Discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of hepatitis C virus replication.

Discovery of sofosbuvir has radically changed hepatitis C treatment and nucleoside/tide NS5B inhibitors are now viewed as one of the key components in combination therapies with other direct-acting antiviral agents. As part of our program to identify new nucleoside inhibitors of HCV replication, we now wish to report on the discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of HCV replication. Although, cytidine analogues have long been recognized to be metabolized to both cytidine and uridine triphosphates through the action of cytidine deaminase, uridine analogues are generally believed to produce exclusively uridine triphosphate. Detailed investigation of the intracellular metabolism of our newly discovered uridine prodrugs, as well as of sofosbuvir, has now revealed the formation of both uridine and cytidine triphosphates. This occurs, not only in vitro in cell lines, but also in vivo upon oral dosing to dogs.

Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS.

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates.

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC(50) values of 44 nM (enzyme assay) and 65 nM (cell-based assay).

Discovery of 4'-azido-2'-deoxy-2'-C-methyl cytidine and prodrugs thereof: a potent inhibitor of Hepatitis C virus replication.

4'-Azido-2'-deoxy-2'-methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC(50) value of 1.2 µM and showing moderate in vivo bioavailability in rat (F=14%). Here we describe the synthesis and biological evaluation of 4'-azido-2'-deoxy-2'-methylcytidine and prodrug derivatives thereof.

Active immunisation against nicotine blocks the reward facilitating effects of nicotine and partially prevents nicotine withdrawal in the rat as measured by dopamine output in the nucleus accumbens, brain reward thresholds and somatic signs.

We recently showed that active immunisation with the nicotine immunoconjugate IP18-KLH reduces the nicotine-induced increase in dopamine (DA) output in the nucleus accumbens (NAC) and prevents reinstatement of nicotine-seeking behaviour in rats. These effects are mediated by altered distribution of nicotine, resulting in reduced amounts of nicotine reaching the brain, thereby interfering with the rewarding properties of the drug. The present study was designed to explore the effect of immunisation against nicotine on mecamylamine-precipitated nicotine withdrawal as assessed by the reduction in DA output in the NAC in rats. Measuring brain reward thresholds and somatic signs of nicotine withdrawal, the effects of immunisation were also tested during chronic nicotine treatment and after its withdrawal. Finally, we examined the effect of immunisation on challenge injections of nicotine on brain reward thresholds after the increases in somatic signs and reward thresholds associated with nicotine withdrawal had dissipated. The results show that immunisation with IP18-KLH prevented the decrease in DA output in the NAC associated with mecamylamine-precipitated nicotine withdrawal. Moreover, immunisation against nicotine did not precipitate a withdrawal syndrome, as measured by brain reward thresholds and somatic signs, in rats chronically exposed to nicotine. Furthermore, the withdrawal syndrome elicited after cessation of chronic nicotine administration was attenuated in immunised rats compared to that of mock-immunised rats. Finally, the lowering in reward thresholds after nicotine challenge injections was attenuated in both naïve and previously nicotine-exposed immunised rats. In conclusion, the present results show that immunisation with IP18-KLH did not precipitate nicotine withdrawal in rats. Thus, immunisation with IP18-KLH may not elicit nicotine withdrawal in smokers either. Furthermore, since the withdrawal syndrome in rats was attenuated by immunisation, the nicotine withdrawal in smokers should not be worsened but may even be ameliorated during a quit attempt.

Active immunization against nicotine alters the distribution of nicotine but not the metabolism to cotinine in the rat.

We have previously shown that active immunization with the nicotine immunoconjugate IP18-KLH attenuates the reinforcing effects of nicotine, i.e., suppresses the nicotine-induced brain dopamine release and prevents reinstatement of the nicotine-seeking behavior in rats. These effects are thought to be due to an alteration of the kinetics of nicotine distribution by the antibodies, resulting in an attenuated nicotine distribution to the brain. In this study, the distribution of nicotine administered at doses corresponding to those used in our previous studies, was investigated in immunized rats and controls. Male Wistar rats received two immunizations with IP18-KLH in Freund's incomplete adjuvant, 21 days apart, and experiments were performed 7-11 days post-immunization under chloral hydrate anesthesia. Blood samples were collected to determine antibody titer and nicotine selectivity using enzyme-linked immunosorbent assay (ELISA) techniques. The animals received an intravenous nicotine dose and were sacrificed either 3 min or 60 min after nicotine administration. Trunk blood was collected and the brains were removed for analysis of nicotine content. The results showed that immunization against nicotine increases the nicotine concentration in blood and significantly decreases the amount of nicotine that reaches the brain. The present findings thus demonstrate an altered distribution of nicotine after immunization with IP18-KLH. Despite the sustained nicotine binding by the antibodies, the active immunization did not alter the metabolism of nicotine to cotinine, the major nicotine metabolite. In conclusion, the attenuation of the reinforcing effect of nicotine after immunization with IP18-KLH, shown previously, is indeed associated with an altered distribution of nicotine.

