Amino-3,5-Dicyanopyridines Targeting the Adenosine Receptors Ranging from Pan Ligands to Combined A1/A2B Partial Agonists

pharmaceuticals

Article

Amino-3,5-Dicyanopyridines Targeting the

Adenosine Receptors. Ranging from Pan Ligands to

Combined A1

/A2B Partial Agonists

Daniela Catarzi1,*, Flavia Varano1, Katia Varani2, Fabrizio Vincenzi2 , Silvia Pasquini2, Diego Dal Ben3 , Rosaria Volpini3 and Vittoria Colotta1

1 _{Dipartimento di Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino, Sezione di Farmaceutica} e Nutraceutica, Università degli Studi di Firenze, Via Ugo Schiff, 6, 50019 Sesto Fiorentino, Italy;

flavia.varano@unifi.it (F.V.); vittoria.colotta@unifi.it (V.C.)

2 _{Dipartimento di Scienze Mediche, Sezione di Farmacologia, Università degli Studi di Ferrara,} Via Fossato di Mortara 17-19, 44121 Ferrara, Italy; vrk@unife.it (K.V.); fabrizio.vincenzi@unife.it (F.V.); silvia.pasquini@unife.it (S.P.)

3 _{Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università degli Studi di Camerino,} via S.Agostino 1, 62032 Camerino (MC); Italy; diego.dalben@unicam.it (D.D.B.);

rosaria.volpini@unicam.it (R.V.)

* Correspondence: daniela.catarzi@unifi.it

Received: 26 September 2019; Accepted: 18 October 2019; Published: 22 October 2019




Abstract:The amino-3,5-dicyanopyridine derivatives belong to an intriguing series of adenosine receptor (AR) ligands that has been developed by both academic researchers and industry. Indeed, the studies carried out to date underline the versatility of the dicyanopyridine scaffold to obtain AR ligands with not only a wide range of affinities but also with diverse degrees of efficacies at the different ARs. These observations prompted us to investigate on the structure–activity relationships (SARs) of this series leading to important previously reported results. The present SAR study has helped to confirm the 1H-imidazol-2-yl group at R2position as an important feature for producing potent AR agonists. Moreover, the nature of the R1 substituent highly affects not only affinity/activity at the hA1 and hA2B ARs but also

selectivity versus the other subtypes. Potent hA1and hA2BAR ligands were developed, and among them,

the 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-[4-(prop-2-en-1-yloxy)phenyl]pyridine-3,5-dicarbonitrile (3) is active in the low nanomolar range at these subtypes and shows a good trend of selectivity versus both the hA2Aand hA3ARs. This combined hA1/hA2Bpartial agonist activity leads to a synergistic effect on

glucose homeostasis and could potentially be beneficial in treating diabetes and related complications.

Keywords: G protein-coupled receptors; adenosine A2Breceptor ligands; adenosine A1receptor

ligands; aminopyridine-3,5-dicarbonitriles; ligand-adenosine receptor modeling studies

1. Introduction

Adenosine is a ubiquitous autacoid that has been reported to play important roles in various tissues by acting through four different adenosine receptors (ARs) classified as A1, A2A, A2B, and A3

receptors [1]. These ARs belong to the G protein–coupled receptors superfamily, and each of them has different functions although with some overlap. Depending on their coupling to G-protein, they can either increase or decrease intracellular cAMP levels by affecting adenylate cyclase (AC) activity. However, other effector systems have been proposed [2]. Adenosine has the highest affinity for the A1

and A2AARs, the lowest for A2BAR, and intermediate affinity for the A3subtype. Selective agonists

and antagonists have been developed for each of the ARs [3,4], and their discovery opened up new avenues for potential drugs treatment of a variety of conditions such as neurodegenerative disorders,

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chronic inflammatory diseases, asthma, and many other pathophysiological states that are known to be associated with changes in adenosine levels [3].

Dysregulated intracellular nucleotide concentrations can be produced by altered and defective cellular metabolism leading to important metabolic pathologies such as diabetes mellitus [3,5,6]. This disease is increasing worldwide due to aging, incorrect lifestyle, and eating habits, and its complications are of great clinical and socioeconomic impact. All four ARs are involved in the regulation of islet hormone secretion and have the potential of being drug targets for the treatment of diabetes [3]. Currently, much attention has been devoted to A2BAR agonists as potential new drugs

for diabetes treatment, even if there is discrepancy in the studies reported in literature, which may be ascribed to the different experimental conditions employed [3,7]. However, it is reported that agonists of this AR subtype may be useful for the therapy of this pathology [3].

