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.

Synthesis and antiviral activity of methylenedifluorocyclopropane analogues of nucleosides.

Synthesis and antiviral activity of methylenedifluorocyclopropane analogues 8a, 8b and 9a, 9b are described.

Identification of colchicine in placental blood from patients using herbal medicines.

While characterizing natural antiinflammatory substances in human placental blood, we discovered a factor that affected human neutrophils and their adherence. Rigorous chemical and stereochemical analyses revealed this factor to be the well-known alkaloid, colchicine. When samples from individual patients were analyzed, significant levels (49-763 microg/L) of colchicine could be found in placental blood of patients using nonprescription herbal dietary supplements during pregnancy. We confirmed the presence of colchicine in commercially available ginkgo biloba. Due to its potential harmful effects, it would appear that such supplements should be avoided by women who are pregnant or are trying to conceive.

Approaches to unsaturated analogues of nucleosides comprising four- and six-membered rings.

Unsaturated nucleoside analogues 21, 22, 46, and 54, comprising four- and six-membered rings, were synthesized using two different approaches. The 2-benzyloxycycloalkanones 23a and 23b served as starting materials for both methods. Conversion to methylenecyclobutanes 29a and 29b was followed by addition of bromine via pyridinium perbromide to give vicinal dibromides 30a and 30b. Reaction of 29a with Br2 gave a ring-contracted cyclopropane derivative 31. Alkylation-elimination of adenine with 30a gave bromoalkene 32 as the major product and adenine-containing unsaturated derivatives 33, 34, and 35 as minor components. Vicinal dibromide 30b gave the Zaitsev cyclohexene 45 as the only product. Epoxidation of 29a and 29b afforded oxiranes 36a and 36b which were used in alkylation of adenine to furnish hydroxy derivatives 37a, 37b, 38a, and 38b. Beta-elimination via mesylates 39a and 40a using tBuOK/DMF gave Z- and E-methylenecyclobutanes 34 and 35. With an excess of base the E-bis-methylenecyclobutane 41 was obtained. Mesylation of cyclohexane derivatives 37b and 38b gave the Z- and E-N6-mesylated product 48. By contrast, the N6-benzoyl derivatives 49 and 50 afforded O-mesyl intermediates 51 and 52. Beta-elimination gave both Hofmann and Zaitsev products 53 and 45. O-Debenzylation of 34 and 35, 45, and 53 afforded analogues 21, 22, 46, and 54. The E-isomer 22 was also obtained by hydroboration procedure from E-bis-methylenecyclobutane 41.

Spiropentane mimics of nucleosides: analogues of 2'-deoxyadenosine and 2'-deoxyguanosine. Synthesis of all stereoisomers, isomeric assignment, and biological activity.

Synthesis of spirocyclic analogues of 2'-deoxyadenosine and 2'-deoxyguanosine (12a-15a and 12b-15b) is described. Rhodium-catalyzed reaction of ethyl diazoacetate with methylenecyclopropane 19, obtained from 2-bromo-2-bromomethylcyclopropane 17 via debromination (16), reduction (18), and acetylation (19), gave a mixture of all four isomeric spiropentanes 20a-20d. Hydrolysis afforded hydroxy carboxylic acids 21a-21d. Acetylation of separated proximal + medial-syn isomers 21a + 21b and medial anti + distal isomers 21c + 21d furnished acetates 22a + 22b and 22c + 22d. Curtius rearrangement effected by diphenylphosphoryl azide in tert-butyl alcohol performed separately with mixtures 22a + 22b and 22c + 22d led to BOC-amino spiropentanes 23a + 23b and 23c + 23d. After deacetylation all isomers 24a-24d were separated and deprotected to give aminospiropentane hydrochlorides 25a-25d. Free bases were of limited stability. The heterocyclic moieties were introduced into individual isomers 25a-25d via 6-chloropurine derivatives 26a-26d or 30a-30d. Ammonolysis of 26a-26d furnished the adenine isomeric series 12a-15a, whereas guanine derivatives 12b-15b were obtained by hydrolysis of 30a-30d with formic acid. The isomeric assignments followed from IR spectra of BOC-aminospiropentanes 24a-24d and NMR spectra of 12a-15a including NOE and (H,H) COSY. The proximal and medial-syn isomers 12a and 12b were modest inhibitors of human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) in culture, whereas the medial-anti isomer 12c was a substrate for adenosine deaminase. The distal isomer 15b was an anti-EBV agent. The medial-syn phosphoralaninate 34 was an effective inhibitor of HCMV replication in vitro. It was also active against herpes simplex virus type 1 (HSV-1), varicella zoster virus (VZV), human immunodeficiency virus (HIV-1), hepatitis B virus (HBV), and EBV with a varying degree of cytotoxicity.

