Synthesis, antiviral activity, and mechanism of drug resistance of D- and L-2',3'-didehydro-2',3'-dideoxy-2'-fluorocarbocyclic nucleosides

Carbocyclic nucleosides have received much attention due to their interesting biological activity and metabolic stability. Among nucleoside analogues, a fluorine substitution on the carbohydrate moiety or introduction of a 2',3'-unsaturated structure motif has been proven to be successful in producing effective antiviral agents. By combining these structural features, both D- and L-2',3'-dideoxy-2',3'-didehydro-2'-fluoro-carbocyclic nucleosides (D- and L-2'F-C-d4Ns) were synthesized as potential anti-HIV agents. The target D- and L-carbocyclic nucleosides were both stereospecifically synthesized from D-ribose. The structure-activity relationships of synthesized compounds against HIV-1 in activated human peripheral blood mononuclear (PBM) cells were studied, from which we found that the L-2',3'-dideoxy-2'3'-didehydro-2'-fluoroadenosine analogue (L-2'F-C-d4A) 46 showed potent anti-HIV activity (EC50 = 0.77 microM), although it is cross-resistant to the lamivudine-resistant variant (HIV-1M184V). Modeling studies demonstrated a good correlation between calculated relative binding energies and activity/resistance data. The modeling study also indicated that an additional hydrogen bond and a favorable van der Waals interaction contribute to the higher antiviral activity of L-2'F-C-d4A in comparison to its D-counterpart. Also, like other L-nucleosides, the unfavorable steric hindrance of the sugar moiety of L-2'F-C-d4A and the side chain of Val184 could explain the cross-resistance of L-2'F-C-d4A with the M184V mutant. The significant difference of antiviral activity between carbovir and its analogue L-2'F-C-d4G 25 may be due to distortion of the phenyl ring of Tyr115 in the L-2'F-C-d4G-TP/HIV-RT complex, which resulted in a poor pi-pi interaction.     

Synthesis and anti-HIV evaluation of 2',3'-dideoxyribo-5-chloropyrimidine analogues: reduced toxicity of 5-chlorinated 2',3'-dideoxynucleosides

In view of the selective anti-HIV activity of 2',3'-dideoxy-3'-fluoro-5-chlorouridine (11), a series of eight 2',3'-dideoxy-5-chloropyrimidines were synthesized and evaluated for their inhibitory activity against human immunodeficiency virus type 1 (HIV-1) replication in MT-4 cells. A marked improvement in selectivity was noted for the 5-chlorouracil derivatives of 2,3-dideoxyribofuranose, 3-azido-2,3-dideoxyribofuranose, and 3-fluoro-2,3-dideoxyribofuranose, mainly due to decreased toxicity of the compounds for the host cells. While chlorination of 2',3'-dideoxycytidine removed the anti-HIV activity, introduction of a chlorine at the C-5 position of 3'-fluoro-, 3'-azido- or 2',3'-didehydro-2',3'-dideoxycytidine led to reduced cytotoxicity with only slightly reduced anti-HIV activity. X-ray analysis showed compound 11 to have two molecules in the asymmetric unit with chi = -168.8 (3) degrees and -131.3 (3) degrees and P = 179 (1) degree and 163 (1) degree, respectively; thus revealing no close resemblance to 3'-azido-3'-deoxythymidine (AZT).     

Fluorinated pyrimidine nucleosides. 4. Synthesis and antitumor testing of a series of 2',5'-dideoxy- and 2',3',5'-trideoxynucleosides of 5-fluorouracil

Dideoxy- and trideoxynucleosides of 5-fluorouracil have been synthesized for antitumor evaluation. 2',5'-Dideoxy-5-fluorouridine (3) was prepared from 2'-deoxy-5-fluorouridine (1) by iodination using methyltriphenoxyphosponium iodide, followed by catalytic reduction. 1-(2',5'-Dideoxy-beta-D-threo-pentofuranosyl)5-fluorouracil (4) was prepared from 3 by mesylation, followed by alkaline hydrolysis. 2',3',5'-Trideoxy-5-fluorouridine (13), a methyl homologue of Ftorafur (17), was synthesized by two routes: Treatment of the 3'-mesylate 8 with potassium tert-butoxide yielded the 2',3'-unsaturated derivative 12, which on hydrogenation yielded 13. Alternatively, treatment of 1 with a large excess of methyltriphenoxyphosphonium iodide produced several products, including two 3'-epimeric diiodo compounds (14 and 15), each of which could be hydrogenated to 13. The major product from this iodination reaction was characterized 3-(2',3'-anhydro-2',5'-dideoxy-5'-iodo-beta-D-threo-pentofuranosyl)-5-fluorouracil (5), presumably produced by rearrangement of the corresponding 1-isomer 9. The dideoxy compounds 3 and 4, as well as the trideoxy compound 13, were tested against sarcoma 180 in mice in comparison with 5-flourouracil, FUDR (1), and Ftorafur (17).     

