Bivalent dopamine agonists with co-operative binding and functional activities at dopamine D2 receptors, modulate aggregation and toxicity of alpha synuclein protein

Abstract

To follow up on our previous report on bivalent compounds exhibiting potent co-operative binding at dopamine D2 receptors, we modified the structure of the linker in our earlier bivalent molecules (S)-6-((9-(((R)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)nonyl)-(propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol (Ia) and (S)-6-((10-(((R)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)decyl)(propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol (Ib) (Figure 1) connecting the two pharmaophoric moieties to observe any tolerance in maintaining similar affinities and potencies. Specifically, we introduced aromatic and piperazine moieties in the linker to explore their effect. Overall, similar activities at D2 receptors as observed in our earlier study was maintained in the new molecules e.g. (6S,6’S)-6,6’-((1,4-phenylenebis(ethane-2,1-diyl))bis(propylazanediyl))bis(5,6,7,8-tetrahydronaphthalen-1-ol) (D-382) (Ki , D2 = 3.88 nM). The aromatic moiety in D-382 was next functionalized by introducing hydroxyl groups to mimic polyhydroxy natural products which are known to interact with amyloidogenic proteins. Such a transformation resulted in development of compounds like 2,5-bis(2-(((S)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl)benzene-1,4-diol (D-666) (Ki , D2 = 7.62 nM) which retained similar affinity and potency at D2 receptors. Such dihydroxyl compounds turned out to be potent inhibitors against aggregation and toxicity of recombinant alpha synuclein protein The work reported here is in line with our overall goal to develop multifunctional dopamine agonist for symptomatic and disease modifying treatment of Parkinson’s disease.

Graphical Abstract

Introduction

Parkinson’s disease (PD) and PD dementia (PDD) are progressive neurodegenerative disorders characterized by degeneration of the nigrostriatal dopaminergic neurons producing the cardinal motor symptoms, cognition decline, and dementia for these disorders(1–3). Although genetic mutations have been implicated in a small minority of patients, PD is mostly sporadic in nature. Multiple pathogenic factors have been implicated in the etiology of the disease process. The pathological hallmark of PD is the presence of Lewy bodies and Lewy neurites consisting primarily of α-synuclein (α-syn) along with Parkin and ubiquitin.(4) Pathological hallmarks of PD are the presence of α-syn aggregates called Lewy bodies (LBs) or Lewy neurites (LN).(5,6) α-Syn is composed of 140 amino acid and it can form a α- helical structure upon lipid binding, and forms thioflavin T (ThT) positive β-sheets upon prolonged period of agitation.(7) Due to its role in the pathogenesis of PD, α-Syn is considered as a rationale target for drug development.(8,9)

Dopamine receptors which belong to the G protein-coupled receptor (GPCR) family of proteins, are classified into two main subfamilies based on their pharmacological properties. They are known as D1-like and D2-like class of receptors. D1-like receptors include D1 and D5 subtypes whereas D2-like is divided into D2, D3 and D4 subtype receptors (10–16). The main difference between the two subtypes lies in their mode of interaction upon receptor activation. Thus, activation of D1-like receptors leads to activation of adenylate cyclase whereas inhibition of adenylate cyclase takes place upon D2-like receptor activation. In the CNS, D2-like receptors are located both pre- and post-synaptically and have high affinity for dopamine whereas D1-like receptors are located post-synaptically with lower affinity for dopamine. (17)

DA receptors systems have been targeted extensively to develop drugs for several neuro-disorders like movement disorders, schizophrenia, mania, depression substance abuse etc.(18) DA receptor agonists have been used more extensively in the treatment of Parkinson’s disease (PD) than any other type of pharmacotherapy.(19,20) The gold standard pharmacological treatment of PD, L-dopa, when given with a peripheral dopamine (DA) decarboxylase inhibitor, dramatically improves the symptoms of the disease by increasing DA in DA depleted neurons.(21,22) However, long term use of L-dopa gives rise to motor fluctuations with dyskinesias characterized by decreased duration of response to a given L-dopa dose (23) and development of “on” and “off” episodes along with, although somewhat controversial, increasing toxicity to DA neurons, hence, accelerating the DA neurodegeneration process. (24) DA agonists have a long history of therapeutic use in PD either as a monotherapy or as an adjunct to L-dopa, particularly, in the early stages of PD and in younger patients to mitigate L-dopa induced side effects.(25–27)

Previously, we have shown a profound influence of linker length in significant co-operative gain for binding and functional activities of bivalent compounds for the D2 receptors. Compounds Ia and Ib (Figure 1) were the most potent compounds in this series. The increase in potency was substantial compared to monovalent agonist, 5-OH-DPAT.(28) We hypothesized that such gain was possibly achieved by participation in interaction of the agonist binding moieties at the two orthosteric binding sites in the D2 homodimer receptor. In our current study, we wanted to observe whether modification of the central linker by inserting either phenyl or piperazine ring would have any impact in activities for the D2/D3 receptors. In addition, we planned to introduce additional functional groups into these molecules with a goal to inhibit aggregation and toxicity of alpha synuclein protein to provide potential disease modifying therapeutic effect in PD.(29,30)

Figure 1:

Molecular structures of dopamine agonists and bivalent molecules

Chemistry

Scheme 1 describes the syntheses of final compounds 4 (D-380), 6 (D-381) and 9 (D-382). At first, piperazine was chloroacetylated at the nitrogen centers in the presence of triethylamine and chloroacetyl chloride to afford 1, which on substitution reaction with (s)-aminotetralin (2) under basic condition afforded bivalent amide 3. Compound 3 was demethylated using boron tribromide to produce 4 (D-380). Compound 6 (D-381) was obtained from 3 by the two-step reactions which included first reduction of the amide bond using borane in THF to yield 5 followed by demethylation using boron tribromide to produce the final compound. Similarly, 9 (D-382) was synthesized by amidation of 1,4-phenylenediacetic acid with (s)-tetralin as the first step, followed by reduction of the intermediate amide 7 and demethylation of both the methoxy groups of tetralin moiety to yield 9. Scheme 2 depicts the synthesis of 13 (D-634). Aminotetralin (2) was reacted with 1,4-phenylene diacetic acid via acid-amine coupling reaction using EDC/HOBT as the zero-order crosslinker to afford monoamide 10, which on further amidation with S-(−)-pramipexole produced diamide 11. Compound 11 was then reduced using borane in THF under refluxing conditions to yield 12, which on demethylation in the presence of 48% aq HBr under reflux afforded 13 (D-634). Schemes 3 and 4 show the synthesis of bivalent compounds 22 (D-666) and 25 (D-673), respectively, where the (s)-5-hydroxy aminotetralines are connected via diethyldihydroxyphenylene linker. As shown in Scheme 3, compound 14 was chloromethylated at 2,5-positions using paraformaldehyde, conc. HCl and acetic acid under sonication at rt to afford 15. Substitution reaction of 15 using sodium cyanide in DMSO produced 16 which on hydrolysis in presence of conc. HCl and acetic acid at 100°C yielded dicarboxylic acid 17. Esterification of 17 using thionyl chloride in methanol produced 18, which was then reduced to di-alcohol 19. Compound 19 was then oxidized to aldehyde 20 using Dess-Martin periodinane in DCM. Aminotetralin 8 was next reductively alkylated with the aldehyde 23 in the presence of NaBH(OAc)₃ to afford the compound 21 which on demethylation produced 22 (D-666) as HBr salt. In Scheme 4, the bis aldehyde (23) was first reductively aminated with S-(−)-pramipexole in the presence of NaBH(OAc)₃, and further demethylated to yield 25 (D-673).

Scheme 1. Reagents and conditions.

a. triethylamine, Chloroacetyl chloride, dichloromethane, 0 °C to RT, 3 h; b. K₂CO₃, acetonitrile, reflux, 3 h; c. BBr₃, dichloormethane, −40 °C to RT, 12 h; d. BH₃ in THF, THF, 80 °C, 5 h; e. 1,4-phenylenediacetic acid, triethyl amine, EDCI, HOBT, dichloromethane, RT, 12 h; f. 48 % aq. HBr, 120 °C, 3 h.

Scheme 2. Reaction and Conditions:

(a) 1,4-phenylene diacetic acid, HOBT, EDC, DMF, rt, 4 h, 54%; (b) S-(−)-pramipexole, HOBT, EDC, DMF, rt, overnight, 53%; (c) BH_3.THF, THF, reflux, 4 h, 21%; (d) 48% aq HBr, reflux 6h.

Scheme 3. Reagents:

(a) paraformaldehyde, conc. HCl, AcOH, sonication, rt, 2 h, 65%; (b) NaCN, DMSO, 50 °C-85 °C, 1 h, 70%; (c) conc. HCl, AcOH, 100 °C, 3 h, 60%; (d) SOCl₂, MeOH, 0 °C-rt, 24 h, 55%; (e) LiAlH₄, THF, 0 °C-rt, overnight, 55%; (f) Dess-Martin periodinane, DCM, 4 h, 60% ; (g) (S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (8), NaBH(OAc)₃, DCM, rt, 40 h, 25%; (h) 48% aqueous HBr, reflux, 6 h, 88%.

