The synthesis of novel 6,5- and 6,6-membered fused heterocyclic compounds derived from thymine

Novel pyrimido[1,2-a]pyrimidinones 14, 15 and 16 and imidazo[1,2-a] pyrimidinones 19 and 20, designed as conformationally constrained analogues of 1-(3-amino-2-hydroxypropyl)thymine and 1-(2-amino-3-hydroxypropyl)thymine, respective ly, were synthesized by the ring-opening/ ring-closure rearrangement of the corresponding byciclic oxygen-containing amino compounds 12 and 17.     

Synthesis of some monodeoxyfluorinated methyl and 4-nitrophenyl alpha-D-mannobiosides and a related 4-nitrophenyl alpha-D-mannotrioside

Treatment of methyl 3,4,6-tri-O-benzyl-2-O-(2,3,4-tri-O-acetyl-alpha-D-mannopyranosyl)-alpha -D- mannopyranoside with N,N-diethylaminosulfur trifluoride (Et2NSF3), followed by O-deacetylation and catalytic hydrogenolysis, afforded methyl 2-O-(6-deoxy-6-fluoro-alpha-D-mannopyranosyl)-alpha-D-mannopyranoside (8). Methyl 6-deoxy-6-fluoro-2-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside (11) was similarly obtained from methyl 3-O-benzyl-2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-alpha-D- mannopyranoside. 1,2,3,4-Tetra-O-acetyl-6-deoxy-6-fluoro-beta-D-mannopyranose (13), used for the synthesis of the 4-nitrophenyl analogs of 8 and 11, as well as their 3-O-linked isomers, was obtained by treatment of 1,2,3,4-tetra-O-acetyl-beta-D-mannopyranose with Et2NSF3. Treatment of 13 with 4-nitrophenol in the presence of tin(IV) chloride, followed by sequential O-deacetylation, isopropylidenation, acetylation, and cleavage of the acetal group, afforded 4-nitrophenyl 4-O-acetyl-6-deoxy-6-fluoro-alpha-D-mannopyranoside (18). Treatment of 13 with HBr in glacial acetic acid furnished the 6-deoxy-6-fluoro bromide 19. Glycosylation of diol 18 with 20 gave 4-nitrophenyl 4-O-acetyl-6-deoxy-6-fluoro-3-O- (21) and -2-O-(2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl)-alpha-D- mannopyranoside (23) in the ratio of approximately 2:1, together with a small proportion of a branched trisaccharide. 4-Nitrophenyl 4,6-di-O-acetyl-alpha-D-mannopyranoside was similarly glycosylated with bromide 19 to give 4-nitrophenyl 4,6-di-O-acetyl-3-O- and -2-O-(2,3,4-tri- O-acetyl-6-deoxy-6-fluoro-alpha-D-mannopyranosyl)-alpha-D-mannopyranosid e. The various di- and tri-saccharides were O-deacetylated by Zemplén transesterification.     

Synthesis of 4,19-disubstituted derivatives of DOC. Radioreceptor assay of some corticosteroid derivatives in human mononuclear leukocytes

Several new 4,19-substituted steroids and previously synthesized corticosteroids were assayed for affinity to type 1 receptors in human mononuclear leukocytes. 11 beta,19-epoxy-4,21-dihydroxypregn-4-ene-3,20-dione (2) was hydrogenated with Pd-C to yield a mixture of all four dihydro derivatives 5, accompanied by 4,21-diacetoxy-11 beta,19-epoxy-3-hydroxypregnan-20-one (6) and 21-acetoxy-11 beta,19-epoxy-4-hydroxypregnane-3,20-dione (7). With hot acetic + p-toluenesulfonic acid 5 underwent rearrangement to 21-acetoxy-11 beta,19-epoxypregn-5-ene-4,20-dione (8) Pd-C hydrogenation of 3,21-diacetoxy-5 beta,19-cyclopregna-2,9(11)-diene-4,20-dione (10) gave 3,21-diacetoxy-5 beta,19-cyclopregn-5-ene-4,20-dione (11) and the 9,11-dihydro derivative of the latter. Treatment of 10 with warm HCl furnished 19-chloro-4,21-dihydroxypregna-4,9(11)-diene-3,20-dione (13). Pd-C hydrogenation of its diacetate 14 afforded the 4,5-dihydro derivative 18, 19-chloro-21-acetoxypregn-9(11)-en-20-one (15), its 4-acetoxy derivative 16 and the 3,4-diacetoxy derivative 17. When tested in a radioreceptor assay in human mononuclear leukocytes the synthesized compounds showed only low relative binding affinities (RBA) to type 1 receptor, the highest being 0.72% for 13 (aldosterone = 100%). For comparison, other RBA in this system were: 19-noraldosterone, 20%; 18-deoxyaldosterone, 5.8%; 18-deoxy-19-noraldosterone, 4.7%; 18,21-anhydroaldosterone, 0.37%; 17-isoaldosterone, 7.6% and apoaldosterone, 4.3%     

