Synthesis of 1-methyl-5-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)uracil and 1-methyl-5-(3-azido-2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)uracil. The C-nucleoside isostere of 3'-azido-3'-deoxythymidine and its 2'-"up"-fluoro analogue

1-Methyl-5-(3-azido-2,3-dideoxy-beta-D-erythro-pentofuranosyl)uracil (C-AZT), a C-nucleoside isostere of the potent anti-AIDS nucleoside 3'-azido-3'-deoxythymidine (AZT), was synthesized. 1-Methyl-2'-deoxy-5'-O-tritylpseudouridine (2a) was oxidized with CrO3/pyridine/Ac2O complex to 1-methyl-5-(5-O-trityl-beta-D-glycero-pentofuranos-3-ulosyl) uracil (12a), which was selectively reduced to 1-methyl-5-(5-O-trityl-beta-D-threo-pentofuranosyl)uracil (13a). Mesylation of 13a to 14a followed by nucleophilic displacement of the mesyloxy group with azide afforded 3'-azido-2',3'-dideoxy-5'-O-trityl-1-methylpseudoridine (15a), which was detritylated to C-AZT. In a similar manner, 1-methyl-5-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil (C-FMAU, a potent antiherpetic nucleoside) was converted into the 3'-azido analogue (3'-azido-C-FMAU). Both C-AZT and 3'-azido-C-FMAU, however, did not exhibit any significant inhibitory activity against HIV in H9 cells.     

A new class of conformationally rigid analogues of 4-amino-5-halopentanoic acids, potent inactivators of gamma-aminobutyric acid aminotransferase

Recently, we found (Qiu, J.; Pingsterhaus, J. M.; Silverman, R. B. J. Med. Chem. 1999, 42, 4725-4728) that conformationally rigid analogues of the GABA aminotransferase (GABA-AT) inactivator vigabatrin were not inactivators of GABA-AT. To determine if this is a general phenomenon of GABA-AT inactivators, several mono- and di-halogen-substituted conformationally rigid analogues (7-15) of other GABA-AT inactivators, 4-amino-5-halopentanoic acids, were synthesized as potential inactivators of GABA-AT. Four of them, (+)-7, (-)-9, (+)-10, and (+)-15, were inactivators, although not as potent as the corresponding open-chain analogues. The maximal inactivation rate constants, k(inact), for the fluoro- and bromo-substituted analogues were comparable, indicating that cleavage of the C-X bond is not rate determining. Consistent with that observation is the finding that [3-(2)H]-10 exhibits a deuterium isotope effect on inactivation of 3.3, suggesting that C-H bond cleavage is the rate-determining step. The rate of inactivation of GABA-AT by the fluorinated analogue 7 is 1/15 that of inactivation by the corresponding open-chain analogue, 4-amino-5-fluoropentanoic acid (3a). Whereas inactivation by 3a releases only one fluoride ion, inactivation by 7 releases 148 fluoride ions, accounting for the less efficient inactivation rate. Inactivation leads to covalent attachment of 2 equiv of inactivator after gel filtration; upon urea denaturation, 1 equiv of radioactivity remains bound to the enzyme. This suggests that, unlike the open-chain anlogue, the conformationally rigid analogue becomes, at least partially, attached to an active-site residue. It appears that the conformational constraint has a larger effect on inactivators that inactivate by a Michael addition mechanism than by an enamine mechanism.     

5'-O-phosphonomethyl-2',3'-dideoxynucleosides: synthesis and anti-HIV activity.

5'-O-Phosphonomethylation of different pyrimidine 2',3'-dideoxynucleosides was accomplished by reaction of the latter with diethyl [(p-toylsulfonyl)oxy]methanephosphonate (1) in the presence of sodium hydride. The base-phosphonomethylated (15-19) and sugar-phosphonomethylated (8-12) derivatives could be readily distinguished by 1H and 13C NMR and MS analysis. Protection of the uracil or thymine residue with a N3-benzoyl group failed to prevent base modification. However, O4-methyl-protected 2',3'-dideoxyuridine readily afforded the 5'-O-phosphonomethylated derivative 12, which was converted to both the 2',3'-didoxyuridine analogue 27 and the 2',3'-dideoxycytidine counterpart 29. The 5'-O-phosphonomethyl derivatives of 3'-deoxythymidine (23), 2',3'-dideoxyuridine, (27), 2',3'-dideoxycytidine (29), 3'-O-methylthymidine (26), and 3'-amino-3'-deoxythymidine (28) did not show an appreciable anti-HIV activity in MT-4 cells. In contrast, the 5'-O-phosphonomethyl derivatives of 3'-deoxy-3'-fluorothymidine (24) and 3'-azido-3'-deoxythymidine (25) inhibited HIV-1 cytopathogenicity by 50% at a concentration of approximately 1 microM.     

