.8S: C, H, N.

4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl)propylamino]ethyl}piperazine-1-carboxylic acid tert-butyl ester [(±)-43]

Aldehyde 6 (2.4 g, 10.5 mmol) was reacted with (±)-pramipexole (2.22 g, 10.5 mmol) and NaBH(OAc)₃ (4.01 g, 18.9 mmol) in CH₂Cl₂ (40 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 20:1) to yield compound (±)-43 (3.34 g, 75%). ¹H NMR (CDCl₃, 400 MHz): δ 0.81 (t, J = 7.2 Hz, 3H), 1.10–1.75 (m, 10H), 1.91 (d, J = 9.6, 1H), 2.10–2.75 (m, 15H), 2.97 (m, 1H), 3.36 (m, 4H), 5.29 (bs, 2H).

(R)-4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl)propylamino]-ethyl}piperazine-1-carboxylic acid tert-butyl ester [(+)-43]

Compound 6 (1.2 g, 5.25 mmol) was reacted with (R)-(+)-pramipexole (1.11 g, 5.25 mmol) and NaBH(OAc)₃ (2.0 g, 9.46 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 20:1) to give compound R-(+)-43 (1.63 g, 73%). ¹H NMR (CDCl₃, 400 MHz): δ 0.83 (t, J = 7.2 Hz, 3H), 1.10–1.75 (m, 10H), 1.91 (d, J = 9.6 Hz, 1H), 2.10–2.75 (m, 15H), 2.98 (m, 1H), 3.36 (m, 4H), 5.29 (bs, 2H).

(S)-4-{2-[(2-Amino-4,5,6,7-tetrahydro-benzothiazol-6-yl)propylamino]ethyl}piperazine-1-carboxylic acid tert-butyl ester [(−)-43]

Compound 6 (1.2 g, 5.25 mmol) was reacted with (S)-(−)-pramipexole (1.11 g, 5.25 mmol) and NaBH(OAc)₃ (2.0 g, 9.46 mmol) in CH₂Cl₂ (25 mL) using procedure B. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 20:1) to give compound S-(−)-43 (1.61 g, 71%). ¹H NMR (CDCl₃, 400 MHz): δ 0.80 (t, J = 7.2 Hz, 3H), 1.10–1.75 (m, 10H), 1.91 (d, J = 9.6 Hz, 1H), 2.12–2.75 (m, 15H), 2.97 (m, 1H), 3.36 (m, 4H), 5.29 (bs, 2H).

N⁶-(2-Piperazin-1-yl-ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine [(±)-44]

Compound (±)-43 (3.34 g, 7.88 mmol) was reacted with TFA (20 mL) in CH₂Cl₂ (20 mL) using procedure C to give compound (±)-44 (2.30 g, 90%). ¹H NMR (CDCl₃, 400 MHz): δ 0.79 (t, J = 7.2 Hz, 3H), 1.25–1.48 (m, 2H), 1.50–1.72 (m, 1H), 1.88 (d, J = 11.2 Hz, 1H), 2.08–3.07 (m, 19H), 5.40 (bs, 2H).

(R)-N⁶-(2-Piperazin-1-yl-ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine [(+)-44]

Compound (+)-43 (1.63 g, 3.84 mmol) was reacted with TFA (15 mL) in CH₂Cl₂ (15 mL) using procedure C to yield compound R-(+)-44 (1.16 g, 93%). ¹H NMR (CDCl₃, 400 MHz): δ 0.80 (t, J = 7.2 Hz, 3H), 1.25–1.48 (m, 2H), 1.52–1.72 (m, 1H), 1.88 (d, J = 11.2 Hz, 1H), 2.08–3.07 (m, 19H), 5.40 (bs, 2H).

(S)-N⁶-(2-Piperazin-1-yl-ethyl)-N⁶-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine [(−)-44]

Compound (−)-43 (1.61 g, 3.80 mmol) was reacted with TFA (15 mL) in CH₂Cl₂ (15 mL) using procedure C to afford compound S-(−)-44 (1.16 g, 94%). ¹H NMR (CDCl₃, 400 MHz): δ 0.79 (t, J = 7.2 Hz, 3H), 1.25–1.48 (m, 2H), 1.50–1.72 (m, 1H), 1.89 (d, J = 11.2 Hz, 1H), 2.08–3.07 (m, 19H), 5.41 (bs, 2H).

