Carbocyclic oxetanocins lacking the C-3' methylene.

Using the observation that the side effects of aristeromycin (carbocyclic adenosine) were reduced by removing the methylene at the center in aristeromycin where phosphorylation occurs, derivatives of carbocyclic oxetanocin A (4a), oxetanocin G (4b), and 2-aminooxetanocin A (16) lacking the 3'-methylene have been prepared in racemic form. The only viruses for which an appreciable inhibitory effect of the compounds (minimum inhibitory concentration ranging from 1 to 40 microg/mL) was noted were herpes simplex virus type 1 (HSV-1) and varicella-zoster virus (VZV). However, when directly compared for their antiviral potency against HSV-1 with their parents oxetanocin A and oxetanocin G, compounds 4a and 4b proved clearly less active.

Does the anti-hepatitis B virus activity of (+)-5'-noraristeromycin exist in its 4'-epimer and 4'-deoxygenated derivatives?

To begin an exploration of the structural parameters responsible for the activity of (+)-5'-noraristeromycin toward hepatitis B virus (HBV), three derivatives varied at the C-4' position have been prepared and evaluated. The syntheses began with a Mitsunobu coupling reaction of an appropriate cyclopentanol with 6-chloropurine. The products of these reactions were synthetically altered by standard ammonolysis and deprotection procedures to give the desired products. Evaluation of the new derivatives indicated that removal of the C-4' hydroxyl of (+)-5'-noraristeromycin increased its potency toward HBV by approximately 10-fold.

(+)-7-Deaza-5'-noraristeromycin as an anti-trypanosomal agent.

The (+)-enantiomer of 7-deaza-5'-noraristeromycin (4) has been found to show IC50 values ranging from 0.16 to 5.3 microM against four strains of African trypanosomes, one Trypanosoma brucei brucei isolate, and several clinical isolates of Trypanosoma brucei rhodesiense (agent of east African sleeping sickness), including a multidrug resistant clone of one isolate. While this compound was originally designed to inhibit S-adenosyl-L-homocysteine hydrolase, it has been found to have no effect on this enzyme.

Inhibition of cyclic nucleotide phosphodiesterases from pig coronary artery by benzo-separated analogues of 3-isobutyl-1-methylxanthine.

The linear and proximal benzo-separated analogues of 7-benzyl-3-isobutyl-1-methylxanthine, 3-isobutyl-1,8-dimethylxanthine, 3-isobutyl-8-tert-butyl-1-methylxanthine, 3-isobutyl-8-(methoxymethyl)-1-methylxanthine , and 1-isoamyl-3-isobutylxanthine have been prepared and assayed as inhibitors of the peak I and peak II forms of cyclic nucleotide phosphodiesterase from pig coronary artery. Most of the benzo analogues were less effective inhibitors of these phosphodiesterases when compared to 3-isobutyl-1-methylxanthine (IBMX) even though the active sites of both enzyme forms tolerated the stretched-out xanthines. Indeed, the linear benzo-separated analogue of 7-benzyl-IBMX was a more potent inhibitor of peak I activity than was IBMX.

Synthesis and antiviral activity of 5'-deoxypyrazofurin.

In searching for derivatives of pyrazofurin that could display antiviral properties by means that do not require C-5' phosphorylation, 5'-deoxypyrazofurin (3) has been synthesized in six steps from methyl5-deoxy-2,3-O-isopropylidene-beta-D-ribofuranoside (4). Compound 3 was evaluated for antiviral activity against a large number of viruses including herpes-, pox-, myxo-, toga-, arena-, rhabdo-, picorna-,reo-, and retroviruses. Compound 3 proved active against respiratory syncytial virus (in HeLa cells), vaccinia virus (in embryonic skin-muscle fibroblast cells), vesicular stomatitis virus (in HeLa cells), and influenza A virus (in Madin-Darby canine kidney cells) at concentrations (ranging from 4 to 20 micrograms/mL) that were nontoxic to the confluent host cell cultures.

Linear and proximal benzo-separated alkylated xanthines as adenosine-receptor antagonists.

