Isolation and Characterisation of (5R, 6S,5′R,8′R)- and (5R,6S,5′R,8′S)-Luteochrome from Brazilian Sweet Potatoes (Ipomoea batatas LAM.)

Luteochrome isolated from the tubers of a white-fleshed variety of sweet potato (Ipomoea batatas LAM .) has been shown by HPLC, ¹H-NMR and CD spectra to consist of a mixture of (5R,6S,5′R,8′R)- and (5R,6S,5′R,8′S)- 5,6:5′,8′-diepoxy-5,6,5′,8′-tetrahydro-β,β-carotene ( 1 and 2 , resp.). Therefore, its precursor is (5R,6S,5′R,6′S)-5,6:5′,6′-diepoxy-5,6,5′,6′-tetrahydro-β,β-carotene ( 4 ). This is the first identification of luteochrome as a naturally occurring carotenoid and, at the same time, gives the first clue to the as yet unknown chirality of the widespread β,β-carotene diepoxide. These facts demonstrate that the enzymic epoxidation of the β-end group occurs from the α-side, irrespective of the presence of OH groups on the ring.     

Syntheses of 6,8-disubstituted-9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphates

A series of 6,8-disubstituted-9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphates were prepared employing preformed 9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphate precursors. Three synthetic approaches were utilized to accomplish the syntheses. The first approach involved a study of the order of nucleophilic substitution, 6 vs 8, of the intermediate 6,8-dichloro-9-β-D-ribofuranosyipurine 3′,5′-cyclic phosphates ( 2 ) with various nucleophilic agents to yield 8-amino-6-chloro-, 8-chloro-6-(diethylamino)-, 6-chloro-8-(diethylamino)-, 6,8-bis-(diethylamino)- and 8-(benzylthio)-6-chloro-9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphate (4, 9, 10, 11, 13) respectively and 6-chloro-9-β-D-ribofuranosylpurin-8-one 3′,5′-cyclic phosphate ( 5 ) and 8-amino-9-β-D-ribofuranosylpurine-6-thione 3′,5′-cyclic phosphate ( 6 ). The order of substitution was compared to similar substitutions on 6,8-dichloropurines and 6,8-dichloropurine nucleosides. The second scheme utilized nucleophilic substitution of 6-chloro-8-substituted-9-β-D-ribofuranosylpurine 3′,5′-cyclic, phosphates obtained from the corresponding 8-subslituted inosine 3′,5′-cyclic phosphates by phosphoryl chloride, 6,8-bis-(benzylthio)-, 6-(diethylamino)-8-(benzylthio),8-(p-chlorophenylthio(-6-(diethylamino)- and 6,8-bis-(methyl-thio)-9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphates ( 14, 12, 20 , and 21 ) respectively, were prepared in this manner. The final scheme involved N¹-alkylation of an 8-substituted adenosine 3′,5′-cyclic phosphate followed by a Dimroth rearrangement to give 6-(benzylamino)-8-(methylthio)- and 6-(benzylamino)-8-bromo-9-β-D-ribofuranosylpurine 3′,5′-cyclic phosphate ( 24 and 25 ).     

2′, 3′-Dideoxy-3′-fluorotubercidin and Related Dideoxyribonucleosides:. Synthesis via a 2′-deoxy-3′-oxoribofuranoside intermediate and conformation studies

