Novel and Convenient Approach to Synthesis of AZT／d4T H-phosphonates

A convenient, efficient and general method has been developed for synthesis of H-phosphonate mono and diesters of AZT and d4T through one-pot reaction of phosphonic acid with AZT or d4T and different alcohols using pivaloyl chloride as condensing agent under mild conditions.     

One-Pot Synthesis of 5′-Diaryl Esters and Diamidates of Phosphate,Phosphorothioate, and Phosphoroselenoate Derivatives of AZT and d4T

Modified nucleosides such as 3′-azido-2′,3′-dideoxythymide (AZT) and 2′,3′-dideoxy-2′,3′-didehydrothymide (d4T) are of importance as antiviral agents and in other medicinal applications. Consequently, there is interest in new analogs of these compounds and in new methods for preparing them. In this article, a simple but efficient, one-pot synthesis of 5′-diaryl esters and diamidates of phosphate, phosphorothioate, and phosphoroselenoate derivatives of AZT and d4T is described. The reaction of AZT/d4T with phosphorus trichloride, followed with phenols, aniline, or amino acid methyl esters in the presence of a base, led to the corresponding tricoordinated phosphite compounds of AZT/d4T, which were then oxidized with 3-chloroperoxybenzoic acid (m-CPBA), sulfur, or selenium powder to finally afford the corresponding target compounds in good yield.     

含长碳烷基核苷氢亚磷酸酯衍生物的合成与表征

通过将抗病毒药物核苷(3'-叠氮-2',3'-二脱氧胸苷AZT或2',3'-二脱氢-2',3'-二脱氧胸苷d4T)与三氯化磷反应, 然后在不同的醇解试剂作用下, 一锅法合成得到6个含长链烷基的核苷氢亚磷酸二酯衍生物, 并采用³¹P NMR, ¹H NMR, ¹³C NMR和ESI-MS对其结构进行了表征.     

An H-phosphonate strategy for the synthesis of 2',3'-dideoxynucleoside triphosphates and homodinucleotides

The triphosphates and homodinucleotides of AZT and d4T have been efficiently synthesized from the corresponding nucleoside 50-H-phosphonate monoesters via the reactive pyridinium phosphoramidate intermediates.     

Synthesis and electrospray ionization mass spectra of AZT/d4T boranophosphates

New anti-HIV prodrugs, conjugates of AZT and d4T with boranophosphates, were prepared by the H-phosphonate method. Their structures were determined by negative ion electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). The fragmentation pathways were investigated, and most of the fragment ions contained the boranophosphate or phosphinate group.     

PHOTOCHEMICAL SENSITIZATION BY AZATHIOPRINE AND ITS METABOLITES—II. AZATHIOPRINE AND NITROIMIDAZOLE METABOLITES

Abstract— The photochemical reactivity of the immunosuppressant drug, azathioprine (AZT) and two representative metabolites of the nitroimidazole moiety, 1-methyl-4-nitroimidazole (MNI) and lmethyl-4-nitro-5-thioimidazole (MNTI) has been examined. No transient species of AZT were observed by laser flash photolysis, inferring a low triplet yield or its rapid deactivation. Consequently AZT, MNI and MNTI displayed little or no photodynamic sensitization via photooxidation reactions. However, AZT and MNI are highly reactive radical scavengers when photoexcited, as determined by reaction with polyacrylamide radicals. The photoreactivity of the metabolites of AZT is greater than that of the parent drug. an observation which may be relevant to the photocarcinogenicity of AZT.     

Syntheses and antitumor and anti-aids activities of polymers containing pyrimidine derivative

The new monomer, 5'-O-methacryloyl-3'-azido-3'-deoxy-thymidine (MAZT), was synthesized from methacryloyl chloride (MAC) and 3'-azido-3'-deoxythymidine (AZT). Poly(MAZT), poly(MAZT-co-AA) and terpoly(MAZT-FUR-MAH) were synthesized by radical polymerizations. The average molecular weights of synthesized polymers were in the range of 8,800 ∼23,000 depending on polymers. The in vivo antitumor activities of polymers at 800mg/kg were increased in the following order: terpoly(MAZT-FUR-MAH) >x poly(MAZT-co-AA) > poly(MAZT) > AZT > 5-FU. The in vitro anti-HIV activities of synthesized polymers were less effective than those of AZT and D4T. But the cytotoxicities of the polymers on the MT-4 cell line were found to be much less toxic than AZT.     

Thio Nucleoside Derivatives as Intermediates or Target Compounds in the Attempt of Finding New Agents Against HIV

Abstract

3′-Alkylthio-2′-,3′-dideoxy nucleosides and D4T are synthesized. An attempt of synthesizing a hybrid between AZT and HEPT is presented.     