Scheme 4. Reagents and conditions:

a) NaBH(OAC)₃, CH₂Cl₂, rt, 48 h; b) 48% aq. HBr, reflux, 5 h.

Scheme 5 depicts the synthesis of 38 (D-679), where two different aminotetralins are connected via diethylphenylene linker. L-aspartic acid was cyclized to the anhydride 26. Friedel–Crafts acylation of veratrole with 26 using anhydrous AlCl₃ produced the ketone 27, which then underwent the carbonyl group reduction in the presence of triethylsilane to yield 28. The reaction of 28 with PCl₅ in dichloromethane followed by SnCl₄ produced the tetralone 29. Next, the selective reduction of the keto group of 29 in the presence of triethylsilane in BF₃.Et₂O produced 30. Amide 30 was then hydrolyzed to amine 31 which underwent N-protection to yield 32. Additional N-protection of 32 in the presence of 1-bromopropane and K₂CO₃ in acetonitrile produced 33 which was then selectively deprotected to 34. (S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine then underwent selective amidation with dicarboxylic acid 1,4-phenylenediacetic acid to afford monoamide 35, which on further amidation with amine 34 afforded 36. Reduction of bis-amide 36 using borane in THF produced 37 which was then demethylated to afford 38 (D-679) in presence of 48% aq HBr under refluxing condition.

Scheme 5. Reagents and conditions:

a) Trifluoroacetic anhydride, CF₃COOH, −60 °C to rt to reflux, 2 h; b) AlCl₃, CH₂Cl₂, rt, 4 days; c) Et₃SiH, CF₃COOH, reflux, 2 h; d) i) PCl₅, CH₂Cl₂, 0 °C, 1 h; ii) SnCl₄, 0 °C to rt, 4 h; e) Et₃SiH, BF_3.Et₂O, rt, 48 h; f) K₂CO₃, MeOH/H₂O, reflux, 4 h; g) 2-Nitrobenzenesulfonyl chloride, Et₃N, THF, −10 °C to rt, 1.5 h; h) 1-Bromopropane, K₂CO₃, CH₃CN, 40 °C, 48 h; i) Thioglycolic acid, K₂CO₃, DMF, 0 °C to rt to 50 °C, 16 h; j) EDC, HOBt, Et₃N, DMF, rt, 4 h; k) EDC, HOBt, Et₃N, DMF, rt, overnight; l) BH_3.THF, THF, rt to 55 °C, 4 h; m) 48% aq. HBr, reflux, 5 h.

Result and Discussion:

Our earlier study in probing the pharmacological properties of bivalent dopamine agonists resulted in development of molecules with significant cooperative gain in affinity and potency for dopamine D2 receptors compared to the corresponding monovalent ligand. Such gain in potency was achieved at an optimum methylene linker length (7–10 methylene unit).(28) The increase in activity is most likely due to its interaction at the two orthosteric binding sites. However, it is not quite clear whether this is due to interaction at the two orthosteric sites in a dimeric receptor structure or at any other two dopamine receptors. We wanted to build on this finding to further modify the structure of the linker by inserting more rigid moieties with a goal to introduce additional functionalities. Thus, several molecules were designed and synthesized. Furthermore, we wanted to introduce functional groups on the linker aromatic moiety to modulate protein aggregation. This is in line with our overall goal for developing potent dopamine agonist with neuroprotective property.

We first wanted to insert a piperazine ring into the linker structure to observe whether it would interfere with receptor interaction. The two compounds designed from this modification are D-380 and D-381. As expected, D-381 turned out to exhibit much higher affinity for the D2 receptor at a low nanomolar level (Ki , D2 = 12 nM, D3 = 3.56 nM). On the other hand, D-380 containing an amide moiety produced little to no activity. The decreased basicity of the piperazine N-atoms in D-380 seems to have impacted its interaction with the DA receptors. The basicity of the N-atoms in the tetralin pharmacophore might also have been influenced by the presence of a beta keto (amide) group in D-380 which, additionally, might have contributed to the loss of activity. Replacement of the piperazine ring by a phenyl moiety as shown in compound D-382 resulted in further enhancement of affinity for the dopamine D2 receptors. Thus, a greater than threefold increase in affinity for D2 receptors was observed in D-382 (Ki, D2 = 3.88 nM, D3 = 3.86 nM) compared to D-381. The affinity of D-382, which approximately represents 7–8 methylene unit linker length, for the D2 receptors compares well with the bivalent compounds containing only 8–9 methylene unit.(28) We next wanted to evaluate whether replacement of one of the phenyl groups in D-382 by a bioisosteric 2-amino thiazole ring as shown in the compound 13 (D-634) could maintain the affinity for the receptors. The results as shown in the Table 1 (Ki, D2 = 4.91 nM, D3 = 2.15 nM) indicates that such replacement resulted in maintenance of a similar activity as D-382.

Table 1.

Inhibition constants determined by competition experiments assessing [³H]spiroperidol binding to cloned rat D_2L and D₃ receptors expressed in HEK-293 cells^a

Compound	Ki (nM)
	D2L, [3H]spiroperidol	D3, [3H]spiroperidol	D2/D3
(−)-5-OH-DPAT	153 ± 32	2.07 ± 0.38	73.9
Ia b	2.5 ± 0.5	0.9 ± 0.3	2.7
Ib b	2.0 ± 0.4	1.8 ± 0.6	1.1
D-380	No Inhibition at 100 μM	114.76 ± 26.24
D-381	12.01 ± 1.42	3.56 ± 0.72	3.37
D-382	3.88 ± 0.67	3.86 ± 0.83	1
D-634	4.91 ± 0.49	2.15 ± 0.51	2.28
D-666	7.62 ± 2.5	5.22 ± 1.22	1.45
D-673	54.7 ± 3.3	6.87 ± 0.11	7.96
D-679	8.34 ± 0.59	1.25 ± 0.24	6.67

^a.Results are means ± SEMs for 3–7 experiments each performed in triplicate.

^b.Data taken from ref. 28

In our next step, we wanted to use the phenyl moiety in the linker as a functional handle for introduction of hydroxyl groups. It is known that phenolic compounds including natural products such as green tea inhibit fibrillation and destabilized preformed α-Syn fibrils.(31,32) Our goal is to deliver multifunctional polyhydroxy dopamine agonist effectively into the brain. Based on this approach, compounds D-666 and D-673 were designed where 1,4 di-hydroxy functional groups were introduced into the phenyl ring embedded in the linker. The central 1,4-dihydroxy phenyl group is flanked by the two 5-hydroxy-tetralin moieties in D-666 and the two bioisosteric 2-amino thiazole rings in D-673. The binding results indicate that the introduction of two hydroxyl groups is tolerated well for their interaction with the DA receptors although D-673 exhibits a lower affinity for D2 receptors. Thus, the affinity for the D2 receptor was exhibited at a low nanomolar level by D-666 (Ki , D2 = 7.62 nM, D3 = 5.22 nM) whereas D-673 exhibited a somewhat lower affinity (Ki , D2 = 54.7 nM, D3 = 6.87 nM). The presence of two thiazole-2-amine moieties in D-673 most likely have contributed towards reduction of some affinity.

In our next design, one of the 5-hydroxy tetralin moieties in D-382 was replaced by a 3, 4-dihydroxy tetralin moiety which bears a closer resemblance to the structure of dopamine molecule, to produce compound D-679. Such a replacement resulted in a high-affinity ligand for both D2 and D3 receptors as shown in Table 1 (Ki , D2 = 8.34 nM, D3 = 1.25 nM). All the compounds reported here with the exception of D-380, exhibited higher affinity at the D2 receptors compared to the reference standard 5-OH-DPAT molecule (Ki, D2 = 153 nM, D3 = 2.07 nM). The most potent compound D-634 exhibited 33-fold higher activity compared to 5-OH-DPAT in this regard.

The functional data of the compounds correspond closely to their binding data. All the compounds turned out to be potent agonists for dopamine D2 and D3 receptors. Compounds D-666 and D-679 showed the highest potency for the D2 receptors (EC50 (GTPγ S); D2 = 7.69 and 5.29 nM, respectively for D-666 and D-679). The presence of the thiazole-2-amine group in D-634 and D-673 had a moderate influence in lowering potency for D2 receptors. However, like the binding data, all the new compounds exhibited higher potency compared to the reference 5-OH-DPAT. This is particularly evident for D-666 and D-679 which displayed a 5–8 fold higher affinity than 5-OHDPAT. With regards to pramipexole, the pramipexole-derived compound D-673 had a more than 7-fold greater potency compared to the functional potency of pramipexole at D2 receptors. These data further indicates co-operative binding of these novel compounds at the orthosteric binding sites.

