N-methylpurine DNA glycosylase and 8-oxoguanine dna glycosylase metabolize the antiviral nucleoside 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole.

The rapid in vivo degradation of the potent human cytomegalovirus inhibitor 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) compared with a structural L-analog, maribavir (5,6-dichloro-2-(isopropylamino)-1-beta-L-ribofuranosyl-1H-benzimidazole), has been attributed to selective glycosidic bond cleavage. An enzyme responsible for this selective BDCRB degradation, however, has not been identified. Here, we report the identification of two enzymes, 8-oxoguanine DNA glycosylase (OGG1) and N-methylpurine DNA glycosylase (MPG), that catalyze N-glycosidic bond cleavage of BDCRB and its 2-chloro homolog, 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole, but not maribavir. To our knowledge, this is the first demonstration that free nucleosides are substrates of OGG1 and MPG. To understand how these enzymes might process BDCRB, docking and molecular dynamics simulations were performed with the native human OGG1 crystal coordinates. These studies showed that OGG1 was not able to bind a negative control, guanosine, yet BDCRB and maribavir were stabilized through interactions with various binding site residues, including Phe319, His270, Ser320, and Asn149. Only BDCRB, however, achieved orientations whereby its anomeric carbon, C1', could undergo nucleophilic attack by the putative catalytic residue, Lys249. Thus, in silico observations were in perfect agreement with experimental observations. These findings implicate DNA glycosylases in drug metabolism.     

Interaction of intestinal nucleoside transporter hCNT2 with amino acid ester prodrugs of floxuridine and 2-bromo-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole

Amino acid ester prodrugs of antiviral and anticancer nucleoside drugs were developed to improve oral bioavailability or to reduce systemic toxicity. We studied the interaction of human concentrative nucleoside transporter (hCNT2) cloned from intestine with various amino acid ester prodrugs of floxuridine (FUdR) and 5,6-dichloro-2-bromo-1-beta-D-ribofuranosylbenzimidazole (BDCRB). Na(+)-dependent uptakes of [(3)H]-inosine and [(3)H]-adenosine were measured in U251 cells transiently expressing intestinal hCNT2. FUdR significantly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine (60-70% of control), while its amino acid ester prodrugs including Val, Phe, Pro, Asp, and Lys esters exhibited markedly decreased inhibition potency (10-30% of control). On the other hand, BDCRB and its amino acid prodrugs markedly inhibited the uptake of both [(3)H]-inosine and [(3)H]-adenosine. Val, Phe, and Pro ester prodrugs of BDCRB showed similar inhibition capacities as parent compound BDCRB (80-90% for adenosine and 60-80% for inosine). The amino acid site of attachment (3'- and 5'-monoesters) and stereochemistry (L- and D-amino acid esters), did not significantly affect the uptake of [(3)H]-inosine and [(3)H]-adenosine. These results demonstrate that the hCNT2 favorably interacts with BDCRB and its amino acid prodrugs, compared to those of FUdR, and that neutral amino acid esters of BDCRB have a high affinity toward this transporter. Therefore, the intestinal hCNT2 may be a target transporter as a factor for modulating oral pharmacokinetics of BDCRB prodrugs.     

Amino acid ester prodrugs of the anticancer agent gemcitabine: synthesis, bioconversion, metabolic bioevasion, and hPEPT1-mediated transport

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 prodrugs exhibited enhanced affinity (IC(50): 0.14-0.16 mM) for the transporter. However, only the 5'-L-valyl and 5'-L-isoleucyl monoester prodrugs exhibited (a) increased uptake (11.25- and 5.64-fold, respectively) in HeLa/hPEPT1 cells compared to HeLa cells and (b) chemical stability in buffers, that were comparable to valacyclovir, a commercially marketed oral amino acid ester prodrug. The widely disparate enzymatic bioconversion profiles of the 5'-L-valyl and 5'-L-isoleucyl prodrugs in Caco-2 cell homogenates along with their significant resistance to deamination by cytidine deaminase suggest that the disposition of gemcitabine following oral administration would be controlled by the rate of bioconversion following transport across the intestinal epithelial membrane. The combined results also suggest that it may be possible to modulate these characteristics by the choice of the amino acid promoiety.     

