Structure–Activity Studies of 1H-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.

Graphical Abstract

INTRODUCTION

Agonists of the four subtypes of adenosine receptors (ARs) are an appealing ligand class for drug development due to the many salutary actions of adenosine, such as tissue repair and protection against ischemia and other organ stress.¹ Activation of the G_i-coupled A₃AR subtype is associated with attenuating chronic neuropathic pain, heart and brain ischemic preconditioning, and anti-inflammatory effects, without causing cardiovascular side effects.^2,3 The A₃AR is overexpressed in immune and cancer cells, adding to its potential as a possible therapeutic target.³ At present, A₃AR agonists are in Phase 2/3 clinical trials for psoriasis, due to their anti-inflammatory properties, and liver diseases, i.e., hepatocellular carcinoma and non-alcoholic steatohepatitis (NASH).⁴

Allosteric modulators bind to sites that are topographically distinct from the orthosteric binding site for native agonists and can exert their effects through receptor conformation changes beyond those induced by orthosteric ligands.⁵ Various types of allosteric modulators differ in their pharmacological effects. Positive allosteric modulators (PAMs) may improve agonist affinity, potency, and/or efficacy, while negative allosteric modulators (NAMs) do the opposite. Some allosteric modulators can induce a characteristic functional response in the absence of an agonist, i.e., ago-PAMs.⁶

The hallmark advantage of PAMs over orthosteric agonists is that they can be event- and site-specific in action.⁷ Because adenosine is endogenously elevated in response to localized distress signals within the body, a pure PAM will enhance the protective function of adenosine only when and where it is elevated, thereby reducing the risk of side effects.⁸ A second advantage of developing AR PAMs over orthosteric ligands is the possibility of achieving a high selectivity for a single AR subtype, since PAMs typically bind to regions that are more variable between receptor subtypes. Third, AR agonists have multiple potential pharmaceutical applications in the central nervous system (CNS),⁹ but current A₃AR orthosteric agonists, mainly nucleosides, tend to have low blood–brain barrier (BBB) permeability, with typically only a few percent or less available in the CNS.^10–13 Therefore, BBB-penetrating PAMs might be preferable over AR nucleoside agonists for CNS applications. Lastly, it might be possible to develop biased PAMs that selectively enhance certain A₃AR-induced signaling pathways.^14,15

At least four heterocyclic classes have been reported to act as A₃AR allosteric modulators (Chart 1): amilorides,¹⁶ represented by 5-(N,N-hexamethylene)amiloride (HMA, 1); PAMs shown are 2 (3-(2-pyridinyl) isoquinolines;¹⁷ 2,4-disubstituted quinolines, represented by 3; and, 1H-imidazo[4,5-c]quinolin-4-amines (4–12),^18–20 including N-(3,4-dichloro-phenyl)-2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-amine (7, LUF6000).²¹

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)^19,20

We previously explored the structure–activity relationship (SAR) of the 1H-imidazo[4,5-c]quinolin-4-amine family of A₃AR PAMs.^19,20,22 Prototypical A₃AR PAM 7, which is now being considered for clinical testing for erectile dysfunction,²³ slowed A₃AR agonist dissociation.^19,20 It enhanced maximal efficacy (E_max) at the human (h) A₃AR (but not the mouse homologue)²⁴ without influencing agonist potency, possibly due to simultaneous competitive interactions at the orthosteric binding site.^17,18 The A₃AR PAM activity of imidazoquinolinamine derivatives is often accompanied by variable degrees of apparent receptor antagonist activity.^19,20 Based on findings with chimeric receptors composed of an A₃AR from a responding (human) and a non-responding (mouse) species, we predict that PAM activity results from binding to an allosteric site located within inner/cytosolic A₃AR regions, exclusive of the orthosteric binding site.²⁴ The chimeric receptors indicated that a 2-cyclopropyl derivative interacts primarily with the orthosteric site as a competitive antagonist, and the corresponding 2-cyclohexyl derivative 7 can interact with both sites. The possibility that 7 might negatively modulate A₃AR agonists by binding to the same intracellular allosteric site, i.e., NAM activity, has been ruled out.²⁴

In this study, we have extended the recent expansion and contraction of the 2-cyclohexyl ring of 7 by Fisher et al.²⁴ with additional 1H-imidazo[4,5-c]quinoline-4-amine derivatives as hA₃AR PAMs through modifications at the 4-arylamino and/or the 2 position of the imidazoquinoline scaffold. We functionalized the rings with polar groups to try to reduce lipophilicity and improve aqueous solubility, as well as introduced bridging groups to determine if there is an A₃AR-preferred conformation of 2 position substituent. We also explored the effect of various 4-arylamino substitutions on positive allosteric enhancement. Here we describe the synthetic methods for the newly prepared analogues, including those reported by Fisher et al.²⁴

The objectives of this study were: to expand the SAR of 1H-imidazo[4,5-c]quinolin-4-amine derivatives related to 7 to identify high efficacy A₃AR PAMs, including those bearing hydrophilic and conformationally constrained groups; to devise a shorter synthetic route to derivatize 1H-imidazo[4,5-c]quinolin-4-amines than the ones previously reported;^18–20 and to obtain a baseline example of the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of potential lead compounds from this series.

RESULTS AND DISCUSSION

Description of Synthetic Compound List.

Four groups of 1H-imidazo[4,5-c]quinolin-4-amine derivatives were synthesized for A₃AR pharmacological characterization, as listed in Table 1 (5–8, 13–39), including six compounds reported earlier (5–8 and 12).¹⁹ The final step reaction yields are shown in Table S1. The first group of derivatives (5–8, 13–23) has hydrophobic alkyl and cycloalkyl substitutions at the 2 position of the 1H-imidazo[4,5-c]quinolin-4-amine scaffold with the 3,4-dichlorophenyl group at the 4-amino position, which was shown in previous SAR studies to improve A₃AR PAM activity and subtype selectivity.^19,20 The second group of derivatives (24–29) includes bridged 2-bicyclic substitutions, based on the favorable PAM activity of compounds 8 and 19, as well as the exo-norbornanyl and adamantan-1-yl derivatives 12a and 12b (Chart 1E). The third group (30–34) has hydrophilic oxygen-containing functionality introduced on a 2-cycloheptyl ring and a 3,4-dichlorophenyl group at the 4-amino position. Lastly, the fourth group (35–39) has a cyclohexyl ring or a heptan-4-yl moiety at the 2 position combined with various p-substituted 4-phenylamino groups. Prior studies showed tolerance of 4-methoxy and 4-chloro substitutions at the 4-phenylamino position (10 and 11, Chart 1D), resulting in promising A₃AR allosteric enhancement based on a slower dissociation rate and increased E_max of agonists.¹⁹ Compounds 29 and 32–34 were racemic mixtures. For most of the analogues, we incorporated the 4-(3,4-dichlorophenylamino) substitution based on previous studies in which this group, compared to other haloaryl groups (3,5- and 2,4-dichloroaniline derivatives), had favorable PAM activity as indicated by effects on agonist dissociation kinetics and efficacy.²⁰

Table 1.

1H-Imidazo[4,5-c]quinolin-4-amine Derivatives Synthesized and Effects on Agonist-Induced A₃AR Activation

Compound	R2	R1	Emaxa(%)
2-alkyl and 2-cycloalkyl derivatives
13	3,4-Cl2		153 ± 12*
14	3,4-Cl2		216 ± 12*
15	3,4-Cl2		159 ±13*
16	3,4-Cl2		150 ± 5*
17	3,4-Cl2		111 ± 16
5	3,4-Cl2		109 ± 3
6	3,4-Cl2		142 ± 5*
7	3,4-Cl2		225 ± 10*
8	3,4-Cl2		175 ± 3*
18	3,4-Cl2		241 ± 9*
19	3,4-Cl2		146 ± 2*
20	3,4-Cl2		241 ± 12*
21	3,4-Cl2		135 ± 7*
22	3,4-Cl2		122 ± 6*
23	3,4-Cl2		101 ± 3
2-bicycloalkyl derivatives
24	3,4-Cl2		187 ± 14*
25	3,4-Cl2		170 ± 6*
26	3,4-Cl2		154 ± 7*
27	3,4-Cl2		237 ± 12*
28	3,4-Cl2		219 ± 16*
29	3,4-Cl2		187 ±8*
2-cycloalkyl derivatives with hydrophilic substitution
30	3,4-Cl2		136 ± 11
31	3,4-Cl2		173 ± 14*
32	3,4-Cl2		160 ± 16*
33	3,4-Cl2		118 ± 14
34	3,4-Cl2		156 ± 10*
2-alkyl and 2-cycloalkyl derivatives with modified 2-arylamino groups
35	4-I		184 ± 9*
36	4-Br		207 ± 17*
37			164 ± 10*
38			170 ± 12*
39	4-I		223 ± 10*
48	4-Sn(CH3)3		ND
49	4-Sn((CH2)3CH3)3		ND

^aEffect of PAM derivative (1.0 μM) on [³⁵S]GTPγS binding induced by 52 using WT hA₃ARs (n = 3).

^*P ≤ 0.05 (one-way ANOVA with Bonferroni-adjusted t test for multiple comparisons) with respect to control in the absence of a PAM. Effects of 0.1 and 10 μM are shown in Table S4. ND, not determined.

Chemical Synthesis.

3,4-Diaminoquinoline intermediate 53 and final compounds 13, 14, and 16 were prepared by literature procedures (Scheme S1).¹⁹ A shorter synthesis than the previously reported 9-step route,¹⁹ requiring only 6 steps, was developed to create a new series of 1H-imidazo[4,5-c]quinolin-4-amine derivatives (Scheme 1). The first four steps closely followed a reported synthetic route used to synthesize a non-adenosine-related series of 2-(p-substituted-phenyl)-4-phenyl-1H-imidazo[4,5-c]quinoline derivatives, which differ from the present compounds in having a 4-phenyl instead of a 4-aminophenyl substitution.²⁵

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%.

In the first step of the 6-step route (Scheme 1), quinoline-2,4-diol 40 was nitrated with nitric acid to produce 3-nitroquinoline-2,4-diol 41. 41 was then chlorinated with phenylphosphonic dichloride to afford 2,4-dichloro-3-nitroquinoline 42. The step-2 product was then aminated in step-3 with 28% aq. ammonia to give regioselectively 2-chloro-3-nitroquinolin-4-amine 43. Subsequently, in step-4 Fe powder and hydrochloric acid reduced the 3-nitro group to an amine to provide the vicinal diamine, 2-chloroquinoline-3,4-diamine 44.²⁵

Two alternative step-5 protocols condensed the vicinal diamine with a carboxylic acid followed by cyclization. The first reaction protocol (see Scheme 1, general procedure A (v)), utilized polyphosphoric acid (PPA) for the condensation between 2-chloroquinoline-3,4-diamine and an appropriate carboxylic acid 45, followed by cyclization to the imidazole 46.19 The second reaction protocol (see general procedure B (vi)), required two steps without isolation of the intermediate. Initially, an acyl imidazolium adduct formed between the coupling agent tetramethylchloroformamidinium hexafluorophosphate (TCFH) and N-methylimidazole (NMI), which provided an electrophile to activate the appropriate carboxylic acid 45.²⁶ The vicinal diamine 44 was acylated to produce an amide intermediate (not shown). A similar published vicinal diamine reaction uses room temperature,²⁶ but we found that heating at 60 °C brought the reaction to completion, increasing the yield. The crude amide was subjected to a subsequent base-catalyzed cyclization reaction to form the imidazole ring in 46 and incorporate a 2 position substitution on the imidazoquinoline scaffold.

The last step, a C–N cross-coupling reaction, was performed using three different reaction protocols. The first protocol used the palladium catalyst tris(dibenzylideneacetone) dipalladium(0) (Pd₂(dba)₃) (general procedure C (viii) in Scheme 1),^27–29 while the second used a water-activated palladium acetate (Pd(OAc)₂) catalyst (general procedure D (ix) in Scheme 1).²⁸ We attempted to improve the cross-coupling reaction yield using Pd(OAc)₂ rather than the Pd₂(dba)₃ catalyst, because the removal of the sizable dba (dibenzylideneacetone) ligand during the palladium catalyst activation might sterically hinder the oxidative addition of the aryl halide during the catalytic cycle.²⁷ However, higher yields were obtained using the step-6 general procedure C than with the general procedure D. The third reaction protocol was a microwave-assisted reaction in ethanol at 130 °C to achieve the final 1H-imidazo[4,5-c]quinolin-4-amine derivative (general procedure E (x) in Scheme 1).¹⁹

Compounds 13–15 have 2-alkyl substitutions, with compound 15 included to investigate the effect of terminal hexafluoro substitution on the pharmacological activity of compound 14.³⁰ The carboxylic acid precursor of compound 15 was prepared by a procedure reported in a patent (Scheme S2).^31,32 Compound 16 introduced a trifluoromethyl group at the 4 position of the cyclohexyl ring of a reference PAM, 7. Final compounds 13–22 were prepared with the step-6 general procedure C, having yields ranging from 5% to 51%. Compound 23 was synthesized using the step-6 general procedure E with the C–N cross-coupling of the 4-chloro in step-5 product to produce 3,4-dichloroaniline in 25% yield. Most carboxylic acids used for the construction of the cycloaliphatic rings in compounds 7 and 17–23 (starting from compounds 46e–o using the synthetic procedures in Scheme 1) were commercially available, except for the cyclononane- and cyclodecanecarboxylic acids, which were synthesized from a Favorskii ring contraction of their respective α-bromo-cycloketones (Scheme S3).

Compounds 24–27 were produced using the step-6 general procedure D, having yields ranging from 2% to 33%. In the cyclopropanation reaction of alkene derivative 18 to form compound 28,³³ only the cis stereoisomer was recovered (Scheme 1, the imidazole group is trans with respect to the cyclopropane), which was confirmed by 1D-NOE NMR.

Bicyclo[2.2.2]oct-2-ene derivative 29 was the only two-carbon bridged derivative with a double bond to provide extra rigidity to the ring system. The precursor bicyclic carboxylic acids were obtained upon saponification of the methyl ester products formed from a Diels–Alder reaction between 1,3-cyclohexadiene and methyl acrylate (Scheme S4).^34,35 Two pairs of enantiomers that are diastereoisomers to one another were isolated from the Diels–Alder reaction, the majority being the endo enantiomeric pair, likely due to the lower transition state energy favoring the formation of the endo over exo stereoisomers. Thus, compound 29 is a racemic mixture of endo enantiomers, which was confirmed by 1D-NOE. The double bond of 2-cycloheptenyl derivative 18 allowed hydrophilic oxygen-containing functional groups to be installed (Scheme 1). Two diastereomeric oxirane compounds, 30 and 31, were produced and resolved from the same epoxidation reaction of compound 18 using m-CPBA.³⁶ The distinguishing characteristic between these two diastereomers is the chemical shift of the ring proton (CH) on the carbon atom connected to the 2 position of the 1H-imidazo[4,5-c]quinolin-4-amine, which was δ2.71 and δ3.07 ppm for 30 and 31, respectively, in the ¹H NMR spectrum. The structures of 27, 29 and 30 were confirmed by 1D and 2D NMR (Supporting Information). Compounds 33 and 34 were made in the same hydroboration–oxidation reaction³⁷ and isolated as racemic mixtures, being enantiomeric pairs that are diastereomers to one another. The assignment of the configuration of compound 33 was similarly confirmed by 1D-NOE (Supporting Information). Compound 32 was made from treating a mixture of compounds 33 and 34 with the oxidizing agent Dess–Martin periodinane,³⁸ producing the racemic carbonyl enantiomers.