Synthesis and biological evaluation of novel 2',3',4'-triply branched carbocyclic nucleosides as potential antiviral agents

Novel 2',3',4'-triply branched carbocyclic nucleosides were synthesized in this study. The introduction of two methyl groups in the 2'- and 3'-position was accomplished by a Horner-Wadsworth-Emmons reaction and isopropenyl magnesium bromide addition, respectively. The construction of the required 4'-quaternary carbon was carried out using a [3,3]-sigmatropic rearrangement. Bis-vinyls were successfully cyclized using a Grubbs' catalyst II. The natural bases (adenine, cytosine) were efficiently coupled using a Pd(0) catalyst. The antiviral activities of the synthesized compounds were evaluated against HIV-1, HSV-1, HSV-2 and HCMV. Compound 30 displayed moderate anti-HCMV activity (EC50 = 30.1 microg/mL), without exhibiting any cytotoxicity at up to 100 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.     

cycloSal-Pronucleotides of 2',3'-dideoxyadenosine and 2', 3'-dideoxy-2',3'-didehydroadenosine: synthesis and antiviral evaluation of a highly efficient nucleotide delivery system

The synthesis, hydrolysis, and antiviral evaluation of novel, lipophilic cycloSal-ddAMP (9a-d) and cycloSal-d4AMP (10a-d) derivatives of the antiviral purine dideoxynucleoside analogues 2', 3'-dideoxyadenosine (ddA) (2) and 2',3'-dideoxy-2', 3'-didehydroadenosine (d4A) (3) are reported. These potential pronucleotides release ddAMP (7) or d4AMP (8) selectively by a controlled, chemically induced tandem reaction. All new compounds 9 and 10a-d were synthesized in good yields using our previously reported phosphorus(III) method starting from substituted salicyl alcohols 14a-h. The phosphotriesters 9 and 10 were obtained with a stereochemical preference of 2:1 with respect to the configuration at the phosphorus center. In an 1-octanol/water mixture phosphotriesters 9 and 10 exhibited 7-43-fold higher lipophilicity than the parent nucleosides ddA (2) and d4A (3) as judged by their log P values. In hydrolysis studies, 9 and 10 decomposed under mild aqueous basic conditions releasing solely ddAMP (7) and d4AMP (8), as well as the diols 14. Further hydrolysis studies under acidic conditions showed a marked increase in stability with respect to the acid-catalyzed cleavage of the glycosyl bond. Phosphotriesters 9 and 10 exhibited antiviral potencies against wild-type HIV-1 and HIV-2 strains in human T-lymphocyte (CEM/O) cells that were, respectively, 100- and 600-fold higher than those of ddA (2) and d4A (3). Furthermore, all triesters 9 and 10 were markedly more active than the corresponding ddI compounds 11 and 12, which supports the concept of the delivery of the adenine nucleotides. Studies with adenosine deaminase (ADA) and adenosine monophosphate deaminase (AMPDA) showed that the triesters were not substrates for enzymatic deamination. The studies reported herein demonstrate conclusively that the cycloSal triesters deliver exclusively the nucleotides ddAMP and d4AMP, not only under chemical-simulated hydrolysis but also under intracellular conditions fulfilling the adenosine deaminase bypass premise.     