An efficient synthesis of the unknown 2′-deoxy-D-threo-tubercidin ( 1b ) and 2′, 3′-dideoxy-3′-fluorotubercidin ( 2 ) as well as of the related nucleosides 9a, b and 10b is described. Reaction of 4-chloro-7-(2-deoxy-β-D-erythro-pentofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine ( 5 ) with (tert-butyl)diphenylsilyl chloride yielded 6 which gave the 3′-keto nucleoside 7 upon oxidation at C(3′). Stereoselective NaBH₄ reduction (→ 8 ) followed by deprotection with Bu₄NF(→ 9a )and nucleophilic displacement at C(6) afforded 1b as well as 7-deaza-2′-deoxy-D-threo-inosine ( 9b ). Mesylation of 4-chloro-7-{2-deoxy-5-O-[(tert-butyl)diphenylsilyl]-β-D-threo-pentofuranosyl}-7H-pyrrolo[2,3-d]-pyrimidine ( 8 ), treatment with Bu₄NF (→ 12a ) and 4-halogene displacement gave 2′, 3′-didehydro-2′, 3′-dideoxy-tubercidin ( 3 ) as well as 2′, 3′-didehydro-2′, 3′-dideoxy-7-deazainosne ( 12c ). On the other hand, 2′, 3′-dideoxy-3′-fluorotubercidin ( 2 ) resulted from 8 by treatment with diethylamino sulfurtrifluoride (→ 10a ), subsequent 5′-de-protection with Bu₄NF (→ 10b ), and Cl/NH₂ displacement. ¹H-NOE difference spectroscopy in combination with force-field calculations on the sugar-modified tubercidin derivatives 1b , 2 , and 3 revealed a transition of the sugar puckering from the ^3′T_2′ conformation for 1b via a planar furanose ring for 3 to the usual ^2′T_3′ conformation for 2.     

Synthesis of certain 6-alkylthio-2,2′-anhydro-5-azauridines

β-D-Arabinofurano[1′,2′:4,5]oxazolo-s-triazin-4-one-6-thione ( 7b ) and its t-butyldimethylsilyl protected counterpart 7a were synthesized by treating the appropriate 2-amino-β-D-arabinofurano[1′,2′:4,5]-2-oxazoline with ethoxycarbonyl isothiocyanate. These 2,2′-anhydro-s-triazine nucleosides were then subjected to alkylation under similar reaction conditions. Alkylation of 3′,5′-bis(O-t-butyldimethylsilyl)-β-D-arabinofurano[1′,2′:-4,5]oxazolo-s-triazin-4-one-6-thione ( 7a ) provided the targeted S-alkylated nucleosides, i.e., the C6-SCH₃ ( 9a ), C6-SCH₂-CH = CH₂ ( 10a ), and C6-S-CH₂-C = CH ( 11a ), in reasonable yields. Attempted deprotection of these nucleosides failed. In order to circumvent this problem, 7b was alkylated with the same reagents. In each case, instead of the expected S-alkylated anhydronucleosides, a mixture of the 5-N-alkylanhydro-s-triazine-4,6-dione and 5-N-alkylanhydro-s-triazin-4-one-6-thione derivatives were obtained. The 2,2′-anhydro linkage of 7a was also found to be more stable than the s-triazine ring to mild base. Basic conditions displaced the C6-sulfur substituent and eventually caused ring opening of the s-triazine aglycone.     

Synthesis of 5-Methyluridine and Its 5′-Mercapto-, 2-Amino-, and 4′,5′-Unsaturated Analogues

The stereospecific cis-hydroxylation of 1-(2,3-dideoxy-β-D -glyceropent-2-enofuranosyl)thymine (1) into 1-β-D -ribofuranosylthymine (2) by osmium tetroxide is described. Treatment of 2′,3′-O, O-isopropylidene-5-methyl-2,5′-anhydrouridine (8) with hydrogen sulfide or methanolic ammonia afforded 5′-deoxy-2′,3′-O, O-isopropylidene-5′-mercapto-5-methyluridine (9) and 2′,3′-O, O-isopropylidene-5-methyl-isocytidine (10) , respectively. The action of ethanolic potassium hydroxide on 5′-deoxy-5′-iodo-2′,3′-O, O-isopropylidene-5-methyluridine (7) gave rise to the corresponding 1-(5-deoxy-β-D -erythropent-4-enofuranosyl)5-methyluracil (13) and 2-O-ethyl-5-methyluridine (14) . The hydrogenation of 2 and its 2′,3′-O, O-isopropylidene derivative 4 over 5% Rh/Al₂O₃ as catalyst generated diastereoisomers of the corresponding 5-methyl-5,6-dihydrouridine ( 17 and 18 ).     