Compounds 35 and 36 have 4-iodophenyl and 4-bromophenyl substitutions, respectively. Both were produced using the step-6 general procedure E microwave-assisted reaction between a 4-chloro-2-cyclohexyl-imidazo[4,5-c]-quinolinamine 46, the step-5 product, with the corresponding p-iodo- or p-bromo-aniline in ethanol (Scheme 1). Compound 37, having a methyl acrylate 4 position substitution on the phenylamino moiety, was generated from a Heck reaction between compound 36 and methyl acrylate using a Pd(OAc)₂ catalyst (Scheme 1)³⁹ as the E-isomer. Compound 38 has a p-(5-chlorothiophen-2-yl)ethynyl substitution, and was prepared by a Sonogashira reaction between compound 35 and 2-chloro-5-ethynylthiophene using a bis(triphenylphosphine) palladium II dichloride catalyst (Scheme 1).^40,41 A reaction using p-bromo derivative 36 was unsuccessful, but using p-iodo derivative 35 provided the desired product 38 (Scheme 1). Compound 39 was produced to study the effect of an I-label and to provide a potential allosteric radioligand. Two trialkylstannyl derivatives, 48 and 49, were synthesized from compound 39 using a bistriphenylphosphine palladium dichloride catalyst with hexamethylditin or hexabutylditin, respectively (Scheme S5).⁴² They are potential precursors for a radio-iodination reaction to produce a ¹²⁵I radioligand (Scheme S6).⁴³

Pharmacological Characterization.

Two assays using fixed PAM concentrations characterized the effects of the new derivatives on hA₃AR agonist radioligand ([¹²⁵I]N⁶-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide 50, [¹²⁵I]I-AB-MECA) binding. Pharmacological results for a subset of these compounds (5–8, 14 and 17–23) were reported by Fisher et al. and are included here for comparison.²⁴ This first assay (Figure 1) consisted of measuring the degree of radioligand dissociation after a standard time delay (3 h) following the addition of an excess of an unlabeled AR agonist (adenosine-5′-N-ethylcarboxamide 51, NECA), reflecting the effect on dissociation rate (as a PAM would slow the dissociation). The second assay (Figure 2) consisted of measuring the influence of each modulator on the relative % binding of the radioligand at the hA₃AR after a long duration of incubation (18 h), at which point equilibrium conditions were achieved. A third assay, a functional assay, consisted of a [³⁵S]GTPγS binding assay to determine the potency of agonist (2-chloro-N⁶-(3-iodobenzyl)-5′-N-methylcarboxamidoadenosine 52, Cl-IB-MECA) as an EC₅₀ value (concentration of half-maximal activation) in hA₃AR activation and its E_max in the presence of the indicated compound. All three assays used membranes prepared from human embryonic kidney (HEK) 293 cells overexpressing the hA₃AR.

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.

In Figure 1, single-point dissociation assays were conducted to measure the relative influence of each derivative on dissociation of 50 from the hA₃AR 60 min after initiation of dissociation by the addition of a competitive AR agonist 51 at a saturating concentration of 100 μM (Table S2). The horizontal dotted line represents the control amount of agonist radioligand that remained bound in the absence of a 1H-imidazo[4,5-c]quinolin-4-amine derivative, i.e., 26% of the amount bound at time 0. Thus, bars significantly above the line represent compounds that had a net effect to increase the amount of radioligand remaining bound, presumably by allosterically slowing the agonist dissociation rate, i.e., PAM activity.

Compounds 13–16 showed varying results, with 2-(heptan-4-yl) derivative 14 slowing agonist dissociation the most, with 71% of the radioligand remaining bound. Despite being the hexa-fluoro equivalent of compound 14, compound 15 did not slow agonist dissociation compared to the control (P-value = 0.958).

Compounds 17 (cyclopropyl), 5 (cyclobutyl) through 8 (cycloheptyl), and 18 (cyclooctyl) through23 (cyclododecyl) represent the 2-cycloalkyl-substituted analogues, ranging from 3 to 12 carbons. As the hydrophobic ring system increased from cyclopropyl 17 to cyclo-octyl 19, there was a steady increase in percent radioligand remaining hA₃AR-bound compared to control. The following compounds displayed the greatest reduction in dissociation (P-values < 0.05, Student’s t test), ranging from 45% to 54% left remaining: 2-cyclohexyl 7, 2-cycloheptyl 8, 2-cyclohept-4-enyl 18, and 2-cyclooctyl 19. With larger rings, radioligand dissociation was not significantly slowed compared to control (P-values = 0.188, 0.786, 0.808, and 0.735, for cyclononyl 20 to cyclododecyl 23, respectively).

exo-Norbornanyl 12a, adamantan-1-yl 12b, and 24–29 represent bi- and tricyclic ring systems of varying sizes. Except for compounds 12a and 29, all compounds slowed the agonist dissociation compared to the control. The percent of radioligand remaining bound in the presence of modulators 12b and 24–28 ranged from 45% to 52%. The compounds with hydrophilic substitutions on the 2-cycloheptyl ring, 30–34, showed no net slowing of radioligand dissociation compared to the control.

Among the p-substituted 4-phenylamino derivatives 35–38, compounds 35 (4-iodophenyl) and 36 (4-bromophenyl) slowed the radioligand dissociation rate the most, with 57% and 56% radioligand remaining, respectively. However, compounds 37 ((E)-methyl-3-(4-aminophenyl acrylate) and 38 (5-chlorothiophen-2-yl)ethynyl)phenyl)) did not significantly slow the agonist dissociation rate compared to the control (P-values = 0.052 and 0.450, respectively). Compound 39, with combined features of compounds 14 and 35, i.e., 2-(heptan-4-yl) and 4-iodophenylamino substitutions, considerably slowed radioligand dissociation with 65% remaining A₃AR-bound.

Figure 2 shows single-point equilibrium binding assay results: the positive, negative, or neutral percent change from the vehicle of the radioligand in the presence of a fixed concentration (10 μM) of each modulator under equilibrium conditions (Table S3). The radioligand bound was measured at 18 h, which ensures equilibrium (k_on = k_off) conditions were achieved.^24,44 Thus, bars above the 0 line represent compounds that positively modulate radioligand binding as PAMs, possibly from increased agonist affinity. Bars significantly below the 0 line represent a net reduction in agonist radioligand binding, presumable due to competitive antagonism (5, 12a, 13, 17, 18 and 28–36). Only three (28, 35 and 36) of these fourteen derivatives slowed agonist radioligand dissociation (Figure 1).²⁴

Comparing open-chain alkyl and fluorinated compounds 13–16, the n-propyl derivative 13 decreased radioligand binding the most with a −45% change from the vehicle. Compound 14 increased radioligand binding the most with a 70% change from the vehicle.

Among the 2-cycloalkyl-substituted derivatives, compound 17 (2-cyclopropyl) decreased radioligand binding by 71% compared to the control, and compound 19 (2-cyclooctyl) increased radioligand binding by 41% compared to the control. Compounds 6–8 and 21–23 did not produce a net change in radioligand equilibrium binding compared to the control.

Among the bridged compounds, i.e., 12a, 12b and 24–29, compounds 12b (2-adamantan-1-yl), 26 (2-bicyclo[3.3.1]-nonan-1-yl), and 27 (2-((1R,3s,5S)-bicyclo[3.3.1]nonan-3-yl)) significantly increased radioligand binding with a 19%, 38% and 41% change from vehicle, respectively. Compounds 24 and 25 had no effect on radioligand binding, while compounds 12a, 28 and 29 decreased radioligand binding with −19%, −37% and −21% change from vehicle, respectively.

All the derivatives with hydrophilic substitutions, 30–34, decreased A₃AR radioligand binding at equilibrium, with compound 33 having the most significant decrease in binding with a 36% change from vehicle when compared to the control (P-value = 0.023).

Of the p-substituted phenylamino derivatives, compound 37 increased agonist equilibrium binding the most with a 15% change from the vehicle. Compounds 35 and 36 decreased agonist equilibrium binding with −31% and −49% change from the vehicle, respectively. Compounds 38 and 39 did not affect radioligand binding at equilibrium compared to control.

In addition to the kinetic binding studies, the ability of each compound to allosterically modulate hA₃AR-dependent G protein activation by agonist 52 was measured in the [³⁵S]GTPγS functional assay (Figure 3 and Table S4). Concentration–response curves were generated for each compound, including a curve for the control (agonist 52 with DMSO) and curves for the agonist 52.²⁴ Compounds 12a and 12b were reported in Kim et al.²⁰ and re-assayed here with a new protocol.

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.

The effect on [³⁵S]GTPγS binding of each modulator (5–8 and 12–39) at concentrations of 0.1, 1.0, and 10 μM was determined (Tables 1 and S4, Figure 3). Compounds 7, 14, 18 and 20 were particularly efficacious and at least doubled the agonist E_max compared to control. Comparing the progression of effects of N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine derivatives at 1.0 μM, there was a trend to increase the E_max from the 2-cyclopropyl derivative 17 to the 2-cyclononyl derivative 20, except for 2-cyclo-octyl 19. While not significant, there was a 2-fold increase in agonist potency in the presence of 1 μM of 2-cyclononyl derivative 20 compared to the agonist alone (P-value = 0.3712, one-way ANOVA with multiple comparisons). As the ring size increased from 2-cyclodecyl 21 to 2-cyclododecyl 23, there was no change in agonist potency but a steady decrease in its E_max. The fluorinated compounds, 15 and 16, at 1 μM moderately improved agonist E_max but did not influence agonist potency.

Of the bridged derivatives, 12b and 26 at 1 μM enhanced the efficacy of agonist 52 to the greatest extent, having an E_max of 237 ± 12 and 219 ± 16 (P-value = 0.0002 and 0.0005, respectively). While neither 12b nor 26 significantly improved agonist potency, 12b at 1.0 μM tended to improve potency almost 4-fold from 46 nM to 10 nM (P-value = 0.058). Compounds 12a, 24, 27 and 28 moderately enhanced the [³⁵S]GTPγS binding in the presence of agonist 52 compared to control. 2-(Bicyclo[2.2.1]heptan-1-yl) derivative 25 was the only bicyclic compound to induce a decrease in agonist potency at 1 μM (4-fold compared to the control, P-value = 0.0257). Compound 29 did not influence agonist potency and led to a moderate increase in agonist E_max (167 ± 12). All bicyclic compounds tended toward inverse agonist activity, and compound 29 at 10 μM decreased the basal efficacy the most, by ~100%, at the lowest agonist concentration.

The derivatives with hydrophilic substitutions, compounds 30–34, moderately allosterically enhanced [³⁵S]GTPγS binding (Figure 3 and Table S4). However, these modulators at concentrations of 0.1 μM and 1.0 μM had no significant effect on agonist potency compared to the control. At 1.0 μM, only compounds 31, 32, and 34 increased agonist E_max compared to the control, ranging from 167% to 186% maximal efficacy of the agonist alone. Like compound 29, compound 34 behaved as an inverse agonist at 10 μM, decreasing basal efficacy at the lowest agonist concentration by ~50%. The decreased lipophilicity of 34 relative to 2-cyclononyl derivative 20 was predicted using Stardrop software⁴⁵ with the following parameters for 34: Log D = 5.53, topological polar surface area (TPSA) = 73.8 Ų, and blood–brain barrier (BBB) log([brain]:[blood]) = −0.442, i.e., not BBB penetrant. The same parameters for 20 were Log D = 7.08, TPSA = 53.6 Ų, and BBB log([brain]:[blood]) ratio = −0.0326, i.e., BBB penetrant. Efficacious PAMs 14, 18 and 39 were also predicted to penetrate the BBB. The calculated parameters for all compounds tested pharmacologically are provided in Table S5.

Of the p-substituted phenylamino derivatives, compounds 35, 37, and 38 at 1.0 μM moderately increased agonist efficacy compared to control (Figure 3 and Table S4). At 1 μM, compound 36 doubled the agonist efficacy compared to control. None of the p-substituted phenylamino derivatives influenced agonist potency compared to control. Compound 39 combined the favorable features of compounds 14 and 35, based on their favorable modulation of dissociation kinetics and [³⁵S]GTPγS binding. Compound 39 contains both the 2-heptan-4-yl substitution of 14 and the 4-iodophenyl substitution of 35. Compared to the control, compound 39 at 1 μM did not improve agonist potency but doubled agonist E_max.

The off-target screening was performed by the NIMH Psychoactive Drug Screening Program (PDSP) (Table 2).⁴⁶ A total of ten derivatives were tested for off-target binding activity against 45 other receptors, transporters, and channels. Only a few weak off-target interactions were observed, the lowest K_i being for compound 39 at the translocator protein (TSPO) with K_i = 0.123 μM. The two 4-iodophenyl derivatives tested, compounds 35 and 39, were the only derivatives interacting with TSPO. Most derivatives interacted with one or two sigma receptors, specifically σ₁ and σ₂. Compound 39 had the lowest K_i observed, 0.891 μM, among the derivatives interacting with a sigma receptor. Compound 27 interacted with the most off-target sites: κ (KOR) and μ (MOR) opioid receptors, σ₁, σ₂, dopamine transporter (DAT), and serotonin 5HT_2B receptors, interacting most strongly with DAT at K_i = 0.467 μM.

Table 2.

Off-Target Analysis of Selected PAM Derivatives at 45 Other Receptors, Transporters, and Channels, As Determined in Radioligand Binding Assays by the PDSP^a

derivative	protein	Ki (μM)
14	5HT2B	1.36
	σ 1	1.48
	σ 2	1.36
18	σ 1	4.64
	σ 2	0.575
20	σ 1	5.15
	σ 2	0.907
21	σ 2	3.62
22	D3	5.28
23	D3	7.63
	σ 1	3.02
	σ 2	2.84
26	σ 1	2.34
	σ 2	1.34
27	MOR	7.22
	σ 1	2.42
	σ 2	3.43
	KOR	5.50
	DAT	0.467
	5HT2B	3.25
35	D3	0.846
	TSPO	0.427
	σ 2	3.01
39	5HT2B	2.60
	TSPO	0.123
	σ 2	0.891

^aThese are the only hits with >50% inhibition in the primary screen at 10 μM.

Experimental ADMET Properties.

