Substituted Adenosine Analogues Research Articles

Synthesis and antiviral evaluation of novel N-6 substituted adenosine analogues

A series of adenosine analogues were synthesized and their biological evaluation was tested against Coxsackie virus B3 (CVB3) and Herpes simplex virus type 1(HSV-1) in HEp-2 cells. The hydrophobic constant, acute toxicity, carcinogenicity and mutagenicity were calculated. Analogues with piperazine derivatives 8b showed promising activities against CVB3 with a lower IC50value and higher selectivity index, their efficacy was better than that of the commercialized medicine, Ribavirin. These described adenosine analogues exhibit potent antiviral activities against several viruses, and offer new leads for further development.

Probing biased/partial agonism at the G protein-coupled A2B adenosine receptor

G protein-coupled A2B adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A2BAR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A2BAR. In cAMP accumulation assays, both 5′-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5′-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A2BAR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A2BAR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative ‘operational model’ characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N6-substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A2BAR antagonist in MIN-6 mouse pancreatic β cells expressing low A2BAR levels, induced insulin release. This is the first relatively systematic study of structure–efficacy relationships of this emerging drug target.

Probing partial/biased agonism at the G protein‐coupled A2B adenosine receptor (662.11)

The A2B adenosine receptor (AR) is involved in many important functions and couples to several G proteins, but the endogenous effects have not been well documented partly due to the lack of selective agonists at this subtype until recently. In the present study, we probed the potential partial and/or biased agonism at the A2B AR in several types of cells, including HEK293 cells and T24 bladder cancer cells, using a series of A2B agonists of adenosine analogs substituted at 5’, N6 and C2 positions, including NECA, MRS5911 and MRS3997, and the non‐nucleoside A2B agonist, BAY60‐6583. All A2B agonists tested induced accumulation of cyclic AMP fully or partially in all cells tested, with NECA being the most efficacious. However, only 5’‐substituted analogs are full agonists in calcium mobilization and ERK1/2 phosphorylation. Over‐expression of the A2B AR in HEK293 cells increased the agonist potency and efficacy in inducing the Gs‐mediated accumulation of cyclic AMP, but not the ability to induce calcium mobilization by the C2‐substituted adenosine derivatives or non‐adenosine A2B agonists. Results from an arrestin translocation assay showed that the 5’‐substituted NECA is more efficacious than adenosine derivatives substituted at C2 and N6 positions and non‐adenosine agonists. BAY actually did not induce arrestin translocation in this assay. The data suggest that 5’‐substituted adenosine analogs are balanced agonists of different pathways, whereas the C2‐ and N6‐substituted adenosine analogs and the class of non‐nucleoside A2B agonists are Gs‐biased agonists. The data also suggest that BAY and some N6‐ and C2‐substituted adenosine analogs may be full agonist, partial agonist or antagonist under various conditions. Indeed, BAY behaves as an A2B antagonist to induce insulin release in MIN6 pancreatic β cells. Thus, differences in efficacy and effector pathways might explain some of the contradictory results reported for A2B AR activation.Grant Funding Source: Supported by NIDDK Intramural Research, NIH

Novel Bacterial NAD + -Dependent DNA Ligase Inhibitors with Broad-Spectrum Activity and Antibacterial Efficacy In Vivo

