Development of Novel Tavapadon Analogs as Dual-targeted Partial Agonists Based on the Dopamine D₁/D₅ Receptors

Abstract

Tavapadon is a potent, selective G protein-biased partial agonist for the dopamine D₁/D₅ receptors, with positive experimental results in phase 3 trials for the treatment of Parkinson's disease (PD). This study aims to study the structure–activity relationship (SAR) of tavapadon to discover novel compounds with improved binding activity to D₁/D₅ receptors. In this work, a series of tavapadon derivatives were designed and synthesized based on the pharmacophores of tavapadon. Their binding activity to D₁/D₅ receptors was evaluated by determining in vitro median effect concentration (EC₅₀). The binding mode was predicted by molecular docking. Our data showed that among those compounds, III-1 exhibited a similar binding pose to tavapadon at D₁ dopamine receptors and demonstrated nanomolar potency for both D₁ and D₅ receptors. Compound III-1 is a potent partial agonist for the D₁/D₅ receptors, and may be a potent alternative to tavapadon for the treatment of PD in further study.

 

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Keywords

dopamine 1 receptor - dopamine 5 receptor - partial agonists - tavapadon - EC₅₀

Introduction

The dopamine receptors are the members of G protein-coupled receptors (GPCRs) and are expressed throughout the nervous system of the human.[1] [2] They are broadly categorized into two families based on their preferential G protein coupling, the D₁-like receptors (D₁ and D₅ subtypes) and the D₂-like receptors (D₂, D₃, and D₄ subtypes). The D₁-like receptors primarily couple to the Gs protein family, activating adenylyl cyclase to increase cAMP production. In contrast, the D₂-like receptors couple to Gi proteins, inhibiting adenylyl cyclase activity and reducing intracellular cAMP levels.[3] [4] The D₁ receptor-mediated dopamine signals are of great significance for brain functions such as reward, cognition, motor coordination, and neuroendocrine function, and their abnormal occurrence is closely related to some neurological and psychiatric disorders, including Parkinson's disease (PD) (characterized by decreased dopamine levels) and schizophrenia (linked to increased dopamine levels), cognitive disorders, drug abuse, and autism.[5] [6] [7] [8] [9] The D₅ receptor, like the D₁ receptor, has a distinct anatomical and functional profile and is abundantly expressed in cortical and hippocampal neurons where it powerfully modulates neuronal oscillations associated with learning and memory.[10] [11] Genetic deletion or knockdown of the D₅ receptor causes pronounced deficits in spatial and recognition memory in rodents. In the striatum, the D₅ receptor is highly enriched in cholinergic interneurons (present in approximately 88% of these cells) and influences motor output.[12] D₅ receptor loss worsens the levodopa (L-DOPA) response and markedly increases L-DOPA-induced dyskinesias (LID) in animal models of Parkinsonism.[13] D₅ receptor contributes to both cognitive and motor pathways, and pharmacologically targeting D₅ receptor may offer new therapeutic benefits for PD. However, there is a lack of highly selective small molecule compounds for D₅ receptor, making it difficult to distinguish the functional differences between D₁ and D₅ receptors in some studies.

D₁/D₅-selective partial dopamine agonists have long been considered an effective method for treating PD; however, most of the marketed dopamine agonists are selective agonists of D₂-like receptors.[14] D₁-like receptor agonists originating from catechol structures are reported, but with defects of fast metabolism, inability to cross the blood–brain barrier, and obvious adverse effects. There is no marketed drug targeting solely the D₁-like receptors in the central nervous system.[15] Therefore, discovering new agents that could activate the D₁-like receptor with minimal side effects and avoid tachyphylaxis has aroused great interest in the pharmaceutical industry.

In 2014, Pfizer Inc. primarily disclosed a series of non-catechol selective D₁ receptor agonists with a pyrimidine scaffold, with tavapadon as a representative compound, which is a partial agonist bias for Gs coupled but not β-arrestin, with a K _i = 8.54 nmol/L for the orthosteric site of D₁ receptor.[16] [17] Tavapadon has good oral pharmacokinetics and brain bioavailability and has been recently evaluated in phase III clinical trials for the treatment of PD. The result showed that tavapadon, as an adjunctive therapy to levodopa (LD), could increase total “on” time by 1.1 hours without troublesome dyskinesia and a significant reduction in “off” time compared with placebo in combination with LD. The trial reached its primary endpoint.[18] Notably, tavapadon has also been identified as a G protein-biased dopamine D₁ receptor agonist, which markedly reduced the recruitment of β-arrestin compared with traditional catechol-based D₁ receptor agonists. This signaling bias preferentially enhances G protein-mediated activation of adenylyl cyclase, resulting in increased intracellular cAMP levels—a pathway closely linked to improved motor function in established animal models of PD.[19] [20] In addition, in contrast to endogenous dopamine, tavapadon is a partial agonist that promotes cAMP accumulation and avoids excessive receptor activation. This moderation may reduce the risk of receptor desensitization and tachyphylaxis, thus supporting sustained therapeutic efficacy.

Previous studies revealed the cryo-electron microscopy structures of activated D₁ receptor-min-Gs-Nb35 complexes bound to tavapadon ([Fig. 1A]).[21] The binding pattern shows that the pyrimidine diketone group interacts with a strong hydrogen bond formed with the backbone of C186^ECL2, S188^ECL2, and the side chain of K81^2.61, while the trifluoromethyl group on the pyridine ring that binds to the OBP pocket forms a hydrogen bond with N292^6.55 through its fluorine atoms. Tavapadon's structure comprises three aromatic rings (A, B, and C) and an oxygen-containing linker region, as illustrated in [Fig. 1B]. Based on the tetracyclic scaffold model (A-ring, B-ring, C-ring, and linker region) proposed by Martini et al[22] [23] for non-catechol D₁ receptor agonists, this study systematically explores the impact of structural modifications across these modules on receptor activation.

Fig. 1 (A) Interactions between tavapadon and D₁ receptors. (Reproduced with permission from Teng et al, copyright 2022 Creative Commons.[21]) (B) Tavapadon regions (A-ring, B-ring, C-ring, and linker) investigated for structure–activity relationship.

