Date of Award
Doctor of Philosophy (PhD)
2',3'-Dideoxynucleoside analogs are commonly used as anti-HIV, anti-HBV, and anti-cancer drugs. Despite of their potent activities, there are some major limitations in using 2',3'-dideoxynucleosides as therapeutic agents. The nucleosides have usually poor cellular uptake because of their hydrophilic nature. Some of the nucleoside analogs, such as anti-HIV agents, become ineffective after multiple administrations because of the development of the drug resistance, and therefore they must be administered in combination therapy. It is hard to deliver the nucleoside analogs to a particular tissue for site specific targeting. Furthermore, nucleoside analogs undergo three intracellular phosphorylation steps to become active. The first phosphorylation step is slow and a rate-limiting process for several compounds.
Herein, we report the synthesis and evaluation of 2',3'-dideoxynucleoside conjugates with fatty acids, peptides, other nucleosides, fatty acyl phosphotriesters, or polymer derivatives. The primary hypothesis of this project was that conjugation of nucleosides with other compounds offers a novel strategy in designing compounds with enhanced anti-HIV activity. This combination may result in development of anti-HIV agents having enhanced lipophilicity, longer duration of action by sustained intracellular release of active substrates at adequate concentrations, higher uptake into infected cells, and/or site specificity. The development of viral resistance to the nucleosides would occur at a slower rate than to either compound alone. Furthermore, some of the compounds may be used to bypass first rate-limiting phosphorylation step.
In the first two chapters, synthesis and anti-HIV activities of fatty acyl derivatives of Zidovudine (AZT), Allovudine (FLT), Emtricitabine (FTC), Lamivudine (3TC), and Stavudine (d4T) are discussed. Among all the compounds, 5'-0-myristoyl derivative of FTC (2.31, EC50 = 70 nM against cell-free virus) exhibited the best anti- HIV profile when compared with other fatty acyl derivatives of other nucleosides and the physical mixture of FTC and myristic acid. 5'-0-Fatty acyl derivatives of FLT, 5'-0-(12-azidododecanoyl) derivative of FLT (KP-1), and 5'-0-(12-thioethyldodecanoyl)thymidine (KP-17), also displayed good activity against cell-free (EC50 values of 0.9 to 1.0 μM, respectively) virus and minimal cellular toxicity. Cellular uptake studies for 5’-O-fatty acyl derivatives of FLT and 3TC were conducted on CCRF-CEM cell line using a 5(6)-carboxyfluorescein derivative attached through 12-aminododecanoic acid as a linker to the nucleosides. The fluorescence-based studies indicated that the fatty acyl derivatives of FLT and 3TC have a higher cellular uptake versus that of the corresponding parent nucleoside substituted with a short alkyl group, such as β-alanine the cellular uptake with concentration-and-time-dependent.
In the third chapter, the synthesis and anti-HIV activities of succinate, suberate, and peptide derivatives of AZT, FLT, and 3TC are discussed. The compounds were designed in such a way to have 1 to 3 nucleosides. The hypothesis underlying this project is that the conjugates are able to deliver 1 to 3 nucleoside analogs to the HIV-infected cells. Some of the nucleoside-peptide conjugates were also substituted with the fatty acids. Peptides conjugated with fatty acids and nucleosides exhibited higher anti-HIV activities when compared with those substituted only with nucleosides. Increasing the number of anti-HIV nucleosides to 2 or 3 on the peptide chain enhanced the anti-HIV potency. A glutamic acid ester derivative, FLT-Succinate-AZT(glutamyl)-3TC, containing three different nucleosides was the most active compound among all the derivatives with an EC50 value of 0.9 μM.
Chapter 4 describes the synthesis of FLT from thymidine using a solid-phase method to circumvent some of the problems associated with the solution-phase methods, such as multiple protecting and deprotecting steps.
Fifth chapter discusses the synthesis and anti-HIV activities of phosphotriesters of AZT and FLT. The conjugates were expected to get hydrolyzed inside the cell, to release nucleoside monophosphates, and to bypass first rate limiting phosphorylation step. The synthesized phosphotriester derivatives showed only modest anti-HIV activity, significantly lower than that of their parent nucleosides.
In chapter 6, synthesis and characterization of dextran prodrug (3TCSD) of the antiviral drug 3TC is discussed. Dextran-3TC conjugate was synthesized to localize 3TC selectively in the liver and provide sustained release of the drug by the action of liver lysosomes. Liver accumulation of conjugated 3TC was enhanced by 50 fold when compared to that of parent drug.
In chapter 7 the synthesis and biological evaluation of double-barreled conjugates of sodium cellulose sulfate (CS) with 2',3'-dideoxynucleosides analogs (AZT, FLT and 3TC) using different linkers are described. Cellulose sulfate is a polyanionic polymer which blocks HIV entry into the cells by interacting with the positive charge of viral gp120 protein. Nucleosides analogs act as reverse transcriptase inhibitors (RTis). Conjugates were expected to undergo enzymatic hydrolysis and \ thereby releasing nucleosides and cellulose sulfate targeting two different strains of virus. Cellulose sulfate conjugates of nucleosides containing an acetate linker showed good activity against both R5 and X4 strains of HIV. For example a CS-AZT conjugate (acetate linker; 1.73% loading) was more effective than CS, especially against the RS HIV-1 lab-adapted strain BaL. Similarly, sodium cellulose sulfate-acetate-FLT and showed better anti-HIV profile than sodium cellulose sulfate and the mixture of sodium cellulose sulfate and FLT.
Overall, the research described in this dissertation demonstrated that conjugation of anti-HIV nucleoside analogs with appropriate compounds (e.g., fatty acids, polymers, peptides groups, or other nucleosides) is an alternative strategy for designing more effective anti-HIV agents that can be further developed as therapeutic or preventative agents.
Agarwal, Hitesh Kumar, "Design and Evaluation of Nucleoside Derivatives for Targeted Drug Delivery and Therapeutic Applications" (2008). Open Access Dissertations. Paper 507.