DNA ligases are indispensable enzymes playing a critical role in DNA replication, recombination, and repair in all living organisms. Bacterial NAD+-dependent DNA ligase (LigA) was evaluated for its potential as a broad-spectrum antibacterial target. A novel class of substituted adenosine analogs was discovered by target-based high-throughput screening (HTS), and these compounds were optimized to render them more effective and selective inhibitors of LigA. The adenosine analogs inhibited the LigA activities of Escherichia coli, Haemophilus influenzae, Mycoplasma pneumoniae, Streptococcus pneumoniae, and Staphylococcus aureus, with inhibitory activities in the nanomolar range. They were selective for bacterial NAD+-dependent DNA ligases, showing no inhibitory activity against ATP-dependent human DNA ligase 1 or bacteriophage T4 ligase. Enzyme kinetic measurements demonstrated that the compounds bind competitively with NAD+. X-ray crystallography demonstrated that the adenosine analogs bind in the AMP-binding pocket of the LigA adenylation domain. Antibacterial activity was observed against pathogenic Gram-positive and atypical bacteria, such as S. aureus, S. pneumoniae, Streptococcus pyogenes, and M. pneumoniae, as well as against Gram-negative pathogens, such as H. influenzae and Moraxella catarrhalis. The mode of action was verified using recombinant strains with altered LigA expression, an Okazaki fragment accumulation assay, and the isolation of resistant strains with ligA mutations. In vivo efficacy was demonstrated in a murine S. aureus thigh infection model and a murine S. pneumoniae lung infection model. Treatment with the adenosine analogs reduced the bacterial burden (expressed in CFU) in the corresponding infected organ tissue as much as 1,000-fold, thus validating LigA as a target for antibacterial therapy.

Fluorine Substituted Adenosines As Probes of Nucleobase Protonation in Functional RNAs

Ionized nucleobases are required for folding, conformational switching, or catalysis in a number of functional RNAs. A common strategy to study these sites employs nucleoside analogues with perturbed pKa, but the interpretation of these studies is often complicated by the chemical modification introduced, in particular modifications that add, remove, or translocate hydrogen bonding groups in addition to perturbing pKa values. In the present study we present a series of fluorine substituted adenosine analogues that produce large changes in N1 pKa values with minimal structural perturbation. These analogues include fluorine for hydrogen substitutions in the adenine ring of adenosine and 7-deaza-adenosine with resulting N1 pKa values spanning more than 4 pKa units. To demonstrate the utility of these analogues we have conducted a nucleotide analogue interference mapping (NAIM) study on a self-ligating construct of the Varkud Satellite (VS) ribozyme. We find that each of the analogues is readily incorporated by T7 RNA polymerase and produces fully active transcripts when substituted at the majority of sites. Strong interferences are observed for three sites known to be critical for VS ribozyme function, most notably A756. Substitutions at A756 lead to slight enhancements in activity for elevated pKa analogues and dramatic interferences in activity for reduced pKa analogues, supporting the proposed catalytic role for this base. The structural similarity of these analogues, combined with their even incorporation and selective interference, provides an improved method for identifying sites of adenosine protonation in a variety of systems.

Ribose-Modified Purine Nucleosides as Ribonucleotide Reductase Inhibitors. Synthesis, Antitumor Activity, and Molecular Modeling of N6-Substituted 3′-C-Methyladenosine Derivatives

A series of cycloalkyl, bicycloalkyl, aryl, and heteroaryl N (6)-substituted derivatives of the antitumor agent 3'- C-methyladenosine (3'-Me-Ado), an inhibitor of the alpha Rnr1 subunit of mammalian ribonucleotide reductase (RR), were synthesized. The cytotoxicity of these compounds was evaluated against a panel of human leukemia and carcinoma cell lines and compared to that of some corresponding N (6)-substituted adenosine analogues. N (6)-cycloalkyl-3'- C-methylribonucleosides 2- 7 and N (6)-phenyl analogue 8 were found to inhibit the proliferation of K562 leukemia cells. N (6)-(+/-)- endo-2-norbornyl-3'- C-methyladenosine ( 7) was found to be the most cytotoxic compound, with GI 50 values slightly higher than that of 3'-Me-Ado against K562 and carcinoma cell lines and 2.7 fold higher cytotoxicity against human promyelocytic leukemia HL-60 cells. The SAR study confirms that an unsubstituted N (6)-amino group is essential for optimal cytotoxicity of 3'-Me-Ado against both K562 and carcinoma cell lines. Computational studies, carried out on the eukaryotic alpha subunit (Rnr1) of RR from Saccharomyces cerevisiae were performed to rationalize the observed structure-activity relationships.

