Synthesis and Biological Evaluation of a Series of Novel 1-(3-((6-Fluoropyridin-3-yl)oxy)propyl) piperazines as Dopamine/Serotonin Receptor Agonists

Evidence suggested that the use of partial dopamine D 2 /D 3 receptor agonists may be a better choice for the treatment of Parkinson ’ s disease (PD), and the stimulation of 5-HT 1A receptors (mainly via nondopaminergic mechanisms) alleviates motor and nonmotor disorders of PD, implying that the multitarget approach may provide a double bonus for the treatment of the disease. In this study, 20 novel 1-(3-((6- ﬂ uoropyridin-3-yl)oxy)propyl)piperazine derivatives were designed and synthesized using a bioisosterism approach, and their activities for D 2 /D 3 /5-HT 1A receptors were further tested. The results showed that several compounds exhibited a multitarget combination of D 2 /5-HT 1A agonism. Compounds 7b and 34c showed agonistic activities on D 2 /D 3 /5-HT 1A receptor. The EC 50 value of 7b for D 2 /D 3 /5-HT 1A receptor were 0.9/19/2.3 nmol/L, respectively; and the EC 50 value of 34c for D 2 /D 3 /5-HT 1A receptor were 3.3/10/ 1.4 nmol/L, respectively. In addition, 34c exhibited good metabolic stability (the half-life T 1/2 ¼ 159.7 minutes) in vitro , which is of great signi ﬁ cance for the further exploration of multitarget anti-PD drugs.


Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease in the elderly with a prevalence of approximately 2% of the population over 60 years old. 1 Patients show motor symptoms such as tremors at rest, bradykinesia, rigidity, and postural instability accompanied by nonmotor symptoms such as autonomic dysfunctions, cognitive impairment, sleep disorders, and mood disorders for the most part. [2][3][4][5] Pathologically, the cardinal motor deficits result from the gradual depletion of dopamine (DA) in the striatum caused by loss of dopaminergic neurons in the substantia nigra pars compacta, and accumulation of presynaptic neuronal protein α-synuclein known as Lewy bodies. 6 The nonmotor symptoms are related to specific dysfunction of cholinergic, noradrenergic, and serotonergic pathways in the brain, together with the dopaminergic pathways. 7,8 Currently, pharmacologic treatments for PD mainly focus on DA-based strategies, including the DA precursor levodopa (L-DOPA), the adjunctive drugs monoamine oxidase B inhibitors, catechol-O-methyl transferase inhibitors, and DA agonists (DAs). 2,9 Despite clear symptomatic benefits, long-term use of L-DOPA often caused motor fluctuations (on-off phenomena of L-DOPA efficacy) and dyskinesias. 10 Although the DAs are less effective than L-DOPA, the motor symptoms of early PD are sufficiently controlled by the DA agonist monotherapy which can also delay the progression of the disease. 9 In the advanced stages, those agents are combined with levodopa to reduce "off" time. 10 DAs can also relieve several bothersome nonmotor symptoms. For example, D 2 / D 3 receptor agonists pramipexole can effectively treat PD depressive symptoms and ropinirole is beneficial for sleep, anxiety, and depression. 11 Unfortunately, this strategy is not devoid of limitations. Over time, patients develop dyskinesias and psychotic-like symptoms, which might be due to the pulsatile stimulation of DA receptors. [12][13][14] In contrast, the use of partial DA D 2 /D 3 receptor agonists may be a better choice. First, D 2 /D 3 receptor partial agonists were also able to elevate locomotion significantly, implying its application in PD therapy. 15 Second, such compounds would hypothetically balance the dopaminergic tone by stimulating DA D 2 /D 3 receptors and counteracting excessive activation of them, 16 thereby reducing the occurrence of side effects.
The 5-HT 1A receptor also plays an important role in PD pharmacotherapy, mainly reflected in three aspects. First, activation of 5-HT 1A receptors can improve L-DOPA-induced dyskinesia (e.g., eltoprazine and NLX-112). 17 Second, they are expected to improve cognitive impairments (e.g., aripiprazole) and relieve symptoms of anxiety and depression. 18 Further, 5-HT 1A receptor agonists have also shown neuroprotective effects (e.g., BAY-639044). Miyazaki et al demonstrated that activation of 5-HT 1A receptor can induce proliferation of astrocytes and increase the level of antioxidant molecules in the striatum, which seems to prevent progressive dopaminergic neurodegeneration. 19 Stimulation of 5-HT 1A receptors alleviates motor and nonmotor disorders mainly via nondopaminergic mecha-nisms, implying that the multitarget approach combining the therapeutic effects of dopaminergic and serotoninergic receptors may provide a double bonus for the treatment of PD. 18,20 Ligands endowed with such a multitarget feature have shown clinical effectiveness. The D 2 /D 3 /5-HT 1A receptor agonist Pardoprunox (SLV-308) displays an anti-PD effect, along with antidepressant and anxiolytic efficacy. 21 In addition, it has a lower propensity to elicit side effects such as dyskinesia compared with other dopaminergic agents and it is now in phase III clinical trials for the treatment of PD. 22,23 Therefore, D 2 /D 3 /5-HT 1A receptor agonists may be of great significance to develop novel potential anti-Parkinson's drugs at present.
This work aims at identifying compounds with D 2 /D 3 R partial agonism and 5-HT 1A R agonism to develop novel anti-Parkinson's active molecules with a lower propensity for side effects. Arylpiperazine is a privileged motif for aminergic receptor ligands. 24 Compounds targeting both DA and serotonin receptors are characterized by an arylpiperazine, comprising a flexible aliphatic spacer and an additional lipophilic moiety serving as secondary pharmacophore. 24 Many studies selected this flexible system as the basic scaffold to achieving a fine balancing of D 2 R/D 3 R and 5-HT 1A R activities. Earlier studies showed that two fragments during I-1 and II, benzamide and phenylacetamide, were developed as new pharmacophores by opening the amide ring of aripiprazole or brexpiprazole (D 2 /D 3 and 5-HT 1A agonist, ►Fig. 1). [25][26][27][28][29][30] Xu et al identified pyridinecarboxamide derivatives III based on bioisosterism of compound I, which showed improved antagonism for D 2 R and agonism for 5-HT 1A R (►Fig. 2). 26 In this article, brexpiprazole and compound II were used as the lead compounds to synthesize 7a for the first time. As shown in ►Fig. 3, compound 7a exhibited higher potency for DR/5-HT 1A R, and its EC 50 values are comparable to that of compound II in terms of activities to the target receptors. Surprisingly, in comparison to brexpiprazole, a partial D 2L / D 3 /5-HT 1A agonist, 7a not only retained partial agonism on D 2 R but also exhibited full agonism on 5-HT 1A R, which may result in stronger efficacy against PD and smaller side effects as previously mentioned.
Starting from 7a, further structural optimizations were conducted to look for more favorable multitarget agonists. Herein, a series of pyridine derivatives were synthesized and their activities on D 2 R, D 3 R and 5-HT 1A R were evaluated. The effects of the substituents of pyridine, spacer, and arylpiperazine moieties on compound activity (structure-activity relationship [SAR]) were also explored (►Fig. 3). At last, compounds with better activities were selected to test for microsomal stabilities in vitro.

Chemistry
The synthesis of 20 target compounds is outlined in Schemes 1 to 4. Their structures have been confirmed by mass spectrometry (MS) and nuclear magnetic resonance (NMR), and their purities have been tested by high-performance liquid chromatography (HPLC).     The preparations of 1-(benzo[b]thiophen-4-yl)piperazine derivatives (7a-7l) are shown in Scheme 1. First, 5-chloro-2,3-difluoropyridine (1) sequentially underwent coupling reaction, boronic esterification, and oxidization reaction to get intermediate 4. At the same time, compound 5 was treated with 1-bromo-2-chloroethane or 1-bromo-3-chloropropane to give intermediates 6a and 6b, which next reacted with 4 or other corresponding pyridinol in the presence of K 2 CO 3 and KI in CH 3 CN to obtain the target compounds 7a-7l, respectively.  (iv) 1-bromo-2-chloroethane or 1-bromo-3-chloropropane, K 2 CO 3 or 25% NaOH, r.t. or 60°C; (v) pyridine derivatives, K 2 CO 3 , CH 3 CN, reflux. Scheme 4 describes the synthesis of target compounds with variations to arylpiperazine moieties (34a-34d). The arylpiperazine fragments were either commercially supplied (34d) or synthetically prepared. In the first step, the hydrogenation reaction of the nitro group of 27 was performed in EtOH at r.t. using Pd/C as the catalyst and ammonium formate as the hydrogen source, leading to the generation of 28. Then arylamines 28 reacted with bis(2-chloroethyl)ethylamine to obtain 29 and aromatic halohydrocarbon 30 reacted with anhydrous piperazine in ethylene glycol to furnish 31. The intermediate 33 was obtained by substitution and the subsequent deprotection of Boc. Finally, those heterocyclic arylpiperazine intermediates were treated with 10a via SN 2 mechanism to yield target compounds 34a-34d.

