Mario Grifantini

2-Chloro-N6-cyclopentyladenosine: a highly selective agonist at A1 adenosine receptors

Summary2-Chloro-N6-cyclopentyladenosine (CCPA) was synthesized as a potential high affinity ligand for A1 adenosine receptors. Binding of [3H]PIA to A1 receptors of rat brain membranes was inhibited by CCPA with a Ki-value of 0.4 nM, compared to a Ki-value of 0.8 nM for the parent compound N6-cyclopentyladenosine (CPA). Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited with a Ki-value of 3900 nM, demonstrating an almost 10,000-fold A1-selectivity of CCPA.CCPA inhibited the activity of rat fat cell membrane adenylate cyclase, a model for the A1 receptor, with an IC50-value of 33 nM, and it stimulated the adenylate cyclase activity of human platelet membranes with an EC50-value of 3500 nM. The more than 100-fold A1-selectivity compares favourably with a 38-fold selectivity of CPA. Thus, CCPA is an agonist at A1 adenosine receptors with a 4-fold higher selectivity and 2-fold higher affinity than CPA, and a considerably higher selectivity than the standard A1 receptor agonist R-N6-phenylisopropyladenosine (R-PIA). CCPA represents the agonist with the highest selectivity for A1 receptors reported so far.

The antinociceptive effect of 2-chloro-2′-C-methyl-N6-cyclopentyladenosine (2′-Me-CCPA), a highly selective adenosine A1 receptor agonist, in the rat

Abstract This study was undertaken in order to investigate the effect of 2‐chloro‐2′‐C‐methyl‐N6‐cyclopentyladenosine (2′‐Me‐CCPA), a potent and highly selective adenosine A1 receptor agonist, on nociceptive responses and on the ongoing or tail flick‐related changes of rostral ventromedial medulla (RVM) ON‐ and OFF‐cell activities. Systemic administrations of 2′‐Me‐CCPA (2.5–5 mg/kg, i.p.) reduced the nociceptive response in the plantar and formalin tests, in a way prevented by DPCPX (3 mg/kg, i.p.), a selective A1 receptor antagonist. Similarly, intra‐periaqueductal grey (PAG) 2′‐Me‐CCPA (0.5–1–2 nmol/rat) reduced pain behaviour in the plantar and formalin tests, in a way inhibited by DPCPX (0.5 nmol/rat). Moreover, when administered systemically (2.5–5 mg/kg, i.p.) or intra‐PAG (0.5–1 nmol/rat) 2′‐Me‐CCPA increased the tail flick latencies, delayed the tail flick‐related onset of the ON‐cell burst and decreased the duration of the OFF‐cell pause in a dose dependent manner. Furthermore, it decreased RVM ON‐cell and increased OFF‐cell ongoing activities. The in vivo electrophysiological effects were all prevented by DPCPX (0.5 nmol/rat). This study confirms the role of adenosine A1 receptors in modulating pain and suggests a critical involvement of these receptors within PAG–RVM descending pathway for the processing of pain.

5'-Chloro-5'-deoxy-(±)-ENBA, a Potent and Selective Adenosine A1 Receptor Agonist, Alleviates Neuropathic Pain in Mice Through Functional Glial and Microglial Changes without Affecting Motor or Cardiovascular Functions

This study was undertaken in order to investigate the effect of chronic treatment with 5′-chloro-5′-deoxy-(±)-ENBA, a potent and highly selective agonist of human adenosine A1 receptor, on thermal hyperalgesia and mechanical allodynia in a mouse model of neuropathic pain, the Spared Nerve Injury (SNI) of the sciatic nerve. Chronic systemic administration of 5′-chloro-5′-deoxy-(±)-ENBA (0.5 mg/kg, i.p.) reduced both mechanical allodynia and thermal hyperalgesia 3 and 7 days post-SNI, in a way prevented by DPCPX (3 mg/kg, i.p.), a selective A1 adenosine receptor antagonist, without exerting any significant change on the motor coordination or arterial blood pressure. In addition, a single intraperitoneal injection of 5′-chloro-5′-deoxy-(±)-ENBA (0.5 mg/kg, i.p.) 7 days post-SNI also reduced both symptoms for at least two hours. SNI was associated with spinal changes in microglial activation ipsilaterally to the nerve injury. Activated, hypertrophic microglia were significantly reduced by 5′-chloro-5′-deoxy-(±)-ENBA chronic treatment. Our results demonstrated an involvement of adenosine A1 receptor in the amplified nociceptive thresholds and in spinal glial and microglial changes occurred in neuropathic pain, without affecting motor coordination or blood pressure. Our data suggest a possible use of adenosine A1 receptor agonist in neuropathic pain symptoms.

Adenosine and 2-Chloroadenosine Deaza-Analogues as Adenosine Receptor Agonists1

Abstract Several deaza-analogues of adenosine and 2-chloro-adenosine have been examined for their adenosine receptor affinity. It was found that the relative contribution of the nitrogen atoms of the purine moiety to binding at A1 rat brain adenosine receptor, follows the order N7 > N3 > N1. The affinity of the adenosine analogues for the adenosine rat brain receptor was besides compared with their activity as inhibitors of platelet aggregation. A synthesis of 2-chloro-1-deazaadenosine by two alternative routes starting from 7-nitroimidazo[4,5-b]pyridine-4-oxide is also reported.

Adenosine deaminase inhibitors. Synthesis and biological activities of deaza analogs of erythro-9-(2-hydroxy-3-nonyl)adenine

Two new deaza analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 1), 7-deaza-EHNA (6) and 1,3-dideaza-EHNA (11), were synthesized and evaluated for adenosine deaminase (ADA) inhibitory activity and compared with EHNA, 1-deaza-EHNA (2), and 3-deaza-EHNA (3). Substitution of a methine group for a nitrogen atom in the 7-position of the purine moiety of EHNA produces a dramatic drop in the inhibitory activity (Ki = 4 X 10(-4) M) whereas compounds 2 and 3 are still good inhibitors (Ki = 1.2 X 10(-7) M and 6.3 X 10(-9) M respectively). EHNA and its deaza analogues so far synthesized were also tested in vitro for their antiviral and antitumor activity in a range of cellular systems. EHNA and 1-deaza-EHNA are equiactive as inhibitors of human respiratory syncytial virus (HRSV) replication (MIC = 6.25 micrograms/mL) while the other compounds are inactive. On the other hand, all the examined compounds displayed an antitumor activity comparable to that of the reference compound 1-beta-D-arabinofuranosyladenine (ara-A), 7-deaza-EHNA being the most active of all. The results obtained showed that there is no correlation between adenosine deaminase inhibition and antiviral or antitumor activity in this series of compounds. 3-Deaza-EHNA, the most active inhibitor of ADA among the EHNA deaza analogues, greatly potentiates the antitumor activity of ara-A in vitro. In vivo activity was observed only when the two compounds were used in combination.

Inhibition of adenosine deaminase from several sources by deaza derivatives of adenosine and EHNA.

Deaza analogues of adenosine and EHNA were tested as inhibitors of the enzyme adenosine deaminase (ADA) obtained from several sources including human erythrocytes, calf intestine, Saccaromices cerevisiae, Escherichia coli and Takadiastase. Ki values of the inhibitors suggest differences among the enzymes both at purine and erythro-nonyl binding site. Among the ribofuranosyl derivatives, 1-deazaadenosine is the best inhibitor, its Ki ranging between 3.5 x 10(-7) and 4 x 10(-5) M for ADA from erythrocytes and Takadiastase respectively. Only ADA from erythrocytes and calf intestine bind EHNA and some of deazaEHNA analogues; 3-deazaEHNA behaves very similarly to EHNA both in affinity and slow binding mechanism, whereas 1-deazaEHNA, though less potent, is a good inhibitor.

A new acyclic heterodinucleotide active against human immunodeficiency virus and herpes simplex virus.

The most common therapies against human herpes virus (HSV-1) and human immunodeficiency virus (HIV-1) infectivity are based on the administration of nucleoside analogues. Acyclovir (ACV) is the drug of choice against HSV-1 infection, while the acyclic nucleoside phosphonate analogue PMPA has shown marked anti-HIV activity in a phase I and II clinical studies. As monocyte-derived macrophages are assumed to be important as reservoirs of both HSV-1 and HIV-1 infection, new approaches able to inhibit replication of both viruses in macrophages should be welcome. ACVpPMPA, a new heterodinucleotide consisting of both an antiherpetic and an antiretroviral drug bound by a phosphate bridge, was synthesized and encapsulated into autologous erythrocytes modified to increase their phagocytosis by human macrophages. ACVpPMPA-loaded erythrocytes provided an effective in vitro protection against both HSV-1 and HIV-1 replication in human macrophages.

A New Tiazofurin Pronucleotide: Synthesis and Biological Evaluation of CycloSaligenyl-Tiazofurin Monophosphate

Abstract Synthesis and biological activities of cyclosaligenyl-tiazofurin monophosphate (CycloSal-TRMP), a new tiazofurin pronucleotide, are reported. CycloSal-TRMP proved to be active in vitro against human myelogenous leukemia K562 cell line and as A1 adenosine receptor agonist.

Inhibition of HIV-1 Replication in Macrophages by Red Blood Cell-Mediated Delivery of a Heterodinucleotide of Lamivudine and Tenofovir

Homo- and heterodimers of nucleoside/nucleotide analogues as reverse transcriptase inhibitors are effective on HIV-1-infected human monocyte-derived macrophages (M/M) compared to the single drugs or their combination. Since the combined treatment of lamivudine (3TC) and tenofovir ((R)PMPA) has an antiretroviral efficacy and a synergic effect respect to separate drugs, the heterodinucleotide 3TCpPMPA was synthesized. A single administration of the dimer as free drug or 3TCpPMPA-loaded RBC selectively targeted to M/M was able to almost completely protect macrophages from “de novo” infection.

Inhibition of HIV-1 Replication in Macrophages by Red Blood Cell-Mediated Delivery of a Heterodinucleotide of Azidothymidine and 9-(R)-2-(Phosphono Methoxypropyl)adenine

Monocyte-derived macrophages (M/M) are considered important in vivo reservoirs for different kinds of viruses, including HIV. Hence, therapeutic strategies are urgently needed to protect these cells from virus infection or to control viral replication. In this paper, we report the synthesis, target delivery and in vitro efficacy of a new heterodinucleotide (AZTpPMPA), able to inhibit HIV-1 production in human macrophages. AZTpPMPA consists of two established anti-HIV drugs [zidovudine (AZT) and tenofovir (PMPA)] chemically coupled together by a phosphate bridge. This drug is not able to prevent p24 production when administered for 18 h to M/M experimentally infected with HIV-1 Bal (inhibition 27%), but can almost completely suppress virus production when given encapsulated into autologous erythrocytes (inhibition of p24 production 97%). AZTpPMPA is slowly converted to PMPA, AZT monophosphate and AZT (36 h half-life at 37°C) by cell-resident enzymes. Thus AZTpPMPA should be considered a new prodrug of AZT and PMPA that is able to provide stechiometric amounts of both nucleoside analogues to macrophage cells and to overcome the low phosphorylating activity of M/M for AZT and the modest permeability of PMPA.