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Gordon L. Amidon - One of the best experts on this subject based on the ideXlab platform.
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enhanced cancer cell growth inhibition by dipeptide prodrugs of floxuridine increased transporter affinity and metabolic stability
Molecular Pharmaceutics, 2008Co-Authors: Yasuhiro Tsume, John M. Hilfinger, Gordon L. AmidonAbstract:Dipeptide monoEster prodrugs of floxuridine were synthesized, and their chemical stability in buffers, resistance to glycosidic bond metabolism, affinity for PEPT1, enzymatic activation and permeability in cancer cells were determined and compared to those of mono Amino Acid monoEster floxuridine prodrugs. Prodrugs containing glycyl moieties were the least stable in pH 7.4 buffer ( t 1/2 < 100 min). The activation of all floxuridine prodrugs was 2- to 30-fold faster in cell homogenates than their hydrolysis in buffer, suggesting enzymatic action. The enzymatic activation of dipeptide monoEster prodrugs containing aromatic promoieties in cell homogenates was 5- to 20-fold slower than that of other dipeptide and most mono Amino Acid monoEster prodrugs ( t 1/2 approximately 40 to 100 min). All prodrugs exhibited enhanced resistance to glycosidic bond metabolism by thymidine phosphorylase compared to parent floxuridine. In general, the 5'-O-dipeptide monoEster floxuridine prodrugs exhibited higher affinity for PEPT1 than the corresponding 5'-O-mono Amino Acid Ester prodrugs. The permeability of dipeptide monoEster prodrugs across Caco-2 and Capan-2 monolayers was 2- to 4-fold higher than the corresponding mono Amino Acid Ester prodrug. Cell proliferation assays in AsPC-1 and Capan-2 pancreatic ductal cell lines indicated that the dipeptide monoEster prodrugs were equally as potent as mono Amino Acid prodrugs. The transport and enzymatic profiles of 5'- l-phenylalanyl- l-tyrosyl-floxuridine, 5'- l-phenylalanyl- l-glycyl-floxuridine, and 5'- l-isoleucyl- l-glycyl-floxuridine suggest their potential for increased oral uptake, delayed enzymatic bioconversion and enhanced resistance to metabolism to 5-fluorouracil, as well as enhanced uptake and cytotoxic activity in cancer cells, attributes that would facilitate prolonged systemic circulation for enhanced therapeutic action.
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molecular basis of prodrug activation by human valacyclovirase an α Amino Acid Ester hydrolase
Journal of Biological Chemistry, 2008Co-Authors: Zhaohui Xu, Jiahai Zhou, Gordon L. AmidonAbstract:Chemical modification to improve biopharmaceutical properties, especially oral absorption and bioavailability, is a common strategy employed by pharmaceutical chemists. The approach often employs a simple structural modification and utilizes ubiquitous endogenous Esterases as activation enzymes, although such enzymes are often unidentified. This report describes the crystal structure and specificity of a novel activating enzyme for valacyclovir and valganciclovir. Our structural insights show that human valacyclovirase has a unique binding mode and specificity for Amino Acid Esters. Biochemical data demonstrate that the enzyme hydrolyzes Esters of-Amino Acids exclusively and displays a broad specificity spectrum for the Aminoacyl moiety similar to tricorn-interacting Aminopeptidase F1. Crystal structures of the enzyme, two mechanistic mutants, and a complex with a product analogue, when combined with biochemical analysis, reveal the key determinants for substrate recognition; that is, a flexible and mostly hydrophobic acyl pocket, a localized negative electrostatic potential, a large open leaving group-accommodating groove, and a pivotal Acidic residue, Asp-123, after the nucleophile Ser-122. This is the first time that a residue immediately after the nucleophile has been found to have its side chain directed into the substrate binding pocket and play an essential role in substrate discrimination in serine hydrolases. These results as well as a phylogenetic analysis establish that the enzyme functions as a specific -Amino Acid Ester hydrolase. Valacyclovirase is a valuable target for Amino Acid Ester prodrugbased oral drug delivery enhancement strategies. Chemical modification through reversible prodrug, modification of a candidate drug, is a frequently employed strategy to improve biopharmaceutical properties of a candidate drug. Notable successes include oseltamivir, enalapril, and capecitabine (1). Membrane transport and absorption are usually thought to be improved by the increased lipophilicity and result in improved passive membrane transport (2). More recently we have shown that prodrugs may be transported by carrier-mediated transport mechanisms (3). A second essential step in effective prodrug therapy is the activation (hydrolysis) of the prodrug to the active therapeutic agent. CarboxylEsterase is a common target for lipophilic approaches to improved membrane permeability (4 – 6). However, often the activation enzymes are unidentified. This manuscript reports the results of structural and biochemical studies on a novel prodrug activating enzyme,
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floxuridine Amino Acid Ester prodrugs enhancing caco 2 permeability and resistance to glycosidic bond metabolism
Pharmaceutical Research, 2005Co-Authors: Christopher P. Landowski, Philip L. Lorenzi, John M. Hilfinger, Xueqin Song, Gordon L. AmidonAbstract:Purpose The aim of this study was to synthesize Amino Acid Ester prodrugs of 5-fluoro-2′-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism.