 

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Results and Discussion

Previous explorations of A-ring modifications focused on pyridine derivatives and pyridine-fused pentacyclic systems.[23] [24] However, molecular docking analysis revealed that the interactions between the pyridine nitrogen of tavapadon and the D₁ receptor binding pocket can be ignored. Therefore, we designed two strategies to optimize the A-ring interactions: (1) replacing pyridine with nitrogen-free aromatics (e.g., benzene) to reduce electronic repulsion; (2) introducing nitrogen-rich rings (e.g., triazole) to target hydrogen bonds with Asp103 and Ser107. Thus, a series of class I compounds were designed. Given that O→N linker substitution (except for unsubstituted amines) results in a marked reduction in agonist potency (EC₅₀ increase >10-fold),[23] we will attempt to introduce methylene-containing flexible linkers to preserve hydrogen bond donor capacity while exploring conformational adaptability for balanced activity and selectivity. A series of class II compounds were then designed. To enhance polar interactions with key residues, a series of class III compounds were designed by introducing nitrogen substitutions at the para- or ortho- positions of tavapadon's B-ring core scaffold.

The synthetic routes and structure–activity relationships for classes I (A-ring modifications), II (linker flexibility), and III (B-ring nitrogen positioning) are elaborated in detail.

Structure–Activity Relationships of Tavapadon Derivatives

To investigate the impact of A-ring substitution on D₁/D₅ receptor pharmacology, we designed tavapadon analogs with diverse aromatic systems ([Table 1]), including monosubstituted benzenes, mono- or bicyclic heterocycles, and 2,6-/2,5-disubstituted derivatives. The compounds (I and II) were prepared through Suzuki-Miyaura coupling of B1/B2 with bromopyrimidine C3, followed by S_NAr with chlorinated arenes to install aromatic substituents to obtain the A − B ether bond ([Scheme 1]). As shown in [Table 1], most monosubstituted A-ring analogs failed to activate the D₁ receptor when it was converted to a benzene ring by a reduction of one nitrogen atom (e.g., I-5, I-6). Among these, analogs bearing trifluoromethyl and amino groups at ortho- position retained partial agonist activity (I-1, I-7), albeit with markedly reduced potency. Further structural exploration involving the introduction of a second substituent on the benzene ring demonstrated that simultaneous substitutions at the 2,6-positions (I-2, I-3, I-11) resulted in a complete loss of D₁ receptor activity. Moreover, dual substitutions at the 2,5-positions led to a substantial decrease in maximal agonist efficacy (I-12 versus I-7). In addition, when the C-ring was replaced with the reported imidazolopyridine (I-10) according to Davoren's exploration of the C-ring structure of tavapadon analogs,[24] the maximal agonist potency of the compounds on D₁ and D₅ receptors could be significantly reduced (D₅ cAMP EC₅₀ = 573.7 nmol/L, E_max = 50.81%) when compared with the control (D₅ cAMP EC₅₀ = 7.36 nmol/L, E_max = 95.72%). When the A-ring was introduced into a five-membered or 6–5 fused bicyclic heterocycle ring rich in nitrogen atoms (I-8 versus I-1), the agonistic activity of the compounds on D₁ and D₅ receptors was completely lost.

Scheme 1 Synthesis route of compounds I and II. Reagents and conditions: (a) MeONa, MeOH, 60°C, 18 hours; then, HCl, 1 hour; (b) NaNO_2, CuBr₂, ACN/H₂O, 0–25°C, 48 hours; (c) chloromethyl pivalate, DBU, ACN, 60°C, 4 hours; (d) PdCl₂(dppf), Cs₂CO₃, 100°C, 6 hours; (e) Pd(OH)₂, MeOH, H₂, 25°C, 12 hours; (f) NaOH, MeOH, 25°C, 1 hour; (g) Cs₂CO₃, DMSO, 130°C, 16 hours; (h) Cs₂CO₃, DMSO, 100°C, 6 hours.

Table 1 Structure–activity relationships of the A-ring of the compounds I

Comp ID	Substituted A-ring	Substituted C-ring	D1 cAMP agonist assay		D5 cAMP agonist assay
			EC50 (nmol/L)	Emax (%)	EC50 (nmol/L)	Emax (%)
I-1			67.29	88.00	7.36	95.72
I-2			>1,000	7.49	>1,000	9.49
I-3			>1,000	4.45	>1,000	25.00
I-4			>1,000	18.19	5.22	38.89
I-5			>1,000	17.76	54.87	89.80
I-6			>1,000	34.64	15.79	93.41
I-7			180.20	75.38	3.43	98.81
I-8			>1,000	6.47	>1,000	6.99
I-9			>1,000	9.33	>1,000	48.53
I-10			>1,000	2.30	573.7	50.81
I-11			>1,000	21.95	55.99	78.12
I-12			419.6	30.61	22.85	97.56
Tavapadon			6.86	86.98	0.34	99.24

In this series, we further assessed the effect of extending the linker (II-1, II-2), the positional effect of the trifluoromethyl group on the extended linker scaffold (II-3), and the nitrogen–oxygen substitution within the extended linker architecture (II-4) on the binding activity of compounds ([Table 2]). We found reduced D₁/D₅ receptor affinity of II-1, II-2, and II-3 when compared with the control (tavapadon). In addition, the N-linker has a higher affinity for the D₅ receptor than the O-linker (II-3 versus II-4), and the D₅ receptor activity was enhanced when the trifluoromethyl group occupied the proximal position (II-1 versus II-3).

To determine the effects of the middle phenyl ring on functional selectivity, we designed a set of compounds (a class III) with differently substituted phenyl rings ([Table 3]), and their synthetic route is outlined in [Scheme 2]. It is interesting to note that when a nitrogen atom is introduced in the middle benzene ring in the para- position of the methyl group (III-1), the agonist activity on D₁ and D₅ receptors were greatly decreased (EC₅₀ = 45.57 and 5.96 nmol/L, respectively) when compared with the control (EC₅₀ = 6.25 and 0.46 nmol/L, respectively). However, the maximum effect is increased, which may contribute to the enhanced intrinsic activity of the compound. When the C-ring structure is bicyclic, ortho-nitrogen-substituted analogs displayed enhanced D₅ receptor agonism when it is in the ortho- position of the methyl group of the intermediate benzene ring (III-4 versus III-7). When the C-ring structure is monocyclic, monocyclic C-ring analogs with para-nitrogen substitution showed better activity when it is in the para- position of the methyl group of the intermediate benzene ring (III-2 versus III-5). Finally, of the series of C-ring structures containing multiple nitrogen atoms that we tried to use, the one with the highest affinity for D₁-like R was 1,5-dimethylpyrimidine-2,4(1H,3H)-dione (C2, e.g., III-1).

