Synthesis of novel (2R,4R)- and (2S,4S)-iso dideoxynucleosides with exocyclic methylene as potential antiviral agents.

Novel (2R,4R)- and (2S,4S)-iso dideoxynucleosides with exocyclic methylene have been designed and synthesized, based on the lead BMS-200475 (3) which exhibited potent anti-HBV activity. For the synthesis of D types of (2R,4R)-nucleosides, L-xylose was converted to the key intermediate 14. The intermediate 14 was converted to the uracil derivative 4a and the cytosine derivative 4b. Compound 14 was also converted to the purine derivatives such as adenine derivative 4c, hypoxanthine derivative 4d, and guanine derivative 4e. The corresponding L types of (2S,4S)-enantiomers were more efficiently synthesized from the commercially available 1,2-isopropylidene-D-xylose (20) than the synthetic method used in the synthesis of (2R,4R)-nucleosides. The key intermediate 25 was converted to the pyrimidine analogues 5a and 5b and the purine derivatives 5c, 5d, and 5e using the similar method used in the preparation of 4c, 4d, and 4e. The synthesized final (2R,4R)- and (2S,4S)-nucleosides were tested against several viruses such as HIV-1, HSV-1, HSV-2, HCMV and HBV. (2R,4R)-Adenine analogue 4c exhibited potent anti-HBV activity (EC(50)=1.5 microM in 2.2.15 cells) among compounds tested, while (2R,4R)-uracil derivative 4a was the most active against HCMV among compounds tested and (2R,4R)-adenine derivative 4c was found to be moderately active against the same virus. However, the corresponding (2S,4S)-isomers were found to be totally inactive against all tested viruses. Both (2R,4R)-adenine derivative 4c and (2S,4S)-adenine analogue 5c were totally resistant to the adenosine deaminase like iso-ddA (1). From the molecular modeling study the hydroxymethyl side chains of BMS-200475 (3) and 4c were almost overlapped, indicating that 4c may be suitable for phosphorylation by cellular kinases like the lead 3, but some discrepancy between two bases was observed, indicating why 4c is less potent against HBV than 3. It is concluded that discovery of (2R,4R)-adenine analogue 4c as potent anti-HBV agent suggested that the sugar moiety of this series can be regarded as a novel template for the development of new anti-HBV agent and oxygen atom can be acted as a bioisostere of C-OH.     

Metabolism of pyrimidine deoxyribonucleosides in Neurospora crassa.

The experiments in this report involve the following series of reactions which were previously demonstrated with purified enzyme preparations from Neurospora crassa: thymidine a yields thymine ribonucleoside b yields thymine c yields 5-hydroxymethyluracil d yields 5-formyluracil e yields uracil-5-carboxylic acid f yields uracil. The evidence for some of the reactions occurring in vivo has been incomplete and for others totally lacking. In this paper intact cells of Neurospora are shown to be capable of converting the substrates of each of the reactions to the corresponding products. Studies are described which were carried out in vivo and in vitro with the pyrimidineless strains pyr-4,uc-1,uc-2 and pyr-4,uc-1,uc-3, developed by Williams and Mitchell. The results reported in the present paper indicate that (reaction a) and the uc-3 mutation affects thymine 7-hydroxylase (reactions c,d, and e). Evidence is presented for the 2'-hydroxylase reaction being the major, if not only, way by which Neurospora can initiate the conversion of thymidine to the pyrimidines of nucleic acids and for the 2'-hydroxylation of thymidine and deoxyuridine being catalyzed by the same enzyme. Deoxycytidine was shown not to be hydroxylated in intact cells but instead deaminated to deoxyuridine, which in turn was converted to uridine. Further studies with the uc-3-carrying strain showed that an enzyme other than thymine 7-hydroxylase can also convert 5-formyluracil to uracil-5-carboxylic acid.     

Synthesis and reactivities of 2,2'-anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl- beta-D-arabinofuranosyl)thymine.

2,2'-Anhydro-1-(3'-deoxy-3'-iodo-5'-O-trityl-beta-D-arabinofuranosyl) thymine (2) was synthesized from 2',3'-didehydro-3'-deoxythymidine (DHT). Compound 2 was readily converted into the 2',3'-anhydrolyxofuranosyl derivatives 4-6. Treatment of 4a with some nucleophiles (N3-, OMe-, Cl-) gave the corresponding 3'-substituted arabinosyl nucleosides (7a,c,e) together with the minor xylosyl isomers (8a,c,d). 7a,c,e were deprotected to 7b,d,f, respectively.     

