Synthesis of 3′-Trifluoromethyl Nucleosides as Potential Antiviral Agents

Abstract

1,2-Di-O-acetyl-3-deoxy-3-trifluoromethyl-5-O-benzoyl-β-D-riboruranose was synthesized from the precursor keto sugar by the use of Ruppert's reagent (CF₃SiMe₃) as the source of a nucleophilic trifluoromethyl group. Coupling of this trifluoromethyl sugar with nucleobases and elaboration gave novel deoxy and dideoxynucleosides. A single crystal X-ray analysis confirmed the structure and stereochemistry. The deoxynucleosides were converted through an elimination reaction to their dideoxydidehydro derivatives.     

Synthesis and antiviral evaluation of 3'-C-trifluoromethyl nucleoside derivatives bearing adenine as the base

3'-deoxy-3'-C-trifluoromethyl- (3), 2',3'-dideoxy-3'-C-trifluoromethyl- (5) and 2',3'-dideoxy-2',3'-didehydro-3'-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

SYNTHESIS AND ANTIVIRAL EVALUATION OF 3′-C-TRIFLUOROMETHYL NUCLEOSIDE DERIVATIVES BEARING ADENINE AS THE BASE

3′-deoxy-3′-C-trifluoromethyl- (3), 2′,3′-dideoxy-3′-C-trifluoromethyl- (5) and 2′,3′-dideoxy-2′,3′-didehydro-3′-C-trifluoromethyladenosine (6) derivatives have been synthesized and their antiviral properties examined. All these derivatives were stereospecifically prepared by glycosylation of adenine with a trifluoromethyl sugar precursor (1), followed by appropriate chemical modifications. The prepared compounds were tested for their activity against HIV, but they did not show an antiviral effect.     

Synthesis of some novel pyrazolo[3,4-d]pyrimidine derivatives as potential antimicrobial agents

The reaction of 4-hydrazino-8-(trifluoromethyl)quinoline (2) with ethoxymethylenecyanoacetate afforded ethyl 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylate (3) and that with ethoxymethylenemalononitrile afforded 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carbonitrile (5). Compounds 3 and 5 were hydrolyzed to get 5-amino-1-[8-(trifluoromethyl)quinolin-4-yl]-1H-pyrazole-4-carboxylic acid and then reacted with acetic anhydride to afford 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]pyrazolo[3,4-d]oxazin-4-one (6), which was condensed with different aromatic amines to give a series of 5-substituted 6-methyl-1-[8-(trifluoromethyl)quinolin-4-yl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones (7). Compounds 3 and 5 also reacted with formamide, urea, and thiourea affording the corresponding pyrazolo[3,4-d]pyrimidines (8-13), respectively. Structures of the products have been determined by chemical reactions and spectral studies. All compounds of the series have been screened for their antibacterial and antifungal activity studies. The results are summarized in Tables 1 and 2.     

Synthesis of trifluoromethyl analogs of vitamin K as substrates for the liver microsomal vitamin K-dependent carboxylase

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of the trifluoromethyl analogs of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) and phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone). The reduced (naphthohydroquinone) forms of the trifluoromethyl analogs of the natural vitamins had no substrate activity but were competitive inhibitors of the reaction with a Ki in the same range as the Km of the normal substrate. The oxidized form of the trifluoromethyl analogs of vitamin K also caused inhibition by a mechanism that could not be established. Under the incubation conditions utilized, fluorine was lost from the trifluoromethyl group by a process that was dithiothreitol and high pH dependent.     

A carbene-yielding amino acid for incorporation into peptide photoaffinity reagents

3-[p-[3-(Trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine has been prepared in 10 steps from p-bromobenzyl alcohol. The alcohol was converted to 3-(alpha-iodo-p-tolyl)-3-(trifluoromethyl)-3H-diazirine, which was used to alkylate N-(diphenylmethylene)glycine ethyl ester. After deprotection the amino acid was obtained with an overall yield of 18%. The D- and L-isomers of 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine have also been resolved, and 3-[p-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenyl]alanine labeled with tritium has been prepared. The advantages of using this amino acid as a building block for peptide photoaffinity reagents is discussed.     

Synthesis and studies of 3'-C-trifluoromethyl nucleoside analogues bearing adenine or cytosine as the base

3'-Deoxy-3'-C-CF3, 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine and cytidine have been synthesized. All these derivatives were prepared by glycosylation of adenine and uracil with a suitable peracylated 3-trifluoromethyl sugar precursor. The resulting protected nucleosides were subject to appropriate chemical modifications to afford the target nucleoside derivatives. Additionally, the chemical stability in acidic and neutral media of the 2',3'-dideoxy-3'-C-CF3 and 2',3'-unsaturated-3'-C-CF3 nucleoside derivatives of adenosine was compared to that of their parent nucleosides 2',3'-dideoxyadenosine (ddA) and 2',3'-dideoxy-2',3'-didehydroadenosine (d(4)A). Our results confirm that addition of a trifluoromethyl group at C-3' on such nucleoside derivatives appears to confer increased chemical stability toward acid-catalyzed cleavage of the glycosidic bond comparatively to their parent counterparts. When evaluated for their antiviral activity in cell culture experiments, two compounds, namely, 2',3'-dideoxy-3'-C-CF3-adenosine and 2',3'-dideoxy-2',3'-didehydro-3'-C-CF3-cytidine exhibited moderate anti-HBV activity with EC50 values of 10 and 5 microM, respectively.     