Synthesis of 2',5'-dideoxy-2-fluoroadenosine and 2',5'-dideoxy-2,5'-difluoroadenosine: potent P-site inhibitors of adenylyl cyclase

Glycosylation of 2-fluoroadenine with the appropriate protected thioglycoside derivatives, followed by deprotection and anomer separation, produced the alpha- and beta-anomers of 2',5'-dideoxy-2-fluoroadenosine (1), 2',5'-dideoxy-2,5'-difluoroadenosine (2), and 2'-deoxy-2-fluoroadenosine (3). These were examined as P-site inhibitors of adenylyl cyclase. The presence of fluorine on the purine ring increased potency of inhibition, and the most potent compound, beta-2',5'-dideoxy-2-fluoroadenosine (1b), was 3 times more potent than beta-2',5'-dideoxyadenosine.     

Fluorinated sugar analogues of potential anti-HIV-1 nucleosides

In order to obtain agents with therapeutic indices superior to those of AZT, FLT, or D4T, several analogues of anti-HIV-1 nucleosides were synthesized. These include 2',3'-dideoxy-2',3' -difluoro-5-methyluridine (13), its arabino analogue 19, arabino-5-methylcytosine analogue 21, 3'-deoxy-2',3'-didehydro-2' -fluorothymidine (25), 3'-azido-2',3'-dideoxy-2'-fluoro-5-methyluridine (29), 2'-azido-3'-fluoro-2',3'-dideoxy-5-methyluridine (31), and 2'3'-dideoxy-2' -fluoro-5-methyluridine (37). These new nucleosides were screened for their activity against HIV and feline TLV in vitro. None of the compounds showed significant activity. It is interesting to note that such a small modification in the sugar moiety of active anti-HIV nucleosides (i.e., displacement of hydrogen by fluorine) almost completely inactivate the agents.     

Synthesis and antiviral activity of various 3'-azido, 3'-amino, 2',3'-unsaturated, and 2',3'-dideoxy analogues of pyrimidine deoxyribonucleosides against retroviruses

Various 3'-azido, 3'-amino, 2',3'-unsaturated, 2',3'-dideoxy, and 5-substituted analogues of pyrimidine deoxyribonucleosides have been prepared and tested against Moloney-murine leukemia virus (M-MULV), a mammalian T-lymphotropic retrovirus in vitro. Among these compounds, the 3'-azido analogues of thymidine, 2'-deoxy-5-bromouridine, and 2'-deoxy-5-iodouridine, the 2',3'-unsaturated analogue of thymidine and and 2'-deoxycytidine, and 2',3'-dideoxycytidine were found to be most active, with ED50 values of 0.02, 1.5, 3.0, 2.5, 3.7, and 4.0 microM, respectively. These active compounds were nontoxic to the host SC-1 cells up to 100 microM concentration. The 3'-azido analogues of thymidine and 2'-deoxy-5-bromouridine were also tested in vitro against HTLV-III/LAV/AAV ("AIDS" virus) and found to be significantly active, with ED50 values of 0.23 and 2.3 microM, respectively. The structure-activity relationships are discussed.     

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine analogues. Inactivation of monoamine oxidase by conformationally rigid analogues of N,N-dimethylcinnamylamine

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent neurotoxin and also an inactivator of monoamine oxidase (MAO). Since MPTP is a conformationally rigid analogue of N,N-dimethylcinnamylamine, other conformationally rigid analogues of N,N-dimethylcinnamylamine were synthesized and tested as inhibitors and inactivators of MAO. (E)-2-(Phenylmethylene)cyclohexanamine (5a), (E)-N,N-dimethyl-2-(phenylmethylene)cyclohexanamine (5b), 3-phenyl-2-cyclohexen-1-amine (6a), N,N-dimethyl-3-phenyl-2-cyclohexen-1-amine (6b), and (E)- and (Z)-N-methyl-3-(phenylmethylene)piperidine (7 and 8) are all inhibitors and time-dependent inactivators of MAO B, but none is as potent as MPTP. alpha-Methylation and methylation of the amino group in all cases increases the Ki value relative to that for the parent compound. Compounds 5a, 5b, 6a and 6b are highly cytotoxic, but cytotoxicity is not prevented by pretreatment of the cells with pargyline. There does not appear to be a correlation between the configuration of the N,N-dimethylcinnamylamine analogue and its potency as a MAO inactivator.     

