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Structural Modifications of Neuroprotective Anti-Parkinsonian (−)-N6-(2-(4-(Biphenyl-4-yl)piperazin-1-yl)-ethyl)-N6-propyl-4,5,6,7-tetrahydrobenzo[d]thiazole-2,6-diamine (D-264): An Effort toward the Improvement of in Vivo Efficacy of the Parent Molecule

In our overall goal to develop multifunctional dopamine D2/D3 agonist drugs for the treatment of Parkinson’s disease (PD), we previously synthesized potent D3 preferring agonist D-264 (1a), which exhibited neuroprotective properties in two animal models of PD. To enhance the in vivo efficacy of 1a, a structure–activity relationship study was carried out. Competitive binding and [35S]GTPγS functional assays identified compound (−)-9b as one of the lead molecules with preferential D3 agonist activity (EC50(GTPγS); D3 = 0.10 nM; D2/D3 (EC50): 159). Compounds (−)-9b and (−)-8b exhibited high in vivo activity in two PD animal models, reserpinized and 6-hydroxydopamine (OHDA)-induced unilateral lesioned rats. On the other hand, 1a failed to show any in vivo activity in these models unless the compound was dissolved in 5–10% beta-hydroxy propyl cyclodextrin solution. Lead compounds exhibited appreciable radical scavenging activity. In vitro experiments with dopaminergic MN9D cells indicated neuroprotection by both 1a and (−)-9b from toxicity of MPP+.


■ INTRODUCTION
Parkinson's disease (PD) is a progressive age-related neurodegenerative disorder of the central nervous system that is characterized by gradual loss of dopaminergic neurons in the substantia nigra region of the brain. 1 It is estimated that PD affects 1−2% of the people older than 65 years of age. According to a statistical analysis published by the Parkinson's Disease foundation, approximately 60 000 Americans are diagnosed with PD each year, and an estimated 7−10 million people worldwide are living with PD. Common symptoms associated with PD include rigidity, bradykinesia, resting tremors, postural instability, and cognitive psychiatric complications. 2−4 The etiology of PD is not clear yet, but it has been shown that both mitochondrial dysfunction and oxidative stress are interdependent, which is thought to play a central role in the pathogenesis of the disease process. 5,6 Oxidative stress and excessive amounts of metals especially iron can lead to the formation of reactive oxygen species (ROS). These mitochondria-derived ROS inhibit mitochondrial respiration and promote the aggregation of alpha synuclein protein (αSN), which ultimately forms Lewy bodies (LBs) and Lewy neuritis (LN). 7 LBs and LN are neuropathological hallmarks of PD and toxic toward dopaminergic neurons. Levodopa (L-DOPA) became available in 1960 for the treatment of PD and is still being considered a main stream therapy. 8 However, prolonged use of L-DOPA gives rise to "on" and "off" episodes along with motor fluctuations, and eventual oxidation of dopamine (DA) derived from L-DOPA further facilitates neurodegeneration. 9 One of the current strategies of PD therapy is to delay the initiation of L-DOPA therapy, by using various combinations of other therapeutic agents including, but not limited to, DA agonists, inhibitors of DA metabolism. 10 However, none of these strategies address the limitations of L-DOPA. Therefore, the need for therapeutic agents with disease-modifying effects is of paramount importance.
