Synthesis and antiviral properties of some 2'-deoxy-5-(fluoroalkenyl)uridines

The following 5-substituted 2,4-dimethoxypyrimidines were synthesized: 5-(2,2,2-trichloro-1-hydroxyethyl), 5-(2,2,2-trichloro-1-fluoroethyl),5-(2,2-dichloro-1-fluorovinyl) (5), and 5-(perfluoropropen-1-yl) (a mixture of E and Z isomers, 6 and 7). Demethylation of 5 gave 5-(2,2-dichloro-1-fluorovinyl)uracil, and demethylation of the mixture of 6 and 7 gave some pure (E)-5-(perfluoropropen-1-yl)uracil. Compound 5 was converted into its 2'-deoxyribonucleoside (12) and its alpha-anomer by standard procedures. 2'-Deoxy-3,5-dilithio-3',5'-O-bis(trimethylsilyl)uridine was reacted with the appropriate fluoroalkene to give the following 5-substituted 2'-deoxyuridines in low yield (6-24%): 5-(2-chloro-1,2-difluorovinyl) (a mixture of E and Z isomers, 15 and 16, which were separated on a small scale), 5-(perfluoropropen-1-yl), 5-(perfluorocyclohexen-1-yl), and 5-(perfluorocyclopenten-1-yl). In these reactions, 2'-deoxy-5-(trimethylsilyl)uridine and 2'-deoxyuridine were also formed. The 5-substituted 2'-deoxyuridines were tested for activity against herpes simplex virus type 1. Compound 12 and the mixture of 15 and 16 had an ID50 of 20-26 micrograms/mL in Vero cells. The activity of the mixture resided in one isomer, which by analogY with the corresponding (Z)- and (E)-5-(2-bromovinyl)-2'-deoxyuridines was concluded to be the Z isomer (16).     

3-Substituted adenines. In vitro enzyme inhibition and antiviral activity.

The direct alkylation of adenine at the 3 position has been extended to produce series of 3-alkyl-, 3-allyl-, and 3-(substituted benzyl)adenines. When these compounds were tested for enzyme inhibition and antiviral activity in vitro, 3-n-pentyladenine was found to be the most active compound in inhibiting the enzyme dopamine-beta-hydroxylase, and 3-(2-bromobenzyl)adenine showed the most striking inhibition of multiplication of Vaccinia virus and of Herpes simplex virus in tissue culture.     

Synthesis and antiviral and cytostatic properties of 3'-deoxy-3'-fluoro- and 2'-azido-3'-fluoro-2',3'-dideoxy-D-ribofuranosides of natural heterocyclic bases

A series of 3'-deoxy-3'-fluoro- and 2'-azido-2',3'-dideoxy-3'-fluoro-D-ribofuranosides of natural heterocyclic bases have been synthesized with the use of universal carbohydrate precursors, viz., 1-O-acetyl-2,5-di-O-benzoyl-3-deoxy-3-fluoro-D-ribofuranose and methyl 2-azido-5-O-benzoyl-2,3-dideoxy-3-fluoro-beta-D-ribofuranoside, respectively. The cytostatic and antiviral activity of the compounds was evaluated against a variety of tumor cell lines and DNA/RNA viruses, respectively. As the most active compound, from both a cytostatic and antiviral activity viewpoint, emerged 3'-deoxy-3'-fluoroadenosine. It inhibited the proliferation of some tumor cell lines (i.e. murine leukemia L1210 and human T-lymphocyte MT-4) at a concentration of 0.2-2 micrograms/mL, and proved inhibitory to the replication of positive-stranded RNA viruses (i.e. polio, Coxsackie, Sindbis, Semliki forest), double-stranded RNA viruses (i.e. reo), and some DNA viruses (i.e. vaccinia) at a concentration of 1-4 micrograms/mL, which is well below the cytotoxicity threshold (40 micrograms/mL).     