The role of the A1AR in diabetes seems to be clearer [3,8]. In fact, improvement of insulin

sensitivity and glucose homeostasis by A1 activation has been well recognized for many years,

thus the promotion of A1AR agonists as drug candidates [5,6]. Since full A1AR agonists are

generally characterized by cardiovascular side effects, partial A1AR agonists could represent the

best choice to avoid cardiovascular complications [9]. For this reason, a synergistic effect on glucose homeostasis targeting both A1and A2BARs could be of interest for treating diabetes and its

complications. It is also worth noting that A1AR is involved in lipid metabolism, so the development

of A1AR agonists as lipolytic agents is reported to be useful in cardiovascular disease–associated

pathologies [10]. While the pathophysiological roles of the A1AR are well known, the A2Bsubtype

is the least characterized because of the paucity of potent and selective agonists. BAY-606583 (2-{[6-Amino-3,5-dicyano-4-(4-(cyclopropylmethoxy)phenyl)pyridin-2-yl]thio}acetamide) is one of the most potent and selective A2BAR agonists known to date [11]. However, we recently

produced a new series of amino-3,5-dicyanopyridines as BAY-606583 analogues [12]; most of the compounds are potent human (h) A2B AR partial agonists. Among them,

the 2-{[(1H-imidazol-2-yl)methyl]thio}-6-amino-4-[4-(cyclopropylmethoxy)phenyl]pyridine-3,5-dicarbonitrile (P453, Figure1) turns out to be three-fold more active than BAY-606583, although it is less selective. Compound P453 was used for experiments in in vitro models of short-term synaptic plasticity and ischemic-like condition [13]. In particular, compound P453 was shown to be a functional A2BAR agonist

whose activation decreases PPF (paired pulse facilitation) by increasing glutamate release at presynaptic terminals and delays A1AR-mediated fEPSP inhibition during a two-minute oxygen and glucose deprivation

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N NH₂ R1 CN CN R2

Reference compounds

_{Currently reported 1-20}

R

_{= aryl, benzyl, methyl}

N H N S

R

₌

LUF series R

₌

R

₌

P453 R

₌

Figure 1. Lead structures and currently reported amino-3,5-dicyanopyridine-based adenosine receptor ligands.

On this basis, the 1H-imidazol-2-yl group was used to design other amino-3,5-dicyanopyridine derivatives in order to deeply investigate the influence of the R1 _{substituent at the 4-position on AR} affinity and selectivity. Moreover, other modifications were performed at R2 _{to enrich what is known} about how the nature of the substituent at this position modulates AR affinity and selectivity.

2. Results

2.1. Chemistry

Compounds 1–4, 6–20, and their corresponding intermediates were synthesized according to the procedure reported in Schemes 1–3. Following the same procedures, the 2-(((1H-Imidazol-2-yl)methyl)thio)-6-amino-4-(4-ethoxyphenyl)pyridine-3,5-dicarbonitrile 5, previously reported in reference [12], was prepared. For the synthesis of derivatives 1–4, 6–13, and 20, the strategic intermediates are represented by the 6-sulfanyl-substituted compounds 28-40 [12,14,18,20–23], which were prepared as depicted in Scheme 1. The suitable aldehyde was reacted with malononitrile and thiophenol, in boiling water, in the presence of a catalytic amount of basic alumina to yield the 4-aryl- and 4-benzyl-6-phenylsulfanyl-derivatives 21–27 [18,24–26], with moderate to good yield. When these latter were treated with anhydrous sodium sulfide in anhydrous DMF at 80 °C, followed by acidification with HCl, the free thiol 28–38 [12,18,20,21], 40 [14]were obtained in high yields. The preparation of the 4-methyl-6-sulfanyl compound 39 [22,23] was performed by reacting the (1-ethoxyethylidene)propanedinitrile 41 [22] with 2-cyanothioacetamide in boiling ethanol containing EtONa. The target dicyanopyridines 1–4, 6–13, and 20 were obtained by reaction of the 6-sulfanyl intermediates 28–40 with 2-(bromomethyl)-1H-imidazole hydrobromide (1–4, 6–13) or methyl chloroacetate (20) in the presence of sodium hydrogen carbonate at room temperature (rt).