Synthesis of 2,2′-Anhydro-1 - (3′ -deoxy -3′ -iodo-β-D-arabino-furanosyl) thymine and Its Derivatives as Versatile Synthetic Intermediates

Abstract

2,2′ -Anhydro-1- (3′ -deoxy-3′ -iodo-5′ -O-trityl-B-D-arabinofuranosyl)-thymine (2) was synthesized from 2′,3′ -didehydro-3′-deoxythymidine (DHT) (1). Compound 2 was readily converted into 2′,3′-anhydro-lyxofuranosyl derivatives 4-6. Reaction of 4a with some nucleophiles (N₃ -, OMe-, Cl-) gave the corresponding 3′-substituted arabinonucleosides (7b,d,f) together with the minor xylosyl isomers (8a,c). Compounds 7b,d,f and 8a were deprotected to 7c,e,g and 8b, respectively.     

Synthesis and β-lactamase inhibitory activity of 6-fluoropenicillanic acids

The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized. The conversion to their free acids 4b, 4b, 6d, 7b, 7e and potassium salts 7c, 7f are described. These acids and salt 7c were evaluated as β- lactamase inhibitors using β-lactamase I from Bacillus cereus. The data indicate that substitution of the 6-hydrogen by a 6- fluorine atom on 6β-bromopenicillanic acid (1), leads to loss of β-lactamase inhibitory activity. In the case of the isomers 6β- and 6-fluoropenicillanic acids the 6β-enantiomer proved to be considerably more potent. Potassium salts of 6β- fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution. The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior of the penicillanic acid sulfone (2) with 0.5 N NaOH.     

Synthesis of a series of ganglioside GM3 analogs containing a deoxy-N-acetylneuraminic acid residue.

Ganglioside GM₃ analogs containing 4-, 7-, 8-, and 9-deoxy-N-acetylneuraminic acids in the place of N-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl O-(6-O-benzoyl-β- - galactopyranosyl)-(1 → 4)-2,6-di-O-benzoyl-β- -glucopyranoside with the methyl 2-thioglycoside derivatives of the respective deoxy-N-acetylneuraminic acids, using dimethyl(methylthio)sulfonium triflate as a promoter, gave the four required 2-(trimethylsilyl)ethyl -sialosyl-(2 → 3b)-β-lactosides. These were converted via O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and subsequent imidate formation, into the corresponding -sialosyl-(2 → 3b)--lactose trichloroacetimidates 15, 17, 19, and 21. Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 15, 17, 19, and 21 in the presence of boron trifluoride etherate afforded the expected β glycosides, which were transformed in good yields, via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and de-esterification, into the target compounds.     

Circular dichroism studies on alpha- and beta-ketosides of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives.

The circular dichroism spectra of a number of N-acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methy, ethyl, and allyl alpha-D-ketosides are characterized by a broad, positive band centered at lambda similar to 195 nm with a slight skew towards the higher wavelengths and weak bands between lambda 225 and 255 nm, whereas the methyl beta-D-ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-nonulopyranose, its methyl beta-D-ketoside, and 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at lambda similar to 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid confirms the beta-D configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O-(N-acetyl-alpha-D-neuraminopyranosyl)-(2yields3)-O-beta-D-galactopyranosyl-(1 yields 4)-D-glucopyranose resembles that of the methyl, ethyl, and allyl alpha-D-ketosides 2-4.     

Studies on Nucleoside Analogs. XXV.1) Synthesis of 5, 7-Dioxopyrimido[5,4-e]-AS-Triazine Glycosides

Abstract

The reaction of glycosyl isothiocyanates (la, b, c, d, e) with 5,6-diamino-1-3-dimethyluracil gave the respective 1-glycosyl-3-(6-amino-1, 3-dimethyl-2, 4-dioxopyrimidine-5-yl) thioureas (2a, b, c, d, e) in excellent yields. Treatment of these thioureas with NBS afforded the respective 5,7-dioxopyrimido-[5,4-e]-as-triazine glycosides (4a, b, c, d, e) in good yields.     