Effect of compounds D-666 and D-673 in inhibiting aggregation of αSyn

In our previous studies, we established that in vitro generated α-syn pre-formed fibrils induce seeding of α-syn monomers to produce aggregates in a time-dependent manner under static conditions in vitro. In contrast, an α-syn solution in the absence of seeding did not show any appreciable aggregation on day 30 indicating the role of pre-formed fibrils (PFF) in recruiting monomeric α-syn.(29) This allowed us to screen test compounds against aggregation of α-syn under similar physiological conditions as opposed to the more aggressive shaking condition that has been applied commonly.(31) We have applied this methodology successfully in identifying compounds with anti-aggregation property both in vitro and in vivo.(29,30) The polyhydroxy compounds D-666 and D-673 were evaluated under these assay conditions to determine their ability to alter aggregation of α-syn and also in modulating toxicity of the aggregates. As we have observed before, α-syn seeded with PFF showed robust aggregation as indicated by enhanced ThT signal (Figure 2). However, presence of both compounds inhibited the aggregation completely (Figure 2). This result is similar to what was found earlier with the catechol derived compounds.(30) In the cell viability assay, the aggregates generated from seeding produced significant toxicity at Day 30 (Figure 3). The compounds D-666 and D-673 were able to significantly reduce this toxicity compared to the untreated group at Day 30 (Figure 3). It seems D-673 by itself shows toxicity at 0D. It might be due to the presence of two thiazole-2-amine moieties in D-673 as we do not see such toxicity in D-666.

Figure 2:

Effect of D-666 and D-673 on the aggregation of α-syn induced by seeding with 0.5% PFFs. 1.25 mg/mL α-syn was incubated with 0.5% PFFs for a period of 30D without shaking in the presence of drugs at a concentration of 172.9 μM. Fibrillation was measured by ThT assay at 30D. Values are represented in terms of % 0D Synuclein. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed, ****p≤0.0001compared to Syn +0.5%−0D; ####p≤0.0001 compared to Syn +0.5%−30D.

Figure 3:

Viability of PC12 cells was measured by MTT assay after 24 h treatment with α-syn seeded samples collected at 30D. Values are represented in terms of % control. Data values shown are means ±SD of three independent experiments. One-way ANOVA analysis followed by Tukey’s multiple comparison post hoc test was performed, ****p≤0.0001compared to Syn +0.5%−0D; ####p≤0.0001 compared to Syn +0.5%−30D.

In brief, our current study describes an expanded SAR study on bivalent compounds we reported earlier. The present study focused on modulation of the central methylene linker structure by inserting aromatic moiety to determine the tolerance against receptor affinity and potency. Bioisosteric replacement was also carried out. The affinity and potency of newly designed compounds were mostly preserved and were similar to our original lead bivalent molecues. Introduction of the phenolic hydroxyl groups into the central phenyl ring in the linker rendered the molecules D-666 and D-673 multifunctional by mimicking the inhibitory effect of natural polyhydroxy phenolic compounds against amyloidogenic protein like alpha synuclein.

Experimental

All reagents and solvents were purchased from commercial suppliers like Sigma Aldrich, Fisher Scientific etc. and used as received unless otherwise stated. Dry solvent was obtained according to the standard procedure. All reactions were performed under inert atmosphere (N₂) unless otherwise indicated. Analytical silica gel 60 F₂₅₄-coated TLC plates were obtained from EMD Chemicals, Inc. and were visualized with UV light or by treatment with phosphomolybdic acid (PMA), Dragendorff’s reagent, or ninhydrin. Flash column chromatographic purifications were performed using Whatman Purasil 60A silica gel 230−400 mesh. ¹H NMR spectra were acquired on a Varian 400 and 600 MHz FT NMR spectrometer using tetramethylsilane (TMS) as the internal standard. The NMR solvent used was CDCl₃ or CD₃OD as indicated. Optical rotations were recorded on PerkinElmer 241 polarimeter. Melting points were recorded using a MEL-TEMP II (Laboratory Devices Inc., Placerville, CA) capillary melting point apparatus and were uncorrected. Elemental analyses were performed by Atlantic Microlab, Inc.

Synthesis of 1,1’-(piperazine-1,4-diyl)bis(2-chloroethanone) (1)

Chloroacetyl chloride (23.2 ml, 290.23 mmol) was added dropwise into a solution of piperazine (5.0 g, 58.05 mmol) and Et₃N (40.0 mL) in anhydrous dichloromethane (300 mL) at −40 °C under N₂ atmosphere. The reaction mixture was stirred at room temperature for 3 h. The reaction was diluted with CH₂Cl₂, washed with water, brine, and the organic layer was dried over Na₂SO₄, evaporated, and purified by flash chromatography. (EtOAc: Hexne=1: 1) to yield compound 1. (9.5 g, 68 %).

¹H NMR (400 MHz, CDCl₃) δ 3.57–3.73 (m, 8H, -N(CH₂CH₂)₂), 4.11 (s, 4H, -NCOCH₂Cl).

Procedure A: Synthesis of (s)-1,1’-(piperazine-1,4-diyl)bis(2-(((s)-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethanone) (3)

To a stirred suspension of 2 (1.38 g, 6.27 mmol) and potassium carbonate (1.45 g, 10.5 mmol) in acetonitrile (25 mL), 1 (0.50 g, 2.09 mmol) was added and the reaction mixture was stirred at 40 °C for 48 h. The reaction mixture was cooled to room temperature, filtered off and the filtrate was evaporated under reduced pressure. The crude reaction mixture was purified by flash chromatography using solvent system ethylacetate: methanol (95:5) to yield compound 3 (1.10 g, 87 %).

¹H NMR (400 MHz, CDCl₃) δ 0.86–0.94 (m, 6H), 1.45–1.52 (m, 4H), 1.60–1.63 (m, 2H), 2.00–2.05 (bs, 2H), 2.52–2.55 (m, 6H), 2.79–2.83 (m, 4H), 2.97–2.99 (m, 4H), 3.43–3.70 (m, 12H), 3.81 (s, 6H), 6.65–6.71 (m, 4H, Ar-H), 7.07–7.11 (t, 2H, J = 8.0 Hz, Ar-H).

Procedure B: Synthesis of (s)-1,1’-(piperazine-1,4-diyl)bis(2-(((s)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethanone) (4) D-380

Boron tribromide (1M solution in dichloromethane) (2.6 mL, 2.6 mmol) was added into a solution of 3 (0.31 g, 0.51 mmol) in anhydrous dichloromethane (15 mL) at −40 °C under nitrogen atmosphere. The reaction mixture was stirred at −40 °C for 2 h and then was continued overnight at room temperature. The reaction was quenched by the addition of saturated NaHCO₃ solution and the aqueous layer was extracted with dichloromethane. The combined organic layer was dried over Na₂SO₄, evaporated under vacuo, and the crude product was purified by flash chromatography using solvent system dichloromethane: methanol = 9: 1 to afford compound 4 (0.07 g, 23.3 %). Optical rotation of 4, [α]_D = −40.2 ° (c=1 in CH₂Cl₂) at Room Temperature (RT). m.p. 240–242 °C. ¹H NMR (400 MHz, CDCl₃) δ 0.86–0.93 (m, 6H), 1.45–1.51 (m, 4H), 1.65–1.68 (m, 2H), 2.06 (bs, 2H), 2.51–2.55 (m, 6H), 2.78–2.97 (m, 8H), 3.44–3.65 (m, 12H), 6.60–6.64 (m, 4H, Ar-H), 6.94–6.97 (m, 2H, Ar-H). ¹³ C NMR (150 MHz, CD3OD): δ 163.96, 154.76, 133.38, 126.64, 121.58, 119.82, 112.02, 62.91, 56.26, 50.44, 50.17, 43.96, 43.67, 41.81, 41.55, 28.38, 23.98, 17.94, 9.80, 9.73. The product was converted into corresponding dihydrochloride salt. Anal. Calcd for (C₃₄H_51.2N₄O_5.6. 2HCl) C, H, N.

Procedure C: Synthesis of (2s,2’s)-N,N’-(2,2’-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine) (5)

Into the solution of 3 (0.79 g, 1.3 mmol) in dry THF was added 13.0 ml (13.0 mmol) solution of borane-THF complex (1 M) with stirring under nitrogen atmosphere. The reaction mixture was refluxed for 5 hours, cooled to room temperature and quenched with methanol. The solvent was evaporated. The white solid complex was suspended in 6N HCl in methanol, stirred for 2 h at 50 °C. Methanol was evaporated under vacuo. Reaction mixture was made alkaline using saturated Na₂CO₃/NaHCO₃ Solution. The aqueous layer was extracted with ethyl acetate (3× 100 ml). The organic layer was dried over Na₂SO₄, concentrated under vacuo and purified by flash chromatography using solvent system dichloromethane: methanol (90:10) to afford compound 5, (0.36 g, 48 %).