Scheme 2 Synthesis of compounds III. Reagents and conditions: (a) Cs₂CO₃, DMSO, 100°C, 6 hours; (b) PdCl₂(dppf), Cs₂CO₃, bis(pinacolato)diboron, 100°C, 6 hours. (c) PdCl₂(dppf), Cs₂CO₃, 125°C, 10 hours.

Fig. 2 The binding pose between compound III-1 and D₁ receptor.

Among the compounds, III-1 showed the best functional activities on D₁/D₅ receptors and was thus chosen for further study.

 

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Docking Results of III-1 and Tavapadon on Selected Frames of D₁ Receptor

We performed a molecular docking experiment of compound III-1 to compare its binding pose with that of tavapadon (PDB ID: 7 × 2D) as a control ligand on D₁ receptor. III-1 has an EC₅₀ value of 45.47 nmol/L for D₁ receptor, and E_max 98.83% compared with tavapadon (86.51%), demonstrating a significantly enhanced maximum agonistic effect, while still maintaining its partial agonism. Compound III-1 has the same binding pose as tavapadon on D₁ receptor ([Fig. 2]). The N atom of the B ring could interact with another hydrogen bond formed with the D103^3.32 compared with the binding of tavapadon on D₁ receptor, which means that III-1 may have a stronger binding ability with D₁ receptor.

 

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Conclusion

In summary, we conducted a comprehensive SAR campaign in four regions of tavapadon. By design, synthesis, and biological characterization of 23 analogs, we found compound III-1 as a partial agonist with a nanomolar potent for D₁/D₅ receptors in vitro. Besides, compound III-1 has the same protein binding pose as tavapadon in D₁ receptors and may be a potent D₁/D₅ receptors partial agonist for further study.

 

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Experimental Section

Material and General Procedures

All solvents and reagents were obtained from commercial sources and were used as received. The ¹H NMR and ¹³C NMR spectra were determined using a Bruker AC-600P spectrometer (Bruker, Germany) (400 MHz for ¹H, and 151 MHz for ¹³C). The solvent used for NMR spectra was CDCl₃, DMSO-d ₆, and MeOH-d ₄ with tetramethylsilane as the internal standard. Chemical shift (δ) is expressed in units of parts per million (ppm). Multiplicities are abbreviated as singlet (s), doublet (d), triplet (t), quartet (q), and multiplet (m). Coupling constants (J) are reported in Hertz (Hz). HRMS data were measured using an Agilent Technologies 6538 UHD accurate-Mass Q-TOF MS spectrometer with electrospray ionization. Melting point (mp) was obtained on a WRS-2A microcomputer melting point meter (Shanghai INESA Physico-Optical Instrument Co., Ltd., Shanghai, China).

 

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Reversed-Phase Analytical HPLC

Analytical HPLC was run on a Waters e2695 LC instrument (Waters, United States) using an analytical column (Welch Ultimate XB-C18 4.6 × 250 mm, 5 μm) with a flow rate of 1.0 mL/min at room temperature. Analytical feeds were monitored at 214 and 254 nm. The mobile phases were 0.1% TFA (v/v) in acetonitrile (solvent A) and 0.1% TFA (v/v) in water (solvent B). A linear gradient of 80 to 20% B was used for 35 minutes at room temperature. The purity of all target compounds (>90%) was confirmed by analytical RP-HPLC (Waters XBridge C18 column, 4.6 × 150 mm, 5 μm) with UV detection at 254 nm. Representative chromatograms are provided in “[Supporting Information]” (available in the online version).

 

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General Synthesis of I-1∼I-10

To a stirred solution of 6-(4-hydroxy-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione (BC-3) (0.405 mmol, 1.0 equiv.) and halogenated aromatic compound (0.607 mmol, 1.5 equiv.) in DMSO (5 mL) was added cesium carbonate (1.214 mmol, 3.0 equiv.). The mixture was heated to 130°C for 16 hours, cooled, and water (30 mL) was added. The aqueous layer was extracted with ethyl acetate (30 mL × 4). The combined organic layers were dried over sodium sulfate, filtered, concentrated, and purified via silica gel chromatography (gradient 30 to 60% ethyl acetate in heptanes) to obtain the target product.

The halogenated aromatic compound used for the synthesis of I-1 was 1-chloro-2-(trifluoromethyl)benzene. Compound I-1, chemically named “1,5-dimethyl-6-(2-methyl-4-(2-(trifluoromethyl)phenoxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a white solid. Yield: 68% (107 mg, 0.275 mmol). mp: 239.6–242.3°C. ¹H NMR (400 MHz, CDCl₃) δ 9.11 (s, 1H), 7.72 (dd, J = 7.9, 1.7 Hz, 1H), 7.54 (td, J = 7.9, 1.7 Hz, 1H), 7.26 (dt, J = 15.3, 0.9 Hz, 1H), 7.06 (dd, J = 13.1, 8.3 Hz, 2H), 7.00 (d, J = 2.5 Hz, 1H), 6.95 (dd, J = 8.3, 2.5 Hz, 1H), 3.02 (s, 3H), 2.16 (s, 3H), 1.65 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.73, 157.96, 154.18, 151.34, 150.94, 137.60, 133.45, 129.36, 127.82, 127.53, 127.48, 123.81, 120.79, 120.23, 116.94, 109.74, 31.78, 16.94, 12.20.