Synthesis and biological activity of 5-fluoro-2',3'-dideoxy-3'-fluorouridine and its 5'-phosphate

5-Fluoro-2',3'-dideoxy-3'-fluorouridine (3'-FFdUrd) and 5-fluoro-2',3'-dideoxy-3'-fluorouridine 5'-phosphate (3'-FFdUMP) have been synthesized, and their interactions with thymidine (dThd) phosphorylase and thymidylate (dTMP) synthetase, respectively, have been examined. 3'-FFdUrd is not a substrate for dThd phosphorylase, but is a weak, noncompetitive inhibitor (Ki = 1.7 mM). 3'-FFdUMP inhibits dTMP synthetase competitively with deoxyuridylate (Ki = 0.13 mM) when both the substrate and inhibitor are present simultaneously. However, in the presence of 5,10-methylenetetrahydrofolate, the inhibition increases with time in a first-order manner (konobsd = 0.029 s-1). A complex is formed between [6-3H]3'-FFdUMP and dTMP synthetase, which is isolable on nitrocellulose filters, and has a dissociation rate (koffobsd = 1.4 X 10(-2) min-1) similar to that of the potent inhibitor 5-fluoro-2'-deoxyuridylate (koffobsd = 1.3 X 10(-2) min-1) from its ternary complex with dTMP synthetase. These results are explained in terms of a two-stage model involving the initial formation of a reversible adsorption complex, followed by a slow conversion to a tight-binding catalytic complex.     

Stereoselective synthesis and antiviral activity of D-2',3'-didehydro-2',3'-dideoxy-2'-fluoro-4'-thionucleosides

As 2',3'-didehydro-2',3'-dideoxy-2'-fluoronucleosides have exhibited interesting antiviral effects against HIV-1 as well as HBV, it is of interest to synthesize the isosterically substituted 4'-thionucleosides in which 4'-oxygen is replaced by a sulfur atom. To study structure-activity relationships, various pyrimidine and purine nucleosides were synthesized from the key intermediate (2R,4S)-1-O-acetyl-5-O-(tert-butyldiphenylsilyl)-2,3-dideoxy-2-fluoro-2-phenylselenyl-4-thio-beta-D-ribofuranoside 8, which was prepared from the 2,3-O-isopropylidene-D-glyceraldehyde 1 in 13 steps. The antiviral activity of the synthesized compounds were evaluated against HIV-1 in human peripheral blood mononuclear (PBM) cells, among which cytidine 17, 5-fluorocytidine 18, adenosine 24, and 2-fluoroadenosine 32 showed moderate to potent anti-HIV activities (EC(50) 1.3, 11.6, 8.1, and 1.2 microM, respectively). It is noteworthy that 2-fluoroadenosine analogue 32 showed antiviral potency as well as high cytotoxicity (IC(50) 1.5, 1.1, and 7.6 microM for PBM, CEM, and Vero, respectively) whereas no other compound showed cytotoxicity up to 100 microM. The cytidine 17 and 5-fluorocytidine 18 analogues showed significantly decreased antiviral activity against the clinically important lamivudine-resistant variants (HIV-1(M184V)), whereas the corresponding D-2'-Fd4 nucleosides showed limited cross-resistance. Molecular modeling studies demonstrated that the larger van der Waals radius as well as the close proximity to Met184 of the 4'-sulfur atom of D-2'-F-4'-Sd4C (17) may be the reasons for the decreased antiviral potency of synthesized 4'-thio nucleosides against the lamivudine-resistant variants (HIV-1(M184V)).     

2'-azido-2',3'-dideoxypyrimidine nucleosides. Synthesis and antiviral activity against human immunodeficiency virus

A series of four 2'-azido-2',3'-dideoxypyrimidine nucleosides were synthesized and their activity against human immunodeficiency virus was explored. 2,2'-Anhydro-5-O-benzoyluridine (6a) was prepared from 5-O-benzoyluridine (5a) and converted into 3'-deoxy analogue 8a by imidazolylthiocarbonylation followed by Bu3SnH reduction. Treatment of 8a with LiN3 in DMF followed by saponification afforded 2'-azido-2',3'-dideoxyuridine (1a). The 5'-O-benzoylated nucleoside 9a was further converted into the 5-bromo and 5-iodo analogues (1b and 1c) by halogenation and debenzoylation. 2',3'-O-Isopropylideneuridine (3) was converted in two steps into the thymine nucleoside, which was benzoylated and de-O-isopropylidenated to afford 5'-O-benzoyl-5-methyluridine (5d). 2'-Azido-2',3'-dideoxy-5-methyluridine (1d) was synthesized from 5d in a similar manner as that used for the synthesis of 1a from 5a. These new nucleosides, closely related to AZT, however, did not exhibit any significant anti-HIV activity in tissue culture using H9 cells.     

