Synthesis, Hydrolysis, and Protonation-Promoted Intramolecular Reductive Breakdown of Potential NRTIs: Stavudine α-P-Borano-γ-P-N-L-tryptophanyltriphosphates.

Phosphorus-modified prodrugs of dideoxynucleoside triphosphates (ddNTPs) have shown promise as pronucleotide strategies for improving antiviral activity compared to their parent dideoxynucleosides. Borane modified NTPs offer a promising choice as nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs). However, the availability of α-P-borano-γ-P-substituted NTP analogs remains limited due to challenges with synthesis and purification. Here, we report the chemical synthesis and stability of a new potential class of NRTI prodrugs: stavudine (d4T) 5'-α-P-borano-γ-P-N-L-tryptophanyltriphosphates. One-pot synthesis of these compounds was achieved via a modified cyclic trimetaphosphate approach. Pure Rp and Sp diastereomers were obtained after HPLC separation. Based on LC-MS analysis, we report degradation pathways, half-lives (5-36 days) and mechanisms arising from structural differences to generate the corresponding borano tri- and di-phosphates, and H-phosphonate, via several parallel routes in buffer at physiologically relevant pH and temperature. Here, the major hydrolysis products, d4T α-P-boranotriphosphate Rp and Sp isomers, were isolated by HPLC and identified with spectral data. We first propose that one of the major degradation products, d4T H-phosphonate, was generated from the d4T pronucleotides via a protonation-promoted intramolecular reduction followed by a second step nucleophilic attack. This report could provide valuable information for pronucleotide-based drug design in terms of selective release of target nucleotides.

Synthesis, and in vitro enzymatic and antiviral evaluation of d4T polyphosphate derivatives as chain terminators.

A series of d4T di- or triphosphate derivatives have been synthesized and evaluated as effective substrates for HIV-1 RT, and also tested for their in vitro anti-HIV activity. The steady-state kinetic study of compounds 1-4 in an enzymatic incorporation assay by HIV-1 RT follows Michaelis-Menten profile. In addition, compounds 2-4 are able to inhibit HIV-1 replication to the same extent as d4T and d4TMP in MT-4 cells, as well as in CEM/0 cells and CEM/TK(-) cells. The data suggests that these d4T polyphosphate derivatives are hydrolyzed to d4T and rephosphorylated to d4TTP before exerting their antiviral activity.

Synthesis and in vitro transdermal penetration of methoxypoly(ethylene glycol) carbonate derivatives of stavudine.

The objective of this study was to synthesize derivatives of the anti-HIV drug stavudine (d4T) with more favourable physicochemical properties for transdermal delivery in an effort to increase transdermal penetration of stavudine and thus reduce the severe side effects associated with the dose-dependent oral therapy. The synthesis, hydrolytic stability, and in vitro human skin permeation flux of a series of novel methoxypoly(ethylene glycol) (MPEG) carbonates of stavudine are reported. The carbonates were synthesized in a two-step process by coupling the MPEG promoiety of various chain lengths to C-5' of d4T. In kinetic studies the carbonates proved to be markedly stable in weakly acidic phosphate medium (pH 5.0) with half-lives ranging from 16 to 58 days. The aqueous solubility increased as the ethylene oxide chain lengthened. However, there was no significant increase in the estimated solubility in octanol. In vitro in the phosphate buffer (200 mM; pH 5.0) almost all carbonates permeate the human skin. However, the most effective penetrant, the derivative with 3 ethylene oxide units in the side chain, exhibited a flux of 26.1 nmol/cm(2)/h as compared to 59.15 nmol/cm(2)/h of the parent drug stavudine. Thus, no permeation enhancement was observed during this study.

Doubly loaded cycloSaligenyl-pronucleotides - 5,5'-Bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates).

Recently, we reported on 3,3'-bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates) (3,3'-bis-(cycloSal-d4TMPs) 4) as the first pronucleotides with a mask-to-drug ratio of 1:2 that is still a novelty in the field of pronucleotides. Here, we report on a new set of compounds of these unique type of cycloSaligenyl prodrugs 5 that bear a biaryl axis at the 5-position of the cycloSal residue. All compounds 5 showed pronounced in vitro activity against HIV-1 and HIV-2 in wild-type CEM cell cultures and better retained their antiviral activities in thymidine kinase-deficient CEM cells than the compound 4 series. Moreover, compound 5b is the first bis-(cycloSal-d4TMP) that even showed complete retention of antiviral activity in TK-deficient CEM cells. The complex hydrolysis behavior of 5 was investigated, and the proposed hydrolysis mechanism was proven by means of (31)P NMR spectroscopy and HPLC analysis.