(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-3-yl)methanone [(±)-45]

Indole-3-carboxylic acid (54 mg, 0.33 mmol) was reacted with compound (±)-44 (90 mg, 0.28 mmol) in the presence of EDCI.HCl (80 mg, 0.42 mmol), TEA (0.12 mL, 0.83 mmol) and HOBt (56 mg, 0.41 mmol) in CH₂Cl₂ (10 mL) using procedure F. The crude product was purified by flash column chromatography (EtOAc/MeOH, 4:1) to afford compound (±)-45 (83 mg, 64%). ¹H NMR (CDCl₃, 400 MHz): δ 0.87 (t, J = 7.6 Hz, 3H), 1.30–1.52 (m, 2H), 1.55–1.80 (m, 1H), 1.95 (d, J = 11.6, 1H), 2.20–2.80 (m, 13H), 2.85–3.10 (m, 1H), 3.50–3.82 (m, 4H), 4.86 (bs, 2H), 7.0–7.38 (m, 4H), 7.60–7.76 (m, 1H), 9.77 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 14.4, 21.3, 22.5, 25.4, 26.1, 26.8, 48.6, 53.7, 54.2, 58.3, 58.8, 60.6, 111.5, 112.1, 117.6, 120.4, 121.2, 122.8, 125.6, 127.5, 135.9, 145.3, 165.8, 167.2. The free base was converted to its hydrochloride salt. M.p. 170–175 °C. Anal. calculated for C₂₅H_38.6Cl₄N₆O_1.3S: C, H, N.

(R)-(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-3-yl)methanone [(+)-45)]

Indole-3-carboxylic acid (54 mg, 0.33 mmol) was reacted with compound (+)-44 (90 mg, 0.28 mmol) in the presence of EDCI.HCl (80 mg, 0.42 mmol), TEA (0.12 mL, 0.83 mmol) and HOBT (56 mg, 0.41 mmol) in CH₂Cl₂ (10 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 4:1) to yield compound R-(+)-45 (87 mg, 67%). ¹H NMR (CDCl₃, 400 MHz): δ 0.89 (t, J = 7.6 Hz, 3H), 1.30–1.52 (m, 2H), 1.57–1.80 (m, 1H), 1.95 (d, J = 11.6 Hz, 1H), 2.20–2.80 (m, 13H), 2.85–3.10 (m, 1H), 3.50–3.82 (m, 4H), 4.87 (bs, 2H), 7.0–7.38 (m, 4H), 7.60–7.76 (m, 1H), 9.77 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 14.4, 21.4, 22.5, 25.4, 26.1, 26.9, 48.6, 53.7, 54.2, 58.3, 58.8, 60.6, 111.6, 112.1, 117.6, 120.4, 121.2, 122.8, 125.6, 127.5, 135.9, 145.3, 165.9, 167.2. [α]²⁵_D = +37.4° (c = 1.0, CH₃OH). The free base was converted to its hydrochloride salt. M.p. 185–190 °C. Anal. calculated for C₂₅H₄₀Cl₄N₆O₂S: C, H, N.

(S)-(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-3-yl)methanone [(−)-45)]

Indole-3-carboxylic acid (54 mg, 0.33 mmol) was reacted with compound (−)-44 in the presence of EDCI.HCl (80 mg, 0.42 mmol), TEA (0.12 mL, 0.83 mmol) and HOBT (56 mg, 0.41 mmol) in CH₂Cl₂ (10 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 4:1) to give compound S-(−)-45 (93 mg, 72%). ¹H NMR (CDCl₃, 400 MHz): δ 0.85 (t, J = 7.6 Hz, 3H), 1.30–1.52 (m, 2H), 1.55–1.80 (m, 1H), 1.95 (d, J = 11.6 Hz, 1H), 2.20–2.80 (m, 13H), 2.85–3.10 (m, 1H), 3.50–3.82 (m, 4H), 4.86 (bs, 2H), 7.0–7.38 (m, 4H), 7.60–7.76 (m, 1H), 9.77 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 14.4, 21.3, 22.5, 25.4, 26.1, 26.8, 48.6, 53.8, 54.2, 58.3, 58.8, 60.6, 111.5, 112.1, 117.7, 120.4, 121.2, 122.8, 125.6, 127.5, 135.9, 145.3, 165.8, 167.2. [α]²⁵_D = −33.6° (c = 1.0, CH₃OH). The free base was converted to its hydrochloride salt. M.p. 205–210 °C. Anal. calculated for C₂₅H_41.₅Cl_4.5N₆O_2.5S: C, H, N.