Synthesis and antiviral activity of 3'-deoxy-3'-C-hydroxymethyl nucleosides

A series of 3'-branched-chain sugar nucleosides, in particular 3'-deoxy-3'-C-hydroxmethyl nucleosides, have been synthesized and evaluated as antiviral agents. Reaction of 1-(2,3-epoxy-5-O-trityl-beta-D-lyxo-pentofuranosyl) derivatives 12 and 13, of uracil and thymine, respectively, with 5,6-dihydro-2-lithio-5-methyl-1,3,5-dithiazine 14 afforded the corresponding 3'-functionalized nucleosides 15 and 16, respectively. Replacement of the trityl group with tertbutyldiphenylsilyl allowed high yielding hydrolysis of the 3'-function to give the 3'-deoxy-3'-C-formyl-beta-D-arabino-pentofuranosyl nucleosides 21 and 22. Desilylation afforded the 1-(3-deoxy-3-C-formyl-beta- D-lyxo-pentofuranosyl) 3',5'-O-hemiacetal nucleosides 33 and 34, respectively. Reduction of the formyl group of 21 and 22, followed by desilylation, yielded the 3'-deoxy-3'-C-(hydroxymethyl)-beta-D-arabino- pentofuranosyl) analogues 7 and 8, respectively. The uracil base moiety of 7 was converted to 5-iodouracil and then to (E)-5-(2-bromovinyl)uracil to furnish an analogue 10 of BVaraU. The 1-(3-deoxy-3-C-(hydroxymethyl)-beta-D-lyxo-pentofuranosyl) and 1-(2,3-dideoxy-3-C-(hydroxymethyl)-beta-D-erythro-pentofuranosyl) derivatives of uracil (31 and 6, respectively) and 5-iodouracil (32 and 9, respectively) were also obtained. All novel, fully deprotected nucleoside analogues were evaluated for antiviral activity against human immunodeficiency virus type-1, herpes simplex virus types-1 and -2, varicella zoster virus, human cytomegalovirus and influenza A. Of the compounds tested only (E)-5-(2-bromovinyl)-1-[3-deoxy- 3-C-(hydroxymethyl)-beta-D-arabino-pentofuranosyl]uracil (10) inhibited VZV (alone), but did so at concentrations well below the cytotoxicity threshold.     

(+-)-carbocyclic 5'-nor-2'-deoxyguanosine and related purine derivatives: synthesis and antiviral properties.

Beginning with 3-cyclopenten-1-ylamine hydrochloride, the 5'-nor derivatives of carbocyclic 2'-deoxyguanosine (2), 2'-deoxyadenosine (3), and 2,6-diaminopurine 2'-deoxyribofuranoside (4) have been prepared. These compounds were evaluated for antiviral potential versus herpes simplex virus, varicella-zoster virus, cytomegalovirus, vaccinia virus, vesicular stomatitis virus, and human immunodeficiency virus and found to lack activity. Also, compounds 2-4 were virtually nontoxic toward the host (human diploid fibroblast ESM and HEL) cells. These biological properties may be due to the inability of 2-4 to be phosphorylated to the requisite nucleotide level that is likely to be necessary for biological activity by correlation to carbocyclic 2'-deoxyguanosine (1), which possesses significant antiviral properties as a result of conversion to its 5'-triphosphate derivative.     

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.     

Design, synthesis, and antiviral activity of 2'-deoxy-2'-fluoro-2'-C-methylcytidine, a potent inhibitor of hepatitis C virus replication

The pyrimidine nucleoside beta-d-2'-deoxy-2'-fluoro-2'-C-methylcytidine (1) was designed as a hepatitis C virus RNA-dependent RNA polymerase (HCV RdRp) inhibitor. The title compound was obtained by a DAST fluorination of N(4)-benzoyl-1-(2-methyl-3,5-di-O-benzoyl-beta-d-arabinofuranosyl]cytosine to provide N(4)-benzoyl-1-[2-fluoro-2-methyl-3,5-di-O-benzoyl-beta-d-ribofuranosyl]cytosine. The protected 2'-C-methylcytidine was obtained as a byproduct from the DAST fluorination and allowed for the preparation of two biologically active compounds from a common precursor. Compound 1 and 2'-C-methylcytidine were assayed in a subgenomic HCV replicon assay system and found to be potent and selective inhibitors of HCV replication. Compound 1 shows increased inhibitory activity in the HCV replicon assay compared to 2'-C-methylcytidine and low cellular toxicity.     

5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.     

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.     

Polymer-bound enzyme inhibitors: synthesis, properties, and physiological relevance.

For the most part, the pharmacological effect of an enzyme inhibitor results from its interaction with a single target molecule--the target enzyme. In many cases, the inhibitor moiety can interact effectively with the enzyme without necessarily being released from the polymer carrier. In the simplest form, i.e., the inhibition of an extracellular enzyme, this interaction can be understood as being two macromolecules in a solution--a reaction that can be readily modeled in vitro. Thus, enzyme inhibitors are very suitable for preparing polymer-bound drugs for investigative purposes or therapeutical applications. The macromolecular properties of polymers impact on the pharmacokinetic behavior of the compound because of the controlled intercompartmental transport. The physiological significance of the polymer-bound enzyme inhibitors is considered herein regarding the factors controlling the biodistribution of polymers among the physiological compartments. Extracellular proteinases and lysosomal enzymes are the most easily approachable targets. Strategies for preparation of polymer-bound inhibitors are discussed.     

