Development of Triazoles and Triazolium Salts Based on AZT and Their Anti-Viral Activity against HIV-1.

We report herein a set of 3'-azido-3'-deoxythymidine (AZT) derivatives based on triazoles and triazolium salts for HIV-1 infection. The compounds were synthesized via click chemistry with Cu(I) and Ru(II) catalysts. Triazolium salts were synthesized by reaction with methyl iodide or methyl triflate in good yields. The antiviral activity of the compounds was tested using two methodologies: In method one the activity was measured on infected cells; in method two a pre-exposure prophylaxis experimental model was employed. For method one the activity of the compounds was moderate, and in general the triazolium salts showed a decreased activity in relation to their triazole precursors. With method two the antiviral activity was higher. All compounds were able to decrease the infection, with two compounds able to clear almost all the infection, while a lower antiviral activity was noted for the triazolium salts. These results suggest that these drugs could play an important role in the development of pre-exposure prophylaxis therapies.

Evaluation of the effect of synthetic compounds derived from azidothymidine on MDA-MB-231 type breast cancer cells.

The present study aimed to investigate the effect of AZT derivates containing tellurium (Te) on human breast cancer cell lines and the mechanisms underlying cell death. The inhibitory effect of AZT and its derivatives (7m and 7r) was determined by the MTT assay (6.25, 12.5, 25, 50 and 100 µM in 24 and 48 h time points), meanwhile the induction of apoptosis and the cell cycle phases was investigated by flow cytometry. The MTT assay showed that AZT derivatives decreased the rate of cell proliferation at concentrations of 12.5 µM, while commercial AZT showed low antitumor potential. In flow cytometric analysis, we demonstrate that the AZT derivatives do not induce apoptosis at the concentration tested and promote the cell cycle arrest in the S phase. Besides, predicted absorption, distribution, metabolization, excretion and toxicity analysis suggest that the compounds possess a good pharmacokinetic profile and possibly less toxicity when compared to conventional AZT. These compounds containing tellurium in their formulation are potential therapeutic agents for breast cancer.

New phosphazine and phosphazide derivatives as multifunctional ligands targeting acetylcholinesterase and ß-Amyloid aggregation for treatment of Alzheimer's disease.

Phosphazine and phosphazide derivatives are described herein as a new class of selective and potent acetylcholinesterase (AChE) inhibitors and ß-amyloid aggregation inhibitors. Phosphazines (5-7) were synthesized smoothly via a redox-condensation reaction of 1,2-bis(diphenylphosphino)ethane with different amines derivatives in the presence of dialkyl azodicarboxylate (Staudinger reaction) while phosphazides (8) via electrophilic attack of azido derivatives. Structures of the synthesized compounds were justified on the basis of compatible elementary and spectroscopic analyses. All the compounds were evaluated for their acetylcholinesterase inhibitory activity. The most three potent compounds (5b-c and 8b) showing AChE IC50 values (29.85-34.96 nM) comparable to that of donepezil (34.42 nM) were subjected to further investigation by testing their butyrylcholinesterase, MMP-2 and self-induced Aß aggregation inhibition activity. Especially, the coumarin phosphazide derivative (8b) presented the best AChE inhibition selectivity index (IC50 = 34.96 nM, AChE/BuChE; 3.81) together with good inhibition ability against MMP-2 (IC50 = 441.33 nM) and self-induced Aß1-42 aggregation (IC50 = 337.77 nM). In addition, the inhibition of metal-induced Aß aggregation by 8b was confirmed by thioflavine T fluorescence. The most potent effect of 8b was observed on the Zn2+-induced Aß42 aggregation. Kinetic study of compound 8b suggested it to be a competitive AChE inhibitor. Also, it specifically chelates metal and is predicted to be permeable to BBB. It also possesses low toxicity on SH-SY5Y neuroblastoma cells with a safety index of 15.37. In addition, it was demonstrated that compound 8b can improve the cognitive impairment of scopolamine-induced model in mice with % alternations and transfer latency time comparable to that of donepezil. Also, a docking study was carried out and it was in accordance with the in vitro results. These promising in vitro and in vivo findings highlight compound 8b as a possible drug candidate in searching for new multifunctional AD drugs.