(±)-5′-Nor ribofuranoside carbocyclic guanosine

The synthesis of the guanine derivative (±)-2-amino-1,9-dihydro-9-[(1′α,2′β,3′β,4′α)-(2′,3′,4′-trihydroxy-1′-cyclopentyl]-6H-purin-6-one ( 2 ) is described. This compound is viewed as the carbocyclic ribofuranoside guanine nucleoside analogue lacking the 5′-methylene.     

Carotinoide mit 7-Oxabicyclo[2.2.1]heptyl-Endgruppen. Teil II. Synthese von (2S,5R,6S,2′S,5′R,6′S)-2,5:2′5′-Diepoxy-5,6,5′,6′-tetrahydro-β,β-carotin

Carotenoids mit 7-Oxabicyclo[2.2.1]heptyl-End Groups. Synthesis of (2S,5R,6S,2′S,5′R,6′S)-2,5:2′5′-Diepoxy-5,6,5′,6′-tetrahydro-β,β-carotene Mukayama's ester 6 (methyl (1S,2R,5S)-2,5-epoxy-2,6,6-trimethylcyclohexane-1-carboxylate) was transformed in a few conventional steps into the title compound 14 . Its CD curve was found to be significantly different from that of the analogous 3,6-epoxide, a fact we tentatively lake as an indication of a (weak) electronic interaction between the ring O-atom and the π-orbitals of the polyene chain.     

New heterocyclic derivatives of 1′- and 3′-amino-5′,6′,7′,8′-tetrahydro-2′-acetonaphthones

The preparation of 1′-and 3′-amino-5′,6′,7′,8′-tetrahydro-2′-acetonaphthones (IIIa and IIIb) is described, by reduction of the low temperature nitration products of 5′,6′,7′,8′-tetrahydro-2′-acetonaphtone (I). The structures of the nitro isomers (IIa and IIb), and the reduction products, IIIa and IIIb, were elucidated spectroscopically. By known reactions, a series of new heterocyclic compounds prepared from the o-aminoketones, IIIa and IIIb, resulted in two series of new heterocyclic compounds.     

Synthesis of 2′-amino-2′-deoxy-5-fluorocytidine

Acetylation of 2′-deoxy-5-fluoro-2′-trifluoroacetamidouridine with acetic anhydride in pyridine, followed by treatment with phosphorus pentasulfide in refluxing dioxane afforded 3′,5′-di-O-acetyl-2′-deoxy-5-fluoro-2′-trifluorothioacetamido-4-thiouridine ( 3 ). Treatment of 3 with methanolic sodium methoxide furnished 2′-deoxy-2′-trifluorothioacetamido-4-thiouridine ( 4 ), whereas its treatment with methanolic ammonia gave 2′-amino-2′-deoxy-5-fluorocytidine ( 5 ). An alternative approach for the preparation of this compound proceeding from 2′-trifluoroacetamidocytidine was unsuccessful, since the use of acetic anhydride in pyridine led to the replacement of the trifluoroacetyl function by an acetyl group, yielding an intermediate unsuitable for obtaining the target compound. The title compound was inactive at 1 × 10^?4 M concentration against HeLa and leukemia L1210 cells in vitro, but inhibited the in vitro growth of E. coli cells at a concentration of 1 × 10^?7 M. It was also found to be a substrate for CR/dCR deaminase partially purified from human liver, with a Km of 128 μM.     