Biological Activity
The functional activities of the obtained pyridine derivatives on D 2L /D 3 /5-HT 1A receptors were further evaluated using cAMP Gi assay. DA and serotonin served as control agents. Cells used in this assay included (1) D 2L Rs, human recombinant (HEK293 cells, genscript); (2) D 3 Rs, human recombi-nant (CHO cells, Jiangsu Enhua Pharmaceutical Co., Ltd.); (3) 5-HT 1A Rs, human recombinant (HEK-293 cells, Jiangsu Enhua Pharmaceutical Co., Ltd.). The concentration of the target compounds was 10 μmol/L and the test results are shown in ►Table 1. 31 The receptor functional activity test in vitro showed that 10 compounds have D 2 /5-HT 1A dual agonism activities, and two compounds having D 2 /D 3 /5-HT 1A triple agonism activities. Compounds 7b and 34c were subjected to rat/human liver microsomes (RLMs/HLMs) to assess their metabolic stability; the results are shown in ►Table 2. The data indicated that compound 34c (the half-life T 1/2 values were 110.8, 85.5, and 159.7 minutes) displayed better metabolic stability than 7b (T 1/2 values were 23.9, 11.1, and 17.6 minutes).

Preliminary Structure-Activity Relationships
In this study, 7b-7k were synthesized to investigate the influence of pyridine N atom and R-groups on the agonism presumably by the conformation constraint that arises from potential hydrogen bond. Varying linkers were conferred on compounds 7l, 11, 16, 21, and 22 to see if the rigidity influenced the activity. In addition, compounds 34a-34d containing diverse base moieties while maintaining pyridine-2-fluorine fragment as the pharmacophore in the pyridine moiety were meant to examine the effect of aryl piperazines.

SAR of the Pyridine Moiety
Results from the SAR analysis of the pyridine moiety showed that: ▪ Replacement of the amide group (7a) with fluorine atom, chlorine, and cyano group yielded compounds 7b, 7c, and 7e, which had higher potency for the D 2 R, and were approximately threefold to 10-fold more potent than 7b for 5-HT 1A R agonism activity. Conversely, introducing bromine or trifluoromethyl to the position, the activities of compounds 7d and 7f for D 2 R or 5-HT 1A R were dramatically decreased. ▪ Changing the relative position between the pyridine N atom and fluorine group (7 g) led to the absence of agonism for D 2L . Compounds 7h, 7i, 7j, and 7k without   one of pre-existing substitutions or with an additional substitution on the 3-position also showed no agonistic activities on D 2L . These data suggested that the pyridine-2-fluorine fragment might be important to the agonism on D 2 R.

SAR of the Linker
Results from the SAR analysis of the linker showed that: ▪ Linker shortening (7l) led to a loss of efficacy for D 2 and 5-HT 1A receptors. Linker lengthening (11), replacing the piperazine group with the homopiperazine group (16), or modifying the classic aliphatic spacer by introducing a cyclopropyl ring (21,22) cannot maintain activities for DA and 5-HT 1A receptors at the same time. ▪ A flexible linker of three carbons may be necessary to maintain the agonistic activity of the three receptors.

