Morphology of the Vasculature and Blood Supply of the Brown Adipose Tissue Examined in an Animal Model by Micro-CT.

We performed micro-CT imaging of the vascular blood supply in the interscapular area of the brown adipose tissue in three mice with the use of intravascular contrast agent Aurovist™. Resulting 3D data rendering was then adapted into 2D resolution with visualization using false color system and grayscale images. These were then studied for the automatic quantification of the blood vessel density within this area. We found the highest most occurring density within arterioles or venules representing smaller blood vessels whereas with the increase of the vessel diameters a lower percentage rate of their presence was observed in the sample. Our study shows that micro-CT scanning in combination with Aurovist™ contrast is suitable for anatomical studies of interscapular area of brown adipose tissue blood vessel supply.

In vitro biological evaluation of the R and S isomers of 1-(Tetrahydrofuran-2-yl)-5-fluorouracil.

The S isomer of Ftorafur was synthesized and the ability of the latter to inhibit growth of cultured human fibroblasts was determined relative to both the R isomer and the racemic mixture (Ftorafur) that is presently used clinically. No significant difference in the cytotoxic effects or the relative abilities to prevent an increase in cell numbers was observed with the three forms. Inhibition of DNA synthesis in murine L1210 leukemia cells by either isomer was observed only after prolonged (18-hr) exposure. The data suggest that Ftorafur is a repository form of 5-fluorouracil and that activity is manifested equally by both isomers.

2'(3')-O-L-(O-methyl-3-phenyllactyl) adenosine. A potential analog of 2'(3')-O-aminoacyl ribonucleosides.

The title compound and the corresponding diacyl derivative has been prepared by the reaction of 5'-O-(4-methoxytrityl)adenosine with L-(O-methyl-3-phenyl)lactic acid in the presence of dicyclohexylcarbodiimide in pyridine and subsequent detritylation in 80% acetic acid. Neither compound accepted the N-acetyl-L-phenylalanyl residue from N-acetyl-L-phenylalanyl-tRNA in an Escherichia coli ribosomal system nor did either inhibit the puromycin reaction in the same system.

2'(3')-O-L-Phenylalanyl derivatives of N2,5'-anhydroformycin and N4,5'-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base.

The 2'(3')-O-L-phenylalanyl-N2,5'-anhydroformycin (1c) and 2'(3')-O-L-phenylalanyl-N4,5'-anhydroformycin (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 x 10(-4) M determined by the release of N-Ac-Phe residue from the 70 S ribosome-poly(U)-N-Ac-[14C]Phe-tRNA complex. The reaction product, 2'(3')-O-(N-acetyl)-L-phenylalanyl-L-phenylalanyl-N2,5'-anhydroformyc in (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 x 10(-4) M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart. Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.

Chemical aminoacylation of transfer ribonucleic acid. The reaction of Escherichia coli tRNA with ethyl N-benzyloxycarbonylorthoglycinate.

The alpha-carbethoxypentadecyltrimethylammonium (Septonex) salt of tRNA (Ib) was condensed with ethyl N-benzyloxycarbonylorthoglycinate (II) in dimethylformamide in vacuo and in the presence of H3PO4 as catalyst. Pancreatic RNAase degradation and phenylalanine acceptor activity showed a 55--60% conversion to the 2',3'-cyclic orthoglycinate derivative of tRNA (IIIb). The orthoester grouping of IIIb was quantitatively hydrolyzed in 80% formic acid at 0 degrees C for 15 min to give 2'(3')-O-(N-benzyloxycarbonyl)glycyl tRNA (IVb). The latter was stripped at pH 8.8 to give tRNA whose behavior on DEAE cellulose column and gel electrophoresis was similar to that of starting tRNA. The phenylalanine acceptor activity amounted to almost 80% of the starting tRNA.

Enantioselectivity of the antiviral effects of nucleoside analogues.