¹H NMR (400 MHz, CDCl₃) δ 0.89–0.92 (t, 6H, J = 7.6 Hz), 1.54–1.63 (m, 6H), 2.10 (bs, 2H), 2.48–3.03 (m, 30H), 3.81 (s, 6H, (−OCH₃)₂), 6.65–6.72 (dd, 4H, J₁ = 21.0 Hz, J₂ = 8.0 Hz, Ar-H), 7.08–7.12 (t, 2H, J = 7.6 Hz, Ar-H).

Synthesis of (6s,6’s)-6,6’-(2,2’-(piperazine-1,4-diyl)bis(ethane-2,1-diyl))bis(propylazanediyl)bis(5,6,7,8-tetrahydronaphthalen-1-ol) (6) D-381

Compound 5 (0.35 g, 0.6 mmol) was reacted with 1M BBr₃ in CH₂Cl₂ (3.0 mL, 3.0 mmol) in CH₂Cl₂ (15 mL) by following the Procedure B. The reaction mixture was purified by flash chromatography using solvent system dichloromethane: methanol = 9: 1 to afford compound 6 (0.09 g, 27%). Optical rotation of 6, [α]_D = −43.6 ° (c= 1 in methanol) at RT. The product was converted into corresponding tetrahydrochloride salt.m.p. 248–251 °C. ¹H NMR (400 MHz, CDCl₃) δ 0.86–0.89 (t, 6H, J = 6.8 Hz), 1.40–1.48 (m, 4H), 1.97–1.99 (m, 2H), 2.24–2.35 (m, 2H), 2.46–3.02 (m, 30H), 6.51–6.56 (m, 4H), 6.92–6.96 (t, 2H, J = 7.6 Hz). 13C NMR (150 MHz, CD3OD): 154.79, 133.31, 126.65, 121.60, 119.86, 112.06, 61.05, 53.15, 50.86, 50.54, 29.10, 23.42, 23.37, 18.21, 9.89. Anal. Calcd for (C₃₅H₅₆N₄O₃. 4HCl) C, H, N.

Procedure D: Synthesis of 2,2’-(1,4-phenylene)bis(N-((s)-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)-N-propylacetamide) (7)

Into the suspension of 1,4-Phenylenediacetic acid (0.24 g, 1.22 mmol) in 15 mL of anhydrous CH₂Cl₂ was added HOBT (0.5 g, 3.64 mmol), EDCI (0.69 g, 3.64 mmol), and triethyl amine (1 mL) after which the reaction mixture was stirred for 1 h at rt. S-(−)- 5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (2) (0.8 g, 3.65 mmol) in DMF (10 mL) was then added slowly and reaction continued for another 4 h at rt. After the reaction, the reaction mixture was concentrated and extracted with ethyl acetate. The reaction mixture was purified by flash chromatography using solvent system ethyl acetate: hexane = 1: 1 to yield compound 7 (0.50 g, 68 %).

¹H NMR (400 MHz, CDCl₃) δ 0.88–0.92 (m, 6H), 1.64–1.78 (m, 6H), 1.89–1.96 (m, 2H), 2.30–2.36 (m, 1H), 2.59–2.68 (m, 2H), 2.83–3.04 (m, 5H), 3.12–3.26 (m, 4H), 3.71–3.80 (m, 10H), 4.02–4.07 (m, 1H), 4.55–4.65 (m, 1H), 6.63–6.68 (m, 4H, Ar-H), 7.06–7.12 (m, 2, Ar-H), 7.17–7.26 (m, 4H, Ar-H).

Synthesis of (2s,2’s)-N,N’-(2,2’-(1,4-phenylene)bis(ethane-2,1-diyl))bis(5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine) (8)

Into the solution of 7 (0.52 g, 0.87 mmol) in dry THF was added 13.1 ml (13.0 mmol) solution of borane-THF complex (1 M) with stirring under nitrogen atmosphere following the Procedure C. The crude reaction mixture obtained after hydrolysis of the borane complex was purified by flash chromatography using solvent system dichloromethane: methanol (100:3) to yield pure compound 8 (0.45 g, 91 %).

¹H NMR (400 MHz, CDCl₃) δ 0.86–0.88 (m, 6H), 1.49 (m, 5H), 1.65–1.68 (m, 1H), 1.86–2.02 (m, 3H), 2.43–2.96 (m, 20H), 3.71–3.74 (m, 6H), 4.45–4.60 (m, 1H), 6.58–6.67 (m, 4H, Ar-H), 7.00–7.04 (m, 2H, Ar-H), 7.11–7.14 (m, 2H, Ar-H), 7.22–7.30 (m, 2H, Ar-H).

Procedure E. Synthesis of (6s,6’s)-6,6’-(2,2’-(1,4-phenylene)bis(ethane-2,1-diyl))bis(propylazanediyl)bis(5,6,7,8-tetrahydronaphthalen-1-ol) (9) D-382

A mixture of compound 8 (0.45 g, 0.79 mmol) and 10 ml 48% aqueous HBr was refluxed under nitrogen atmosphere for 3h. The reaction mixture was then evaporated to dryness. The resulted solid was then crystallized from ethanol to get white solid of 9 as HBr salt. (0.10 g, 18 %). [α]_D = −46.2 ° (c=0.5 in methanol) at RT. m.p. 230–232 °C.¹H NMR (400 MHz, CD₃OD) δ 1.01–1.15 (m, 6H), 1.85–2.05 (brm, 6H), 2.40–2.45 (m, 2H), 2.6–2.75 (m, 2H), 3.11–3.68 (m, 16H), 3.83 (brm, 4H), 6.64–6.71 (m, 4H, -Ar-H), 7.00–7.04 (t, 2H, J = 7.6 Hz, -Ar-H), 7.42 (brs, 4H, -Ar-H). ¹³C NMR (150 MHz, CD3OD): 154.73, 135.47, 133.32, 132.45, 130.23, 129.18, 126.64, 121.63, 119.83, 113.75, 113.22, 112.06, 60.57, 52.34, 51.97, 30.52, 29.48, 23.61, 23.33, 22.81, 22.27, 18.54, 14.04, 9.52. Anal. Calcd for the HBr salt (C₃₆H_53.2N₂O_4.6. 2HBr) C, H, N.

(S)-2-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)-2-oxoethyl)phenyl)acetic acid (10).

1,4-Phenylenediacetic acid (1.41g, 7.26 mmol) was reacted with HOBT (0.29 g, 2.17 mmol), EDC (0.42 g, 2.17 mmol), and triethyl amine (0.35 mL, 2.54 mmol) in DMF was subsequently reacted with S-(−)- 5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (2) (0.4 g, 1.81 mmol) by the following the Procedure D. The organic layer was evaporated in vacuo and dried over Na₂SO₄. The crude obtained was purified by column chromatography using 3% MeOH in DCM to provide compound 10 (0.39 g, 54%). ¹H NMR (600 MHz, CDCl₃): δ ppm 7.20–7.24 (m, 2H), 7.18 (d, J = 8.4 Hz, 1H), 7.15 (d, J = 8.4 Hz, 1H), 7.05–7.09 (m, 1H), 6.64 (t, J = 7.8 Hz, 1H), 6.60 (d, J = 7.8 Hz, 1H), 3.98–4.04 (m, 1H), 3.78 (d, J = 3.6 Hz, 3H), 3.73 (d, J = 16.2 Hz, 2H), 3.59 (d, J = 8.4 Hz, 2H), 3.11–3.21 (m, 2H), 2.82–3.00 (m, 2H), 2.57–2.63 (m, 1H), 2.33–2.39 (m, 1H), 1.88–1.97 (m, 1H), 1.60–1.73 (m, 3H), 0.88 (t, J = 7.8 Hz, 3H).

N-((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)-2-(4-(2-(((S)-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)-2-oxoethyl)phenyl)-N-propylacetamide (11).

Compound 10 (0.26 g, 0.67 mmol), HOBT (0.096 g, 0.71 mmol), EDC (0.136 g, 0.71 mmol), and triethyl amine (98 μL, 0.71 mmol) in DMF (20 mL) were reacted with S-(−) pramipexole (0.15 g, 0.71 mmol) in DMF (5 mL) according to the Procedure D. The reaction mixture was stirred at rt for 24 h. After the reaction, saturated NaHCO₃ (20 mL) was added to the reaction mixture and extracted with ethyl acetate (3 × 50 mL). The organic layer was evaporated in vacuo and dried over Na₂SO₄. The crude obtained was purified by column chromatography using 3–5% MeOH in DCM to afford compound 11 (0.21 g, 53%). ¹H NMR (600 MHz, CDCl₃): δ ppm 7.14–7.22 (m, 4H), 7.03–7.08 (m, 1H), 6.60–6.64 (m, 2H), 4.54 (bs, 1H), 3.96–3.99 (m, 1H), 3.73 (s, 3H), 3.66–3.72 (m, 4H), 3.08–3.20 (m, 4H), 2.79–2.98 (m, 4H), 2.56–2.65 (m, 3H), 2.30–2.39 (m, 2H), 1.82–1.94 (m, 2H), 1.53–1.72 (m, 6H), 0.88 (m, 6H).