Nicotine hapten structure, antibody selectivity and effect relationships: results from a nicotine vaccine screening procedure.

The aim of the present study was to synthesise and screen a set of novel nicotine hapten immunogens used for the treatment of nicotine dependence. In the screening process we studied the amount of antibodies generated and their selectivity, using ELISA techniques, and their effects on nicotine-induced dopamine release in the NAC(shell) of the rat, assessed by in vivo voltammetry. We conclude that even small changes such as the linker attachment on the nicotine molecule as well as the structure of the linker may greatly influence the selectivity of the antibodies and the central neurobiological effects of nicotine that are considered critical for its dependence producing properties.

The design, synthesis, and antiviral activity of 4'-azidocytidine analogues against hepatitis C virus replication: the discovery of 4'-azidoarabinocytidine.

4'-Azidocytidine 3 (R1479) has been previously discovered as a potent and selective inhibitor of HCV replication targeting the RNA-dependent RNA polymerase of hepatitis C virus, NS5B. Here we describe the synthesis and biological evaluation of several derivatives of 4'-azidocytidine by varying the substituents at the ribose 2' and 3'-positions. The most potent compound in this series is 4'-azidoarabinocytidine with an IC(50) of 0.17 microM in the genotype 1b subgenomic replicon system. The structure-activity relationships within this series of nucleoside analogues are discussed.

The design, synthesis, and antiviral activity of monofluoro and difluoro analogues of 4'-azidocytidine against hepatitis C virus replication: the discovery of 4'-azido-2'-deoxy-2'-fluorocytidine and 4'-azido-2'-dideoxy-2',2'-difluorocytidine.

The discovery of 4'-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett. 2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC(50) = 1.28 microM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of several monofluoro and difluoro derivatives of 4'-azidocytidine. The most potent compounds in this series were 4'-azido-2'-deoxy-2',2'-difluorocytidine and 4'-azido-2'-deoxy-2'-fluoroarabinocytidine with antiviral EC(50) of 66 nM and 24 nM in the HCV replicon system, respectively. The structure-activity relationships within this series were discussed, which led to the discovery of these novel nucleoside analogues with the most potent compound, showing more than a 50-fold increase in antiviral potency as compared to 4'-azidocytidine (3).

2'-deoxy-4'-azido nucleoside analogs are highly potent inhibitors of hepatitis C virus replication despite the lack of 2'-alpha-hydroxyl groups.

RNA polymerases effectively discriminate against deoxyribonucleotides and specifically recognize ribonucleotide substrates most likely through direct hydrogen bonding interaction with the 2'-alpha-hydroxy moieties of ribonucleosides. Therefore, ribonucleoside analogs as inhibitors of viral RNA polymerases have mostly been designed to retain hydrogen bonding potential at this site for optimal inhibitory potency. Here, two novel nucleoside triphosphate analogs are described, which are efficiently incorporated into nascent RNA by the RNA-dependent RNA polymerase NS5B of hepatitis C virus (HCV), causing chain termination, despite the lack of alpha-hydroxy moieties. 2'-deoxy-2'-beta-fluoro-4'-azidocytidine (RO-0622) and 2'-deoxy-2'-beta-hydroxy-4'-azidocytidine (RO-9187) were excellent substrates for deoxycytidine kinase and were phosphorylated with efficiencies up to 3-fold higher than deoxycytidine. As compared with previous reports on ribonucleosides, higher levels of triphosphate were formed from RO-9187 in primary human hepatocytes, and both compounds were potent inhibitors of HCV virus replication in the replicon system (IC(50) = 171 +/- 12 nM and 24 +/- 3 nM for RO-9187 and RO-0622, respectively; CC(50) >1 mM for both). Both compounds inhibited RNA synthesis by HCV polymerases from either HCV genotypes 1a and 1b or containing S96T or S282T point mutations with similar potencies, suggesting no cross-resistance with either R1479 (4'-azidocytidine) or 2'-C-methyl nucleosides. Pharmacokinetic studies with RO-9187 in rats and dogs showed that plasma concentrations exceeding HCV replicon IC(50) values 8-150-fold could be achieved by low dose (10 mg/kg) oral administration. Therefore, 2'-alpha-deoxy-4'-azido nucleosides are a new class of antiviral nucleosides with promising preclinical properties as potential medicines for the treatment of HCV infection.