(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-5-yl)methanone [(±)-46]

Indole-5-carboxylic acid (60 mg, 0.37 mmol) was reacted with compound (±)-44 (100 mg, 0.31 mmol) in the presence of EDCI.HCl (89 mg, 0.46 mmol), TEA (0.13 mL, 0.93 mmol) and HOBT (63 mg, 0.46 mmol) in CH₂Cl₂ (15 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 4:1) to yield compound (±)-46 (102 mg, 71%). ¹H NMR (CDCl₃, 400 MHz): δ 0.88 (t, J = 7.2 Hz, 1H), 1.40–1.54 (m, 2H), 1.60–1.81 (m, 1H), 1.97 (d, J = 11.2 Hz, 1H), 2.20–2.72 (m, 14H), 2.90–3.15 (m, 1H), 3.30–4.02 (m, 4H), 4.89 (bs, 2H), 6.57 (s, 1H), 7.15–7.29 (m, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 8.84 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 22.5, 25.3, 26.0, 26.7, 48.5, 53.7, 58.3, 58.7, 103.1, 111.4, 117.3, 120.4, 121.4, 125.9, 127.0, 127.5, 136.7, 145.1, 166.1, 172.2. The free base was converted to its hydrochloride salt. M.p. 185–190 °C. Anal. calculated for C₂₅H₄₁Cl₄N₆O_2.5S: C, H, N.

(R)-(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-5-yl)methanone [(+)-46]

Indole-5-carboxylic acid (60 mg, 0.37 mmol) was reacted with compound (+)-44 (100 mg, 0.31 mmol) in the presence of EDCI.HCl (89 mg, 0.46 mmol), TEA (0.13 mL, 0.93 mmol) and HOBT (63 mg, 0.46 mmol) in CH₂Cl₂ (15 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 4:1) to furnish compound R-(+)-46 (94 mg, 65%). ¹H NMR (CDCl₃, 400 MHz): δ 0.89 (t, J = 7.2 Hz, 1H), 1.40–1.54 (m, 2H), 1.60–1.81 (m, 1H), 1.97 (d, J = 11.2 Hz, 1H), 2.20–2.72 (m, 14H), 2.90–3.15 (m, 1H), 3.30–4.03 (m, 4H), 4.89 (bs, 2H), 6.57 (s, 1H), 7.16–7.29 (m, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 8.84 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.0, 22.5, 25.3, 26.0, 26.7, 48.5, 53.7, 58.3, 58.7, 103.1, 111.4, 117.3, 120.4, 121.4, 125.9, 127.0, 127.5, 136.7, 145.1, 166.1, 172.2. [α]²⁵_D = +38.6° (c = 1.0, CH₃OH). The free base was converted to its hydrochloride salt. M.p. 200–205 °C. Anal. calculated for C₂₅H₄₀Cl₄N₆O₂S: C, H, N.

(S)-(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-5-yl)methanone [(−)-46]

Indole-5-carboxylic acid (60 mg, 0.37 mmol) was reacted with compound (−)-44 (100 mg, 0.31 mmol) in the presence of EDCI.HCl (89 mg, 0.46 mmol), TEA (0.13 mL, 0.93 mmol) and HOBT (63 mg, 0.46 mmol) in CH₂Cl₂ (15 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 4:1) to yield compound S-(−)-46 (95 mg, 66%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 7.2 Hz, 1H), 1.40–1.54 (m, 2H), 1.62–1.81 (m, 1H), 1.97 (d, J = 11.2 Hz, 1H), 2.20–2.72 (m, 14H), 2.90–3.15 (m, 1H), 3.30–4.02 (m, 4H), 4.89 (bs, 2H), 6.58 (s, 1H), 7.15–7.29 (m, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.71 (s, 1H), 8.84 (s, 1H). ¹³C (CDCl₃, 100 MHz): δ 12.2, 22.5, 25.3, 26.1, 26.7, 48.5, 53.7, 58.3, 58.9, 103.1, 111.4, 117.3, 120.4, 121.4, 125.9, 127.1, 127.5, 136.7, 145.1, 166.1, 172.2. [α]²⁵_D = −34.4° (c = 1.0, CH₃OH). The free base was converted to its hydrochloride salt. M.p. 200–205 °C. Anal. calculated for C₂₅H_42.1Cl_4.5N₆O_2.8S: C, H, N.