Synthesis and antiviral evaluation of trisubstituted indole N-nucleosides as analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB)

2,5,6-Trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)benzimidazole (BDCRB) are nucleosides that exhibit strong and selective activity against human cytomegalovirus (HCMV). Selected polyhalogenated indole nucleosides have now been synthesized as 3-deaza analogues of the benzimidazole nucleosides using the sodium salt glycosylation method. 2-Benzylthio-1-?2-deoxy-3, 5-bis-O-(4-methylbenzoyl)-beta-D-erythro-pentofuranosyl-5, 6-dichloroindole (8) was prepared stereoselectively via the coupling of a 2-deoxyribofuranosyl alpha-chloride derivative with the sodium salt of 2-benzylthio-5,6-dichloroindole (5). Compound 8 was then elaborated into the targeted 2-benzylthio-1-(beta-D-ribofuranosyl)-5, 6-dichloroindole (18) in five steps. 2,5, 6-Trichloro-(1-beta-D-ribofuranosyl)indole (19) was prepared using the same synthetic route with 2,5,6-trichloroindole (6) as the starting material. We were subsequently able to prepare 19 in three steps using a modification of the sodium salt glycosylation method. 2-Bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)indole (25) was also prepared using the same procedures. Target compounds were tested for activity against HCMV, herpes simplex virus type 1 (HSV-1), and human herpes virus six (HHV-6) and for cytotoxicity. All of the compounds were less active against HCMV than TCRB and weakly active or inactive against HSV-1 and HHV-6.     

Synthesis and antiviral evaluation of polyhalogenated imidazole nucleosides: dimensional analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole

A series of polyhalogenated imidazole nucleosides were designed and synthesized as ring-contracted analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) and its 2-bromo analogue (BDCRB) in an effort to explore the spatial limitation of the active pocket(s) in the target protein(s). 2,4,5-Trichloro-, 2-bromo-4,5-dichloro-, and 2,4,5-tribromoimidazole nucleosides were prepared by a condensation of the preformed heterocycles with the appropriate sugar precursors. The ribofuranosyl and xylofuranosyl analogues were prepared by a direct glycosylation using the Vorbruggen's silylation method and provided exclusively the beta-anomers. The arabinofuranosyl analogues were prepared by the sodium salt method to give both the alpha- and beta-anomers. The absolute configurations were established by 1H NMR spectroscopy. Alkylation of the polyhalogenated imidazoles with the appropriate bromomethyl ethers gave the acyclic acyclovir and ganciclovir analogues. In general, the parent polyhalogenated imidazoles showed some activity against human cytomegalovirus (HCMV) (IC50 approximately 35 microM). However, with the exception of two tribromo analogues (7c, 13c-beta), most of their nucleoside derivatives were inactive against both HCMV and herpes simplex virus type-1 (HSV-1) and were not cytotoxic. The results suggest that the ring-contracted nucleoside analogues of TCRB and BDCRB interacted weakly or not at all with viral and cellular targets.     

Ibuprofen is a non-competitive inhibitor of the peptide transporter hPEPT1 (SLC15A1): possible interactions between hPEPT1 substrates and ibuprofen

BACKGROUND AND PURPOSE

Recently, we identified etodolac as a possible ligand for the human intestinal proton-couple peptide transporter (hPEPT1). This raised the possibility that other non-steroidal anti-inflammatory drugs, and especially ibuprofen, could also interact with hPEPT1. Here, we have assessed the interactions of ibuprofen with hPEPT1.

EXPERIMENTAL APPROACH

The uptake of [¹⁴C]Gly-Sar, [³H]Ibuprofen and other radio-labelled compounds were investigated in Madin–Darby canine kidney cells (MDCK)/hPEPT1, MDCK/Mock, LLC-PK₁ or Caco-2 cells. The transepithelial transport of ibuprofen and hPEPT1 substrates was investigated in Caco-2 cell monolayers.