Natural D-nucleosides are no longer the sole basis for designing effective antiviral analogues. Many antivirals with an opposite (L) configuration were reported, with lamivudine being the most notable example. In contrast, carbocyclic nucleoside analogues are significantly more enantioselective, and enantiomers with a configuration corresponding to D-nucleosides are usually favored. In the series of acyclic nucleoside analogues, the antiviral potency resides in a single enantiomer. Allenic analogues with an axial dissymmetry are R-enantioselective, in contrast to structurally similar methylenecyclopropanes, where the enantioselectivity strongly depends on the type of virus. Enantioselectivity of acyclic nucleotide analogues exhibits a more complex pattern. The overall enantioselectivity of the antiviral effects is determined by responses of activating (phosphorylating) enzymes, as well as target DNA polymerases (reverse transcriptase), toward enantiomers of active analogues.

Synthesis and biological properties of 9-(trans-4-hydroxy-2-buten-1-yl)adenine and guanine: open-chain analogues of neplanocin A.

Alkylation of adenine (5a) or 2-amino-6-chloropurine (5b) with excess trans-1,4-dichloro-2-butene (4), effected by K2CO3 in dimethyl sulfoxide or tetra-n-butylammonium fluoride in tetrahydrofuran, led in 90-95% regioselectivity to 9-alkylpurines 6a and 6b. The title compounds 2a and 2b were obtained by refluxing intermediates 6a and 6b in 0.1 M NaOH or HCl. Adenine derivative 2a is a substrate for adenosine deaminase whereas both 2a and 2b exhibit 50% inhibition of the growth of murine leukemia L 1210 cell culture at 1 mM concentration.

Analogues of 2'(3')-O-L-phenylalanyladenosine as substrates and inhibitors of ribosomal peptidyltransferase.

The chemical syntheses of 2'(3')-O-(L-3-amino-3-phenylpropionyl)adenosine (2e), the corresponding D stereoisomer 2f, 2'(3')-O-(DL-phenylglycyl)adenosine (2g), 2'(3')-O-(N-benzylglycyl)adenosine (2h), and 9(2-O-L-phenylalanyl-beta-D-xylofuranosyl)adenine (3b) are described. Compounds 2e-h were obtained by acylation of 5'-O-(4-methoxytrityl)adenosine with the appropriate N-benzyloxycarbonyl or N-tert-butoxycarbonyl amino acids with dicyclohexylcarbodiimide in pyridine. The corresponding reaction of N-(benzyloxycarbonyl)-D-phenylglycine led to an almost complete racemization of the aminoacyl residue (compounds 2c and 2g). Subsequent chromatographic separation and deprotection of intermediates 2a-d afforded the desired target derivatives 2e-h. Product 3b was obtained by a similar acylation of 9-(3,5-O-isopropylidene-beta-D-xylofuranosyl)adenine with N-(benzyloxycarbonyl)-L-phenylalanine, followed by deblocking. The NMR spectra of 2' and 3' isomers of stereoisomers 2a and 2b are discussed. Compounds 2g and 3b are both substrates and inhibitors of Escherichia coli ribosomal peptidyltransferase, although the activity of 3b is low. Derivatives 2e,f,h do not accept AcPhe from N-AcPhe-tRNA in a peptidyltransferase-catalyzed reaction, but they inhibit the puromycin reaction in the same system. The order of inhibitory activity is 2e greater than 2f greater than 2h. The implications of these findings for the mechanism of peptidyltransferase and comparison of the latter with the action of chymotrypsin are discussed.

Phosphodiester amidates of allenic nucleoside analogues: anti-HIV activity and possible mechanism of action.