Synthesis and antiviral activity of some 3'-C-difluoromethyl and 3'-deoxy-3'-C-fluoromethyl nucleosides

The synthesis and antiviral activity of a number of 3'-C-difluoromethyl and 3'-deoxy-3'-C-fluoromethyl nucleosides are reported. The 3'-C-difluoromethyl nucleosides 26a and 26b were obtained by treatment of the corresponding 2',5'-di-O-protected-3'-C-formyl nucleosides 25a and 25b with (diethylamino)sulfur trifluoride (DAST). Removal of the 2'-O-protecting group from 26a and subsequent reaction with DAST furnished the 2'-deoxy-2'-fluoro-beta-D-ribo-pentofuranosyl nucleoside 29. Selective fluorination with DAST of the 5'-O-protected analogues 3'-deoxy-3'-C-hydroxymethyl derivatives 13a and 13b gave the 3'-deoxy-3'-C-fluoromethyl derivatives 30a and 30b, while nonselective fluorination afforded the 2',3'-dideoxy-2'-fluoro-3'-C-fluoromethyl analogues 31a and 31b. The deprotected uracil analogue 17a was iodinated to the 5-iodouracil derivative 18. The fully deprotected fluorinated 3'-C-branched nucleosides 14-18 and 32 were evaluated for their antiviral activity. None were active against human immunodeficiency virus type-1 (HIV-1) at concentrations up to 100 microM. However, 5-iodouracil analogue 18 showed activity, comparable to that of acyclovir, against varicella zoster virus without observed cytotoxicity.     

Synthesis of a 2'-deoxyguanosine adduct of cis-2-butene-1,4-dial, a reactive metabolite of furan

Furan is a liver and kidney toxicant and a hepatocarcinogen in rodents. Its reactive metabolite, cis-2-butene-1,4-dial, reacts with nucleosides to form adducts in vitro. The reaction with 2'-deoxyguanosine generates 3-(2'-deoxy-beta-D-erythropentafuranosyl)-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one as the major reaction product. A synthetic approach to this adduct is presented in this report. The key step in this synthesis is the preparation of 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(1,2,5,6-tetrahydroxyhexan-3-yl)guanosine. Treatment of this intermediate with sodium periodate gave three reaction products: a one-substituted adduct, 2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-1-(2,5-dihydroxy-tetrahydrofuran-3-yl)guanosine; a 1,N(2)-cyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-6-hydroxy-8-formyl-5,6,7,8-tetrahydropyrimidino[1,2-alpha]purin-10(3H)-one; and the 1,N(2)-bicyclic adduct, 3-[2'-deoxy-3',5'-O-bis(tert-butyldimethylsilyl)-beta-D-erythropentafuranosyl]-3,5,6,7-tetrahydro-6-hydroxy-7-(ethane-2"-al)-9H-imidazo[1,2-alpha]purine-9-one. The one-substituted and 1,N(2)-cyclic reaction products were unstable and rearranged over time to yield the 1,N(2)-bicyclic 2'-deoxyguanosine adducts. The desired reaction product was obtained as a mixture of four diastereomers by removing the tert-butyldimethylsilyl groups with hydrogen fluoride. This synthetic approach to the cis-2-butene-1,4-dial-derived dGuo adducts confirms our previous structural characterization of the in vitro cis-2-butene-1,4-dial-dGuo reaction product. These studies demonstrate that the observed 1,N(2) bicyclic structure is the thermodynamically stable isomer, supporting our previous observations that this adduct is the major product formed in vitro. Finally, these studies provide the necessary groundwork for the preparation of oligonucleotides with site specifically incorporated cis-2-butene-1,4-dial-derived adducts.     

Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.     