We chose 2-cyclohept-4-enyl 18 and 2-heptan-4-yl-N-4-iodophenyl 39 derivatives as relatively potent (estimated) and efficacious A₃AR PAMs having only weak off-target interactions, to assess the pharmacological and ADMET properties in vivo and in vitro, as determined by reported methods.⁴⁷ In fasted Wistar rats, the plasma concentration of 18 and 39 was determined following three different doses administered by oral gavage (p.o.) or one dose administered intravenously (i.v.). Multiple in vitro assays assessed drug metabolism and pharmacokinetics (DMPK): plasma stability, HepG2 cytotoxicity, human ether-à-go-go-related gene (hERG) potassium channel inhibition, cytochrome P450 (CYP) inhibition, microsomal stability, pION solubility, plasma protein binding, and chemical stability in simulated gastric and intestinal fluids.

In vitro and in vivo pharmacokinetic parameters of compounds 18 and 39 are shown in Table 3 and Tables S6 and S7. When dissolved in simulated intestinal and gastric fluids, 88.1% and 69.1% of compound 18 remained after 2 h, respectively, and 39 was more stable in gastric fluid. Out of the five CYP enzymes tested, the inhibition with CYP1A2 was the most concerning, with IC₅₀ values of 6.99 (18) and 1.31 (39) μM. Both compounds had IC₅₀ values above 30 μM for the other four CYP enzymes measured. The plasma stability was satisfactory, with compound 18 having 88.7% remaining in rat plasma after 120 min. The highest half-life of compound 18 in plasma was achieved in the mouse plasma at 695 min. Both compounds were relatively stable in liver microsomal assays, having the most degradation with rat microsomes, i.e., 52% of 18 remaining after 120 min. In the microsomal stability assays, compound 18 had a greater % remaining in all species than the reference compound, testosterone. In the hERG potassium ion channel inhibition assay, compounds 18 and 39 displayed IC₅₀ values of 6.06 and >30 μM, respectively. Both compounds were not toxic to HepG2 liver cells, evidenced by an IC₅₀ greater than 30 μM. In all three species, human, rat, and mouse, compound 18 was strongly bound to plasma protein, having % bound values of ~100, 99.1, and ~100, respectively. 39 was less water-soluble but had a much higher free fraction (93.6% bound) than 18 in human plasma. The measured concentration of compound 18 in the pION solution buffer (pH 7.4) was 0.39 μg/mL (0.92 μM), i.e., low aqueous solubility.

Table 3.

In Vitro and In Vivo Experimental Pharmacokinetic Parameters of Compounds 18 and 39

test	reference compound(s)	compound 18	compound 39
simul. intestinal fluid % remaining at 120 min (t1/2, min)	Verapamil 100 (>240)	100 (573)	86.2 (533)
simul. gastric fluid % remaining at 120 min t1/2, min)	Omeprazole 0.0 (15.3)	69.1 (204)	99.7 (>240)
plasma stability % remaining at 120 min t1/2, min, 3 species)	0.0 (0.11, h, procaine); 83.9 (414, r, diltiazem); 0.0 (0.11, m, procaine)	69.7 (159, h); 88.7 (580, r); 83.5 (695, m)	53.2 (140, h); ~ 100 (>240, r); ~ 100 (>240, m);
CYP1A2, IC50 (μM)	Miconazole, 4.55	6.99	1.31
CYP2C9, IC50 (μM)	Miconazole, 0.38	>30	32.3
CYP2C19, IC50 (μM)	Miconazole, 0.00002	>30	83.6
CYP2D6, IC50 (μM)	Miconazole, 1.64	>30	60.8
CYP3A4 (IC50, μM)	Miconazole (0.0010)	>30	61.6
microsomal stability % remaining at 120 min (t1/2, min, 3 species)	Testosterone 5.44 (15.6, h); 0 (1.43, r); 0 (4.33, m)	81.2 (200, h); 52.1 (70, r); 76.4 (194, m)	92.1 (>120, h); 86.3 (>120, r); 75.2 (>120, m);
plasma protein binding % bound (3 species)	Warfarin, 96.3 (h); 100 (r); 83.6 (m)	~100 (h);99.1 (r);~100 (m)	93.6 (h);100 (r); 100 (m)
hERG, IC50 (μM)a	n/a	6.06	>30b
HepG2 cell toxicity, IC50 (μM)	Verapamil, 65.2	>30	35.4
aqueous solubility pH 7.4 (μg/mL)	n/a	0.39	<0.01
oral bioavailability, t1/2 (h) (3 mg/kg, p.o.)	n/a	28.7%F, 1.29	64.0%F, 3.44

^aFluorescence polarization hERG assay.

^b27% inhibition at 30 μM.

Caco-2 permeability assay assessed intestinal permeability of compounds 18 and 39 (Table S7). Compound 18 had no measurable permeability (P_app) for either apical to basolateral (A to B) and basolateral to apical (B to A) and was therefore classified as having low permeability. An efflux ratio was not calculated due to low P_app in A to B and B to A. B to A had a relatively large % recovery of 78.0%, which was substantially more than the 48.1% seen for the A to B % recovery.

A comparison of the in vivo pharmacokinetics of 18 and 39 (Figure 4, Table 3, Table S8), indicated that 39 had considerably longer in vivo half-life and improved oral bioavailability (3.44 h, 64.0%F at 3 mg/kg; 3.84 h, 61.5%F at 10 mg/kg) than 18, indicating substantial oral bioavailability. The longest half-life of 2.60 h of 18 was with an oral dose of 10 mg/kg, similar to the half-life of 2.38 h seen with the i.v. dose of 0.5 mg/kg 18. 28.7%F and 47.5%F were determined for compound 18. The source of the discrepancy between the low passive drug absorption predicted using Caco-2 cells⁴⁸ and the moderate oral bioavailability remains unexplored.

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).

An oral dose of 10 mg/kg of compound 18 produced higher plasma concentrations (C_max at 2 h of 1780 ng/mL, equal to 4.2 μM) compared to the other groups during each time point measured. All groups of rats administered compound 18 orally, i.e., at 1, 3, and 10 mg/kg, achieved the maximum plasma concentration at ~2 h. However, other parameters were similar for 18 and 39. Rats given 3 mg/kg had almost double the clearance rate (1580 mL/(h·kg)) of compound 18 from the plasma compared to 10 mg/kg (975 mL/(h·kg)) and roughly three times the clearance rate compared to 1 mg/kg (447 mL/(h·kg)) (Table S8). A 3 mg/kg dose had double the elimination rate constant, k_el, compared to other p.o. doses, having ~50% of the remaining compound in the body excreted every h. A 3 mg/kg dose produced the longest mean residence time (MRT, length of time a compound remains in the system before elimination) of 3.98 h.

Molecular Modeling.

In general a mixture of positive and negative modulatory hA₃AR effects was observed for this class of PAMs, with the negative modulation likely due to competitive antagonism at the orthosteric site.²⁴

As reported in Fisher et al.,²⁴ the 2-cyclopropyl analogue 17 was proposed to function predominantly as a competitive antagonist of the orthosteric site of the hA₃AR, because it retained the ability to right-shift the concentration–response curve of agonist 52 in [³⁵S]GTPγS binding assays with a chimeric mouse_in/human_out A₃AR that is non-responsive to the efficacy-enhancing effect of the imidazoquinolinamine PAMs. Chimeric receptor studies proved that compound 7 interacts with both the allosteric and the orthosteric site. The binding affinity (K_B) of 17 for the orthosteric site of the chimeric receptor was estimated by Schild analysis to be 140 nM (Figure S1B). The affinity of 7, which also reduces potency of Cl-IB-MECA in [³⁵S]GTPγS binding assays at the mouse_in/human_out A₃AR, was estimated to be 799 nM at this chimeric receptor (Figure S1A). This conclusion was further supported in equilibrium binding studies with the antagonist radio-iodinated ligand [¹²⁵I]2-(4-(3-(4-amino-3-iodobenzyl)-2,6-dioxo-1-propyl-2,3,6,7-tetrahydro-1H-purin-8-yl)phenoxy)-acetic acid ([¹²⁵I]I-ABOPX), where 7 and 17 were found to displace specific binding to the WT hA₃AR (Figure S2). Compounds 7 and 17 were roughly equipotent in inhibiting orthosteric radioligand binding (K_i ≈ 2.0 μM), whereas compound 14 was slightly less potent (~5.40 μM). Compound 17 did not inhibit orthosteric antagonist binding to the hA₁, hA_2A, or hA_2B ARs at concentrations as high as 10 μM (Supporting Information).

Molecular modeling studies to rationalize the PAM activity and predict the allosteric binding site(s) are still ongoing and not part of this study, because further pharmacological characterization is needed. A PAM-bound hA₁AR experimental structure (PDB ID: 7LD3) is available,⁶ with 2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)-methanone (MIPS521, an hA₁AR PAM) bound to an intramembrane region at the interface among TM1, TM6, and TM7. Homology modeling of hA₃AR using structure 7LD3⁶ as template (46% hA₁AR and hA₃AR sequence identity, computed with GPCRdb),⁶⁶ followed by docking of compound 7 to a binding pocket equivalent to the site of MIPS521 in the A₁AR, did not provide any reasonable poses (data not reported). This is possibly due to a more hindered pocket in hA₃AR, where the bulkier A273^7.44 and M276^7.47 replace the non-conserved G279^7.44 and A282^7.47 of hA₁AR (alignment reported in Figure S3). This, together with ongoing pharmacological experiments,²⁴ suggests that hA₁AR and hA₃AR allosteric sites are distinct, at least for MIPS521 at hA₁AR and the series of compounds of this manuscript at hA₃AR.

Conversely, we could model the ability of 17 to be accommodated in hA₃AR orthosteric site, which is well known for ARs. Given the lack of an experimental hA₃AR structure, we employed a previously reported homology model built on the experimental structure of an antagonist-bound hA₁AR X-ray structure.⁴⁹ The compound was docked to the orthosteric site through an induced fit docking (IFD) procedure⁵⁰ and refined by minimization through molecular mechanics with a generalized Born and surface area solvation (MM-GBSA).⁵¹ The selected hA₃AR pose of compound 17 (Figure 5) was compared to other known A₃AR ligands. In the suggested binding mode, the compound’s 1H-imidazo[4,5-c]pyridin-4-amine moiety mimics the adenine scaffold of adenosine-like agonists⁴⁷ and the [1,2,4]triazolo[1,5-c]-pyrimidin-5-amine scaffold of the known antagonist N-(9-chloro-2-(furan-2-yl)-[1,2,4]triazolo[1,5-c]quinazolin-5-yl)-2-phenylacetamide (64, MRS1220) in its previously predicted pose.⁴⁹ A conserved π–π stacking interaction is observed between the aromatic scaffold of 17, similar to 64, and F168^EL2, while nitrogen N3 and the exocyclic amino group at position 4 are engaged in a bidentate H-bond with N250^6.55. The 2-cyclopropyl moiety is located deep into the orthosteric binding pocket, surrounded by T94^3.36, M177^5.38, S181^5.42, I186^5.47, W243^6.48, L246^6.51, and N250^6.55 (residues located within 3 Å), while the 3,4-dichlorophenyl group points toward the extracellular portion of the receptor, surrounded by the tip of TM6, TM7, EL2, and EL3, and specifically by residues Q167^EL2, F168 ^EL2, I249^6.54, N250^6.55, I253^6.58, V259^EL3, L264^7.35, and I268^7.39.

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.

Considering that compound 7 shares with 17 the ability to inhibit orthosteric antagonist radioligand binding, we assessed its ability to fit the orthosteric binding pocket. We applied induced fit docking and MM-GBSA minimization and selected by visual inspection a possible orthosteric binding mode. Poses with the dichlorophenyl group anti respect to atom C5 (Figure S4B) were discarded as they are less favored and require overcoming a high energy barrier, as suggested by a dihedral scan performed with the semiempirical quantum mechanical methods GFNn-xTB (Figure S5). Compound 7 was thus predicted to bind the hA₃AR orthosteric binding pocket similarly to compound 17 (Figure S4A), which is in agreement with the experimentally demonstrated antagonist effect of the two compounds. This suggest that the two compounds’ differential ability to enhance agonist activity (present for 7 and absent for 17) is not due to a major antagonist character of 17 as compared to 7, since their antagonist behavior is similar, but to a major PAM effect of compound 7.

This study extended previous SAR studies of hA₃AR PAMs having an imidazoquinolinamine scaffold.^14,52 A ligand structure-guided design approach was pursued given that structural information for the A₃AR is not yet available to enable a target-guided structural approach for PAMs. Although modeling predictions can be made for ligand recognition at the hA₃AR orthosteric binding site by close similarity to the hA₁AR and hA_2AAR,⁵³ prediction of the precise allosteric ligand binding site is uncertain.

This class of PAMs is selective for the A₃AR with respect to allosteric modulation at other AR subtypes.^18–20,22 However, a mixture of positive and negative modulatory A₃AR effects is generally observed in this family. The relatively low competitive binding of 4 and 7^19,20 and the lack of allosteric enhancement by 4 and 7^17,54 at other AR subtypes were reported. Based on chimeric receptors, we now know that the site of PAM action is on the cytosolic side of the receptor, while the negative effects in this series appear to arise from the canonical orthosteric site binding.²⁴ The A₃AR orthosteric antagonism by 2-cyclopropyl derivative 17 and prototypical A₃AR PAM 7 was quantified here using functional data from the mouse_in/human_out chimeric A₃AR and radioligand binding at the WT receptor,²² and their orthosteric binding was modeled. The ability of these derivatives and compound 14 to bind to varying degrees to the orthosteric site is also consistent with the fact that this 1H-imidazo[4,5-c]quinolin-4-amine scaffold was initially characterized as potent A₁AR antagonists.⁵⁵ Curiously, the topical anti-tumor agent imiquimod, which contains the same scaffold, was reported to antagonize ARs with μM affinity.⁵⁶

Like past studies, this series of derivatives showed variable allosteric modulatory effects, with positive effects on [¹²⁵I]50 dissociation, potency and/or maximal efficacy of 52 in functional assays. Although PAM effects were demonstrated here with synthetic agonists [¹²⁵I]50 and 52, the enhancing effects apply similarly to endogenous adenosine, as 7 increased the maximal efficacy of adenosine in [³⁵S]GTPγS binding.²⁴

Prior 2-(n-pentyl) substitution (9) of the 1H-imidazo[4,5-c]quinolin-4-amine scaffold with the 3,4-dichlorophenylamino group at the 4 position did not significantly enhance A₃AR activity.¹⁹ However, another acyclic analogue, 2-heptan-4-yl derivative 14, originating from the synthetic precursor valproic acid, at 1 μM, enhanced agonist E_max compared to control (E_max = 216 ± 12%, P-value < 0.0001). This 2-fold increase in E_max of 52 at the receptor was comparable to the enhancement by 2-cyclohexyl derivative 7. Yet, it did not increase agonist potency compared to the control (P-value = 0.2041). Thus, for the first time we demonstrated that acyclic alkyl substitutions at the 2 position provides pharmacological advantages.