Lipophilic phosphodiester amidates 2a, 2b, 4a, 4b, and 6 derived from anti-HIV agent adenallene 1a, 3a, inactive hypoxallene 1b, 3b, and 9-(4-hydroxy-2-butyn-1-yl)adenine (5) were synthesized and studied as inhibitors of HIV-1 in ATH8 cell system. All phosphodiester amidates were more biologically active than their parent nonphosphorylated compounds. Analogues 2a and 4a derived from (+/-)-adenallene 1a and (R)-enantiomer 3a are effective anti-HIV agents with EC50 approximately 0.88 and 0.21 microM, respectively. Both analogues are 16 and 28 times more effective than parent compounds 1a and 3a, respectively. Some anti-HIV activity of hypoxallene derivatives 2b and 4b was noted in the range of 0.1-10 microM but the dose-response relationship was poor. Phosphodiester amidate analogue 6 also exhibited anti-HIV activity in the range of 0.1-100 microM, but this effect was accompanied by cytotoxicity. Hydrolytic studies performed at pH 9.8 and with pig liver esterase at pH 7.4 have shown that analogue 2a gives adenallene 4'-phosphoralaninate (10a) as the major product. These results can be interpreted in terms of initial hydrolysis of phosphodiester amidates 2a, 2b, 4a, 4b, and 6 catalyzed by intracellular esterase(s) to give stable phosphomonoester amidate intermediates with a free carboxyl group. The results obtained with hypoxallene phosphoramidates 2b and 4b indicate that the aminosuccinate-fumarate enzyme system responsible for activation of AIDS drug ddIno (didanosine, Videx) can also, albeit less efficiently, activate hypoxallene 4'-phosphate (9b) and the respective (R)-enantiomer released inside the HIV-infected cells.

Analogues of chloramphenicol: circular dichroism spectra, inhibition of ribosomal peptidyltransferase, and possible mechanism of action.

Circular dichroism spectra of series of chloramphenicol derivatives la-r were measured in water at pH 7. Compounds 1a-o exhibit two positive Cotton effects at 310--340 and 240--260 nm, respectively, and a weaker negative Cotton effect at 280--300 nm. In analogues 1c, 11, and 1m there is only a minimum between the two positive Cotton effects. Derivatives 1p--r possess a strong negative Cotton effect at ca. 280 nm. Compounds 1a--r were examined as inhibitors of the puromycin reaction with Escherichia coli 70S ribosome-poly(U)-N-AcPhe-tRNA complex. Analogues 11, 1n, lo, and lq are potent competitive inhibitors of puromycin comparable to or better than chloramphenicol (1b). Compounds 1k and 1m are less active, whereas 1d--g and 1j are only moderately effective. The rest of the analogues have marginal or no activity. The results are compared with previous biological data and discussed in terms of a retro-inverso relationship of chloramphenicol (1b) to the aminoacyl moiety of puromycin (aminoacyl-tRNA) and to a hypothetical transition state of peptide bond formation.

Phosphodiester amidates of unsaturated nucleoside analogues: synthesis and anti-HIV activity.

The effect of introduction of a lipophilic phosphodiester amidate moiety on the HIV activity of inactive unsaturated nucleoside analogues was investigated. Phosphodiester alaninates 5a, 5b, and 6 derived from unsaturated nucleoside analogues 3b, 3c, and 4a were synthesized and investigated as inhibitors of cytopathic effect and replication of HIV-1 in ATH-8 cells. Compound 5a is an inhibitor of HIV-1 whereas analogue 6 is inactive with cytotoxicity appearing above 10 microM and 5b is both inactive and nontoxic. Alkaline or enzymic hydrolysis of 5a gave phosphomonoester alaninate 14, a putative product of intracellular metabolism. Compound 14 as well as adenallene derivative 15c were devoid of anti-HIV activity, and they also failed to inhibit HIV reverse transcriptase. A new regioselective method for preparation of (Z)-4-(benzoyloxy)-1-hydroxy-2-butene, 7, a key intermediate for the synthesis of unsaturated nucleoside analogues of cis configuration such as 3a, 3b, and 3c, is also described.

Methylene-gem-difluorocyclopropane analogues of nucleosides: synthesis, cyclopropene-methylenecyclopropane rearrangement, and biological activity.

Alkylation-elimination of adenine and 2-amino-6-chloropurine with gem-difluorocyclopropane dibromide 10 gave E- and Z-methylene-gem-difluorocyclopropanes 11a, 11b, 12a, and 12b and gem-difluorocyclopropenes 13a and 13b. Debenzylation of intermediates 11a, 11b, 12a, and 12b afforded E- and Z-methylenecyclopropanes 4a, 4b, 5a, and 5b. Hydrolysis of 2-amino-6-chloropurine derivatives 4b and 5b afforded guanine analogues 4c and 5c. Composition of products (except 14b) obtained from alkylation-elimination reflects thermodynamically controlled cyclopropene-methylenecyclopropene rearrangement. The E-isomer 4a was moderately active against human cytomegalovirus and along with the Z-isomer 5a was active against leukemia L1210 and solid tumors in vitro.

