Synthesis and evaluation of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purines and 2-amino-9-(3-alkoxycarbonyloxymethyl-4-alkoxycarbonyloxybut-1- yl)purines as potential prodrugs of penciclovir.

A series of 2-amino-9-(3-acyloxymethyl-4-alkoxycarbonyloxybut-1-yl)purin es (1-8) and 2-amino-9-(3-alkoxycarbonyl-oxymethyl-4-alkoxycarbonyloxybut -1-yl)purines (9-12) were synthesized as potential prodrugs of penciclovir. Treatment of 6-deoxypenciclovir with trimethyl orthoacetate or triethyl orthopropionate (1.2 equiv) in DMF in the presence of p-TsOH.H2O (0.1 equiv) followed by quenching with excess H2O gave the corresponding mono-O-acetyl or mono-O-propionyl compound, 17 or 18, in excellent yields of 95 and 92%, respectively. Reactions of 17 or 18 with an appropriate alkyl (Me, Et, n-Pr, and i-Pr) 4-nitrophenyl carbonate (1.2 equiv) in pyridine in the presence of a catalytic amount of DMAP (0.1 equiv) at 80 degrees C afforded the monoacyl, monocarbonate derivatives of 6-deoxypenciclovir, 1-8, in 86 94% yields. Similar reactions of 6-deoxypenciclovir with 2.1 equiv of alkyl 4-nitrophenyl carbonate produced the dicarbonate derivatives 9 12 in 81-83% yields. Of the prodrugs tested in rats, 2-amino-9-(3-acetoxymethyl-4-isopropoxycarbonyloxybut-1-yl)purine (4) achieved the highest mean urinary recovery of penciclovir (36%), followed in order by compounds 2 (35%), 6 (35%), 7 (34%), 10 (34%), 8 (32%), 3 (32%), and famciclovir (31%). The mean urinary recovery of penciclovir and concentrations of penciclovir in the blood from 4 in mice were also slightly higher than those from famciclovir. The in vivo antiviral efficacy of 4 in HSV-1-infected normal BALB/c mice was higher than those of famciclovir and valaciclovir in terms of mortality (100, 80, and 40%) and mean survival time ( > 21, 13+/-5.0 (SEM), and 13+/-1.6 days). Compound 4 demonstrated an effective anti-hepadnaviral response with intrahepatic viral load being reduced by 90%, the viral supercoiled DNA levels reduced by 70% and Pre-S expression inhibited by 30% against duck hepatitis B virus (DHBV) in vivo, and did not cause any significant hepatotoxicity after 4 weeks of treatment.     

Phytoalexin resveratrol attenuates the mutagenicity of the heterocyclic amines 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline

Resveratrol is a phytoalexin, that belongs to a family of naturally occurring stilbenes. It has been reported that resveratrol can inhibit chemical carcinogenesis in experimental animals and although the mechanisms involved are unknown, an anti-mutagen mechanism has been proposed. We have explored this hypothesis using mutagenicity assays based on bacterial (Salmonella typhimurium) and eukaryotic cells (Chinese hamster V79 cells). We found resveratrol to be potent in both systems, blocking the mutagenicity of the food-derived heterocyclic amines (HA) 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at micromolar concentrations. Furthermore, in cells capable of activating 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine to cytotoxic derivatives, resveratrol was able to attenuate cytotoxicity. Paradoxically, in cells lacking the ability to activate PhIP, resveratrol itself was toxic and co-incubation with PhIP reduced this toxicity. Our data confirm the potent anti-mutagenic activity of resveratrol and support its potential as a chemopreventative.     

Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin.

Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.     

Synthesis and evaluation of 2-amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine esters as potential prodrugs of acyclovir

2-Amino-6-fluoro-9-(2-hydroxyethoxymethyl)purine (2) and its ester derivatives 4a–d were synthesized as potential prodrugs of acyclovir, and were evaluated for their oral acyclovir bioavailability in rats and in vivo antiviral efficacy in HSV-1-infected mice. Treatment of 2-amino-6-chloro-9-(2-hydroxyethoxymethyl)purine (3) with trimethylamine in THF/DMF (4:1) followed by a reaction of the resulting trimethylammonium chloride salt 5 with KF in DMF gave 2 in 78% yield. Esterification of 2 with an appropriate acid anhydride (Ac₂O, (EtCO)₂O, (n-PrCO)₂O, or (i-PrCO)₂O) in DMF in the presence of a catalytic amount of DMAP at room temperature produced the esters 4a–d in 90–98% yields. Of the prodrugs tested in rats, the isobutyrate 4d achieved the highest mean urinary recovery of acyclovir (51%) that is 5.7-fold higher than that of acyclovir (9%) and comparable to that of valacyclovir (50%). The prodrug 4d protected dose-dependently the mortality of HSV-1-infected mice, and the group treated with 4d at a dose of 400 mg/kg showed the longest mean survival day (14.6 ± 3.1 days) (mean ± S.D.).     