(S)-N⁶-(4-(2-(((S)-5-methoxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl)phenethyl)- N⁶-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (12).

To a stirring cold solution of compound 11 (0.2 g, 0.34 mmol) in THF (10 mL), BH₃.THF (3.40 mL, 3.40 mol) solution was added according to the Procedure C. The reaction mixture was refluxed for 4 h after which 1 mL of MeOH was added and the solvent evaporated in vacuo. The organic layer was washed with sat. NaHCO₃ solution (50 mL) and dried over Na₂SO₄. The crude obtained was purified by column chromatography using 5–7% of MeOH in DCM to give compound 12 (0.04 g, 21%). ¹H NMR (600 MHz, CDCl₃): δ ppm 7.14–7.22 (m, 4H), 7.03–7.08 (m, 1H), 6.60–6.64 (m, 2H), 4.54 (bs, 1H), 3.96–3.99 (m, 1H), 3.73 (s, 3H), 3.66–3.72 (m, 4H), 3.08–3.20 (m, 4H), 2.79–2.98 (m, 4H), 2.56–2.65 (m, 3H), 2.30–2.39 (m, 2H), 1.82–1.94 (m, 2H), 1.53–1.72 (m, 6H), 0.88 (m, 6H).

(S)-6-((4-(2-(((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)ethyl)-phenethyl)propyl)amino)-5,6,7,8-tetrahydronaphthalen-1-ol. (13) D-634

To a stirring solution of methoxy compound 12 (0.04 g, 0.07 mmol) was added 48% aq. HBr (3 mL) and reaction mixture was refluxed for 6 hours. The reagent was removed in vacuo and crude product which was purified by preparative TLC using 10% MeOH in DCM and product obtained was washed with diethylether to furnish HBr salt of amine (−)- 13. [α]_D = −26.0 ° (c=1.0 in CH₃OH) at RT; Mp 158–161 °C, ¹H NMR (600 MHz, CDCl₃): δ ppm 7.34–7.42 (bs, 4H), 6.95 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 7.2 Hz, 1H), 6.58 (d, J = 7.8 Hz, 1H), 3.86 (bs, 1H), 3.76 (bs, 1H), 3.46 (bs, 3H), 3.33 (m, 6H), 3.00–3.14 (m, 8H), 2.90–2.94 (m, 1H), 2.60–2.72 (m, 3H), 2.31–2.35 (m, 2H), 2.04–2.07 (m, 1H), 1.83–1.88 (m, 5H), 1.05 (m, 6H). ¹³C NMR (150 MHz, CDCl₃): δ 9.91, 18.51, 22.25, 23.12, 24.28, 29.40, 30.49, 51.89, 52.45, 60.30, 60.60, 60.86, 72.12, 112.07, 119.83, 121.62, 126.64, 129.15, 129.19, 133.30, 135.45, 154.68. Anal. HRMS (M+): calcd for C₃₃H₄₆N₄OS, 547.34. Found: C₃₃H₄₆N₄OS, 547.3481.

1,4-Bis(chloromethyl)-2,5-dimethoxybenzene (15):

Paraformaldehyde (3.3 g, 109 mmol) was added to a slurry of compound 14 (5.0 g, 36.2 mmol) in conc. HCl (15 mL) and acetic acid (15 mL) at rt under inert gas atmosphere. The mixture was sonicated for 2h. The mixture was filtered and the solid was washed with hexane (3 X 50 mL) followed by acetone (15 mL). The crude product was purified by column chromatography using 5% ethyl acetate in hexane to afford compound 15 (5.53 g, 23.53 mmol, 65%) as white solid. ¹H NMR (600 MHz, CDCl₃): δ 6.92 (s, 2H), 4.63 (s, 4H), 3.85 (s, 6H).

2,2’-(2,5-Dimethoxy-1,4-phenylene)diacetonitrile (16):

To a mechanically stirred suspension of NaCN (3.45 g, 70.5 mmol) in anhydrous dimethylsulfoxide was added in small portions of 15 (7.11 g, 30.2 mmol). The reaction mixture was stirred at 50 °C for 1h. Temperature was increased to 85 °C and the reaction mixture was allowed to stir at that temperature for additional 5 minutes. The mixture was cooled to rt. The reaction mixture was diluted with water (100 mL) and extracted with ethyl acetate (3 X 150 mL). The combined organic layer was washed with water, brine, dried over Na₂SO₄, and solvent was removed under vacuum. Crude product was purified by column chromatography using 20% ethyl acetate in hexane to give compound 16 (4.57 g, 21.2 mmol, 70%) as white solid. ¹H NMR (600 MHz, CDCl₃): δ 6.92 (s, 2H), 3.85 (s, 6H), 3.70 (s, 4H),.

2,2’-(2,5-Dimethoxy-1,4-phenylene)diacetic acid (17):

To a suspension of compound 16 (0.40 g, 1.85 mmol) in conc. HCl (10 mL) was added acetic acid (1 mL). The mixture was allowed to stir at 100 °C for 3 h. After completion of the reaction as indicated by TLC, the mixture was cooled to rt. The mixture of acids was removed under low pressure. The crude residue was extracted with ethyl acetate (70 mL) and washed with water (3 X 20 mL). The organic layer was washed with brine and dried over Na₂SO₄. The solvent was removed under vacuum. Crude product was purified by column chromatography using 10% methanol in dichloromethane to give compound 17 (0.28 g, 1.11 mmol, 60%) as brown solid. ¹H NMR (600 MHz, d₆-DMSO): δ 12.14 (brs, 2H), 6.82 (s, 2H), 3.66 (s, 6H), 3.65 (s, 4H).

Dimethyl 2,2’-(2,5-dimethoxy-1,4-phenylene)diacetate (18):

To a suspension of the acid 17 (1.0 g, 3.93 mmol) in methanol (25 mL) was slowly added thionyl chloride (4.85 mL, 66.9 mmol) at 0 °C. The mixture was allowed to stir at that temperature for 1 h. Then the reaction temperature was gradually increased to rt and the stirring was continued for 24h. Solvent and excess reagents were removed under low pressure. The crude residue was extracted with ethyl acetate (120 mL) and washed with saturated aqueous NaHCO₃ solution (40 mL). The organic layer was washed with water (3 X 30 mL), brine and dried over Na₂SO₄. The solvent was removed under vacuum. Crude product was purified by column chromatography using 3% methanol in dichloromethane to give compound 18 (0.61 g, 2.16 mmol, 55%) as white solid. ¹H NMR (600 MHz, CDCl₃): δ 6.74 (s, 2H), 3.77 (s, 6H), 3.68 (s, 6H), 3.61 (s, 4H).

2,2’-(2,5-Dimethoxy-1,4-phenylene)diethanol (19):

To a stirring solution of the ester 18 (0.56 g, 2.0 mmol) in THF (20 mL) was added LiAlH₄ (0.30 g, 8.0 mmol) in portions at 0 °C under inert gas atmosphere and the stirring was continued for 1 h. The reaction temperature was increased to rt and the mixture was allowed to stir for overnight. The reaction was quenched with the addition of water (3 mL) followed by 2 N aqueous NaOH (3–5 mL) at 0 °C. The mixture was filtered through celite and the solid residue was washed with THF (2 X 20 mL) followed by hot ethylacetate (40 mL). The combined solution was evaporated under vacuum. Crude product was purified by column chromatography using 3% methanol in dichloromethane to give compound 19 (0.25 g, 1.10 mmol, 55%) as white solid. ¹H NMR (600 MHz, CDCl₃): δ 6.71 (s, 2H), 3.82 (t, J = 6.6 Hz, 4 H), 3.78 (s, 6 H), 2.87 (t, J = 6.6 Hz, 4 H).

2,2’-(2,5-Dimethoxy-1,4-phenylene)diacetaldehyde (20):

To a stirring solution of 19 (50 mg, 0.22 mmol) in DCM (10 mL) was added Dess-Martin periodinane (280 mg, 0.66 mmol) at rt. After stirring the reaction mixture for 4 h the volume of the mixture was reduced to half under low pressure. It was then passed through celite and the solid was washed with DCM (2 X 7 mL). The combined filtrate was evaporated to produce a solid residue which was purified by column chromatography using 25% ethylacetate in hexane to give compound 20 (29 mg, 0.13 mmol, 60%). ¹H NMR (600 MHz, CDCl₃): δ 9.68 (t, J = 1.8 Hz, 2H), 6.71 (s, 2H), 3.77 (s, 6H), 3.65 (d, J = 1.8 Hz, 4H).