SAR of the Arylpiperazine Moiety
Results from the SAR analysis of the arylpiperazine moiety showed that:

Conclusion
In summary, 20 new compounds of pyridyl alkylarylpiperazines were synthesized based on bioisosterism which were also biophysically evaluated for D 2 /D 3 and 5-HT 1A receptors. Most of these derivatives were D 2 /5-HT 1A receptor agonists, and compounds 7b and 34c behaved as partial D 2 /D 3 R agonists and potent full 5-HT 1A R agonists. Reactive molecules with these pharmacological profiles could effectively address motor and nonmotor disorders with a lower propensity for side effects. Compound 34c also exhibited good metabolic stability in vitro, so it was confirmed as the optimal compound. Besides, preliminary SAR between the designed compounds and three targets was further discussed, which could provide insights into the development of novel multi-target anti-PD molecules.

Experimental Section
Unless specified otherwise, all starting materials, reagents, and solvents were commercially available. All reactions were monitored by thin-layer chromatography (TLC) on silica gel plates (GF-254) and visualized with ultraviolet (UV) light (Shanghai Heqi Glass Instrument Co., Ltd.). Column chromatographic purification was performed using silica gel (Greagent). NMR spectra were recorded in DMSO-d 6 or D 2 O on a 400 MHz or 600 MHz spectrometer (Unity Inova) with tetramethylsilane as an internal reference. All chemical
To a stirred suspension of 2 (3.00 g, 18.90 mmol) in toluene (20 mL) were added pinacol (2.20 g, 18.90 mmol) and anhydrous magnesium sulfate (15.00 g). The mixture was stirred at r.t. overnight and filtered. The filtrate was concentrated in vacuo, and the residue was purified by silica gel column chromatography to give 3 (2.43 g, 54% yield) as oil.

Procedures for the Preparation of Compound 29
The nitro compounds 27 (2.00 g, 12.30 mmol) and Pd/C (0.30 g, 10 wt % palladium on activated carbon paste and 55% moisture) were dissolved in EtOH (30 mL) and stirred at r.t. The mixture was bubbled with nitrogen. This is followed by the addition of HCO 2 NH 4 (3.2 g, 50.7 mmol) and the mixture was stirred at r.t. overnight. The reaction was finished and filtered. The filtrate was concentrated in vacuo and the residue was purified by silica gel column chromatography to provide 28 (1.60 g, 99% yield) as oil.

Procedure for the Preparation of Compound 31
To a solution of anhydrous piperazine (5.10 g, 59.00 mmol) in ethylene glycol (100 mL), 7-chlorofuro[2,3-c]pyridine (30, 1.00 g, 5.90 mmol) was added, and the mixture was stirred at 140°C for 9 hours. After cooling down, the mixture was washed with saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The organic layer was dried over anhydrous magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was purified by silica gel column chromatography to provide 31 (0.80 g, 75% yield) as oil.
To a stirred solution of p-TsCl (6.30 g, 33.30 mmol), triethylamine (7.80 g, 76.80 mmol), and DMAP (313 mg, 2.56 mmol) in dry CH 2 Cl 2 (75 mL), the solution of 9a (3.00 g, 25.6 mmol) in dry CH 2 Cl 2 (25 mL) was added slowly at 0°C. The reaction mixture was stirred for 1 hour at r.t., washed with water and saline water, dried over anhydrous Na 2 SO 4 , filtered, and concentrated. The residue was purified by silica gel column chromatography to give 10a (5.92 g, 71% yield) as a white solid. Following the same procedure for compound 10a, compound 10b was obtained with 8 and 4-bromo-1-butanol being used as the starting materials.

Synthesis of Target Compounds
General Procedures for the Preparation of Compound 7a-7i Compound 5 (6.00 g, 27.23 mmol) was dissolved in acetone (60 mL), and potassium carbonate (K 2 CO 3 , 11.29 g, 81.69 mmol) was added, followed by dropwise addition of 1-bromo-2-chloroethane (7.81 g, 54.46 mmol). The reaction mixture was stirred at 60°C for 12 hours, cooled to r.t., filtered, and concentrated. The residue was purified by silica gel column chromatography to give compound 6a (1.02 mg, 13.5% yield) as a clear liquid.
A solution of the crude 13 (6.00 g, 18.10 mmol) was dissolved in methanol (20 mL), then a 4 mol/L hydrogen chloride methanol solution (20 mL) was added dropwise. The mixture was stirred at r.t. overnight and H 2 O (20 mL) was added. The aqueous solution was extracted with CH 2 Cl 2 (30 mL Â 2). The aqueous layer was basified with 10% NaHCO 3 until pH ¼ 11 and continuously extracted with CH 2 Cl 2 . The organic phase was dried (Na 2 SO 4 ) and the solvent was removed to give 14 (0.8 g, 19% yield) as a white solid. Following the same procedure for 6b, compound 15 was obtained. Following the same procedure for 7, compound 16