Synthesis and evaluation of 3-hydroxy-3-phenylpropanoate ester­AZT conjugates as potential dual-action HIV-1 Integrase and Reverse Transcriptase inhibitors.

Novel 3-hydroxy-3-phenylpropanoate ester-azidothymidine (AZT) conjugates have been prepared using Baylis-Hillman methodology, and their potential as dual-action HIV-1 Integrase and Reverse Transcriptase inhibitors has been explored using enzyme inhibition and computer modelling techniques; their activity and HeLa cell toxicity have been compared with those of their cinnamate ester analogues.

Synthesis and cytotoxic evaluation of novel ester-triazole-linked triterpenoid-AZT conjugates.

Betulinic acid and analogous naturally occurring triterpenoid acids were transformed into the corresponding propargyl esters and subsequently deployed as substrates for a click chemistry-mediated coupling with azidothymidine (AZT) en route to novel 1,2,3-triazole-tethered triterpenoid-AZT conjugates. Twelve new hybrids were thus prepared and assessed in terms of their cytotoxic activity, revealing an interesting anticancer activity of five triterpenoid-AZT hybrids on KB and Hep-G2 tumor cell lines.

Toward hyperpolarized molecular imaging of HIV: synthesis and longitudinal relaxation properties of (15) N-Azidothymidine.

Previously unreported (15) N labeled Azidothymidine (AZT) was prepared as an equimolar mixture of two isotopomers: 1-(15) N-AZT and 3-(15) N-AZT. Polarization decay of (15) N NMR signal was studied in high (9.4 T) and low (~50 mT) magnetic fields. (15) N T1 values were 45 ± 5 s (1-(15) N-AZT) and 37 ± 2 s (3-(15) N-AZT) at 9.4 T, and 140 ± 16 s (3-(15) N-AZT) at 50 mT. (15) N-AZT can be potentially (15) N hyperpolarized by several methods. These sufficiently long (15) N-AZT T1 values potentially enable hyperpolarized in vivo imaging of (15) N-AZT, because of the known favorable efficient (i.e., of the time scale shorter than the longest reported here (15) N T1 ) kinetics of uptake of injected AZT. Therefore, 3-(15) N-AZT can be potentially used for HIV molecular imaging using hyperpolarized magnetic resonance imaging.

Efficient syntheses of [¹¹C]zidovudine and its analogs by convenient one-pot palladium(0)-copper(I) co-mediated rapid C-[¹¹C]methylation.

The nucleosides zidovudine (AZT), stavudine (d4T), and telbivudine (LdT) are approved for use in the treatment of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) infections. To promote positron emission tomography (PET) imaging studies on their pharmacokinetics, pharmacodynamics, and applications in cancer diagnosis, a convenient one-pot method for Pd(0)-Cu(I) co-mediated rapid C-C coupling of [(11)C]methyl iodide with stannyl precursor was successfully established and applied to synthesize the PET tracers [(11)C]zidovudine, [(11)C]stavudine, and [(11)C]telbivudine. After HPLC purification and radiopharmaceutical formulation, the desired PET tracers were obtained with high radioactivity (6.4-7.0 GBq) and specific radioactivity (74-147 GBq/µmol) and with high chemical (>99%) and radiochemical (>99.5%) purities. This one-pot Pd(0)-Cu(I) co-mediated rapid C-[(11)C]methylation also worked well for syntheses of [methyl-(11)C]thymidine and [methyl-(11)C]4'-thiothymidine, resulting twice the radioactivity of those prepared by a previous two-pot method. The mechanism of one-pot Pd(0)-Cu(I) co-mediated rapid C-[(11)C]methylation was also discussed.

Synthesis and evaluation of coumarin derivatives as potential dual-action HIV-1 protease and reverse transcriptase inhibitors.

Baylis-Hillman-derived 3-(benzylaminomethyl)coumarins have been treated, sequentially, with chloroacetyl chloride and propargylamine to afford alkynylated coumarins as substrates for Click Chemistry reactions with azidothymidine (AZT) in the presence of a Cu(I) catalyst. The dual-action HIV-1 protease (PR) and reverse transcriptase (RT) inhibition potential of the resulting N-benzylated cycloaddition products, and a series of non-benzylated analogues, has been explored using saturation transfer difference (STD) NMR, computer modelling and enzyme inhibition techniques.

