Identification of a New Series of Potent Adenosine A2A Receptor

Aug 28, 2016 - *(X.C.Z.) Mailing address: Soochow University College of Pharmaceutical Sciences, 199 Ren Ai Road, Suzhou, Jiangsu, P.R.China 215123. E...
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Identification of a New Series of Potent Adenosine A2A Receptor Antagonists Based on 4‑Amino-5-carbonitrile Pyrimidine Template for the Treatment of Parkinson’s Disease Zhaohui Yang, Linlang Li, Jiyue Zheng, Haikuo Ma, Sheng Tian, Jiajun Li, Hongjian Zhang, Xuechu Zhen,* and Xiaohu Zhang* Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psychiatric-Diseases and College of Pharmaceutical Sciences, Soochow University, Su Zhou, Jiangsu 215021, P. R. China S Supporting Information *

ABSTRACT: Adenosine receptor A2A antagonists have emerged as potential treatment for Parkinson’s disease in the past decade. We have recently reported a series of adenosine receptor antagonists using heterocycles as bioisosteres for a potentially unstable acetamide. These compounds, while showing excellent potency and ligand efficiency, suffered from moderate cytochrome P450 inhibition and high clearance. Here we report a new series of adenosine receptor A2A antagonists based on a 4amino-5-carbonitrile pyrimidine template. Compounds from this new template exhibit excellent potency and ligand efficiency with low cytochrome P450 inhibition. Although the clearance remains moderate to high, the leading compound, when dosed orally as low as 3 mg/kg, demonstrated excellent efficacy in the haloperidol induced catalepsy rat model for Parkinson’s disease. KEYWORDS: Adenosine receptor, antagonist, Parkinson’s disease, GPCR, lead identification

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receptors on striatopallidal output neuron in the striatum.10 Preclinical animal models as well as clinical studies have demonstrate that adenosine receptor A2A antagonists can improve motor dysfunctions of PD while reducing side effects such as dyskinesia.11 Furthermore, it is suggested that adenosine receptor A2A antagonists may have neuron protective functions and therefore can delay the onset and progression of PD.12 Numerous adenosine A2A receptor antagonists have been tested clinically and some examples are shown in Figure 1.7,8 Istradefylline (KW-6002), developed by Kyowa Hakko, is the most advanced A2A receptor antagonist.13 On March 25, 2013, istradefylline received marketing approval in Japan.14 However, istradefylline was not approved in the US market.15 The clinical development of another promising A2A receptor antagonist, preladenant,16 was discontinued due to lack of efficacy.17 The mixed clinical results underscore the challenge for the development of novel therapeutics for PD.

he current clinical treatments for Parkinson’s disease (PD) almost exclusively focus on the dopaminergic pathway.1,2 Dopamine replacement therapies such as the prodrug levodopa (L-Dopa) and dopamine agonists are highly effective in providing symptomatic relief for patients with motor disorders such as bradykinesia, muscle rigidity, and tremor. However, these treatments cannot stop or delay the disease progression. Long-term usage of these drugs is associated with diminished efficacy and side effects such as dyskinisia.3,4 As the disease progresses, nonmotor symptoms such as depression, pain, and sleep disturbance become more prevalent. Unfortunately, the currently available dopaminergic drugs are not very effective on treating these symptoms.5 Consequently, there is an urgent need for the development of long-term disease-modifying therapies for Parkinson’s disease. Adenosine receptor A2A antagonists have emerged as potential treatment for PD in the past decade.6−8 Adenosine receptor A2A is one of the four known adenosine receptors (A1, A2A, A2B, and A3). Adenosine receptors are members of the seven trans-membrane G-protein coupled receptor (GPCR) super family.9 In the central nervous system, adenosine receptor A2A is highly expressed and colocalized with dopamine D2 © XXXX American Chemical Society

Received: July 26, 2016 Accepted: August 28, 2016

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DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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between 1 and A2A receptor in Figure 3b. Two important hydrogen bonds, formed between the oxygen and the nitrogen atoms of the acetamide of 1 and residues Asn253 and Glu169, respectively, were detected. In addition, the 5 position of the amino pyrimidine of 1 was surrounded by multiple residues, which could serve as potential hydrogen bond donors or acceptors in the binding pocket. Furthermore, 3-methoxypyridine on the right side of 1 was in close vicinity of a number of residues (Ala63, Tyr9, Ile66, and Ser67). Given these observations and the potential instability of the acetamide of 1, we decided to remove the acetamide group and add several electron withdrawing groups/moieties on the 5 position of the amino pyrimidine core of 1, with the aim of fostering favorable interactions with nearby residues in the binding pocket. For instance, the addition of a fluorine or cyano group resulted in compounds 10 and 14, respectively. The binding mechanism between compound 14 and adenosine receptor was investigated using SP scoring function of Glide docking.27 The interaction spectra of compound 14 with adenosine receptor using MOE molecular simulation package29 was shown in Figure 4. The key residue Asn253 formed hydrogen bond interaction with the cyano group of 14, instead of with the oxygen atom of the acetamide of 1, while the interaction between Glu169 and the amino group of 14 persists. In addition, residue Leu249 can form interaction with the pyridine group of 14 (Figure 4). This would be in agreement with SAR results from a previous series of compounds based on an aminopyrimidine template. In summary, our docking study indicated that the 4-amino-5carbonitrile pyrimidine template could be a good starting point for exploring a new series of A2A antagonists. Chemistry. Scheme 1 outlines the synthetic approach to compound 10. Treatment of 4,6-dichloro-5-fluoro-2(methylthio)pyrimidine with ammonia in THF at reflux provided intermediate 5, which was oxidized by m-CPBA to give sulfonyl compound 6. The methylsulfonyl group of 6 was replaced with 3,5-dimethyl-pyrazole under the influence of NaH to yield intermediate 7. Meanwhile, 3-bromo-5-methoxypyridine was reacted with isopropyl borate in the presence of n-BuLi and the resulting borate was fluorinated with potassium hydrogen fluoride aqueous solution to give intermediate 9. Finally, a Suzuki coupling was carried out to produce the desired compound 10. The general synthesis of compounds 14−37 is outlined in Scheme 2. Commercially available aromatic aldehydes 11a,b were treated with methyl carbamimidothioate sulfate and malononitrile in the presence of NaOH in ethanol at reflux to give pyrimidines 12a,b, or reacted with the desired arylimidamide hydrochloride in similar conditions to give compounds 17−19. Oxidation of 12a,b with oxone yielded the

Figure 1. Representatives of advanced A2A receptor antagonists.

