Novel Irreversible Agonists Acting at the A1 Adenosine Receptor

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Novel Irreversible Agonists Acting at the A Adenosine Receptor Manuela Jörg, Alisa Glukhova, Alaa Abdul-Ridha, Elizabeth A. Vecchio, Anh T.N. Nguyen, Patrick M. Sexton, Paul J. White, Lauren T. May, Arthur Christopoulos, and Peter J. Scammells J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01561 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Journal of Medicinal Chemistry

Novel Irreversible Agonists Acting at the A1 Adenosine Receptor

Manuela Jörg†,§, Alisa Glukhova,‡,§, Alaa Abdul-Ridha,‡ Elizabeth A. Vecchio,‡ Anh T.N. Nguyen,‡ Patrick M. Sexton,‡ Paul J. White,‡ Lauren T. May,‡ Arthur Christopoulos,‡,* Peter J. Scammells†,*

†

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences; Monash University, Parkville, ‡

VIC 3052, Australia; Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences Biology & Department of Pharmacology, Monash University, Parkville, VIC 3052, Australia §

These authors contributed equally to this manuscript

ABSTRACT: The A1 adenosine receptor (A1AR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the A1AR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesised and four compounds, 8b, 15a, 15b and 15d, were shown to posses similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the A1AR. Of these compounds, 15b and 15d were more directly validated as irreversible agonists of the A1AR using membrane-based [3H]DPCPX and [35S]GTPγS binding experiments.

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INTRODUCTION Adenosine receptors (ARs) are a family of G protein-coupled receptors (GPCRs) that are distributed broadly throughout the body and are linked to a number of physiological functions.1,

2

The

initiation of these functions occurs upon the activation of four different AR subtypes; namely the A1, A2A, A2B and A3ARs.3, 4 The A1AR is found in spinal cord, central nervous system, heart, and adipose tissue, and is associated with a wide range of effects including the ability to slow down heart rate, inhibit neurotransmitter release and inhibit lipolysis.1, 5-8 The A1AR has been recognized as a promising drug target for a variety of disorders, such as neuropathic pain9,

10

and cardiac

ischemia-reperfusion injury.11, 12 Despite recent breakthroughs in GPCR structural biology,13-17 and numerous crystal structures solved for the A2AAR, there is surprisingly limited information available about the structural and functional features of the A1AR.

Irreversible probes/ligands are valuable tools for studying GPCRs both in vitro and in vivo.18-22 Of note, recent work has demonstrated advantages that covalent ligands could hold for GPCR structural biology studies, which is based on the irreversible probe’s ability to form a stable and long living ligand-receptor complex with the targeted GPCR.23-27

Irreversible agonists and

antagonists targeting the A1AR subtype have been reported in the past, and the chemical structures of the best-studied ligands are illustrated in Figure 1. The xanthine-based irreversible antagonists 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl(2-aminoethylaminocarbonyl(4-methyloxy (phenyl)))xanthine (DITC-XAC) and 8-cyclopentyl-3-(3-((4-fluorosulfonylbenzoyl)oxy)propyl)-1propylxanthine (FSCPX), as well as variants thereof, have been part of extensive investigations.28-30 Interrogation of linker attachment positions, irreversible groups, as well as a range of linker lengths and types, suggested that the 3- and 8-postions of the xanthine scaffold are suitable for the attachment of the irreversible group to maintain high affinity and selectivity for the A1AR subtype.


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selective A1AR irreversible antagonists O O O N O

NH HN

H N O

N

H N

N

N

N

O

S NH

O NCS

N

O O FO2S

m- or p-DITC-XAC

FSCPX

selective A1AR irreversible agonists H N O

HN N N HO

OH

O N H

N

H N

S

HN

HN

N

N

NCS N

N

HO

O

N

N3

O

OH OH

OH OH m- or p-DITC-ADAC

R-AHPIA

Figure 1. Chemical structures of irreversible xanthine-based A1AR antagonists DITC-XAC and FSCPX as well as the adenosine-based agonists DITC-ADAC and R-AHPIA that haven been previously described in the literature (blue: pharmacophore; red: reactive group; orange: linker).