[Synthesis, properties and anti-HIV activity of novel lipophilic 3'-azido-3'-deoxythymidine conjugates containing functional phosphoric linkages].

One of the approaches to enhance bioavailability of nucleoside reverse transcriptase HIV inhibitors consists in design of their prodrugs based on 1,3-diacylglycerols, which may simulate nature lipids metabolic pathways promoting the improvement of drug delivery to the target cells. Glycerolipidic AZT conjugates with different functional phosphoric centers were synthesized by H-phosphonate technique in the present work. Study of prepared prodrugs sensibility to the chemical and enzymatic hydrolysis (in buffer solution and under the influence of pancreatic lipase) and also study of their anti-HIV activity on the T-lymphoid human MT-4 cells in regarding to virus HIV-1(899A) strain were carried out.

Diastereoselective synthesis of cyclosaligenyl-nucleosyl-phosphotriesters.

A diastereoselective synthesis of cycloSal-phosphotriesters (cycloSal=cycloSaligenyl) based on chiral auxiliaries has been developed that allows the synthesis of single diastereomers of the cycloSal-pronucleotides. In previously described synthesis routes, the cycloSal-compounds were always obtained as 1:1 diastereomeric mixtures that could be separated in only rare cases. However, it was shown that the diastereomers have different antiviral activity, toxicity, and hydrolysis stabilities. Here, first a chiral thiazoline derivative was used to prepare nonsubstituted and 5-methyl-cycloSal-phosphotriesters in 48 and ≥95% de (de=diastereomeric excess). However, this approach failed to give the important group of 3-substituted cycloSal-nucleotides. Therefore, two other chiral groups were discovered that allowed the synthesis of (R(P))- and (S(P))-3-methyl-cycloSal-phosphotriesters as well. The antiviral activity was found to be five- to 20-fold different between the two individual diastereomers, which proved the importance of this approach.

Design and synthesis of α-carboxy phosphononucleosides.

Rhodium catalyzed O-H insertion reactions employing α-diazophosphonate 20 with appropriately protected thymidine, uridine, cytosine, adenosine and guanosine derivatives leads to novel 5'-phosphononucleoside derivatives. Deprotection led to a novel series of phosphono derivatives bearing a carboxylic acid moiety adjacent to the phosphonate group with potential antiviral and/or anticancer activity. The phosphononucleosides bearing an α-carboxylic acid group are envisaged as potential diphosphate mimics. Conversion to mono- and diphosphorylated phosphononucleosides has been effected for evaluation as nucleoside triphosphate mimics. Most of the novel phosphononucleosides proved to be inactive against a variety of DNA and RNA viruses. Only the phosphono AZT derivatives 56-59 showed weak activity against HIV-1 and HIV-2.

Synthesis and anticancer activity of 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (AZT).

A series of novel N-alkyl 5'-chloromethylphosphonates of 3'-azido-3'-deoxythymidine (6-15) was synthesized by means of phosphonylation of 3'-azido-3'-deoxythymidine (4) with P-chloromethylphosphonic ditriazolide (3) followed by a reaction with the appropriate amine. The synthesized phosphonamidates 6-15 were evaluated for their cytotoxic activity in two human cancer cell lines: oral (KB) and breast (MCF-7) using the sulforhodamine B (SRB) assay. The highest activity in KB human cancer cells was displayed by phosphonamidate 8 (IC(50)=5.8 µg/mL), however, this compound was less potent than the parent AZT (IC(50)=3.1 µg/mL). Phosphonamidate 10 showed only moderate activity (IC(50)=12.1 µg/mL) whereas the other phosphonamidates proved inactive. Similarly, the highest activity in MCF-7 human cancer cells was displayed by phosphonamidate 8 (IC(50)=3.7 µg/mL) but it proved somewhat less active than AZT (IC(50)=2.6 µg/mL). Some activity was also displayed by phosphonamidate 10 (IC(50)=12.8 µg/mL) but the other phosphonamidates were found inactive. Hydrolysis studies indicate that the synthesized phosphonamidates are likely to act as prodrugs of the parent nucleoside (AZT). Transport measurements showed that the most active phosphonamidates (8 and 10) were able to permeate across the intestinal epithelium in vitro. The apparent permeability coefficients determined in Caco-2 cell monolayers indicated that these compounds could be moderately absorbed in humans.

