Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A(3)AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of beta-D-apio-D-furano- and alpha-D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N-6 of beta-D-apio-D-furanoadenosine afforded an A(3)AR antagonist (10c, K = 0.98 mu M), while a similar modification of an alpha-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, K-i = 3.07 mu M). The structural basis for this difference was examined by docking to an A(3)AR model; the antagonist lacked a crucial interaction with Thr94

Gao, Zhan-Guo

Jacobson, Kenneth A

Moss, Steven M

Paoletta, Silvia

Toti, Kiran

Van Calenbergh, Serge

English

PubMed

Ghent University Academic Bibliography

  biblio.ugent.be  The UGent Institutional Repository is the electronic archiving and dissemination platform for all UGent research publications. Ghent University has implemented a mandate stipulating that all academic publications of UGent researchers should be deposited and archived in this repository. Except for items where current copyright restrictions apply, these papers are available in Open Access.  This item is the archived peer‐reviewed author‐version of: Title:  Synthesis and Evaluation of N6‐Substituted Apioadenosines as Potential Adenosine A3 Receptor Modulators Author(s):  Kiran S. Toti, Steven M. Moss, Silvia Paoletta, Zhan‐Guo Gao, Kenneth A. Jacobson, and Serge Van Calenbergh  Source: Bioorg. Med. Chem. 2014, 22, 4257-68. 1  Synthesis and Evaluation of N6-Substituted Apioadenosines as Potential Adenosine A3 Receptor Modulators Kiran S. Toti,a Steven M. Moss,b Silvia Paoletta,b Zhan-Guo Gao,b Kenneth A. Jacobson,b and Serge Van Calenbergh a,*  a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University Harelbekestraat 72, B-9000 Gent, Belgium. b Molecular Recognition Section, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.   Keywords: G protein-coupled receptor, apionucleosides, Adenosine A3 receptor  * correspondence to Prof. Serge Van Calenbergh, Ghent University, Faculty of Pharmaceutical Sciences, Laboratory for Medicinal Chemistry, Harelbekestraat 72, B-9000 Gent, Belgium, Tel +32 9 264 81 24, Fax +32 9 264 81 46, email: serge.vancalenbergh@ugent.be.  Abbreviations: AR, adenosine receptor; BAIB, bis-acetoxyiodobenzene; cAMP, adenosine 3,5-cyclic phosphate; CDI, carbonyldiimidazole; Cl-IB-MECA = 2-chloro-N6-(3-iodobenzyl)-5-N-methylcarboxamidoadenosine; CHO, Chinese hamster ovary; DMF, N,N-dimethylformamide; DMEM, Dulbecco’s modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; I-AB-MECA, N6-(4-amino-3-iodobenzyl)adenosine-5-N-methyl-uronamide; NECA, 5′-N-ethylcarboxamidoadenosine; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRMS, high resolution mass spectroscopy; NMR, nuclear magnetic resonance; R-PIA, N6-R-phenylisopropyladenosine; TBDMS, tert-butyldimethylsilyl; TCA, trichloroacetic acid; TEMPO, (2,2,6,6-tetramethylpyperidin-1yl)-oxyl; THF, tetrahydrofuran; TLC, thin layer chromatography; TM, transmembrane helical domain.     2  ABSTRACT Adenosine receptors (ARs) trigger signal transduction pathways inside the cell when activated by extracellular adenosine. Selective modulation of the A3AR subtype may be beneficial in controlling diseases such as colorectal cancer and rheumatoid arthritis. Here, we report the synthesis and evaluation of β-D-apio-D-furano- and -D-apio-L-furanoadenosines and derivatives thereof. Introduction of a 2-methoxy-5-chlorobenzyl group at N6 of β-D-apio-D-furanoadenosine afforded an A3AR antagonist (10c, Ki = 0.98 µM), while a similar modification of an α-D-apio-L-furanoadenosine gave rise to a partial agonist (11c, Ki = 3.07 µM). The structural basis for this difference was examined by docking to an A3AR model; the antagonist lacked a crucial interaction with Thr94.  NONNNNH2OH OHHOAdenosine (1)NNNNHNOHOOH OHOMeCl10c: A3AR antagonistA3 ARaffinityNNNNHNOHOOHOMeCl11c: A3AR partial agonistHONONNNNH2R R1HO-D-apio-D-furanoadenosines4: R = R1 = OH5: R = H; R1 = OH6: R = R1 = HNORNNNNH2R1-D-apio-L-furanoadenosines7: R = R1 = OH8: R = H; R1 = OH9: R = R1 = HHO    3  INTRODUCTION Transmembrane receptors coupled to G-proteins (heterotrimeric guanine nucleotide binding proteins) constitute a large receptor family, referred to as G protein-coupled receptors (GPCRs) or seven-transmembrane domain (7TM) receptors. Triggered by messenger molecules or signals outside the cell, GPCRs activate different signal transduction pathways inside the cell and, ultimately, cellular responses. The extracellular messengers range from photons to biogenic amines and other neurotransmitters to proteins. GPCRs are ubiquitous and involved in processes varying from directed chemotaxis[1] of small organisms (e.g. searching food for survival) to triggering apoptosis (programmed cell death) in large animals. They are the targets of almost 50% of marketed active pharmaceutical ingredients.[2]  GPCRs activated by extracellular adenosine are classified as adenosine receptors (ARs), which are divided in four different subtypes, i.e. A1, A2A, A2B and A3 ARs.