Structure-activity relationships of pyrimidine nucleosides as antiviral agents for human immunodeficiency virus type 1 in peripheral blood mononuclear cells

The structure-activity relationships of several pyrimidine nucleosides related to 3'-azido-3'-deoxythymidine (AZT) were determined in human immunodeficiency virus type 1 (HIV-1) infected human peripheral blood mononuclear cells. These studies indicated that nucleosides with a 3'-azido group on the sugar ring exhibited the most potent antiviral activity. Substitution at C-5 with H, CH3, and C2H5 produced derivatives with the highest potency, whereas alkyl functions greater than C2, including bromovinyl substitution reduced the antiviral potency significantly. Changing the 3'-azido function to an amino or iodo group reduced the antiviral activity. Replacement of the uracil ring by cytosine or 5-methylcytosine produced analogues with high potency and low toxicity. Modification of the 5'-hydroxy group markedly reduced the antiviral activity. Similarly, various C-nucleoside analogues related to AZT and 2',3'-dideoxycytidine were inactive and nontoxic. From these systematic studies 3'-azido-2',3'-dideoxyuridine (5a), 3'-azido-5-ethyl-2',3'-dideoxyuridine (5c), and 3'-azido-2',3'-dideoxycytidine (7a) and its 5-methyl analogue (7b) were identified as potent and selective anti-HIV-1 agent in primary human lymphocytes.     

Fluorocarbocyclic nucleosides: synthesis and antiviral activity of 2'- and 6'-fluorocarbocyclic 2'-deoxy guanosines.

A series of four isomeric 2'- and 6'-fluorocarbocyclic guanosine analogues have been prepared and evaluated as potential anti-herpes agents. The racemic 2' beta-fluoro isomer 2-amino-1,9-dihydro- 9-[(1 alpha, 2 alpha, 3 beta, 4 alpha)-2-fluoro-3-hydroxy-4- (hydroxymethyl)cyclopentyl]-6H-purin-6-one (11a, C-AFG) and its 2' alpha-fluoro epimer 11b plus the chiral 6' beta-fluoro isomer 2-amino-1,9-dihydro-9-[[1S-(1 alpha, 2 alpha, 3 alpha, 4 beta)]- 2-fluoro-4-hydroxy-3-(hydroxymethyl)cyclopentyl]-6H-purine-6-one (11c) and its 6' alpha-fluoro epimer 11d were prepared from their respective fluoro amino diol hydrochlorides (6a,d). For comparison, the furanosyl compound 9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)guanine (17, AFG) was prepared by coupling 2-amino-6-chloropurine with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha- D-arabinofuranosyl bromide followed by base hydrolysis. The 6' alpha-fluoro derivative 11d exhibited comparable activity to that of acyclovir (ACV) against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in vitro but was greater than 30-fold more active than ACV against HSV-1 and HSV-2 in vivo in the mouse systemic model. The 2' beta-fluoro derivative (11a, C-AFG) was extremely potent in vitro against HSV-1 and HSV-2 (ID50 0.006 and 0.05 micrograms/mL) and in vivo it was greater than 2 orders of magnitude more potent than ACV against HSV-1 and 70-fold more potent against HSV-2. The 2' alpha-fluoro 11b and 6' beta-fluoro 11c isomers were much less active.     

Structure-activity relationships of gamma-MSH analogues at the human melanocortin MC3, MC4, and MC5 receptors. Discovery of highly selective hMC3R, hMC4R, and hMC5R analogues

It has been shown by extensive studies that melanotropin bioactivities are critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, and in alpha-MSH it has been demonstrated further that a reverse-turn type conformation exists at this pharmacophore. To probe the receptor active conformation of the pharmacophore His-Phe-Arg-Trp in gamma-MSH, two different series of gamma-MSH analogues have been designed and synthesized and their biological activities determined at hMC3R, hMC4R, and hMC5R. The 1st series consists of a cyclic scan using different disulfides or lactam bridges. It was found that cyclization of the native gamma-MSH around the highly conserved sequence can lead to shifts in affinity and selectivity for hMC4R instead of the hMC3R as seen in the native peptide. Furthermore, a 23-membered ring is desirable for potency (e.g., analogues 6 and 10) whereas a 26-membered ring (analogue 1, H-Tyr-Val-c[Cys-Gly-His-Phe-Arg-Trp-Cys]-Arg-Phe-Gly-NH(2) with Gly(4)) is more important for selectivity. The 2nd series is made of d-2-naphthylalanine (d-Nal(2')) scan of the gamma-MSH sequence at position 6 and 8 and the replacement of His(5) with Pro (analogue 13). Analogue 12, H-Tyr-Val-Nle-Gly-His-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a potent and selective antagonist at the hMC4R, and analogue 15, H-Tyr-Val-Nle-Gly-Aib-Phe-Arg-d-Nal(2')-Asp-Arg-Phe-Gly-NH(2), is a highly selective and potent agonist of the hMC5R. A most promising analogue is 13, H-Tyr-Val-Nle-Gly-Pro-d-Nal(2')-Arg-Trp-Asp-Arg-Phe-Gly-NH(2), which is a very potent agonist of the hMC4R, and this analogue can be further evaluated for feeding behavior and the regulation of fat stores.     