Our efforts in pursuit of novel A2A receptor antagonists have centered on an aminopyrimidine template.18−25 We recently reported a series of adenosine receptor antagonists using heterocycles as bioisosteres for a potentially unstable acetamide.24,25 This series of compounds, while showing excellent potency and ligand efficiency, suffered from moderate cytochrome P450 inhibition and high clearance. Here we report a new series of adenosine receptor A2A antagonists based on a 4-amino-5-carbonitrile pyrimidine template. The evolution of the current series is demonstrated in Figure 2. Docking studies indicate that the relatively unexplored 5 position of the amino pyrimidine template may provide additional manipulation for receptor binding affinity, as well as overall physicochemical properties of a new series of compounds. Because of its intrinsic low clogP, the 4-amino-5-carbonitrile pyrimidine template is of particular interest to address the moderate cytochrome P450 inhibition associate with the old series. Here we report the results of the exploration structure−activity relationship campaign initiated from docking studies.

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RESULTS AND DISCUSSION Docking Studies. As reported in our previous work,24,25 by replacing a potentially unstable acetamide with heterocycles, we obtained a series of A2A receptor antagonists with excellent potency and ligand efficiency. Unfortunately, that series of compounds suffered from moderate cytochrome P450 inhibition and high clearance. In order to understand the interaction patterns, we docked compound 1 into the crystal structure of the adenosine receptor (PDB ID: 3VGA26) by using Glide docking.27 The docking pose of 1 and antagonist− residue interaction spectra derived from Glide docking were depicted in Figure 3 using DS2.5.28 As shown in Figure 3a, a surface pore area near the 5 position of the amino pyrimidine core of compound 1 was evident. We further analyzed the antagonist-residue interaction spectra

Figure 2. Evolution path of the current series. B

DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Figure 3. (a) Docking pose of 1 derived from Glide docking in adenosine receptor. (b)Aantagonist−residue interaction spectra between 1 and A2A receptor (hydrogen bonds are depicted in the dotted green line).

corresponding sulfonyl compounds 13a,b. The sulfonyl compounds 13a,b were then treated with pyrazole or 3,5dimethyl-pyrazole using NaH as base in anhydrous THF to give compounds 14−16, respectively. Displacement of the bromo atom of 16 with corresponding amines in NMP using N,Ndiisopropylethylamine as base at 120 °C provided final compounds 20−37. Pharmacological Assays and Structure−Activity Relationship (SAR). We recently reported a series of adenosine receptor antagonists using heterocycles as bioisosteres for a potentially unstable acetamide.24,25 This series of compounds, while showing excellent potency and ligand efficiency, suffered from moderate cytochrome P450 inhibition and high clearance. For example, compound 3 exhibited 64% inhibition of CYP3A4 at 10 μM; it also inhibited other CYP isoforms. Furthermore, its high clearance in rat liver microsomal tests prevented us from dosing compound 3 orally in the haloperidol induced catalepsy rat model of PD. In order to address these problems, we decided to explore other regions of the amino pyrimidine template. As guided in the docking studies, the relatively unexplored 5 position of the amino pyrimidine template may provide additional manipulation for receptor binding affinity, as well as overall physicochemical properties of a new series of compounds. Based on its intrinsic low clogP, the 4-amino-5carbonitrile pyrimidine template is of particular interest to address the moderate cytochrome P450 inhibition associate with the old series. Here we report the results of the preliminary SAR campaign based on the 4-amino-5-carbonitrile pyrimidine template. Table 1 outlines the SAR of the synthesized compounds. Our previous studies of the amino pyrimidine template demonstrated that the acetamide is beneficial to the binding affinity for the adenosine A2A receptor antagonists.17−22 We therefore were not surprised that compound 10 (370 nM) exhibited decreased binding affinity toward to the adenosine A2A receptor. We were pleased to see that when the fluorine was replaced by a cyano group, the binding affinity was much improved (compound 14, 58 nM). This result was in agreement with the initial docking study and we decided to focus our work on the 4-amino-5-

Figure 4. Schematic representation of the interaction between 14 and A2A receptor.

Scheme 1. Synthesis of 4-Amino-5-fluoropyrimidinea

Reagents and conditions: (a) NH4OH, THF, 60 °C, 3 h, 95%; (b) mCPBA, CH2Cl2, r.t., overnight, 95%; (c) 3,5-dimethyl-1H-pyrazole, NaH, THF, 0 °C, overnight, 59%; (d) (i) isopropyl borate, n-BuLi, THF/toluene, −40 °C, 2 h; (ii) KHF2(aq), r.t., 4 h; (e) K2CO3, Pd(PPh3)4, dioxane/H2O, 100 °C, overnight, 92%. a

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ACS Chemical Neuroscience Scheme 2. Synthesis of 4-Amino-5-carbonitrile Pyrimidinesa

a

Reagents and conditions: (a) methyl carbamimidothioate sulfate, malononitrile, NaOH, EtOH, reflux, overnight, 14−62%; (b) oxone, THF/H2O, r.t., 4 h; (c) for 14: 3,5-dimethyl-1H-pyrazole, NaH, THF, 0 °C, 6 h, 28%; for 15: 1H-pyrazole, NaH, THF, 0 °C, 6 h, 23%; for 16: (i) hydrazine hydrate, EtOH, reflux, 4 h, (ii) acetylacetone, EtOH, reflux, overnight, 81% for two steps; (d) corresponding arylimidamide hydrochloride, malononitrile, NaOH, EtOH, reflux, overnight, 6−49%; (e) corresponding amine, DIPEA, NMP, 120 °C, overnight, 9−64%.