Other PAMs that significantly enhanced agonist efficacy at 0.1 μM were 7, 8, 12b, 16, 18, 20, 27, 37 and 39 (Table S4). When evaluating other cycloalkyl derivatives compared to compound 7, compounds 18 (2-cyclohept-4-enyl), and 20 (2-cyclononyl) stand out. Both potentiated the maximal efficacy of agonist 52 similarly to compound 7 (225 ± 10%) at 1 μM, having E_max values of 241 ± 9% and 242 ± 9%, respectively. Compounds 18 and 20 also showed PAM characteristics by slowing the rate of agonist radioligand dissociation from the receptor.

Use of bridged alicyclic rings at the 2 position for steric constraint failed to identify a hypothetical receptor-preferred conformation. All bridged modulators at 1 μM increased agonist efficacy, with an approximately 2-fold increase by compounds 12b, 24 and 28. The tricyclic 2-(adamantan-1-yl) substitution enhanced agonist E_max and trended toward increasing potency (P-value = 0.058). Thus, none of the bicyclic derivatives improved agonist potency compared to control in the [³⁵S]GTPγS assay, but uniformly acted as PAMs by slowing the radioligand dissociation and improving the maximal efficacy of the agonist. The binding of bridged derivatives at both the orthosteric and allosteric binding sites is the most likely explanation for lack of substantial improvement in agonist potency. Another potential explanation is that the activation and binding cooperativities of these compounds are separate parameters controlling both agonist E_max and potency changes. Interestingly, the effect on agonist EC₅₀ of bicyclic alkene 29 was similar to competitive antagonist 17, significantly decreasing the potency in a concentration-dependent manner.

We installed different hydrophilic groups on compound 18 to potentially improve PAM water solubility while maintaining enhancement of the agonist dissociation and functional effects at the A₃AR. These derivatives with various oxygen substitutions (i.e., oxirane, alcohol, and carbonyl groups) did not influence agonist potency, but had similar improvements in agonist efficacy compared to the bicyclic library of derivatives. Thus, they act as PAMs,⁵⁷ but with limited cooperativity due to their mediocre slowing of radioligand dissociation, and they reduced radioligand binding at equilibrium, suggestive of orthosteric site binding.⁵⁸ Although efficacy enhancement compared to control was moderate, these derivatives were the first example of A₃AR PAM activity with any hydrophilic substitution, in light of the failure of previously tested polar heterocyclic derivatives.²⁰

In prior studies, certain halogenated and 4-substituted phenylamino derivatives were tolerated with respect to A₃AR PAM effects.²⁰ Overall, all p-phenylamino substitutions were tolerated here, but none having exceptional binding or functional effects compared to compound 7. At 1 μM concentration, potency remained unchanged and E_max doubled. Compounds 35 and 36, with the 4-iodo- and 4-bromophenylamino substitutions, respectively, had a similar non-optimal influence on potency and efficacy as compounds 37 and 38; though, they both considerably slowed the radioligand dissociation (58% and 56% remaining, respectively) compared to control (P-values = 0.010 and 0.015, respectively). Although compounds 35 and 36 slowed radioligand dissociation, they both considerably decreased the radioligand binding at equilibrium by 31% and 49% compared to the vehicle (P-values = 0.040 and 0.036, respectively). Thus, 35 and 36 bound to both orthosteric and allosteric sites, with greater inhibition of radioligand binding compared to other PAMs. Important to note is the substantial increase in agonist E_max at 1 μM between compounds 35 and 39, (184 ± 9% and 223 ± 10%, P-value = 0.044), correlating with the 2-heptan-4-yl substitution of 39, which was also evident in improved equilibrium radioligand binding.

Interestingly, compound 37, having a methyl acrylate p-phenylamino substitution, slowed radioligand dissociation and had favorable equilibrium binding results, with low % competitive inhibition of radioligand binding at the orthosteric binding site. These are promising results because 37 has more hydrophilic character (predicted Log D = 5.12, TPSA = 79.7 Ų, and BBB log([brain]:[blood]) = −0.450, i.e., not BBB penetrant) than the rest of the library due to the 4-aminophenyl substitution. Of the compounds with hydrophilic substitutions, compound 37 most represents a PAM. A hydrophilic p-phenylamino substitution might achieve a hydrophilic PAM, suggested by the differences in SAR between 37 and the derivatives with hydrophilic substitutions at the 2 position. Altogether, it would be worth investigating the effects of other polar p-phenylamino substitutions.

Compounds 18 and 39 proved to be orally bioavailable, despite having low A-B and B-A permeability in Caco-2 cells. Neither was strongly depleted by simulated digestive fluid, while 39 and to a lesser extent 18 inhibited CYP1A2. 18 displayed extremely high plasma protein binding in all three species tested, attributed to its hydrophobic nature. It is conceivable that the high plasma protein binding could provide a reservoir of the PAM in the body for prolonged activity, as it would release gradually from a bound to unbound state. On the other hand, this could limit the amount of compound available to traverse to the cellular environment for event- and site-specific action.

CONCLUSIONS

This study aimed to further explore the SAR of A₃AR PAMs and to determine the suitability of the new analogues for translational development. We achieved our objectives of creating a shorter 6-step synthetic route for 1H-imidazo[4,5-c]quinolin-4-amine PAM derivatives, preparing a new series of 1H-imidazo-[4,5-c]quinolin-4-amine PAM derivatives, determining the SAR of the series of derivatives, and obtaining the first reported baseline ADMET of this structural family.

Although we did not discover a PAM derivative that induced a greater agonist efficacy-enhancement than compound 7, we did reveal a trend toward higher efficacy with certain 2- and 4-amino substitutions, including 12, 14, 18, 20, 30, and 39. The heptan-4-yl 2 position substitution slowed agonist dissociation from the A₃AR the most, with both 3,4-dichlorophenyl and 4-iodophenyl substitutions at the 4-amino position. In addition to the 2-heptan-4-yl substitution, the 2-cyclohept-4-enyl and 2-cyclononyl substitutions improved agonist efficacy comparable to the reference 2-cyclohexyl. Other 2 position substitutions such as bicyclic and hydrophilic groups did not significantly enhance PAM effects, although these hydrophilic substitutions were more successful than previous attempts to introduce polar groups.

The human/mouse chimeric A₃AR data showed the dependence on the 2 position substitution of orthosteric site affinity, which is spatially distinct from the allosteric binding site located on the cytosolic half of the receptor. Thus, fine-tuning of this family of PAMs is achievable, by separating distinct orthosteric (undesired) and allosteric binding effects. These separate activities could be further probed using the chimeric A₃ARs as screening tools.

Developing a radioligand specific for the 1H-imidazo[4,5-c]quinolin-4-amine PAM binding site would significantly aid the pharmacological characterization of this PAM family. We prepared a precursor for introducing a ¹²⁵I label and will perform future labeling studies.

Thus, we have developed a series of 1H-imidazo[4,5-c]quinolin-4-amine modulators with promising characteristics. Selected compounds will be utilized in future preclinical studies once an animal disease model is identified, furthering the allosteric approach to developing drugs to activate the A₃AR that are event- and site-specific in action.

EXPERIMENTAL METHODS

Reagents and Instrumentation.

All glassware and stir bars were oven-dried before use in a reaction. All reactions were conducted in a ventilated hood. Pyrophoric reagents were handled and administered under a nitrogen gas atmosphere to reactions in an evacuated oven-dried glass round-bottom flask. Room temperature (rt) refers to 25 ± 5 °C. All final compounds were stored at 4 °C in a parafilm-sealed vial. Unless otherwise stated, all the reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). Unless noted, the reagents and solvents were of reagent grade and used without further purification. The following suppliers supplied the following commercial compounds: TCFH was purchased from TCI (Portland, OR), bicyclo[3.3.1]nonane-1-carboxylic acid was purchased from Chemspace US (Monmouth Junction, NJ); bicyclo[3.3.1]nonane-3-carboxylic acid was purchased from AstaTech (Bristol, PA); cyclohept-4-ene carboxylic acid was purchased from Ambeed (Arlington Heights, IL); bicyclo[2.2.1]heptane-1-carboxylic acid was purchased from Enamine (Kyiv, Ukraine). All reagents used were of commercial grade.

NMR spectra were recorded on a Bruker 400, 500, or 600 MHz spectrometer at 25 °C under an optimized parameter setting for each sample (Supporting Information). For compounds 14, 17, 18–22, 24–39, 48, and 49 ¹H NMR chemical shifts were measured relative to the residual solvent peak of 7.26 ppm for CDCl₃ or CDCl₃/CD₃OD. For compounds 15 and 23, ¹H NMR chemical shifts were measured to the residual solvent peak of 2.5 ppm for DMSO-d₆. For compound 13, ¹H NMR chemical shifts were measured to the residual solvent peak of 3.34 ppm for CD₃OD. For compounds 16, ¹H NMR chemical shifts were measured to the residual solvent peak of 1.94 for CD₃CN. ¹H NMR chemical shifts were measured relative to tetramethylsilane at 0.00 ppm in CDCl₃ and the residual water peak at 3.30 ppm in CD₃OD. ¹⁹F NMR spectra were recorded for derivatives 15, 16, and 20–23.

Analytical TLC was performed on 0.2 mm silica-coated sheets with an F254 indicator (Sigma-Aldrich). TLC visualization of the products was aided using UV light or by staining with a solution of potassium permanganate (1.5 g of KMnO₄, 10 g K₂CO₃, and 1.25 mL 10% NaOH in 200 mL water). Column chromatography was performed on 230–400 mesh silica gel (pore size of 60 Å, Sigma-Aldrich).

Accurate mass data were obtained using a Xevo G2-XS QTof mass spectrometer (Supporting Information). The instrument was operated in positive-ion ESI mode at a resolution of 25,000. The ESI capillary voltage was 2.8 kV and the desolvation temperature was 280 °C. Accurate masses were determined using trifluoroacetic acid (TFA) sodium salt as an internal standard. An Acquity I-class Ultra Performance Liquid Chromatography (Waters, Milford, MA) was the liquid chromatography system. Solvent A was 100% water and solvent B was an 80:20 mixture of ACN:MeOH with 0.1% TFA and 0.2% formic acid added. The column was a ProSwift RP-4H 1 × 50 mm monolithic (ThermoFisher, Waltham, MA). The LC gradient was 0% B to 100% B in 10 min at a flow rate of 0.250 mL per min. Accurate mass high-resolution LC/ESI/MS at the Mass Spectrometry Facility, NIDDK, NIH.

HPLC analysis was carried out with Agilent 1100 Series HPLC equipped with Agilent Eclipse 5 μm XDB-C18 analytical column (250 × 4.6 mm; Agilent Technologies Inc., Palo Alto, CA, USA). Mobile phase: linear-gradient solvent system, 10 mM TEAA (triethylammonium acetate): CH₃CN from 50:50 to 0:100 in 20 min, and then 100% CH₃CN for 5 min; the flow rate was 1.0 mL min, unless noted. The RP-HPLC was performed using a Phenomenex Luna 5 μm C18(2)100A, AXIA, 21.2 mm × 250 mm column. The peaks were detected by UV absorption with a diode array detector at 210, 230, 254, and 280 nm. All derivatives tested for biological activity showed ≥95% purity in the HPLC systems, unless noted.

General Procedure for the Synthesis of Compounds 13–22.

Procedure C.

An oven-dried 10 mL round-bottom flask (cooled to rt under nitrogen) equipped with a stir bar was charged with the appropriate 2-substituted 4-chloro-1H-imidazo[4,5-c]quinoline derivative (46a–n) (1.0 equiv), 3,4-dichloroaniline (1.5 equiv), Pd₂(dba)₃ (5–10 mol%), tBuXPhos (20 mol%), and sodium butoxide (1.5–2.0 equiv). The flask was evacuated for 2 min and backfilled with N₂ (gas). Dioxane (~1 mL) was added via syringe, and the reaction mixture was flushed/purged with nitrogen for 5 min while stirring. The reaction mixture was stirred at preheated oil bath (95–100 °C) under nitrogen for 16–24 h. Subsequently, the reaction mixture was cooled to rt and diluted with EtOAc (5 mL). The solution was then filtered through a silica plug. The filtrate was concentrated by rotary evaporation to obtain the crude product, purified by silica column with 10–25% ethyl acetate in hexane as the eluent system to obtain the product. Few products were further purified using reverse-phase high-pressure liquid chromatography.

2-Propyl-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (13).

Compound 13 was synthesized following the general procedure C described above using 46a (15.0 mg, 0.061 mmol), Pd₂(dba)₃ (5 mol%) and sodium t-butoxide (1.5 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 6.0 mg (23%) of a white solid (HPLC t_R = 14.9 min): ¹H NMR (400 MHz, methanol-d₄) δ 8.60–8.55 (m, 1H), 8.00 (d, J = 8.1 Hz, 1H), 7.84 (dd, J = 19.6, 8.8 Hz, 2H), 7.55 (t, J = 7.9 Hz, 1H), 7.48–7.30 (m, 2H), 2.99 (t, J = 7.5 Hz, 2H), 1.96 (h, J = 7.4 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H); HRMS calcd for C₁₉H₁₇N₄Cl₂ (M+H)⁺ 371.0830, found 371.0827.

2-(Heptan-4-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (14).

Compound 14 was synthesized following the general procedure C described above using 46b (26.0 mg, 0.086 mmol), Pd₂(dba)₃ (5 mol%) and sodium t-butoxide (1.5 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 4.2 mg (11%) of a white solid (HPLC t_R = 19.7 min): ¹H NMR (400 MHz, chloroform-d) δ 8.50 (d, J = 2.5 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.87–7.77 (m, 3H), 7.56 (t, J = 7.8 Hz, 1H), 7.44–7.30 (m, 2H), 3.03 (p, J = 7.3 Hz, 1H), 1.81 (td, J = 8.9, 8.3, 4.4 Hz, 4H), 1.43–1.26 (m, 4H), 0.92 (t, J = 7.3 Hz, 6H); HRMS calcd for C₁₃H₂₅N₄Cl₂ (M+H)⁺ 427.1456, found 427.1462.

2-(1,1,1,7,7,7-Hexafluoroheptan-4-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (15).

Compound 15 was synthesized following the general procedure C described above using 46c (4.8 mg, 0.012 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv) gave 15 (3.2 mg, 50%) as a white solid. The crude was purified by silica gel column (10–20% ethyl acetate/hexane) followed by semipreparative HPLC (RP-HPLC linear gradient solvent system: ACN:H₂O from 80:20 to 100:0 in 40 min, at a rate of 5 mL/min. t_R = 29 min) to afford 3.2 mg (50%) of a white solid (HPLC purity: 91%; t_R = 18.1 min): ¹⁹F NMR (DMSO-d₆) δ −64.75; ¹H NMR (400 MHz, DMSO-d₆) δ 8.27–8.09 (m, 1H), 8.09–7.94 (m, 1H), 7.85 (s, 1H) 7.62 (d, J = 11.5 Hz, 2H), 7.49 (s, 2H), 2.34 (d, J = 14.0, 2H), 2.10 (d, J = 7.5 Hz, 6H); HRMS calcd for C₂₃H₁₉N₄F₆Cl₂ (M+H)⁺ 535.0891, found 535.0901.