Although not as well-studied, some irreversible probes capable of activating the A1AR have also been

developed;

namely,

N6[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl]

amino]ethyl]amino]carbonyl]methyl]aniline]carbonyl]methyl]phenyl]adenosine (p- and m-DITCADAC)31 and R-2-azido-N6-p-hydroxyphenylisopropyladenoisine (R-AHPIA).32, 33 Both probes are based on an adenosine scaffold; however, they are structurally and functionally different from each other. R-AHPIA forms its irreversible bond with the receptor upon photoactivation by UV light, due to the incorporation of a photoreactive azide group in the 2-postion of the adenosine scaffold. In contrast, p- and m-DITC-ADAC form a direct covalent bond with the receptor via a chemoreactive isothiocyanate group attached to a lengthy 2-(4-aminophenyl)acetic acid-based linker that is incorporated into the N6-position of the adenosine scaffold. Both p- and m-DITCACS Paragon Plus Environment


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ADAC exhibited high (sub-nanomolar) affinity at the A1AR, as well as, 200- to 600-fold selectivity over the A2ARs.31 In addition, both ligands were capable of activating the A1AR to yield persistent cAMP responses in functional assays,34 but limited progress has been made since these early studies, and it remains unknown as to whether such probes can also be used as useful tools to study A1AR structural biology. R-AHPIA possesses an aryl azide reactive group, which needs to be activated by photolysis to generate a covalent bond with the receptor. Even though p- and m-DITC-ADAC are known irreversible probes based on a chemoreactive isothiocyanate group, these probes have a relatively long and flexible linker. In view of the current interest in development of probes to facilitate GPCR structural biology studies, we have chosen to investigate the effects of different linkers in length and flexibility on the pharmacology of novel A1AR irreversible agonists, as well as their effects on receptor thermostability.

Specifically, we present the synthesis, characterization and

pharmacological evaluation of 15 novel adenosine analogs containing a reactive functional group capable of forming a covalent bond with the receptor. A range of chemoreactive groups with different types of action were incorporated; namely bicyclo[2.2.2]octa-2,5-dienes (Michael acceptors), isothiocyanates and fluorosulfonylphenyl groups. The reactive groups were introduced via the N6-position of the adenosine scaffold, since structure-activity relationship data35-38 as well as the irreversible ligands p/m-DITC-ADAC31 have shown that changes to this position are well tolerated and have no detrimental effect on the affinity and selectivity at the A1AR. Since the formation of a covalent bond with chemoreactive irreversible probe is only possible by positioning the chemoreactive functionality in close proximity to a nucleophilic amino acid residue, three series of irreversible probes were synthesised incorporating linear linkers with 2-, 6- and 10-carbon in length. In addition, given that many high affinity A1AR ligands have either an aromatic or another cyclic moiety at the N6-position of the adenosine scaffold, an aromatic linker was also examined.35, 37, 38


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RESULTS AND DISCUSSION Chemistry. The synthesis of the putative irreversible A1AR agonists was based on a two-step procedure; the attachment of a linker unit to the adenosine scaffold, followed by the introduction of a chemoreactive group to the linker unit (Scheme 1). The linear linkers were introduced via a substitution reaction of the respective diaminoalkane 2-4 with 6-chloropurine riboside (1) in ethanol and triethylamine. The resulting intermediates 5-7 were obtained in good yields (68-90%) after 3 hours at reflux. In the case of the 4-aminobenzylamine linker (11), the more nucleophilic aliphatic amino group was first protected with a Boc group under standard conditions. Next, the Bocprotected linker 12 was attached to the adenosine scaffold in 88% yield using the previously described conditions for the substitution reaction with 6-chloropurine riboside (13). The lower nucleophilicity of this aryl amine contributed to the increased reaction time of 26 h compared to 3 h for the alkyl linkers.

The Boc protecting group was removed with trifluoroacetic acid in

dichloromethane to furnish 14, noting that it was important to avoid hydrochloric acid since it initiated cleavage of the sugar moiety from the purine unit. The chemoreactive bicyclo[2.2.2]octa-2,5-diene and 4-fluorosulfonylphenyl moieties were introduced using standard peptide bond coupling conditions (BOP and triethylamine).

The

bicyclo[2.2.2]octa-2,5-diene (8a-10a, 15a) analogs were obtained in reasonable yields ranging between 11-42%, whereas the reactions with 4-(fluorosulfonyl)benzoic acid resulted in the formation of several by-products which made the purification extremely challenging. Accordingly, compounds 9d-10d and 15d were only isolated in very low yield (1-10%).