KEY RESULTS

Ibuprofen concentration dependently inhibited hPEPT1-mediated uptake of Gly-Sar in MDCK/hPEPT1 cells (K_i^app= 0.4 mM) but uptake of ibuprofen in Caco-2 cells and MDCK/hPEPT1 cells was not inhibited by hPEPT1 substrates. The maximum uptake rate for Gly-Sar uptake was reduced from 522 pmol·min⁻¹·cm⁻² to 181 pmol·min⁻¹·cm⁻² and 78 pmol·min⁻¹·cm⁻² in the presence of 0.5 mM and 1 mM ibuprofen, respectively. The interaction between ibuprofen and hPEPT1 was thus non-competitive. In LLC-PK1 cells, ibuprofen (1 mM) did not influence the transporter-mediated uptake of glycine or α-methyl-D-glycopyranoside. In Caco-2 cell monolayers the absorptive transport of δ-aminolevulinic acid was reduced by 23% and 48% by ibuprofen (1 and 10 mM), respectively. Likewise the transport of Gly-Sar was reduced by 23% in the presence of ibuprofen (1 mM).

CONCLUSIONS AND IMPLICATIONS

Ibuprofen is a non-competitive inhibitor of hPEPT1. As ibuprofen reduced the transepithelial transport of δ-aminolevulinic acid, drug–drug interactions between ibuprofen and hPEPT1 drug substrates at their site of absorption are possible if administered together.     

Carbamate ester derivatives as potential prodrugs of the presynaptic dopamine autoreceptor agonist (-)-3-(3-hydroxyphenyl)-N-propylpiperidine

Twenty derivatives bearing substituents on the phenolic function of (-)-3-(3-hydroxyphenyl)-N-propylpiperidine [(-)-3-PPP] were synthesized and tested as prodrugs. The carbamate ester derivatives were found to be the most suitable prodrugs, and especially the 4-isopropylphenylcarbamate 20 was capable of escaping the first-pass metabolism and still generating high plasma levels of the parent compound. Four hours after an oral dose of 100 mumol/kg to rats, a plasma level of 2400 nmol/L of (-)-3-PPP was detected by an HPLC method. This was 90 times the level reached after 4 h (27 nmol/L) when (-)-3-PPP itself was given orally at the same dose.     

CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs

The present study characterized Chinese hamster ovary cells overexpressing a human intestinal peptide transporter, CHO/hPEPT1 cells, as an in vitro model for peptidomimetic drugs. The kinetic parameters of Gly-Sar uptake were determined in three different cell culture systems such as untransfected CHO cells (CHO-K1), transfected CHO cells (CHO/hPEPT1) and Caco-2 cells. Vmax in CHO/hPEPT1 cells was approximately 3-fold higher than those in Caco-2 cells and CHO-K1 cells, while Km values were similar in all cases. The uptake of beta-lactam antibiotics in CHO/hPEPT1 cells was three to twelve fold higher than that in CHO-K1 cells, indicating that CHO/hPEPT1 cells significantly enhanced the peptide transport activity. However, amino acid drugs also exhibited high cellular uptake in both CHO-K1 and CHO/hPEPT1 cells due to the high background level of amino acid transporters. Thus, cellular uptake study in CHO/hPEPT1 cells is not sensitive enough to distinguish the peptidyl drugs from amino acid drugs. The potential of CHO/hPEPT1 cells as an in vitro model for peptidomimetic drugs was also examined through the inhibition study on Gly-Sar uptake. Peptidomimetic drugs such as beta-lactam antibiotics and enalapril significantly inhibited Gly-Sar uptake whereas the nonpeptidyl compounds, L-dopa and alpha-methyldopa, did not compete with Gly-Sar for cellular uptake within the therapeutic concentrations. In conclusion, the present study demonstrates the further characterization of CHO/hPEPT1 cells as an uptake model as well as inhibition study and suggests their utility as an alternative in vitro model for drug candidates targeting the hPEPT1 transporter.     