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Amino Acid Ester prodrugs of 2 bromo 5 6 dichloro 1 β d ribofuranosyl benzimidazole enhance metabolic stability in vitro and in vivo
Journal of Pharmacology and Experimental Therapeutics, 2005Co-Authors: Philip L. Lorenzi, Christopher P. Landowski, John M. Hilfinger, Xueqin Song, John C Drach, Leroy B Townsend, Katherine Z Borysko, Julie M Breitenbach, Jae Seung Kim, Gordon L. AmidonAbstract:2-Bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV), but it lacks clinical utility due to rapid in vivo metabolism. We hypothesized that Amino Acid Ester prodrugs of BDCRB may enhance both in vitro potency and systemic exposure of BDCRB through evasion of BDCRB-metabolizing enzymes. To this end, eight different Amino Acid prodrugs of BDCRB were tested for N-glycosidic bond stability, Ester bond stability, Caco-2 cell uptake, antiviral activity, and cytotoxicity. The prodrugs were resistant to metabolism by BDCRB-metabolizing enzymes, and Ester bond cleavage was rate-limiting in metabolite formation from prodrug. Thus, BDCRB metabolism could be controlled by the selection of promoiety. In HCMV plaque-formation assays, l-Asp-BDCRB exhibited 3-fold greater selectivity than BDCRB for inhibition of HCMV replication. This potent and selective antiviral activity in addition to favorable stability profile made l-Asp-BDCRB an excellent candidate for in vivo assessment and pharmacokinetic comparison with BDCRB. In addition to rapid absorption and sufficient prodrug activation after oral administration to mice, l-Asp-BDCRB exhibited a 5-fold greater half-life than BDCRB. Furthermore, the sum of area under the concentration-time profile (AUC)(BDCRB) and AUC(prodrug) after l-Asp-BDCRB administration was roughly 3-fold greater than AUC(BDCRB) after BDCRB administration, suggesting that a reservoir of prodrug was delivered in addition to parent drug. Overall, these findings demonstrate that Amino Acid prodrugs of BDCRB exhibit evasion of metabolizing enzymes (i.e., bioevasion) in vitro and provide a modular approach for translating this in vitro stability into enhanced in vivo delivery of BDCRB.
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targeted delivery to pept1 overexpressing cells Acidic basic and secondary floxuridine Amino Acid Ester prodrugs
Molecular Cancer Therapeutics, 2005Co-Authors: Christopher P. Landowski, Balvinder S. Vig, Xueqin Song, Gordon L. AmidonAbstract:Floxuridine is a clinically proven anticancer agent in the treatment of metastatic colon carcinomas and hepatic metastases. However, prodrug strategies may be necessary to improve its physiochemical properties and selectivity and to reduce undesirable toxicity effects. Previous studies with Amino Acid Ester prodrugs of nucleoside drugs targeted to the PEPT1 transporter coupled with recent findings of the functional expression of the PEPT1 oligopeptide transporter in pancreatic adenocarcinoma cell lines suggest the potential of PEPT1 as therapeutic targets for cancer treatment. In this report, we show the feasibility of achieving enhanced transport and selective antiproliferative action of Amino Acid Ester prodrugs of floxuridine in cell systems overexpressing PEPT1. All prodrugs exhibited affinity for PEPT1 (IC50, 1.1–2.3 mmol/L). However, only the prolyl and lysyl prodrugs exhibited enhanced uptake (2- to 8-fold) with HeLa/PEPT1 cells compared with HeLa cells, suggesting that the aspartyl prodrugs are PEPT1 inhibitors. The selective growth inhibition of Madine-Darby canine kidney (MDCK)/PEPT1 cells over MDCK cells by the prodrugs was consistent with the extent of their PEPT1-mediated transport. All Ester prodrugs hydrolyzed to floxuridine fastest in Caco-2 cell and MDCK homogenates and slower in human plasma and were most chemically stable in pH 6.0 buffer. Prolyl and lysyl prodrugs were relatively less stable compared with aspartyl prodrugs in buffers and in cell homogenates. The results suggest that optimal design for targeted delivery would be possible by combining both stability and transport characteristics afforded by the promoiety.