[3,4] The amino acid sequence similarity between the human (h) A3AR and hA1, hA2A, hA2B AR is 54 %, 48 % and 44 %, respectively.[5] The ARs use different signaling pathways; the A1 AR and A3 AR are preferentially coupled to Gi-proteins, and upon activation lead to adenylate cyclase inhibition, while the A2A AR and A2B AR are preferentially coupled to Gs-proteins and lead to adenylate cyclase activation. In some cells (e.g., mast cells), the A2BAR is dually coupled to Gs and Gq and consequently also mobilizes calcium and activates phospholipase C and MAPK. [3,6]  Besides adenosine (1) itself, which is used clinically for the treatment of supraventricular tachycardia and in myocardial perfusion imaging,[7] only one AR-specific agent, the A2AAR agonist regedenoson, has so far been approved by the FDA. However, a relatively large group of AR ligands is currently under clinical evaluation.[8]  4  With the exception of compound 2, which was reported in the mid-1980s as being inactive at A1 and A2ARs,[9] and the recently reported carbocyclic analogue 3,[10] a weak A3AR agonist, 4-hydroxymethyl transposed nucleosides have not been investigated as AR ligands.  This led us to employ a new and convenient method for the synthesis of apionucleosides from 1,2-O-isopropylidene-α-L-threose,[11] for the construction of suitably modified 9-(3-C-hydroxymethyl-β-D-erythrofuranosyl)adenines (aka β-D-apio-D-furanoadenosines) (4-6) and 9-(3-C-hydroxymethyl--L-threofuranosyl)adenines (aka -D-apio-L-furanoadenosines) (7-9) as potential A3AR modulators (Figure 1).  On the one hand, we envisaged to substitute the N6 position of β-D-apio-D-furanoadenosine 4 with substituted benzyl groups known to enhance A3AR affinity;[12] on the other hand we planned to substitute the well-known ethylcarboxamide moiety in place of the 4-CH2OH group.[13] We decided to introduce a benzyl moiety at the N6 position and an N-alkylcarboxamide moiety at the 5 position because this combination was shown to be conducive to selectivity at the A3AR.  For example, N6-(3-iodobenzyl)-5-N-methylcarboxamidoadenosine and its 2-chloro analogue (Cl-IB-MECA) display Ki values at A3AR of ~1 nM and ≥50-fold selectivity for this subtype. [14] Subsequently, an N6-(3-chloro-5-methoxybenzyl) substitution was shown to be beneficial for A3AR selectivity,[12] and we incorporated this moiety in the current target compounds.  However, synthetic problems in introducing an ethylcarboxamide moiety motivated us to introduce a N-methyl or N-ethylcarbamoyloxymethyl group instead. Analogue modifications were carried out on α-D-apio-L-furanoadenosine 7. 5   Figure 1. Apio-type nucleosides previously tested for modulation of ARs (1-3) and target analogues of the present study (4-15). 6   RESULTS AND DISCUSSION Chemistry The β-D-apio-D-furanoadenosines 4-6 and the α-D-apio-L-furanoadenosines 7-9 were prepared by microwave assisted synthesis as described in ref. [11].  Scheme 1. Synthesis of N6 substituted apioadenosines. Reagents and conditions: (a) (i) appropriate benzyl bromide, DMF, 50 °C, 48 h; (ii) 25% NH4OH, 50 °C, 48 h or 90 °C, 3 h, 20-56% over two steps; (b) (i) appropriate benzyl bromide, DMF, rt, 48 h; (ii) 25% NH4OH, 50 °C, 24 h, 8-21% over two steps.   7  The L-threo analogue 7 was used as a model substrate for N6-derivatisation reactions. Treatment of 7 with the appropriate benzyl bromide first afforded the N1-benzyl derivative, which was isolated in the case of the 3-chlorobenzyl derivative 16 (Scheme 1). Upon prolonged heating with ammonia solution, the N1-benzyl intermediates were converted to the desired N6-benzylated compounds 11a-11c via Dimroth rearrangement.[14] Although in many cases this method suffered from low yields, it allowed gaining fast access to the target molecules. Using similar conditions β-D-apio-D-furanoadenosine 4 was benzylated at N6 affording the desired derivatives in low yield after purification by RP-HPLC or preparative TLC. In the case of 10c, using excess of the benzyl bromide led to degradation of the starting material and afforded bis-(2-methoxy-5-chlorobenzyl)adenine as observed by HRMS. By limiting the amount of this benzyl bromide to one equivalent, 10c could be obtained in 21% yield.  8   Scheme 2. Synthesis of sugar modified α-D-apio-L-furanoadenosines. Reagents and conditions: (a) TBDMSCl, imidazole, DMF, 100 °C, 3 days, 80%; (b) 7N NH3 in MeOH, rt, 18 h, 73%; (c) HF.pyridine, pyridine, THF, rt or TCA-H2O, THF, rt; (d) TBDMSCl, imidazole, DMF, rt, 18 h, 75-83%; (e) 22, TCA-H2O, THF, 0°C, 1 h, rt, 1 h, 87%; 23, HF.pyridine, pyridine, THF, 0°C, 1 h, rt, 1 h, 82%; (f) (i) CDI, THF, rt, 3h; (ii) EtNH2, rt, 16 h, 55-95%; (g) (i) RuCl3, NaIO4, AcCN-CCl4-H2O, rt, 7 h; (ii) CDI, THF, rt, 3 h; (iii) EtNH2, rt, 18 h, 9% over three steps; (h) NH4F, MeOH, 50 °C, 48 h, 70-94%.  

Synthesis and evaluation of N⁶-substituted apioadenosines as potential adenosine A₃ receptor modulators

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Synthesis and evaluation of N⁶-substituted apioadenosines as potential adenosine A₃ receptor modulators

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