Synthesis and anti-HIV activity of several 2'-fluoro-containing pyrimidine nucleosides

Several 2'-fluoroarabino-2',3'-dideoxy- and 2'-fluoro-2',3'-unsaturated 2',3'-dideoxy pyrimidine nucleoside analogues are reported. The saturated analogues 1-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)thymine (2'-threo-FddT, 33), 1-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)uracil (2'-threo-FddU, 22) were readily prepared from the corresponding 2'-deoxy-2'-fluoroarabinosyl nucleoside analogue by radical deoxygenation of the 3'-OH. The unsaturated compounds 1-(2,3-dideoxy-2-fluoro-beta-D-glycero-pent-2-enofuranosyl)thymine (2'-Fd4T, 40) and 1-[5-O-(mono-methoxytrityl)-2-fluoro-2,3-dideoxy-beta-D-glycero-pen t-2- enofuranosyl]uracil (39) were synthesized by an elimination reaction of the O-2,3'-anhydro-2'-fluoro-lyxo derivatives under basic conditions. The cytidine analogues 28 and 41 were prepared by amination of the corresponding uridine derivatives; compounds 28 and 41 were deprotected to give 1-(2,3-dideoxy-2-fluoro-beta-D-arabinofuranosyl)cytidine (2'-threo-FddC, 29) and 1-(2,3-dideoxy-2-fluoro-beta-D-glycero-pent-2- enofuranosyl)cytosine (2'-Fd4C, 42), respectively. All of these novel compounds were evaluated in vitro against human immunodeficiency virus (HIV) (LAV isolate). 2'-threo-FddC (29) was the most active of the newly synthesized substances against HIV with an ID50 of 0.8 microgram/mL; ddC had an ID50 of 0.007 micrograms/mL. Because of its potency in the initial tests, 29 was further evaluated in both T cells and macrophage/monocyte cell lines, with several different isolates of HIV. Although 2'-threo-FddC (29) exhibited good antiviral activity in these systems it was less active than AZT in these assays. At 1 microM the inhibition of CFU-GM by 29 was found to be 35-40%; this is slightly higher than seen with AZT.     

Synthesis and structure-activity relationships of 6-substituted 2',3'-dideoxypurine nucleosides as potential anti-human immunodeficiency virus agents

In order to study the structure-activity relationships of 2',3'-dideoxypurine nucleosides as potential anti-HIV agents, various 6-substituted purine analogues have been synthesized and examined in virus-infected and uninfected human peripheral blood mononuclear cells. N6-methyl-2',3'-dideoxyadenosine (D2MeA, 7a) was initially synthesized from adenosine via 2',3'-O-bisxanthate 3. As extension of this reaction to other N6-substituted compounds failed, a total synthetic method utilizing 2',3'-dideoxyribose derivative 9 was used for the synthesis of other purine nucleosides. An acid-stable derivative of N6-methyl-2',3'-dideoxyadenosine, 2'-fluoroarabinofuranosyl analogue 32 (D2MeFA), has been synthesized from the appropriate carbohydrate 24 by condensation with N6-methyladenine 23. Among these compounds, N6-methyl derivative (D2MeA) 7a proved to be one of the most potent antiviral agents. The order of potency for the 6-substituted compounds was NHMe greater than NH2 greater than Cl approximately N(Me)2 greater than SMe greater than OH approximately NHEt greater than SH greater than NHBn approximately H. The results suggest that a bulk tolerance effect at the 6-position of the 2',3'-dideoxypurine nucleoside may dictate the antiviral activity of these compounds. Acid-stable analogue 32 (D2MeFA) was found to be 20-fold less potent than the parent compound. Both D2MeA and D2MeFA were resistant to calf intestine adenosine deaminase. The presence of a fluorine atom in the carbohydrate moiety greatly increased stability to acid, making D2MeFA a potential orally active antiviral agent that could be useful for the treatment of retroviral infections in humans.     