Unsaturated acyclic analogues of 2'-deoxyadenosine and thymidine containing fluorine: synthesis and biological activity.

The syntheses and biological activities of fluorobutynol 11 and (E)- and (Z)-fluorobutenols 8a,d and 9a,d are described. Alkylation of adenine with bromofluorobutyne 13a afforded intermediate 14 which was converted to fluorobutynol 11. Aldehyde 16a and (carbethoxyfluoromethyl)-triphenylphosphonium bromide furnished (E)- and (Z)-fluorobutenoates 19a and 20a accompanied by regioisomer 21a. A similar reaction of compound 16d afforded Z- and E-esters 19d and 20d. Reduction of the mixture of 19a and 20a with DIBALH gave (E)- and (Z)-fluoroalkenols 8a and 9a. Similarly, the Z-ester 19d gave (Z)-fluoroalkenol 9d. Both 19d and 20d were reduced with NaBH4 to give (Z)- and (E)-fluoroalkenols 9d and 8d. Hydrogenation of 19a and 20a afforded fluoro ester 23. A similar reduction of 8a and 9a led to fluoro alcohol 24 and the defluorinated product 25 which were separated by chromatography on a Bio-Rad AG 1-X2 (OH-) column. (Z)-Fluorobutenol 9a is a substrate for adenosine deaminase, whereas the E-isomer 8a is inert toward the enzyme. By contrast, analogue 8a inhibited the replication and cytopathic effect of HIV-1 in ATH8 cells with an IC50 of approximately 100 microM, but the Z-isomer 9a was inactive. This effect was accompanied by 36% cytotoxicity at 100 microM. Compounds 11 and 8d inhibited the growth of murine leukemia L1210 culture with IC50 = 89 and 60 microM, respectively.

(R)-(-)- and (S)-(+)-adenallene: synthesis, absolute configuration, enantioselectivity of antiretroviral effect, and enzymic deamination.

Synthesis of optically pure (-)- and (+)-adenallene 2 and 3 is described. Racemic adenallene (1a) was subjected to deamination with adenosine deaminase monitored by HPLC using a Chiralcel CA-1 column to give (-)-adenallene (2) and (+)-hypoxallene (4). The latter compound was converted to acetate 5. The reaction of 5 with trifluoromethanesulfonic anhydride and pyridine followed by ammonolysis furnished acetate 6 or (+)-adenallene (3) depending on the solvent used in the last step. Acetate 5 was smoothly transformed to the 6-chloro derivative 7, but an attempted ammonolysis led only to racemization and decomposition. Single crystal X-ray diffraction established the R-configuration of (-)-enantiomer 2. The latter forms a pseudosymmetric dimer in the lattice with the adenine moiety in an anti-like conformation. The torsional angles of the allenic bonds show departures from 90 degrees (91 and 97 degrees, respectively) and rotameric preference of the hydroxymethyl groups is different in both molecules of the dimer. The R-enantiomer 2 inhibited the replication and cytopathic effect of human immunodeficiency virus (HIV-1) in ATH8 cell culture with an IC50 of 5.8 microM, whereas the S-enantiomer 3 was less active (IC50 > 200 microM). The enantioselectivity of the anti-HIV effect is significantly lower than that of 2',3'-dideoxyadenosine. Kinetics of deamination of R- and S-enantiomers 2 and 3 catalyzed by adenosine deaminase gave the following parameters: Km values of S-form 3 and R-form 2 were 0.41 and 0.52 mM with Vmax being 530 and 18.5 mumol/min, respectively [corrected]. Again,, a much lower level of enantioselectivity of deamination was observed than that of D- and L-adenosine. These results indicate (i) different enantioselectivity of enantiomers 2 and 3 as HIV inhibitors and adenosine deaminase substrates and (ii) both R- and S-enantiomers 2 and 3 can function as nucleoside analogues with varied enantioselectivity for different enzymes or receptors.