The DA receptors, belonging to a class of G-protein-coupled receptor (GPCR) family, are mainly found in the central nervous system (CNS) (controlling neuronal signaling thereby modulating many important behaviors) and in the periphery (to affect cardiovascular and renal functions). 10 The D 1 -like receptors (D 1 and D 5 subtypes) and the D 2 -like receptors (D 2 , D 3 , and D 4 subtypes) transduce signals via adenylate cyclase, an effectors molecule. Upon receptor activation, D 1 -like receptors activate adenylate cyclase, whereas D 2 -like receptors inhibit it. Interestingly, the DA D 3 receptor has a different distribution in the brain compared to the D 2 receptor. 11,12 The D 3 receptor is found to be densest in the limbic region of the brain, whereas the highest level of D 2 expression is in the striatum of the midbrain. 13 It is important to mention that D 2 and D 3 receptor subtypes exhibit 50% homology in their amino acid sequence, which increases to 75−80% in the helical transmembrane spanning domains, where agonist binding sites are believed to be located. 14,15 This makes the task of developing D 3 -selective ligands challenging. Interestingly, DA D 3 preferring agonists were shown to provide an additional neuroprotective effect compared to the DA D 2 receptor agonist, probably via the production of neurotrophic factor. 16,17 An enormous amount of work has been done to develop D 3 selective agonists and to identify key pharmacophoric features responsible for selectivity for D 3 receptor over D 2 . 18−27 It is important to mention that D 3 receptor bound to an antagonist was recently crystallized to provide a detailed molecular structure. 28 The research from the past two decades in the PD area has provided more insights into the basic pathogenetic factors of PD such as roles of oxidative stress, aggregation of αSN proteins in the form of soluble toxic aggregates and fibrils, and increased concentration of iron in the PD brain. 29−31 αSN is a component of Lewy bodies, a pathological hallmark of PD. αSN along with oxidized DA (DA-quinone) could have a synergistic effect in terms of disease susceptibility and progression. 32−35 It is increasingly evident that drugs aimed at a single target may be inadequate for the treatment of complex diseases such as PD, which is multifactorial in nature. Thus, it is hypothesized that multifunctional drugs exhibiting multiple pharmacological activities addressing underlying pathogenic factors of PD will be effective as disease modifying agents. 36 With this in mind, we initiated our drug discovery approach aimed at identifying novel multifunctional agents possessing D 2 /D 3 agonist or D 3 preferring agonist activity along with antioxidant, iron chelating, and modulation of αSN aggregation activities. In this regard, we have designed and explored a novel, hybrid molecular template by combining known D 2 /D 3 agonists with D 2 /D 3 antagonist fragments, which led to the development of a number of potent D 3 preferring and D 2 /D 3 agonists and lead molecules exhibiting potent in vivo activity in PD animal models. 37−44 One such lead compound, D-264 (1a, Figure 1), exhibited potent in vivo activity in PD animal models and also exhibited neuroprotective properties in two different PD animal neuroprotection models. 45 Although its neuroprotective action is an important feature, 1a suffers from poor in vivo efficacy probably due to lack of sufficient brain penetration, although additionally high plasma protein binding and possibly binding to adipose tissue can also potentially contribute to less efficacy. In vivo activity of 1a was enhanced significantly when 1a was solubilized in 5−10% β-hydroxy-propyl-cyclodextrin (BHPC) solution presumably by encapsulating the molecule leading to enhanced blood brain barrier penetration of 1a. The present structure−activity study with 1a-related compounds has been designed to enhance the in vivo efficacy without compromising their multifunctional agonist and neuroprotective properties. Introduction of different polar hydroxyl group(s) will contribute toward reducing the lipophilicity of the parent 1a, which will help bring the compounds, for example, (−)-9b, more in compliance with Lipinski's rule of five compared to the parent 1a. 46 Consequently, this should contribute toward a higher in vivo efficacy.

■ RESULTS AND DISCUSSION
Potency and Agonism at DA D 2 and D 3 Receptors. Our first-generation hybrid compound 1a, a potent and D 3 preferring agonist with multifunctional properties for potential PD treatment, was the starting point for designing compounds with enhanced in vivo efficacy without compromising agonist potency. The structural modifications are mainly centered on the introduction of methoxy and hydroxyl groups at various positions on the biphenyl moiety of this hybrid molecule.
Methoxy and hydroxyl substitutions also should help us to examine the possible contribution of any hydrogen-bonding interaction originating from this region of the molecule with D 2 and D 3 receptors. Apart from these modifications, other molecular alterations involving bioisosteric replacement of the thiazolidium moiety by aminotetraline or quanazoline rings, change of ethylene linker length, and incorporation of amide bond at the piperazine nitrogen atom distal to the agonist headgroup have also been incorporated.
First, the influence of methoxy and hydroxyl substitutions on the biphenyl ring of 1a was tested in binding assays with rat DA D 2 and D 3 (rD 2 and rD 3 ) receptors expressed in HEK-293 cells.