To replace the thiomethyl linker at the 6-position with an aminomethyl bridge, compounds 14–

16 were synthesized (Scheme 2) starting from the 6-chloro derivative 42, obtained with a reported

procedure [27], and the suitable commercially available amine. The reaction was carried out in the presence of triethylamine and in a refluxing mixture of THF/EtOH. The same procedure was applied for the synthesis of the 6-(1H-imidazol-1-yl)-derivative 17, which was obtained in high yield.

Figure 1. Lead structures and currently reported amino-3,5-dicyanopyridine-based adenosine receptor ligands.

From a structure–activity point of view, the new findings confirm the 1H-imidazol-2-yl group in the R2 pendant as an important feature for producing potent AR agonists. Certain 2-amino-4-aryl-6-(1H-imidazol-2-yl-methylsulfanyl)-pyridine-3,5-dicarbonitrile compounds, belonging to the LUF series (Figure1), were already reported to display nanomolar affinity for all the ARs, including the A2B subtype for which they showed, in general, considerable affinity and

efficacy [14]. The growing interest in this class is also dictated by the fact that non-nucleoside AR agonists seem to be more versatile for pharmacological studies, showing fewer species differences than the adenosine-like ones [4]. Moreover, the studies carried out to date are sufficient to underline the versatility of the amino-3,5-dicyanopyridine scaffold for producing AR ligands with not only a wide range of affinities but, interestingly, with different degrees of efficacy at the different ARs [11,12,14–19].

On this basis, the 1H-imidazol-2-yl group was used to design other amino-3,5-dicyanopyridine derivatives in order to deeply investigate the influence of the R1substituent at the 4-position on AR affinity and selectivity. Moreover, other modifications were performed at R2_{to enrich what is known}

about how the nature of the substituent at this position modulates AR affinity and selectivity.

2. Results

2.1. Chemistry

Compounds 1–4, 6–20, and their corresponding intermediates were synthesized according to the procedure reported in Schemes 1–3. Following the same procedures, the 2-(((1H-Imidazol-2-yl)methyl)thio)-6-amino-4-(4-ethoxyphenyl)pyridine-3,5-dicarbonitrile 5, previously reported in reference [12], was prepared. For the synthesis of derivatives 1–4, 6–13, and 20, the strategic intermediates are represented by the 6-sulfanyl-substituted compounds 28–40 [12,14,18,20–23], which were prepared as depicted in Scheme1. The suitable aldehyde was reacted with malononitrile and thiophenol, in boiling water, in the presence of a catalytic amount of basic alumina to yield the 4-aryl- and 4-benzyl-6-phenylsulfanyl-derivatives 21–27 [18,24–26], with moderate to good yield. When these latter were treated with anhydrous sodium sulfide in anhydrous DMF at 80◦C, followed by acidification with HCl, the free thiol 28–38 [12,18,20,21], 40 [14] were obtained in

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high yields. The preparation of the 4-methyl-6-sulfanyl compound 39 [22,23] was performed by reacting the (1-ethoxyethylidene)propanedinitrile 41 [22] with 2-cyanothioacetamide in boiling ethanol containing EtONa. The target dicyanopyridines 1–4, 6–13, and 20 were obtained by reaction of the 6-sulfanyl intermediates 28–40 with 2-(bromomethyl)-1H-imidazole hydrobromide (1–4, 6–13) or methyl chloroacetate (20) in the presence of sodium hydrogen carbonate at room temperature (rt).

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Elimination of the 6-substituent was realized in compound 18 which was synthesized by catalytic reduction (10% Pd/C) of 42 with hydrogen at 30 Psi in absolute EtOH.