(2S,2’S)-N,N’-((2,5-dimethoxy-1,4-phenylene)bis(ethane-2,1-diyl))bis(5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine) (21):

Into a stirring solution of compound 20 (47 mg, 0.21 mmol) in DCM (6 mL), (S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (13) (93 mg, 0.42 mmol) was added at room temperature. The reaction mixture was stirred for 1 h, and then NaBH(OAc)₃ (161 mg, 0.76 mmol) was added into the solution. After stirring for 40 h, saturated solution of NaHCO₃ (6 mL) was added into the reaction mixture and the compound was extracted with DCM (3 × 10 mL). The combined organic layer was washed with water and brine and finally purified by silica gel column chromatography (60% EtOAc in hexane) to yield compound 21 (33 mg, 0.05 mmol, 25%). ¹H NMR (600 MHz, CDCl₃): δ 7.08 (dd, J₁ = 8.4 Hz, J₂ = 7.8 Hz, 2H), 6.71 (d, J = 7.8 Hz, 2H), 6.66 (s, 2H), 6.65 (d, J = 8.4 Hz, 2H), 3.80 (s, 6H), 3.77 (s, 6H), 3.01–2.86 (m, 6H), 2.77–2.72 (m, 8H), 2.56–2.50 (m, 6H), 2.11 (m, 2H), 1.66–1.55 (m, 8H), 0.91 (t, J = 7.2 Hz, 6H)

2,5-Bis(2-(((S)-5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-yl)(propyl)amino)ethyl)benzene-1,4-diol (22) D-666:

A mixture of compound 21 (30 mg, 0.05 mmol) and 48% aqueous HBr (6 mL) was refluxed for 6 h. The reaction mixture was then evaporated to dryness in vacuum. The crude mixture was then washed with diethyl ether to afford compound 22 as a brown solid HBr salt (31 mg, 0.04 mmol, 88%). Optical rotation of 22, [α]_D = −40.2 °(c=1.0 in CH₃OH) at RT, Mp 212–215 °C. ¹H NMR (600 MHz, CD₃OD): δ 6.96 (t, J = 7.8 Hz, 2H), 6.70–6.69 (m, 2H), 6.63–6.60 (m, 4H), 3.79–3.76 (m, 2H), 3.52–3.46 (m, 2H), 3.39–3.32 (m, 4H), 3.24–3.16 (m, 4H), 3.09–2.95 (10H), 2.66–2.56 (m, 2H), 2.40–2.32 (m, 2H), 1.89–1.78 (m, 6H), 1.05 (t, J = 7.2 Hz, 8H). ¹³C NMR (150 MHz, CD3OD): 154.57, 148.02, 133.54, 126.63, 122.69, 121.71, 120.07, 117.18, 112.20, 60.39, 52.69, 52.49, 50.35, 50.27, 29.69, 29.61, 29.56, 26.22, 26.10, 23.63, 23.51, 22.33, 18.38, 18.26, 10.29,10.25. Anal. HRMS (M+): calcd for C₃₆H₄₈N₂O₄ Calculated 572.36, Found 573.3687.

2,5-bis(2-(((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)-ethyl)benzene-1,4-diol (24).

Into a stirring solution of aldehyde 23 (0.09 g, 0.41 mmol) in CH₂Cl₂ (10 mL) was added (S)-N⁶-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (0.15 g, 0.73 mmol). After the mixture was stirred for 1.5 h, NaBH(OAc)₃ (0.34 g, 1.6 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 CH₂Cl₂ (3 × 40 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 8:1) to afford compound 24 (0.085 g, 34%). ¹H NMR (600 MHz, CDCl₃): δ 6.65 (s, 2H), 5.19 (bs, 4H), 3.77 (s, 6H), 3.13‒3.07 (m, 2H), 2.73–2.67 (m, 12H), 2.59‒2.49 (m, 8H), 2.01‒1.99 (m, 2H), 1.74–1.68 (m, 2H), 1.54‒1.48 (m, 4H), 0.90 (t, J = 7.2 Hz, 6H);

2,5-bis(2-(((S)-2-amino-4,5,6,7-tetrahydrobenzo[d]thiazol-6-yl)(propyl)amino)-ethyl)benzene-1,4-diol (25) (D-673).

A mixture of compound 24 (0.075 g, 0.12 mmol) and 48% aqueous HBr (15 mL) was refluxed at 130 °C for 5 h. The reaction mixture was evaporated to dryness, washed with ether followed by vacuum drying to yield HBr salt of 25 (0.105 g, 95%). [α]_D = −42.0 °(c=1.0 in CH₃OH) at RT, Mp 232–235 °C ¹H NMR (600 MHz, CD₃OD): δ 6.77 (s, 2H), 4.03 (s, 2H), 3.55–3.33 (m, 8H), 3.27‒3.23 (m, 2H), 3.14 (t, J = 10.2 Hz, 2H), 3.04 (t, J = 7.8 Hz, 4H), 3.00–2.94 (m, 2H), 2.78 (bs, 4H), 2.47‒2.41 (m, 2H), 2.18–2.11 (m, 2H), 1.94‒1.83 (m, 4H), 1.04 (t, J = 7.2 Hz, 6H); ¹³C NMR (150 MHz, CD₃OD): δ 173.95, 151.96, 136.64, 126.43, 120.98, 115.79, 63.10, 62.86, 57.37, 56.82, 54.90, 52.45, 29.99, 27.01, 26.16, 25.54, 22.26, 13.99; [α]_D²⁵= −42.0 (c=1.0 in CH₃OH); Anal. Calcd for C₃₀H₅₁Br₅N₆O₃S₂: C, 35.77; H, 5.10; N, 8.34. Found: C, 36.08; H, 5.42; N, 8.27.

N-(2,5-Dioxo-tetrahydro-furan-3-yl)-2,2,2-trifluoro-acetamide (26).

Trifluoroacetic anhydride (13.1 mL, 93.91 mmol) was added to D-aspartic acid (5.0 g, 37.6 mmol) at −60 °C and stirred for 10 min. The reaction mixture was allowed to warm to room temperature and then to 40 °C during which a vigorous exothermic reaction took place. Trifluoroacetic acid (10 mL) was added next and the reaction mixture was stirred under refluxing condition for 2 h. After cooling, petroleum ether was added and the solid thus formed was collected by filtration, washed successively with petroleum ether and ether and finally dried under vacuum to afford compound 26 (7.7 g, 97%). ¹H NMR (600 MHz, Acetone-D₆): δ 9.41 (bs, 1H), 5.30–5.26 (m, 1H), 3.53 (q, J = 10.2 Hz, 1H), 3.27 (dd, J = 12.0, 6.6 Hz, 1H).

4-(3,4-Dimethoxy-phenyl)-4-oxo-2-(2,2,2-trifluoro-acetylamino)-butyric acid (27).

To a stirring suspension of compound 26 (4.0 g, 18.95 mmol) and AlCl₃ (6.3 g, 47.4 mmol) in CH₂Cl₂ (90 mL), veratrole (3.62 mL, 28.4 mmol) was added and the reaction mixture was stirred at room temperature for 4 days. 6M HCl was added to the reaction vessel and the layers were separated. Aqueous layer was extracted with ether (3 × 50 mL) and the combined organic portions were dried over Na₂SO₄, filtered and evaporated under reduced pressure. The crude material was purified by silica gel column chromatography (hexane:EtOAc = 1:1) to give compound 27 (5.3 g, 80%). ¹H NMR (600 MHz, CDCl₃): δ 7.61 (bs, 1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 (s, 1H), 6.92 (d, J = 8.4 Hz, 1H), 5.02–5.00 (m, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.89–3.86 (m, 1H), 3.58 (dd, J = 14.4, 4.2 Hz, 1H).

4-(3,4-Dimethoxy-phenyl)-2-(2,2,2-trifluoro-acetylamino)-butyric acid (28).

Triethylsilane (9.7 mL, 60.7 mmol) was added to a magnetically stirred solution of keto acid 27 (5.3 g, 15.2 mmol) in trifluoroacetic acid (22.5 mL, 303.5 mmol). This solution was boiled under reflux under N₂ for 2 h after which it was cooled and carefully neutralized to pH 8 with NaHCO₃ solution at 0 °C. The aqueous solution was washed twice with Et₂O and acidified (pH <5) by the dropwise addition of 6N HCl at 0 °C. The product was extracted with Et₂O (3 × 30 mL), the organic layers were dried over MgSO₄, and the ether was removed by rotary evaporation to yield a yellow oil, which solidified on standing. This solid was recrystallized from EtOAc/hexane to afford carboxylic acid 28 (4.7 g, 92%). ¹H NMR (600 MHz, CDCl₃): δ 6.81 (d, J = 8.4 Hz, 1H), 6.74–6.71 (m, 2H), 6.70 (d, J = 1.2 Hz, 1H), 4.73–4.70 (m, 1H), 3.87 (s, 3H), 3.86 (s, 3H), 2.68 (t, J = 7.2 Hz, 2H), 2.37–2.31 (m, 1H), 2.21–2.15 (m, 1H).