Synthesis, structure-activity relationships, and drug resistance of beta-d-3'-fluoro-2',3'-unsaturated nucleosides as anti-HIV Agents

Our recent studies demonstrated that d- and l-2'-fluoro-2',3'-unsaturated nucleosides (d- and l-2'-F-d4Ns) display moderate to potent antiviral activities against HIV-1 and HBV. As an extension of these findings, beta-d-3'-fluoro-2',3'-unsaturated nucleosides were synthesized as potential antiviral agents. The key intermediate (2S)-5-(1,3-dioxolan)-1-benzoyloxy-3,3-difluoropentan-2-ol 6 was prepared from 2,3-O-isopropylidene-d-glyceraldehyde 1, which was converted to 5-O-benzoxy-d-2-deoxy-3,3-difluoropentofuranosyl acetate 7 by the ring-closure reaction under acidic conditions. The acetate 7 was condensed with silylated purine and pyrimidine bases, which produced the alpha and beta isomers. The 3',3'-difluoro nucleosides were then treated with t-BuOK to give the desired 3'-fluoro-unsaturated nucleosides. We studied the structure-activity relationships of d-3'-fluoro-2',3'-unsaturated nucleosides against HIV-1 in human peripheral blood mononuclear cells, from which we found that the cytosine derivative 26 was the most potent among the synthesized compounds. To understand the mode of action and drug resistance profile, with particular regard to the role of fluorine, we performed the molecular modeling studies of the cytidine analogue d-3'F-d4C and found a good correlation between calculated relative binding energies and activity/resistance data. Our model also shows interactions of the 3'-fluorine and the 2',3' double bond, which can be correlated to the observed biological data. Differences between fluorine substitution at the 3' and 2' positions may account for the higher cross-resistance with lamivudine observed in the 2'-fluorinated series.     

Synthesis and in vitro evaluation of some modified 4-thiopyrimidine nucleosides for prevention or reversal of AIDS-associated neurological disorders

Oxygen-sulfur exchange at the C-4 carbonyl of several modified pyrimidine nucleosides, including 3'-azido-3'-deoxythymidine (AZT), is described in an effort to enhance the lipophilicity and, thereby, the delivery to the central nervous system of the sulfur analogues without compromising the anti-HIV activities of the parental structures. Preparation of 3'-azido-3'-deoxy-4-thiothymidine (3) proceeded from 4-thiothymidine (1) and utilized the same methodology developed for the initial synthesis of AZT. Thiation of 2',3'-didehydro-3'-deoxythymidine (4a) and 2',3'-didehydro-2',3'-dideoxyuridine (4c) was carried out with Lawesson's reagent on the corresponding 5'-O-benzoate esters, 4b and 4d, to give 5a and 5c, respectively. The latter, on alkaline hydrolysis, gave 2',3'-didehydro-3'-deoxy-4-thiothymidine (5b) and 2',3'-didehydro-2',3'-dideoxyuridine (5d), respectively. The same series of reactions were applied to the 5'-O-benzoate esters of 2',3'-dideoxyuridine (6a) and 3'-deoxythymidine (6b) to give 2',3'-dideoxy-4-thiouridine (7d) and 3'-deoxy-4-thiothymidine (7b), respectively. Characterization of the saturated and unsaturated thionucleosides included mass spectrometric studies. Under electron impact conditions, the thiated analogues gave more intense parent ions than the corresponding oxygen precursors. The lipophilicity of thymidine and the 3'-deoxythymidine derivatives are enhanced significantly, as indicated, by increases in corresponding P values (1-octanol-0.1 M sodium phosphate) upon replacement of the 4-carbonyl oxygens by sulfur. Compounds 5b, 5d, 7b, and 7d were evaluated for their effects on HIV-induced cytopathogenicity of MT-2 and CEM cells. Only 5b and 7b were moderately active in protecting both cell lines against the cytolytic effect of HIV. The inhibitory effects of analogues 5b, 5d, 7b, and 7d on thymidine phosphorylation by rabbit thymus thymidine kinase were evaluated. Only 3 showed moderate affinity (Ki = 54 microM) for the enzyme. The generally weak anti-HIV activities of the remaining thio analogues are consistent with correspondingly low susceptibilities to thymidine kinase phosphorylation as estimated from the respective Ki values of the synthetic nucleosides. However, the phosphorylation of the 5'-monophosphate derivatives to their respective 5'-triphosphates must also be considered in connection with the weak in vitro anti-HIV effects of these thiated compounds.     