R1 N CN CN S NH₂ R R1 OHC a R1 N NH₂ CN CN S 21-27 b R1 N CN CN S H NH₂ 28-40 c 1-4, 6-13 R = 20 R = COOCH₃ N N H d EtO CN CN 41

R1 _{compd R}1 _{compd R}1 _compd

O _{1, 28} Cl 7, 33, 21 12, 38, 26 O 2, 29 F 8, 34, 22 CH3 13, 39 O _{3, 30} CF3 9, 35, 23 OCH3 20, 40, 27 O _{4, 31} CH3 10, 36, 24 NH O 6, 32 SCH3 11, 37, 25

Scheme 1. Reagents and conditions. (a) malononitrile, thiophenol, basic Al2O3, H2O, 100 °C; (b) Na2S,

anhydrous DMF, 80 °C; 1N HCl, room temperature (rt); (c) 2-cyanothioacetamide, EtONa, absolute EtOH, reflux; d) RCH2Cl, NaHCO3, anhydrous DMF, rt.

Scheme 1. Reagents and conditions. (a) malononitrile, thiophenol, basic Al2O3, H2O, 100◦C; (b) Na2S, anhydrous DMF, 80◦C; 1N HCl, room temperature (rt); (c) 2-cyanothioacetamide, EtONa, absolute EtOH, reflux; d) RCH2Cl, NaHCO3, anhydrous DMF, rt.

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a N NH₂ CN CN Cl b N CN CN R2 NH₂ 14 R2 = 15 R2 = NHCH2CH2OH 16 R2 = 17 R2 = N CN CN NH₂ 18 N H N NHCH2 N _N OCH3 NHCH2CH2 42

Scheme 2. Reagents and conditions. (a) 1-(1H-imidazol-2-yl)methanamine for 14, ethanolamine for 15, 2-(4-methoxyphenyl)ethanamine for 16, or imidazole (for 17), triethylamine, THF/EtOH 2:1, reflux;

(b) H2, 10% Pd/C, absolute EtOH, 30 Psi.

Finally, the 6-[(2-hydroxyethyl)amino]-derivative 19 was synthesized (Scheme 3) starting from the intermediate 27 [24] by exploiting the good property of the 6-phenylthio function as the leaving group in nucleophilic substitution [15]. The reaction was performed at 100 °C in DMF using an excess of 2-aminoethanol as nucleophile. a N CN CN S NH₂ OCH₃ 27 N CN CN NH NH₂ O H OCH₃ 19

Scheme 3. Reagents and conditions. (a) 2-aminoethanol, DMF, 100 °C.

2.2. Pharmacological Assays

The amino-3,5-dicyanopyridines 1–20 were tested for their affinity at hA1, hA2A, and hA3 ARs, stably transfected in Chinese Hamster Ovary (CHO) cells, and were also studied as hA2B agonists by evaluating their stimulatory effect on cAMP production in CHO cells, stably expressing the hA2B AR. Compounds 3, 8, and 11, the most interesting in terms of combined affinity/activity at hA1 and hA2B ARs, were evaluated for their A1 pharmacological profile in the cAMP assay, where each compound was tested to assess its capability to modulate Forskolin-stimulated cAMP levels in the absence or presence of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (1 nM). All pharmacological data are reported in Tables 1–2.

2.3. Molecular Docking Studies

To simulate the binding mode of the herein reported dicyanopyridines at hA1 and hA2B ARs, molecular docking studies were performed on the cryo-EM structure of the agonist-bound hA1AR (pdb code: 6D9H; 3.6-Å resolution [28]) and on a homology model of the hA2BAR developed by using the crystal structure of agonist-bound hA2AAR as a template (pdb code: 2YDO; 3.0-Å resolution [29]). The obtained hA2BAR homology model was checked using the Protein Geometry Monitor application within MOE (Molecular Operating Environment 2019.0101) [30] by inspecting the structural quality of the protein model (backbone bond lengths, angles, and dihedrals, Ramachandran φ-ψ dihedral plots, and quality of side chain rotamer and non-bonded contact). The two AR structures were then

Scheme 2. Reagents and conditions.(a) 1-(1H-imidazol-2-yl)methanamine for 14, ethanolamine for 15, 2-(4-methoxyphenyl)ethanamine for 16, or imidazole (for 17), triethylamine, THF/EtOH 2:1, reflux; (b) H2, 10% Pd/C, absolute EtOH, 30 Psi.