Thomas R M Barends - One of the best experts on this subject based on the ideXlab platform.
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Acetobacter turbidans alpha-Amino Acid Ester hydrolase: merohedral twinning in P21 obscured by pseudo-translational NCS.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Thomas R M Barends, Bauke W. DijkstraAbstract:The structure elucidation of the alpha-Amino Acid Ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After re-evaluation of the data, it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage.
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Acetobacter turbidans alpha-Amino Acid Ester hydrolase
Acta Crystallographica Section D Biological Crystallography, 2003Co-Authors: Thomas R M Barends, Bauke W. DijkstraAbstract:The structure elucidation of the alpha-Amino Acid Ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After re-evaluation of the data, it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage.
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the sequence and crystal structure of the alpha Amino Acid Ester hydrolase from xanthomonas citri define a new family of beta lactam antibiotic acylases
Journal of Biological Chemistry, 2003Co-Authors: Thomas R M Barends, Jolanda Polderman Tijmes, Cmh Hensgens, Peter A Jekel, Erik De Vries, Dick B. Janssen, Bebauke W DijkstraAbstract:Abstract α-Amino Acid Ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of Esters and amides with an α-Amino group. As such, they can synthesize β-lactam antibiotics from acyl compounds and β-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-A resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an α/β-hydrolase fold domain, a helical cap domain, and a jellyroll β-domain. Structural homology was observed to the Rhodococcus cocaine Esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a β-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an α-Amino group on the substrate, and explains the low specificity toward the β-lactam nucleus.
Bauke W. Dijkstra - One of the best experts on this subject based on the ideXlab platform.
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Acetobacter turbidans alpha-Amino Acid Ester hydrolase
Acta Crystallographica Section D Biological Crystallography, 2003Co-Authors: Thomas R M Barends, Bauke W. DijkstraAbstract:The structure elucidation of the alpha-Amino Acid Ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After re-evaluation of the data, it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage.
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Acetobacter turbidans alpha-Amino Acid Ester hydrolase: merohedral twinning in P21 obscured by pseudo-translational NCS.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Thomas R M Barends, Bauke W. DijkstraAbstract:The structure elucidation of the alpha-Amino Acid Ester hydrolase from Acetobacter turbidans by molecular replacement is described. In the monoclinic crystal, the molecules are related by both rotational and pseudo-crystallographic translational NCS (non-crystallographic symmetry). Refinement of the structure converged at unacceptably high R factors. After re-evaluation of the data, it was found that the crystal was merohedrally twinned, with a high twinning fraction. It is shown that the pseudo-crystallographic NCS causes aberrant behaviour of conventional twinning indicators, which explains why the twinning was only realized at the refinement stage.
Andreas Liese - One of the best experts on this subject based on the ideXlab platform.
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development of a continuously operating process for the enantioselective synthesis of a β Amino Acid Ester via a solvent free chemoenzymatic reaction sequence
Advanced Synthesis & Catalysis, 2013Co-Authors: Simon Strompen, Markus Weis, Harald Groger, Lutz Hilterhaus, Andreas LieseAbstract:A sequential, chemoenzymatic process for a continuously operating production of the chiral -Amino Acid Ester ethyl (S)-3-(benzylAmino)butanoate was developed. The reactor set-up combined a plug-flow reactor for the thermal aza-Michael addition of benzylamine to trans-ethyl crotonate coupled to a subsequent packed-bed reactor for the lipase (Novozym 435)-catalyzed kinetic resolution of the racemic intermediate product, which was formed in the initial step. The coupled reactors were operated continuously for a time period of 4 days without significant loss of enzyme activity. The target -Amino Acid Ester was obtained with 92% conversion in the plug-flow reactor and 59% conversion in the packed bed reactor at high enantiomeric excess of >98%. A space-time yield of 0.4kgL(-1)d(-1) was calculated for the total reactor system and 1.8kgL(-1)d(-1) based solely on the volume of the packed bed reactor. A total turnover number of 158,000 was calculated for the biocatalyst assuming the same deactivation rate as observed in batch experiments. The continuously operating, solvent-free process thus represents an efficient method for the enantioselective production of a value added (S)âAmino Acid Ester starting from cheap substrates.