The halogenated aromatic compound used for the synthesis of I-2 was 2-bromo-1,3-bis (trifluoromethyl)benzene. Compound I-2, chemically named “6-(4-(2,6-bis(trifluoromethyl)phenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a white solid. Yield: 74% (137 mg, 0.300 mmol). mp: 147.3–149.2°C. ¹H NMR (400 MHz, CDCl₃) δ 8.36 (s, 1H), 7.98 (d, J = 7.9 Hz, 2H), 7.56 (t, J = 7.9 Hz, 1H), 6.98 (d, J = 8.5 Hz, 1H), 6.80 (d, J = 2.6 Hz, 1H), 6.66 (dd, J = 8.5, 2.6 Hz, 1H), 2.97 (s, 3H), 2.12 (s, 3H), 1.26 (d, J = 1.7 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.40, 151.08, 137.05, 131.77, 128.82, 126.77, 126.05, 118.00, 114.03, 109.71, 32.64, 19.20, 11.59.

The halogenated aromatic compound used for the synthesis of I-3 was 1-bromo-2-fluoro-3-trifluoromethylbenzene. Compound I-3, chemically named “6-(4-(2-bromo-6-(trifluoromethyl)phenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a white solid. Yield: 67% (127 mg, 0.271 mmol). mp: 172.2–173.9°C. ¹H NMR (400 MHz, CDCl₃) δ 7.87 (dd, J = 8.0, 1.6 Hz, 1H), 7.72 (dd, J = 8.0, 1.5 Hz, 1H), 7.29 (td, J = 8.0, 0.9 Hz, 1H), 6.98 (dd, J = 8.4, 4.5 Hz, 1H), 6.82 (d, J = 2.6 Hz, 1H), 6.69 (dt, J = 8.5, 2.1 Hz, 1H), 5.60 (s, 1H), 3.02 (s, 2H), 2.13 (s, 3H), 1.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.50, 157.25, 150.82, 150.19, 149.06, 148.22, 137.07, 136.26, 128.01, 125.76, 125.73, 118.11, 116.93, 112.92, 108.37, 65.39, 32.32, 31.69, 18.32, 11.04.

The halogenated aromatic compound used for the synthesis of I-4 was 2-bromo-3-fluorobenzotrifluoride. Compound I-4, chemically named “6-(4-(2-bromo-3-(trifluoromethyl)phenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione”, is a white solid. Yield: 81% (153 mg, 0.328 mmol). mp: 268.3–270.2°C. ¹H NMR (400 MHz, CDCl₃) δ 8.97 (s, 1H), 7.56 (dd, J = 7.9, 1.1 Hz, 1H), 7.44 (t, J = 8.0 Hz, 1H), 7.23 (dd, J = 8.1, 1.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.4, 2.5 Hz, 1H), 3.02 (s, 3H), 2.16 (s, 3H), 1.65 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.61, 156.64, 153.18, 150.26, 149.80, 136.77, 128.49, 127.57, 126.78, 123.30, 122.67, 122.61, 120.28, 118.78, 114.94, 113.11, 108.74, 31.78, 18.25, 10.74.

The halogenated aromatic compound used for the synthesis of I-5 was 3-chlorobenzotrifluoride. Compound I-5, chemically named “1,5-dimethyl-6-(2-methyl-4-(3-(trifluoromethyl)phenoxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a yellow solid. Yield: 74% (117 mg, 0.300 mmol). mp: 248.2–250.2°C. ¹H NMR (400 MHz, CDCl₃) δ 8.86 (s, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.33 (s, 1H), 7.23 (d, J = 8.1 Hz, 1H), 7.08 (d, J = 8.3 Hz, 1H), 6.98 (d, J = 1.9 Hz, 1H), 6.95 (dd, J = 8.3, 2.3 Hz, 1H), 3.03 (s, 3H), 2.16 (s, 3H), 1.66 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.59, 157.89, 156.41, 151.25, 150.91, 137.73, 130.63, 129.49, 127.75, 122.68, 120.85, 120.81, 120.53, 116.73, 116.58, 116.54, 109.75, 32.81, 19.26, 11.76.

The halogenated aromatic compound used for the synthesis of I-6 was 1-chloro-2-nitrobenzene. Compound I-6, chemically named “1,5-dimethyl-6-(2-methyl-4-(2-nitrophenoxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a yellow solid. Yield: 80% (119 mg, 0.324 mmol). ¹H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H), 8.01 (dd, J = 8.2, 1.6 Hz, 1H), 7.61 (td, J = 8.3, 1.6 Hz, 1H), 7.36–7.29 (m, 1H), 7.16 (dd, J = 8.3, 1.0 Hz, 1H), 7.09 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 6.96 (dd, J = 8.3, 2.4 Hz, 1H), 3.02 (s, 3H), 2.16 (s, 3H), 1.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.74, 157.49, 151.36, 150.75, 149.23, 142.00, 137.81, 134.46, 129.50, 128.16, 126.00, 124.54, 122.01, 120.35, 116.49, 109.77, 32.82, 19.26, 11.76.

The halogenated aromatic compound used for the synthesis of I-8 was 3-chloro-[1,2,4]triazolo[4,3-a]pyridine. Compound I-8, chemically named “6-(4-([1,2,4]triazolo[4,3-a]pyridin-3-yloxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a light yellow solid. Yield: 35% (51 mg, 0.142 mmol). mp: 226.6–228.6°C. ¹H NMR (400 MHz, CDCl₃) δ 8.92 (s, 1H), 7.97 (d, J = 6.9 Hz, 1H), 7.69 (d, J = 9.4 Hz, 1H), 7.53 (d, J = 2.5 Hz, 1H), 7.48 (dd, J = 8.4, 2.5 Hz, 1H), 7.31–7.24 (m, 1H), 7.18 (d, J = 8.4 Hz, 1H), 6.88 (t, J = 6.7 Hz, 1H), 3.02 (s, 3H), 2.22 (s, 3H), 1.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.57, 154.91, 151.20, 150.55, 137.83, 129.47, 127.44, 120.99, 120.47, 116.92, 116.66, 113.73, 109.76, 32.86, 19.34, 11.71.