Bis-cycloSal-d4T-monophosphates: drugs that deliver two molecules of bioactive nucleotides.

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their "monomeric" counterparts and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated via 31P NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong dependence of the pronucleotides' properties on the stereochemistry at the phosphorus centers. Both 6 and 7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in vitro. These compounds were significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells, indicating an efficient TK-bypass.

Application of the cycloSal-prodrug approach for improving the biological potential of phosphorylated biomolecules.

Pronucleotides represent a promising tool to improve the biological activity of nucleoside analogs in antiviral and cancer chemotherapy. The cycloSal-approach is one of several conceptually different pronucleotide systems. This approach can be applied to various nucleoside analogs. A salicyl alcohol as a cyclic bifunctional masking unit is used, and shown to afford a chemically driven release of the particular nucleotide from the lipophilic phosphate triester precursor molecule. A conceptual extension of the cycloSal-approach results in the design of "lock-in"-cycloSal-derivatives. The cycloSal-approach is not restricted to the delivery of bioactive nucleotides but also useful for the intracellular delivery of hexose-1-phosphates.

Synthesis, separation and anti-HIV activity of distereoisomers of N-[p-(4-bromophenyl) -2',3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester (stampidine).

The distereoisomers of stampidine (STAMP, DDE-113, HI-113, N-[p-(4-bromophenyl)-2'3'-didehydro-3'-deoxy-5'-thymidylyl]-L-alanine methyl ester, CAS 217178-62-6) were separated using two different procedures. The first method involved separation of the isomers by fractional crystallization, and the second method utilized a preparative HPLC. Both isomers were active against the HIV-1 strain HTLV(IIIB) and neither isomer was more or less active than distereoisomeric mixture of stampidine.

"Lock-in" modified cyclosal nucleotides--the second generation of cyclosal prodrugs.

A new generation of cycloSal-pronucleotides is presented. CycloSal-d4TMPs have been modified by introduction of an esterase-cleavable site in order to trap them inside cells. Hydrolysis studies in different media (PBS, CEM/0- and liver extracts) and anti-HIV evaluation of separated diastereomers revealed unexpected differences between the isomers.

A new approach to the synthesis of 4'-carbon-substituted nucleosides: development of a highly active anti-HIV agent 2', 3'-didehydro-3'-deoxy-4'-ethynylthymidine.

Oxidation of 3'-O-TBDMS-4',5-unsaturated thymidine 3 with dimethyldioxirane (DMDO) allowed the isolation of the epoxide 4. Upon reacting with organosilicon reagents in the presence of SnCl4, 4 underwent stereoselective ring opening to give 4'-alpha-allyl (6), 4'-alpha-(2-bromoallyl) (7), 4'-alpha-(cyclopenten-3-yl) (8), and 4'-alpha-cyano (9) derivatives of thymidine. Reactions of the 3'-epimer 12 with organoaluminum reagents gave 4'-alpha-methyl (13), 4'-alpha-vinyl (14), and 4'-alpha-ethynyl (15) analogues. Compounds 13-15 were transformed into corresponding 2',3'-didehydro-3'-deoxy derivatives. Evaluation of their ability to inhibit the replication of HIV in cell culture showed that 4'-ethynyl-d4T (19) is more potent and less toxic than the parent compound d4T.

Synthesis of stavudine amino acid ester prodrugs with broad-spectrum chemotherapeutic properties for the effective treatment of HIV/AIDS.

A series of prodrugs of stavudine were synthesized in an effort to enhance spectrum of chemotherapeutic properties for the effective treatment of HIV/AIDS. The 5'-OH function of stavudine was esterified with ciprofloxacin, norfloxacin, isoniazide, pyrazinamide, piperazine and dimethylamine acetic acid. The anti-HIV-1 activity of the esters was determined in CEM cell line and stavudine ester bearing piperazine acetic acid was found to be the most potent compound with a selective index of >15,723. Stavudine prodrug bearing ciprofloxacin and norfloxacin acetic acid showed 100% inhibition against Mycobacterium tuberculosis H(37)Rv at 6.25 microg/mL. The prodrugs also exhibited antibacterial activity against 24 pathogenic bacteria. In vitro hydrolysis of the various esters in human plasma indicated that these agents were relatively stable toward plasma esterases with t(1/2) ranging from 20-240 min.

Synthesis of 5-alkynylated d4T analogues as potential HIV-1 reverse transcriptase inhibitors.