Synthesis and biological evaluation of 5′-O-dicarboxylic fatty acyl monoester derivatives of anti-HIV nucleoside reverse transcriptase inhibitors

A number of 5′-O-dicarboxylic fatty acyl monoester derivatives of 3′-azido-3′-deoxythymidine (zidovudine, AZT), 2′,3′-didehydro-2′,3′-dideoxythymidine (stavudine, d4T), and 3′-fluoro-3′-deoxythymidine (alovudine, FLT) were synthesized to improve the lipophilicity and potentially the cellular delivery of parent polar 2′,3′-dideoxynucleoside (ddN) analogs. The compounds were evaluated for their anti-HIV activity. Three different fatty acids with varying chain length of suberic acid (octanedioic acid), sebacic acid (decanedioic acid), and dodecanedioic acid were used for the conjugation with the nucleosides. The compounds were evaluated for anti-HIV activity and cytotoxicity. All dicarboxylic ester conjugates of nucleosides exhibited significantly higher anti-HIV activity than that of the corresponding parent nucleoside analogs. Among all the tested conjugates, 5′-O-suberate derivative of AZT (EC₅₀ = 0.10 nM) was found to be the most potent compound and showed 80-fold higher anti-HIV activity than AZT without any significant toxicity (TC₅₀ >500 nM).     

Simultaneous analysis of azidothymidine and its mono-, di- and triphosphate derivatives in biological fluids, tissue and cultured cells by a rapid high-performance liquid chromatographic method.

A rapid high-performance liquid chromatographic (HPLC) method for the simultaneous analysis of the antiviral drug azidothymidine (AZT), AZT monophosphate, AZT diphosphate and AZT triphosphate, with ultraviolet detection in the nanomolar range, is described. Determination of these compounds in vitro in the human MT-4 lymphocyte cell line did not require a prior extraction, and AZT and its phosphorylated derivatives could be accurately analysed in one HPLC run. However, plasma, bile, liver homogenate and urine samples could not be injected directly into the chromatograph. Therefore, a solid-phase extraction procedure was developed, using azidodideoxyinosine as internal standard. The extractions of the compounds of interest from all but urine samples were reproducible, with recoveries between 65% (AZT triphosphate from plasma) and 100% (AZT from plasma).     

The Use of Zidovudine Pharmacophore in Multi-Target-Directed Ligands for AIDS Therapy

The concept of polypharmacology embraces multiple drugs combined in a therapeutic regimen (drug combination or cocktail), fixed dose combinations (FDCs), and a single drug that binds to different targets (multi-target drug). A polypharmacology approach is widely applied in the treatment of acquired immunodeficiency syndrome (AIDS), providing life-saving therapies for millions of people living with HIV. Despite the success in viral load suppression and patient survival of combined antiretroviral therapy (cART), the development of new drugs has become imperative, owing to the emergence of resistant strains and poor adherence to cART. 3′-azido-2′,3′-dideoxythymidine, also known as azidothymidine or zidovudine (AZT), is a widely applied starting scaffold in the search for new compounds, due to its good antiretroviral activity. Through the medicinal chemistry tool of molecular hybridization, AZT has been included in the structure of several compounds allowing for the development of multi-target-directed ligands (MTDLs) as antiretrovirals. This review aims to systematically explore and critically discuss AZT-based compounds as potential MTDLs for the treatment of AIDS. The review findings allowed us to conclude that: (i) AZT hybrids are still worth exploring, as they may provide highly active compounds targeting different steps of the HIV-1 replication cycle; (ii) AZT is a good starting point for the preparation of co-drugs with enhanced cell permeability.     

Characterization of novel nucleoside analogs by electrospray ionization mass spectra

In this paper, we synthesized a novel nucleoside analog by coupling thymine with dimethyl dicarboxylate biphenyl (DDB). The structure of the target compound was determined using 1H nuclear magnetic resonance (NMR) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). The fragmentation pathways were studied in details through ESI-MS/MS. By comparing with unsubstituted nucleosides, such as AZT, MCI, d4T and DDI, it was found that the nucleoside analog coupled with DDB would not yield the daughter ions corresponding to the fragments of the nucleoside base and arabinofuranose analogs, but would lose a neutral molecule HF and DDB easily. However, the unsubstituted nucleosides could lightly yield the fragment ions of the nucleoside base and sugar ring. Hence, electrospray ionization mass spectrometry combined with tandem mass spectrometry (MS/MS) provides a convenient method to recognize the substituted and unsubstituted nucleosides.     

Analysis of 3'-azido-3'-deoxythymidine levels in tissues and milk by isocratic high-performance liquid chromatography

A sensitive high-performance liquid chromatographic (HPLC) assay was established to analyze levels of the antiretroviral agent 3'-azido-3'-deoxythymidine (AZT, zidovudine) in serum, milk and tissue extracts. After methanol precipitation, serum samples could be injected directly into the HPLC apparatus, whereas tissue extracts required further clarification. Recovery of AZT was virtually complete. Isocratic elution with a mobile phase consisting of 6% acetonitrile and 0.1 M ammonium acetate, pH adjusted to 4.5 with glacial acetic acid, resulted in good resolution of AZT and its metabolites; retention times for AZT and the internal standard, p-nitrophenol, were 20 and 37 min, respectively. Using this method, we have demonstrated that AZT crosses both the blood-brain and placental barriers and is excreted into milk at high levels.     