The 4-

fluorosulfonylphenyl product with the diaminoethyl linker could not be isolated with high purity (>95%) due to interference from other side products. It is worth mentioning that the introduction of chemoreactive groups with acid halides instead of carboxylic acids failed due to side reactions on the adenosine scaffold. Nevertheless, by simply changing the base from triethylamine to 2,6lutidine we were able significantly improve the yield of 15d from 1% to 68%, as no side products were formed under this conditions and purification by column chromatography was not necessary. ACS Paragon Plus Environment


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To afford the 1-(4-isothiocyanatophenyl)thiourea (8b-10b, 15b) and isothiocyanate (8c-10c, 15c) analogs, either 1,4-phenylene diisothiocyanate or N,N'-thiocarbonyldiimidazole was reacted with the amine linker of intermediates 5-7 and 14 in dichloromethane at room temperature. In case of the isothiocyanate analogs 8c-10c and 15c it was important to use only one equivalent of the reagent

to

avoid

the

formation

of

further

side

products,

while

for

the

1-(4-

isothiocyanatophenyl)thiourea analogs (8b-10b, 15b), an excess of reagent (3 equivalents) was used without the earlier described implications. The yields of the synthesized isothiocyanates were generally very modest, especially the two analogs with the 4-aminobenzylamine linker (15b and 15c) which were obtained in extremely poor yields, possibly due to the low solubility of intermediate 14 in dichloromethane. The synthesis of 15b was optimized by using DMF as the solvent to improve the conversion of the starting material to product as well as 1.1 equivalent of reagent and a reaction concentration of 10 mg of starting material/mL of solvent to minimize the formation of side products, including the dimer product. Preparative RP-HPLC was used for purification avoiding silica gel column chromatography (normal phase), which contributed to the degradation of 15b. The optimized protocol allowed the isolation of 15b in 17% yield (previously 1%).


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Scheme 1. Synthesis of the adenosine A1 receptor irreversible agonistsa

a

Reagents and conditions: (a) respective diaminoalkane, Et3N, EtOH, reflux, 3 h, 68-90%; (ba)

bicyclo[2.2.2]octa-2,5-diene-2-carboxylic acid, BOP reagent, Et3N, DMF, rt, 20 h, 11-42%; (bb) 1,4-phenylene diisothiocyanate, CH2Cl2, rt, 5-22 h, 18-35%; (bc) N,N'-thiocarbonyldiimidazole, CH2Cl2, rt, 43 h, 3-35%; (bd) 4-(fluorosulfonyl)benzoic acid, BOP reagent, Et3N, DMF, rt, 20 h, 810%; (c) Boc anhydride, THF, rt, 5 h, 94%; (d) 12, Et3N, EtOH, reflux, 26 h, 88%; (e) TFA, CH2Cl2, rt, 2 h, followed by 1 M NaOH, 70%; (fb) 1,4-phenylene diisothiocyanate, DMF, rt, 2 h, 17%; (fd) 4-(fluorosulfonyl)benzoic acid, BOP reagent, 2,6-lutidine, DMF, rt, 1 h, 68% .

Pharmacology. To assess the biological activity of the newly synthesised compounds, assays of ERK1/2 phosphorylation (pERK1/2), a downstream signalling pathway linked to receptor activation, were performed in FlpInCHO cells stably expressing the human A1AR.39 No effects on pERK1/2 were noted for any of the compounds in parental FlpInCHO cells not transfected with the A1AR (data not shown). Concentration-response curves were constructed at the peak-response time, identified from time-course experiments as 5 min of agonist exposure.


Analysis of


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concentration-response curves allowed for the estimation of agonist potency and maximal pERK1/2 stimulated in the presence of each agonist (Table 1 and Figure 2). The maximal agonist response was not significantly different from that of NECA for any of the compounds tested (p>0.05, oneway ANOVA, Dunnett’s post hoc test), indicating that these ligands acted as full agonists of the A1AR in the pERK1/2 assay. Despite most compounds being essentially equally effective, greater variations were noted in their potencies. Only four compounds, 8b, 15a, 15b and 15d, exhibited low-nanomolar potencies that were not significantly different to NECA (Table 1 and Figure 2), while the potencies of all other compounds were significantly lower (p

Novel Irreversible Agonists Acting at the A1 Adenosine Receptor

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