the basic amine improved binding affinity (compounds 33, 34, and 37; 1.0, 5.9, and 4.5 nM, respectively), while larger substitutions proved less favorable (compounds 35 and 36, 10 and 17 nM, respectively). In order to confirm that these new compounds are functional antagonists for the A2A receptor, the representative compounds 20, 24, 25, and 33 were profiled in a cell based cAMP assay. Preladenant was used as a control compound (IC50 3.1 nM). Compounds 20, 24, 25, and 33 inhibited cAMP production (IC50 9.1, 1.1, 19, and 0.6 nM, respectively). In summary, the current SAR campaign yielded numerous compounds with exceptional receptor binding affinity (e.g., compounds 24 and 33, 1.0 nM) and functional potency (e.g., compounds 24 and 33, 1.1 and 0.6 nM, respectively). They also exhibited desirable physical-chemical properties and ligand efficiency. The overall SAR was consistent with a previously established series22 and the initial docking study. In order to evaluate the performance of the molecular docking based on 3VGA26 as docking template, the correlation between the docking scores and the experimental pKi values was calculated (Supporting Information, Table S1 and Figure S1). The Glide docking exhibited satisfactory capability to rank the binding affinities of the designed compounds listed in Table 1, as demonstrated by high correlation coefficient (r2 = 0.63). The results indicated that the molecular docking could serve as a powerful tool for rational design of promising A2A receptor antagonists. DMPK Evaluation. In order to assess potential drug/drug interactions and liver safety, compounds 20, 24, 30, and 33 were evaluated in a CYP screening assay. Compounds 20, 24, and 30 demonstrated minimal inhibitions of CYP3A4 and

carbonitrile pyrimidine template. Replacement of the dimethyl pyrazole with pyrazole, phenyl and pyridyl groups on the R1 position of the 4-amino-5-carbonitrile pyrimidine template all led to diminished receptor binding affinity (compounds 15, 17−19). We therefore decided to keep dimethyl pyrazole on the R1 position. The SAR thus far seemed to follow the trend of a well established series of adenosine receptor A2A antagonists that we had previously worked on,22 and was in agreement with the initial docking study. Encouraged, we decided to introduce the privileged structural element obtained from the previous SAR work.22 We were very pleased to see that when the 5methoxyl-3-pyridyl on R2 was replaced by the 3-(4-methoxypiperidyl)-2-pyridyl group, a 15-fold improvement of binding affinity was achieved (compound 20, 4.1 nM vs compound 14, 58 nM). Compound 20 confirmed our suspicion that the current template may follow a previously established SAR and was the first compound to reach low nanomolar binding affinity derived from the new template. Based on this result, we introduced a diverse set of substitutions on R2 to manipulate binding affinity. For the cyclic amino substitutions, both polar and nonpolar groups can be tolerated, as demonstrated by compounds 20−22 (4.1, 6.4, and 1.9 nM, respectively). Interestingly, stereo preference was evident, as compound 25 (70 nM) was 70-fold less active compared to its enantiomer 24 (1.0 nM). This trend was consistent with the more polar counterparts, albeit to a smaller degree (compound 26, 3.2 nM vs compound 27, 15 nM, respectively). Acyclic amines were also active (compound 30, 4.7 nM), but the extended hydroxyl group was less tolerated (compound 31, 80 nM). A basic center could be introduced without compromising much of the binding affinity (compound 32, 13 nM). Small substitutions on D

DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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CYP2D6 at 10 μM, consistent with their low clogP values within this aminopyrimidine structural class (Table 2, CYP2C9 and CYP2C19, Supporting Information, Table S2). This is in contrast to the previous series that we worked on, which exhibited moderate inhibitions on multiple CYP isoforms. In spite of the low clogP value, compound 33 demonstrated moderate inhibition (53%) for CYP3A4 at 10 μM. This moderate inhibition might be due to the basic center in 33. Nevertheless, considering the excellent binding affinity and functional potency of compound 33, the moderate CYP3A4 inhibition of compound 33 was not particularly worrisome. In summary, the initial choice to work on the 4-amino-5carbonitrile pyrimidine template due to its intrinsic low clogP value led us to numerous potent A2A antagonists with minimal drug/drug interaction liabilities. Compounds 20, 24, 30, and 33 were evaluated in human, rat, and mouse liver microsomal tests in order to assess their metabolic stability. Compounds 20, 24, and 30 exhibited relatively high clearance and short half-lives in human, rat and mouse liver microsomes. Since all compounds had clogP values under 1.5, it was evident that the high clearance was not directly related to the lipophilicity of these compounds. We suspect that these compounds might contain potential metabolic soft spots which led to the high clearance. Preliminary metabolic profiling of compound 24 in rat liver microsomes was performed. A metabolite from demethylation was observed and counted for over 10% of all metabolites at 30 min. The site of demethylation was confirmed to have happened on the pyrrolidine, resulting in compound 26, which shows a Ki of 3.6 nM (Supporting Information, Figure S2). Interestingly, compound 33 demonstrated good metabolic stability in human liver microsomes. Again, this might due to the basic center presented in compound 33. Unfortunately, compound 33 remained poorly stable in rat and mouse liver microsomes (Table 2). Plasma protein binding of compounds 24 and 33 was evaluated. Although both compounds bind significantly to plasma proteins, compound 33 showed slightly better unbound fractions, especially in rat (Table 2). Haloperidol Induced Catalepsy. Haloperidol induced catalepsy (HIC) in rat is a classical pharmacological model for PD. Based on the overall available data, we chose to test compound 24 in this animal model in order to validate the observed in vitro profiles. Because compound 24 demonstrated suboptimal metabolic stability in rat liver microsomes, we decided to use both per oral (p.o.) and intraperitoneal injection (i.p.) routes as dosing regimen in the HIC test. KW-6002 3 mg/kg (p.o. and i.p.) was used as control. The results were presented in Figure 5. When dosed by i.p., compound 24 exhibited significant inhibition of catalepsy at all three doses (1, 3, 10 mg/kg), validating its observed excellent in vitro receptor binding affinity and functional potency. When dosed by p.o., compound 24 again demonstrated inhibition of catalepsy at all three doses (1, 3, and 10 mg/kg), however, the extent of reversal was significantly compromised at 1 mg/kg by oral dosing. This was an indication that compound 24 might suffer from high clearance and/or suboptimal absorption. Finally, we determined the plasma and brain exposures of compound 24 after 1 h of dosing at 10 mg/kg with LC-MS-MS (Table 3). Compound 24 could cross the blood brain barrier. Its brain/ plasma (B/P) ratio was 1.1 and 1.6 for i.p. and p.o. dosing, respectively. The relatively low exposure of compound 24 by p.o. dosing as compared to i.p. dosing confirmed our concerns that it might suffer from high clearance and/or suboptimal