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kinetic investigation of a solvent free chemoenzymatic reaction sequence towards enantioselective synthesis of a β Amino Acid Ester
Biotechnology and Bioengineering, 2012Co-Authors: Simon Strompen, Markus Weis, Harald Groger, Lutz Hilterhaus, Thomas Ingram, Irina Smirnova, Andreas LieseAbstract:A solvent-free, chemoenzymatic reaction sequence for the enantioselective synthesis of β-Amino Acid Esters has been kinetically and thermodynamically characterized. The coupled sequence comprises a thermal aza-Michael addition of cheap starting materials and a lipase catalyzed Aminolysis for the kinetic resolution of the racemic Ester. Excellent ee values of >99% were obtained for the β-Amino Acid Ester at 60% conversion. Kinetic constants for the aza-Michael addition were obtained by straightforward numerical integration of second-order rate equations and nonlinear fitting of the progress curves. A different strategy had to be devised for the biocatalytic reaction. Initially, a simplified Michaelis–Menten model including product inhibition was developed for the reaction running in THF as an organic solvent. Activity based parameters were used instead of concentrations in order to facilitate the transfer of the kinetic model to the solvent-free system. Observed solvent effects not accounted for by the use of thermodynamic activities were incorporated into the kinetic model. Enzyme deactivation was observed to depend on the ratio of the applied substrates and also included in the kinetic model. The developed simple model is in very good agreement with the experimental data and allows the simulation and optimization of the solvent-free process. Biotechnol. Bioeng. 2012; 109:1479–1489. © 2012 Wiley Periodicals, Inc.
Philip L. Lorenzi - One of the best experts on this subject based on the ideXlab platform.
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floxuridine Amino Acid Ester prodrugs enhancing caco 2 permeability and resistance to glycosidic bond metabolism
Pharmaceutical Research, 2005Co-Authors: Christopher P. Landowski, Philip L. Lorenzi, John M. Hilfinger, Xueqin Song, Gordon L. AmidonAbstract:Purpose The aim of this study was to synthesize Amino Acid Ester prodrugs of 5-fluoro-2′-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism.
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Amino Acid Ester prodrugs of 2 bromo 5 6 dichloro 1 β d ribofuranosyl benzimidazole enhance metabolic stability in vitro and in vivo
Journal of Pharmacology and Experimental Therapeutics, 2005Co-Authors: Philip L. Lorenzi, Christopher P. Landowski, John M. Hilfinger, Xueqin Song, John C Drach, Leroy B Townsend, Katherine Z Borysko, Julie M Breitenbach, Jae Seung Kim, Gordon L. AmidonAbstract:2-Bromo-5,6-dichloro-1-(beta-d-ribofuranosyl)benzimidazole (BDCRB) is a potent and selective inhibitor of human cytomegalovirus (HCMV), but it lacks clinical utility due to rapid in vivo metabolism. We hypothesized that Amino Acid Ester prodrugs of BDCRB may enhance both in vitro potency and systemic exposure of BDCRB through evasion of BDCRB-metabolizing enzymes. To this end, eight different Amino Acid prodrugs of BDCRB were tested for N-glycosidic bond stability, Ester bond stability, Caco-2 cell uptake, antiviral activity, and cytotoxicity. The prodrugs were resistant to metabolism by BDCRB-metabolizing enzymes, and Ester bond cleavage was rate-limiting in metabolite formation from prodrug. Thus, BDCRB metabolism could be controlled by the selection of promoiety. In HCMV plaque-formation assays, l-Asp-BDCRB exhibited 3-fold greater selectivity than BDCRB for inhibition of HCMV replication. This potent and selective antiviral activity in addition to favorable stability profile made l-Asp-BDCRB an excellent candidate for in vivo assessment and pharmacokinetic comparison with BDCRB. In addition to rapid absorption and sufficient prodrug activation after oral administration to mice, l-Asp-BDCRB exhibited a 5-fold greater half-life than BDCRB. Furthermore, the sum of area under the concentration-time profile (AUC)(BDCRB) and AUC(prodrug) after l-Asp-BDCRB administration was roughly 3-fold greater than AUC(BDCRB) after BDCRB administration, suggesting that a reservoir of prodrug was delivered in addition to parent drug. Overall, these findings demonstrate that Amino Acid prodrugs of BDCRB exhibit evasion of metabolizing enzymes (i.e., bioevasion) in vitro and provide a modular approach for translating this in vitro stability into enhanced in vivo delivery of BDCRB.