Synthesis and antiviral activities of novel 2', 4'- or 3', 4'-doubly branched carbocyclic nucleosides as potential antiviral agents

In this study, a series of 2', 4-' or 3', 4'-doubly branched carbocyclic nucleosides (11, 12, 19, and 20) were synthesized from simple acyclic ketone derivatives as starting materials. The installation of the 4'-quaternary carbon needed was carried out using a [3, 3]-sigmatropic rearrangement. In addition, the introduction of a methyl group in the 2'- or 3'-position was accomplished by either Grignard reaction or Horner-Wadsworth-Emmons reaction with triethyl-2-phosphonopropionate, respectively. Bis-vinyl was successfully cyclized using a Grubbs' catalyst II. The natural bases (adenine, cytosine) were coupled efficiently using a Pd(0) catalyst. Although all the synthesized compounds were assayed against several viruses, only the cytosine analogue showed moderate antiviral activity against the human cytomegalovirus.     

Asymmetric synthesis and antiviral activities of L-carbocyclic 2', 3'-didehydro-2',3'-dideoxy and 2',3'-dideoxy nucleosides.

Asymmetric syntheses of L-carbocyclic 2',3'-didehydro-2',3'-dideoxy- and 2',3'-dideoxypyrimidine and purine nucleoside analogues were accomplished, and their anti-HIV and anti-HBV activities were evaluated. The key intermediate, (1S, 4R)-1-benzoyloxy-4-(tert-butoxymethyl)cyclopent-2-ene (7), was prepared by benzoylation of the alcohol 2, selective deprotection of the isopropylidene group of 3, followed by thermal elimination via cyclic ortho ester or deoxygenation via cyclic thionocarbonate. The target compounds were also synthesized by thermal elimination via cyclic ortho esters from protected nucleosides. It was found that L-carbocyclic 2',3'-didehydro-2',3'-dideoxyadenosine (34) exhibited potent anti-HBV activity (EC(50) = 0.9 microM) and moderate anti-HIV activity (EC(50) = 2.4 microM) in vitro without cytotoxicity up to 100 microM.     

Synthesis and antiviral evaluation of nucleosides of 5-methylimidazole-4-carboxamide

Due to the antiviral activity of certain 5-substituted imidazole nucleosides related to ribavirin, 5-methylimidazole-4-carboxamide nucleosides having beta-D-ribofuranosyl, 2-deoxy-beta- and -alpha-D-ribofuranosyl, and (2-hydroxyethoxy)methyl moieties have been prepared and tested as antiviral agents. 1-beta-D-Ribofuranosyl-5-methylimidazole-4-carboxamide was obtained by deacetylation of the corresponding tri-O-acetyl nucleoside 11 or by deacetylation and ammonolysis of the blocked ethyl 5-methylimidazole-4-carboxylate nucleoside 10, which was prepared from the stannic chloride catalyzed condensation of the trimethylsilyl derivative of ethyl 4(5)-methylimidazole-5(4)-carboxylate. Glycosylation of 4(5)-methylimidazole-5(4)-carboxamide with 3,5-di-O-p-toluoyl-2-deoxy-D-erythro-pentofuranosyl chloride via mercuric cyanide method provided an anomeric mixture of the blocked 5-methylimidazole-4-carboxamide deoxynucleoside 14 along with an anomeric mixture of the 4-methyl 5-carboxamide isomer 15. Separation of compound 14 into the corresponding beta and alpha anomers was achieved by conversion to the 3',5'-di-O-acetyl derivatives 17 and 18, which after chromatographic separation were deacetylated to give 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-5-methylimidazole-4-carboxa mid e and its alpha anomer 20. 1-[(2-Hydroxyethoxy)methyl]-5-methylimidazole-4-carboxamide was prepared by alkylation of the imidazole 13 with (2-acetoxyethoxy)methyl bromide followed by treatment with methanolic ammonia. All these imidazole nucleosides were tested in HeLa cell cultures against type 1 herpes simplex and vesicular stomatitis viruses. The ribofuranosyl derivative 12 showed a significant activity against type 1 herpes simplex virus.     

Synthesis and antiviral evaluation of novel cyclopropyl nucleosides, phosphonate nucleosides and phosphonic acid nucleosides

Novel phenyl-branched cyclopropyl nucleosides, phosphonates, and phosphonic acid analogues were designed and synthesized as potential antiviral agents. Coupling of the mesylate 10 with natural bases (U, T, C, A) and desilylation/hydrolysis afforded a series of novel cyclopropyl nucleosides 15-18. Phosphonate and phosphonic acid nucleosides 22-29 were also readily synthesized from the mesylate 21. The synthesized compounds were evaluated for their antiviral activity against various viruses such as HIV-1, HSV-1, HSV-2, and HCMV.     