(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(5-methoxy-1H-indol-3-yl)methanone (47)

5-methoxy-1H-indole-3-carboxylic acid (213 mg, 1.11 mmol) was reacted with compound (±)-44 (300 mg, 0.93 mmol) in the presence of EDCI.HCl (267 mg, 1.40 mmol), TEA (0.39 mL, 2.78 mmol) and HOBT (188 mg, 1.40 mmol) in CH₂Cl₂ (25 mL) using procedure F. The crude material was purified by column chromatography over silica gel (EtOAc/MeOH, 6:1) to give compound 47 (280 mg, 61%). ¹H NMR (CDCl₃, 400 MHz): δ 0.85 (t, J = 7.2 Hz, 3H), 1.32–1.48 (m, 2H), 1.56–1.76 (m, 1H), 1.93 (d, J = 10 Hz, 1H), 2.26–2.78 (m, 1H), 2.90–3.10 (m, 1H), 3.52–3.77 (m, 4H), 3.77 (s, 3H), 5.21 (bs, 2H), 6.77 (d, J = 8.4 Hz, 1H), 6.90–7.22 (m, 3H), 10.3 (bs, 1H).

(4-{2-[(2-Amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(5-hydroxy-1H-indol-3-yl)methanone (48)

Compound 47 (140 mg, 0.28 mmol) was brought to −78 °C in CH₂Cl₂ (15 mL), followed by dropwise addition of BBr₃ (0.13 mL, 1.41 mmol). The reaction mixture was allowed to stir for 12 h, while gradually attaining room temperature. The reaction was quenched by the addition of a saturated solution of NaHCO₃ and extracted with CH₂Cl₂ (2 × 100 mL). The organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The crude material was purified by flash column chromatography over silica gel (EtOAc/MeOH, 2:1) to give compound 48 (80 mg, 59%). ¹H NMR (CD₃OD, 400 MHz): δ 0.91 (t, J = 7.6 Hz, 3H), 1.42–1.58 (m, 2H), 1.62–1.80 (m, 1H), 2.00 (d, J = 8.8 Hz, 1H), 2.40–2.85 (m, 15H), 3.00–3.22 (m, 1H), 3.60–3.88 (m, 4H), 6.74 (dd, J = 8, 1.6 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.24 (s, 1H), 7.53 (s, 1H). The free base was converted to its hydrochloride salt. M.p. 210–215 °C. Anal. calculated for C_25.8H_43.4Cl₄N₆O_3.9S: C, H, N.

N⁶-{2-[4-(1H-Indol-5-ylmethyl)piperazin-1-yl]ethyl}-N⁶-propyl-4,5,6,7-tetrahydrobenzothiazole-2,6-diamine (49)

Amine (±)-44 (80 mg, 0.25 mmol) was reacted with 1H-indole-5-carbaldehyde (36 mg, 0.25 mmol) and NaBH(OAc)₃ (94 mg, 0.45 mmol) in CH₂Cl₂ (15 mL) using procedure B. The crude residue was purified by column chromatography (EtOAc/MeOH, 4:1) to afford compound 49 (71 mg, 63%). ¹H NMR (CDCl₃, 400 MHz): δ 0.86 (t, J = 7.2 Hz, 3H), 1.32–1.54 (m, 2H), 1.60–1.78 (m, 1H), 1.95 (d, J = 10.4 Hz, 1H), 2.20–2.80 (m, 18H), 2.90–3.10 (m, 1H), 3.63 (s, 2H), 4.87 (bs, 2H), 6.51 (s, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.19 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.55 (s, 1H), 8.45 (s, 1H). The free base was converted to its hydrochloride salt. M.p. 230–235 °C. Anal. calculated for C₂₇H₄₉Cl₅N₆O₂S: C, H, N.