Electrochemical bromination of cholest-5-enes

Four 5,6-unsaturated steroids--3beta-chlorocholest-5-ene (1a), cholesterol (1b) and its acetate (1c) and benzoate (1d)-were subjected to constant current electrolysis (50 mA, 2 F mol(-1)) in an electrolytic cell divided by a ceramic membrane, using a platinum foil as the anode and a graphite stick as the cathode. When electrolysis was carried out in a solution of tetraethylammonium bromide in aprotic solvents (dichloromethane, acetonitrile or acetic anhydride), the addition of electrochemically-generated elemental bromine onto the double bond of the cholesterol derivatives gave their corresponding 5alpha,6beta-dibromosteroids--3beta-chloro-5alpha,6beta-dibromocholestane (2a), 5alpha,6beta-dibromocholestan-3beta-ol (2b), 5alpha,6beta-dibromocholestan-3beta-yl acetate (2c) and 5alpha,6beta-dibromocholestan-3beta-yl benzoate (2d)--as the sole products, and in good yields (58-91%). However, the electrolysis of steroids 1a-c in a solution of tetraethylammonium bromide with methanol as the solvent proceeded to give, in addition to dibromides 2a-c, the corresponding diastereomeric pairs of 5-bromo-6-methoxysteroids: 5alpha-bromo-3beta-chloro-6beta-methoxycholestane (3a) and 5beta-bromo-3beta-chloro-6alpha-methoxycholestane (4a), 5alpha-bromo-6beta-methoxycholestan-3beta-ol (3b) and 5beta-bromo-6alpha-methoxycholestan-3beta-ol (4b) and 5alpha-bromo-6beta-methoxycholestan-3beta-yl acetate (3c) and 5alpha-bromo-6beta-methoxycholestan-3beta-yl acetate (4c). The benzoate 1d was not soluble enough in methanol, even with heating. The products were characterized by physical and spectral data (IR, 1H NMR and 13C NMR). Single crystal X-ray structure determinations of compounds 2a and 3a are also reported.     

Synthesis of some C-8-modified 3-deoxy-beta-D-manno-2-octulosonic acid analogs as inhibitors of CMP-Kdo synthetase

Selective C-8 modifications of 2,6-anhydro-3-deoxy-D-glycero-D-talo-octonic acid ("2,3-dideoxy-beta-D-manno-2-octulosonic acid", 1a) were effected via the protected 8-hydroxy derivatives 2d and 2e. Swern oxidation of 2d and 2e gave the aldehydes 3a and 3b, respectively. Compounds 3a and 3b were converted into the oxime 13b and the O-methyloxime 13c derivatives, respectively. Methodology was developed for selective cleavage of the protecting groups of 13b and 13c to give the deprotected oxime 12m and the deprotected O-methyloxime 12n, respectively. Side chain-extended products were prepared from the aldehyde 3a utilizing Wittig methodology. The branched chain allylic amine 12p was prepared from 3a in a sequence the keys steps of which were preparation of the methyl ketone 19a using LiCuMe2, followed by Swern oxidation, methylenation of 19a using CH2I2-Zn-TiCl4 to give the alkene 19b, followed by Wohl-Ziegler bromination of 19b to give the allylic bromide 19c, and conversion of the latter to the allylic azide 19d. A number of the analogs showed significant activities vs CMP-Kdo synthetase. The most active of these was the side-chain extended alkene 12d, which proved second in activity only to the 9-amino analog (1c).     

Systematic synthesis of purine 8,5'-imino and substituted imino cyclonucleosides

To achieve a systematic synthesis of purine 8,5'-imino and substituted imino cyclonucleosides, 2',3'-O-isopropylidene-purinenucleosides substituted with a methylamino (4a,b), benzyl-amino (4c,d,g and h) and allylamino group (4e,f,i and j) at the C8 were synthesized. With these substrates in hand, extensive 8,5'-cyclization reactions were carried out using diphenyl carbonate/Et3N (Method A), N,N'-carbonyldiimidazole (Method B) and the Mitsunobu reaction (Method C) to give 8,5'-substituted imino cyclonucleosides (5a,c,d,e,f and g). The yields of cyclization by Method C are generally higher than by the other two methods. 5a, b,c,d,e,f,g and h were deprotected to the corresponding mother compounds 8 through one or two steps. In guanosine series, a new cyclic system comprising an 8,5'-carbamate ester bridge (6a-c) has been introduced.     

