First Potent Macrocyclic A3 Adenosine Receptor Agonists Reveal G-Protein and β-Arrestin2 Signaling Preferences

Abstract

(N)-Methanocarba adenosine derivatives (A₃ adenosine receptor (AR) agonists containing bicyclo[3.1.0]hexane replacing furanose) were chain-extended at N⁶ and C2 positions with terminal alkenes for ring closure. The resulting macrocycles of 17-20 atoms retained affinity, indicating a spatially proximal orientation of these receptor-bound chains, consistent with molecular modeling of 12. C2-Arylethynyl-linked macrocycle 19 was more A₃AR-selective than 2-ether-linked macrocycle 12 (both 5'-methylamides, human (h) A₃AR affinities (K_i): 22.1 and 25.8 nM, respectively), with lower mouse A₃AR affinities. Functional hA₃AR comparison of two sets of open/closed analogues in β-arrestin2 and G_i/o protein assays showed certain signaling preferences divergent from reference agonist Cl-IB-MECA 1. The potencies of 1 at all three Gα_i isoforms were slightly less than its hA₃AR binding affinity (K_i: 1.4 nM), while the Gα_i1 and Gα_i2 potencies of macrocycle 12 were roughly an order of magnitude higher than its radioligand binding affinity. Gα_i2-coupling was enhanced in macrocycle 12 (EC₅₀ 2.56 nM, ∼40% greater maximal efficacy than 1). Di-O-allyl precursor 18 cyclized to form 19, increasing the Gα_i1 potency by 7.5-fold. The macrocycles 12 and 19 and their open precursors 11 and 18 potently stimulated β-arrestin2 recruitment, with EC₅₀ values (nM) of 5.17, 4.36, 1.30, and 4.35, respectively, and with nearly 50% greater efficacy compared to 1. This example of macrocyclization altering the coupling pathways of small-molecule (nonpeptide) GPCR agonists is the first for potent and selective macrocyclic AR agonists. These initial macrocyclic derivatives can serve as a guide for the future design of macrocyclic AR agonists displaying unanticipated pharmacology.

Chart 1. Reference High Affinity A₃AR Nucleoside Ligands Containing N⁶, C2, and 5′ Position Substitutionsa

Figure 1

Hypothetical binding mode of compound 5 (green) (A) and 12 (pink) (B) at the hA₃AR (grey) orthosteric binding site. The poses were obtained by molecular docking followed by three replicates of 30 ns MD simulations, followed by clustering of the MD frames on the basis of the RMSD of the N⁶-(2-phenylethyl) substituent (after aligning the trajectories on the ligands’ adenine scaffold). Key interacting residues are shown by grey sticks.

Scheme 1. Synthesis of Ether-Linked Macrocycle (2 Position, 12) Containing a 5′-N-Methyluronamide and Related Non-cyclized Derivative 11

Reagents and conditions: (i) 4-bromobutene, K₂CO₃, DMF, 90 °C; (ii) but-3-en-1-ol, NaH, 90 °C; (iii) Grubb’s 2nd gen. catalyst, CH₂Cl₂, rt; (iv) 10% TFA (aqueous): MeOH (1:1), 70 °C. Compound 23 was synthesized as reported by Tosh et al.

Scheme 2. Synthesis of an Ether-Linked Macrocycle (2 Position, 14) Containing a 5′-CH₂OH Group and Related Non-cyclized Derivative 13

Reagents and conditions: (i) 5-(3-aminopropyl)-2-methoxyphenol, DIPEA, 2-propanol, rt; (ii) 4-bromobutene, K₂CO₃, DMF, 80 °C; (iii) but-3-en-1-ol, NaH, 90 °C; (iv) 10% TFA (aqueous)/MeOH (1:1), 70 °C; (v) Grubb’s 2nd gen. catalyst, CH₂Cl₂, rt.

Scheme 3. Synthesis of Alkyne-Linked Macrocycles (2 Position) Containing a 5′-Ethyl Ester (16) or a 5′-N-Methyluronamide (21) and Related Non-cyclized Derivatives 15 and 20

Reagents and conditions: (i) 3-iodobenzylamine, DIPEA, 2-propanol, rt; (ii) hex-1-en-5-yne, PdCl₂(Ph₃P)₂, CuI, Et₃N, DMF, rt; (iii) Grubb’s 2nd gen. catalyst, CH₂Cl₂, rt; (iv) 10% TFA (aqueous)/MeOH (1:1), 70 °C; (v) 40% MeNH₂, MeOH, rt.

Scheme 4. Synthesis of Arylalkyne-Linked Macrocycles (2 Position) Containing a 5′-N-Methyluronamide (19) and Related Non-cyclized Derivatives 17 and 18

Reagents and conditions: (i) 3-ethynylphenol, PdCl₂(Ph₃P)₂, CuI, Et₃N, DMF, 60 °C, 2 h; (ii) allyl bromide, K₂CO₃, DMF, 70 °C; (iii) Grubb’s 2nd gen. catalyst, CH₂Cl₂, 60 °C; (iv) 10% TFA (aqueous)/MeOH (1:1), 70 °C. Compound 23 (structure shown in Scheme 1) was synthesized as reported by Tosh et al.

Figure 2

Pharmacological results comparing activation of various G proteins. Data are presented as the change in net BRET² values compared to vehicle. E_max and EC₅₀ values are provided in Table 3.

Figure 3

Pharmacological results comparing activation of β-arrestin2. Data are presented as the change in net BRET¹ compared to vehicle. E_max and EC₅₀ values are provided in Table 3.

Figure 4

Structures of the two most potent macrocyclic derivatives, 12 (MRS7735) and 19 (MRS8033). Predicted interactions between 12 and TMs 3, 6, and 7 and ELs 2 and 3 of the hA₃AR are shown.