The linear and proximal benzo-separated derivatives of 8-phenyltheophylline, 1,3-diethyl-8-phenylxanthine, 1,3-dipropylxanthine, 1,3-dibutylxanthine, 3-isobutyl-1-methylxanthine, theophylline, caffeine, and isocaffeine have been synthesized and evaluated for affinity at the A1 and A2 adenosine receptors. Although structure-activity relationships in the benzo-separated series differed from the relationships in the simple xanthines, the most potent of the benzo-separated xanthines were about equal in affinity to the most potent of the corresponding xanthines. On the basis of the present results and the diverse structures reported in the literature as non-xanthine adenosine antagonists, it appears that the primary requirement for adenosine-receptor affinity in nonnucleosides is a flat, neutral, fused-ring heterocycle and that once this requirement is met there are numerous potential binding modes.

(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.

Synthesis and anti-trypanosomal activity of various 8-aza-7-deaza-5'noraristeromycin derivatives.

A recent observation that (+)-7-deaza-5'-noraristeromycin (1), as an L-like analogue of aristeromycin, possessed meaningful anti-trypanosomal properties has prompted a search of other 7-deazapurines with similar or improved anti-trypanosomal responses. In that direction a series of pyrazolo[3,4-d]pyrimidines (that is, 8-aza-7-deaza-5'-noraristeromycin derivatives, 2-11) related to 1 have been prepared. These derivatives were evaluated against bloodstream forms of Trypanosoma brucei brucei and Trypanosoma brucei rhodesiense grown in vitro. Of these compounds, the parent L-like derivative 2 was less potent (IC50 40-70 microM) than 1 (IC50 0.165-5.3 microM) whereas the D-like analogue 3 was inactive, which is the same trend observed previously with 7-deaza-5'-noraristeromycin. Interestingly, some moderate activity (IC50 12.2-16.8 microM) was seen in the D-like 4'-methyl derivative 7 while its L-like partner was inactive.

Synthesis and xanthine oxidase inhibitory analysis of 1H-pyrrolo[3,2-c]pyridine-4,6(5H,7H)-dione (3,7-dideazaxanthine) and two of its derivatives.

The synthesis of 1H-pyrrolo[3,2-c]pyridine-4,6(5H,7H)-dione (3,7-dideazaxanthine) (1), 5-methyl-1H-pyrrolo-[3,2-c]pyridine-4,6(5H,7H)-dione (1-methyl-3,7-dideazaxanthine) (2), and 1,7-dihydropyrano[4,3-b]pyrrole-4,6-dione (1-oxa-1,3,7-trideazaxanthine) (3) has been accomplished from 3-alkoxycarbonylpyrrole-2-acetates (4, 11, and 12 for 1 and 2) and from 3-carboxypyrrole-2-acetic acid (6 for 3). Compounds 1 and 2 have been found to be weak inhibitors of the noncompetitive type for xanthine oxidase while 3 showed no inhibitory properties toward this enzyme.

Antiviral enantiomeric preference for 5'-noraristeromycin.

In order to determine if the potent antiviral properties of (+/-)-5'-noraristeromycin reside in one of its enantiomers, an analysis of each enantiomer has been carried out. A five-step route to the (+)-stereoisomer is described from (+)-(1R,4S)-4-hydroxy-2-cyclopenten-1-yl acetate, whereas the synthesis of the (-)-enantiomer had been reported previously from the same starting material. The (-)-2 and (+)-2 enantiomers were evaluated for antiviral activity against a large number of viruses and found to display an antiviral activity spectrum characteristic of (S)-adenosyl-L-homocysteine hydrolase inhibitors. The (-)-enantiomer retained the significant anticytomegalovirus properties previously reported for the racemic 2 and was, on the average, 10-fold more potent than (+)-2 in inhibiting virus replication, tumor cell growth, and (S)-adenosyl-L-homocysteine hydrolase activity.

An epimer of 5'-noraristeromycin and its antiviral properties.