Table 1. SAR of Designed Compounds

a Displacement of [3H]-ZM 241385 binding at human A2A receptors expressed in HEK293 cells. Data are expressed as geometric mean values of at least two runs ± the standard error measurement (SEM). b HEK-293 cells transfected with A2A receptor were incubated with test compound, and the cAMP production was measured by a Cisbio kit. Data are expressed as geometric mean values of at least two runs ± the standard error measurement (SEM). cCalculated by Molinspiration. d LE (ligand efficiency) = 1.37pKi/HA (heavy atom).

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ACS Chemical Neuroscience Table 2. CYP, Metabolic, and Plasma Protein Binding Profiles of Leading Compounds CYP inhibition (%)b compd

clogP

20 24 30 33

1.0 1.5 1.4 1.3

a

Clint (mL/min/kg)c

3A4

2D6

HLM

11 9 15 53

17 12 2 18

69 34 43 7.5

d

RLM

e

90 89 163 44

t1/2 (min)c MLM 400 305 462 350

f

PPB (%)g

HLM

RLM

MLM

26 53 42 239

22 22 12 44

15 20 13 17

human

rat

mouse

99

98

98

97

89

95

Calculated by Molinspiration. All compounds were tested at 10 μM concentration. All compounds were tested at 1 μM concentration. HLM = human liver microsomes. eRLM = rat liver microsomes. fMLM = mouse liver microsomes. gPPB = plasma protein binding. a

b

c

gel column chromatography. 1H NMR spectroscopic data were recorded on a Varian 400 MHz spectrometer. 13C NMR spectroscopic data were recorded on a Varian 300 MHz spectrometer or a Varian 600 MHz spectrometer. Tetramethylsilane was used as internal standard and chemical shifts were given in ppm. Mass spectra were recorded on an Agilent 1100 LC/MSD Trap SL version mass spectrometer. 6-Chloro-5-fluoro-2-(methylthio)pyrimidin-4-amine (5). A sealed tube was charged with 4 (1.50 g, 7.04 mmol) and ammonia (5 mL, 70.4 mmol) in 50 mL THF. The reaction was stirred at 60 °C for 3 h. After cooling to room temperature, 20 mL of water was added. The aqueous phase was extracted with ethyl acetate for three times. The combined organic layer was washed with brine and dried over anhydrous Na2SO4. The solvent was removed by vacuum to give the title product (1.28 g, 95%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 7.71 (br s, 2H), 2.40 (s, 3H). 6-Chloro-5-fluoro-2-(methylsulfonyl)pyrimidin-4-amine (6). To a solution of 5 (1.38 g, 7.11 mmol) in CH2Cl2. (50 mL) was added mCPBA (3.08 g, 85%, 17.8 mmol) in batches. The mixture was stirred at room temperature overnight, before sat. aqueous Na2S2O3 and NaHCO3 were added successively. The mixture was stirred for another 2 h, and then filtered. The cake was washed with water and dried under vacuum to give the title product (1.50 g, 95%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (br s, 2H), 3.27 (s, 3H). 6-Chloro-2-(3,5-dimethyl-1H-pyrazol-1-yl)-5-fluoropyrimidin-4amine (7). To a solution of 6 (1.50 g, 6.64 mmol) and 3,5-dimethyl1H-pyrazole (765 mg, 7.96 mmol) in anhydrous THF (50 mL) at 0 °C was added NaH (480 mmol, 80%, 16.0 mmol) in batches. After stirred at room temperature overnight, the reaction mixture was quenched with water (200 mL). The resulting precipitate was filtered and dried under vacuum to give the title product (950 mg, 59%) as a gray solid. 1 H NMR (400 MHz, CDCl3) δ 5.99 (s, 1H), 5.54 (br s, 2H), 2.61 (s, 3H), 2.31 (s, 3H). Potassium Trifluoro(5-methoxypyridin-3-yl)borate (9). To a solution of 8 (4.40 g, 22.7 mmol) and isopropyl borate (5.20 g, 27.2 mmol) in anhydrous THF/toluene (40 mL/10 mL) under N2 atmosphere at −40 °C, was added n-BuLi (2.5 M in hexane, 10.8 mL, 27.2 mmol) dropwise. The reaction was stirred at −40 °C for another 2 h, and then a solution of KHF2 (5.40 g, 68.2 mmol) in H2O (20 mL) was added. The mixture was stirred at room temperature for another 4 h before the solvent was removed by vacuum. The residue was suspended in MeOH and filtered. The filtrate was concentrated under vacuum, and the resulting residue was washed with ether and resuspended in acetone. The suspension was filtered, and the filtrate was concentrated under vacuum to give the title product (3.50g, crude) as a brown solid, which was used directly in the next step without further purification. 2-(3,5-Dimethyl-1H-pyrazol-1-yl)-5-fluoro-6-(5-methoxypyridin3-yl)pyrimidin-4-amine (10). To a solution of 7 (50 mg, 0.21 mmol) and 9 (67 mg, 0.31 mmol) in dioxane/H2O (5 mL/1 mL) were added of K2CO3 (114 mg, 0.83 mmol) and Pd(PPh3)4 (24 mg, 0.02 mmol). The flask was vacuumed and then filled with N2 three times. The reaction was carried out at 100 °C overnight before water (10 mL) was added. The resulting precipitate was filtered, washed with water and dried under vacuum to give the title product (60 mg, 92%) as a gray solid. 1H NMR (400 MHz, CDCl3) δ 8.93 (s, 1H), 8.42 (d, J = 2.4 Hz, 1H), 7.92 (s, 1H), 6.04 (s, 1H), 5.57 (s, 2H), 3.94 (s, 3H), 2.71 (s,

Figure 5. Effect of compound 24 in the haloperidol induced catalepsy bar test. Mean descent latency (±standard error) in seconds. The vehicle bar represents the mean and standard error from four experiments. For i.p., *** p < 0.001 vs vehicle controls. For p.o., ###p < 0.001 vs vehicle controls.