2-(4-(Trifluoromethyl)cyclohexyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (16).

Compound 16 was synthesized following the general procedure C described above using 46d (14.0 mg, 0.040 mmol), Pd₂(dba)₃ (5 mol%) and sodium t-butoxide (2.0 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 4.0 mg (21%) of a white solid (HPLC t_R = 18.2 min): ¹⁹F NMR (acetonitrile-d₃) δ −74.30; ¹H NMR (400 MHz, acetonitrile-d₃) δ 8.65 (d, J = 2.5 Hz, 1H), 8.14 (s, 1H), 8.01 (dd, J = 8.8, 2.6 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.55 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.50–7.38 (m, 2H), 3.05–2.94 (m, 1H), 2.31 (d, J = 12.9 Hz, 2H), 2.13 (d, J = 22.1 Hz, 4H), 1.80–1.71 (m, 1H), 1.61–1.49 (m, 2H); HRMS calcd for C₂₃H₂₀N₄F₃Cl₂ (M +H)⁺ 479.1017, found 479.1019.

2-(Cyclopropyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (17).

Compound 17 was synthesized following the general procedure C described above using 46e (11.0 mg, 0.045 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 3.0 mg (18%) of a white solid (HPLC t_R = 14.4 min): ¹H NMR (400 MHz, chloroform-d) δ 8.48 (s, 1H), 7.91 (d, J = 8.3 Hz, 1H), 7.86–7.71 (m, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.39–7.27 (m, 2H), 2.12 (p, J = 6.7 Hz, 1H), 1.14 (d, J = 6.5 Hz, 4H); HRMS calcd for C₁₉H₁₅N₄Cl₂ (M+H)⁺ 369.0674, found 369.0676.

2-(Cyclohept-4-en-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (18).

Compound 18 was synthesized following the general procedure C described above using 46j (9.0 mg, 0.030 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 3.0 mg (23%) of a white solid (HPLC t_R = 18.4 min): ¹H NMR (400 MHz, chloroform-d) δ 8.49 (s, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 8.4 Hz, 2H), 7.60–7.52 (m, 1H), 7.43–7.34 (m, 2H), 5.91 (dd, J = 4.9, 2.4 Hz, 2H), 3.27 (dq, J = 6.8, 3.3 Hz, 1H), 2.43 (d, J = 15.8 Hz, 2H), 2.37–2.20 (m, 4H), 1.85 (dt, J = 13.5, 10.9 Hz, 2H); HRMS calcd for C₂₃H₂₁N₄Cl₂ (M +H)⁺ 423.1143, found 423.1137.

2-(Cyclooctyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (19).

Compound 19 was synthesized following the general procedure C described above using 46k (40.0 mg, 0.13 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 15 mg (27%) of a white solid (HPLC t_R = 20.5 min): ¹H NMR (400 MHz, chloroform-d) δ 8.43 (s, 1H), 8.04–7.65 (m, 3H), 7.61–7.47 (m, 1H), 7.46–7.29 (m, 2H), 3.27 (t, J = 9.6 Hz, 1H), 2.21–2.10 (m, 2H), 2.09–1.95 (m, 2H), 1.84 (s, 2H), 1.67 (s, 8H); HRMS calcd for C₂₄H₂₅N₄Cl₂ (M+H)⁺ 439.1456, found 439.1452.

2-(Cyclononyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (20).

Compound 20 was synthesized following the general procedure C described above using 46l (6.0 mg, 0.018 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The product was obtained as a white solid (TFA salt) after lyophilization (2.0 mg, 5%; RP-HPLC, linear gradient solvent system: ACN:0.1% aq. TFA from 70:30 to 100:0 in 40 min, t_R = 21.7 min): ¹H NMR (400 MHz, chloroform-d) δ 8.01 (s, 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.60–7.54 (m, 1H), 7.48 (s, 1H), 7.36 (s, 1H), 7.29 (dd, J = 8.5, 2.5 Hz, 2H), 3.29 (s, 1H), 1.97 (d, J = 4.6 Hz, 5H), 1.77–1.53 (m, 9H), 1.26 (s, 2H); HRMS calcd for C₂₅H₂₇N₄Cl₂ (M+H)⁺ 453.1613, found 453.1616.

2-(Cyclodecyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (21).

Compound 21 was synthesized following the general procedure C described above using 46m (13.0 mg, 0.038 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The product was obtained as a white solid (TFA salt) after lyophilization (2.8 mg, 16%; RP-HPLC, linear gradient solvent system: ACN:0.1% aq. TFA from 70:30 to 100:0 in 40 min, at a rate of 5 mL/min; HPLC purity: 77%; t_R = 23.8 min)): ¹H NMR (400 MHz, chloroform-d) δ 8.44 (s, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 7.8 Hz, 1H), 7.37–7.27 (m, 2H), 3.53 (s, 1H), 2.05 (t, J = 10.1 Hz, 1H), 1.94 (s, 2H), 1.59 (s, 8H), 0.81 (s, 4H), 0.71–0.57 (m, 3H); HRMS calcd for C₂₆H₂₉N₄Cl₂ (M+H)⁺ 467.1769, found 467.1770.

2-(Cycloundecyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (22).

Compound 22 was synthesized following the general procedure C described above using 46n (14.0 mg, 0.039 mmol), Pd₂(dba)₃ (10 mol%) and sodium t-butoxide (2.0 equiv). The product was obtained as a white solid (TFA salt) after lyophilization (2.82 mg, 15%; RP-HPLC, linear gradient solvent system: ACN:0.1% aq. TFA from 70:30 to 100:0 in 40 min, at a rate of 5 mL/min; HPLC purity: 80%; t_R = 22.7 min): ¹H NMR (400 MHz, chloroform-d) δ 8.10 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.60–7.42 (m, 2H), 7.36 (d, J = 9.0 Hz, 2H), 3.19 (s, 1H), 1.88 (td, J = 13.1, 12.7, 6.8 Hz, 2H), 1.41 (d, J = 11.0 Hz, 6H), 1.27–1.14 (m, 10H), 0.78 (d, J = 7.0 Hz, 2H); HRMS calcd for C₂₇H₃₁N₄Cl₂ (M+H)⁺ 481.1926, found 481.1921.

General Procedure for the Synthesis of Compounds 23, 29, 35, 36, and 39.

Procedure E.

The appropriate 4-chloro-2-substituted 1H-imidazo[4,5-c]quinoline (46b,h,o,t) starting material (0.05 mmol, 1 equiv) and the appropriate halogenated aniline compound (47a–c) (0.15–0.25 mmol, 3–5 equiv) were added to 1 mL of ethanol in a 2 to 5 mL microwaveable vial. The reaction contents were degassed with N₂(g) for 15 min, and the reaction was set up in an Initiator microwave reactor (Biotage, Charlotte, NC) at 130 °C for 6 h. The reaction mixture was filtered through a silica plug. The filtrate was evaporated in vacuo, and the product was purified by flash chromatography with a 15% ethyl acetate in hexane eluent system.

2-(Cyclododecyl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (23).

Compound 23 was synthesized following the general procedure E described above using 46o (6.0 mg, 0.016 mmol) and 47a (13.0 mg, 0.08 mmol, 5.0 equiv). The product was obtained as a white solid (TFA salt) after lyophilization (2.0 mg, 25%; RP-HPLC acetonitrile/0.1% aq. TFA, 70/30 in 40 min at a rate of 5 mL/min, HPLC t_R = 21.6 min): ¹H NMR (400 MHz, DMSO-d₆) δ 8.22 (d, J = 7.4 Hz, 1H), 7.87 (s, 1H), 7.61 (s, 2H), 7.50 (s, 2H), 2.97 (s, 1H), 1.98 (s, 3H), 1.83 (s, 3H), 1.43 (d, J = 32.5 Hz, 17H), 1.27 (s, 3H); HRMS calcd for C₂₈H₃₃N₄Cl₂ (M+H)⁺ 495.2082, found 495.2085.

General Procedure for the Synthesis of Compounds 24–27.

Procedure D.

To a 10 mL round-bottom flask (cooled to rt under N₂(g)) equipped with a stir bar were added Pd(OAc)₂ (1 mol%), tBuXPhos (3 mol%), and H₂O (40 mol %) and dissolved in 1.0 mL of 1,4-dioxane. The solution was degassed with nitrogen for 15 min and allowed to stir at 80 °C for 5 min. The appropriate 2-substituted 4-chloro-1H-imidazo-[4,5-c]quinoline derivative (46p–s) (1.0 equiv), 3,4-dichloroaniline (1.2 equiv), and sodium butoxide (2.0 equiv) were dissolved in 3 mL of dry 1,4-dioxane in a separate 25 mL round-bottom flask. The reaction mixture in the 25 mL round-bottom flask was degassed at rt with nitrogen for 15 min. The activated catalyst from the 10 mL flask was cooled and then transferred to the 25 mL reaction mixture using a cannula. The 25 mL flask was slightly immersed in a 100 °C oil bath, and the reaction continued for 16–20 h. The reaction mixture was diluted with ethyl acetate (5 mL) and filtered through a short silica plug. A rotary evaporator concentrated the filtrate to obtain a residue, purified by silica column chromatography with 10% ethyl acetate in hexane as the eluent system to afford the product.

2-(Bicyclo[1.1.1]heptan-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (24).

Compound 24 was synthesized following the general procedure D described above using 46p (47.0 mg, 0.17 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 9.0 mg (13%) of a white solid (HPLC t_R = 16.1 min): ¹H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 8.00 (s, 1H), 7.80 (d, J = 24.6 Hz, 2H), 7.57 (s, 1H), 7.41 (d, J = 9.6 Hz, 2H), 2.68 (s, 1H), 2.40 (s, 6H); HRMS calcd for C₂₁H₁₇N₄Cl₂ (M+H)⁺ 395.0830, found 395.0835.

2-(Bicyclo[2.2.1]heptan-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (25).

Compound 25 was synthesized following the general procedure D described above using 46q (383 mg, 1.29 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 179 mg (33%) of a white solid (HPLC t_R = 18.3 min): ¹H NMR (400 MHz, chloroform-d) δ 8.49 (d, J = 2.6 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H), 7.82 (dd, J = 8.7, 2.7 Hz, 2H), 7.56 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.43–7.34 (m, 2H), 2.53 (d, J = 4.8 Hz, 1H), 1.95–1.84 (m, 6H), 1.57 (d, J = 11.2 Hz, 2H), 1.26 (t, J = 7.1 Hz, 2H); HRMS calcd for C₂₃H₂₁N₄Cl₂ (M +H)⁺ 423.1143, found 423.1140.

2-(Bicyclo[3.3.1]nonan-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (26).

Compound 26 was synthesized following the general procedure D described above using 46r (333 mg, 1.02 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 101 mg (22%) of a white solid (HPLC t_R = 21.0 min): ¹H NMR (400 MHz, chloroform-d) δ 8.49 (d, J = 2.6 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.82 (dd, J = 11.5, 8.5 Hz, 2H), 7.55 (t, J = 7.7 Hz, 1H), 7.44–7.34 (m, 2H), 2.27–2.08 (m, 6H), 1.79 (q, J = 8.4, 6.6 Hz, 6H), 1.26 (t, J = 7.1 Hz, 3H); HRMS calcd for C₂₅H₂₅N₄Cl₂ (M+H)⁺ 451.1456, found 451.1452.

2-((1R,3s,5S)-Bicyclo[3.3.1]nonan-3-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (27).

Compound 27 was synthesized following the general procedure D described above using 46s (255 mg, 0.78 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 5.7 mg (2%) of a white solid (HPLC t_R = 19.4 min): ¹H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.85–7.75 (m, 3H), 7.60–7.51 (m, 1H), 7.37 (t, J = 8.3 Hz, 2H), 3.23 (ddt, J = 19.1, 12.9, 5.7 Hz, 1H), 2.37 (td, J = 12.7, 5.7 Hz, 2H), 2.23 (d, J = 11.4 Hz, 2H), 1.93–1.81 (m, 2H), 1.72–1.62 (m, 3H), 1.51 (dd, J = 8.3, 4.8 Hz, 5H); HRMS calcd for C₂₅H₂₅N₄Cl₂ (M+H)⁺ 451.1456, found 451.1460.

Synthesis of 28 by the Cyclopropanation of 18.

2-((1R,4r,7S)-bicyclo[5.1.0]octan-4-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (28).

2-(Cyclohept-4-en-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (18, 30.0 mg, 0.071 mmol) was added to 1 mL of dichloromethane in a 10 mL round-bottom flask. The reaction mixture was cooled to 0 °C and degassed with N₂(g) for 15 min. Diethylzinc (249 μL, 0.25 mmol) was slowly added to the reaction vessel, followed by the slow addition of diiodomethane (29 μL, 0.35 mmol). The reaction mixture was stirred at 0 °C for 30 min and then the silicone stopper was replaced with a plastic stopper and wrapped with parafilm. The reaction mixture was stirred at rt overnight. Saturated ammonium chloride (0.5 mL) was added to the reaction vessel, and the reaction continued to stir for 30 min. The product was extracted with ethyl acetate, and the organic layer was washed with water (1 × 5 mL) and brine (1 × 5 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated by a rotary evaporator to obtain a residue purified by flash chromatography using 15% ethyl acetate in hexane eluent system to provide 4.0 mg (13%) of product as a white solid (HPLC t_R = 19.0 min): ¹H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.86 (s, 1H), 7.79–7.72 (m, 1H), 7.50 (t, J = 7.7 Hz, 1H), 7.38 (d, J = 8.7 Hz, 1H), 7.31 (t, J = 7.5 Hz, 1H), 2.95–2.84 (m, 1H), 2.33 (dt, J = 13.3, 5.7 Hz, 2H), 2.17 (dt, J = 9.0, 5.3 Hz, 2H), 1.88 (q, J = 12.5 Hz, 2H), 1.13–1.00 (m, 2H), 0.95 (q, J = 7.3, 6.3 Hz, 2H), 0.86 (d, J = 7.7 Hz, 1H), 0.77 (td, J = 7.8, 4.5 Hz, 1H). HRMS calcd for C₂₄H₂₃N₄Cl₂ (M+H)⁺ 437.1300, found 437.1304.

2-((1R,2R,4R)- and (1S,2S,4S))-Bicyclo[2.2.2]oct-5-en-2-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (29).