S-Adenosyl-L-homocysteine Hydrolase as an Attractive Target for Antimicrobial Drugs

S-Adenosyl-L-homocysteine (SAH) hydrolase catalyzes breakdown of SAH, which arises after S-adenosylmethionine-dependent methylation, into adenosine and homocysteine. The enzyme activity is required for both metabolic pathway of sulfur-containing amino acids and a variety of biological methylations. Because of the essential roles of SAH hydrolase for living cells, inhibitors of SAH hydrolase are expected to be antimicrobial drugs, especially for viruses and malaria parasite. Our research focused on the development of new antimalarials based on the SAH hydrolase inhibition. Malaria parasite employs SAH hydrolase of itself for coping with the toxicity of SAH, so that the target offers opportunities for chemotherapy if structural differences are exploited between the parasite and human enzymes. In vitro screens of nucleoside analogs resulted in moderate but selective inhibition for recombinant SAH hydrolase of malaria parasite, Plasmodium falciparum, by 2-position substituted adenosine analogs. Similar selectivity was observed in the growth inhibition assay of cultured cells. Following crystal structure analysis of the parasite SAH hydrolase discovered an additional space, which is located near the 2-position of the adenine-ring, in the substrate binding pocket. Mutagenic analysis of the amino acid residue forming the additional space confirmed that the inhibition selectivity is due to the difference of only one amino acid residue, between Cys59 in P. falciparum and Thr60 in human. For developing antimalarial drugs, it might be suitable to select target from pathways that are present in the parasite but absent from humans; nevertheless, even if the target was common in parasite and host, slight structural difference such as single amino acid variation is likely to be available for improving inhibitor selectivity.

Structure of the N-terminal Domain of GRP94: BASIS FOR LIGAND SPECIFICITY AND REGULATION

GRP94, the endoplasmic reticulum (ER) paralog of the chaperone Hsp90, plays an essential role in the structural maturation or secretion of a subset of proteins destined for transport to the cell surface, such as the Toll-like receptors 2 and 4, and IgG, respectively. GRP94 differs from cytoplasmic Hsp90 by exhibiting very weak ATP binding and hydrolysis activity. GRP94 also binds selectively to a series of substituted adenosine analogs. The high resolution crystal structures at 1.75-2.1 A of the N-terminal and adjacent charged domains of GRP94 in complex with N-ethylcarboxamidoadenosine, radicicol, and 2-chlorodideoxyadenosine reveals a structural mechanism for ligand discrimination among hsp90 family members. The structures also identify a putative subdomain that may act as a ligand-responsive switch. The residues of the charged region fold into a disordered loop whose termini are ordered and continue the twisted beta sheet that forms the structural core of the N-domain. This continuation of the beta sheet past the charged domain suggests a structural basis for the association of the N-terminal and middle domains of the full-length chaperone.

Blood–brain barrier transport of synthetic adenosine A1 receptor agonists in vitro: structure transport relationships