To this end, racemic derivatives 8a−c, 9a−c, and 22 were synthesized and characterized. It is evident from Table 1 that most of these compounds displayed high affinity for D 3 and moderate affinity for D 2 receptors. Among this series of analogues, compound 9b with monohydroxyl substitution on the meta position of the phenyl ring distal to the piperazine was found to be the most potent and selective for D 3 tested at this  receptor compared to 9b, while D 3 affinity decreased approximately 2-fold in comparison to 9b (K i , D 2 = 464, D 3 = 2.11 nM, D 2 /D 3 = 220 for 8b). These results indicated that introduction of monomethoxy and monohydroxyl groups is well tolerated on the distal phenyl ring of 1a and actually increases selectivity for the D 3 receptor.
In our next series of compounds, aminotetraline and amino pyrimidine moieties were incorporated as bioisosteric replacement of the thiazolidium moiety of pramipexole in 9b or 1a, resulting in (−)-11, 26a, and 27. 50 It was hypothesized that in both cases H-bonding interaction of the parent amino group with serine-192 at the DA receptor should be maintained. 51 Specifically, the (−) isomer of 5-hydroxy aminotetraline was synthesized, as we have shown in our previous reports that the (−)-enantiomer exhibits the highest affinity compared to the (+)-isomer for both D 2 and D 3 receptors. As expected, the 5-hydroxy aminotetraline analogue (−)-11 exhibited higher affinity, compared to (−)-9b, for both D 2 and D 3 receptors with overall less selectivity for D 3 receptor (K i , D 2 = 27.8, D 3 = 0.77 nM, D 2 /D 3 = 36). In our previous report, the phenolic moiety of 5-hydroxy aminotetraline was replaced by an amino pyrimidine moiety, which is a known bioisostere of a phenolic group. Here we wanted to explore this further with linearly fused biphenyl rings at the other side of the molecule. Incorporation of amino pyrimidine to this moiety in compound 26a resulted in reduced potency for both D 2 /D 3 receptors (K i , D 2 = 735 nM, D 3 3.65 nM) with decreased in selectivity (D 2 /D 3 = 201) compared to 9b. Next, in compound 27 we introduced a hydroxyl group on the biphenyl ring of 26a (targeting the accessory binding domain of the receptor). Compound 27, which is a bioisosteric analogue compound 9b, exhibited significantly decreased binding affinity, compared to 9b, at both D 2 and D 3 receptors (K i , D 2 = 13121 nM, D 3 = 67 nM, vs D 2 = 235 nM, D 3 0.70 nM for 27 vs 9b, respectively). This suggests the combination of either 2-aminothiazole or hydroxytetralin, and a linearly fused biphenyl moiety gives rise to D 2 and D 3 potency and D 2 /D 3 selectivity. Next, we increased the length of the two-carbon linker in 9b to four carbons in compound 9d. In agreement with our previous results with a four-methylene linker at this position, compound 9d (K i , D 2 = 567 nM, D 3 = 9.43 nM) displayed lower potency at both D 2 and D 3 receptors compared to 9b. 38 The reason behind characterizing bio-isosteric ((−)-11, 26a, and 27) and higher chain length (9d) compound is to expand the SAR study to better understand molecular interaction of our hybrid molecules with D 2 /D 3 receptors.
Finally, in an earlier publication, we reported compound (S)-(4-{2-[(2-amino-4,5,6,7-tetrahydrobenzothiazol-6-yl)propylamino]ethyl}piperazin-1-yl)-(1H-indol-2-yl)methanone (D-440) as one of the most potent and selective agonists for D 3 receptor known to date, and this compound contains a carbonyl bond between the piperazine nitrogen atom and 5-position of indole, distal to the agonist headgroup. 44 So, to probe the impact of introduction of a carbonyl group on D 3 receptor selectivity in our first generation hybrid compound 1a, we incorporated a carbonyl bond between the piperazine nitrogen and the accessory binding biphenyl ring of 1a. This modification generated compound 35, Scheme 4, which exhibited lower binding affinity for D 2 /D 3   (Table 1). On the other hand, compound (−)-8b turned out to be functionally 2-fold less potent at D 3 receptor (EC 50 = 3.42 nM) in comparison to 1a and was a partial agonist at D 3 but full agonist at D 2 receptor. The functional potency of compound (−)-8b for D 2 was comparable to 1a (EC 50 = 36.8 nM vs 33.1 nM for (−)-8b vs 1a, respectively).