Etoposide: a new approach to the synthesis of 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-O- demethyl-4-epipodophyllotoxin

Synthesis of 3-O-acetyl-2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene- alpha-(7 alpha) and-beta-D-glucopyranose (7 beta) and their 3-O-chloroacetyl analogues (11 alpha and 11 beta) are described. Condensation (BF3-etherate, ethyl acetate, -20 degrees) of 7 alpha with 4'-O-benzyloxycarbonyl-4'-O-demethyl-4-epipodophyllotoxin (8) afforded mainly the beta-glycoside 9 beta (alpha, beta-ratio 1:9). Condensation of 11 alpha beta with 8 or the 4'-O-chloroacetyl analogue 13 gave mainly the 4-O-(2-benzyloxycarbonylamino-3-O-chloroacetyl-2-deoxy-4,6-O-ethyl idene-beta-D- glucopyranosyl)-epipodophyllotoxin 12 beta or 15 beta. Glycosidation of podophyllotoxin (14) with 11 alpha beta (during which the aglycon epimerized at C-4 under the action of BF3-etherate) afforded alpha- (16 alpha) and beta-glycoside (16 beta) in the ratio 1:5. Removal of the chloroacetyl groups from 12 beta, its alpha analogue 12 alpha, and 15 beta gave the 4-O-(2-benzyloxycarbonylamino-2-deoxy-4,6-O-ethylidene-alpha-(17 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-epipodophyllotoxins (17 beta and 20 beta), respectively. Hydrogenolysis of the benzyloxycarbonyl groups then gave 4-O-(2-amino-2-deoxy-4,6-O-ethylidene-alpha- (18 alpha) and -beta-D-glucopyranosyl)-4'-O-demethyl-4-epipodophyllotoxin (18 beta). Reductive alkylation of 18 beta and 18 alpha afforded the 2"-deoxy-2"-dimethylamino-etoposide 3 and its alpha analogue 19 alpha.     

Exomethylene pyranonucleosides: efficient synthesis and biological evaluation of 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine

A new series of unsaturated pyranonucleosides with an exocyclic methylene group and thymine as heterocyclic base have been designed and synthesized. d-Galactose (1) was readily transformed in three steps into the corresponding 1-(beta-d-galactopyranosyl)thymine (2). Selective protection of the primary hydroxyl group of 2 with a t-butyldimethylsilyl (TBDMS) group, followed by specific acetalation, and oxidation gave 1-(6-O-t-butyldimethylsilyl-3,4-O-isopropylidene-beta-d-lyxo-hexopyranosyl-2-ulose)thymine (5). Wittig reaction of the ketonucleoside 5, deprotection and tritylation of the 6'-hydroxyl group gave 1-(2-deoxy-2-methylene-6-O-trityl-beta-d-lyxo-hexopyranosyl)thymine (9). Exomethylene pyranonucleoside 9 was converted to the olefinic derivative 10, which after detritylation afforded the title compound 1-(2,3,4-trideoxy-2-methylene-beta-d-glycero-hex-3-enopyranosyl)thymine (11). These novel synthesized compounds were evaluated for antiviral activity against rotaviral infection and cytotoxicity in colon cancer. As compared to AZT, compounds 1-(2-deoxy-2-methylene-beta-d-lyxo-hexopyranosyl)thymine (7) and 1-(beta-d-lyxo-hexopyranosyl-2-ulose)thymine (8) showed to be more efficient, in rotavirus infections and in treatment of colon cancer.     

An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents

1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-alpha-D-xylofuranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) uracil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically active against rotavirus infection and the growth of tumor cells.     