General Procedures for the Preparation of Compounds 21 and 26
A solution of 17 (4.00 g, 21.48 mmol) in ethanol (12 mL) was heated at reflux. Aqueous 14 mol/L NaOH (1.5 mL, 21.48 mmol) was added for 2 minutes, and the mixture was continued to reflux for 5 minutes, cooled down, and added water (40 mL). The aqueous solution was extracted with CH 2 Cl 2 (30 mL Â 2). The aqueous layer was acidified with 3 mol/L HCl (aq) until pH ¼ 0.7, and continuously extracted with CH 2 Cl 2 . The new organic phase was dried with Na 2 SO 4 and concentrated to give 18 (2.50 g, 74% yield) as oil.
To a stirred solution of 19 (3.10 g, 8.60 mmol) in dry THF (30 mL) was added slowly a suspension of LiAlH 4 (1.96 g, 25.80 mmol) in dry THF (50 mL) at 0°C. The reaction was stirred at r.t. for 5 hours and quenched with 10% NaOH solution. The mixture was extracted with DCM and washed with water and saline water. The organic layer was dried over anhydrous MgSO 4 , filtered, and concentrated. The residue was purified by silica gel column chromatography to give 20 as a yellow oil (1.10 g, 42.3% yield).
A solution of 20 (1.10 g, 3.6 mmol), 6-fluoropyridin-3-ol (497 mg, 4.4 mmol), and PPh 3 (1.1 g, 4.2 mmol) was stirred in dry THF (30 mL) at 0°C under a N 2 atmosphere. To this mixture was added dropwise DIAD (0.77 g, 4.4 mmol) for 10 minutes, then the reaction was allowed to warm to r.t. and monitored by TLC. After completion of the reaction, the solvent was evaporated under reduced pressure and the resulting oil was purified by silica gel column chromatography to give 21. The compound 21 was dissolved in ethyl acetate (10 mL), then hydrogen chloride ethyl acetate solution (2 N, 2 mL) was added dropwise. The mixture was stirred at r.t. for 1 hour, then filtered. The residue was washed with EtOAc or EtOH, dried in vacuo to give 21 hydrochloride (782 mg). Following the same procedure, compound 26 was obtained.

Microsomal Metabolic Stability Assay
Microsomal metabolic stability assay was performed to determine the metabolic stability of the optimal compound using human, rat, and mouse liver microsomes in vitro according to a reported study. 32 Human liver microsomes were obtained from Corning Inc., Corning, New York, United States with CAS No. 452117; SD rat liver microsomes were obtained from Research Institute for Liver Diseases (Shanghai) Co. Ltd. with CAS No. LM-DS-02M; and CD-1 mouse liver microsomes were obtained from Research Institute for Liver Diseases (Shanghai) Co. Ltd. with CAS No. LM-XS-02M. The final incubation contained 0.5 mg/mL microsomal protein, 1 µmol/L test article/positive control, 1.3 mmol/L NADP, 3.3 mmol/L glucose 6 phosphate, and 0.6 U/mL glucose 6 phosphate dehydrogenase. The mixtures were incubated in a 37°C for 10, 30, and 90 minutes before quenching with acetonitrile containing tolbutamide and propranolol (serve as internal standard). LC-MS/MS was used for analysis. The aqueous mobile phase consisted of 0.1% formic acid; and the organic mobile phase consisted of 0.1% formic acid and 99.9% acetonitrile. The flow rate was set as 0.5 mL/min. The C18 trapping cartridge was a polymer-based column. A multiple reaction monitoring method was used to analyze each molecule. And the data were analyzed by Analyst 7.1 (Sciex, Framingham, Massachusetts, United States). The ratio of the peak area response of each compound to that of an internal standard was used to calculate the half-life (T 1/2 ) of the tested compounds, as determined by the slope of the corresponding lines.

Ethics Statement
This article does not contain any studies with human participants or animals performed by any of the authors.