N-(6,7-Dimethoxy-1-oxo-1,2,3,4-tetrahydro-naphthalen-2-yl)-2,2,2-trifluoro-acetamide (29).

To an ice-cooled solution of compound 28 (4.5 g, 13.4 mmol) in 40 mL of CH₂C1₂ was added solid PC1₅ (3.35 g, 16.1 mmol). Stirring was continued for 1 h at 0 °C, and SnC1₄ (3.14 mL, 26.8 mmol) was added. The mixture was stirred for 0.5 h at 0 °C and then allowed to warm to room temperature and stirred for an additional 4 h. The mixture was poured into ice water and vigorously stirred for 15 min. The layers were separated, and the aqueous phase was extracted with CH₂C1₂ (3 × 50 mL). The organic portions were dried over MgSO₄ and rotary evaporated to furnish white solid, which was purified by silica gel column chromatography (hexane:EtOAc = 3:2) to give colorless tetralone 29 (2.5 g, 59%). ¹H NMR (600 MHz, CDCl₃): δ 7.58 (bs, 1H), 7.46 (dd, J = 3.6, 1.2 Hz, 1H), 6.69 (s, 1H), 4.58–4.54 (m, 1H), 3.96 (s, 3H), 3.93 (s, 3H), 3.26–3.20 (m, 1H), 2.99–2.96 (m, 1H), 2.89–2.85 (m, 1H), 1.95 (dq, J = 9.0, 4.8 Hz, 1H).

N-(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-2,2,2-trifluoro-acetamide (30).

Triethylsilane (4.53 mL, 28.4 mmol) was added to a solution of compound 29 (2.25 g, 7.09 mmol) in 17.5 mL (141.8 mmol) of BF₃.Et₂O, and the resulting solution was stirred at room temperature under N₂ for 48 h. After basification of the mixture by addition to saturated NaHCO₃, the layers were separated, and the product was extracted with Et₂O (3 × 50 mL). The combined organic layers were dried over Na₂SO₄ and concentrated on a rotary evaporator. The crude residue was purified by silica gel column chromatography (hexane:EtOAc = 7:3) to afford compound 30 (1.65 g, 77%). ¹H NMR (600 MHz, CDCl₃): δ 6.59 (s, 1H), 6.55 (s, 1H), 6.34 (bs, 1H), 4.37–4.30 (m, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.11 (dd, J = 11.4, 4.8 Hz, 1H), 2.90–2.85 (m, 1H), 2.83–2.78 (m, 1H), 2.67 (dd, J = 8.4, 7.8 Hz, 1H), 2.11–2.06 (m, 1H), 1.91–1.85 (m, 1H).

6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamine (31).

To a stirred suspension of K₂CO₃ (1.37 g, 9.9 mmol) in 30 mL of MeOH containing 1.5 mL of H₂O was added trifluoroacetamide 30 (1.0 g, 3.3 mmol), and the mixture was boiled under reflux for 4 h. The mixture was allowed to cool to room temperature, and the undissolved K₂CO₃ was removed by filtration through a cotton plug. The filtrate was concentrated in vacuo, and the dark residue was diluted with water. The product was extracted with EtOAc (3 × 40 mL), the organic layers were dried over Na₂SO₄ and the volatiles were removed by rotary evaporator to furnish compound 31 (0.65 g, 95%). ¹H NMR (600 MHz, CD₃OD): δ 6.62 (s, 1H), 6.60 (s, 1H), 4.87 (bs, 2H), 3.75 (s, 6H), 3.03–2.99 (m, 1H), 2.88 (dd, J = 10.8, 4.8 Hz, 1H), 2.79–2.72 (m, 2H), 2.45 (dd, J = 9.6, 6.0 Hz, 1H), 2.00–1.95 (m, 1H), 1.55–1.49 (m, 1H).

N-(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-nitro-benzenesulfonamide (32).

2-Nitrobenzenesulfonyl chloride (0.64 g, 2.89 mmol) was dissolved in 20 mL of THF, and the solution was cooled to approximately −10°C. Et₃N (1.82 mL, 13.03 mmol) and compound 31 (0.6 g, 2.89 mmol) were added and the resulting suspension was heated during mixing to approximately 25°C, and allowed to react for 1.5 h. Precipitated triethylammonium chloride was filtered off, and the filtrate was concentrated. Water was added and extracted with EtOAc (3 × 30 mL). The combined organic layer was dried using Na₂SO₄, and the solvent was removed under reduced pressure to obtain sulfonamide 32 (1.05 g, 93%). ¹H NMR (600 MHz, CDCl₃): δ 8.21–8.19 (m, 1H), 7.90–7.88 (m, 1H), 7.78–7.74 (m, 2H), 6.54 (s, 1H), 6.41 (s, 1H), 5.40 (d, J = 7.2 Hz, 1H), 3.83 (s, 3H), 3.80 (s, 3H), 2.95‒2.91 (m, 1H), 2.81–2.74 (m, 2H), 2.69‒2.65 (m, 1H), 2.01–1.97 (m, 1H), 1.83‒1.77 (m, 1H); [α]_D²⁵= +76.6 (c=1.0 in CH₂Cl₂).

N-(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-2-nitro-N-propyl-benzene-sulfonamide (33).

To a stirred suspension of 32 (1.0 g, 2.55 mmol) and potassium carbonate (2.47 g, 17.84 mmol) in acetonitrile (25 mL), 1-bromopropane (0.69 mL, 7.65 mmol) was added and the reaction mixture was stirred at 40 °C for 48 h. The reaction mixture was cooled to room temperature, filtered off and the filtrate was evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (hexane:EtOAc = 7:3) to afford compound 33 (0.99 g, 89%). ¹H NMR (600 MHz, CDCl₃): δ 8.09 (dd, J = 6.0, 1.8 Hz, 1H), 7.71–7.66 (m, 2H), 7.63 (dd, J = 6.0, 1.8 Hz, 1H), 6.55 (s, 1H), 6.50 (s, 1H), 4.12–4.06 (m, 1H), 3.83 (s, 3H), 3.82 (s, 3H), 3.28 (t, J = 7.8 Hz, 2H), 2.96‒2.78 (m, 4H), 2.01‒1.98 (m, 1H), 1.87–1.80 (m, 1H), 1.74‒1.64 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H); [α]_D²⁵= +75.4 (c=1.0 in CH₂Cl₂).

(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amine (34).

Into a mixture of potassium carbonate (1.63 g, 11.81 mmol) in 20 mL of DMF, thioglycolic acid (0.47 mL, 6.56 mmol) was added slowly at 0 °C. The mixture was stirred at room temperature for approximately 1 h, followed by addition of compound 33 (0.57 g, 1.31 mmol, in 10 mL of DMF). The reaction mixture was heated during stirring to about 50 °C and allowed to react for an additional 15 h, after which it was quenched by addition of 1N NaOH and extracted with EtOAc (4 × 25 mL). The combined organic layer was dried using Na₂SO₄, and the solvent was concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (0–10% MeOH in CH₂Cl₂) to give compound 34 (0.22 g, 67%). ¹H NMR (600 MHz, CDCl₃): δ 6.56 (d, J = 5.4 Hz, 2H), 3.84 (s, 3H), 3.82 (s, 3H), 2.99‒2.91 (m, 2H), 2.82–2.76 (m, 2H), 2.71 (t, J = 7.8 Hz, 2H), 2.60–2.56 (m, 2H), 2.08–2.06 (m, 1H), 1.66‒1.55 (m, 3H), 0.95 (t, J = 7.2 Hz, 3H); [α]_D²⁵= +68.6 (c=1.0 in CH₂Cl₂).

Procedure A. (4-{[(5-Methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-carbamoyl]-methyl}-phenyl)-acetic acid (35).

To a solution of 1,4-phenylenediacetic acid (1.0 g, 5.15 mmol) in DMF (20 mL) were added EDC (0.30 g, 1.54 mmol), HOBt (0.21 g, 1.54 mmol) and Et₃N (0.25 mL, 1.80 mmol) and the resulting mixture was stirred at room temperature for 1 h. (S)-5-methoxy-N-propyl-1,2,3,4-tetrahydronaphthalen-2-amine (0.28 g, 1.29 mmol) in DMF (5 mL) was added next to the reaction mixture and stirred at room temperature for an additional 3 h after which DMF was evaporated under vacuum. Water was added and extracted with CH₂Cl₂ (3 × 25 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 (hexane:EtOAc = 2:3) to yield compound 35 (0.325 g, 64%). ¹H NMR (600 MHz, CDCl₃): δ 7.24–7.15 (m, 4H), 7.08 (q, J = 7.8 Hz, 1H), 6.66–6.60 (m, 2H), 4.58–4.55 (m, 1H), 4.04–3.99 (m, 1H), 3.80 (s, 3H), 3.75 (s, 1H), 3.72 (s, 1H), 3.24–3.12 (m, 2H), 3.02–2.82 (m, 3H), 2.63–2.57 (m, 1H), 2.40–2.34 (m, 1H), 1.97‒1.94 (m, 1H), 1.74–1.59 (m, 3H), 0.91–0.87 (m, 3H).