Synthesis of 18-hydroxy-19-norcorticosterone and 18-deoxy-19-noraldosterone. Structure determination of related 19-nor steroids by means of 2-D 1H nmr

The compounds named in the title have been synthesized from the di-(ethylene ketal) of 21-hydroxy-3,20-dioxo-19-norpregn-5-ene-18, 11 beta-lactone and its 5(10)-ene isomer. Reduction of this mixture 1 with sodium aluminum bis-(methoxyethoxy)hydride furnished the 11 beta, 18, 21-triol 2a. Conversion to the 18,21-diacetate 2b, followed by deketalization to the free dione 3 and hydrolysis, afforded 18-hydroxy-19-norcorticosterone 4a which, in the solid state and probably in solution, has the 18,20-hemiacetal structure. Periodate oxidation of 4a gave 11 beta-hydroxy-3-oxo-19-norandrost-4-ene-17 beta, 18-carbolactone 5a, and acid treatment of 4a or its precursor 2a yielded 18-deoxy-19-noraldosterone 6a. The structure of 5a was confirmed by mass spectrometry and 1H nmr, and compared with that of its C-19 methyl homolog 5b and 19-noraldosterone-gamma-etiolactone 8. In particular, 2-D nmr COSY 45 experiments, affording full 1H line assignments, have rigorously established the "natural" beta (axial) configuration of the C-10 hydrogen in the 19-nor lactones 5a and 8, and therefore also in the related 4a, 6a and 19-noraldosterone 7.     

Synthesis of glycosides of 3-deoxy-4-thiopentopyranosid-2-uloses and their reduction products: 3-deoxy-4-thiopentopyranosides

Michael addition of common thiols to the enone system of (2S)-2-benzyloxy-2H-pyran-3(6H)-one (1) afforded the corresponding 3-deoxy-4-thiopentopyranosid-2-ulose derivatives (2-4). The reaction was highly diastereoselective, and the addition was governed by the quasiaxially disposed 2-benzyloxy substituent of the starting pyranone. As expected from the enantiomeric excess of 1 (ee > 86%) the corresponding thiouloses 2-4 exhibited the same optical purity. However, the enantiomerically pure thioulose 5 was obtained by reaction of 1 with the chiral thiol, N-(tert-butoxycarbonyl)-L-cysteine methyl ester. The thio derivative 7 was also synthesized by reaction of 6 (enantiomer of 1) with the same chiral thiol. Alternatively, 4-thiopent-2-uloses 9-12 were prepared in high optical purity by 1,4-addition of thiols to (2S)-[(S)-2'-octyloxy]dihydropyranone 8. Similarly, reaction of 13 (enantiomer of 8) with benzenemethanethiol afforded 14 (enantiomer of 10). This way, the stereocontrol exerted by the anomeric center on the starting dihydropyranone led to 4-thiopentuloses of the D and L series. Sodium borohydride reduction of the carbonyl function of uloses 10 and 12 gave the corresponding 3-deoxy-4-thiopentopyranosid-2-uloses (16-19). The diastereomers having the beta-D-threo configuration (16, 18) slightly predominated over the beta-D-erythro (17, 19) analogues. However, the reduction of the enantiomeric pyranones 10 and 14 with K-Selectride was highly diastereofacial selective in favor of the beta-D- and beta-L-threo isomers 16 and 20, respectively.     

Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity

Steroidal epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives have been prepared using 3beta,17beta-dihydroxy-17alpha-picolyl-androst-5-ene (1), 3beta-acetoxy-17-picolinylidene-androst-5-ene (2), and 3beta-hydroxy-17-picolinylidene-androst-5-ene (3) as synthetic precursors. The compounds 2 and/or 3 were reacted with m-chloroperoxybenzoic acid (MCPBA). The compounds synthesized from 2 were 17-picolinylidene-N-oxide 4, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 5 and 6, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 7 and 8. Starting from compound 3, a mixture of 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene 9 and 10, 5alpha,6alpha-epoxy and 5beta,6beta-epoxy-17-picolinylidene-N-oxide 11 and 12, and 5alpha,6alpha:17alpha,20alpha- and 5beta,6beta:17alpha,20alpha-diepoxy-N-oxide 13 and 14 were obtained. From compounds 15 and 18, obtained from 1 and 3 by the Oppenauer oxidation, the 4alpha,5alpha-epoxy and 4beta,5beta-epoxy derivatives 16, 17 and 20, 21 were prepared by oxidation with 30% H(2)O(2). Oxidation of 18 with MCPBA yielded only the N-oxide 19. The structures of compounds 15 and 18 were proved by the X-ray analysis. Compounds 1-6, 9, 15, 17, 18, and 21 were tested on activity against the enzyme aromatase. Antitumor activity against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER-, MDA-MB-231, and prostate cancer PC3) was evaluated. Three tested compounds (1, 4, and 19) showed strong activity against PC3, the IC(50) values being in the range 0.55-10microM, whereas compound 17 showed strong activity against MDA-MB-231 (IC(50) 10.4microM).     