Oxoferryl-porphyrin radical catalytic intermediate in cytochrome bd oxidases protects cells from formation of reactive oxygen species

The quinol-linked cytochrome bd oxidases are terminal oxidases in respiration. These oxidases harbor a low spin heme b(558) that donates electrons to a binuclear heme b(595)/heme d center. The reaction with O(2) and subsequent catalytic steps of the Escherichia coli cytochrome bd-I oxidase were investigated by means of ultra-fast freeze-quench trapping followed by EPR and UV-visible spectroscopy. After the initial binding of O(2), the O-O bond is heterolytically cleaved to yield a kinetically competent heme d oxoferryl porphyrin π-cation radical intermediate (compound I) magnetically interacting with heme b(595). Compound I accumulates to 0.75-0.85 per enzyme in agreement with its much higher rate of formation (~20,000 s(-1)) compared with its rate of decay (~1,900 s(-1)). Compound I is next converted to a short lived heme d oxoferryl intermediate (compound II) in a phase kinetically matched to the oxidation of heme b(558) before completion of the reaction. The results indicate that cytochrome bd oxidases like the heme-copper oxidases break the O-O bond in a single four-electron transfer without a peroxide intermediate. However, in cytochrome bd oxidases, the fourth electron is donated by the porphyrin moiety rather than by a nearby amino acid. The production of reactive oxygen species by the cytochrome bd oxidase was below the detection level of 1 per 1000 turnovers. We propose that the two classes of terminal oxidases have mechanistically converged to enzymes in which the O-O bond is broken in a single four-electron transfer reaction to safeguard the cell from the formation of reactive oxygen species.     

Stereoselective hydrolysis of 16 alpha-halo-17-keto steroids and long-range substitution effects on the hydrolysis of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones

Epimerizations of 16 alpha-chloro- (1a), bromo- (1b), and iodo-3 beta-hydroxy-5-androsten-17-one (1c) by a brief treatment with 0.2 equiv NaOH in aqueous pyridine reached equilibrium between 16 alpha- and 16 beta-halo ketones. 16 alpha-/16 beta-Halo ketone ratios at equilibrium were 1.5 for Cl, 1.25 for Br, and 1.0 for I. Kinetic analysis showed that compounds 1a-c were stereoselectively converted to the corresponding 16 alpha-hydroxy derivative 3 by an SN2 mechanism, in which the order of the apparent reactivity was Br greater than I greater than Cl. The hydrolysis of a number of 16 alpha-bromo-17-ketones and 2 alpha-bromo-3-ketones was carried out. The yields of the corresponding alcohols were found to depend on remote structural features in the steroids.     

New nordihydroguaiaretic acid derivatives as anti-HIV agents

Reaction of nordihydroguaiaretic acid with various alkyl chloride, 1-piperidinecarbonyl chloride, methyl chloroformate, or 1,1'-carbonyldiimidazole under alkaline conditions produced the corresponding phenol ethers, carbamates and carbonates, respectively, in 67-83% yields. Among these derivatives, the nitrogen-containing compounds were converted to the corresponding hydrochloride salts. Having good solubility, these NDGA derivatives were found stable in aqueous solution. These new compounds exerted appealing activity against HIV Tat-regulated transactivation in human epithelial cells. The most potent compound meso-2,3-dimethyl-1,4-bis(3,4-[2-(piperdino)ethoxyphenyl])butane tetrakishydrochloride salt (5b) showed IC(50) value of 0.88 microM.     

Oligonucleotides containing pyrazolo[3,4-d]pyrimidines: 8-Aza-7-deazaadenines with bulky substituents in the 2- or 7-position

The synthesis of the 2'-deoxyadenosine analogues 1b, 2b, and 3c modified at the 7- and/or 2-position is described. The effect of 7-chloro and 2-methylthio groups on the duplex stability is evaluated. For that, the nucleosides 1b, 2b, and 3c were converted to the corresponding phosphoramidites 15, 19, and 22, which were employed in the solid-phase oligonucleotide synthesis. In oligonucleotide duplexes, compound 1b forms stable base pairs with dT, of which the separated 1b-dT base pairs contribute stronger than that of the consecutive base pairs. Compound 2b shows universal base pairing properties while its N8 isomer 3c forms duplexes with lower stability.     