Table 3. Plasma and Brain Exposure for Compound 24, at 1 h after Dosing, in Rats Used in the HIC Model route

dose (mg/kg)

plasma (ng/mL)

brain (ng/g)

B/P ratio

i.p. p.o.

10 10

340 ± 59a 24 ± 4b

361 ± 37a 39 ± 2b

1.1 1.6

Data are expressed as geometric mean values of three runs ± the standard error measurement (SEM). bData are expressed as geometric mean values of two runs ± the standard error measurement (SEM). a

absorption. Given the high plasma protein binding of compound 24 and the limited plasma/brain exposure after oral dosing, we suspect that the observed efficacy of compound 24 in HIC resulted from both the parent compound and active metabolites (e.g., demethylation product 26). We are currently working on the optimization of the DMPK profiles based on this new template and the results will be communicated in due course. Summary. By applying a rational design strategy, we discovered a series of adenosine receptor A2A antagonists derived from a 4-amino-5-carbonitrile pyrimidine template. Compounds from this new template exhibit excellent potency and ligand efficiency with low cytochrome P450 inhibition. Although the clearance remains moderate to high, when dosed orally as low as 3 mg/kg, the leading compound demonstrated significant reversal of the haloperidol induced catalepsy in rats. Optimization to improve metabolic stability and DMPK profiles of this template is ongoing. The results will be presented in future communication.

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d

METHODS

Chemistry. In most cases, analytical thin layer chromatography (TLC) performed on silica gel HSGF254 precoated plates was used to monitor reaction progress. Final compounds were purified with silica F

DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience 3H), 2.34 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 156.3 (d, J = 14.7 Hz), 155.7 (s), 152.1 (d, J = 4.5 Hz), 148.9 (s), 143.5 (d, J = 8.4 Hz), 141.8 (s), 141.7 (s), 141.3 (d, J = 257 Hz), 139.2 (s), 130.5 (d, J = 5.1 Hz), 120.1 (d, J = 4.5 Hz), 109.3 (s), 56.1 (s), 15.0 (s), 13.8 (s). LCMS (ESI): m/z 315.0. HRMS (ESI) calcd for C15H15FN6O (M + H)+, 315.1364; found, 315.1368. General Procedure for the Preparation of 12a,b. A mixture of methyl carbamimidothioate sulfate (1.86 g, 10.0 mol), malononitrile (990 mg, 15.0 mmol), NaOH (200 mg, 5.00 mol), and corresponding aromatic aldehyde (10.0 mmol) in anhydrous EtOH (50 mL) was heated at reflux overnight. After cooling to room temperature, the resulting precipitate was filtered, washed with EtOH, and dried under vacuum to give desired compounds. 4-Amino-6-(5-methoxypyridin-3-yl)-2-(methylthio)pyrimidine-5carbonitrile (12a). Compound 12a (380 mg, 14%) as a white solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 8.48 (s, 1H), 7.78 (s, 1H), 4.00 (s, 3H), 2.51 (s,3H). 4-Amino-6-(6-bromopyridin-2-yl)-2-(methylthio)pyrimidine-5carbonitrile (12b). Compound 12b (2.00 g, 62%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 7.6 Hz, 1H), 7.72 (dd, J = 8.0, 7.6 Hz, 1H), 7.64 (d, J = 8.0 Hz, 1H), 5.78 (br s, 2H), 2.59 (s,3H). General Procedure for the Preparation of 13a,b. To a stirred solution of 12a,b (6.21 mmol) in THF/H2O (10 mL/1 mL) was added oxone (4.80 g, 15.6 mmol). The mixture was stirred at room temperature overnight before water (200 mL) was added. The resulting precipitate was filtered, washed with H2O, and dried under vacuum to give the desired compounds. 4-Amino-6-(5-methoxypyridin-3-yl)-2-(methylsulfonyl)pyrimidine-5-carbonitrile (13a). Compound 13a (1.60 g, crude) as a white solid. 4-Amino-6-(6-bromopyridin-2-yl)-2-(methylsulfonyl)pyrimidine5-carbonitrile (13b). Compound 13b (1.83 g, crude) as a white solid. General Procedure for the Preparation of 14 and 15. To a solution of 13a (45 mg, 0.15 mmol) and corresponding pyrazole (0.16 mmol) in 5 mL anhydrous THF was added NaH (18 mg, 80%, 0.60 mmol) at 0 °C. The mixture was then warmed up to room temperature and stirred at room temperature for 6 h. The reaction mixture was poured into water (4 mL). The resulting precipitate was filtered, washed with H2O, and dried under vacuum to give the desired compounds. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(5-methoxypyridin3-yl)pyrimidine-5-carbonitrile (14). Compound 14 (13 mg, 28%) as a gray solid. 1H NMR (400 MHz, CDCl3) δ 8.87 (s, 1H), 8.50 (d, J = 2.8 Hz, 1H), 7.85 (s, 1H), 6.14 (s, 2H), 6.09 (s, 1H), 3.95 (s, 3H), 2.72 (s, 3H), 2.36 (s, 3H). LCMS (ESI): m/z 322.0. HRMS (ESI) calcd for C16H15N7O (M + H)+, 322.1411; found, 322.1414. 4-Amino-6-(5-methoxypyridin-3-yl)-2-(1H-pyrazol-1-yl)pyrimidine-5-carbonitrile (15). Compound 15 (10 mg, 23%) as a white solid. 1H NMR (400 MHz, CDCl3) δ 8.89 (s, 1H), 8.65 (s, 1H), 8.52 (s, 1H), 7.87 (s, 1H), 7.86 (s, 1H), 6.54 (s, 1H), 6.14 (br s, 2H), 3.97 (s, 3H). LCMS (ESI): m/z 294.0. 4-Amino-6-(6-bromopyridin-2-yl)-2-(3,5-dimethyl-1H-pyrazol-1yl)pyrimidine-5-carbonitrile (16). A mixture of 13b (1.83 g, 5.17 mmol) and hydrazine hydrate (1.22 g, 85%, 20.7 mmol) in EtOH (50 mL) was stirred at reflux for 2 h. The solvent was removed by vacuum and the residue was dissolved in anhydrous EtOH (50 mL). Acetyl acetone (620 mg, 6.20 mmol) was added, and the mixture was heated to reflux overnight. After cooling to room temperature, the precipitate was filtered, washed with EtOH, and dried under vacuum to give the title product (1.55 g, 81%) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 8.0 Hz, 1H), 8.02 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 6.18 (s, 1H), 5.73 (s, 2H), 2.65 (s, 3H), 2.20 (s, 3H). General Procedure for the Preparation of 17−19. A mixture of corresponding arylimidamide hydrochloride (0.30 mmol), malononitrile (30 mg, 0.45 mmol), NaOH (12 mg, 0.30 mmol) and 5methoxynicotinaldehyde (41 mg, 0.30 mmol) in anhydrous EtOH (5 mL) was heated to reflux overnight. After cooling to room temperature, the precipitate was filtered, washed with EtOH and dried under vacuum to give desired compounds. 4-Amino-6-(5-methoxypyridin-3-yl)-2-phenylpyrimidine-5-carbonitrile (17). Compound 17 (4 mg, 6%) as a white solid. 1H NMR