Reversal of Reserpine-Induced Hypolocomotion in Rats by 1a, (−)-8b, (−)-9b, and Ropinirole. Reserpine induces depletion of catecholamine in nerve terminals, resulting in a cataleptic condition in rats, which is a well established animal model for PD. 52,53 Significant inhibition of locomotion of rats was observed 18 h after the administration of reserpine (5 mg/kg, s.c.) which indicated the development of akinesia. Compounds (−)-8b and (−)-9b at a dose of 5 μmol/kg, i.p., in DI water, were highly efficacious in reversing akinesia (Figure 3), while 1a at the same dose (5 μmol/kg, i.p., in DI water) failed to produce any significant effect in reversing akinesia in reserpine-treated rats. However, 1a was more effective when it was dissolved in 10% beta-hydroxy cyclodextrin solution ( Figure 3). The reference drug Ropinirole exhibited much shorter duration of action compared to the test compounds ( Figure 3). The locomotor activity of (−)-8b at the end of 6h remained high compared to (−)-9b. It is evident from the result that compounds (−)-8b and (−)-9b were more efficacious in producing reversal of akinesia than 1a ((−)-8b > (−)-9b > 1a). Thus, the results indicate that compounds (−)-8b and (−)-9b exhibited higher in vivo efficacy   Horizontal activity was measured as described under materials and methods. The plots are the representation of horizontal locomotor activity at discrete 30-min intervals after the administration of (−)-9b (i.p.), (−)-8b (i.p.), ropinirole (s.c) and 1a (i.p.) at the dose of 5 μmol/kg compared to control reserpine treated rats in 18 h post reserpine treatment. One way ANOVA analysis demonstrates significant effect among treatments F (5,95) = 14.16 (P < 0.0001). Dunnett's analysis following ANOVA showed that the effects of (−)9b (P < 0.01), (−)-8b (P < 0.01), and ropinirole (P < 0.01) were significantly different compared to reserpine control. which might be due to efficient crossing of the blood brain barrier. Compound 1a was able to produce in vivo activity only if dissolved in 10% beta-hydroxy propyl cyclodextrin solution, indicating limitations in brain uptake for this compound when administered by itself. Interestingly, all three compounds displayed a long duration of action ( Figure 3). In Vivo Pharmacology in 6-OHDA Lesioned Rats. On the basis of the above locomotor data, compounds (−)-8b and (−)-9b as well as the reference Ropinirole were selected for in vivo evaluation in rats carrying an unilateral lesion in the medial forebrain bundle; the lesion was induced by application of the neurotoxin 6-hydroxydopamine (6-OHDA), resulting in the production of supersensitized DA receptors on the lesioned side. Such rats, when challenged with direct acting DA agonists, respond with contralateral rotations away from the lesioned side. This rat model is considered to be one of the standard models for preclinical screening of drugs for possible antiparkinsonian activity. 54 Both compounds (−)-8b and (−)-9b produced potent rotational activity in a dose-dependent manner when administered intraperitoneally (i.p). At a 10 μmol/kg dose, both (−)-8b (6.56 mg/kg) and (−)-9b (9.28 mg/kg) produced potent rotation that lasted for more than 10 h (Figure 4). Compound (−)-8b was more efficacious in producing rotation compared to (−)-9b (total of 5866 vs 2653 rotations for (−)-8b vs (−)-9b, respectively). Peak effect of both compounds was reached at 7.5 h. This is an indication of long duration of action of both compounds in producing contralateral rotation. When tested at a lower doses (5 μmol/kg), both compounds, (−)-8b (3.28 mg/kg) and (−)-9b (4.64 mg/kg), produced a lower total number of rotations (3333 and 1839 for (−)-8b and (−)-9b, respectively) than at the 10 μmol/kg dose. The rotation in this case lasted for more than 7 h (Figure 4). Interestingly, both compounds produced initial increase of rotational activity followed by a brief decrease of activity before exhibiting a steady increase of rotational activity. At present, the reason for such biphasic activity is unknown. At both tested doses (−)-8b was more efficacious than (−)-9b in the rotation test, just as in the locomotor activity study with reserpinized rats (Figure 3). The reference drug Ropinirole at a higher dose (10 μmol/kg) exhibited much shorter duration of action. As we have reported in the Supporting Information section, pretreatment studies with the potent DA receptor antagonist haloperidol demonstrated block of the production of rotation by our hybrid D 2 /D 3 agonist, indicating site-specific interaction at the target D 2 /D 3 receptor sites.