A series of 2',3'-didehydro-2',3'-dideoxynucleosides substituted with an alkynylhydroxy- (6, 7, 12 and 13) and alkynylamino- (20) groups at the C-5 position were synthesized. All these five target modified nucleosides were tested for anti-human immunodeficiency virus type 1 activity in CEM-SS and MT-4 cells and unfortunately displayed no improvement in antiviral activity.

Study of different substituted cyclic and acyclic benzylpronucleotides of d4T relative to their hydrolytic stability and antiviral activity.

CycloSal-d4TMP and two different bis(benzyl) phosphate triesters of the antivirally active nucleoside analog d4T were studied with regard to their chemical hydrolysis behavior at pH 7.3, in CEM/0 cell extracts, and their anti-HIV activity. In contrast to triesters 2-4, bis-(o-AB)-d4TMP 1 was found to be chemically exquisitely stable. All compounds led to the formation of d4TMP in cell extracts and all triesters achieved the TK-bypass.

Synthesis and anti-HIV activity of [d4U]-[trovirdine analogue] and [d4T]-[trovirdine analogue] heterodimers as inhibitors of HIV-1 reverse transcriptase.

A series of eleven heterodimers containing both a nucleoside analogue (d4U, d4T) and a non-nucleoside type inhibitor (Trovirdine analogue) were synthesized and evaluated for their ability to inhibit HIV replication. Unfortunately, the (N-3)d4U-Trovirdine conjugates (9a-e) and (N-3)d4T-Trovirdine conjugates (10a-f) were found to be inactive suggesting that the two individual inhibitor compounds do not bind simultaneously in their respective sites.

Phosphoramidate derivatives of d4T as inhibitors of HIV: the effect of amino acid variation.

Phosphoramidate derivatives of the nucleoside analogue, 2',3'-dideoxy-2',3'-didehydro thymidine (d4T) have been prepared as potential membrane-soluble pro-drugs of the big-active free phosphate forms. In particular phenyl phosphates, linked via nitrogen to methyl-esterified amino acids, were studied. All compounds were fully characterised by a range of methods (high-field multinuclear NMR, mass spectrometry and high performance liquid chromatography (HPLC)) and were subjected to in vitro evaluation of their anti-HIV efficacy. The nature of the amino acid appeared to be extremely important for the eventual antiviral action. Of the amino acids studied, L-alanine was the most efficacious, whilst L-proline and glycine were particularly poor. However, an unnatural amino acid moiety, dimethylglycine, could substitute for alanine with little or no loss of activity.

[Which is the strategy? The latest in the use of antiretrovirals]. / Cual es la estrategia? Lo mas reciente en el uso de antiretrovirales.

AIDS: The four main antiretroviral drugs, or nucleoside analogs, used in AIDS treatment are AZT, ddI, ddC and d4T. AZT is generally prescribed first, adding combinations of ddC of ddI if resistance or intolerance appear. Researchers are determining, in vitro, the effects of combining three of these drugs to significantly reduce the reproduction of HIV. The combination of AZT and 3TC in studies has produced a potent antiretroviral combination, showing prolonged suppression of HIV production, and an increase in T4 cells. A new group of drugs, protease inhibitors, shows promise because protease is needed for HIV to reproduce in infected cells. In preliminary studies, these drugs reduce virus levels in blood and may be more effective and less toxic than current drugs. Saquinavir, L-524, and ABT538 are in advanced stages of development. Studies which measure their impact on T4 cells, viral reproduction, or AIDS progress are not available. Some problems include rapid development of viral resistance, limited availability, and difficulty in production of these medications.^ieng

Prodrugs of 2',3'-didehydro-3'-deoxythymidine (D4T): synthesis, antiviral activity, and rapid pharmacokinetic evaluation.

A series of 5'-derivatives and modified pyrimidine analogues of 2',3'-didehydro-3'-deoxythymidine (d4T, stavudine, 1) were synthesized to determine their potential as oral prodrugs of d4T. Utilizing a screen developed for the rapid evaluation of a variety of prodrugs in mice, it was determined that 5'-acetate 2 provided comparable plasma levels of d4T after oral administration of the prodrug to that when d4T was administered alone. The relative oral bioavailability of methoxy acetate 3 and cyclohexyl carbonate 5 was 79 and 41%, respectively. Dihydropyridine ester 6 did not provide detectable levels of d4T up to 1 h after oral administration of 6. Thiopyrimidines 8 and 9, as well as aminopyrimidine 10 also failed to provide measurable levels of d4T after oral administration. 5'-Derivatives 3, 5, and 6 showed similar activity to that of d4T against HIV and MuLV, as did 5'-benzoyl-4-thio derivative 8. However, the corresponding 4-thio 5'-alcohol 9 was inactive.