Stereoselective synthesis of 9-beta-d-arabianofuranosyl guanine and 2-amino-9-(beta-d-arabianofuranosyl)purine

9-beta-d-Arabianofuranosyl guanine (6) and 2-amino-9-(beta-d-arabianofuranosyl)purine (8) were prepared from 2-amino-6-chloro-9-(2,3,5-triphenylmethoxyl-beta-d-arabianofuranosyl)purine (4), a key intermediate which was stereoselectively prepared from 2,3,5-triphenylmethoxyl-d-arabianofuranose and 2-amino-6-chloro-purine. The yield of the intermediate was obviously improved and only beta-isomer was formed by using the activated molecular sieve as environmental friendly catalyst, overcoming the defect that a 1:1 mixture of alpha- and beta-isomers was formed, which was difficult to separate, when toxic mercury cyanide was previously used as catalyst.     

Synthesis of 9-alkyl and 9-heteroalkyl substituted 2-amino-6-guanidinopurines and their influence on the NO-production in macrophages

9-Alkyl and 9-heteroalkyl substituted derivatives of the 2-amino-6-guanidinopurine were synthesized by alkylation of 2-amino-6-chloropurine and subsequent guanidinolysis. The activity of the thus prepared compounds on murine macrophages was examined. Compounds 4a, 4b, and 4d inhibit the LPS+IFN-gamma-induced NO production in murine macrophages while compound 4h stimulates this production.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

Quantification of the carcinogens 2-amino-3,8-dimethyl- and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in food using a combined assay based on gas chromatography—negative ion mass spectrometry

A gas chromatographic—mass spectrometric assay has been developed for the measurement of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in food. Stable isotope-labelled analogues of MeIQx and PhIP are used as internal standards and the synthesis of deuterated PhIP is described. The mass spectrometer is operated in the electron-capture negative ion chemical ionisation mode and the amines are chromatographed as their di-3,5-bistrifluoromethylbenzyl derivatives. All three compounds can be measured in a single chromatographic run and detection limits of 0.05, 0.1 and 0.2 ng/g for MeIQx, DiMeIQx and PhIP, respectively, in food are obtained. Various home-cooked and commercially prepared foodstuffs were analysed with this assay and several were found to contain measurable amounts of one or more of the three amines. These results are presented and discussed.     

Synthesis and evaluation of 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine mono- and diesters as potential prodrugs of penciclovir

2-Amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (7), and its mono- and diesters 8-15 were prepared and evaluated for their potential as prodrugs of penciclovir. Treatment of 2-amino-6-chloro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (5) with trimethylamine in THF followed by a reaction of the resulting trimethylammonium chloride salt 6 with KF in DMF afforded 2-amino-6-fluoro-9-(4-hydroxy-3-hydroxymethylbut-1-yl)purine (7) in 80% yield. Esterification of 7 with an appropriate acid anhydride [Ac2O, (EtCO)2O, (n-PrCO)2O, or (i-PrCO)2O] in DMF in the presence of a catalytic amount of DMAP produced the mono-esters 8-11 in 42-45% yields and diesters 12-15 in 87-99% yields. Of the prodrugs tested in rats, the monoisobutyrate 11 was the most efficiently absorbed and metabolized to 7, showing the mean maximum total concentration of penciclovir (5.5 microg/mL) and 7 (10.8 microg/mL) in the blood was much higher than the mean maximum concentration of penciclovir (11.5 microg/mL) from famciclovir. However, the mean concentrations of penciclovir from 11 were lower than those from famciclovir because of the limited conversion of a major metabolite 7 to penciclovir by adenosine deaminase.     

Microbial transformation of 2-amino-4-methyl-3-nitropyridine

Biotransformation of the highly substituted pyridine derivative 2-amino-4-methyl-3-nitropyridine by Cunninghamella elegans ATCC 26269 yielded three products each with a molecular weight of 169?Da which were identified as 2-amino-5-hydroxy-4-methyl-3-nitropyridine, 2-amino-4-hydroxymethyl-3-nitropyridine, and 2-amino-4-methyl-3-nitropyridine-1-oxide. Biotransformation by Streptomyces antibioticus ATCC 14890 gave two different products each with a molecular weight of 169?Da; one was acid labile and converted to the other stable product under acidic conditions. The structure of the stable product was established as 2-amino-4-methyl-3-nitro-6(1H)-pyridinone, and that of the less stable product was assigned as its tautomer 2-amino-6-hydroxy-4-methyl-3-nitropyridine. Four of the five biotransformation products are new compounds. Several strains of Aspergillus also converted the same substrate to the lactam 2-amino-4-methyl-3-nitro-6(1H)-pyridinone. Microbial hydroxylation by C. elegans was found to be inhibited by sulfate ion. In order to improve the yield and productivity of the 5-hydroxylation reaction by C. elegans, critical process parameters were determined and Design of Experiments (DOE) analyses were performed. Biotransformation by C. elegans was scaled up to 15-l fermentors providing 2-amino-5-hydroxy-4-methyl-3-nitropyridine at ca. 13?% yield in multi-gram levels. A simple isolation process not requiring chromatography was developed to provide purified 2-amino-5-hydroxy-4-methyl-3-nitropyridine of excellent quality.     