(400 MHz, CDCl3) δ 8.96 (s, 1H), 8.50 (d, J = 7.6 Hz, 3H), 7.91 (s, 1H), 7.56−7.49 (m, 3H), 5.79 (s, 2H), 3.98 (s, 3H). LCMS (ESI): m/ z 304.0. 4-Amino-6-(5-methoxypyridin-3-yl)-2-(pyridin-3-yl)pyrimidine-5carbonitrile (18). Compound 18 (25 mg, 31%) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.50 (d, J = 1.4 Hz, 1H), 8.78−8.71 (m, 2H), 8.66 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 2.8 Hz, 1H), 7.92 (s, 1H), 7.58 (dd, J = 8.0, 5.0 Hz, 1H), 3.94 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 166.3 (s), 164.7 (s), 163.2 (s), 155.5 (s), 152.6 (s), 150.1 (s), 141.6 (s), 140.1 (s), 136.3 (s), 133.3 (s), 132.2 (s), 124.1 (s), 120.7 (s), 116.3 (s), 86.3 (s), 56.35 (s). LCMS (ESI): m/z 305.0. HRMS (ESI) calcd for C16H12N6O (M + H)+, 305.1145; found, 305.1144. 4-Amino-6-(5-methoxypyridin-3-yl)-2-(pyridin-4-yl)pyrimidine-5carbonitrile (19). Compound 19 (45 mg, 49%) as a pale yellow solid. 1 H NMR (400 MHz, DMSO-d6) δ 8.79 (d, J = 5.8 Hz, 2H), 8.73 (s, 1H), 8.52 (d, J = 2.8 Hz, 1H), 8.23 (d, J = 5.8 Hz, 2H), 7.92 (s, 1H), 3.94 (s, 3H). LCMS (ESI): m/z 305.0. General Procedure for the Preparation of 20−37. A mixture of 16 (48 mg, 0.15 mmol), corresponding amine (0.45 mmol) and DIPEA (120 mg, 1.00 mmol) in NMP (1 mL) was stirred at 120 °C overnight. After cooling to room temperature, the mixture was diluted with ethyl acetate and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1−6% MeOH/CH2Cl2) to give the desired compounds. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-methoxypiperidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (20). Compound 20 (19 mg, 32%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.65− 7.63 (m, 2H), 6.85 (d, J = 7.0 Hz, 1H), 6.10 (s, 1H), 6.08 (s, 2H), 4.20−4.17 (m, 2H), 3.53−3.44 (m, 1H), 3.40 (s, 3H), 3.41−3.65 (m, 2H), 2.78 (s, 3H), 2.36 (s, 3H), 2.03−2.00 (m, 2H), 1.67−1.62 (m, 2H). LCMS (ESI): m/z 405.0. HRMS (ESI) calcd for C21H24N8O (M + H)+, 405.2146; found, 405.2148. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-hydroxypiperidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (21). Compound 21 (25 mg, 64%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (br s, 1H), 7.74−7.70 (m, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 6.17 (s, 1H), 4.76 (d, J = 4.0 Hz, 1H), 4.20 (d, J = 13.6 Hz, 2H), 3.74−3.71 (m, 1H), 3.21 (t, J = 11.8 Hz, 2H), 2.64 (s, 3H), 2.19 (s, 3H), 1.86−1.72 (m, 2H), 1.41−1.34 (m, 2H). LCMS (ESI): m/z 391.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(piperidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (22). Compound 22 (17 mg, 45%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (br s, 1H), 7.70 (d, J = 11.2 Hz, 1H), 7.48 (d, J = 4.0 Hz, 1H), 7.04−7.00 (m, 1H), 6.17 (s, 1H), 3.73−3.68 (m, 4H), 2.64 (s, 3H), 2.19 (s, 3H), 1.67−1.59 (m, 2H), 1.59−1.48 (m, 4H). LCMS (ESI): m/z 375.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-morpholinopyridin-2-yl)pyrimidine-5-carbonitrile (23). Compound 23 (20 mg, 35%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.69 (d, J = 4.2 Hz, 2H), 6.88−6.79 (m, 1H), 6.09 (s, 3H), 3.92−3.84 (m, 4H), 3.76−3.69 (m, 4H), 2.79 (s, 3H), 2.37 (s, 3H). LCMS (ESI): m/z 377.1. (R)-4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(3-methoxypyrrolidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (24). Compound 24 (17 mg, 29%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.63−7.56 (m, 2H), 6.55 (d, J = 8.0 Hz, 1H), 6.08 (s, 3H), 4.14 (s, 1H), 3.90−3.87 (m, 1H), 3.76−3.70 (m, 1H), 3.70−3.61 (m, 2H), 3.40 (s, 3H), 2.79 (s, 3H), 2.36 (s, 3H), 2.24−2.16 (m, 1H), 2.18−2.09 (m, 1H). 13C NMR (75 MHz, DMSO-d6) δ 166.8 (s), 166.6 (s), 156.9 (s), 156.5 (s), 151.1 (s), 150.4 (s), 143.2 (s), 138.5 (s), 116.5 (s), 111.0 (s), 110.7 (s), 110.1 (s), 83.1 (s), 79.6 (s), 56.2 (s), 52.3 (s), 45.3 (s), 30.8 (s), 15.9 (s), 13.9 (s). LCMS (ESI): m/z 391.0. HRMS (ESI) calcd for C20H22N8O (M + H)+, 391.1989; found, 391.1993. (S)-4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(3-methoxypyrrolidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (25). Compound 25 (24 mg, 41%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.66−7.54 (m, 2H), 6.55 (d, J = 7.8 Hz, 1H), 6.08 (s, 3H), 4.15−4.14 (m, 1H), 3.91−3.87 (m, 1H), 3.75−3.73 (m, 1H), 3.68− G

DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

(22 mg, 35%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.70− 7.58 (m, 2H), 6.82 (dd, J = 5.6, 2.8 Hz, 1H), 6.08 (s, 3H), 3.77 (t, J = 4.4 Hz, 4H), 2.78 (s, 3H), 2.76−2.72 (m, 1H), 2.68 (t, J = 4.4 Hz, 4H), 2.36 (s, 3H), 1.10 (d, J = 6.4 Hz, 6H). LCMS (ESI): m/z 418.1. 4-Amino-6-(6-(4-cyclopropylpiperazin-1-yl)pyridin-2-yl)-2-(3,5-dimethyl-1H-pyrazol-1-yl)pyrimidine-5-carbonitrile (36). Compound 36 (27 mg, 43%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.70−7.59 (m, 2H), 6.82 (dd, J = 6.8, 2.4 Hz, 1H), 6.08 (s, 3H), 3.73(t, J = 4.8 Hz, 4H), 2.78 (s, 3H), 2.76 (t, J = 4.8 Hz, 4H), 2.36 (s, 3H), 1.70−1.63 (m, 1H), 0.49−0.48 (m, 4H). 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-(2methoxyethyl)piperazin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile(37). Compound 37 (26 mg, 40%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.70−7.58 (m, 2H), 6.83 (dd, J = 5.6, 2.8 Hz, 1H), 6.11 (br s, 2H), 6.09 (s, 1H), 3.80 (d, J = 4.4 Hz, 4H), 3.58 (t, J = 5.6 Hz, 2H), 3.39 (s, 3H), 2.78 (s, 3H), 2.71−2.57 (m, 6H), 2.37 (s, 3H). LCMS (ESI): m/z 434.0. Pharmacological Assays. The cell and membrane preparations were performed as previously described.30,31 The detailed experimental procedures for the radioligand binding assay, cAMP assay, and haloperidol induced catalepsy animal assay can be found in the Supporting Information. DMPK Assays. The detailed experimental procedures for the CYP inhibition assays, liver microsomal assays, and LC-MS/MS analysis methods can be found in the Supporting Information.