A derivative of 5'-noraristeromycin epimeric at the 5'-nor center ((-)-3) has been prepared enantiospecifically in three steps from (+)-((1R,4S)-4-hydroxy-2-cyclopenten- 1-yl acetate. Compound (-)-3 was evaluated for antiviral activity against a large number of viruses and found to display marked activity against varicella-zoster virus, vaccinia virus, vesicular stomatitis virus, parainfluenza virus, reovirus, and cytomegalovirus. A similar antiviral activity spectrum was shown by the S-adenosylhomocysteine hydrolase inhibitors neplanocin A and carbocyclic 3-deazaadenosine. While equally potent as neplanocin A against most of the viruses tested, compound (-)-3 was significantly less cytotoxic. The results of this study suggest that (-)-3 should be pursued for the treatment of those virus infections [that is, pox (VV), rhabdo (VSV), paramyxo (parainfluenza), and reo] that appear to be exquisitively sensitive to the compound.

Synthesis and antiviral properties of (+/-)-5'-noraristeromycin and related purine carbocyclic nucleosides. A new lead for anti-human cytomegalovirus agent design.

(+/-)-5'-Noraristeromycin (3) has been prepared in three steps beginning with the 2,3-O-isopropylidene derivative of (+/-)-(1 alpha, 2 beta, 3 beta, 4 alpha)-4-amino-1,2,3-cyclopentanetriol (7). Also prepared from the same starting material were the related hypoxanthine (4), guanine (5), and 2,6-diaminopurine (6) analogues. Compounds 3-6 were evaluated for antiviral activity against a large number of viruses with marked activity being observed for 3 towards vaccinia virus, human cytomegalovirus, vesicular stomatitis virus, parainfluenza (type 3) virus, measles virus, respiratory syncytial virus, reovirus (type 1), and the arenaviruses Junin and Tacaribe. None of the compounds showed cytotoxicity to the host cell monolayers used in the antiviral studies. Both 3 and 6 have been found to be inhibitors of S-adenosyl-L-homocysteine hydrolase (AdoHcy hydrolase), which likely accounts for their antiviral activity. Inhibition of AdoHcy hydrolase represents a new approach to human cytomegalovirus drug design that should be pursued. Also, the activity of 3 should be further scrutinized for the treatment of pox-, rhabdo-, paramyxo-, reo-, and arenavirus infections.

3-deaza- and 7-deaza-5'-noraristeromycin and their antiviral properties.

An enantiospecific synthesis of 3-deaza-5'-noraristeromycin as its dihydrochloride ((-)-6) has been accomplished in six steps beginning with the reaction of (+)-(1R,4S)-4-hydroxy-2-cyclopenten-1-yl acetate with 4-chloro-1H-imidazo[4,5-c]pyridine. The preparation of 7-deaza-5'-noraristeromycin ((-)-7) was described previously. Compounds (-)-6 and (-)-7 were evaluated for antiviral activity against a large number of viruses. Compound (-)-6 produced an antiviral activity pattern similar to 5'-noraristeromycin but was less potent. Compound (-)-6 inhibited CEM cell proliferation at a 50% inhibitory concentration of 27 micrograms/mL but proved not inhibitory to HEL cell proliferation and not toxic to E6SM, HeLa, Vero, and MDCK cells at concentrations up to 200 micrograms/mL. While (-)-6 showed inhibition of S-adenosyl-L-homocysteine (AdoHcy) hydrolase, it was less inhibitory than 5'-noraristeromycin. Compound (-)-7 displayed no antiviral properties or inhibitory effects toward AdoHcy hydrolase.

Biological activity and a modified synthesis of 8-amino-3-beta-D-ribofuranosyl-1,2,4-triazolo[4,3-a]pyrazine, an isomer of formycin.

A two-step synthesis of 8-amino-3-beta-D-ribofuranosyl-1,2,4-triazolo[4,3-a]pyrazine (3), which is an isomer of formycin that resembles 3-deazaadenosine, is reported. Compound 3 is also described as as being a very poor substrate for adenosine deaminase and to be both a competitive and an irreversible inhibitor of S-adenosylhomocysteinase in the synthesis direction. L1210 cell growth in culture was inhibited by 3. Compound 3 was not converted to the nucleotide level in erythrocytes but was found to inhibit both the cellular uptake of nucleic acid precursors and their incorporation into the nucleic acids of L1210 cells. Finally, 3 was found to be a weak antiviral agent and coronary vasodilator.