Synthesis of (Z)- and (E)-9-[(2-hydroxyethylidene)cyclopropyl]adenine--new methylenecyclopropane analogues of adenosine and their substrate activity for adenosine deaminase.

Synthesis of Z- and E-methylenecyclopropane analogues of adenosine 3 and 4 by alkylation of adenine with novel alkylating agent 5 is described. The E-isomer 4 is a substrate for adenosine deaminase. Compounds 3 and 4 were tested for antiviral activity against HCMV, HSV-1, HSV-2, EBV, VZV, HBV and HIV-1.

15N NMR and electronic properties of S-nitrosothiols.

Investigation of the 15N NMR of S-nitrosothiols showed that primary and tertiary RSNOs have distinct 15N chemical shifts around 730 and 790 ppm, respectively. Using 15N NMR technique, the equilibrium constant of NO transfer between SNAP and GSH was found to be 0.74. For primary RSNOs, linear relationships exist among 15N NMR chemical shifts, reduction potentials, and the pK(a)s of their parent thiols.

Conformational analysis of an alpha-galactosyl trisaccharide epitope involved in hyperacute rejection upon xenotransplantation.

alpha-Galactosyl epitopes are carbohydrate structures bearing an alpha-Gal-(1-->3)-Gal terminus (alpha-Gal epitopes). The interaction of these epitopes on the surface of animal cells with anti alpha-Gal antibodies in human serum is believed to be the main cause in antibody-mediated hyperacute rejection in xenotransplantation. In this paper, conformational analysis of an N-linked alpha-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp trisaccharide epitope was conducted in terms of each monosaccharide residue conformation, primary hydroxymethyl group configuration, and interglycosidic conformations. Selective 2D J-delta INEPT experiments have been carried out at three different temperatures to evaluate three-bond, long-range 13C-1H coupling constants for the crucial alpha-(1-->3) linkage. The NMR experimental data were complemented by theoretical calculations. The flexibility and dynamics of the trisaccharide have been studied by Metropolis Monte Carlo simulations. Ensemble-averaged three-bond, long-range 13C-1H coupling constants and nuclear Overhauser effects were in good agreement with the experimental data. The alpha-(1-->3) glycosidic linkage has shown a restricted flexibility as indicated by NMR spectroscopy and molecular modeling.

(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.

Caparratriene, an active sesquiterpene hydrocarbon from Ocotea caparrapi.

Caparratriene (1), a new sesquiterpene hydrocarbon with significant growth inhibitory activity (IC50 = 3.0 +/- 0.5 x 10(-6) M) against CEM leukemia cells, was isolated from the oil of Ocotea caparrapi (Nates) Dugand. The structure of 1, determined by spectroscopic techniques, corresponded to (E,E)-3,7,11-trimethyl-2,4,10-dodecatriene (C15H26).

Arylamination and arylation of 4,4,4-trifluoro-1-phenyl-1,3-butanedione with N-acetoxy derivatives of 2-aminofluorene.

The present study investigated the reaction of carcinogenic electrophiles with beta-diketones which possess an active methylene group. N-Acetoxy-2-(acetylamino)fluorene bound to tRNA at 37 degrees C, pH 7.0, and this reaction was inhibited by 4,4,4-trifluoro-1-phenyl-1,3-butanedione or 2-thenoyltrifluoroacetone. N-Acetoxy-2-(acetylamino)fluorene reacted with these active methylene compounds to form transitory 3-substituted 2-(acetylamino)fluorene intermediates, which, following cleavage of the trifluoroacetyl group, yielded 3-phenacyl-2-(acetylamino)fluorene or 3-(2-thenoylmethyl)-2-(acetylamino)fluorene. N-Acetoxy-2-[(trifluoroacetyl)amino]fluorene reacted with 4,4,4-trifluoro-1-phenyl-1,3-butanedione to yield two products, N-phenacyl-1-(trifluoroacetyl)-2-aminofluorene and N-phenacyl-3-(trifluoroacetyl)-2-aminofluorene. The ratio of these two products was approximately 1:2. The same N-phenacyl products were produced by incubation of the beta-diketone, N-hydroxy-2-(acetylamino)fluorene, and rat liver cytosol which catalyzed the formation of N-acetoxy-2-aminofluorene. Thus, the inhibitions by beta-diketones of nucleic acid-binding and bacterial mutagenesis of carcinogens are likely due to their trapping of the carcinogens.