Synthesis and biological activity of O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses

Benzoylation of benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-α-d-glucopyranoside, benzyl 2-deoxy-2-(dl-3-hydroxytetradecanoylamino)-4,6-O-isopropylidene-α-d-glucopyranoside, and benzyl 2-deoxy-4,6-O-isopropylidene-2-octadecanoylamino-β-d-glucopyranoside, with subsequent hydrolysis of the 4,6-O-isopropylidene group, gave the corresponding 3-O-benzoyl derivatives (4, 5, and 7). Hydrogenation of benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-glucopyranoside, followed by chlorination, gave a product that was treated with mercuric actate to yield 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucopyranose (11). Treatment of 11 with ferric chloride afforded the oxazoline derivative, which was condensed with 4, 5, and 7 to give the (1→6)-β-linked disaccharide derivatives 13, 15, and 17. Hydrolysis of the methyl ester group in the compounds derived from 13, 15, and 17 by 4-O-acetylation gave the corresponding free acids, which were coupled with l-alanyl-d-isoglutamine benzyl ester, to yield the dipeptide derivatives 19–21 in excellent yields. Hydrolysis of 19–21, followed by hydrogenation, gave the respective O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses in good yields. The immunoadjuvant activity of these compounds was examined in guinea-pigs.     

Keto-fluorothiopyranosyl nucleosides: a convenient synthesis of 2- and 4-keto-3-fluoro-5-thioxylopyranosyl thymine analogs

A novel series of fluorinated keto-β-d-5-thioxylopyranonucleosides bearing thymine as the heterocyclic base have been designed and synthesized. Deprotection of 3-deoxy-3-fluoro-5-S-acetyl-5-thio-d-xylofuranose (1) and selective acetalation gave the desired isopropylidene 5-thioxylopyranose precursor 3. Acetylation and isopropylidene removal followed by benzoylation led to 3-deoxy-3-fluoro-1,2-di-Ο-benzoyl-4-O-acetyl-5′-thio-d-xylopyranose (6). This was condensed with silylated thymine and selectively deacetylated to afford 1-(2′-Ο-benzoyl-3′-deoxy-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (8). Oxidation of the free hydroxyl group in the 4′-position of the sugar led to the formation of the target 4′-keto compound together with the concomitant displacement of the benzoyl group by an acetyl affording, 1-(2′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-4′-ulose)thymine (9). Benzoylation of 3 and removal of the isopropylidene group followed by acetylation, furnished 3-deoxy-3-fluoro-1,2-di-Ο-acetyl-4-O-benzoyl-5′-thio-d-xylopyranose (12). Condensation of thiosugar 12 with silylated thymine followed by selective deacetylation led to the 1-(4′-Ο-benzoyl-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (14). Oxidation of the free hydroxyl group in the 2′-position and concomitant displacement of the benzoyl group by an acetyl gave target 1-(4′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-2′-ulose)thymine (15).     

Synthesis and biological activity of O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses

Benzoylation of benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-α-d-glucopyranoside, benzyl 2-deoxy-2-(dl-3-hydroxytetradecanoylamino)-4,6-O-isopropylidene-α-d-glucopyranoside, and benzyl 2-deoxy-4,6-O-isopropylidene-2-octadecanoylamino-β-d-glucopyranoside, with subsequent hydrolysis of the 4,6-O-isopropylidene group, gave the corresponding 3-O-benzoyl derivatives (4, 5, and 7). Hydrogenation of benzyl 2-acetamido-4,6-di-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-α-d-glucopyranoside, followed by chlorination, gave a product that was treated with mercuric actate to yield 2-acetamido-1,4,6-tri-O-acetyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucopyranose (11). Treatment of 11 with ferric chloride afforded the oxazoline derivative, which was condensed with 4, 5, and 7 to give the (1→6)-β-linked disaccharide derivatives 13, 15, and 17. Hydrolysis of the methyl ester group in the compounds derived from 13, 15, and 17 by 4-O-acetylation gave the corresponding free acids, which were coupled with l-alanyl-d-isoglutamine benzyl ester, to yield the dipeptide derivatives 19–21 in excellent yields. Hydrolysis of 19–21, followed by hydrogenation, gave the respective O-(N-acetyl-β-muramoyl-l-alanyl-d-isoglutamine)-(1→6)-2-acylamino-2-deoxy-d-glucoses in good yields. The immunoadjuvant activity of these compounds was examined in guinea-pigs.     

Synthesis, spectral analysis and in vitro microbiological evaluation of novel ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates and 4,5-dihydro-6-(napthalen-2-yl)-4-aryl-2H-indazol-3-ols

A series of ethyl 4-(naphthalen-2-yl)-2-oxo-6-arylcyclohex-3-enecarboxylates 8-14 and 4,5-dihydro-6-(naphthalen-2-yl)-4-aryl-2H-indazol-3-ols 15-21 were synthesised and characterised by their spectroscopic data. In vitro microbiological evaluations were carried out for all the newly synthesised compounds 8-21 against clinically isolated bacterial and fungal strains. Compounds 9, 12 and 20 against Staphylococcus aureus, 10, 12, 20 against β-haemolytic streptococcus, 11, 17 against Bacillus subtilis, 12, 16 and 20 against Vibreo cholerae, 13, 16 against Escherichia coli, 13, 16, 18, 19 against Salmonella typhii, 12, 18 against Shigella flexneri, 10 against Salmonella typhii, 10, 13, 17, 18 against Aspergillus flavus, 12, 17, 21 against Aspergillus niger, 12, 15, 17, 18, 20 against Mucor, Rhizopus and Microsporeum gypsuem exhibit potent antimicrobial activity.     

Synthesis of non-glycosidic 4,6'-thioether-linked disaccharides as hydrolytically stable glycomimetics

Michael addition of 1,2:3,4-di-O-isopropylidene-6-thio-alpha-D-galactose (2) to 2-propyl 6-O-acetyl-3,4-dideoxy-alpha-D-glycero-hex-3-enopyranosid-2-ulose (1) afforded, as the major diastereoisomer, 2-propyl 6-O-acetyl-3-deoxy-4-S-(6-deoxy-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranos-6-yl)-4-thio-alpha-D-threo-hexopyranosid-2-ulose (3, 91% yield). Reduction of the carbonyl group of 3, followed by O-deacetylation gave the two epimers 7 (alpha-D-lyxo) and 8 (alpha-D-xylo) in a 1:2 ratio. On removal of the protecting groups of 8 by acid hydrolysis, formation of an 1,6-anhydro bridge was observed in the 3-deoxy-4-thiohexopyranose unit (10). The free non-glycosidic thioether-linked disaccharide 3-deoxy-4-S-(6-deoxy-alpha,beta-D-galactopyranos-6-yl)-4-thio-alpha,beta-D-xylo-hexopyranose (11) was obtained by acetolysis of 10 followed by O-deacetylation. A similar sequence starting from the enone 1 and methyl 2,3,4-tri-O-benzoyl-6-thio-alpha-D-glucopyranoside (12) led successfully to 2-propyl 3-deoxy-4-S-(methyl 6-deoxy-alpha-D-glucopyranos-6-yl)-4-thio-alpha-D-lyxo-hexopyranoside (17) and its alpha-D-xylo analog (19, major product). In this synthetic route, orthogonal sets of protecting groups were employed to preserve the configuration of both reducing ends and to avoid the formation of the 1,6-anhydro ring.     