Synthesis and biological evaluation of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine).

The synthesis of 6-amino-1H-pyrrolo[3,2-c]pyridin-4(5H)-one (3,7-dideazaguanine, 2) has been accomplished from 3-(ethoxycarbonyl)pyrrole-2-acetonitrile. In contrast to 3-deazaguanine, compound 2 did not show any antitumor, antiviral, or antibacterial properties. Furthermore, it was not a substrate for hypoxanthine-guanine phosphoribosyltransferase or purine nucleoside phosphorylase.

Search for new purine- and ribose-modified adenosine analogues as selective agonists and antagonists at adenosine receptors.

The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2'-, 3'-, and 5'-deoxy; 2'- and 3'- O-methyl; 2'-deoxy 2'-fluoro; 6'-thio; 5'-uronamide; carbocyclic; 4'- or 3'-methyl; and inversion of configuration). (-)- and (+)-5'-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6'-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(beta-L-2'-deoxy-6'- thiolyxofuranoside) displayed a Ki value of 8 microM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropyl-xanthine). 2-Chloro-2'-deoxyadenosine and 2-chloroadenin-9-yl(beta-D-6'-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 microM, respectively. The A2a selective agonist 2-(1-hexynyl)-5'-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4'-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4'-methyladenosine-5'-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5'-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.

Synthesis and antiviral properties of carbocyclic 3'-oxa-2',3'-dideoxyguanosine and its 7-deazaguanosine analogue.

To evaluate analogues of the antiviral agent (R)-9-(3,4-dihydroxybutyl)guanine in which the side-chain C-3 hydroxyl oxygen is part of a five-membered ring, carbocyclic 3'-oxa-2',3'-dideoxyguanosine (4) and carbocyclic 3'-oxa-2',3'-dideoxy-7-deazaguanosine (5) have been synthesized in 17 and 14 steps, respectively, from 5-O-acetyl-1,2-O-isopropylidene-alpha-D-xylofuranose. Compounds 4 and 5 and their 6-chloro precursors were evaluated against a wide variety of DNA and RNA viruses. Only 4 showed any marginal activity and this was limited to HSV-1 and HSV-2. Even though 4 was less potent towards these latter two viruses than acyclovir, its mechanism and target of action is proposed to resemble that of acyclovir. The only toxicity observed for these compounds was observed in the cell growth assay with human embryonic lung cells.

Structural aspects of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of L1210 cell growth and ribonucleotide reductase activity.

Previous studies have shown that N-hydroxy-N'-aminoguanidine (HAG) derivatives [RCH = NNHC(= NH)NHOH-tosylate] inhibit ribonucleotide reductase activity and block the growth of leukemia L1210 cells and human colon carcinoma, HT-29, cells in culture. In the current studies, the role of the side chains and the location of the bond of the side chain moiety to HAG were investigated using a new series of HAG derivatives which contained as the R-group--cyclohexyl, phenyl-, pyridyl- or napthyl moieties. The effects of these compounds as inhibitors of L1210 cell growth and ribonucleotide reductase activity were compared with the parent compound. N-hydroxy-N'-aminoguanidine was less inhibitory to ribonucleotide reductase activity and L1210 cell growth than hydroxyurea. The phenyl-HAG compounds which included 1-benzyloxybenzylidene- and 4-cyclohexylmethoxybenzylidene-HAG inhibited CDP reductase with IC50s which ranged from 50-110 microM. 1-Naphthylmethylene-HAG was more inhibitory than 2-naphthylmethylene-HAG and more inhibitory than the phenyl-HAG compounds. 2-Pyridylmethylene-HAG was more inhibitory than 3-pyridylmethylene- or 4-pyridylmethylene-HAG. While HAG inhibited CDP and ADP reductase activities essentially to the same extent, the HAG-derivatives inhibited ADP reductase activity to a greater extent than CDP reductase activity. Cyclohexylmethylene-HAG did not inhibit either L1210 cell growth or ribonucleotide reductase activity. There was good correlation between the inhibition of ribonucleotide reductase activity and L1210 cell growth by these HAG-derivatives. These data indicate that not only is the nature of the side chain substitution important, but also the location of the HAG-moiety on the ring position.