Synthesis and preliminary evaluation of anti-HIV agent AZT prodrug.

In this research, phosphate and thiophosphate prodrugs 3a, 3b of anti-HIV agent AZT were synthesized, and their anti-HIV activities and cytotoxicities were investigated in vitro. Results showed that the prodrugs 3a and 3b with an IC50 value of 11.0 and 4.0 micromol x L(-1), respectively, were less toxic than AZT (1.0 micromol x L(-1)). Although the EC50 values of both 3a (0.04 micromol x (L(-1) and 3b (0.16 micromol x L(-1)) were lower than that of AZT (0.01 micromol x L(-1)), the therapeutic index (IC50/EC50) of prodrug 3a (275) was much higher than that of both AZT (100) and prodrug 3b (25). This indicated that the prodrug 3a merited further investigation as an anti-HIV agent.

Conjugates of betulin derivatives with AZT as potent anti-HIV agents.

Fourteen novel conjugates of 3,28-di-O-acylbetulins with AZT were prepared as anti-HIV agents, based on our previously reported potent anti-HIV triterpene leads, including 3-O-acyl and 3,28-di-O-acylbetulins. Nine of the conjugates (49-53, 55, 56, 59, and 60) exhibited potent anti-HIV activity at the submicromolar level, with EC(50) values ranging from 0.040 to 0.098muM in HIV-1(NL4-3) infected MT-4 cells. These compounds were equipotent or more potent than 3-O-(3',3'-dimethylsuccinyl)betulinic acid (2), which is currently in Phase IIb anti-AIDS clinical trial.

Potential application of thymidylate kinase in nucleoside analogue activation in Plasmodium falciparum.

Plasmodium falciparum thymidylate kinase (PfTMPK) shows a broad range of substrate tolerance when compared to the corresponding human enzyme. Besides 2'-deoxythymidine monophosphate (dTMP), PfTMPK can phosphorylate 3'-azido-2',3'-dideoxythymidine monophosphate (AZTMP) very efficiently. In contrast, human thymidylate kinase (hTMPK) is 200 times less active towards AZTMP. We were interested to see if we could use PfTMPK to activate 3'-azido-2',3'-deoxythymidine (AZT) derivatives as a strategy to treat malaria. P. falciparum lacks a pyrimidine nucleoside kinase which usually activates nucleoside and nucleoside analogues to the corresponding monophosphates. Therefore, several prodrug analogues of AZT and related nucleoside monophosphates were prepared and analysed for antiparasitic activity. The prodrugs showed an increase in activity over the parent nucleoside analogues, which showed no inhibition of parasite growth at the concentration tested. The evidence here reported provides a strategy that could be exploited for further anti-malarial design.

3'-(1,2,3-Triazol-1-yl)-3'-deoxythymidine analogs as substrates for human and Ureaplasma parvum thymidine kinase for structure-activity investigations.

The pathogenic mycoplasma Ureaplasma parvum (Up) causes opportunistic infections and relies on salvage of nucleosides for DNA synthesis and Up thymidine kinase (UpTK) provides the necessary thymidine nucleotides. The anti-HIV compound 3 -azido-3'-deoxythymidine (AZT) is a good substrate for TK. Methods for a rapid and efficient synthesis of new 3'-alpha-[1,2,3]triazol-3'-deoxythymidine analogs from AZT under Huisgen conditions are described. Thirteen 3'-analogues were tested with human cytosolic thymidine kinase (hTK1) and UpTK. The new analogs showed higher efficiencies (K(m)/V(max) values) in all cases with UpTK than with hTK1. Still, hTK1 was preferentially inhibited by 9 out of 10 tested analogs. Structural models of UpTK and hTK1 were constructed and used to explain the kinetic results. Two different binding modes of the nucleosides within the active sites of both enzymes were suggested with one predominating in the bacterial enzyme and the other in hTK1. These results will aid future development of anti-mycoplasma nucleosides.