Nucleotides. Part XLV. Synthesis of new (2′–5′)adenylate trimers,containing 5′-amino-5′-deoxyadenosine residues at the 5′-end of the oligoadenylate chain,and of its analogues,carrying a 9-[(2-hydroxyethoxy)methyl]adenine residue at the 2′-terminus

The 5′-amino-5′-deoxy-2′,3′-O-isopropylideneadenosine ( 4 ) was obtained in pure form from 2′,3′-O-isopropylideneadenosine ( 1 ), without isolation of intermediates 2 and 3 . The 2-(4-nitrophenyl)ethoxycarbonyl group was used for protection of the NH₂ functions of 4 (→7) . The selective introduction of the palmitoyl (= hexadecanoyl) group into the 5′-N-position of 4 was achieved by its treatment with palmitoyl chloride in MeCN in the presence of Et₃N (→ 5 ). The 3′-O-silyl derivatives 11 and 14 were isolated by column chromatography after treatment of the 2′,3′-O-deprotected compounds 8 and 9 , respectively, with (tert-butyl)dimethylsilyl chloride and 1H-imidazole in pyridine. The corresponding phosphoramidites 16 and 17 were synthesized from nucleosides 11 and 14 , respectively, and (cyanoethoxy)bis(diisopropylamino)phosphane in CH₂Cl₂. The trimeric (2′–5′)-linked adenylates 25 and 26 having the 5′-amino-5′-deoxyadenosine and 5′-deoxy-5′-(palmitoylamino)adenosine residue, respectively, at the 5′-end were prepared by the phosphoramidite method. Similarly, the corresponding 5′-amino derivatives 27 and 28 carrying the 9-[(2-hydroxyethoxy)methyl]adenine residue at the 2′-terminus, were obtained. The newly synthesized compounds were characterized by physical means. The synthesized trimers 25–28 were 3-, 15-, 25-, and 34-fold, respectively, more stable towards phosphodiesterase from Crotalus durissus than the trimer (2′–5′)ApApA.     

8-Aza-7-deaza-2′,3′-dideoxyguanosine: Deoxygenation of its 2′deoxy-β-D-ribofuranoside

The synthesis of 6-amino-1-(2′,3′-dideoxy-β-D -glycero-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one ( =8-aza-7-deaza-2′,3′-dideoxyguanosine; 1 ) from its 2′-deoxyribofuranoside 5a by a five-step deoxygenation route is described. The precursor of 5a, 3a , was prepared by solid-liquid phase-transfer glyscosylation which gave higher yields (57%) than the liquid-liquid method. Ammonoloysis of 3b furnished the diamino nucleoside 3c . Compound 1 was less acid sensitive at the N-glycosydic bond than 2′,3′-dideoxyguanosine ( 2 ).     

Synthesis of various 5-substituted 2′-deoxy-3′-5′-di-O-acetyluridines

The Pd(0)-catalyzed coupling reaction of β-5-iodo-2′-deoxy-3′,5′-di-O-acetyluridine with various heteroaryltrimethylstannyl compounds gave the corresponding β-5-heteroaryl-2′-deoxy-3′,5′-di-O-acetyluridines in moderate yields. This direct coupling approach for nucleosides represented an interesting alternative to the 5-heteroaryl functionalization of pyrimidines followed by the Hilbert-Johnson glycosylation reaction which often yields mixtures of the α and β anomers.     

Carbon-13 NMR spectra of nucleoside 3′,5′-cyclic phosphates. Proposition for revised signal assignments

On the basis of the data obtained from ¹³C NMR spectra of 8,2′-S-cycloadenosine 3′,5′-cyclic phosphate and other nucleoside 3′,5′-cyclic phosphate analogues, it is suggested that the published assignments of the C-3′ and C-4′ signals in nucleoside 3′,5′-cyclic phosphates should be reversed. According to the revised assignments, C-4′, which is fixed very closely to the diesterified phosphate group is markedly shielded (?12.5 to ?15 ppm), and the C-3′ signal shows a downfield shift (+6 to +8 ppm) which is comparable to that for the C-5′ signal, for all compounds so far measured when compared with the chemical shifts for the corresponding nucleosides. The 3′,5′-cyclic phosphates of thymidine and 8,2′-S-cycloadenosine, which have no α-OH group on C-2′, show similar chemical shift changes for the corresponding sugar carbons which are different from those observed for ribonucleoside derivatives.     