Optimization of lipase-catalyzed glucose ester synthesis in ionic liquids

Lipase-catalyzed esterification of glucose with fatty acids in ionic liquids (ILs) mixture was investigated by using supersaturated glucose solution. The effect of ILs mixture ratio, substrate ratio, lipase content, and temperature on the activity and stability of lipase was also studied. The highest yield of sugar ester was obtained in a mixture of 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim][TfO]) and 1-methyl-3-octylimidazolium bis[(trifluoromethyl)-sulfonyl]amide ([Omim][Tf₂N]) with a volume ratio of 9:1, while Novozym 435 (Candida antarctica type B lipase immobilized on acrylic resin) showed the optimal stability and activity in a mixture of [Bmim][TfO] and [Omim][Tf₂N] with a 1:1 volume ratio. Reuse of lipase and ILs was successfully carried out at the optimized reaction conditions. After 5 times reuse of Novozym 435 and ILs, 78% of initial activity was remained.     

Discovery,synthesis, and biological evaluation of piperidinol analogs with anti-tuberculosis activity

Direct anti-tuberculosis screening of commercially available compound libraries identified a novel piperidinol with interesting anti-tuberculosis activity and drug like characteristics. To generate a structure activity relationship about this hit a 22 member optimization library was generated using parallel synthesis. Products of this library 1-((R)-3-(4-chlorophenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl)piperidin-4-ol and 1-((S)-3-(4-(trifluoromethyl) phenoxy)-2-hydroxypropyl)-4-(4-chloro-3-(trifluoromethyl) phenyl) piperidin-4-ol demonstrated good anti-tuberculosis activity. Unfortunately, side effects were observed upon in vivo anti-tuberculosis testing of these compounds precluding their further advancement, which may be in part due to the secondary pharmacology associated with the aryl piperidinol core.     

Amino substituted analogs of 1-phenyl-3-phenylimino-2-indolones with potent galanin Gal3 receptor binding affinity and improved solubility

A series of amino analogs of 1,3-dihydro-1-phenyl-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one (1) were synthesized to improve aqueous solubility, while retaining high affinity for the human galanin Gal3 receptor. A very potent analog (9e, 1,3-dihydro-1-[3-(2-pyrrolidinylethoxy)phenyl]-3-[[3-(trifluoromethyl)phenyl]imino]-2H-indol-2-one, Ki=5 nM) shows good selectivity and solubility of 48 microg/mL at pH 7.4.     

Trifluoromethyldiazirine-Containing dUTP: Synthesis and Application in DNA/Protein Crosslinking

Abstract

The 5-[N-(4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoyl)-3-aminoallyl]-2′-deoxyuridine-5′-triphosphate was synthesized via acylation of 5-aminoallyl-2′-deoxyuridine-5′-triphosphate with 4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoate N-hydroxysuccinimide. It was used for the preparation of 30 bp ATFMD-DNA coding for promoter sequence. UV-Irradiation (365 nm) of the specific complex of this duplex and E. coli RNA polymerase leads to the effective crosslinking DNA with all protein subunits.     

Photolysis of 3-aryl-3-(trifluoromethyl)diazirines: a caveat regarding their use in photoaffinity probes.

The photolysis of 3-(4-tolyl)-3-(trifluoromethyl)diazirine in the presence of benzene, methanol, carbon tetrachloride, cyclohexane, triethylsilane, or diethylamine led to photoproducts consistent with the intermediacy of a singlet carbene. In the case of diethylamine, the photoinsertion into the N-H bond of diethylamine produced the expected adduct, 1-(diethylamino)-2,2,2-trifluoro-1-(4-tolyl)ethane. However, the base-catalyzed elimination of hydrogen fluoride from this adduct afforded an enamine, alpha-(diethylamino)-beta,beta-difluoro-4-methylstyrene, and the subsequent hydrolysis of this enamine furnished diethylamine and 2,2-difluoro-1-(4-tolyl)ethanone. This elimination and hydrolysis sequence effectively reversed the photoinsertion process. A similar photoinsertion and hydrolysis process using 3-(4-n-octylphenyl)-3-(trifluoromethyl)diazirine also produced 2,2-difluoro-1-(4-n-octylphenyl)ethanone in modest yield. These results suggest that the photoinsertion products from 3-aryl-3-(trifluoromethyl)-diazirines in biological systems may suffer similar fates limiting, in part, their utility in obtaining primary sequence data.     