Neighboring group participation. Part 16. Stereoselective synthesis and receptor-binding examination of the four stereoisomers of 16-bromomethyl-3,17-estradiols

The four possible isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a-4a) with proven configurations were converted into the corresponding 3-benzyloxy-16-bromomethylestra-1,3,5(10)-triene-3,17-diols (5e-8e). Depending on the reaction conditions the cis isomers of 3-benzyloxy-16-hydroxymethylestra-1,3,5(10)-trien-17-ol (1a and 2a) were transformed into 3-benzyloxy-16-bromomethylestra-1,3,5(10)-trien-17-yl acetate (5b and 6b) or 16-bromomethyl-3-hydroxyestra-1,3,5(10)-trien-17-yl acetate (5c and 6c) on treatment with HBr and acetic acid. The mechanism of the process can be interpreted as involving front-side neighboring group participation. Under similar experimental conditions, the trans isomers (3a and 4a) yielded only 3-benzyloxy-16-acetoxymethylestra-1,3,5(10)-trien-17-yl acetates (3b and 4b) or 16-acetoxymethylestra-1,3,5(10)-triene-3,17-diyl diacetates (3d and 4d). Both the cis (1a and 2a) and the trans (3a, and 4a) isomers were transformed into 16-bromomethylestra-1,3,5(10)-trien-17-ol (5a-8a) by the Appel reaction on treatment with CBr4/Ph3P. Debenzylation of 5a-8a was carried out with HBr and acetic acid to yield 5e-8e. The debenzylation process in the presence of acetic anhydride produces the diacetates 5d-8d. The structures of the compounds were determined by means of MS, 1H NMR and 13C NMR spectroscopic methods. Compounds 5c-8c and 5e-8e were tested in a radioligand-binding assay. Except for the affinity of 7e for the estrogen receptor (Ki=2.55 nM), the affinities of the eight compounds (5c-8c and 5e-8e) for the estrogen, androgen and progesterone receptors are low (Ki > 0.55, 0.52 and 0.21 microM, respectively).     

Circular dichroism studies on α- and β-ketosides of 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives

The circular dichroism spectra of a number of N-acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methyl, ethyl, and allyl α- -ketosides are characterized by a broad, positive band centered at λ ≈ 195 nm with a slight skew towards the higher wavelengths and weak bands between λ 225 and 255 nm, whereas the methyl β- -ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy- -glycero-β- -galacto-nonulopyranose, its methyl β- -ketoside, and 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at λ ≈ 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy- -glycero- -galacto-nonulopyranosonic acid confirms the β- configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O-(N-acetyl-α- -neuraminopyranosyl)-(2→3)-O-β- -galactopyranosyl-(1→4)- -glucopyranose resembles that of the methyl, ethyl, and allyl α- -ketosides 2-4.     

Synthesis of ( + )-Methyl Phaseate and Its Isomer from ( — )-β-Pinene

The total synthesis of ( + )-methyI phaseate (2b) and its epimer (25) is described. The known β- ketoester (8), which was prepared from ( — )-/f-pinene (4), was converted to a key intermediate (5) via a 1, 4-dioxoester (7). The reaction of 5 with a lithium reagent of the acetylene TBDMS ether (6) in THF-HMPA at — 70°C afforded the desired acetylene alcohol (17) and its epimer (18) in high yields. 17 was transformed into ( + )-methyl phaseate (2b). From this synthetic work, the absolute configuration of natural ( — )-phaseic acid (2a) was confirmed.     

Synthesis and anti-HIV activity of beta-D-3'-azido-2',3'-unsaturated nucleosides and beta-D-3'-azido-3'-deoxyribofuranosylnucleosides

Since the discovery of 3'-azido-3'-deoxythymidine (AZT) and 2',3'-didehydro-2',3'-dideoxythymidine (d4T) as potent and selective inhibitors of the replication of human immunodeficiency virus (HIV), there has been a growing interest for the synthesis of 2',3'-didehydro-2',3'dideoxynucleosides with electron withdrawing groups on the sugar moiety. Here we described an efficient method for the synthesis of such nucleoside analogs bearing structural features of both AZT and d4T The key intermediate, 3-azido-1,2-bis-O-acetyl-5-O-benzoyl-3-deoxy-D-ribofuranose, 5 was synthesized from commercially available D-xylose in five steps, from which a series of pyrimidine and purine nucleosides were synthesized in high yields. The resultant protected nucleosides were converted to target nucleosides using appropriate chemical modifications. The final nucleosides were evaluated as potential anti-HIV agents.     

Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.