Synthesis of racemic and enantiomerically pure 2′-thia-2′,3′-dideoxycytidine as potential anti-hepatitis B virus agents

A novel class of nucleosides with the C₁, atom bonded to three hetero atoms was synthesized. 2′-Thia-2′,3′-dideoxycytidine was the pilot compound of this series. (±)-β-2′-Thia-1′,3′-dideoxycytidine ( 6 ) and (±)-α-2′-thia-2′,3′-dideoxycytidine ( 7 ) were synthesized from (±)-3-mercapto-1,2-propanediol. The synthesis of the enantiomerically pure 2′-thia-2′,3′-dideoxycytidines (α-D-form, β-D-form, α-1-form and β-L-form) from optically pure (S)-(2,2-dimethyl-1,3-dioxalan-yl)methyl p-toluenesulfonate ( 8 ) and its (R)-isomer 18 was also described. The preliminary biological results showed that (+)-β-D-2′-thia-2′,3′-dideoxycytidine ( 26 ) was the most active against human hepatitis B virus with an ED₅₀ of 3 μM.     

Aziridination of the uracil 5,6-olefinic bond of 3-N-3′,5′-Di-O-tribenzoyl-5-vinyl-2′-deoxyuridine

Oxidation of N-aminophthalimide with lead tetra-acetate at -50° gives N-acetoxyaminophthalimide ( 3 ) which selectively aziridinates the 5,6-double bond present in 3-N-3′,5′-di-O-tribenzoyl-5-vinyl-2′-deoxyuridine ( 1a ) to yield 2-[1′-(2′-deoxy-β-D-ribofuranosyl)]-7-(1-phthalimido)-4-N-3′,5′-di-O-tribenzoyl-6-vinyl-2,4,7-triazabicyclo[4.1.0]heptan-3,5-dione ( 5 ).     

Synthesis of 1,2′,3,3′,5′,6′-hexahydro-3-phenylspiro[isobenzofuran-1,4′-thiopyrans] and 1,2′,3,3′,5′,6′-hexahydro-3-phenylspiro-[isobenzofuran-1,4′-pyrans]

The 1,2′,3,3′,5′,6′-hexahydro-3-phenylspiro[isobenzofuran-1,4′-thiopyran] ring system ( 2a ) has been prepared from o-bromobenzoic acid. The 1,2′,3,3′,5′,6′-hexahydro-3-phenylspiro[isobenzofuran-1,4′-pyran] ring system ( 3a ) has been prepared from 2-bromobenzhydrol methyl ether. Several 3-(dimethylaminoalkyl) derivatives of both 2a and 3a were prepared by lithiation followed by alkylation.     

Folic acid analogs. Modifications in the benzene-ring region. 5. 2′,6′-diazafolic acid

The synthesis of 2′,6′-diazafolic acid was accomplished by the condensation of 2-acetylamino-4(3H)pteridinone-6-earboxaldehyde (XIV) with diethyl N-[(5-amino-2-pyrimidinyl)carbonyl]-L-glutamate (XIII) followed by reduction of the anil double bond and alkaline hydrolylic cleavage of the N²-acetyl and ethyl ester protecting groups. Intermediate XIII was prepared by starling with 5-nitro-2-styrylpyrimidine (VI) and proceeding via 5-arnino-2-styrylpyrimidine (IX). The henzyloxycarbonyl derivative of IX was prepared and oxidized to the corresponding 5-benzyloxycarbonylaminopyrimidine-2-carboxylic acid (XI). The coupling of XI with diethyl L-glutamate followed by hydrogenolysis of the henzyloxycarbonyl function afforded the desired intermediate XIII. 2′,6′-Diazafolic acid was a potent inhibitor of Streptococcus faecium and displayed marginal activity against leukemia 1,1210 in mice.