The halogenated aromatic compound used for the synthesis of I-9 was 5-chloro-1-methyl-1H-1,2,4-triazole. Compound I-9, chemically named “1,5-dimethyl-6-(2-methyl-4-((1-methyl-1H-1,2,4-triazol-5-yl)oxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a light brown solid. Yield: 32% (42 mg, 0.130 mmol). mp: 226.4–229.2°C. ¹H NMR (400 MHz, CDCl₃) δ 9.42 (s, 1H), 7.63 (s, 1H), 7.32 (d, J = 7.9 Hz, 2H), 7.18–7.12 (m, 1H), 3.81 (s, 3H), 3.00 (s, 3H), 2.20 (s, 3H), 1.62 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.81, 156.45, 154.62, 151.39, 150.53, 148.14, 137.68, 129.51, 129.37, 121.13, 117.50, 109.78, 33.59, 32.85, 19.30, 11.71.

The materials used for synthesis of I-10 were 4-(imidazo[1,2-a]pyridin-5-yl)-3-methylphenol (BC-7) and 1-chloro-2-(trifluoromethyl)benzene. Compound I-10, chemically named “5-(2-methyl-4-(2-(trifluoromethyl)phenoxy)phenyl)imidazo[1,2-a]pyridine,” is a little brown solid. Yield: 67% (101 mg, 0.271 mmol). mp: 189.3–190.6°C. ¹H NMR (400 MHz, CDCl₃) δ 7.73 (d, J = 7.7 Hz, 1H), 7.65 (s, 1H), 7.57–7.44 (m, 2H), 7.35–7.24 (m, 3H), 7.15 (s, 1H), 7.10 (d, J = 8.2 Hz, 1H), 7.04 (d, J = 1.8 Hz, 1H), 6.96 (dd, J = 8.3, 2.1 Hz, 1H), 6.76 (d, J = 6.5 Hz, 1H), 2.07 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.10, 154.40, 145.57, 139.39, 137.50, 133.43, 132.15, 132.06, 131.29, 128.58, 128.46, 127.48, 127.43, 123.67, 120.72, 120.26, 116.56, 116.36, 111.27, 19.30.

 

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General Synthesis of I-7, I-11, I-12

To a stirred solution of BC-3 (0.405 mmol, 1.0 equiv.) and halogenated aromatic compound (0.607 mmol, 1.5 equiv.) in DMSO (5 mL) was added cesium carbonate (1.214 mmol, 3.0 equiv.). The mixture was heated to 130°C for 16 hours, cooled, and water (30 mL) was added. The aqueous layer was extracted with ethyl acetate (30 mL × 4). The combined organic layers were dried over sodium sulfate, filtered, concentrated, and purified via silica gel chromatography (gradient 30% to 60% ethyl acetate in heptanes) to obtain the key intermediate, which was dispersed in methanol in the presence of palladium 10% on carbon (wetted with ca. 55% water) (0.1 equiv., wt./wt. relative to the key intermediate), and stirred under an atmosphere of hydrogen at 25°C for 16 hours. The reaction mixture was then filtered through a pad of Celite, concentrated, and purified by silica gel chromatography (gradient 2 to 10% MeOH in DCM) to obtain the target compound.

The halogenated aromatic compound used for the synthesis of I-7 was 1-chloro-2-nitrobenzene. Compound I-7, chemically named “6-(4-(2-aminophenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a light yellow solid. Yield: 92% (25 mg, 0.074 mmol). mp: 176.4–179.6°C. ¹H NMR (400 MHz, CDCl₃) δ 8.70 (s, 1H), 7.05 (td, J = 7.8, 1.4 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.94 (dd, J = 8.0, 1.1 Hz, 2H), 6.90 (ddd, J = 7.7, 5.6, 1.9 Hz, 2H), 6.79 (td, J = 7.8, 1.5 Hz, 1H), 3.01 (s, 3H), 2.12 (s, 3H), 1.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.55, 157.71, 150.25, 150.20, 141.12, 137.44, 136.26, 128.19, 125.50, 124.73, 119.92, 118.29, 117.63, 116.01, 113.99, 108.68, 31.78, 18.25, 10.75.

The halogenated aromatic compound used for the synthesis of I-11 was 2-chloro-1-nitro-3-(trifluoromethyl)benzene. Compound I-11, chemically named “6-(4-(2-amino-6-(trifluoromethyl)phenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a yellow oil. Yield: 47% (77 mg, 0.190 mmol). ¹H NMR (400 MHz, DMSO-d ₆) δ 11.41 (s, 1H), 7.17 (d, J = 8.5 Hz, 1H), 6.98–6.91 (m, 3H), 6.86 (dd, J = 8.8, 2.9 Hz, 1H), 6.80 (dd, J = 8.4, 2.6 Hz, 1H), 5.59 (s, 0H), 2.83 (s, 3H), 2.09 (s, 3H), 1.44 (s, 3H). ¹³C NMR (101 MHz, DMSO-d ₆) δ 164.10, 159.53, 151.64, 150.88, 146.33, 142.17, 137.56, 129.93, 127.24, 123.93, 119.20, 118.60, 114.93, 111.37, 111.32, 108.20, 32.66, 19.11, 11.97.

The halogenated aromatic compound used for the synthesis of I-12 was 2-chloro-1-nitro-4-(trifluoromethyl)benzene. Compound I-12, chemically named “6-(4-(2-amino-5-(trifluoromethyl)phenoxy)-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione,” is a yellow oil. Yield: 44% (72 mg, 0.178 mmol). ¹H NMR (400 MHz, DMSO-d ₆) δ 11.42 (s, 1H), 7.28 (dd, J = 8.6, 2.1 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.13 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 2.5 Hz, 1H), 6.93 (d, J = 8.4 Hz, 1H), 6.85 (dd, J = 8.4, 2.6 Hz, 1H), 5.76 (s, 2H), 2.82 (s, 2H), 2.11 (s, 3H), 1.45 (s, 3H). ¹³C NMR (101 MHz, DMSO-d ₆) δ 164.10, 158.08, 151.64, 150.89, 145.04, 140.48, 137.59, 130.02, 127.57, 126.47, 123.24, 118.94, 118.07, 115.95, 115.67, 115.03, 108.20, 32.69, 19.17, 11.98.