Neuroprotection against MPP+ Toxicity. The dosedependent effect of treatment of 1a and (−)-9b in reversing the toxicity of MPP+ to dopaminergic MN9D cells is demonstrated in Figure 5. From our previous dose−effect experiment with MPP+, we chose 100 μM of MPP+ which can induce 50−60% cell death, for our study. 55 To test whether 1a and (−)-9b can protect dopaminergic MN9D cells from MPP+ induced toxicity, the cells were pretreated with various concentrations of (20, 10, 5, 1, 0.1, 0.01, and 0.001 μM) of either 1a or (−)-9b for 1 h and then cotreated with 100 μM MPP+ for an additional 24 h. The data from the MTT assay indicated that both 1a and (−)-9b are able to protect the MN9D cells in a dose-dependent manner. For 1a, significant protection from toxicity of MPP+ was conferred by 1, 5, 10, and 20 μM doses, and this result correlates well with in vivo neuroprotection result that we published earlier. 45 For (−)-9b, significant neuroprotection was conferred at 5 and 10 μM doses. It seems 1a is relatively more potent and efficacious than (−)-9b in this neuroprotection assay. Interestingly, (−)-8b did not show any neuroprotection when the assay was carried out under identical condition (see Supporting Information).

■ CONCLUSION
In this paper, we describe an SAR study based on our earlier lead molecule 1a, with some highly potent agonist molecules for D 2 and D 3 receptors with enhanced blood brain barrier crossing ability compared to the parent molecule 1a. SAR results have demonstrated that hydroxyl derivatives of 1a have higher affinity for the D 3 receptor. In both binding and functional assays, compound (−)-9b exhibited the highest selectivity for D 3 over D 2 receptors. Lead molecules also exhibited potent free radical quenching property, indicating their antioxidant property. Furthermore, lead molecules were tested in two PD animal models and compared with parent molecule 1a.

■ EXPERIMENTAL SECTION
Reagents and solvents were purchased from commercial suppliers and used as received unless otherwise indicated. Dry solvent was obtained according to the standard procedure. All reactions were performed under inert atmosphere (N 2 ) unless otherwise noted. Analytical silica gel 60 F254-coated TLC plates were obtained from EMD Chemicals, Inc. and were visualized with UV light or by treatment with phosphomolybdic acid (PMA), Dragendorff's reagent, or ninhydrin. Flash column chromatographic purifications were performed using Whatman Purasil 60A silica gel 230−400 mesh. The proton nuclear magnetic resonance ( 1 H NMR) spectra were measured on a Varian 400 MHz FT NMR spectrometer using tetramethylsilane (TMS) as an internal standard. The NMR solvent used was CDCl 3 or CD 3 OD as indicated. Optical rotations were recorded on Perkin-Elmer 241 polarimeter. Melting points were recorded using MEL-TEMP II Dunnett's analysis showed that the effect of (−)-9b, (−)-8b and ropinirole on rotations at two doses was significantly different compared to vehicle (P < 0.01).
(Laboratory Devices Inc., USA) capillary melting point apparatus and were uncorrected. Elemental analyses were performed by Atlantic Microlab, Inc.
t-Butyl 4-(4-Iodophenyl)piperazine-1-carboxylate (1). Into a stirring solution of 1-phenylpiperazine (21.8 g, 134.0 mmol) in acetic acid/water (3:1, 42 mL), a suspension of iodine monochloride (24.0 g, 148.0 mmol) in acetic acid/water (3:1, 42 mL) was added at 55°C. The reaction was stirred at 55°C for 1 h and then at room temperature for another 1 h. The solution was poured into 400 mL of crushed ice, and the pH was adjusted to 13 with 4 N NaOH. The product was then extracted with dichloromethane (3 × 100 mL). The combined organic layer was dried over Na 2 SO 4 , filtered, and evaporated in vacuo to provide the free amine of compound 1 as a pale yellow solid (28.69 g, 74%) which was converted to t-Boc derivative without further purification.