Orally active 1-(cyclohexyloxycarbonyloxy)alkyl ester prodrugs of cefotiam

Orally active 1-(alkyl substituted cyclohexyloxycarbonyloxy)alkyl ester prodrugs (9b-h) of 7 beta-[2-(2-aminothiazol-4-yl)acetamido]-3- [[[1-(2-dimethylaminoethyl)-1H-tetrazol-5-yl]thio]-methyl]ceph+ ++-3- em-4-carboxylic acid (cefotiam, CTM) have been studied as well as the thia (9i) and aza (9j) analogs. These represent derivatives of the 1-(cyclohexylacetoxy)ethyl ester (2) of CTM. The syntheses and oral bioavailability (BA) in mice are described. Among them, the 1-(cyclohexyloxycarbonyloxy)butyl ester (9h) gave the highest BA, 93.5%; the esters having a cyclohexyloxy group in the ester moiety gave BAs of more than 75%, although the BA of the 1-(ethoxycarbonyloxy)ethyl ester (9a) was only 23.9%. The thia analog showed a moderate BA, 46%, but the aza analog, 9j, did not show a BA of CTM. These results indicate that the 1-(substituted cyclohexyloxycarbonyloxy)alkyl group was the suitable promoiety to improve the oral BA of CTM. Chiral 1-(alkoxycarbonyloxy)alkyl groups used as the ester moiety, gave an almost 1: 1 mixture of diastereoisomeric esters. These were tested as such. However, an experiment in which the separated isomers of the 1-(cyclohexyloxycarbonyloxy)ethyl ester (9d) were administered orally confirmed that both diastereoisomers gave identical BAs.     

Synthesis of fluorosugar analogues of 2,5,6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole as antivirals with potentially increased glycosidic bond stability

The metabolic instability in vivo of the glycosidic bond of 2,5, 6-trichloro-1-(beta-D-ribofuranosyl)benzimidazole (TCRB) prompted us to design and synthesize the hitherto unreported fluorinated benzimidazole nucleosides 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole , 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole, and 2-bromo-5, 6-dichloro-1-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)benzimidazole. TCRB was converted into the 2',5'-ditrityl and 3',5'-ditrityl derivatives, which were fluorinated with DAST and deprotected to yield 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole and 2,5, 6-trichloro-1-(3-deoxy-3-fluoro-beta-D-xylofuranosyl)benzimidazole. The resulting low overall yield (5%) of 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole encouraged us to develop an alternative route. The heterocycle 2,5, 6-trichlorobenzimidazole was condensed with 1-bromo-3, 5-di-O-benzoyl-2-deoxy-2-fluoro-alpha-D-arabinofuranose to give, after deprotection, 2,5, 6-trichloro-1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)benzimidazole in a 50% overall yield. The 2'-deoxy-2'-fluoro-beta-D-ribofuranosyl compounds were prepared using 2'-deoxy-2'-fluorouridine, N-deoxyribofuranosyl transferase, and 5,6-dichlorobenzimidazole. Functionalization of the C2 position then gave the desired derivatives. Antiviral and cytotoxicity testing revealed that the deoxy fluoro arabinofuranosyl, xylofuranosyl, and ribofuranosyl derivatives were less active against human cytomegalovirus and more cytotoxic than TCRB.     

Studies on the mechanism of antiviral action of 1-(beta-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin).

Syntheses are described for the 5'-phosphates of the 2'- and 3'-O-methylribavirins (2a and 2b) and the methyl ester of ribavirin 5'-phosphate (3). The 5'-phosphate of 1-beta-(D-ribofuranosyl)-1,2,4-triazole (2d) was obtained via the 3-carboxyl derivative of ribavirin 5'-phosphate (2c). Compounds 2a, 2b, and 2d were inactive as inactive as inhibitors of IMP dehydrogenase under conditions where the parent ribavirin 5'-phosphate (2) was an effective inhibitor. Weak inhibitory activity was exhibited by 3 (Ki approximately 200 muM) and 2c (K1 approximately 70 muM). Under conditions where ribavirin (1) is effectively phosphorylated by rat liver nucleoside kinase, the 2'- and 3'-O-methylribavirins (1a and 1b), the 3-carboxylate of ribavirin (1c), and the riboside of 1,2,4-triazole (1d) were totally inactive. The overall results are fully consistent with the lack of antiviral activity of 1a and 1b, while the specificity of ribavirin as an antiviral agent is further underlined by the behavior of the methyl ester 3.     