 

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General Synthesis of II-1∼II-4

To a stirred solution of BC-3 (0.405 mmol, 1.0 equiv.) and halogenated aliphatic compound (0.607 mmol, 1.5 equiv.) in DMSO (5 mL) was added cesium carbonate (1.214 mmol, 3.0 equiv.). The mixture was heated to 100°C for 6 hours, cooled, and water (30 mL) was added. The aqueous layer was extracted with ethyl acetate (30 mL × 4). The combined organic layers were dried over sodium sulfate, filtered, concentrated, and purified via silica gel chromatography (gradient 30 to 60% ethyl acetate in heptanes) to obtain the target product.

The halogenated aliphatic compound used for the synthesis of II-1 was 2-chloromethyl-3-(trifluoromethyl)pyridine. Compound II-1, chemically named “1,5-dimethyl-6-(2-methyl-4-((3-(trifluoromethyl)pyridin-2-yl)methoxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a light brown solid. Yield: 92% (151 mg, 0.373 mmol). mp: 247.4–248.9°C. ¹H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H), 8.89–8.81 (m, 1H), 8.06 (dd, J = 7.9, 1.6 Hz, 1H), 7.47 (dd, J = 8.0, 4.9 Hz, 1H), 7.08–6.87 (m, 3H), 5.37 (s, 2H), 2.99 (s, 3H), 2.13 (s, 3H), 1.62 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.91, 159.43, 157.57, 153.78, 152.32, 151.54, 151.45, 136.89, 134.83, 134.78, 128.97, 125.43, 123.15, 117.24, 113.09, 109.76, 68.85, 68.83, 32.77, 19.31, 11.72.

The halogenated aliphatic compound used for the synthesis of II-2 was 2-(trifluoromethyl)benzyl chloride. Compound II-2, chemically named “1,5-dimethyl-6-(2-methyl-4-((2-(trifluoromethyl)benzyl)oxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a light brown solid. Yield: 96% yield (157 mg, 0.389 mmol). ¹H NMR (400 MHz, CDCl₃) δ 7.74 (dd, J = 12.0, 7.8 Hz, 2H), 7.60 (t, J = 7.6 Hz, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 6.98–6.88 (m, 2H), 5.29 (s, 2H), 3.00 (s, 3H), 2.15 (s, 3H), 1.63 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.73, 159.28, 151.46, 151.32, 137.03, 132.25, 129.09, 128.73, 128.02, 126.10, 126.04, 125.41, 117.10, 113.09, 113.09, 109.74, 66.24, 66.21, 32.76, 19.34, 11.73.

The halogenated aliphatic compound used for the synthesis of II-3 was 2-chloromethyl-6-(trifluoromethyl)pyridine. Compound II-3, chemically named “1,5-dimethyl-6-(2-methyl-4-((6-(trifluoromethyl)pyridin-2-yl)methoxy)phenyl)pyrimidine-2,4(1H,3H)-dione,” is a light brown solid. Yield: 96% (157 mg, 0.324 mmol). ¹H NMR (400 MHz, CDCl₃) δ 9.29 (s, 1H), 7.95 (t, J = 7.8 Hz, 1H), 7.78 (d, J = 7.9 Hz, 1H), 7.65 (dd, J = 7.8, 1.0 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 6.94 (dd, J = 8.4, 2.6 Hz, 1H), 5.29 (s, 2H), 2.99 (s, 3H), 2.15 (s, 3H), 1.62 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 162.87, 157.98, 156.82, 150.45, 150.31 137.38, 136.14, 128.17, 124.61, 122.91, 118.49, 118.46, 116.04, 112.05, 108.76, 69.08, 31.72, 18.32, 10.69.

The materials used for synthesis of II-4 were 6-(4-amino-2-methylphenyl)-1,5-dimethylpyrimidine-2,4(1H,3H)-dione (BC-5) and 2-chloromethyl-6-(trifluoromethyl)pyridine. Compound II-4, chemically named “1,5-dimethyl-6-(2-methyl-4-(((6-(trifluoromethyl)pyridin-2-yl) methyl) amino) phenyl) pyrimidine-2,4(1H,3H)-dione,” is a light brown solid. Yield: 55% (91 mg, 0.224 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.93 (s, 1H), 7.87 (t, J = 7.8 Hz, 1H), 7.58 (dd, J = 24.1, 7.8 Hz, 2H), 6.86 (d, J = 8.0 Hz, 1H), 6.68–6.56 (m, 2H), 4.56 (s, 2H), 3.00 (s, 3H), 2.07 (s, 3H), 1.64 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 163.91, 158.91, 152.30, 151.46, 148.56, 147.98, 147.64, 138.13, 136.30, 128.81, 124.33, 121.74, 119.03, 114.61, 111.11, 109.76, 48.59, 32.73, 19.30, 11.78.

 

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Synthesis of III-1∼III-7

To a stirred solution of 2-chloro-3-(trifluoromethyl)pyridine (100 mg, 0.551 mmol) and 5-bromo-2-hydroxy-4-methylpyridine (86 mg, 0.459 mmol) in DMSO (5 mL) was added cesium carbonate (448 mg, 1.377 mmol). The reaction mixture was heated to 100°C for 6 hours, cooled, and water (30 mL) was added. The organic layer was extracted with ethyl acetate and concentrated in vacuo to give a residue of AB-1.

To an oven-dried Schleck tube with a stirring bar was added [1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium (17 mg, 0.03 mmol), cesium carbonate (222 mg, 0.684 mmol), 6-bromo-1,5-dimethylpyrimidine-2,4(1H,3H)-dione (C2) (50 mg, 0.228 mmol), and a solution of AB-1 residue (91 mg, 0.273 mmol) in dioxane (1 mL) and water (0.2 mL). The mixture was exchanged with nitrogen, heated at 125°C for 14 hours, and subsequently filtered through Celite. The filter was washed with ethyl acetate and extracted with ethyl acetate. The combined organic layers were washed with brine and saturated aqueous NaHCO₃, dried over anhydrous Na₂SO₄, concentrated, and purified via silica gel chromatography (gradient 30% to 60% ethyl acetate in heptanes) to give the target product.