Transport of 11 structurally related adenosine A(1) receptor agonists was determined in an in vitro BBB model of brain-capillary-endothelial-cells and astrocytes. Inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI) was used to quantify the contribution of the es nucleoside transporter to the overall transport. The N(6)-substituted adenosine analogues N(6)-cyclobutyladenosine (CBA), N(6)-cyclopentyladenosine (CPA) and N(6)-cyclohexyladenosine (CHA) showed concentration-dependent clearance and their transport could be inhibited by NBTI. The V(max) was 1.5+/-0.2 pmol min(-1) and the Km values were 2.2+/-0.2, 1.8+/-0.3 and 15+/-4 microM for CBA, CPA and CHA, respectively. Further chemical modification such as substitution in the C8-position or modification at the ribose-moiety resulted in loss of affinity for the es nucleoside transporter. Transport by passive diffusion was slow with clearances ranging from 0.21+/-0.01 microl min(-1) for 8-(methylamino)-CPA (MCPA) to 1.8+/-0.18 microl min(-1) for 5'-deoxy-CPA (5'dCPA). Regression analysis showed no relationship between transport clearance by passive diffusion and the GTP-shift, a non-linear relationship between the transport clearance by passive diffusion and the dynamic polar surface area (Cl=0.469e(-0.071DPSA); R2=0.88) and a linear relationship between transport clearance and prediction of BBB transport on basis of the Abraham equation (logCl=1.53logBB-1.56; R2=0.83). It is concluded that the transport of synthetic A(1) adenosine derivatives across the blood-brain barrier is generally quite slow. In addition, transport by the es nucleoside transporter may contribute to the transport of certain structurally distinct analogues.

Adenosine analogues as inhibitors of Trypanosoma brucei phosphoglycerate kinase: elucidation of a novel binding mode for a 2-amino-N(6)-substituted adenosine.

As part of a project aimed at structure-based design of adenosine analogues as drugs against African trypanosomiasis, N(6)-, 2-amino-N(6)-, and N(2)-substituted adenosine analogues were synthesized and tested to establish structure-activity relationships for inhibiting Trypanosoma brucei glycosomal phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and glycerol-3-phosphate dehydrogenase (GPDH). Evaluation of X-ray structures of parasite PGK, GAPDH, and GPDH complexed with their adenosyl-bearing substrates led us to generate a series of adenosine analogues which would target all three enzymes simultaneously. There was a modest preference by PGK for N(6)-substituted analogues bearing the 2-amino group. The best compound in this series, 2-amino-N(6)- [2''(p-hydroxyphenyl)ethyl]adenosine (46b), displayed a 23-fold improvement over adenosine with an IC(50) of 130 microM. 2-[[2''-(p-Hydroxyphenyl)ethyl]amino]adenosine (46c) was a weak inhibitor of T. brucei PGK with an IC(50) of 500 microM. To explore the potential of an additive effect that having the N(6) and N(2) substitutions in one molecule might provide, the best ligands from the two series were incorporated into N(6),N(2)-disubstituted adenosine analogues to yield N(6)-(2''-phenylethyl)-2-[(2'' -phenylethyl)amino]adenosine (69) as a 30 microM inhibitor of T. brucei PGK which is 100-fold more potent than the adenosine template. In contrast, these series gave no compounds that inhibited parasitic GAPDH or GPDH more than 10-20% when tested at 1.0 mM. A 3.0 A X-ray structure of a T. brucei PGK/46b complex revealed a binding mode in which the nucleoside analogue was flipped and the ribosyl moiety adopted a syn conformation as compared with the previously determined binding mode of ADP. Molecular docking experiments using QXP and SAS program suites reproduced this "flipped and rotated" binding mode.

3′-Amidated 3′-Deoxyxylofuranose Analogues of N 6-−Cyclopentyladenosines: a New Class of Non-Xanthine Antagonists at the Adenosine A1 Receptor.

Full adenosine A1 receptor agonists like CPA and other N 6-−substituted adenosine analogs have previously been shown to become partial agonists upon deletion of the 3′-hydroxyl moiety. The present study further explored the C-3′ site for modification. The modest affinity at A1 and A2a receptors found in the 3′-amido-3′-deoxyxyIofuranosyladenine series prompted us to synthesize the corresponding N 6-−cyclopentyl derivatives, which proved to exhibit potent antagonistic behaviour at the A1 receptors. This represents a new perspective in the purinergic field.

5'-substituted adenosine analogs as new high-affinity partial agonists for the adenosine A1 receptor.