Compound 29 was synthesized following the general procedure E described above using 46t (8.0 mg, 0.026 mmol) and 47a (13 mg, 0.077 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 4.0 mg (36%) of a white solid (HPLC t_R = 17.9 min): ¹H NMR (400 MHz, chloroform-d) δ 8.50 (d, J = 2.6 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.1 Hz, 3H), 7.55 (ddd, J = 8.4, 7.0, 1.5 Hz, 1H), 7.44–7.33 (m, 2H), 6.70 (t, J = 7.5 Hz, 1H), 6.40 (t, J = 7.4 Hz, 1H), 3.47 (ddd, J = 10.3, 5.4, 2.1 Hz, 1H), 2.97–2.91 (m, 1H), 2.87–2.78 (m, 1H), 2.25 (ddd, J = 13.1, 10.2, 2.7 Hz, 1H), 1.86–1.76 (m, 1H), 1.71–1.62 (m, 2H), 1.49–1.34 (m, 2H); HRMS calcd for C₂₄H₂₁N₄Cl₂ (M+H)⁺ 435.1143, found 435.1136.

Synthesis of 30 and 31 by the Epoxidation of 18.

m-Chloroperoxybenzoic acid (17.0 mg, 0.10 mmol, 2.0 equiv) was added to a 10 mL round-bottom flask containing a solution of 2-(cyclohept-4-en-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine 18 (21.0 mg, 0.05 mmol, 1.0 equiv) in 2 mL of dichloromethane. The reaction mixture was stirred at rt for 3 h. Once the starting material disappeared via TLC, 1 mL of acetone and 1 mL of 10% aq. sodium bicarbonate were added, and the mixture was allowed to stir for 30 min. The product was extracted with ethyl acetate (5 mL), and the organic layer was washed with water (10 mL) and brine (10 mL). The organic layer was dried using magnesium sulfate and then filtered. A rotary evaporator evaporated the solvent to obtain a crude residue, first purified by flash chromatography using a 30% acetone in hexane eluent system to obtain a mixture of 30 and 31. A second flash chromatography column used a 0.75–1.0% methanol in dichloromethane eluent system to separate and isolate 30 (2.0 mg, 6%, red solid, analytical HPLC t_R = 14.1 min) and 31 (4.0 mg, 12%, red solid, analytical HPLC t_R = 14.3 min).

2-((1R,4s,7S)-8-Oxabicyclo[5.1.0]octan-4-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (30).

¹H NMR (400 MHz, chloroform-d) δ 8.47 (s, 1H), 7.93 (t, J = 8.7 Hz, 2H), 7.76 (dd, J = 8.7, 2.6 Hz, 1H), 7.56–7.49 (m, 1H), 7.41–7.31 (m, 2H), 3.27 (dd, J = 4.4, 1.8 Hz, 2H), 2.71 (s, 1H), 2.46 (d, J = 15.4 Hz, 2H), 2.09–1.80 (m, 6H); HRMS calcd for C₂₃H₂₁N₄OCl₂ (M+H)⁺ 439.1092, found 439.1093.

2-((1R,4r,7S)-8-Oxabicyclo[5.1.0]octan-4-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (31).

¹H NMR (400 MHz, chloroform-d) δ 8.50 (s, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.86 (d, J = 8.0 Hz, 1H), 7.79 (dd, J = 8.9, 2.6 Hz, 1H), 7.56–7.48 (m, 1H), 7.35 (dd, J = 15.7, 8.2 Hz, 2H), 3.15 (q, J = 4.6 Hz, 2H), 3.11–3.02 (m, 1H), 2.41 (dt, J = 13.8, 6.4 Hz, 2H), 2.14–2.05 (m, 3H), 2.01–1.88 (m, 3H); HRMS calcd for C₂₃H₂₁N₄OCl₂ (M+H)⁺ 439.1092, found 439.1090.

Synthesis of 32 by the Oxidation of a Mixture of Compounds 33 and 34.

(R)- and (S)-4-(4-((3,4-dichlorophenyl)amino)-1H-imidazo[4,5-c]quinolin-2-yl)cycloheptan-1-one (32). A 10 mL round-bottom flask containing a solution of (1R,4S)-, (1S,4R)-, (1R,4R)-, and (1S,4S)-4-(4-((3,4-dichlorophenyl)amino)-1H-imidazo[4,5-c]quinolin-2-yl)cycloheptan-1-ol (17.0 mg, 0.039 mmol) 33, and 34 in 1.5 mL dichloromethane, Dess–Martin periodinane (25.0 mg, 0.058 mmol) was added in one portion. The reaction mixture was stirred at rt until the starting material disappeared (monitored by TLC). The reaction mixture was quenched with saturated aq. NaHCO₃ (2 mL). The product was extracted with dichloromethane (5 mL) and the organic layer washed with water (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered, and concentrated by rotary evaporator to obtain a crude residue, which was purified by flash chromatography using a 0.75 to 1.0% methanol in dichloromethane eluent system to provide 3.9 mg (23%) of compound 32 as a red solid (HPLC purity: 95%; t_R = 13.2 min): ¹H NMR (400 MHz, chloroform-d) δ 8.46 (s, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.79 (s, 2H), 7.60–7.53 (m, 1H), 7.42–7.34 (m, 2H), 3.27–3.18 (m, 1H), 2.77 (t, J = 6.1 Hz, 2H), 2.67 (q, J = 5.1 Hz, 2H), 2.38 (d, J = 17.5 Hz, 2H), 2.15 (td, J = 12.9, 10.8, 7.3 Hz, 2H), 2.04–1.88 (m, 2H); HRMS calcd for C₂₃H₂₁N₄OCl₂ (M+H)⁺ 439.1092, found 439.1100.

Synthesis of Hydroxy Derivatives 33 and 34 by the Hydroboration–Oxidation of 18.

2-(Cyclohept-4-en-1-yl)-N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine 18 (20 mg, 0.05 mmol, 1.0 equiv) was added to 0.5 mL of dry tetrahydrofuran (THF) in a 25 mL round-bottom flask and flushed with N₂(g). The reaction mixture was cooled to 0 °C, and borane dimethyl sulfide complex solution in 2.0 M THF (47 μL, 0.1 mmol) was added to the reaction vessel. The reaction mixture was stirred for 30 min at 0 °C and then overnight at rt. The reaction flask was placed in a 0 °C ice–water bath when all the starting material was consumed. 10% aq. NaOH (0.5 mL, 0.15 mmol) and H₂O₂ (1 mL, 0.26 mmol) were added successively to the reaction vessel. The reaction mixture reacted at 0 °C for 3 h. The product was extracted with ethyl acetate (5 mL), and the organic layer was washed with water (10 mL) and brine (10 mL). The organic layer was dried over magnesium sulfate, filtered, and then evaporated by a rotary evaporator. Compounds 33 (1.6 mg, 8%, red solid, analytical HPLC t_R = 12.2 min) and 34 (2.6 mg, 12%, red solid (analytical HPLC t_R = 12.4 min) were isolated and purified from the crude residue by flash chromatography using a 0.75–1.0% methanol in dichloromethane eluent system.

(1R,4S)- and (1S,4R)-4-(4-((3,4-Dichlorophenyl)amino)-1H-imidazo[4,5-c]quinolin-2-yl)cycloheptan-1-ol (33).

¹H NMR (400 MHz, chloroform-d) δ 8.47 (d, J = 2.6 Hz, 1H), 7.93 (t, J = 7.7 Hz, 2H), 7.77 (dd, J = 8.8, 2.6 Hz, 1H), 7.50 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.38–7.28 (m, 2H), 4.06 (dq, J = 9.3, 4.5 Hz, 1H), 3.15 (tt, J = 9.6, 4.4 Hz, 1H), 2.22–2.03 (m, 3H), 2.00–1.79 (m, 5H), 1.63 (dddd, J = 13.7, 10.3, 8.0, 2.1 Hz, 1H), 1.52–1.39 (m, 1H); HRMS calcd for C₂₃H₂₃N₄OCl₂ (M+H)⁺ 441.1249, found 441.1247.

(1R,4S)- and (1S,4R)-4-(4-((3,4-Dichlorophenyl)amino)-1H-imidazo[4,5-c]quinolin-2-yl)cycloheptan-1-ol (34).

¹H NMR (400 MHz, chloroform-d) δ 8.48 (d, J = 2.7 Hz, 1H), 7.93 (d, J = 8.3 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.78 (dd, J = 8.8, 2.6 Hz, 1H), 7.50 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.40–7.28 (m, 2H), 3.97 (h, J = 4.3 Hz, 1H), 3.18–3.09 (m, 1H), 2.13 (pd, J = 8.0, 7.5, 4.5 Hz, 3H), 2.03–1.62 (m, 7H); HRMS calcd for C₂₃H₂₃N₄OCl₂ (M+H)⁺ 441.1249, found 441.1252.

2-Cyclohexyl-N-(4-iodophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (35).

Compound 35 was synthesized following the general procedure E described above using 46h (10 mg, 0.035 mmol) and 47b (38.0 mg, 0.17 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 10.0 mg (61%) of a white solid (HPLC t_R = 16.7 min): ¹H NMR (400 MHz, chloroform-d) δ 7.97 (d, J = 8.3 Hz, 1H), 7.90 (d, J = 8.4 Hz, 2H), 7.80–7.62 (m, 3H), 7.54 (t, J = 7.8 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 3.06–2.94 (m, 1H), 2.22 (d, J = 12.8 Hz, 2H), 1.95 (d, J = 12.8 Hz, 2H), 1.82 (d, J = 13.0 Hz, 1H), 1.69 (q, J = 12.2 Hz, 2H), 1.56–1.31 (m, 3H); HRMS calcd for C₂₂H₂₂N₄I (M+H)⁺ 469.0889, found 469.0898.

2-Cyclohexyl-N-(4-bromophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (36).

Compound 36 was synthesized following the general procedure E described above using 46h (15.0 mg, 0.052 mmol) and 47c (49.0 mg, 0.28 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 2.0 mg (10%) of a white solid (HPLC t_R = 15.7 min): ¹H NMR (400 MHz, chloroform-d) δ 7.98 (t, J = 9.4 Hz, 2H), 7.81–7.68 (m, 2H), 7.58–7.51 (m, 1H), 7.46 (d, J = 2.0 Hz, 1H), 7.36 (t, J = 7.5 Hz, 1H), 3.00 (tt, J = 11.9, 3.6 Hz, 1H), 2.26–2.17 (m, 2H), 1.94 (dt, J = 12.3, 3.8 Hz, 2H), 1.82 (d, J = 13.1 Hz, 1H), 1.69 (qd, J = 12.3, 3.4 Hz, 2H), 1.55–1.31 (m, 4H); HRMS calcd for C₂₂H₂₂N₄Br (M+H)⁺ 421.1028, found 421.1029.

Synthesis of 37 from a Heck Reaction Using 36.

Methyl (E)-3-(4-((2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-yl)amino)phenyl)acrylate (37).

2-Cyclohexyl-N-(4-bromophenyl)-1H-imidazo[4,5-c]quinolin-4-amine 36 (15.0 mg, 0.036 mmol), methyl acrylate (9 μL, 0.10 mmol, 1 equiv), palladium acetate (1.0 mg, 4.4 μmol, 2.8 equiv), and triethylamine (15 μL, 0.11 mmol, 3 equiv) were added successively to 2 mL of DMF in a 50 mL sealed tube. The reaction mixture was purged with N₂(g) at rt for 30 min and then stirred at 140 °C for 24 h. The product was diluted with ethyl acetate (5 mL) and filtered through a short silica plug. The filtrate was concentrated by rotary evaporator and then co-evaporated with toluene (2 × 2 mL) to obtain a crude residue, which was purified by flash chromatography using a 0.75–1.0% methanol in dichloromethane solvent system to afford 2.8 mg (18%) of compound 37 as a white solid (E-isomer, HPLC purity: 98%; t_R = 13.3 min): ¹H NMR (400 MHz, CDCl₃) δ 9.74 (s, 1H), 8.12 (s, 2H), 8.00 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.70 (d, J = 15.9 Hz, 1H), 7.55 (dd, J = 8.0, 5.9 Hz, 3H), 7.37 (t, J = 7.5 Hz, 1H), 6.37 (d, J = 15.9 Hz, 1H), 3.81 (s, 3H), 2.99 (d, J = 17.2 Hz, 1H), 2.22 (d, J = 12.8 Hz, 2H), 1.94 (d, J = 12.9 Hz, 3H), 1.88–1.74 (m, 1H), 1.69 (td, J = 12.4, 3.3 Hz, 2H), 1.48 (q, J = 12.6 Hz, 2H). HRMS calcd for C₂₆H₂₇N₄O₂ (M+H)⁺ 427.2134, found 427.2133.

Synthesis of 38 by a Sonogashira Reaction of 35.

2-Cyclohexyl-N-(4-((5-chlorothiophen-2-yl)ethynyl)phenyl)-1H-imidazo[4,5-c]quinolin-4-amine (38).

2-Chloro-5-ethynylthiophene (141 mg, 1.06 mmol, 5.0 equiv), 2-cyclohexyl-N-(4-iodophenyl)-1H-imidazo[4,5-c]quinolin-4-amine 35 (100 mg, 0.214 mmol, 1.0 equiv), bis(triphenylphosphine) palladium(II) dichloride (30.0 mg, 42.8 μmol, 5 mol%), copper(I) iodide (4.0 mg, 21.4 μmol, 2.5 mol%), and triethylamine (298 μL, 2.14 mmol, 10 equiv) were added successively to 4 mL of dry DMF in a round-bottom flask. The reaction mixture was purged with N₂(g) at rt for 30 min and then stirred at 80 °C for 4 h under N₂(g). The reaction mixture was cooled to the rt, diluted with ethyl acetate (10 mL), and filtered through a silica plug. The filtrate was concentrated by rotary evaporator and then co-evaporated with toluene (2 × 3 mL) to obtain a crude residue, which was purified by flash chromatography using a 15% ethyl acetate in hexane eluent system to provide 10 mg (10%) of compound 38 as a white solid (HPLC purity: 96%; t_R = 16.3 min): ¹H NMR (400 MHz, chloroform-d) δ 8.07 (d, J = 7.5 Hz, 2H), 8.00 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 7.1 Hz, 1H), 7.57–7.45 (m, 3H), 7.35 (s, 1H), 7.02 (d, J = 3.9 Hz, 1H), 6.81 (d, J = 3.9 Hz, 1H), 2.97 (tt, J = 11.8, 3.6 Hz, 1H), 2.19 (d, J = 11.8 Hz, 2H), 1.92 (d, J = 13.0 Hz, 2H), 1.80 (d, J = 12.7 Hz, 1H), 1.68 (d, J = 12.2 Hz, 2H), 1.45 (d, J = 12.7 Hz, 4H); HRMS calcd for C₂₈H₂₄N₄SCl (M+H)⁺ 483.1410, found 483.1412.

2-(Heptan-4-yl)-N-(4-iodophenyl)-1H-imidazo[4,5-c]quinolin-4-amine (39).