Compound III-1, chemically named “1,5-dimethyl-6-(4-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)pyrimidine-2,4(1H,3H)-dione,” is a yellow oil. Yield: 32% (29 mg, 0.073 mmol). ¹H NMR (400 MHz, MeOH-d ₄) δ 8.46 (dd, J = 5.0, 1.8 Hz, 1H), 8.27 (dd, J = 7.7, 1.8 Hz, 1H), 8.13 (s, 1H), 7.44 (dd, J = 7.8, 5.0 Hz, 1H), 7.29 (s, 1H), 4.59 (s, 1H), 3.05 (s, 3H), 2.32 (s, 3H), 1.65 (s, 3H). ESI-HRMS (m/z): calcd. for C₁₈H₁₆F₃N₄O₃ ⁺ [M + H]⁺ 393.11690, found 393.11653, error ppm −0.94.

Compound III-2 was prepared using the same procedure as outlined above for the preparation of III-1, starting with the 5-bromo-4,6-dimethylpyrimidine (50 mg, 0.267 mmol) and the key intermediate AB-1 (107 mg, 0.320 mmol). Compound III-2, chemically named “4,6-dimethyl-5-(4-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)pyrimidine,” is a clear oil. Yield: 53% (51 mg, 0.142 mmol). ¹H NMR (400 MHz, CDCl₃) δ 9.13 (s, 1H), 8.58 (dd, J = 5.0, 1.9 Hz, 1H), 8.21 (dd, J = 7.6, 1.9 Hz, 1H), 8.10 (s, 1H), 7.42–7.36 (m, 2H), 2.38 (s, 6H), 2.22 (s, 3H), 1.37 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 165.44, 161.53 160.07, 157.52, 151.35, 149.54, 147.45, 137.37, 137.32, 129.28, 129.07, 119.63, 116.70, 116.31, 115.49, 29.65, 22.82, 19.41.

Compound III-3 was prepared using the same procedure as outlined above for the preparation of III-1, starting with the 5-bromo-6-methylimidazo[1,2-a]pyrazine (100 mg, 0.472 mmol) and the key intermediate AB-1 (189 mg, 0.566 mmol). Compound III-3, chemically named “6-methyl-5-(4-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)imidazo[1,2-a]pyrazine,” is a yellow oil. Yield: 40% (73 mg, 0.189 mmol). ¹H NMR (400 MHz, CDCl₃) δ 9.12 (s, 1H), 8.46 (dd, J = 4.8, 1.4 Hz, 1H), 8.10 (dd, J = 7.6, 1.4 Hz, 1H), 7.76 (d, J = 0.8 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.28 (dd, J = 7.6, 5.0 Hz, 1H), 7.13 (t, J = 4.1 Hz, 2H), 2.36 (s, 3H), 2.20 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.54, 158.71, 157.22, 142.34, 141.44, 140.11, 137.45, 137.40, 136.73, 135.87, 125.18, 123.06, 119.81, 112.54, 112.18, 21.76, 20.12.

Compound III-4 was prepared using the same procedure as outlined above for the preparation of III-1, starting with the 3-bromo-6-hydroxy-2-methylpyridine (100 mg, 0.534 mmol) and 5-bromoimidazo[1,2-a]pyridine (107 mg, 0.445 mmol). Compound III-4, chemically named “5-(2-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)imidazo[1,2-a]pyridine,” is a light yellow oil. Yield: 67% (110 mg, 0.298 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.46–8.40 (m, 1H), 8.08 (dd, J = 7.7, 1.9 Hz, 1H), 7.75 (d, J = 8.2 Hz, 2H), 7.65 (s, 1H), 7.30 (dd, J = 9.1, 6.8 Hz, 1H), 7.27–7.23 (m, 1H), 7.18 (s, 1H), 7.07 (d, J = 8.2 Hz, 1H), 6.76 (dd, J = 6.8, 1.0 Hz, 1H), 2.24 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 161.12, 158.93, 156.97, 151.17, 141.30, 137.43, 137.38, 135.36, 133.58, 125.91, 119.58, 117.14, 113.78, 112.15, 111.06, 36.64, 36.50, 27.71, 27.63, 22.13.

Compound III-5 was prepared using the same procedure as outlined above for the preparation of III-1, starting with 3-bromo-6-hydroxy-2-methylpyridine (100 mg, 0.534 mmol) and 5-bromo-4,6-dimethylpyrimidine (83 mg, 0.445 mmol). Compound III-5, chemically named “4,6-dimethyl-5-(2-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)pyrimidine,” is a clear oil. Yield: 69% (110 mg, 0.307 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.99 (s, 1H), 8.41 (dd, J = 5.1, 1.9 Hz, 1H), 8.07 (dd, J = 7.7, 1.9 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.23 (dd, J = 7.6, 5.1 Hz, 1H), 7.03 (d, J = 8.2 Hz, 1H), 2.26 (s, 6H), 2.18 (s, 3H). 2.26 (s, 6H), 2.18 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 164.97, 160.25, 159.04, 157.30, 155.36, 151.13, 140.28, 137.34, 137.30, 130.85, 127.84, 123.93, 119.33, 116.18, 115.85, 112.43, 22.80, 22.19.

Compound III-6 was prepared using the same procedure as outlined above for the preparation of III-1, starting with 3-bromo-6-hydroxy-2-methylpyridine (100 mg, 0.534 mmol) and 2-amino-6-bromo-5-methylpyrazine (84 mg, 0.445 mmol). Compound III-6, chemically named “5-methyl-6-(2-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)pyrazin-2-amine,” is a yellow oil. Yield: 60% (96 mg, 0.267 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.36 (dd, J = 5.1, 1.9 Hz, 1H), 8.03 (dd, J = 7.7, 1.9 Hz, 1H), 7.94 (s, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.22–7.17 (m, 1H), 6.97 (d, J = 8.2 Hz, 1H), 4.71 (s, 2H), 2.29 (s, 3H), 2.25 (s, 3H). ¹³C NMR (101 MHz, CDCl₃) δ 158.85, 158.25, 158.23, 154.75, 151.18, 150.04, 147.34, 139.38, 139.35, 136.19, 126.96, 129.86, 118.07, 110.83, 21.30, 19.45.