Design and synthesis of novel small molecule CCR2 antagonists: Evaluation of 4-aminopiperidine derivatives

A novel N-(2-oxo-2-(piperidin-4-ylamino)ethyl)-3-(trifluoromethyl)benzamide series of human CCR2 chemokine receptor antagonists was identified. With a pharmacophore model based on known CCR2 antagonists a new core scaffold was designed, analogues of it synthesized and structure–affinity relationship studies derived yielding a new high affinity CCR2 antagonist N-(2-((1-(4-(3-methoxyphenyl)cyclohexyl)piperidin-4-yl)amino)-2-oxoethyl)-3-(trifluoromethyl)benzamide.     

1H NMR conformational study of antiherpetic C5-substituted 2'-deoxyuridines: insight into the nature of structure-activity relationships

1H NMR study and conformational analysis of a broad series of biologically important C5-substituted 2'-deoxyuridines, including alkyl, halogen, vinyl, hydroxymethyl, and hydroxy derivatives as well as nitro, formyl, trifluoromethyl, and dimethylamino substituents, is presented. A thorough analysis of chemical shifts in correlation with C5-substituent electronegativity as well as calculations by SCF semi-empirical method of the formal charge localized on C6 carbon is discussed in terms of charge distribution for electron attracting and electron donating groups. Conformation of the sugar ring is determined from proton-proton coupling constants and described in terms of pseudorotation between two main puckering domains C2'endo (S) and C3'endo (N). Generally, electron donating groups destabilise the N conformation, simultaneously decreasing the mean pseudorotation amplitude. Absolute assignments of the H5' and H5' methylene protons in 1H NMR spectra permitted the unequivocal determination of molar fractions of the three classical exocyclic C4'-C5' rotamers gauche+, trans, and gauche-, and correlation of them with the sugar ring puckering domains. Conformation about the glycosidic bond is described in terms of equilibrium between two conformational regions, anti and syn. Finally, the role of the C5-substituent in the creation of cytotoxic activity is considered on the basis of a simplified model assuming that compound activity is a function of substituent polar surface, its molecular volume, and its molecule polarity defined at the relative partition of the polar atoms.     

Phosphodiesterase inhibitors. Part 3: Design, synthesis and structure-activity relationships of dual PDE3/4-inhibitory fused bicyclic heteroaromatic-dihydropyridazinones with anti-inflammatory and bronchodilatory activity

(-)-6-(7-Methoxy-2-trifluoromethylpyrazolo[1,5-a]pyridin-4-yl)-5-methyl-4,5-dihydro-3-(2H)-pyridazinone (KCA-1490) is a dual PDE3/4 inhibitor that exhibits potent combined bronchodilatory and anti-inflammatory activity. A survey of potential bicyclic heteroaromatic replacement subunits for the pyrazolo[1,5-a]pyridine core of KCA-1490 has identified the 4-methoxy-2-(trifluoromethyl)benzo[d]thiazol-7-yl and 8-methoxy-2-(trifluoromethyl)quinolin-5-yl analogues as dual PDE3/4-inhibitory compounds that potently suppress histamine-induced bronchoconstriction and exhibit anti-inflammatory activity in vivo.     

Incorporation of 2'-deoxy-5-(trifluoromethyl)uridine and 5-cyano-2'-deoxyuridine into DNA.

In an attempt to synthesize DNA containing 2'-deoxy-5-(trifluoromethyl)uridine (1) using previously published protocols, we found that the trifluoromethyl group converted into a cyano group, resulting in DNA containing 5-cyano-2'-deoxyuridine (3). We show that nucleoside 1 can be incorporated into DNA using phosphoramidite 2 in combination with acetyl-protected deoxycytidine and phenoxyacetyl-protected purine phosphoramidites. Replacing thymidine in DNA with 1 caused a slight decrease in DNA duplex stability at pH 6.9.     

The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., B?chi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.     

Identification of new non-carboxylic acid containing inhibitors of aldose reductase

Non-carboxylic acid containing bioisosteres of (5-arylidene-2,4-dioxothiazolidin-3-yl)acetic acids, which are active as aldose reductase (ALR2) inhibitors, were designed by replacing the carboxylic group with the trifluoromethyl ketone moiety. The in vitro evaluation of the ALR2 inhibitory effects of these trifluoromethyl substituted derivatives led to the identification of two inhibitors effective at low micromolar doses. It was further confirmed that a carboxylic chain on N-3 of the thiazolidinedione scaffold is a determining requisite to obtain the highest efficacy levels; however, it is not essential for the interaction with the target enzyme and it can be replaced by different polar groups, thus obtaining less ionised or unionised inhibitors.