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a N NH₂ CN CN Cl b N CN CN R2 NH₂ 14 R2 = 15 R2 = NHCH₂CH₂OH 16 R2 ₌ 17 R2 ₌ N CN CN NH₂ 18 N H N NHCH₂ N _N OCH3 NHCH2CH2 42

Scheme 2. Reagents and conditions. (a) 1-(1H-imidazol-2-yl)methanamine for 14, ethanolamine for 15, 2-(4-methoxyphenyl)ethanamine for 16, or imidazole (for 17), triethylamine, THF/EtOH 2:1, reflux;

(b) H2, 10% Pd/C, absolute EtOH, 30 Psi.

Finally, the 6-[(2-hydroxyethyl)amino]-derivative 19 was synthesized (Scheme 3) starting from the intermediate 27 [24] by exploiting the good property of the 6-phenylthio function as the leaving group in nucleophilic substitution [15]. The reaction was performed at 100 °C in DMF using an excess of 2-aminoethanol as nucleophile. a N CN CN S NH₂ OCH₃ 27 N CN CN NH NH₂ O H OCH₃ 19

Scheme 3. Reagents and conditions. (a) 2-aminoethanol, DMF, 100 °C.

2.2. Pharmacological Assays

The amino-3,5-dicyanopyridines 1–20 were tested for their affinity at hA1, hA2A, and hA3 ARs, stably transfected in Chinese Hamster Ovary (CHO) cells, and were also studied as hA2B agonists by evaluating their stimulatory effect on cAMP production in CHO cells, stably expressing the hA2B AR. Compounds 3, 8, and 11, the most interesting in terms of combined affinity/activity at hA1 and hA2B ARs, were evaluated for their A1 pharmacological profile in the cAMP assay, where each compound was tested to assess its capability to modulate Forskolin-stimulated cAMP levels in the absence or presence of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (1 nM). All pharmacological data are reported in Tables 1–2.

2.3. Molecular Docking Studies

To simulate the binding mode of the herein reported dicyanopyridines at hA1 and hA2B ARs, molecular docking studies were performed on the cryo-EM structure of the agonist-bound hA1AR (pdb code: 6D9H; 3.6-Å resolution [28]) and on a homology model of the hA2BAR developed by using the crystal structure of agonist-bound hA2AAR as a template (pdb code: 2YDO; 3.0-Å resolution [29]). The obtained hA2BAR homology model was checked using the Protein Geometry Monitor application within MOE (Molecular Operating Environment 2019.0101) [30] by inspecting the structural quality of the protein model (backbone bond lengths, angles, and dihedrals, Ramachandran φ-ψ dihedral plots, and quality of side chain rotamer and non-bonded contact). The two AR structures were then

Scheme 3.Reagents and conditions. (a) 2-aminoethanol, DMF, 100◦_C.

To replace the thiomethyl linker at the 6-position with an aminomethyl bridge, compounds

14–16 were synthesized (Scheme2) starting from the 6-chloro derivative 42, obtained with a reported procedure [27], and the suitable commercially available amine. The reaction was carried out in the presence of triethylamine and in a refluxing mixture of THF/EtOH. The same procedure was applied for the synthesis of the 6-(1H-imidazol-1-yl)-derivative 17, which was obtained in high yield. Elimination of the 6-substituent was realized in compound 18 which was synthesized by catalytic reduction (10% Pd/C) of 42 with hydrogen at 30 Psi in absolute EtOH.

Finally, the 6-[(2-hydroxyethyl)amino]-derivative 19 was synthesized (Scheme3) starting from the intermediate 27 [24] by exploiting the good property of the 6-phenylthio function as the leaving group in nucleophilic substitution [15]. The reaction was performed at 100◦C in DMF using an excess of 2-aminoethanol as nucleophile.

2.2. Pharmacological Assays

The amino-3,5-dicyanopyridines 1–20 were tested for their affinity at hA1, hA2A, and hA3ARs,

stably transfected in Chinese Hamster Ovary (CHO) cells, and were also studied as hA2Bagonists by

evaluating their stimulatory effect on cAMP production in CHO cells, stably expressing the hA2BAR.

Compounds 3, 8, and 11, the most interesting in terms of combined affinity/activity at hA1and hA2B

ARs, were evaluated for their A1pharmacological profile in the cAMP assay, where each compound

was tested to assess its capability to modulate Forskolin-stimulated cAMP levels in the absence or presence of the A1AR agonist 2-chloro-N6-cyclopentyladenosine (CCPA) (1 nM). All pharmacological