Determination of Azidothymidine – an Antiproliferative and Virostatic Drug by Square‐Wave Voltammetry

The 3′‐azido‐3′‐deoxythymidine (AZT, Zidovudine) is an antiproliferative and virostatic drug widely used in human immunodeficiency virus type 1 (HIV‐1) infection treatment. With respect to side effects of high doses and a short half‐life of AZT, a fast and simple detection method for this agent could be helpful. The aim of our study was to determine AZT levels in natural samples (urine, serum, whole blood, and cell cultures, such as the HaCaT line of keratinocytes) without their mineralization and/or purification, by means of electrochemical methods using hanging mercury drop electrode (HMDE). On this electrode, AZT undergoes irreversible reduction at the peak potential near E_p?1.1 V (vs. Ag/AgCl/3 M KCl). Reduction AZT signals were measured by cyclic voltammetry (CV), differential pulse voltammetry (DPV), square‐wave voltammetry (SWV), and constant current chronopotentiometric stripping analysis (CPSA). In phosphate buffer (pH 8) the SWV yielded the best AZT signal with the detection limit of 1 nM. The determination of AZT concentration in biological materials is affected by electroactive components, such as proteins and DNA. For monitoring the influence of these compounds, AZT reduction was performed in the presence of 10 μg/mL calf thymus ssDNA and/or 100 μg/mL bovine serum albumin. In these cases, the detection limit increased to 0.25 μM. Also studied was the AZT concentration in keratinocyte cells (HaCaT line) during cell cultivation. It has been shown that the SWV may be considered as a useful tool for the determination of AZT concentration in cell cultures, and for monitoring AZT pharmacokinetics.     

Quantitative thin-layer chromatographic method of analysis of azithromycin in pure and capsule forms

A validated stability-indicating thin-layer chromatographic (TLC) method of the analysis of azithromycin (AZT) in bulk and capsule forms is developed. Both AZT potential impurity and degradation products can be selectively and accurately estimated in both raw material and product onto one precoated silica-gel TLC plate 60F254. The development system used is n-hexane-ethyl acetate-diethylamine (75:25:10, v/v/v). The separated bands are detected as brown to brownish red spots after spraying with modified Dragendorff's solution. The Rf values of AZT, azaerythromycin A, and the three degradation products are 0.54, 0.35, 0.40, 0.20, and 0.12, respectively. The optical densities of the separated spots are found to be linear in proportion to the amount used. The stress testing of AZT shows that azaerythromycin A is the major impurity and degradation product, accompanied by three other unknown degradation products. The stability of AZT is studied under accelerated conditions in order to provide a rapid indication of differences that might result from a change in the manufacturing process or source of the sample. The forced degradation conditions include the effect of heat, moisture, light, acid-base hydrolysis, sonication, and oxidation. The compatibility of AZT with the excipients used is also studied in the presence and absence of moisture. The amounts of AZT and azaerythromycin A are calculated from the corresponding linear calibration curve; however, the amounts of any other generated or detected unknown impurities are calculated as if it were AZT. This method shows enough selectivity, sensitivity, accuracy, precision, linearity-range, and robustness to satisfy Federal Drug Administration/International Conference of Harmonization regulatory requirements. The method developed can also be used for the purity testing of AZT raw material and capsules, content uniformity testing, dissolution testing, and stability testing of AZT capsules. The potential impurity profiles of both active AZT material and capsule forms are found comparable. The linear range of AZT is between 5 and 30 mcg/spot with a limit of quantitation of 2 mcg/spot. The intraassay relative standard deviation percentage is not more than 0.54%, and the day-to-day variation is not more than 0.86%, calculated on the amounts of AZT RS recovered using different TLC plates.     

d(C4)相关序列形成四分子i-Motif结构的电喷雾质谱研究

设计了与富含胞嘧啶(C)的DNA序列d(C4)相关的DNA序列d(C4), d(TC4), d(AC4), d(T2C4), d(A2C4), d(C4T), d(C4A)和d(TC4T); 采用电喷雾质谱测定发现这些序列形成四分子非共价复合物离子, 根据离子的相对丰度可确定形成四链i-motif结构的数量和可能性; 同时考察了腺嘌呤(A)和胸腺嘧啶(T)在d(C4)序列的5'和3'端对其形成四分子i-motif结构的影响. 结果表明, 在d(C4)的5'端增加A碱基或T碱基更易形成四分子复合物; 5'端含T碱基比含A碱基更利于形成四分子复合物; 而在d(C4)序列中增加2个A碱基或T碱基比增加相应的单个碱基形成了更高丰度的四分子离子峰.