Synthesis, absolute configuration, and enantioselectivity of antiretroviral effect of (R)-(-)- and (S)-(+)-cytallene. Lipase-catalyzed enantioselective acylations of (+/-)-N4-acylcytallenes.

Enantioselectivity of acylations of (+/-)-cytallene (1b), (+/-)-N4-acetylcytallene (11a), (+/-)-N4-benzoylcytallene (11b), and (+/-)-N4-(9-fluorenylmethoxycarbonyl)cytallene (11c) using vinyl butyrate or acetate catalyzed by lipases in organic solvents was investigated. Reactions with 1b, 11a, and adenallene (1a) did not display a high enantioselectivity but all resulted in a predominant acylation of the (-)-enantiomers. Application of the Lowe-Brewster rule led to a tentative assignment of the R-configuration to all acylated products. Studies of the time course of acylation of (+/-)-N4-benzoylcytallene (11b) in chloroform, tetrahydrofuran (THF), tetrahydropyran (THP), tetrahydrothiophene (THT), and dioxane with lipase PS30 and/or AK showed that the reaction in THF catalyzed by lipase AK was the most promising for resolution of 11b. Indeed, a large-scale acylation afforded, after separation and deprotection of intermediates 3e and 10d, (+)- and (-)-cytallene (3c and 2b) in high yield and enantioselectivity. Acylation of 11c in THF led also to formation of 3c and 2b in high enantioselectivity. Single crystal X-ray diffraction established the S-configuration of (+)-cytallene (3c), thus confirming the assignment made on the basis of Lowe-Brewster rule. An improved large-scale synthesis of (+/-)-cytallene (1b) is also described. The R-enantiomer 2b inhibited the replication of a primary human immunodeficiency virus (HIV-1) isolate in phytohemagglutinin-activated peripheral blood mononuclear cells (PHA-PBM) with IC50 0.4 and IC90 1.7 microM. (+/-)-Cytallene (1b) exhibited IC50 0.8 and IC90 3.4 microM. Both compounds completely suppressed replication of HIV-1 at 10 microM with no detectable cytotoxicity. The S-enantiomer (3c) was inactive.

Hybrids of antibiotics inhibiting protein synthesis. Synthesis and biological activity.

Four hybrid antibiotics combining structural features of chloramphenicol (1a), sparsomycin (2b), lincomycin (5c), and puromycin (6d)--lincophenicol (1c), chloramlincomycin (5a), sparsolincomycin (5b), and sparsopuromycin (6b)--were synthesized. They were investigated as inhibitors of several partial reactions of procaryotic and eucaryotic protein synthesis as well as potential antimicrobial agents. Lincophenicol (1c) was active as inhibitor of Escherichia coli ribosomal peptidyltransferase-catalyzed puromycin reaction. Both lincophenicol (1c) and sparsophenicol (1b) inhibited the binding of the iodophenol analogue of sparsomycin to E. coli ribosomes. The results are discussed in terms of a retro-inverso hypothesis advanced earlier for interpretation of biological activity of chloramphenicol (1a) and sparsophenicol (1b). Chloramlincomycin (5a) suppressed the growth of Streptococcus pyogenes with MIC 6.25 micrograms/mL.

(Z)- and (E)-2-((hydroxymethyl)cyclopropylidene)methyladenine and -guanine. New nucleoside analogues with a broad-spectrum antiviral activity.