Compound III-7 was prepared using the same procedure as outlined above for the preparation of III-1, starting with 5-bromoimidazo[1,2-a]pyridine (50 mg, 0.254 mmol) and the key intermediate AB-1 (102 mg, 0.305 mmol). Compound III-7, chemically named “5-(4-methyl-6-((3-(trifluoromethyl)pyridin-2-yl)oxy)pyridin-3-yl)imidazo[1,2-a]pyridine,” is a clear oil. Yield: 38% (36 mg, 0.096 mmol). ¹H NMR (400 MHz, CDCl₃) δ 8.48 (dd, J = 5.0, 1.9 Hz, 1H), 8.25 (s, 1H), 8.10 (dd, J = 7.6, 2.0 Hz, 1H), 7.67 (s, 1H), 7.32–7.27 (m, 2H), 7.17 (s, 2H), 6.79 (s, 1H), 2.17 (s, 3H). ESI-HRMS (m/z): calcd. for C₁₉H₁₅F₃N₄O⁺ [M + H]⁺ 371.11142, found 371.10993, error ppm −4.02.

 

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D₁/D₅ Receptors Functional Activity Assay

Human dopamine D₁/D₅ receptor agonist activity of the compounds was measured using the Cisbio Dynamic 3′-5′-cyclic adenosine monophosphate (cAMP) homogeneous time-resolved fluorescence (HTRF) competitive immunoassay detection kit (Cisbio International 62AM4PEJ) (Revvity, Shanghai, China) to determine dopamine cAMP levels according to the manufacturer's suggested protocol with minor amendments. Suspended CHO-K1/D1 cells and CHO-K1/D5/Gα15 cells (GenScript, Nanjing, Jiangsu, China) were added to a centrifuge tube containing 10 mL Hanks' Balanced Salt Solution (HBSS) and centrifuged at 750 rpm for 5 minutes. The supernatant was discarded, the precipitate was resuspended in an appropriate amount of experimental buffer, and 20 μL was taken and counted with a cell counter (Countstar, Shanghai, China). An appropriate amount of cell suspension (10 μL) was added to each well of the cell plate, and centrifuged at 1,000 rpm for 1 minute. The compound was added using Tecan-D3000 (Tecan, Männedorf, Swiss). The cell plate was centrifuged at 1,000 rpm for 1 minute, sealed, and incubated at room temperature for 45 minutes. An appropriate amount of cAMP-D2 storage solution and Anti-cAMP-Cryptate storage solution (Cisbio, Shanghai, China) was taken, diluted with lysis buffer at a ratio of 1:20, and then the two solutions were mixed upside down in a 1:1 ratio, avoiding vortexing. After adding 10 μL of prepared detection reagent to the cell plate, centrifugation was done at 1,000 rpm for 1 minute. The cell plate was incubated at room temperature in the dark for 1 hour. After centrifuging the cell plate at 1,000 rpm for 1 minute, the plate was read using Envision (PerkinElmer, Shanghai, China). The measurements were performed with an excitation wavelength of 340 nm and emission wavelengths of 620 and 665 nm. The ratio of two channel signals (665 nm/620 nm) was multiplied by 10,000 as the final raw data for analysis. EC₅₀ values were determined using a logistic 4-parameter fit model to a concentration–response curve with half-log increments. The percentage efficacy for each curve was determined by the maximum asymptote of that fitted curve and expressed as a percent of the maximum response produced by the positive controls (dopamine) on each plate. Reported values are the mean of results obtained across at least three independent experiments (n ≥ 3), each assayed in triplicate.

 

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Molecular Docking

To provide some structural insights into the activities of these molecules in the D₁ receptor, we implemented molecular docking with recent cryo-electron microscopy structures of the D₁ receptor. Molecular docking was performed using Schrödinger Maestro 11.5 (https://www.schrodinger.com/platform/products/maestro/). The activated D₁ receptor in complex with the downstream Gs protein was obtained from the Protein Data Bank (PDB ID: 7 × 2D). The D₁ receptor structure was pre-processed using the protein preparation wizard module, including hydrogen addition, disulfide bond formation, and protonation-state adjustment. Subsequently, the energy minimization was performed using an OPLS3 force field with a Root Mean Square Deviation (RMSD) constraint of heavy atoms to converge within 0.30Å.

The ligands were initially sketched in three dimensions using the ChemDraw software and subjected to structural optimization via the LigPrep module with a CHARMm force field. The receptor grid box for docking was defined based on the crystallographic coordinates of the co-crystallized ligand tavapadon (PDB: 7 × 2D). Glide SP docking was conducted as an initial estimation of binding modes with a shorter computation time. Subsequently, CDOCKER docking was employed for more accurate and high-precision prediction of binding modes.

 

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Supporting Information

Spectroscopic characterization processes (¹H NMR, ¹³C NMR, or HR-MS) and chemical purity analysis of HPLC for all synthesized compounds are included in the “[Supplementary Material]” section of this article's webpage.

Table 2 Structure–activity relationships of the linker of the compounds II

Comp ID	Substituted Linker + A ring	D1 cAMP agonist assay		D5 cAMP agonist assay
		EC50 (nmol/L)	Emax (%)	EC50 (nmol/L)	Emax (%)
II-1		>1,000	11.64	262.70	83.37
II-2		>1,000	59.10	>1,000	85.13
II-3		>1,000	6.93	>1,000	42.00
II-4		>1,000	17.86	645.00	71.79
Tavapadon		3.69	97.07	0.36	99.30

Table 3 Structure–activity relationships of the B- and C-rings of the compounds III

Comp ID	Substituted B ring	Substituted C ring	D1 cAMP agonist assay		D5 cAMP agonist assay
			EC50 (nmol/L)	Emax (%)	EC50 (nmol/L)	Emax (%)
III-1			45.57	98.83	5.96	99.15
III-2			>1,000	7.40	304.80	71.78
III-3			>1,000	12.42	568.60	96.71
III-4			>1,000	5.84	358.40	68.53
III-5			>1,000	10.21	>1,000	37.97
III-6			>1,000	3.93	>1,000	7.37
III-7			>1,000	10.62	620	70.76
Tavapadon			6.21	86.51	0.46	97.42

 

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Conflicts of Interest

None declared.

^# These authors contributed equally to this work.

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Publication History

Received: 24 February 2025

Accepted: 16 June 2025

Article published online:
28 July 2025