5'-(Alkylthio)-, 5'-(methylseleno)-, and 5'-(alkylamino)-substituted analogues of N6-cyclopen-tyladenosine (CPA) were synthesized in 30-50% overall yields. The affinities of these compounds for the adenosine A1 and A2A receptors were determined in rat brain membranes. The 5'-substituted CPA analogues proved selective for the adenosine A1 receptors, displaying affinities in the nanomolar range. The compounds were also evaluated for their ability to stimulate [35S]GTP gamma S binding, also in rat brain membranes. The Ki values in receptor binding studies corresponded well to the EC50 values thus obtained. Intrinsic activities of the compounds were tested in vitro by determining the GTP shift in receptor binding studies as well as the maximal binding of [35S]GTP gamma S. It appeared that the 5'-thio and 5'-seleno derivatives in particular behaved as partial agonists.

Synthesis of complex ethynyladenosines using organic triflic enolates in palladium-catalyzed reactions: Potential agonists for the adenosine A2 receptor

Many high affinity adenosine A2 receptor agonists contain substituents at position 2 of the purine base. In the search for new methodology to develop C-2 substituted adenosine analogues, we have applied organic triflates in palladium-catalyzed cross-couplings that resulted in new 2-cycloalkylated ethynyladenosines. The organic triflates are derived from commercially available ketones and, when used in the cross-couplings, result generally in clean reactions with good yields. These are the first examples of the utilization of organic triflates in palladium-catalyzed cross-coupling reactions in the synthesis of modified nucleosides.

Identification of an adenosine receptor domain specifically involved in binding of 5'-substituted adenosine agonists.

The bovine A1 adenosine receptor (A1AR) and rat A3 adenosine receptor (A3AR) display distinct agonist and antagonist binding properties. To identify regions involved in ligand recognition, A1AR/A3AR chimeric receptors were created, expressed in COS-7 cells, and analyzed by radioligand binding. A chimeric receptor in which the third intracellular loop of the A1AR was replaced with that of the A3AR bound agonists and the antagonist, [3H]xanthine amine congener, with affinities identical to wild-type A1AR. A chimeric receptor with the fifth transmembrane domain (TM5) and third intracellular loop of the A1AR replaced with that of the A3AR displayed antagonist affinity similar to wild-type A1AR. However, relative to the A1AR, this chimeric demonstrated much greater affinity for 5'-substituted adenosine analogs, whereas affinity for N6-substituted compounds was unaffected. Substitution of a 6-amino acid cassette of the exofacial half of TM5 of the A3AR into the A1AR produced enhanced binding of exclusively a 5'-substituted analog, indicating involvement of this specific region in ligand recognition. These findings suggest that the 5'- and N6-substituents of adenosine agonists bind to distinct regions of ARs and that TM5 of the A3AR interacts more favorably with 5'-substituted compounds than does that of the A1AR.

2'-Substituted adenosine analogues as trypanosomal glyceraldehyde phosphate dehydrogenase (GAPDH) inhibitors

2'-Substituted adenosine analogues as trypanosomal glyceraldehyde phosphate dehydrogenase (GAPDH) inhibitors

Agonist activity of 2- and 5′-substituted adenosine analogs and their N6-cycloalkyl derivatives at A 1 − and A 2 −adenosine receptors coupled to adenylate cyclase

The activity of N 6-cycloalkyl derivatives of adenosine, 2-chloroadenosine, 5′-chloroadenosine and N-ethylcarboximidoadenosine (NECA) and of 2-fluoroadenosine and 5-methylthioadenosines were compared at the A 1-adenosine receptor inhibitory to adenylate cyclase in rat fat cell membranes and at the A 2A-adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. The N 6-cycloalkyl derivatives in all cases were more potent (4- to 23-fold) than the parent compound at the A 1 receptor, and were less potent (1.6- to 11-fold) than the parent compound at the A 2A receptor. N 6-Cyclopentyl-5′-chloroadenosine was the most selective agonist (900-fold) for the A 1 receptor, while 2-fluoroadenosine was the only agonist with some selectivity (4.8-fold) for the A 2A receptor. 5′-Methylthioadenosine was a weak agonist at both adenosine receptors. A 2-fluoro derivative of 5′-methylthioadenosine was somewhat more potent. Affinities of these analogs for inhibition of binding of radioligands to rat brain A 1 and A 2A receptors are presented.