Stereospecific gas chromatographic/mass spectrometric assay of the chiral labetalol metabolite 3-amino-1-phenylbutane.

We previously identified 3-amino-1-phenylbutane (APB) as an oxidative N-dealkylated, metabolite of the antihypertensive agent labetalol. Labetalol has two asymmetric centers and is used clinically as a mixture of the four possible stereoisomers; APB has one asymmetric center. We now report an enantiospecific gas chromatographic/mass spectrometric assay for APB in urine. After adding the internal standard 1-methyl-2-phenoxyethylamine and alkalinizing, the urine samples were extracted with ether. The extracts were derivatized with the optically active acid chloride prepared from (S)-alpha-methoxy-alpha-trifluoromethylphenylacetic acid. The derivatives were separated by capillary gas chromatography and detected by electron capture negative ion chemical ionization mass spectrometry with selected ion monitoring. The derivative of the R enantiomer eluted first, and the [M--32]- ions were monitored for both the drug and the internal standard. The method was linear in the 0.05-2.5 micrograms enantiomer-1 ml-1 range and had inter-assay and intra-assay coefficients of variation of less than 6%. The assay was used in the analysis of urine samples from a patient in labetalol therapy and no interference was found. Further studies are needed to elucidate the oxidative metabolism of labetalol and its stereochemical aspects.     

Fluorescent 7- and 8-methyl etheno derivatives of adenosine and 6-amino-9-ethylpurine: syntheses and fluorescence properties

Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethylpurine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and 3-chloropropyne as reagents. The structures of the products were determined by spectroscopic and spectrometric methods (1H-, 13C- and 2D NMR, MS, UV and fluorescence spectrometry). Their fluorescence properties were determined and found to be similar to those of ethenoadenosine. Also, the stabilites of 5 and 7 in aqueous solutions were determined and found to be higher than that of the etheno derivative of adenosine.     

Modified metabolism of a carcinogen, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by liver S9 from Schistosoma japonicum-infected mice.

Schistosoma japonicum infection has been associated with an increased incidence of liver and colorectal cancers in humans. To explore the mechanisms underlying this association, we investigated the carcinogen-metabolizing properties of liver S9 preparations from S. japonicum-infected mice and compared them with those of S9 from uninfected animals. When the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) was incubated with these S9s and the products were analyzed by high-performance liquid chromatography, we observed that the S9 from infected mice had a lower ability to convert Trp-P-2 into 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), an activated form of promutagenic Trp-P-2, than the S9 from uninfected mice. We found that both of these S9 preparations have a high ability to reduce Trp-P-2(NHOH) into Trp-P-2; however, the infected-mouse S9 showed a significantly greater reducing power than the control S9. This difference appears to be responsible for the observed lower mutagen-activating potential of the infected mouse S9. These results suggest that hepatic enzyme activities of S. japonicum-infected mice are quantitatively different from those of normal mice.     

Friedländer reaction on 2-amino-3-cyano-4H-pyrans: Synthesis of derivatives of 4H-pyran [2,3-b] quinoline, new tacrine analogues

The unprecedented Friedländer reaction of densely functionalized 2-amino-3-cyano-4H-pyrans (1) with cyclohexanone has afforded in one step and good yield 5-amino-4-aryl-3-ethoxycarbonyl-2-methyl-6,7,8,9-tetrahydro-4H-pyran[2,3-b]quinolines (2), novel amino-substituted fused pyran derivatives. These compounds are new tacrine analogues.     

Discovery of (E)-1-amino-4-phenylbut-3-en-2-ol derivatives as novel neuraminidase inhibitors

Neuraminidase has been considered as an important target for designing agents against influenza viruses. In a discovery of anti-influenza agents with epigoitrin as the initial lead compound, a series of 1-amino-2-alkanols were synthesized and biologically evaluated. The in vitro evaluation indicated that (E)-1-amino-4-phenylbut-3-en-2-ol (C1) had better inhibitory activities than 2-amino-1-arylethan-1-ol derivatives. To our surprise, sulfonation of C1 with 4-methoxybenzenesulfonyl chloride afforded more active inhibitor II with up to 6.4?μM IC₅₀ value against neuraminidase. Furthermore, docking of inhibitor II into the active site of NA found that the H atoms in both NH₂ and OH groups of inhibitor II were the key factors for potency. Molecular docking research did not explained very well the observed structure-activity relationship (SAR) from amino acid residue level, but also aided the discovery of (E)-1-amino-4-phenylbut-3-en-2-ol derivatives as novel and potent NA inhibitors.     

Identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors

Inhibition of the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway is one of the most intensively studied approaches to cancer therapy. Rational design led to the identification of novel 7-amino-5-methyl-1,6-naphthyridin-2(1H)-one derivatives as potent PI3K/mTOR dual inhibitors. Design, synthesis and structure activity relationship are reported.     

Assay for proteolytic activity using a new fluorogenic substrate (peptidyl-3-amino-9-ethyl-carbazole); quantitative determination of lipopolysaccharide at the level of one picogram.

A new sensitive fluorimetric assay has been developed using peptidyl-3-amino-9-ethyl-carbazole as substrate. The fluorescence intensity of free 3-amino-9-ethyl-carbazole (AEC) at 460 nm is between two and three orders of magnitude higher than the fluorescence intensity of acyl-AEC. The release of AEC from a peptidyl derivative by proteases may be monitored continuously during the hydrolysis step or may be quantified upon addition of a general inhibitor such as benzamidinium chloride. Using N-benzoyl-arginyl-AEC as substrate, as little as 1 ng trypsin may be detected. Using t-butyloxycarbonyl-Val-Leu-Gly-Arg-AEC and the amoebocyte lysate of Limulus polyphemus, as little as 1 pg lipopolysaccharide can be detected. This fluorimetric method allows detection of trace amounts of lipopolysaccharide (endotoxins) in various biological materials, including sera.     

Synthesis of a novel C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo-[3,2-d]pyrimidin-4-one (2'-deoxy-9-deazaguanosine).

A synthesis of the C-nucleoside, 2-amino-7-(2-deoxy-beta-D-erythro- pentofuranosyl)-3H,5H-pyrrolo[3,2-d]pyrimidin-4-one (9-deaza-2'-deoxyguanosine) was achieved starting from 2-amino-6-methyl-3H-pyrimidin-4-one (5) and methyl 2-deoxy-3,5-di-O-(p-nitrobenzoyl)-D-erythro-pento-furanoside (11). The anomeric configuration of the C-nucleoside was established by 1H NMR, NOEDS and ROESY. This C-nucleoside did not inhibit the growth of T-cell lymphoma cells.     

Synthesis and immunobiological activity of base substituted 2-amino-3-(purin-9-yl)propanoic acid derivatives

2-Amino-3-(purin-9-yl)propanoic acids substituted at position 6 of the purine base moiety by dimethylamino, cyclopropylamino, pyrrolidin-1-yl, hydroxy, and sulfanyl group as well as their 2-aminopurine analogues were prepared from corresponding 9-(2,2-diethoxyethyl)purines and 2-aminopurines, respectively, by the Strecker synthesis. 2-Aminopropanoic acid derivatives were tested for their immunostimulatory and immunomodulatory potency. Some of these compounds significantly enhanced secretion of chemokines RANTES and MIP-1alpha, the most potent was 2-amino-6-sulfanylpurine derivative. Most of these compounds also augmented NO biosynthesis triggered primarily by IFN-gamma.     

Synthesis,structural studies,and biological evaluation of some purine substituted 1-aminocyclopropane-1-carboxylic acids and 1-amino-1-hydroxymethylcyclopropanes

The novel purine derivatives of 1-aminocyclopropane-1-carboxylic acid (8 and 9) and 1-amino-1-hydroxymethylcyclopropane (12 and 13) with methylene spacer between the base and the cyclopropane ring were prepared by multistep synthetic route involving alkylation of adenine and 6-(N-pyrrolyl)purine with 2-hydroxy-methyl-1-aminocyclopropane-1-carboxylic acid derivative 3 as a key reaction. All novel compounds were racemic. The N-9 substitution of the purine ring and the Z-configuration of the cyclopropane ring in 4-13 were deduced from their 1H and 13C NMR spectra by analyses of chemical shifts, H-H coupling constants and connectivities in two-dimensional homo- and heteronuclear correlation spectra. An unequivocal proof of the stereostructure of 1, 4 and 5 was obtained by their X-ray structure analysis. The novel compounds were evaluated on cytostatic and antiviral activities in several cell lines. The 6-(N-pyrrolyl)purine derivative of 1,2-aminocyclopropane alcohol 12 exhibited a more pronounced inhibitory activity against the proliferation of cervical carcinoma (HeLa) and human fibroblast (WI-38) cells than other types of tumor cell lines. None of the compounds showed inhibitory activities against cytomegalovirus, varicella-zoster virus or other viruses.