Structure-Activity Studies of 1 H-Imidazo[4,5- c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators

Abstract

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A₃ adenosine receptor (A₃AR) positive allosteric modulators (PAMs). A₃AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A₃AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [³⁵S]GTPγS binding E_max, with only 12b trending toward decreasing the agonist EC₅₀. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A₃AR-activating drugs that are potentially event- and site-specific in action.

Figure 1.

Effect of 1H-imidazo[4,5-c]quinolin-4-amine derivatives on the dissociation of [¹²⁵I]50 using hA₃ARs. HEK-293 membranes stably overexpressing the hA₃AR were incubated with ~0.3 nM [¹²⁵I]50 and 10 μM of the indicated modulator for 3 h. The addition of 100 μM 51 initiated dissociation. The amount of radioligand left remaining after 60 min was measured. Statistical significance was calculated by a two-tailed Student’s t test (n = 3; * denotes P < 0.05). Data are presented as mean ± SEM.

Figure 2.

Effect of PAM derivatives on equilibrium binding of [¹²⁵I]50 at the hA₃AR. HEK-293 cell membranes stably overexpressing the hA₃AR were incubated with ~0.3 [¹²⁵I]50 and 10 μM of the indicated modulator for ~18 h to reach equilibrium. The amount of specific binding was determined, and % change from the vehicle was calculated. Statistical significance was determined by two-tailed paired Student’s t test of raw counts per min values for modulator compared to vehicle (n = 3; * denotes P < 0.05). Data are presented as mean ± SEM.

Figure 3.

Effect of 2-alkyl- and 2-cycloalkyl-substituted PAM derivatives (5–8, 13–23), bridged PAM derivatives (12a, 12b, 24–29), PAM derivatives bearing hydrophilic substituents (30–34), and p-phenylamino-substituted PAM derivatives (35–39) on hA₃AR activation by 52 determined using [³⁵S]GTPγS binding (n = 3). HEK-293 cell membranes expressing hA₃AR were pretreated with 52 and modulators for 1 h, followed the addition of radiolabeled [³⁵S]GTPγS to initiate the reaction, and incubated for an additional 2 h at room temperature. AR antagonists 8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine (62, PSB-603) and 4-[2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5-a]-[1,3,5]triazin-5-yl-amino]ethyl]phenol (63, ZM241385) were added (0.3 μM each final concentration) to block any endogenously expressed A_2BARs. 1 unit/mL of adenosine deaminase (ADA) was added to degrade endogenous adenosine. The incubation was terminated by rapid filtration through a Whatman GF/B filter and membrane-bound radioactivity was measured by liquid scintillation spectrometry. Non-specific binding of [³⁵S]GTPγS was measured in the presence of 10 μM of unlabeled GTPγS. 52 potency and maximal efficacy was measured using DMSO, 0.1 μM, 1.0 μM, and 10 μM of the modulator. Results were normalized to concentration–response curve E_max values obtained by 52 with DMSO and expressed as % activation.

Figure 4.

Mean plasma concentrations of compound 18 (A) and compound 39 (B) in Wistar rats vs multiple time points. The dose and administration route are specified above for each of the four groups (three rats in each group).

Figure 5.

Predicted favored pose of compound 17 (orange) at hA₃AR orthosteric binding site (gray). The pose was generated though IFD followed by MM-GBSA minimization.

Chart 1.

(A–D) Four Classes of Heterocyclic A₃AR Allosteric Modulators [Amiloride Derivative 1 (A), 3-(2-Pyridinyl)isoquinoline 2 (B), 2,4-Disubstituted Quinoline 3 (C), and 1H-Imidazo[4,5-c]quinolin-4-amines with 2-Alkyl and 2-Cycloalkyl Substitutions (4–12) (D), Including Alternative 4-Substituted Phenylamino Derivatives and the Bulky 2-Bicycloalkyl (exo-Norbornanyl, 12a) and 2-Tricycloalkyl (Adamantan-1-yl, 12b) Derivatives (E)^,

Scheme 1.. Six-Step Synthetic Route for 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives with 2 Position and 4-Arylamino Substitutions (B), Including the Synthesis of a Common Diamino Intermediate 44 (A)^a

^aReagents and conditions: (i) HNO₃, 75 °C, 95%; (ii) PhPOCl₂, 135 °C, 87%; (iii) 28% aq. ammonia, CH₃CN, 50 °C, 97%; (iv) Fe powder, HCl, CH₃CH₂OH/H₂O, 75 °C, 70%; (v) PPA, 120 °C; (vi) TCFH, NMI, ACN, 60 °C; (vii) NaOH, H₂O:MeOH (1:1), 90 °C; (viii) Pd₂(dba)₃, tBuXPhos, t-BuONa, 1,4-dioxane, 100 °C, 5–51%, (ix) Pd(OAc)₂, tBuXPhos, t-BuONa, H₂O, 1,4-dioxane, 100 °C, 2–33%; (x) EtOH, microwave, 130 °C, 10–61%; (xi) 18, Et₂Zn, CH₂I₂, 0 °C, 13%; (xii) 18, m-CPBA, CHCl₃, 6–12%, 30 and 31 separated chromatographically; (xiii) 18, (CH₃)₂S·BH₃, THF, NaOH, H₂O₂, 0 °C, 8–12%, 33 and 34 separated chromatographically; (xiv) 33/34, DMP, CHCl₃, 23%; (xv) 36, Pd(OAc)₂, CH₂═CHCOOCH₃, Et₃N, 140 °C, 18%; (xvi) 35, Pd(Ph₃P)₂Cl₂, 5-Cl-thien-2-yl-acetylene, CuI, Et₃N, 80 °C, 10%.