Compound 39 was synthesized following the general procedure E described above using 46h (195 mg, 0.64 mmol) and 47b (424 mg, 1.94 mmol). The crude was purified by silica gel column (10–20% ethyl acetate/hexane) to afford 33 mg (11%) of a white solid (HPLC t_R = 18.6 min): ¹H NMR (400 MHz, chloroform-d) δ 8.14–7.98 (m, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.67 (t, J = 7.4 Hz, 2H), 7.59–7.46 (m, 3H), 7.32 (t, J = 7.5 Hz, 1H), 3.05 (tt, J = 9.0, 5.7 Hz, 1H), 1.90–1.67 (m, 4H), 1.34–1.15 (m, 4H), 0.86 (t, J = 7.3 Hz, 6H). HRMS calcd for C₂₃H₂₆N₄I (M+H)⁺ 485.1202, found 485.1200.

6-Step Synthesis Protocol for 1H-Imidazo-[4,5-c]quinolin-4-amine Derivatives.

3-Nitroquinoline-2,4-diol (41).

According to the literature procedure,¹⁹ quinoline-2,4-diol (2.0 g, 12.41 mmol) was added to 12 mL of concentrated nitric acid in a 50 mL round-bottom flask. The reaction mixture was stirred for 10 min at rt and heated in an oil bath at 75 °C for 15 min. The reaction mixture was cooled to rt and poured on a crushed ice–water mixture to obtain a precipitate. The suspension was filtered, and the solid was washed with cold water and dried to obtain 2.43 g (95%) product as a nitrate salt (yellow solid).

2,4-Dichloro-3-nitroquinoline (42).¹⁹

The nitrate salt of 3-nitroquinoline-2,4-diol (41, 2.0 g, 9.7 mmol) was added to 17 mL of phenylphosphonic dichloride in a 50 mL round-bottom flask. The flask was fitted with a condenser and stirred the reaction mixture in an oil bath at 135 °C for 3 h. The reaction mixture was cooled to rt, poured slowly on a crushed ice–water mixture, and stirred vigorously to obtain the light brown clay-like precipitate. The mixture was filtered using a fritted funnel, and the precipitate was washed with cold water and dried under air to afford 2.05 g (87%) of crude product as an orange solid.

2-Chloro-3-nitroquinolin-4-amine (43).¹⁹

2,4-Dichloro-3-nitroquinoline (42, 2.0 g, 8.0 mmol) and 25% aq. ammonia (6 mL, 80 mmol, 10 equiv) were added to 20 mL of acetonitrile in a 100 mL glass pressure vessel. The mixture was stirred at 50 °C for 6 to 7 h. The reaction mixture was diluted with water (20 mL) and extracted with a mixture of ethyl acetate and methanol (95:5, 2 × 50 mL). The organic layer was separated, dried over MgSO₄, filtered, and concentrated by rotary evaporation to obtain 1.7 g (97%) of crude product as a yellow solid.

2-Chloroquinoline-3,4-diamine (44).¹⁹

To a 100 mL sealed vessel, 2-chloro-3-nitroquinolin-4-amine (43, 2.0 g, 9.0 mmol, 1.0 equiv), 30 mL of EtOH: H₂O (4:1), 4 N hydrochloric acid (~10 mL) and Fe powder (2.5 g, 45 mmol, 5 equiv) were added, and the reaction mixture was stirred at 75 °C for 3 h. The reaction mixture was cooled to rt and filtered through a short silica plug, and the plug was washed with EtOH:H₂O (95:5, 20 mL). The combined filtrate was neutralized with aq. NaOH/KOH until pH ~7.0. The product was extracted with a mixture of ethyl acetate and methanol (95:5, 2 × 100 mL). The organic layer was separated, dried over MgSO₄, filtered, and concentrated by rotary evaporation, leaving a residue. The residue was purified by silica chromatography with 2% methanol in dichloromethane as an eluent to obtain 1.2 g (70%) product as a brown solid.

Synthesis of Commercially Unavailable Carboxylic Acids (45c,l,m,t).

5,5,5-Trifluoro-2-(3,3,3-trifluoropropyl)-pentanoic Acid (45c).

³² In a 50 mL round-bottom flask equipped with a stir bar were added ethyl 2-cyanoacetate (0.91 mL, 8.54 mmol, 1.0 equiv, Scheme S2), 3-bromo-1,1,1-trifluoropropane (2.0 mL, 18.8 mmol, 2.2 equiv), potassium carbonate (2.48 g, 17.9 mmol, 2.1 equiv) and 21 mL of DMF (~0.4 M). The reaction mixture was flushed with nitrogen and stirred at 60 °C for 48 h. The reaction mixture was cooled to rt, and the solvent was removed under reduced pressure by rotary evaporation to obtain a crude residue as a mixture of ethyl 2-cyano-5,5,5-trifluoropentanoate and ethyl 2-cyano-5,5,5-trifluoro-2-(3,3,3-trifluoropropyl)pentanoate 57. The residue (1.37 g, 4.49 mmol, 1.0 equiv) was dissolved in aq. NaOH (21 mL, ~9.0 g, 225 mmol, 50 equiv), and tetrabutylammonium bromide (303 mg, ~21 mol%) was added to the flask. The reaction mixture was stirred under reflux at 90 °C for 36 h. 1 M aq. HCl was added to the reaction mixture until pH ~7. The mixture was extracted with ethyl acetate, and the organic layer was washed with water. The organic layer was dried over MgSO₄, filtered, and solvent evaporated in vacuo to provide the product as a black-brown oil, which was distilled under high vacuum at 170–180 °C to afford 300 mg (16%) crude product, assuming purity of ~50% (by ¹⁹F and ¹H NMR): ¹⁹F NMR (chloroform-d) δ −66.65; ¹H NMR (400 MHz, chloroform-d) δ 10.91–10.86 (m, 1H), 3.06–2.98 (m, 1H), 2.46 (ddt, J = 14.1, 9.6, 5.4 Hz, 1H), 2.27–2.01 (m, 3H), 1.97–1.83 (m, 2H), 1.75 (ddt, J = 13.7, 10.8, 5.5 Hz, 2H), 1.68–1.55 (m, 1H), 1.45–1.29 (m, 1H), 0.94 (td, J = 7.4, 1.2 Hz, 2H).

General Method for Favorskii Rearrangement^59,60 to Prepare 45l,m.

The appropriate cyclic ketone (1.25 mmol, 1.0 equiv, Scheme S3), N-bromosuccinimide (1.40 mmol, 1.0–1.1 equiv) and p-toluenesulfonic acid (0.13 mmol, 10 mol%)⁶¹ were added to 5 mL of dichloromethane in a 15 mL round-bottom flask. The reaction mixture was stirred at rt for 16 h. The solvent was removed under reduced pressure by rotary evaporation to obtain a residue. The residue was dissolved in 10% ethyl acetate in hexane and then passed through a short silica plug. The filtrate was concentrated by rotary evaporation to obtain the α-brominated cyclic ketone as an oily residue, which was used for the next step without further purification.

In a 25 mL round-bottom flask, the crude α-brominated cyclic ketone 59a,b (0.43 mmol) and sodium methoxide (232 mg, 4.3 mmol) were dissolved in 8 mL of methanol. The reaction mixture was stirred overnight and then refluxed for 30 min. 1 M aq. HCl was added to the reaction mixture until pH ~7. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (2 × 20 mL). The organic layer was separated, dried over MgSO₄, filtered, and concentrated by rotary evaporation to get a crude product. Purification by silica gel chromatography using a 20% ethyl acetate in hexane eluent system to provide white solid products.

Cyclononanecarboxylic acid (45l).

Compound 45l was synthesized following the Favorskii rearrangement procedure described above using 58a (100 mg, 0.65 mmol). The crude was purified by silica gel column (20% ethyl acetate in hexane) to afford 79 mg (27%) of a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 11.97 (s, 1H), 2.38 (ddq, J = 12.7, 8.6, 4.5 Hz, 1H), 1.74 (d, J = 4.0 Hz, 2H), 1.44 (d, J = 7.0 Hz, 14H). Alternatively, a solution of crude α-brominated cyclic ketone (59a, 145 mg, 0.622 mmol, 1.0 eq.) in 3 mL of 10% aq. KOH (10 eq.) was refluxed for 3 h. The reaction mixture was cooled to the room temperature and was acidified with dil. HCl until the pH ~ 5–6. The reaction mixture was diluted with EtOAc (20 mL), and separated the layers. The aqueous phase was washed with EtOAc (2×10 mL), and the combined EtOAc layer was dried over Na₂SO₄ and filtered. The filtrate was concentrated by rotary evaporation to obtain the crude material, which was purified by silica column to obtain a colorless oil. Yield: 410 mg (76%).

Cyclodecanecarboxylic acid (45m).

Compound 45m was synthesized following the Favorskii rearrangement procedure described above using 58b (50 mg, 0.30 mmol). The crude was purified by silica gel column (20% ethyl acetate in hexane) to afford 89 mg (29%) of a white solid: ¹H NMR (400 MHz, DMSO-d₆) δ 12.01 (s, 1H), 2.57 (d, J = 6.4 Hz, 1H), 1.82–1.45 (m, 18H).

Synthesis of 45t by a Diels–Alder Reaction and a Subsequent Hydrolysis Reaction.

((1R,2R,4R)- and (1S,2S,4S)-bicyclo[2.2.2]oct-5-ene carboxylic acid (45t). Toluene (2 mL) was added to a 15 mL glass pressure tube containing 1,3-cyclohexadiene 60 (0.50 mL, 5.2 mmol, 1.0 equiv, Scheme S4) and methyl acrylate (0.52 mL, 5.78 mmol, 1.1 equiv). The solution was purged with N₂(g) and then sealed. The reaction mixture was stirred at 180 °C for 20 h. The reaction mixture was cooled to rt, and the solvent evaporated by rotary evaporation to afford the mixture of endo and exo isomers as a clear oil. The racemic endo product (carboxylate methyl ester) was isolated as a clear oil (402 mg, 49% yield) by silica gel chromatography using 5% ethyl acetate in hexane as the eluent system.

The endo carboxylate methyl ester (80.0 mg, 0.48 mmol) was dissolved in 5 mL of methanol in a 25 mL round-bottom flask, and an aq. NaOH solution (1.5 M, 5 mL) was added. The reaction mixture was stirred at rt for 1 h. 1 M aq. HCl was added to the reaction mixture until pH ~ 7. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (2 × 20 mL). The organic layer was separated, dried over MgSO₄, filtered, and concentrated by rotary evaporation to obtain the desired 400 mg (54%) of the carboxylic acid 45t: ¹H NMR (400 MHz, DMSO-d₆) δ 11.91 (s, 1H), 6.25 (s, 1H), 6.10 (s, 1H), 2.85 (d, J = 3.4 Hz, 1H), 2.53 (d, J = 7.4 Hz, 2H), 1.65 (s, 1H), 1.60–1.48 (m, 2H), 1.48–1.39 (m, 1H), 1.16 (ddd, J = 13.4, 8.9, 3.5 Hz, 2H).

General Procedures for 2-Substituted-4-chloro-1H-imidazo[4,5-c]quinolines (46b,c,e,h,j–t).

Procedure A.

2.0 g PPA (per 50 mg of 2-chloroquinoline-3,4-diamine) was weighed in a 5 mL round-bottom flask. 2-Chloroquinoline-3,4-diamine (1.0 equiv) and the appropriate carboxylic acid 45c,e,h,k–n (1.2 equiv) were added to the flask. The reaction mixture was stirred at 120 °C for 5 h. The reaction mixture was quenched by pouring on a crushed ice–water mixture. The ice–water mixture was neutralized with aq. K₂CO₃ (2 M) until pH 8–9 under stirring. The reaction mixture was extracted with ethyl acetate and then washed with water and brine several times. The organic layer was dried over MgSO₄, filtered, and removed the solvent by rotary evaporation to obtain the crude product, purified by silica column to obtain the 1H-imidazo[4,5-c]quinoline derivative with 15–25% ethyl acetate in hexane as the eluent system.

Procedure B.

The appropriate carboxylic acid 45b,j,o–t (1.4 equiv) and NMI (3.5 equiv), were added to 4 mL acetonitrile in a 15 mL round-bottom flask with stirring, followed by 2-chloroquinoline-3,4-diamine (1 equiv) and TCFH (1.5 equiv). The reaction mixture was stirred at 60 °C for 5 h. The cooled reaction mixture was diluted with ethyl acetate, and the organic layer was washed with brine (3×). The organic layer was dried over MgSO₄ and filtered, transferred to a 50 mL round-bottom flask, and the solvent evaporated by a rotary evaporator. A 1:1 solution of aq. NaOH (15 equiv of NaOH in 10 mL of H₂O):methanol (10 mL) was added with stirring. The reaction mixture was refluxed at 90 °C for 3 h. The cooled reaction mixture was diluted with ethyl acetate, and the organic layer was washed with brine (3 × 10 mL) followed by water (3 × 10 mL). The organic layer was dried over MgSO₄, evaporated in vacuo, and the resulting residue was used in the final reaction step without further purification.

General Stannylation Procedure to Prepare 48 and 49 from 39.

2-(Heptan-4-yl)-N-(4-iodophenyl)-1H-imidazo-[4,5-c]quinolin-4-amine 39 (0.10 mmol, 1.0 equiv), bis(triphenylphosphine)palladium(II) dichloride (20 μmol, 20 mol%), and hexamethylditin or hexabutylditin (0.5 mmol, 5.0 equiv) were added to 2 mL of 1,4-dioxane in a 10 mL round-bottom flask. The reaction mixture was purged with N₂(g) at rt for 30 min and stirred at 70 °C for 2.5 h or until the starting material disappeared (monitored by TLC). The reaction mixture was cooled to the rt, diluted with ethyl acetate (10 mL), and filtered through a short silica plug. The filtrate was concentrated by rotary evaporator and then co-evaporated with toluene (2 × 3 mL) to obtain a crude residue, purified by flash chromatography using a 10% ethyl acetate in hexane eluent system.

2-(Heptan-4-yl)-N-(4-(trimethylstannyl)phenyl)-1H-imidazo[4,5-c]quinolin-4-amine (48).

Compound 48 was synthesized following the stannylation procedure described above using 39 (49.0 mg, 0.10 mmol) and hexamethylditin (105 μL, 0.50 mmol). The crude was purified by silica gel column (20% ethyl acetate in hexane) to afford 5.0 mg (10%) of a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.04 (s, 2H), 7.83 (s, 1H), 7.57–7.47 (m, 3H), 7.40–7.28 (m, 2H), 3.11–2.95 (m, 1H), 1.87–1.70 (m, 4H), 1.40–1.28 (m, 4H), 0.89 (d, J = 7.3 Hz, 6H), 0.39–0.17 (m, 9H); HRMS calcd for C₂₆H₃₅N₄Sn (M+H)⁺ 523.1884, found 523.1882.

2-(Heptan-4-yl)-N-(4-(tributylstannyl)phenyl)-1H-imidazo[4,5-c]quinolin-4-amine (49).