Characteristics of adenosine binding sites in atrial sarcolemmal membranes

The studies reported here involve an exploration of the sites on atrial myocyte membranes with which adenosine interacts to produce its potent physiological effects in atrial muscle. Specific, high affinity binding of the stable adenosine analogs 2-chloro[ 3H]adenosine (2-ClAdo) and [ 3H]adenosine 5′-N- ethylcarboxamide (NECA) to atrial sarcolemmal membranes was measured in kinetic and equilibrium studies at 4°C and 35°C. Analysis of the [ 3H]2-ClAdo binding isotherm indicated the presence of two classes of binding site with equilibrium K assoc values estimated to be 5.7 · 10 7 M −1 and 2.7 · 10 6 M −1. Displacement of bound [ 3H]2-ClAdo by adenosine 5′-N- cyclopropylcarboxamide (NCPCA) and by several N 6- substituted adenosine analogs confirmed the presence of two classes of binding site. Analysis of the [ 3H]NECA binding also revealed the presence of two types of binding site for this ligand. The methylxanthines isobutylmethylxanthine and theophylline displaced bound [ 3H]2-ClAdo whereas adenosine uptake inhibitors and several other purines showed little activity. These atrial membrane binding sites exhibit many of the characteristics of the physiological adenosine receptors studied in intact atria. Furthermore, the [ 3H]2-ClAdo binding sites were sensitive to treatment with proteolytic enzymes, suggesting that these sites exist on sarcolemmal membrane proteins.

Subclasses of adenosine receptors in brain membranes from adult tissue and from primary cultures of chick embryo.

Abstract: Membranes from adult chicken brain have high‐affinity binding sites for N6‐cyclohexyl[3H]adenosine (CHA) (KD= 4 nM, Bmax= 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A1 type, since substituted adenosine analogs, e.g. N6‐(l‐2‐phenylisopropyl)adeno sine (IC50= 60 nM), were very potent displacers. Binding sites for 1,3‐diethyl‐ 8‐[3H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (KD= 50 nM, Bmax= 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8‐phenyltheophylline (IC50= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A1 receptors and the other half are of the A2 type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [3H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (Bmax= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (KD= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A1 receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8‐phenyltheophylline, CHA, theophylline, caffeine, and 3‐isobutyl‐I‐methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A2 type.

Additions and Corrections - Some Short-Chain N6-Substituted Adenosine Analogues with Antitumor Properties.

Additions and Corrections - Some Short-Chain N⁶-Substituted Adenosine Analogues with Antitumor Properties.

Cytotoxicity of N6-substituted adenosine analogs to cultured trophoblastic tumor cells

Cultured trophoblastic tumor cells were found to have an elevated adenosine kinase activity relative to levels in normal trophoblasts and those derived from a hydatidiform mole. The effect of N 6 substituted adenosine analogs on the various cell lines was investigated to determine whether the differential enzyme levels might, in turn. result in differential sensitivity of the cell lines to the compounds. When cells were exposed to appropriate concentrations of N 6-methyladenosine or N 6, N 6-dimethyladenosine, it was found that the nucleosides were only cytostatic to non-malignant trophoblastic cells derived from placenta or hydatidiform mole, and cell lines of non-trophoblastic origin. However, the same concentrations of the nucleosides were cytotoxic to the malignant trophoblastic cell lines. The primary effect of exposure of cells to N 6, N 6-dimethyladenosine appeared to be inhibition of DNA synthesis. A third adenosine analog, N 6-( Δ 2-isopentenyl)aden was toxic to almost all cell lines regardless of origin.