In Vivo Rotational Experiment with 6-OH-DA Lesioned Rats. In the first 14 days post lesion challenge with apomorphine was done with lesioned animals to observe a complete rotation session post administration. In the second challenge with apomorphine (0.05 mg/kg) 21 days postlesion, contralateral rotations were recorded for 30 min; apomorphine produced rotations in all four rats (average rotation of >250) indicating successful unilateral lesion. In these rats, lesion was performed on the left side of the medial forebrain bundle in the brain, and the coordinates used from Bregma are the following: AP, −4.3; ML, p1.2; DV, −8.3. The rotations produced upon agonist challenge were clockwise. In this study, apomorphine was also used as a reference compound. The test drugs were dissolved in saline. The drugs (−)-8b, 1a, and (−)-9b were administered i.p. The rotations were measured over 7−12 h. For control, vehicle was administered alone. Rotations were measured in the Rotomax rotometry system (AccuScan Instruments, Inc. Columbus, OH) equipped with Rotomax analyzer, high resolution sensor, and animal chambers with harnesses. Data were analyzed with Rotomax Windows software program. The rotations were measured in a rotational chamber immediately after administration of drugs. The data were collected at every 30 min. Data were analyzed by the GraphPad (version 4, San Diego, CA) program. All drugs produced contralateral rotations in all lesioned rats, which lasted over 3−10 h.
Cell Culture and Treatments. The hybridoma dopaminergic MN9D cells are derived from the somatic infusion of rostral mesencephalic neurons from embryonic C57BL/6J (E14) mice with N18TG2 mouse cells. They were cultured in T-75 flask (Greiner Bio One, Frickenhausen, Germany) coated with 1 mg/mL poly-L-lysine and maintained in DMEM (high glucose with phenol red) supplemented with 10% Fecal Clone III serum, penicillin (50 units/mL), and streptomycin (50 μg/mL) at 37°C under 5% CO 2 atmosphere. Stock solution of 1a and (−)-9b were prepared in DMSO and stored at −20°C for the period of experiments. MN9D cells were pretreated with various concentrations of drugs for 1 h and then cotreated with 100 μM MPP+ (prepared freshly before addition from a stock solution in DMSO stored at −20°C) for 24 h. The control cells were treated with the above medium having 0.01% DMSO only.
Assessment of Cell Viability. To evaluate the neuroprotection ability of the test compounds in the presence of the neurotoxins MPP+, the quantitative and colorimetric MTT (3−4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide) tetrazolium salt assay was used to assess cell viability. MN9D cells were seeded into poly-L-lysine coated 96-well plates at 1 × 10 4 cells/well in 100 μL medium. After the plate was equilibrated for 40 h, old medium was taken out from each well, and 160 μL of fresh medium (containing 0.01% DMSO) was added to control wells and wells which were to be treated with MPP+. A solution of 160 μL of 1a or (−)-9b in the above medium without DMSO in 20, 10, 5, 1, 0.1, 0.01, 0.001 μM were added to wells which would be cotreated with MPP+. The plate was incubated for 1 h at 37°C under 5% CO 2 atmosphere. At the end of incubation, required amount of MPP+ was added to each well (except the control wells) to maintain a final concentration of 100 μM. The plate was then incubated for 24 h at 37°C under 5% CO 2 atmosphere. Next, 20 μL of MTT stock solution (prepared in Dulbecco's phosphate-buffered saline) was added to each well to maintain a final concentration of 0.5 mg/mL, and the plate was incubated for another 3 h at 37°C under 5% CO 2 atmosphere. Next, the plate was centrifuged at 1500 rpm for 10 min, and the supernatants were removed carefully. The formazan crystals were dissolved in 100 μL of a 1:1 mixture of DMSO/methanol solution by shaking gently at 400 rpm for 30 min at room temperature on a Thermomix R shaker (Eppendorf, Hamburg, Germany). Then, the absorbance was measured at 570 nM and 690 nM using an Epoch microplate reader (BioTek, Winooski, VT, USA). Background corrected values (570−690 nM) were used to plot the graph. Data from at least three experiments were analyzed using GraphPad software (Version 4, San Diego, USA).