New nucleoside analogues 14-17 based on a methylenecyclopropane structure were synthesized and evaluated for antiviral activity. Reaction of 2,3-dibromopropene (19) with adenine (18) led to bromoalkene 20, which was benzoylated to give N6,N6-dibenzoyl derivative 23. Attempts to convert 20 or 23 to bromocyclopropanes 21 and 22 by reaction with ethyl diazoacetate catalyzed by Rh2(OAc)4 were futile. By contrast, 2,3-dibromopropene (19) afforded smoothly (E)- and (Z)-dibromocyclopropane carboxylic esters 24 + 25. Alkylation of adenine (18) with 24 + 25 gave (E)- and (Z)-bromo derivatives 21 + 22. Base-catalyzed elimination of HBr resulted in the formation of (Z)- and (E)-methylenecyclopropanecarboxylic esters 26 + 27. More convenient one-pot alkylation-elimination of adenine (18) or 2-amino-6-chloropurine (30) with 24 + 25 afforded (Z)- and (E)-methylenecyclopropane derivatives 26 + 27 and 31 + 32. The Z-isomers were always predominant in these mixtures (Z/E approximately 2/1). Reduction of 26 + 27 and 31 + 32 with DIBALH afforded (Z)- and (E)-methylenecyclopropane alcohols 14 + 16 and 33 + 34. The latter were resolved directly by chromatography. Compounds 14 + 16 were converted to N6-(dimethylamino)methylene derivatives 28 and 29 which were separated and deprotected to give 14 and 16. Reaction of 33 and 34 with HCO2H led to guanine analogues 15 and 17. The 1H NMR spectra of the Z-analogues 14 and 15 are consistent with an anti-like conformation of the nucleobases. By contrast, 1H NMR and IR spectra of bromo ester 21 are indicative of syn-conformation of adenine. Several Z-(hydroxymethyl)methylenecyclopropanes exhibited in vitro antiviral activity in micromolar or submicromolar range against human and murine cytomegalovirus (HCMV and MCMV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV-6), varicella zoster virus (VZV), and hepatitis B virus (HBV). Analogues 14, 15, and 33 were the most effective agents against HCMV (IC50 1-2.1, 0.04-2.1, and 0.8-5.6 microM), MCMV (IC50 2.1, 0.3, and 0.3 microM) and EBV in H-1 (IC50 0.2, 0.3, and 0.7 microM) and Daudi cells (IC50 3.2, 5.6, and 1.2 microM). Adenine analogue 14 was active against HBV (IC50 2 microM), VZV (IC50 2.5 microM), and HHV-6 (IC50 14 microM). Synadenol (14) and the E-isomer (16) were substrates of moderate efficiency for adenosine deaminase from calf intestine. The E-isomer 16 was more reactive than Z-isomer 14. The deamination of 14 effectively stopped at 50% conversion. Synadenol (14) was also deaminated by AMP deaminase from aspergillus sp.

(R)-(-)- and (S)-(+)-Synadenol: synthesis, absolute configuration, and enantioselectivity of antiviral effect.

Synthesis of (R)-(-)- and (S)-(+)-synadenol (1a and 2a, 95-96% ee) is described. Racemic synadenol (1a + 2a) was deaminated with adenosine deaminase to give (R)-(-)-synadenol (1a) and (S)-(+)-hypoxanthine derivative 5. Acetylation of the latter compound gave acetate 6. Reaction with N, N-dimethylchloromethyleneammonium chloride led to 6-chloropurine derivative 7. Ammonolysis furnished (S)-(+)-synadenol (2a). Absolute configuration of 1a was established by two methods: (i) synthesis from (R)-methylenecyclopropanecarboxylic acid (8) and (ii) X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. Racemic methylenecyclopropanecarboxylic acid (10) was resolved by a modification of the described procedure. The R-enantiomer 8 was converted to ethyl ester 13 which was brominated to give vicinal dibromides 14. Reduction with diisobutylaluminum hydride then furnished alcohol 15 which was acetylated to the corresponding acetate 16. Alkylation-elimination procedure of adenine with 16 yielded acetates 17 and 18. Deprotection with ammonia afforded a mixture of Z- and E-isomers 1a and 19 of the R-configuration. Comparison with products 1a and 2a by chiral HPLC established the R-configuration of (-)-synadenol (1a). These results were confirmed by X-ray diffraction of a single crystal of (-)-synadenol hydrochloride. The latter forms a pseudosymmetric dimer with adenine-adenine base pairing in the lattice with the nucleobase in an anti-like conformation. Enantiomers 1a and 2a exhibit varied enantioselectivity toward different viruses. Both enantiomers are equipotent against human cytomegalovirus (HCMV) and varicella zoster virus (VZV). The S-enantiomer 2a is somewhat more effective than R-enantiomer 1a in herpes simplex virus 1 and 2 (HSV-1 and HSV-2) assays. By contrast, enantioselectivity of antiviral effect is reversed in Epstein-Barr virus (EBV) and human immunodeficiency virus type 1 (HIV-1) assays where the R-enantiomer 1a is preferred. In these assays, the S-enantiomer 2a is less effective (EBV) or devoid of activity (HIV-1).