Analogues of N-hydroxy-4-acetylaminobiphenyl as substrates and inactivators of hamster hepatic acetyltransferases

1. A series of analogues of N-hydroxy-4-acetylaminobiphenyl (1, N-OH-AAB) has been synthesized and evaluated in vitro as substrates and inactivators of hamster hepatic N,N-acetyltransferase (N,N-AT) activity. The analogues of 1 are N-arylhydroxamic acids in which an atom or small functional group has been incorporated between the phenyl rings of 1. 2. The structural and molecular properties of the atoms between the two phenyl rings had little influence on the ability of the compounds to serve as acetyl donors in the N-arylhydroxamic acid-dependent transacetylation of 4-aminoazobenzene (AAB) catalysed by N,N-AT. An exception was the SO2 analogue (6) which was inactive. 3. All of the compounds except 6 were mechanism-based inactivators (suicide inhibitors) of hamster hepatic N,N-AT. The inhibition of N,N-AT by the hydroxamic acids was irreversible. The properties of the atom or functional group between the phenyl rings had a substantial influence on the relative effectiveness of the compounds as inactivators of N,N-AT. trans-N-Hydroxy-4-acetylaminostilbene (N-OH-AAS, 7) was the most potent and effective mechanism-based inactivator among the compounds studied. The ketone analogue (2) was the least effective among the compounds that exhibited inactivating activity. 4. The presence of the nucleophile cysteine in the incubation mixtures reduced the extent of inactivation of N,N-AT by 1 and by the ether (4) analogue but had little effect on the inactivation caused by 7. The inactivation of N,N-AT by N-OH-AAS (7) does not appear to involve electrophiles that are released from the active site and subsequently become covalently bound to the enzyme.     

2',3'-Dideoxy-N6-cyclohexyladenosine: an adenosine derivative with antagonist properties at adenosine receptors

The 2',3'-dideoxy analogue of the potent A1 receptor agonist, N6-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A1 adenosine receptor. In studies on adenylate cyclase 2',3'-dideoxy-N6-cyclohexyladenosine (ddCHA) did not show agonist properties at A1 or at A2 receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A1 receptors with a Ki value of 13 microM. ddCHA competed for the binding of the selective A1 receptor antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX), to rat brain membranes with a Ki value of 4.8 microM; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A1 receptor for the alpha- and the natural beta-anomer of adenosine, the alpha-anomer of ddCHA showed a comparable affinity for the A1 receptor (K1 value 13.9 microM). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required for agonist activity at and high affinity binding to A1 adenosine receptors and for the distinction between the alpha- and beta-forms.     

Glucosamine-glycerophospholipids that activate cell-matrix adhesion and migration

Two new analogues derived from the platelet activating factor (PAF), containing glucosamine instead of the acetyl group, were synthesized, and their effect on the human keratinocyte cell line HaCaT was evaluated with respect to cytotoxicity, proliferation, adhesion, and migration. Starting with (R)-1,2-isopropylideneglycerol (3), the glycosylation acceptor 1-O-octadecyl-3-O-tert-butyldimethylsilyl-sn-glycerol (6) was synthesized in three steps. Glycosylation of 6 with the already known O-(3,4,6-tri-O-acetyl-2-deoxy-2-dimethylmaleimido-beta-D-glycopyranosyl)trichloracetimidate gave 1-O-octadecyl-2-O-(3',4',6'-tri-O-acetyl-2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-3-O-tert-butyldimethylsilyl-sn-glycerol (7). After removing the (tert-butyldimethyl)silyl (TBDMS) group with FeCl3x6H2O, phosphoryl choline was introduced, yielding [1-O-octadecyl-2-O-(2'-deoxy-2'-dimethylmaleimido-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine (2) (glucosimide-PAF). pH controlled cleavage of the amino protection group gave [1-O-octadecyl-2-O-(2'-deoxy-2'-amino-beta-D-glucopyranosyl)-sn-glycero(3)]phosphorylcholine hydrochloride (1) (glucosamine-PAF). 2 inhibited proliferation of HaCaT cells by 26% at nontoxic concentrations, while 1 increased the proliferation rate by 30% at low concentrations. At higher concentrations, both compounds showed cytotoxic properties with LD50 = 30 micromol/L (1) and LD50 = 5-6 micromol/L (2). Both 1 and 2 were potent promoters of cell adhesion and migration of HaCaT cells.