2-(4-{[(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-carbamoyl]-methyl}-phenyl)-N-(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-N-propyl-acetamide (36).

Compound 35 (0.28 g, 0.71 mmol), 34 (0.17 g, 0.71 mmol), EDC (0.14 g, 0.71 mmol), HOBt (0.096 g, 0.71 mmol) and Et₃N (0.1 mL, 0.71 mmol) were reacted in DMF (15 mL) overnight according to procedure A. The crude product was purified by silica gel column chromatography (hexane:EtOAc = 2:3) to afford compound 36 (0.325 g, 73%). ¹H NMR (600 MHz, CDCl₃): δ 7.26–7.17 (m, 4H), 7.11–7.06 (m, 1H), 6.67–6.61 (m, 2H), 6.56–6.50 (m, 2H), 4.63–4.57 (m, 1H), 4.06–3.99 (m, 1H), 3.83–3.78 (m, 9H), 3.76–3.71 (m, 4H), 3.24–3.11 (m, 4H), 3.03–2.75 (m, 5H), 2.73–2.58 (m, 3H), 2.42–2.31 (m, 1H), 1.97‒1.87 (m, 2H), 1.81–1.63 (m, 5H), 0.92–0.87 (m, 6H); [α]_D²⁵= +12.4 (c=1.0 in CH₂Cl₂).

(6,7-Dimethoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-[2-(4-{2-[(5-methoxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amino]-ethyl}-phenyl)-ethyl]-propyl-amine (37).

To a solution of compound 36 (0.31 g, 0.5 mmol) in anhydrous THF (10 mL), BH₃.THF complex (4.95 mL, 4.95 mmol, 1.0 M in THF) was added dropwise at room temperature. Reaction temperature was raised to 55 °C and the mixture was stirred for 4 h. After cooling, water (1 mL) and concentrated HCl (2 mL) were added at 0 °C and then THF was evaporated under vacuum. 25% NaOH solution (20 mL) was added to the aqueous phase and extracted with EtOAc (3 × 25 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 (hexane:EtOAc = 7:3) to furnish compound 37 (0.205 g, 69%). ¹H NMR (600 MHz, CDCl₃): δ 7.18 (s, 4H), 7.11–7.08 (m, 1H), 6.71 (q, J = 7.8 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 6.60–6.56 (m, 2H), 3.84–3.79 (m, 9H), 3.30–3.19 (m, 3H), 3.18–3.04 (m,7H), 3.01–2.73 (m, 10H), 2.57–2.38 (m, 3H), 1.91‒1.71 (m, 7H), 0.97–0.93 (m, 6H);

6-{[2-(4-{2-[(5-Hydroxy-1,2,3,4-tetrahydro-naphthalen-2-yl)-propyl-amino]-ethyl}-phenyl)-ethyl]-propyl-amino}−5,6,7,8-tetrahydro-naphthalene-2,3-diol (38) (D-679).

A mixture of compound 37 (0.2 g, 0.33 mmol) and 48% aqueous HBr (15 mL) was refluxed at 130 °C for 5 h. The reaction mixture was evaporated to dryness, washed with ether followed by vacuum drying to yield HBr salt of 38 (0.22 g, 89%). [α]_D²⁵= +7.8 (c=0.5 in CH₃OH) at RT, Mp: 220–222 ^oC ¹H NMR (600 MHz, CD₃OD): δ 7.31 (t, J = 7.8 Hz, 4H), 6.85 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 7.8 Hz, 1H), 6.57–6.52 (m, 2H), 6.47 (s, 1H), 3.64–3.62 (m, 2H), 3.46–3.32 (m, 4H), 3.27–3.13 (m, 9H), 3.01–2.88 (m, 4H), 2.68 (bs, 2H), 2.54–2.46 (m, 1H), 2.32‒2.26 (m, 2H), 1.84–1.76 (m, 6H), 1.02 (t, J = 7.2 Hz, 6H); ¹³C NMR (150 MHz, CD₃OD): δ 154.53, 143.73, 143.41, 135.52, 133.56, 129.27, 126.61, 125.78, 122.99, 121.73, 120.09, 115.39, 114.66, 112.24, 60.88, 60.52, 52.57, 52.31, 52.14, 51.82, 30.46, 29.47, 28.66, 27.31, 24.11, 23.48, 22.36, 18.54, 10.31; Anal. Calcd for C₃₆H₅₃Br₂N₂O_4.5: C, 57.99; H, 7.16; N, 3.76. Found: C, 57.94; H, 7.03; N, 3.81.

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

Binding potency was monitored by inhibition of [³H]spiroperidol (16.2 Ci/mmole, Perkin-Elmer) binding to DA rD2 and rD3 receptors expressed in HEK-293 cells, in a buffer containing 0.9% NaCl under conditions corresponding to our ‘high [radioligand] protocol’ as described by us previously.(33,34) Observed IC₅₀ values were converted to inhibition constants (K_i) by the Cheng–Prusoff equation.(33) 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.(33)

Assessment of α- synuclein aggregation inhibition and toxicity activity by D-666 and D-673:

The experimental conditions were followed as described in our earlier publication.(29) Briefly, α-syn pre-formed fibrils (PFF) were formed by incubating purified α-syn at a concentration of 5 mg/mL at 37 °C under constant agitation at 1000 rpm in a Thermomix R shaker (Eppendorf, Hamburg, Germany) for a period of 5 days. These fibrils were then added at a concentration of 0.5% to seed the aggregation of α-syn at 1.25 mg/ml (86.45 μM) with or without compounds D-666 or D-673 without agitation at 37°C for a period of 30 days. Samples were collected at 0D and 30D. The compounds were used at a concentration of 172.9 μM giving rise to drug to α-syn (2:1) ratio. Thioflavin T (ThT) assay was performed using these samples as described previously to evaluate the extent of aggregation and inhibition of aggregation.(29,30)

Next, the cytotoxicity of the α-syn aggregates formed above was evaluated in PC12 cells ((ATCC CRL1721.1)) as described by us previously.(29) Briefly, PC12 cells were culture in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum, and 1% Penicillin Streptomycin (100X, 10,000 Units/ml penicillin, 10,000 g/ml streptomycin) at 37 °C in 5% CO2 atmosphere. All reagents were purchased from Gibco. PC12 cells were seeded at 17000 cells/ well density in a 96-well plate. After 24 h the cells were treated with α-syn aggregates incubated either in presence or absence of the compounds as described above.

The final concentration of α-syn in the well is 10 μM and the concentration of the drugs are 20 μM. These solutions were prepared after appropriate dilution of the above drug and α-syn solution collected at different time points. 24h following treatments, viability was measured by MTT assay as described previously.(29)

Statistial analysis:

The data was analyzed by Prism software (GraphPad Prism 6.0) using one way ANOVA . p<0.05 was considered significant.

Table 2.

Stimulation of [³⁵S]GTPγS binding to cloned human D₂ and D₃ receptors expressed in CHO cells^a

Compound	hCHO-D2		hCHO-D3
	[35S]GTPγS EC50 (nM)	Emax (%)	[35S]GTPγS EC50 (nM)	Emax (%)	D2/D3
DA	200.41 ± 21.59	100	6.13 ± 1.78	100	32.69
(–)-5-OH-DPAT	41 ± 6	80 ± 4	0.63 ± 0.08	75 ± 4	65
Ia	1.7 ± 0.1	100 ± 3	0.53 ± 0.04	92 ± 1	3.2
Ib	0.44 ± 0.09	97 ± 4	0.94 ± 0.28	74 ± 5	0.46
(–)-Pramipexole	251 ± 16	96.6 ± 4.9	4.08 (6) ± 1.00	96.9 ± 0.8	61.5
D-666	7.69 ± 1.08	59.1 ± 5.6	1.53 (4) ± 0.31	83.7 ± 5.3	5
D-673	34.37 ± 6.17	85.5 ± 7.6	5.36 ± 1.47	108 ± 4	6.41
D-634	13.4 ± 2.8	88.9 ± 11.7	13.3 ± 2.7	96.6 ± 7.7	1
D-679	5.29 ± 0.91	98.0 ± 1.7	0.86 ± 0.09	95.0 ± 6.4	6.15

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

^b.Data taken from Ref 28. triplicate.