Effective treatment of murine cytomegalovirus infections with methylenecyclopropane analogues of nucleosides.

A number of new nucleoside analogues with a Z- or E-methylenecyclopropane structure exhibited significant activity against human and murine cytomegaloviruses (HCMV, MCMV) in tissue culture that was generally comparable to, or greater than, 9-[(1-3-dihydroxy-2-propoxy)methyl]guanine (ganciclovir, GCV). Several of these analogues were chosen for further evaluation of therapeutic efficacy utilizing a MCMV infection. Intraperitoneal (i.p.) inoculation of 3-week-old Balb/c mice with 2.0 x 10(5) plaque forming units (pfu) of MCMV results in an acute, lethal infection with rapid virus replication in visceral and glandular tissue, thus, making it an ideal model for identifying compounds that have potential for use in humans. Synadenol (QYL-284A) and synguanol (QYL-438) were administered i.p. once daily for 5 days initiated 6, 24, or 48 h post-viral infection. Significant protection was demonstrated at 50 and 16.7 mg/kg compared to placebo, with efficacy comparable to GCV. When delivered orally once or twice daily at 100 mg/kg per day, QYL-438 was active, but less effective than GCV. In addition, 2-amino-6-methoxypurine analogue (QYL-941) was active at 60 mg/kg administered orally twice daily, comparable to GCV, while it's prodrug (QYL-972) was as effective as GCV at 40 mg/kg when delivered twice daily for 5 days. Additionally, analogue 2-amino-6-cyclopropylaminopurine (QYL-769) was found to be highly efficacious when given orally twice daily for 5 days. Mortality of 0% and 13% was observed at 60 and 20 mg/kg, respectively, which was similar to GCV. Oral treatment with QYL-769 or GCV reduced virus replication in target organs, but neither resulted in complete clearance of MCMV. These data indicate that these new analogues have activity comparable to GCV when given orally to mice and should be evaluated further to assess their potential for use in humans.

Synthesis and antiviral activity of phosphoralaninate derivatives of methylenecyclopropane analogues of nucleosides.

Phenylmethylphosphoro-L-alaninate prodrugs of antiviral Z-methylenecyclopropane nucleoside analogues and their inactive E-isomers were synthesized and evaluated for their antiviral activity against HCMV, HSV-1, HSV-2, HHV-6, EBV, VZV, HIV-1 and HBV. The adenine Z-analogue was a potent inhibitor of all these viruses but it displayed cellular toxicity. The guanine Z-derivative was active against HCMV, HBV, EBV and VZV and it was not cytotoxic. The 2,6-diaminopurine analogue was the most potent against HIV-1 and HBV and somewhat less against HHV-6, HCMV, EBV and VZV in a non-cytotoxic concentration range. The 2-amino-6-cyclopropylamino and 2-amino-6-methoxypurine prodrugs were also more active than parent analogues against several viruses but with a less favorable cytotoxicity profile. In the E-series of analogues, adenine derivative was active against HIV-1, HBV and EBV, and it was non-cytotoxic. The guanine analogue exhibited a significant effect only against HBV. The 2,6-diaminopurine E-analogue was inactive with the exception of a single EBV assay. The 2-amino-6-methoxypurine Z-methylenecyclopropane nucleoside analogue was an effective inhibitor of HCMV, MCMV and EBV. The 2,6-diaminopurine Z-prodrug seems to be the best candidate for further development.