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.     

Reactions of 2-acyloxyisobutyryl halides with nucleosides. Synthesis and biological evaluation of some 3"-acyl and 3',5'-diacyl derivatives of 1-beta-D-arabinofuranosylcytosine.

Previous papers in this series have described efficient syntheses of 3'-O-acyl and 3',5'-di-O-acyl and 3',5'-di-O-acyl derivatives of 2,2'-anhydro-1-(beta-D-arabinofuranosyl)cytosine hydrochloride (1,3). It has now been shown that the 2,2'-anhydro linkage in 1 and 3 can be selectively and efficiently cleaved by treatment with a mixture of pyridine and methanol giving the corresponding 3'-O-acyl derivatives of 1-beta-D-arabinofuranosylcytosine (2,4). The selective hydrolysis of the more soluble derivatives can also be achieved using either aqueous pyridine or a mixture of sodium carbonate and sodium bicarbonate in aqueous dioxane. Using the above procedures 3'-O-acyl araCs and 3',5'-di-O-acyl araCs with saturated or unsaturated ester groups containg from 2 to 22 carbon atoms have been prepared, and these substances have been evaluated for cytotoxicity and antiviral activity in tissue culture and for antitumor activity these substances have been evaluated for cytotoxicity and antiviral activity in tissue culture and for antitumor activity against L1210 leukemia in mice. Many of the compounds show high anti-L1210 activity relative to araC itself.     

Synthesis and antiviral activity of several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine, 3'-azido-2',3'-dideoxyuridine, 3'-azido-2',3'-dideoxy-5-halouridines, and 3'-deoxythymidine against human immunodeficiency virus and Rauscher-murine leukemia virus

Several 2,5'-anhydro analogues of 3'-azido-3'-deoxythymidine (AZT), 3'-azido-2'3'-dideoxyuridine (AZU), 3'-azido-2'3'-dideoxy-5-bromouridine, 3'-azido-2',3'-dideoxy-5-iodouridine, and 3'-deoxythymidine and the 3'-azido derivative of 5-methyl-2'-deoxyisocytidine have been synthesized for evaluation as potential anti-HIV (human immunodeficiency virus) agents. These 2,5'-anhydro derivatives, compounds 13-17, demonstrated significant anti-HIV-1 activity with IC50 values of 0.56, 4.95, 26.5, 27.1, and 48 microM, respectively. Compared to that of the parent compounds AZT and AZU, the respective 2,5'-anhydro analogues, compounds 13 and 14, were somewhat less active. Whereas AZT was cytotoxic with a TCID50 of 29 microM, the toxicity of the 2,5'-anhydro derivative of AZT, compound 13, was reduced considerably to a TCID50 value of greater than 100 microM. The 2,5'-anhydro analogue of 5-methyl-2'-deoxyisocytidine also demonstrated anti-HIV-1 activity with an IC50 value of 12 microM. These compounds were also evaluated against Rauscher-Murine leukemia virus (R-MuLV) in cell culture. Among them, AZT, 3'-azido-2',3'-dideoxy-5-iodouridine, 3'-azido-2',3'-dideoxy-5-bromouridine, and 2,5'-anhydro-3'-azido-3'-deoxythymidine (13) were found to be most active, with IC50 values of 0.023, 0.21, 0.23, and 0.27 microM, respectively.     

Studies on enzyme-cleavable dialkoxymethyl-cyclosaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates

Recently we reported on conceptually new enzymatically activated cycloSal-pronucleotides. Now, we developed this concept further with new compounds of this type. The basic idea is fast intracellular cleavage of a functionalized group at the cycloSal residue that results in a rapid delivery of the nucleotide and thus an intracellular enrichment of the nucleotide. The introduction of a higher alkylated acylal group, the di- iso-butyryloxymethyl group, to the aromatic ring led to the expected higher stability of these prodrugs against enzymatic cleavage but also entailed surprisingly a decrease in hydrolysis stabilities and solubility problems. For some compounds, a separation of the two diastereomeric forms ( R P or S P) was achieved. By X-ray structure analysis, the absolute configuration at the P-atom was assigned. For all separated diastereomers the ( S P) form showed better antiviral activity than the ( R P) form.