Synthesis and evaluation of the anti-hepatitis B virus activity of 4'-Azido-thymidine analogs and 4'-Azido-2'-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent.

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4'-Azido-thymidine/4'-Azido-2'-deoxy-5-methylcytidine derivatives (6, 10-15) were synthesized, and their anti-HBV activities evaluated. Compounds 10-15 were synthesized via an SNAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11-15 showed no antiviral activity. However, 4'-Azido thymidine (6) and 4'-Azido-2'-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC50 = 0.63 and 5.99 µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100 µM.

Antibacterial AZT derivative regulates metastasis of breast cancer cells.

Antimicrobial peptides (AMP) with anticancer activity have drawn remarkable attention in modern treatments. However, long peptide length and protease instability are the most addressing factors, which hampers their further development as therapeutic agents. In view of this, herein, we designed and synthesized a series of AZT-based cationic small molecule incorporating a variety of hydrophobic groups and cationic charges, including amine and guanidine groups to mimic the amphipathic structure of AMPs. These compounds were evaluated for their antibacterial activity against Gram-positive and Gram-negative bacteria. Through an extensive structure activity relationship study (SAR), we identified ADG-2e as the most potent antibacterial agent, which exhibited remarkable potency against drug resistant bacterial strains such as MRSA and MDRPA. Further, ADG-2e was examined for their anti-metastatic ability by investigating the cancer cell migration and invasiveness through scratch wound-healing assay and transwell invasive assay, respectively. In addition, time-lapse cell tracking analysis also performed for analyzing the cell movement pattern. Treatment of ADG-2e against metastatic breast cancer cells (MDA-MB-231) suppressed tumor cell migration by multi-directional lamellipodium formation, indicating their anti-metastatic potential. Thus, our cationic AZT based small molecules may evolve as an appealing class of antibacterial agents with anti-metastasis potential.

Synthesis, characterization, and in vitro assay of folic acid conjugates of 3'-azido-3'-deoxythymidine (AZT): toward targeted AZT based anticancer therapeutics.

Conjugates of three components namely folic acid, poly(ethyleneglycol) and 3 '-azido-3 '-deoxythymidine (AZT) are presented. Folate-PEG units were coupled to AZT to facilitate delivery of the nucleoside into the cell. A convenient separation of the polydisperse PEGylated-folic acid regioisomers produced upon conjugation is described. This is to select for the active gamma-regioisomer over the inactive alpha-regioisomer. In vitro cytotoxicity assays were conducted against an ovarian cell line (A2780/AD) that overexpresses the folate receptor (FR) and compared to a FR free control cell line. Compared to AZT a approximately 20-fold greater potency against the resistant ovarian line was observed for the conjugates.

A novel anti-HIV dextrin-zidovudine conjugate improving the pharmacokinetics of zidovudine in rats.

The aim of this study was to investigate a newly synthesized dextrin-zidovudine (AZT) conjugate designed as a sustained release prodrug of AZT for parenteral administration. AZT was first reacted with succinic anhydride to form a succinoylated AZT which was subsequently coupled with dextrin to yield the dextrin-AZT conjugate. The structure of the conjugate was characterized by FT-IR and (1)H-NMR spectroscopy. The drug content of the conjugate was 18.9 wt.%. The release in vitro of free AZT and succinoylated AZT was investigated in buffer solutions at pH 5.5 and 7.4 and in human plasma. AZT and succinoylated AZT release from the conjugate was 1.4% (pH 5.5), 41.7% (pH 7.4) and 78.4% in human plasma after 24 h. Release was complete in human plasma after 48 h. A pharmacokinetic study in rats following intravenous administration of the conjugate showed prolonged plasma levels of AZT compared to free AZT. The use of the conjugate extended the plasma half-life of AZT from 1.3 to 19.3 h and the mean residence time from 0.4 to 23.6 h. Furthermore, the conjugate provided a significant greater area under the plasma concentration-time curve and reduced the systemic clearance of AZT. This study suggested the potential of this novel dextrin-AZT conjugate as a new intravenous preparation of AZT.