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Amino Acid Ester prodrugs of the anticancer agent gemcitabine synthesis bioconversion metabolic bioevasion and hpept1 mediated transport
Molecular Pharmaceutics, 2005Co-Authors: Xueqin Song, Balvinder S. Vig, Philip L. Lorenzi, Christopher P. Landowski, John M. Hilfinger, Gordon L. AmidonAbstract:Gemcitabine, a clinically effective nucleoside anticancer agent, is a polar drug with low membrane permeability and is administered intravenously. Further, extensive degradation of gemcitabine by cytidine deaminase to an inactive metabolite in the liver affects its activity adversely. Thus, strategies that provide both enhanced transport and high metabolic bioevasion would potentially lead to oral alternatives that may be clinically useful. The objective of this study was to evaluate whether Amino Acid Ester prodrugs of gemcitabine would (a) facilitate transport across intestinal membranes or across cells that express hPEPT1 and (b) provide resistance to deamination by cytidine deaminase. 3‘-MonoEster, 5‘-monoEster, and 3‘,5‘-diEster prodrugs of gemcitabine utilizing aliphatic (l-valine, d-valine, and l-isoleucine) and aromatic (l-phenylalanine and d-phenylalanine) Amino Acids as promoieties were synthesized and evaluated for their affinity and direct hPEPT1-mediated transport in HeLa/hPEPT1 cells. All pr...
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Amino Acid Ester prodrugs of the antiviral agent 2 bromo 5 6 dichloro 1 β d ribofuranosyl benzimidazole as potential substrates of hpept1 transporter
Journal of Medicinal Chemistry, 2005Co-Authors: Xueqin Song, Balvinder S. Vig, Philip L. Lorenzi, John C Drach, Leroy B Townsend, Gordon L. AmidonAbstract:Amino Acid Ester prodrugs of 2-bromo-5,6-dichloro-1-(β-d-ribofuranosyl)benzimidazole (BDCRB) were synthesized and evaluated for their affinity for hPEPT1, an intestinal oligopeptide transporter. Assays of competitive inhibition of [3H]glycylsarcosine (Gly-Sar) uptake in HeLa/hPEPT1 cells by the Amino Acid Ester prodrugs of BDCRB suggested their 2- to 4-fold higher affinity for hPEPT1 compared to BDCRB. Further, promoieties with hydrophobic side chains and l-configuration were preferred by the hPEPT1 transporter.
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Amino Acid Ester Prodrugs of Floxuridine: Synthesis and Effects of Structure, Stereochemistry, and Site of Esterification on the Rate of Hydrolysis
Pharmaceutical research, 2003Co-Authors: Balvinder S. Vig, Philip L. Lorenzi, Sachin Mittal, Christopher P. Landowski, Ho-chul Shin, Henry I. Mosberg, John M. Hilfinger, Gordon L. AmidonAbstract:Purpose. To synthesize Amino Acid Ester prodrugs of floxuridine (FUdR) and to investigate the effects of structure, stereochemistry, and site of Esterification of promoiety on the rates of hydrolysis of these prodrugs in Caco-2 cell homogenates. Methods. Amino Acid Ester prodrugs of FUdR were synthesized using established procedures. The kinetics of hydrolysis of prodrugs was evaluated in human adenocarcinoma cell line (Caco-2) homogenates and pH 7.4 phosphate buffer. Results. 3′-MonoEster, 5′-monoEster, and 3′,5′-diEster prodrugs of FUdR utilizing proline, L-valine, D-valine, L-phenylalanine, and D-phenylalanine as promoieties were synthesized and characterized. In Caco-2 cell homogenates, the L-Amino Acid Ester prodrugs hydrolyzed 10 to 75 times faster than the corresponding D-Amino Acid Ester prodrugs. Pro and Phe Ester prodrugs hydrolyzed much faster (3- to 30-fold) than the corresponding Val Ester prodrugs. Further, the 5′-monoEster prodrugs hydrolyzed significantly faster (3-fold) than the 3′,5′-diEster prodrugs. Conclusions. Novel Amino Acid Ester prodrugs of FUdR were successfully synthesized. The results presented here clearly demonstrate that the rate of FUdR prodrug activation in Caco-2 cell homogenates is affected by the structure, stereochemistry, and site of Esterification of the promoiety. Finally, the 5′-Val and 5′-Phe monoEsters exhibited desirable characteristics such as good solution stability and relatively fast enzymatic conversion rates.