Compound 49 was synthesized following the stannylation procedure described above using 39 (50.0 mg, 0.10 mmol) and hexabutylditin (300 μL, 0.50 mmol). The crude was purified by silica gel column (20% ethyl acetate in hexane) to afford 9.0 mg (13%) of a white solid: ¹H NMR (400 MHz, chloroform-d) δ 8.05 (d, J = 8.8 Hz, 1H), 7.97 (s, 1H), 7.79 (d, J = 21.8 Hz, 2H), 7.57–7.44 (m, 2H), 7.38 (t, J = 7.7 Hz, 1H), 7.32 (s, 1H), 3.04 (s, 1H), 1.80 (dq, J = 12.3, 7.1, 6.3 Hz, 4H), 1.65 (tt, J = 8.2, 6.5 Hz, 6H), 1.56 (ddt, J = 10.4, 8.0, 3.7 Hz, 4H), 1.38–1.28 (m, 19H), 0.91 (dd, J = 8.9, 7.3 Hz, 18H); HRMS calcd for C₃₅H₅₃N₄Sn (M+H)⁺ 649.3292, found 649.3293.

Pharmacological Methods.

Cell Culture.

HEK-293 cell lines expressing wild-type human, mouse, or chimeric A₃AR were cultured and maintained in Dulbecco’s modified eagle medium (DMEM) along with 10% fetal bovine serum, 1% penicillin/streptomycin, and 0.6 mg/mL G418.

Membrane Preparation.

HEK-293 cells stably expressing wild-type human, mouse, or chimeric A₃ARs were washed in phosphate-buffered saline followed by homogenization in hypotonic lysis buffer containing 10 mM Na⁺-HEPES, 10 mM EDTA, and 1 mM benzamidine (pH 7.4) and then centrifuged at 27000g for 30 min at 4 °C. Cell pellets were subsequently resuspended in He buffer containing 10 mM Na⁺-HEPES, 1 mM EDTA, and 1 mM benzamidine (pH 7.4) and re-centrifuged. Supernatant was discarded and remaining pellets were resuspended in He buffer containing 10% sucrose and stored at −20 °C.

Single-Point Dissociation Study.

~0.3 nM of [¹²⁵I]50 was incubated with 50 μg of HEK-293 cell membranes expressing A₃ARs for 2 h at rt in 100 μL of binding buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgCl₂, 1 mM EDTA, and 1 unit/mL adenosine deaminase). The assay was started by adding 100 μM of the nonselective agonist 51, along with 10 μM of the PAM or vehicle (DMSO). After 60 min, the bound [¹²⁵I]50 was measured following rapid filtration using GF/C filters. Data are expressed as the amount of radioligand left remaining after 60 min as a percent of the vehicle. A gamma counter measured [¹²⁵I]50-bound receptors in the filters. Nonspecific binding was determined for all assays by incubation of membranes in the presence of 100 μM 51. All experiments were performed in technical and biological triplicate. Raw CPM values for agonist and modulator tubes and control tubes were used to calculate statistical significance using a two-tailed paired Student’s t test.

Single-Point Equilibrium Binding Study.

HEK-293 membranes (50 μg) were incubated at rt with ~0.3 nM [¹²⁵I]50 and 10 μM modulator of vehicle (DMSO) for 18 h in 100 μL of binding buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgCl₂, 1 mM EDTA and 1 unit/mL adenosine deaminase). At that point, the amount of radioligand was measured by rapid filtration using GF/C filters. Data are expressed as the amount of specific binding as a % change from vehicle. Nonspecific binding was determined for all assays by incubation in the presence of 100 μM 51. All experiments were performed in technical and biological triplicate. Binding data of agonists influenced by modulators were statistically compared to the control using a two-tailed unpaired Student’s t test.

GTPγS Binding Method for Measuring Receptor Activation.

[³⁵S]GTPγS binding assays were conducted to assess the direct effects of modulators on receptor activation. Five μg membranes overexpressing the indicated A₃AR were pretreated with modulators for 1 h in 100 μL GTPγS binding buffer (50 mM Tris-HCl [pH 7.4], 10 mM MgCl₂, 1 mM EGTA, 100 mM NaCl, 0.004% CHAPS, an 0.5% BSA). To block endogenously expressed A_2BARs found in HEK-293 cells, AR antagonists 62 and 63 were added, with each at a final concentration of each of 300 nM. 1 Unit/mL of ADA was added to break down any endogenous adenosine that might have been present. Reactions were initiated by the addition of ~0.2 nM [³⁵S]GTPγS and agonist in 100 μL GTPγS binding buffer and incubated for 2 h. Reactions were stopped with membranes being harvested by rapid filtration through Whatman GF/B filters presoaked for 2 h in GTPγS binding buffer containing an additional 0.02% CHAPS using a cell harvester (Brandel, Gaithersburg, MD). Radioactivity trapped in the filters was measured by liquid scintillation counting. Nonspecific binding of [³⁵S]GTPγS was measured in the presence of 10 μM unlabeled GTPγS. Agonist potency and maximal efficacy at 0.1 μM, 1.0 μM, and 10 μM were statistically compared to the control using one-way ANOVA with multiple comparisons and Bonferroni post hoc test.

ADMET Methods.

Both in vitro and in vivo ADMET properties were determined by JRF India of JRF Global (Gujarat, India).

In Vivo Assays.⁴⁷

The animal breeding facility, Jai Research Foundation, provided healthy adult male rats (Rattus norvegicus) of Wistar strain (RccHan:WIST). Rats were 6 to 10 weeks old at the start of acclimation. The weight variation of rats was ±20% of mean body weight (b.wt.) at the start of acclimation. Table S6 shows the experimental outline for the in vivo study of pharmacokinetics using rats.

Two routes of administration, intravenous and oral, were used to determine A₃AR PAM bioavailability. The dose level for the G₁ intravenous route was 0.5 mg/kg b.wt., and the dose levels for the G_2–4 oral route were 1, 3, and 10 mg/kg b.wt. The A₃AR PAM-DMSO solution was diluted with an aq. solution of 20% 2-hydroxypropyl-β-cyclodextrin (HPBCD) for the intravenous administration, creating a final volume ratio of DMSO to HPBCD, 10:90. An equal volume of Kolliphor EL (polyoxyl castor oil) was added to the A₃AR PAM-DMSO solution for oral administration, followed by phosphate buffer saline (PBS). The final volume ratio of DMSO:Kolliphor:PBS for the oral administration was 15:15:70.

200 μL blood was collected from the jugular vein from each rat group on the day of dosing at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post dosing to assess the A₃AR PAM PK profile through intravenous administration. Blood samples were collected in prelabeled microcentrifuge tubes with the anticoagulant heparin (20 IU/mL of blood). After collection, samples were inverted 4 to 5 times, placed on ice, and centrifuged at 9000 rpm for 10 min. Plasma was frozen at −70 °C.

Pharmacokinetic analysis of the frozen plasma concentration–time data was performed using the non-compartmental model of the WinNonlin software. Estimated parameters were the maximum plasma concentration (C_max), the time to achieve peak plasma concentration (T_max), the area under the plasma concentration–time curve until the last measured time point (AUC_0-last), the area under the plasma concentration–time curve extrapolated to infinity (AUC_0−∞), the terminal elimination half-life (T_1/2), the mean residence time (MRT), volume of distribution (V_d), the elimination rate constant (k_el), the bioavailability (%F), and the clearance (Cl).

In Vitro Assays.

Plasma stability was measured in three species: human, rat, and mouse at 0 to 120 min by liquid chromatography mass spectroscopy–mass spectroscopy (LCMS-MS). % A₃AR PAM remaining was provided at 120 min and t_1/2 (min). The methods were as described.⁴⁷

HepG2 cytotoxicity was measured using the CellTiter-Glo Luminescent Cell Viability assay. This assay determined the number of live cells in culture by measuring the ATP metabolism of active cells. 10 to 40 K cells were placed in a 96-well plate. The A₃AR PAMs were incubated with live cells for 48 h. Eight dilutions were made with varying concentrations of A₃AR PAM ranging from 30 μM to 0.2 μM and incubated. Verapamil/Rifampicin were used as a reference compound. Analysis was done via luminometry. Data was reported as an IC₅₀ value, the concentration of A₃AR PAM that reduces cell viability by 50% (ATP measurement).

Human Ether-à-go-go Related Gene (hERG) potassium channels are involved in cardiac action potential re-polarization. hERG assay measures the % inhibition of the potassium channels by the A₃AR PAM. The assay used HEK-293 cells stably transfected with the hERG potassium channel. A₃AR PAM concentrations were prepared using serial half-log dilutions, starting at 30 μM. A₃AR PAMs were incubated with the hERG-expressing cells for 2 h. A₃AR PAMs bound to the hERG ion channel were identified by their ability to displace the tracer (Predictor hERG Tracer Red), which resulted in a lower fluorescence polarization using a TAMRA fluorescent polarization filter. E-4031 is a reference compound. % inhibition was reported as the ratio of half-maximum inhibitory concentration of the hERG channel (hERG IC_50, μM) to the peak serum concentration of unbound drug (C_max).

CYP inhibition was determined with a cocktail approach. A₃AR PAMsat six concentrations in duplicate (30 μM to 0.12 μM)were incubated with liver microsomes (human, rat, mice) containing the CYP panel with known substrates (1A2, phenacetin (10 μM); 2C9, diclofenac (5 μM); 2C19, omeprazole (5 μM); 2D6, dextromethorphan (5 μM); 3A4, midazolam (2 μM)), 1 mM of NADPH cofactor, 1 μM miconazole (pan inhibitor) in plasma. Analysis of substrates was done by LC-MS/MS. This approach reported A₃AR PAM concentrations which produced 50% inhibition of CYPs (IC₅₀ value, μM). LCMS-MS quantified metabolites by area ratio: acetaminophen (1A2), 4-OH-diclofenac (2C9), 5-OH-omeprazole (2C19), dextrorphan (2D6), and 1-OH-midazolam (3A4).

Microsomal stability assays were done using human, rat, and mouse liver microsomes containing the following enzymes: CYPs, flavin monooxygenases, carboxylesterases, and epoxide hydrolase. A₃AR PAMs were in 3 μM concentrations duplicates and mixed with 1 mM NADPH and 0.5 g/mL of human, rat, or mouse liver microsomes. Samples were taken from five time periods (0.5, 15, 30, 60, 90, 120 min) and analyzed by LC-MS/MS. The average half-life (min) and % remaining at 120 min was reported.

Caco-2 cell permeability assay measured the rate of transport of a compound across the Caco-2 cell line (21-day process consisting of bidirectional monitoring of absorptive (A-B) and secretory (B-A) fluxes). The Caco-2 cell line originated from a human colon carcinoma, having a polarized monolayer, an apical surface, and intercellular junctions. The cells were exposed to the A₃AR PAM (10 μM) in an HBSS buffer with 2% BSA. Samples were gathered at 0- and 120 min. Samples were analyzed by LCMS-MS. Positive controls were atenolol, digoxin, and propranolol. Apparent permeability (P_app – 10⁶ cm/sec), efflux ratio, and % recovery were reported.

pION solubility of each A₃AR PAM at pH 7.4 was measured using the pION buffer method. Each A₃AR PAM was added to the pION buffer in sufficient amount to reach 500 μM (hypothetically). Sampling was done at 18 h. Positive controls were albendazole and flurbiprofen. Solubility was reported for each A₃AR PAM as mean solubility (μg/mL).

Plasma protein binding was determined and expressed as the % unbound A₃AR PAM in the plasma of three species (rat, mouse, and human). Cellulose membranes in potassium phosphate buffer (100 mM, pH 7.4) were exposed to 10 μM of A₃AR PAM and 500 μL of plasma. Samples were loaded into dialysis cells and maintained in an incubator at 37 °C at 100 rpm. Samples were collected into prelabeled microfuge tubes at 0 an 5 h. The samples were vortexed and centrifuged. Supernatants were analyzed by LC-MS/MS. % binding reported: 1–40% low bound, 41–70% medium bound, 71–100% highly bound.

Simulated gastrointestinal fluids were used to determine whether A₃AR PAMs are chemically stable in the stomach at low pH 1–2 or in the intestine pH 6–8. Five μM of each A₃AR PAM was added to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Sampling was done at 0- and 120 min time points. Method of analysis was LC-MS/MS by area ratio. % A₃AR PAM at 120 min was reported.

Prediction of Log D, TPSA, and BBB log([brain]:[blood]) was performed using the StarDrop software (v. 7.2), https://www.optibrium.com/stardrop-installers/.⁴⁵

Molecular Docking.

Maestro (Maestro 2021–2) of the Schrödinger (New York, NY, USA) suite was used for basic molecular modeling operations. Ligands were drawn and minimized with OPLS4⁶² force field with water solution model. The model of hA₃AR was generated in a previous work,⁴⁹ using an antagonist-bound hA₁AR X-ray structure as a template and a refinement with Induced Fit Docking of the antagonist 64. The following tautomeric states were employed for histidines: protonation at N_δ nitrogen for H272^7.43 (HSD according to the CHARMM nomenclature) and at the Nε nitrogen for H793.21, H953.37, H1244.39, H158EL2, and H3048.65. The Ballesteros–Weinstein sequence-based numbering scheme⁶³ is indicated as superscript for all residues throughout the text.

Compounds 17 and 7 were docked to the receptor structure using the IFD tool of the Schrödinger suite.^50,65 An inner box of 10 Å box and outer box of 30 Å, centered on the barycenter of residues F168^EL2 and N250^6.55, were employed. Residues within 3 Å of the ligand (with the addition of Q167^EL2 and V169^EL2) were optimized, and the XP scoring function⁶⁷ was used for the refinement stage. A maximum of 20 poses was generated for each ligand, and the 5 top scoring ones (according to the IFDScore) were minimized with Prime MM-GBSA tool, using implicit membrane, VSGB solvation model, OPLS4 force field, and minimizing residues within 3 Å from the ligand.

The Semiempirical Extended Tight-Binding Program Package, XTB,⁶⁴ was used for the dihedral scan. The geometry of the common N-(3,4-dichlorophenyl)-1H-imidazo[4,5-c]quinolin-4-amine scaffold was optimized with the GFN2-xTB method, and then a relaxed dihedral scan of the torsional angle defined by atoms N5–C4–N(amino)–Cp(phenyl) was performed from 0° to 360° in 72 steps (freezing the scanned dihedral with a force constant of 50 E_h/a₀²).

ABBREVIATIONS USED

ADA

adenosine deaminase

AR

adenosine receptor

DAT

dopamine transporter

DIPEA

diisopropylethylamine

DPFGSE

Double Pulsed Field Gradient Selective Echo

HBSS

Hanks’s balanced salt solution

HEK-293

human embryonic kidney 293

HMA

hexamethylene amiloride

HPBCD

2-hydroxypropyl-β-cyclodextrin

IFD

induced fit docking

KOR

κ opioid receptor

MOR

μ opioid receptor

NAM

negative allosteric modulator

NMI

N-methylimidazole

PAM

positive allosteric modulator

PDSP

NIMH Psychoactive Drug Screening Program

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

PPA

polyphosphoric acid

RMSF

root-mean-square-fluctuation

TCFH

tetramethylchloroformamidinium hexafluorophosphate

TPSA

topological polar surface area

TSPO

translocator protein