Additional minor ecdysteroid components of Leuzea carthamoides

Seventeen additional minor ecdysteroid compounds were isolated and identified from the roots of Leuzea carthamoides (Wild.) DC. Eight of them are new phytoecdysteroids: carthamoleusterone (13) is a new side-chain cyclo-ether with five-membered ring; 14-epi-ponasterone A 22-glucoside (12) is a rare and unusual natural 14 beta-OH epimer; 15-hydroxyponasterone A (11) is also new and rare with its C-15 substituted position, as well as 22-deoxy-28-hydroxymakisterone C (18) possessing secondary hydroxyl in position C-28 and 26-hydroxymakisterone C (20) with hydroxy groups in positions 25 and 26. New are also 1 beta-hydroxymakisterone C (21) and 20,22-acetonides of inokosterone (8) and integristerone A (10). Series of already known ecdysteroids: ecdysone (1), 20-hydroxyecdysone 2- and 3-acetates (3 and 4), turkesterone (6), inokosterone (7), 24-epi-makisterone A (14), and amarasterone A (22) are reported here as new constituents of L. carthamoides. Seven earlier reported Leuzea ecdysteroids: 20-hydroxyecdysone (2), ajugasterone C (5), integristerone A (9), 24(28)-dehydromakisterone A (15), 24(28)-dehydroamarasterone B (16), (24Z)-29-hydroxy-24(28)-dehydromakisterone C (17) and makisterone C (19) are also included because they are now better characterized.     

Synthesis of purine and pyrimidine 2′-deoxynucleosides from a 1,2-dithio sugar precursor

9-(2-S-Ethyl-2-thio- and α-D-mannofuranosyl)adenine (8α and 8β) were synthesized from ethyl 3,5,6-tri-O-acetyl-2-S-ethyl-1,2-dithio-α-D-mannofuranoside (1) by bromination followed by coupling of the resultant bromide (2) with 6-benzamido-(chloromercuri)purine. The 2-chloro analogues (10α and 10β) of 8α and 8β were obtained by way of a fusion reaction between 1,3,5,6-tetra-O-acetyl-2-S- ethyl-2-thio-α-D-mannofuranose (5) and 2,6-dichloropurine. Fusion of the bromide 2 with 2,4-bis(trimethylsilyloxy)pyrimidine and its 5-methyl derivative led to 1-(2-S- ethyl-2-thio-β-D-mannofuranosyl)uracil (16) and its thymine analogue (15). The action of Raney nickel led to rapid dechlorination of 10α and 10β, and all of the 2′-thio-nucleosides underwent desulfurization to give the corresponding 2′-deoxynucleosides. Sequential periodate oxidation-borohydride reduction converted the hexofuranosyl nucleosides into their pentofuranosyl analogues. Thus prepared were 9-(2-deoxy-α-and β-D-arabino-hexofuranosyl)adenine (11α and 11β) and their 2-deoxy-D-threo-pentofuranosyl counterparts (6α and 2′-deoxy-3′-epiadenosine, 6β), and 1-(2-deoxy- β-D-arabino-hexofuranosyl)-thymine (17) and -uracil (18) and their 2-deoxy-D-threo-pentofuranosyl counterparts (3′-epithymidine, 21, and 2′-deoxy-3′-epiuridine, 20). Detailed n.m.r.-spectral correlations are described for the series, and various derivatives of the nucleosides are reported.     

Bicarbonate is a recycling substrate for cyanase

Cyanase is an inducible enzyme in Escherichia coli that catalyzes bicarbonate-dependent decomposition of cyanate to ammonia and bicarbonate. Previous studies provided evidence that carbamate is an initial product and that the kinetic mechanism is rapid equilibrium random (bicarbonate serving as substrate as opposed to activator); the following mechanism was proposed (Anderson, P. M. (1980) Biochemistry 19, 2282-2888; Anderson, P. M., and Little, R. M. (1986) Biochemistry 25, 1621-1626). (formula; see text) Direct evidence for this mechanism was obtained in this study by 1) determining whether CO2 or HCO3- serves as substrate and is formed as product, 2) identifying the products formed from [14C]HCO3- and [14C] OCN-, 3) identifying the products formed from [13C] HCO3- and [12C]OCN- in the presence of [18O]H2O, and 4) determining whether 18O from [18O]HCO3- is incorporated into CO2 derived from OCN-. Bicarbonate (not CO2) is the substrate. Carbon dioxide (not HCO3-) is produced in stoichiometric amounts from both HCO3- and OCN-. 18O from [18O]H2O is not incorporated into CO2 formed from either HCO3- or OCN-. Oxygen-18 from [18O]HCO3- is incorporated into CO2 derived from OCN-. These results support the above mechanism, indicating that decomposition of cyanate catalyzed by cyanase is not a hydrolysis reaction and that bicarbonate functions as a recycling substrate.