Deuterium enrichment of plasma determined by nuclear magnetic resonance spectroscopy: dilution kinetics of 2H2O and estimation of total body water.

We demonstrate the feasibility of quantifying the abundance of 2H in plasma by nuclear magnetic resonance (NMR) spectroscopy. After adding internal standard (tert-butyl-d9 alcohol) to deproteinized plasma samples containing 2H2O, we determined the ratio of NMR peak areas for 2H2O and tert-butyl-d9 alcohol. This peak-area ratio was directly proportional to the exogenous 2H enrichment of plasma (difference between measured and naturally occurring 2H) between 0 and 0.272 atom % (r = 0.999). The coefficient of variation was 1.34% at an exogenous enrichment of 0.136 atom %. We applied this method to a study of the dilution kinetics of 2H2O to determine the optimal time and method of blood sampling for estimation of total body water content. The 2H enrichment of plasma stabilized by 4 h after intravenous injection of 2H2O, 1 g/kg of body weight, and fluctuated within 2-4% of the 4- to 8-h mean thereafter.

Investigation of the adducts formed by reaction of malondialdehyde with adenosine.

Malondialdehyde (MDA) forms oligomeric adducts with DNA bases. It has been proposed that the 2:1 MDA-guanosine (M2G) and 3:1 MDA-adenosine (M3A) adducts result from sequential addition of MDA molecules to the nucleoside base. Reaction of 1:1 MDA-guanosine (M1G) and 1:1 MDA-adenosine (M1A) adducts with MDA does not produce the multimeric adducts. This suggests they arise by reaction of the nucleoside bases with oligomers of MDA. If so, the proposed structure for M3A is inconsistent with the addition of an MDA trimer to adenosine. Therefore, we investigated the structure of this molecule. Two-dimensional double quantum filtered COSY and hetero-COSY NMR experiments were performed, and a series of insensitive nuclei assignment by polarization transfer (INAPT) NMR spectra were also recorded. The results of these experiments revealed the presence of a propano group and two alpha,beta-unsaturated aldehydes. The UV spectrum of M3A displayed a maximum at 326 nm, similar to that of N6-[3-oxo-1(E)-propenyl]adenosine (M1A). The adducts were reduced with sodium borohydride for comparison of the UV and NMR spectra. On the basis of our results, a new structure for M3A is proposed which is tentatively named 6(5*,7*-diformyl-2*H-3*,6*-dihydro-2*,6*-methano-1*,3*-oxazocin -3*-yl)-9-beta-D-ribofuranosylpurine.

A new spermatostatic glycoside from the soft coral Sinularia crispa.

The steroid glycoside 1 has been isolated from the Sri Lankan soft coral Sinularia crispa. The structure has been determined by spectroscopic (1H and 13C nmr) techniques. Glycoside 1 showed spermatostatic activity on rat cauda epididymal spermatozoa.

Sesquiterpene furans and thiosesquiterpenes from the nudibranch Ceratosoma brevicaudatum.

Six furanosesquiterpenes have been isolated from an Australian nudibranch, Ceratosoma brevicaudatum, and their structures were determined by spectral analysis. The metabolites include the known terpenes dehydrodendrolasin [1], dehydrolasiosperman [3], and thiofurodysinin acetate [7]. The remaining metabolites were determined to be an unreported cis isomer 2 of dehydrodendrolasin, (methylthio) furodysinin [4], and dithiofurodysinin disulfide [5], derivatives of thiofurodysinin acetate that had been isolated earlier from a sponge. 13C-nmr data were obtained for all compounds.

11 beta, 12 beta-Epoxypukalide, a furanocembranolide from the gorgonian Leptogorgia setacea.

A furanocembranolide, 11 beta, 12 beta-epoxypukalide (1), C21H24O7, was isolated in trace quantities from the gorgonian Leptogorgia setacea. The structure was determined from detailed 1H-nmr and 13C-nmr analyses and comparison of these spectral data with those of the closely related compounds pukalide (2) and lophotoxin (3). The ultimate origin of 1 is discussed briefly.

24 epsilon-Methyl-5 alpha-cholestane-3 beta,5,6 beta,22r,24-pentol 6-acetate: new polyhydroxylated sterol from the soft coral Asterospicularia randalli.

A monoacetylated pentahydroxylated sterol has been isolated from the soft coral Asterospicularia randalli collected at Guam Is. in the Pacific. The unusual feature of this sterol is the presence of a hydroxyl group at C-22. The structure was determined by spectral analyses of the new sterol and several transformation products. Proton-carbon correlated spectra were used to make 13C chemical shift assignments.