In vitro evaluation of N-methyl amide tripeptidomimetics as substrates for the human intestinal di-/tri-peptide transporter hPEPT1.

Oral absorption of tripeptides is generally mediated by the human intestinal di-/tri-peptide transporter, hPEPT1. However, the bioavailability of tripeptides is often limited due to degradation in the GI-tract by various peptidases. The aim of the present study was to evaluate the general application of N-methyl amide bioisosteres as peptide bond replacements in tripeptides in order to decrease degradation by peptidases and yet retain affinity for and transport via hPEPT1. Seven structurally diverse N-methyl amide tripeptidomimetics were selected based on a principal component analysis of structural properties of 6859 N-methyl amide tripeptidomimetics. In vitro extracellular degradation of the selected tripeptidomimetics as well as affinity for and transepithelial transport via hPEPT1 were investigated in Caco-2 cells. Decreased apparent degradation was observed for all tripeptidomimetics compared to the corresponding natural tripeptides. However, affinity for and transepithelial transport via hPEPT1 were only seen for Gly-Sar-Sar, AsnPsi[CONCH(3)]PhePsi[CONCH(3)]Trp, and Gly-Sar-Leu. This implies that tripeptidomimetics originating from tripeptides with neutral side chains are more likely to be substrates for hPEPT1 than tripeptidomimetics with charged side chains. The results of the present study indicate that the N-methyl amide peptide bond replacement approach for increasing bioavailability of tripeptidomimetic drug candidates is not generally applicable to all tripeptides. Nevertheless, retained affinity for and transport via hPEPT1 were shown for three of the evaluated N-methyl amide tripeptidomimetics.     

(Z)-1-溴-2-甲基-1-丁烯的合成

以丁酮为原料,与由亚磷酸三乙酯和氯乙酸乙酯反应得到的二乙氧基磷酰基乙酸乙酯进行Wittig-Horner反应后,再经碱性水解、溴加成反应和脱溴脱羧反应制得(z)-1-溴-2-甲基-1-丁烯,总收率53.2%(以丁酮计).     

In vivo metabolism of the anti-inflammatory agent 2-(5-ethylpyridin-2-yl)benzimidazole

The metabolic fate of anti-inflammatory agent 2-(5-ethylpyridin-2-yl)benzimidazole (KB-1043) was studied in rats after oral administration. An average of 12.2 +/- 1.5% of the dose was excreted in the urine in the course of 0-48 h; 56.7 +/- 2.6% with feces. Two metabolites were also detected in the urine and isolated by reverse phase HPLC. Structures have been given after identification by comparison with authentic samples. The more abundant metabolite proved to be 2-(5-(1-hydroxyethyl)pyridin-2-yl)benzimidazole, a benzylic oxidation product, which was also excreted as glucuronic acid conjugate; the other metabolite was confirmed to be 2-(5-acetylpyridin-2-yl)benzimidazole. Carrageenin edema and gastric ulcerogenic activity were also tested for the two identified metabolites.     

Antiarrhythmic activity of the hydrochloride of 1-methoxymethyl-2-(1-perhydroazepinyl)ethyl ester of 2-(n)-pentyloxycarbanilic acid

Antiarrhythmic effects of the hydrochloride of 1-methoxymethyl-2-(1-perhydroazepinyl)ethyl ester of 2-(n)-pentyloxycarbanilic acid (BK 129) was studied in pentobarbital anesthetized dogs. The dose of 1 mg kg-1 i.v. suppressed ouabain-induced ventricular tachycardia for approx. 15 min. The minimal effective dosis was 0.72 +/- 0.09 mg kg-1 in comparison with 4.56 +/- 1.90 mg kg-1 of trimecaine and 6.80 +/- 0.86 mg kg-1 of quinidine. The doses of 1 and 2 mg kg-1 decreased ectopic activity 24-48 h after coronary artery ligation without complete suppression of extrasystoles. Epinephrine-induced arrhythmias were not prevented with the dose of 1 mg kg-1. The results demonstrate effectiveness of BK 129 in suppression of some types of arrhythmias.