5'-Nor carbocyclic 5'-deoxy-5'-(isobutylthio)adenosine and a 2',3'-dideoxy-2',3'-didehydro derivative.

The inhibition of biochemical processes requiring S-adenosylmethionine as a co-factor have led to many nucleoside-based medicinal agents. Included in this group are 5'-deoxy-5'-(isobutylthio)adenosine (SIBA), a nucleoside with antiparasitic, antiviral and antiproliferative effects, and 5'-noraristeromycin, a carbocyclic-derived nucleoside with potent antiviral properties. This report brings together the structural components of these two compounds by describing both enantiomers of carbocyclic 5-nor SIBA (3 and 4). Owing to the recent interest in 2',3'-dideoxy-2',3'-didehydro nucleosides as antiviral agents, this derivative of 3 (5) is also described. All three compounds were screened against a variety of viruses and were found to be inactive at high concentrations or at limiting concentrations for the screening methods. The viruses subjected to 3-5 were herpes simplex virus types 1 and 2, human cytomegalovirus, vaccinia virus, vesicular stomatitis virus, respiratory syncytial virus, varicelIa zoster virus, coxsackie virus, parainfluenza-3 virus, sindbis virus, punta toro virus, reovirus-1, human immunodeficiency virus, influenza virus types A and B, adenovirus type 1 and measles virus. These results suggest that the C-5' methylene of the C-5' thio-based carbocyclic nucleosides is important for their antiviral properties.

Inhibition of measles virus replication by 5'-nor carbocyclic adenosine analogues.

Despite intense efforts to increase vaccine coverage, measles virus (MV) still causes significant morbidity and mortality in the world, sometimes as the result of severe, chronic, lethal disease. In an effort to develop therapies to supplement immunization strategies, a number of 5'-nor carbocyclic adenosine analogues were evaluated for anti-MV activity in CV-1 monkey kidney cells. Of those compounds tested, those either unsubstituted at C4 or possessing a hydroxyl, azido or amino substituent at that position were the most active, with particularly significant inhibition of MV, strain Chicago-1. The EC50 values against this strain ranged from <0.1 to 1 mg/ml, as determined by cytopathic effect reduction assay, and confirmed by neutral red uptake. By virus yield reduction assay (+)-(1S,25,3R,4S)-4-(6'-amino-9'H-purin-9'-yl)cyclopentane-1,2,3-triol (2) (-)-(1R,2S,3R)-1-(6'-amino-9'H-purin-9'-yl)-2,3-dihydoxycyclopent-4-ene (3) (-)-(1R,2S,3R)-1-(6'-amino-9'H-purin-9'-yl)cyclopentane-2,3-dihydoxycyclopentane (5) and (-)-(1R,2R,3R,4S)-4-amino-1-(6'-amino-9'H-purin-9'-yl)cyclopentane-2,3-diol (8) were the most potent compounds tested, all with EC90 values of < or =0.4 mg/ml. Compounds 3 and 5 were also tested against other MV strains, and similarly inhibited those strains except for four designated as Bil, Edmonston, SA and X-1108. Compound 8 did not potently inhibit these other MV strains. In addition, 3, 5 and 8 demonstrated synergistic (additive) inhibition of MV replication in combination with ribavirin at several concentrations. Compounds 3, 5 and 8 were also potent MV inhibitors even when added to infected cells 24 h after virus exposure. None of these three compounds was virucidal at concentrations that inhibited viral replication as determined by virus yield reduction assay. Most compounds tested were also not toxic at concentrations >100 mg/ml in actively growing and stationary-phase cells. Results suggest that these compounds may be clinically useful anti-MV virus agents.