3.65 (m, 2H), 3.40 (s, 3H), 2.79 (s, 3H), 2.36 (s, 3H), 2.24−2.14 (m, 2H). LCMS (ESI): m/z 391.0. (R)-4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(3-hydroxypyrrolidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (26). Compound 26 (17 mg, 30%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.67−7.55 (m, 2H), 6.55 (d, J = 8.0 Hz, 1H), 6.09 (s, 3H), 4.67 (s, 1H), 3.88−3.76 (m, 2H), 3.72−3.69 (m, 2H), 2.79 (s, 3H), 2.37 (s, 3H), 2.26−2.10 (m, 2H). LCMS (ESI): m/z 377.0. (S)-4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(3-hydroxypyrrolidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (27). Compound 27 (12 mg, 21%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.66−7.54 (m, 2H), 6.55 (d, J = 7.8 Hz, 1H), 6.08 (s, 3H), 4.15−4.14 (m, 1H), 3.91−3.87 (m, 1H), 3.75−3.73 (m, 1H), 3.68− 3.65 (m, 2H), 2.79 (s, 3H), 2.36 (s, 3H), 2.24−2.14 (m, 2H). LCMS (ESI): m/z 377.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(pyrrolidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (28). Compound 28 (10 mg, 18%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.61−7.59 (m, 1H), 7.54 (d, J = 7.6 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 6.09 (s, 1H), 6.07 (s, 2H), 3.62 (s, 4H), 2.78 (s, 3H), 2.36 (s, 3H), 2.05 (s, 4H). LCMS (ESI): m/z 361.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(3-hydroxyazetidin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (29). Compound 29 (5 mg, 9%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (br s, 1H), 7.75−7.71 (m, 1H), 7.51 (d, J = 7.2 Hz, 1H), 6.60 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.69 (d, J = 6.0 Hz, 1H), 4.61 (d, J = 5.2 Hz, 1H), 4.27 (t, J = 7.4 Hz, 2H), 3.88−3.71 (m, 2H), 2.65 (s, 3H), 2.19 (s, 3H). LCMS (ESI): m/z 363.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-((2-methoxyethyl) (methyl)amino)pyridin-2-yl)pyrimidine-5-carbonitrile (30). Compound 30 (16 mg, 28%) as a yellow solid.1H NMR (400 MHz, CDCl3) δ 7.65−7.57 (m, 2H), 6.71 (d, J = 8.8 Hz, 1H), 6.14 (br s, 2H), 6.07 (s, 1H), 3.95 (t, J = 5.2 Hz, 2H), 3.67 (t, J = 5.2 Hz, 2H), 3.36 (s, 3H), 3.21 (s, 3H), 2.78 (s, 3H), 2.36 (s, 3H). LCMS (ESI): m/z 379.0. HRMS (ESI) calcd for C19H22N8O (M + H)+, 379.1989; found, 379.1992. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-((2-hydroxyethyl) (methyl)amino)pyridin-2-yl)pyrimidine-5-carbonitrile (31). Compound 31 (25 mg, 46%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 1H), 7.70 (d, J = 4.2 Hz, 1H), 7.54 (s, 1H), 7.46 (d, J = 6.8 Hz, 1H), 6.85 (s, 1H), 6.16 (s, 1H), 4.62 (s, 1H), 3.70 (d, J = 4.0 Hz, 2H), 3.62 (d, J = 4.0 Hz, 2H), 3.18 (s, 3H), 2.65 (s, 3H), 2.19 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 167.0 (s), 166.6 (s), 158.0 (s), 156.9 (s), 150.8 (s), 143.1 (s), 138.7 (s), 116.6 (s), 111.2 (s), 110.7 (s), 109.6 (s), 82.8 (s), 70.9 (s), 58.6 (s), 49.6 (s), 40.5 (s), 37.7 (s), 15.6 (s), 13.7 (s). LCMS (ESI): m/z 365.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(piperazin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (32). Compound 32 (6 mg, 11%) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.48 (br s, 1H), 7.84−7.80 (m, 1H), 7.60 (d, J = 7.4 Hz, 1H), 7.14 (d, J = 8.8 Hz, 1H), 6.18 (s, 1H), 3.82 (s, 4H), 3.07 (s, 4H), 2.65 (s, 3H), 2.19 (s, 3H). LCMS (ESI): m/z 376.0. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-methylpiperazin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (33). Compound 33 (16 mg, 27%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.70− 7.59 (m, 2H), 6.83 (dd, J = 6.2, 2.8 Hz, 1H), 6.08 (s, 1H), 6.06 (br s, 2H), 3.79 (t, J = 4.4 Hz, 4H), 2.78 (s, 3H), 2.58 (t, J = 4.4 Hz, 4H), 2.37 (s, 3H), 2.36 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 166.9 (s), 166.6 (s), 158.6 (s), 156.9 (s), 150.9 (s), 150.5 (s), 143.2 (s), 139.1 (s), 116.7 (s), 112.5 (s), 110.7 (s), 110.6 (s), 82.9 (s), 55.3 (s), 54.8 (s), 46.2 (s), 45.2 (s), 40.5 (s), 15.8 (s), 13.9 (s). LCMS (ESI): m/z 390.0. HRMS (ESI) calcd for C20H23N9 (M + H)+, 390.2149; found, 390.2154. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-ethylpiperazin1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (34). Compound 34 (19 mg, 30%) as a yellow solid. 1H NMR (400 MHz, CDCl3) δ 7.71−7.60 (m, 2H), 6.82 (s, 1H), 6.08 (s, 1H), 6.07 (s, 2H), 3.79 (t, J = 4.6 Hz, 4H), 2.78 (s, 3H), 2.61 (t, J = 4.6 Hz, 4H), 2.49 (q, J = 7.2 Hz, 2H), 2.36 (s, 3H), 1.15 (t, J = 7.2 Hz, 3H). LCMS (ESI): m/z 404.1. 4-Amino-2-(3,5-dimethyl-1H-pyrazol-1-yl)-6-(6-(4-isopropylpiperazin-1-yl)pyridin-2-yl)pyrimidine-5-carbonitrile (35). Compound 35

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschemneuro.6b00218. H and 13C NMR files of synthetic intermediates and final products (PDF) 1

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AUTHOR INFORMATION

Corresponding Authors

*(X.C.Z.) Mailing address: Soochow University College of Pharmaceutical Sciences, 199 Ren Ai Road, Suzhou, Jiangsu, P.R.China 215123. E-mail: [email protected]. Telephone: (86)512 65880369. Fax: (86)512 65880369. *(X.H.Z.) Mailing address: Soochow University College of Pharmaceutical Sciences, 199 Ren Ai Road, Suzhou, Jiangsu, P.R.China 215123. E-mail: [email protected]. Telephone: (86)512 65880380. Fax: (86)512 65880380. Author Contributions

Z.Y. and L.L. contributed equally to this paper. X.H.Z. conceived the project. Z.Y., J.Z., and H.M. performed synthetic chemistry work. S.T. performed molecular modeling and computational work. J.L. and H.Z. performed and interpreted in vitro DMPK experiments. L.L. performed the pharmacology and in vivo experiments, and X.C.Z. interpreted the data. Funding

This work was supported by National Natural Science Foundation of China (Grant Nos. 81273372, 81473090, 81473278, 81130023, 31373382, 21502133, and 81502982); China Postdoctoral Science Foundation (2015T80586, 2015M581862), BM2013003, and PAPD (A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions). Notes

The authors declare no competing financial interest. H

DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

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ABBREVIATIONS B/P, brain/plasma; m-CPBA, meta-chloroperoxybenzoic acid; CYP, cytochrome P450; DIPEA, N,N-diisopropylethylamine; DMPK, drug metabolism and pharmacokinetics; L-Dopa, levodopa; GPCR, G protein-coupled receptor; HA, heavy atom; HIC, haloperidol induced catalepsy; HLM, human liver microsomes; i.p, intraperitoneal injection; LE, ligand efficiency; MLM, mouse liver microsomes; NMP, N-methyl-2-pyrrolidone; PD, Parkinson’s disease; p.o., per oral; PPB, plasma protein binding; RLM, rat liver microsomes; SAR, structure− activity relationship; SEM, standard error of the mean; THF, tetrahydrofuran

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DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acschemneuro.6b00218 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX