A series of ring-expanded ("fat") heterocycles, nucleoside and nucleotide analogues (RENs) containing the imidazo[4,5-e][1,3]diazepine ring system (9, 14, 15, 18, 24-26, 28, 31, and 33) and imidazo[4,5-e][1,2,4]triazepine ring systems (30b, 30c, 32, and 34), have been synthesized as potential inhibitors of NTPases/helicases of Flaviviridae, including the West Nile virus (WNV), hepatitis C virus (HCV), and Japanese encephalitis virus (JEV). An amino-terminal truncated form of human enzyme Suv3(delta1-159) was also included in the study so as to assess the selectivity of RENs against the viral enzymes. The analogues of RENs included structural variations at position 1 of the heterocyclic base and contained changes in both the type of sugar moieties (ribo, 2'-deoxyribo, and acyclic sugars) and the mode of attachment (alpha versus beta anomeric configuration) of those sugars to the heterocyclic base. The target RENs were biochemically screened separately against the helicase and ATPase activities of the viral NTPases/helicases. A number of RENs inhibited the viral helicase activity with IC50 values that ranged in micromolar concentrations and exhibited differential selectivity between the viral enzymes. In view of the observed tight complex between some nucleosides and RNA and/or DNA substrates of a helicase, the mechanism of action of RENs might involve their interaction with the appropriate substrate through binding to the major or minor groove of the double helix. The REN-5'-triphosphates, on the other hand, did not influence the above unwinding reaction, but instead exerted the inhibitory effect on the ATPase activity of the enzymes. The activity was found to be highly dependent upon the low concentration levels of the substrate ATP. At concentrations >500 microM of RENs and the ATP concentrations >10 times the Km value of the enzyme, a significant activation of NTPase activity was observed. This activating effect underwent further dramatic enhancement (>1000%) by further increases in ATP concentration in the reaction mixture. A tentative mechanistic model has been proposed to explain the observed results, which includes an additional allosteric binding site on the viral NTPases/helicases that can be occupied by nucleoside/nucleotide-type molecules such as RENs.

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4‐Hydrazinothieno[2,3‐d]pyrimidines were cyclized with triethyl orthoformate and formic acid to give 1,2,4‐triazolo[4,3‐c]thieno[3,2‐e]pyrimidines and 1,2,4‐triazolo[2,3‐c]thieno[3,2‐e]pyrimidines depending on the reaction conditions employed.

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A significant number of patients with AIDS and AIDS-related complex develop neurological complications. Therefore, it is critical that anti-HIV agents penetrate the blood-brain barrier and suppress viral replication in the brain. In an effort to increase the brain delivery of anti-HIV nucleosides, in vitro and in vivo pharmacokinetics of dihydropyridine derivatives of 3'-azido-2',3'-dideoxyuridine (AzddU, AZDU, or CS-87) and 3'-azido-3'-deoxythymidine (AZT, Zidovudine) have been studied. In vitro studies of the prodrugs (AzddU-DHP and AZT-DHP) in human serum, mouse serum, and mouse brain homogenate indicated that the rates of serum conversion from prodrugs to parent drugs are species dependent: mouse brain homogenate greater than mouse serum greater than human serum. Half-lives in human serum, mouse serum, and mouse brain homogenate are 4.33, 0.56, 0.17 h, respectively, for AzddU and 7.70, 1.40, and 0.18 h, respectively, for AZT. In vivo studies of AzddU-DHP and AZT-DHP showed that the prodrugs have areas under the serum concentration-time curves (AUC) similar to those of the parent drugs. The AUC in serum for AzddU following prodrug administration is 25.79 micrograms h/mL, which is similar to the value of 25.83 micrograms h/mL when AzddU was administered. Analogously, the serum AUCs for AZT when AZT-DHP and AZT were administered are 25.38 and 26.64 micrograms h/mL, respectively. However, the brain AUCs for both AzddU and AZT derived from prodrugs, being 11.43 and 11.28 micrograms h/mL, respectively, are greater than the brain AUCs for AzddU (2.09 micrograms h/mL) and AZT (1.21 micrograms h/mL) when the parent drugs were administered. Thus, the relative brain exposure (re) for AzddU (5.47) and AZT (9.32) indicate a significant increase in exposure to the anti-HIV nucleosides following prodrug administrations. The results of extended half-lives of the synthesized prodrugs in human serum along with the higher re values in vivo warrant studies in larger animals to determine the potential usefulness of the prodrugs in humans.