Evaluation of potency in binding to and activating dopamine D2 and D3 receptors

Binding potency was monitored by inhibition of [³H]spiroperidol (15.0 Ci/mmole, Perkin-Elmer) binding to DA rD2 and rD3 receptors expressed in HEK-293 cells, in a buffer containing 0.9% NaCl. Functional activity of test compounds in activating dopamine hD2 and hD3 receptors expressed in CHO cells was measured by stimulation of [³⁵S]GTPγS (1250 Ci/mmole, Perkin-Elmer) binding in comparison to stimulation by the full agonist DA as described by us previously.²⁹

Evaluation of antioxidant activity

DPPH Radical Scavenging Assay

To a 96 well plate, 100 µL of methanolic drug solutions ranging from 20 µM to 250 µM were added. Next, 100 µL of 200 µM methanolic solution of DPPH (1,1-diphenyl-2-picryl-hydrazyl) was added and the plate was shaken vigorously at 30 °C for 20 min. Control wells received 100 µL of methanol and 100 µL of 200 µM methanolic DPPH solution. Wells containing only 200 µL of methanol served as a background correction. The change in absorbance of all samples and standard (ascorbic acid) were 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)] × 100.⁴⁰

Animal Experiment

Drugs and chemicals

The following commercially available drugs were used in the experiment: reserpine hydrochloride (Alfa Aesar) and ropinirole (Sigma Aldrich). The trifluoroacetate salt of (−)-19 and (−)-40 and the hydrochloride salt of (−)-46 and ropinirole were dissolved in water. 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 subcutaneously (sc), in a volume of 0.1–0.3 mL to each rat.

Animals

In rodent studies, animals were male and female, Sprague-Dawley rats from Harlan (Indianapolis, IN) weighing 220–225 g, unless otherwise specified. Animals were maintained in sawdust-lined cages, in a temperature and humidity controlled environment at 22 ± 1°C and 60 ± 5% humidity, respectively. A 12 h light/dark cycle was maintained, with lights on from 6:00 AM to 6:00 PM. 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 induces catalepsy in rodents, primarily by blocking the vesicular monoamine trasporter (VMAT), which assists in the internalization of monoamines into vesicles. VMAT inhibition results in metabolism of unprotected monoamines in the cytosol, ultimately causing depletion of monoamines in the synapses of the peripheral sympathetic nerve terminals.^{41, 43} The ability of compounds (−)-19, (−)-40 and (−)-46 to reverse reserpine-induced, hypolocomotion was investigated.⁴⁴ Ropinirole was used as a standard, reference compound in this study. Reserpine (5.0 mg/kg, sc) was administered 18 h before the injection of drug or vehicle (sc). The rats were placed individually in the chambers for 1 h for acclimatization, before 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 activity (HACTV). The effect of individual doses of drugs on locomotor activity was compared with respect to saline treated controls (mean ± S.E.M.). 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 the control group was observed at p<0.05.

Supplementary Material

001

NIHMS381896-supplement-001.pdf^{(83.8KB, pdf)}

Effects of different drugs (administered sc) upon reserpine (5.0 mg/kg, sc, 18 h pretreatment)-induced hypolocomotion in rats. Each point represents the mean ± S.E.M for three to six rats. Horizontal activity was measured as described under materials and methods section. Representation of horizontal locomotor activity at discrete 30 min intervals after the administration of (−)-19 (5 µmol/kg), (−)-46 (10 µmol/kg), (−)-40 (10 µmol/kg) and ropinirole (5 µmol/kg) compared to control rats, 18 h post reserpine treatment. Differences among treatments were significant by one-way ANOVA analysis (F (4.95) = 6.69 (P< 0.0001)). ** P< 0.01 ((−)-**19)** or * P< 0.05 (ropinirole) compared to reserpine control (Dunnett’s analysis post one-way ANOVA).

Acknowledgements

This work is supported by National Institute of Neurological Disorders and Stroke/ National Institute of Health (NS047198, AKD). We are grateful to Dr. K. Neve, Oregon Health and Science University, Portland, OR, for D2L and D3 expressing HEK cells. We are also grateful to Dr. J. Shine, Garvan Institute for Medical Research, Sydney, Australia, for D2L expressing CHO cells. We are grateful to Dr. Sanjib Gogoi and Mr. Gyan Modi for help with synthesis and in vivo animal experiments.

Abbreviations

GTPγS: guanosine 5’-[g-thio]triphosphate
5-OH-DPAT: 5-hydroxy-2-(dipropylamino)tetralin
CHO: chinese hamster ovary
HEK: human embryonic kidney
L-DOPA: (S)-(3,4-dihydroxyphenyl) alanine
DPPH: 1,1-diphenyl-2-picryl-hydrazyl
PD: Parkinson’s disease
DA: Dopamine
SC: Sub Cutaneous

Footnotes

Supporting Information Available: Elemental analysis data for all final targets is available. This material is available free of charge via the internet at http://pubs.acs.org.

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