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.     

Diastereoselective synthesis of aryloxy phosphoramidate prodrugs of 3'-deoxy-2',3'-didehydrothymidine monophosphate

The first diastereoselective synthesis of aryloxy phosphoramidate prodrugs of 3'-deoxy-2',3'-didehydrothymidine monophosphate (d4TMP) is reported. In our approach, (S)-4-isopropylthiazolidine-2-thione 1 was used as a chiral auxiliary to introduce the stereochemistry at the phosphorus atom. In the last step of the developed reaction sequence, the nucleoside analogue d4T was introduced to a stereochemically pure phosphordiamidate which led to the formation of the almost diastereomerically pure phosphoramidate prodrugs 8a-d (≥95% de). As expected, the individually prepared diastereomers of the phosphoramidate prodrugs showed significant differences in the antiviral activity. Moreover, the difference was strongly dependent on the aryl substituent attached to the phosphoramidate moiety.     

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.     

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 antitumor and antiviral properties of 5-alkyl-2'-deoxyuridines, 3',5'-cyclic monophosphates, and neutral cyclic triesters

A series of 5-alkyl-2'-deoxyuridine 3',5'-cyclic monophosphates (5-R-cdUMP's, R = Et, i-Pr, n-Pr, n-Bu, n-Pent, n-Hex, n-Oct) was prepared and tested in culture systems as antitumor and antiviral agents in comparison to the 5-alkyl-2'-deoxyuridines (5-R-dUrd's) themselves. Only the 5-Et- and 5-n-Bu-cdUMP showed appreciable cytostatic activities against murine L1210 and human lymphoblast Raji cells (ID50 range: 28-82 micrograms/mL). 5-Et-dUrd itself was much more active (ID50 = 1.6 and 2.9 micrograms/mL). The 5-i-Pr-, and 5-n-Bu-dUrd's were inactive, but activity increased again for groups with chain lengths of five carbons or greater. 5-Et-cdUMP and 5-Et-dUrd had greatly reduced activities against deoxythymidine kinase deficient (TK-) L1210 and Raji cells. 5-Et-cdUMP evidently is not an efficient prodrug source of the corresponding 5'-monophosphate where the TK- cells are concerned. Of the 5-R-cdUMP's, 5-Et-cdUMP displayed reasonably good antiviral potency against herpes simplex types 1 and 2 (MIC50, mostly 7-70 micrograms/mL) and vaccinia virus (MIC, 70 micrograms/mL). The activity was nonetheless 10- to 100-fold less than that for 5-Et-dUrd. The other 5-R-dUrd's generally showed decreasing antiviral activity with increasing 5-R chain length. Methyl and/or benzyl neutral triesters of certain 5-R-cdUMP's were inactive as antivirals and largely inactive against tumor cells in culture. In contrast to the 5'-monophosphates, the 5-R-cdUMP's failed to inhibit thymidylate synthetase from L1210 cells.     

Improved synthesis and in vitro antiviral activities of 5-cyanouridine and 5-cyano-2'-deoxyuridine.

In order to evaluate the influence of the cyano group on the antiviral activity of pyrimidine deoxyribonucleosides, a moderate yield, unified approach to the synthesis of both 5-cyanouridine and 5-cyano-2'-deoxyuridine was developed. Thus, treatment of the appropriate acetylated 5-bromouracil nucleoside with NaCN or KCN in Me2SO at 90-110 degrees C gave, after deblocking, 35-45% yields of the corresponding 5-cyanouracil nucleosides. 5-Cyanouridine was devoid of significant activity against vaccinia virus, herpes simplex-1, and vesicular stomatitis virus, but 5-cyano-2'-deoxyuridine, while lacking activity against herpes simplex, showed significant inhibition of vaccina virus; for instance, 5-cyano-2'-deoxyuridine inhibited vaccinia virus replication at concentrations 10-20 times that required for inhibition by the known antivirals, 5-iodo-2'-deoxyuridine and 1-(beta-D-arabinofuranosyl)adenine. Replacement of the 5-halogeno substituents of pyrimidine deoxyribonucleosides thus decreases, but does not abolish, antiviral activity.